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Clinical Studies |
INSERM U-403, Hôpital E. Herriot (P.V., P.G., P.J.M., P.D.D.), Lyon; and INSERM U-149 (G.B.), Villejuif, France; and Takara Shuzo Co. (K.K.), Shiga, Japan
Address all correspondence and requests for reprints to: Prof. Pierre D. Delmas, INSERM U-403, Hôpital E. Herriot, Pavilon F, 69437 Lyon Cedex 03, France.
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Abstract |
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The aim of this study was to confirm these findings in a prospective cohort study (EPIDOS prospective study) of 7598 healthy, independently living women over 75 yr of age. One hundred and four women who sustained a hip fracture during a 22-month follow-up period were age matched with 255 controls who did not fracture. Baseline samples were collected before hip fracture for measurement of total OC and ucOC, assessed either with the HAP binding assay or directly with a new enzyme-linked immunosorbent assay (ELISA). This direct ELISA uses human recombinant noncarboxylated OC as a standard and two monoclonal antibodies, one of which was raised against the 14–30 Glu synthetic peptide. We found that the intra- and interassay variations are less than 11%, and this assay exhibits a 5% cross-reactivity with purified human bone OC, used as a source of carboxylated OC. ucOC levels measured with this ELISA correlated well with the HAP binding assay in the population of 359 elderly women (r = 0.82; P < 0.0001).
We estimated the risk of hip fracture for women with levels of ucOC in the highest quartile of values for the 255 controls. We found that increased levels of ucOC measured by ELISA were associated with increased hip fracture risk with an odds ratio (OR) of 1.9 (95% confidence interval, 1.2–3.0), and the ELISA had a greater sensitivity than the HAP assay. In contrast, total OC was not associated with hip fracture risk. After adjustment for femoral neck bone mineral density (BMD) and mobility status assessed by gait speed, ucOC still predicted hip fracture with an OR of 1.8 (1.0–3.0). Women with both femoral neck BMD in the lowest quartile and ucOC in the highest quartile were at higher risk of hip fracture, with an OR of 5.5 (2.7–11.2), than those with only low BMD or high ucOC levels.
In conclusion, we have developed a new specific ELISA for serum ucOC, with low cross-reactivity with carboxylated OC and increased specificity and sensitivity over the HAP assay. Using this new ELISA, we found that ucOC, but not total OC, predicts hip fracture risk independently of femoral neck BMD in elderly women drawn from the general population. Thus, ucOC measurement could be combined with bone mass determination to improve the assessment of hip fracture risk in elderly women.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-carboxyglutamic acid (Gla) at positions 17, 21, and 24
of the amino acid sequence, which are responsible for its high affinity
for hydroxyapatite (HAP) (3, 4). The synthesis of OC is vitamin D and
vitamin K dependent. Indeed, the promoter of the OC gene contains a
vitamin D-responsive element that directly stimulates the transcription
of OC (5), and vitamin K is necessary for the posttranslational
-carboxylation of glutamic acid residues in proosteocalcin (6).
In a cross-sectional study, serum concentrations of vitamin
K1 and menaquinones 7 and 8, two major components of
circulating vitamin K2, were significantly lower in elderly
women with hip fracture than in age-matched healthy controls (7).
Patients with spinal crush fracture were also reported to have very low
concentrations of circulating menaquinones (8). These findings suggest
that vitamin K deficiency could result in alterations in bone strength
and thus in increased risk of fracture. Warfarin, a potent vitamin K
antagonist, impairs the -carboxylation of glutamic acid, which is
reflected by an increase in the fraction of circulating
undercarboxylated OC (ucOC) (4). Interestingly, we have previously
shown that ucOC is significantly increased with age, especially in
elderly women (9). Levels return to normal values for young adults
after treatment with low doses of vitamin K1 (10). We also
showed that ucOC negatively correlated with hip bone mineral density
(BMD) (11) and that the fraction of ucOC is a powerful predictor of hip
fracture risk in a group of elderly women (12, 13). However, these
results were obtained in a small group of elderly institutionalized
women who are known to be under nutritional deficiency and, therefore,
are not representative of the general population.
In addition, in all previous studies, ucOC was assessed with indirect assays, based on the different affinities of normal carboxylated OC and ucOC for HAP or barium sulfate (3, 14, 15). These methods do not give accurate levels of the undercarboxylated fraction of OC, as a significant fraction of fully decarboxylated OC binds nonspecifically to these chemical compounds used to precipitate carboxylated OC. In addition, these techniques are cumbersome, their long term precisions are somewhat variable, and there is a clear need for a more convenient, accurate, and precise method to be used in clinical studies.
