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Articles |
1
Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland.
2
Centre for Biotechnology, University of Turku and Åbo
Akademi University, FIN-20520 Turku, Finland.
3
Department of Orthopaedics, Malmö University
Hospital, S-20502 Malmö, Sweden.
4
Institute of Biomedicine, Department of Anatomy,
University of Turku, FIN-20520 Turku, Finland.
a Address correspondence to this author at: University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Endocrinology, 7703 Floyd Curl Dr., San Antonio, TX 78284-7877. Fax 210-567-6693; e-mail kakonen{at}uthscsa.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: The two-site IFMAs were based on previously
characterized monoclonal antibodies. Assay 2 recognized intact hOC,
assays 4 and 9 measured the NH2-terminal mid-fragment and
the intact hOC. In addition, assay 9 required hOC to be
-carboxylated.
Results: A 76–79% increase of serum immunoreactive hOC was found in the postmenopausal group compared with the premenopausal group with all IFMAs. With EDTA-plasma samples, the observed increases were lower (49–65%). The hOC concentration in the postmenopausal group receiving hormone replacement therapy was 42–44% lower than that in the postmenopausal control group in both serum and EDTA-plasma samples. The depressed carboxylation in warfarin-treated patients was accompanied by lower results in assay 9. The ratio of assay 9 to assay 4 totally discriminated the warfarin-treated patients from the controls. Assay 9 showed the smallest decreases in measured hOC after storage of serum or plasma for 4 weeks at 4 °C, followed by assay 4 and assay 2. Results from the last assay were <17% of their initial values after 4 weeks of storage. No diurnal variation was observed with assay 9 as opposed to the two other IFMAs.
Conclusion: The three assays with their distinct specificity profiles (intact vs fragmented and carboxylated vs decarboxylated hOC) may provide valuable tools for investigating the significance of different hOC forms in various bone-related diseases.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-carboxylation of hOC has been indicated by Poser et al.
(1). When Ca2+ binds to the
-carboxylated glutamic acid residues in hOC, an 
-helix
structure is formed that affects the conformation
(12)(13). hOC can also interact with other
Ca2+-binding proteins or complexes in circulation
(12). In this report, we describe three hOC assays with different specificities for circulating hOC forms. The detailed characterization of the monoclonal antibodies (Mabs) used has been reported previously (14). We here report the validation of the assays using samples from pre-, peri-, and postmenopausal women as well as postmenopausal women receiving hormone replacement therapy (HRT). A panel of warfarin-treated patients was also included. The correlation to commercially available hOC assays is described. We also report on the in vitro stability and diurnal variation of the hOC immunoreactivity as measured by the three assays.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Samples and calibrators (10 µL of each) were added into wells of streptavidin-coated microtiter plates (Wallac Oy), followed by a mixture of biotinylated and Eu3+-labeled Mabs in 50 µL of Delfia® Assay Buffer (Wallac Oy). For all three assays, 200 ng/well of capture Mab was used; 200 ng/well of the appropriate tracer Mab was used for assay 4, and 100 ng/well was used for assays 2 and 9. In assays 4 and 9, the Delfia Assay Buffer contained 5 mmol/L EDTA. After shaking at 35 °C for 1 h, the plates were washed, and the fluorescence was measured as described previously (14).
The between- and within-assay reproducibility, precision profile, and analytical detection limit were determined for each assay. Studies of serum sample linearity and recovery of calibrator antigen were performed.
In the long-term stability test, serum, EDTA plasma, and lithium heparin samples from six in-house volunteers were collected and stored at -70, 4, 22, and 35 °C for 1, 2, and 4 weeks. After storage, all samples were frozen and stored at -70 °C until measured simultaneously. The freeze-thaw stability samples were frozen (-70 °C) and thawed one or four times at room temperature for 2 h, after which they were refrozen.
subjects and blood collection
Blood sample collections were in accordance with the Helsinki
Declaration of 1975 as revised in 1996. Serum and EDTA-plasma samples
were taken from 91 apparently healthy women, were allowed to stand for
30 min at room temperature, and then were centrifuged at
3000g for 20 min. The samples were aliquoted within 1 h
and stored at -70 °C. The women were divided into premenopausal
(42 ± 4 years; n = 47), perimenopausal (48 ± 3 years;
n = 11), and postmenopausal (56 ± 5 years; n = 31)
groups according to menstrual status, based on questionnaire responses,
and follicle-stimulating hormone concentration in serum samples
measured by Delfia hFSH assay (Wallac Oy). The postmenopausal group was
further divided into subjects receiving (56 ± 7 years; n =
16) or not receiving (56 ± 3 years; n = 15) HRT. All HRT
patients had received treatment for at least 6 months.
The response to treatment affecting -carboxylation of hOC was
studied using a cohort of 37 patients receiving warfarin treatment (19
women and 18 men; 73 ± 8 years) and 30 untreated controls (12
women and 18 men; 72 ± 6 years). Warfarin was taken for recurrent
thromboembolism or chronic heart disease for at least 12 months, and
the weekly dosage varied between 8.75 and 78.75 mg (mean, 33.6 mg). All
but one of the patients had therapeutic prothrombin concentrations with
an international normalized ratio of 2.0–3.8 as measured by the
Prothrombin complex test (Diagnostic Stago). Detailed
information of this panel has been reported by Obrant et al.
(15).
For diurnal variation, samples were taken from six healthy premenopausal women who worked at the University of Oulu, Finland, at 0600 (0609 ± 021, mean ± SD), at 1200 (1207 ± 012), at 1800 (1819 ± 024), and at 2400 (2402 ± 011); hOC concentrations in these samples were measured with assays 2, 4, and 9.
commercial osteocalcin assays
A subset of samples from the pre- (n = 15) and postmenopausal
(n = 15) groups and the postmenopausal group receiving HRT (n
= 6) were selected for a correlation study between two commercially
available hOC two-site IRMAs: ELSA-OST-NAT and ELSA-OSTEO (CIS bio
international) and the immunofluorometric assays (IFMAs) of this study.
statistical methods
The hOC values are given as mean ± SD. A weight of 5930
g/mol was used to calculate the molar concentrations of hOC.
ANOVA followed by the Bonferroni test was used for subject
groups in the panel of healthy controls. The Student
t-test was used for comparisons of results from the warfarin
panel, and repeated measures ANOVA was used for the stability and
diurnal variation studies. Linear regression was used to analyze the
relationship between two variable groups. P <0.05 was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The detection limits, defined as the concentration
corresponding to the mean value of 12 determinations of the zero
calibrator + 2 SD, were 0.01, 0.008, and 0.012 nmol/L for IFMAs 2, 4,
and 9, respectively. The within- and between-assay variations were
calculated with three serum samples (mean concentrations, 0.81, 3.30,
and 5.28 nmol/L, respectively, as measured with assay 2). The
within-assay CVs were <5% (n = 12) and the between-assay CVs
(n = 12) were <8% for each of the assays. The linearity of the
assays was estimated by two-, four-, and eightfold dilutions of five
serum samples with the zero calibrator (Fig. 2
). The observed increase in intact hOC may be caused by
nonspecific matrix effects or decreased proteolysis of hOC in samples
diluted with the bovine serum albumin-containing diluent used for the
calibrators. The analytical recoveries of hOC calibrator (1.69, 3.37,
and 8.43 nmol/L) added to human sera (n = 9) were 92% ± 8.2%,
91% ± 5.3%, and 95% ± 4.6% for IFMAs 2, 4, and 9, respectively.
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The results of long-term stability studies of hOC in serum,
EDTA-plasma, or lithium heparin-plasma samples are shown in Table 1
. In addition to poor stability of hOC measured by assay 2,
there was a remarkable variation between individual samples after
long-term storage. The concentration of total (assay 4) and
-carboxylated (assay 9) hOC was unchanged or slightly increased
during 4 weeks of storage at 4 °C. However, during storage at 22 or
35 °C, the stability of immunoreactive hOC as determined by assay 9
was superior to that of the total hOC assay (assay 4). The
concentration of intact hOC was significantly decreased in serum
(P = 0.037) and heparin-plasma (P =
0.0049) samples after one cycle of freezing and thawing. In EDTA-plasma
samples, this reduction was observed after four cycles
(P = 0.017). Significant reduction of hOC
immunoreactivity was observed after four cycles in heparin-plasma
samples as measured with assay 4 (P = 0.010). In other
samples, hOC immunoreactivity remained stable as measured with assays 4
and 9.
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clinical performance of the assays with healthy subjects
The mean hOC concentrations (± SD) in serum samples as measured
by IFMAs 2, 4, and 9 were 0.99 ± 0.29, 1.38 ± 0.37, and
1.21 ± 0.30 nmol/L in the premenopausal group (n = 47);
1.06 ± 0.29, 1.48 ± 0.39, and 1.31 ± 0.32 nmol/L in
the perimenopausal group (n = 11); and 1.75 ± 0.61,
2.46 ± 0.81, and 2.17 ± 0.67 nmol/L in the postmenopausal
group not receiving HRT (n = 16). On average, the hOC
concentrations in the EDTA samples were slightly lower (5.4%, 7.8%,
and 4.7%, as measured with assays 2, 4, and 9, respectively) than the
concentrations measured in the serum samples of the same individuals.
Compared with the premenopausal group, the serum hOC
concentrations in the postmenopausal group were 76%, 77%, and 79%
higher for assays 2, 4, and 9, respectively (P <0.0001).
With EDTA-plasma samples, the differences were smaller (58%, 65%, and
49%, respectively). The mean hOC concentrations for both serum and
EDTA plasma were 42–44% (P <0.0001) lower in the group
receiving HRT (n = 15) than in the postmenopausal group not
receiving HRT. The perimenopausal and postmenopausal groups differed
significantly from each other, but no significant differences between
the pre- and perimenopausal groups were observed with either serum or
EDTA-plasma samples. The correlation coefficients between assays 4 and
2 and between assays 4 and 9 were 0.97 [assay 4 = 1.346(assay 2)
+ 0.077; Sy|x = 0.156; and assay 4 =
1.147(assay 9) - 0.016; Sy|x = 0.144], and
the correlation coefficient between assays 2 and 9 was 0.96 [assay
2 = 0.813(assay 9) - 0.013; Sy|x =
0.134] when samples from the healthy control panel (n =
89) were included. The corresponding values obtained with the
EDTA-plasma samples were 0.96 [assay 4 EDTA = 1.247(assay 2 EDTA)
+ 0.138; Sy|x = 0.165], 0.97 [assay 4 EDTA
= 1.194(assay 9 EDTA) - 0.199; Sy|x =
0.140], and 0.92 [assay 2 EDTA = 0.87(assay 9 EDTA) - 0.9;
Sy|x = 0.166].
The performance of the IFMAs was also compared to two commercially available assays. The hOC concentration was 1.67 ± 0.42 nmol/L (n = 15) in the premenopausal group and 2.6 ± 0.89 nmol/L (n = 15) in the postmenopausal group as measured with ELSA-OST-NAT. The corresponding values measured by ELSA-OSTEO assay were 2.77 ± 0.66 nmol/L (n = 15) and 4.91 ± 1.5 nmol/L (n = 15), respectively. The hOC concentration obtained in the postmenopausal group was 57% higher with ELSA-OST-NAT (P = 0.0009) and 79% higher with ELSA-OSTEO (P <0.0001), and the values were decreased in the HRT group (n = 6) by 38% (P = 0.0048) and 40% (P = 0.0006), respectively. The regressions between IFMAs and IRMAs were all highly significant (P <0.0001), but clear differences in slopes were observed [assay 2 = 0.662(ELSA-OST-NAT) - 0.074; r = 0.93; Sy|x = 0.212; assay 4 = 0.51(ELSA-OSTEO) - 0.073; r = 0.99; Sy|x = 0.136; and assay 9 = 0.428(ELSA-OSTEO) + 0.027; r = 0.96; Sy|x = 0.195].
Significant diurnal variation was observed in the hOC concentrations
measured with the intact hOC-specific assay (assay 2) and with the
total hOC-specific assay (assay 4), whereas no significant variation
was measured by assay 9 (Fig. 3
).
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warfarin panel
The concentration of hOC as measured with assay 9 was lower in the
warfarin-treated patients (0.83 ± 0.64 nmol/L; n = 37) than
in the control patients (2.2 ± 1.6 nmol/L; n = 30;
P <0.0001). There were no significant differences between
the warfarin-treated patients and the controls in the amount of total
hOC measured by assay 4 (1.59 ± 1.5 vs 2.14 ± 1.79 nmol/L;
P = 0.173) or full-length hOC measured by assay 2
(1.25 ± 1.33 vs 1.31 ± 0.86 nmol/L; P =
0.816). The proportion of carboxylated hOC/total hOC was lower in the
warfarin-treated patients compared with the controls (P
<0.0001) and a single cutoff ratio of 0.8 separated the two
patient groups (Fig. 4
.). In addition, a significant (P <0.0001)
difference was observed between the carboxylated hOC/full-length hOC
ratios for the two groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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clinical performance of IFMAs
Despite the distinct specificity profiles of the three assays,
their performance in measuring hOC in the menopausal sample panel was
highly similar. The observed increase of serum hOC during the change in
menopausal status is consistent with results of other studies
(8)(23)(24). The hOC concentrations
obtained with the ELSA-OST-NAT and ELSA-OSTEO assays were higher than
the results obtained with the IFMAs. Differences in assay design or hOC
calibrator preparations may be contributing factors. Reductions of 53%
± 5% (n = 4) and 56% ± 2% (n = 4) were observed when
ELSA-OST-NAT calibrators were measured with IFMA 2 and ELSA-OSTEO
calibrators were measured with IFMA 4, respectively, compared with the
nominal values given by the manufacturer.
The diurnal rhythm of hOC has important implications for the selection of sampling times, e.g., in the monitoring of hOC changes after various pharmaceutical interventions. In agreement with previous reports, the hOC concentration was high at night and early morning and lower during the late morning and afternoon (25)(26)(27). Significant variations in the hOC concentration were observed with IFMAs 2 and 4. A similar but nonsignificant trend was seen with assay 9. This could explain the differences between the reported peak and nadir values (10–50%) with different hOC immunoassays (25)(26)(27).
the clinical significance of -carboxylated hOC
Osteocalcin binds, through its -carboxylated glutamic acid
residues, to hydroxyapatite, which has been used to distinguish the
fully -carboxylated form from the undercarboxylated (uchOC) forms
(2). The hydroxyapatite- binding capacity of circulating hOC
is abnormally low in the elderly (28). Szulc and co-workers
(29)(30) have shown that circulating uchOC is a
marker of hip fracture risk in a population of institutionalized women
and that the bone mineral density is decreased in women with increased
uchOC. Separation of the hOC forms using hydroxyapatite is difficult
because of inconsistent binding (29)(31).
Vergnaud et al. (32) have developed an ELISA for uchOC based
on Mabs with low cross-reactivity to carboxylated hOC. They showed that
uchOC predicted hip fracture risk independently of bone mass in elderly
women. However, the uchOC concentrations measured with the same assay
were not statistically different between the warfarin-treated patients
and control subjects, whereas the ratio of uchOC to intact hOC differed
significantly between the two groups (31). In our study, the
concentration of -carboxylated hOC was significantly lower in
warfarin-treated patients compared with controls (P
<0.0001). The ratio of -carboxylated to total hOC fully
distinguished patients on long-term warfarin treatment from age-matched
controls. We recently reported that the ratio of -carboxylated hOC
to total hOC as measured with IFMAs 9 and 4, respectively, predicts the
occurrence of fractures in older community-dwelling adults [Luukinen
et al., submitted for publication; and Ref. (33)].
The described three hOC IFMAs show clear differences in their recognition of various circulating hOC forms. These assays are suitable for monitoring circulating hOC in several situations affecting bone metabolism.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical structure in osteocalcin. Biochemistry 1982;21:2538-2547.
[Medline]
[Order article via Infotrieve]
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), 4 (•), and 9
(
) given as fluorescence counts vs concentrations of the hOC
calibrator over the range 0.017–13.5 nmol/L, and the corresponding
within-assay CVs (open symbols) calculated from 12
replicates.