The aims of this study were to develop a direct and specific enzyme-linked immunosorbent assay (ELISA) for measuring ucOC in serum and to determine the value of serum ucOC to prospectively predict the risk of hip fracture in a large cohort of healthy independently living women over 75 yr of age (EPIDOS study). Results obtained with direct ELISA were compared to the predictive value of ucOC, assessed indirectly with the conventional HAP binding assay.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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This study is part of a large prospective study on the risk factors for hip fracture in elderly French women, named EPIDOS. During a period of 2 yr (January 1992 through December 1993), 7598 healthy female volunteers, 75 yr of age or older, were recruited from population-based lists in 5 French cities (Amiens, Lyon, Montpellier, Paris, and Toulouse). All women were ambulatory, and 90% of them were living independently. At the baseline visit, a fasting serum sample was collected, and they were given a lifestyle questionnaire and mobility tests, such as gait speed. Gait speed was assessed by the time required to perform a 6-m walk at the usual pace (average of 2 attempts). During an average 22-month follow-up, 124 women sustained a hip fracture. We then performed a nested case control study where each hip fracture patient was matched with 3 controls (C) from the same cohort according to age (±1 yr) and time of recruitment (±1.5 months). None of the controls sustained any fracture during the follow-up period. Of the 496 elderly women, 5 patients were excluded because of the presence of primary hyperparathyroidism (2 hip fracture and 1 C) or chronic hemodialysis (2 C). Patients receiving the following treatments known to influence calcium metabolism were also excluded: bisphosphonate (n = 13), fluoride (n = 6), and calcium and/or vitamin D3 (n = 52). For each hip fracture patient excluded, the 3 age-matched controls were also excluded to perform the statistical analysis. A total of 104 patients with hip fracture and 255 elderly controls were analyzed. After exclusion, 53% of hip-fractured patients were still matched with 3 controls, the remaining were associated with 2 age-matched controls.
This study was approved by the local ethical committee, and written informed consent was obtained from all participants.
Methods
Serum calcium, creatinine, albumin, and total alkaline phosphatase activity were measured by standard laboratory methods. Serum 25-hydroxyvitamin D (25OHD) was measured by a RIA after extraction (Incstar, Stillwater, MN).
Total OC
Levels were measured using an immunoradiometric assay (IRMA; ELSA-Osteo, CIS BioInternational, Gif-sur-Yvette, France) that recognizes a large N-terminal midfragment in addition to the intact molecule. This assay recognizes fully carboxylated and noncarboxylated OC with the same affinity (16).
Measurements of ucOC by the HAP binding assay
This method is based on the lower affinity of ucOC for HAP compared to fully carboxylated OC and has been previously described (14). Briefly, 250 µL serum were incubated with 7.5 mg HAP (calcium phosphate tribasic type IV, Sigma Chemical Co., St. Louis, MO) in an Eppendorf tube, mixed end over end for 1 h at 4 C, and then centrifuged (3000 rpm, 15 min). This concentration of HAP has been shown to provide the best discrimination between fully carboxylated human OC and thermically decarboxylated human OC. OC measured in the supernatant with IRMA for total OC represents a valid estimate of the undercarboxylated fraction of OC. The ucOC level was expressed either as an absolute value (nanograms per mL) or as a percentage of the total OC.
Measurements of ucOC by ELISA
Standard. Recombinant human Glu-OC (Biotechnology Research Laboratory, Takara Shuzo Co., Otsu, Shiga, Japan) was used as a standard. This recombinant protein was calibrated by amino acid analysis and by IRMA for total OC.
Monoclonal antibodies. Two monoclonal antibodies (OC4–5 and OCG3) were used in the ELISA. OC 4–5 has been obtained from immunization of BALB/c mice with 14–30 GluOC synthetic peptide linked to nitrocellulose as a carrier. OC G3 was obtained by the same method, using bovine OC linked to keyhole limpet hemocyanin as an immunogene. This antibody recognizes the 21–31 sequence of human OC, but does not distinguish Gla-OC from Glu-OC (17).
Procedure
Two micrograms of OC 4–5 in 200 µL 0.1 mol/L phosphate-buffered saline (PBS) were added in each well of a microtiter plate (MaxiSorp F16, Nunc, Roskilde, Denmark) and incubated overnight at 4 C. The wells were then washed three times with PBS and saturated with 200 µL BlockAce, Snow Brand Milk Products Co., Ltd., Sapporo, Japan (mainly consisting of skim milk) at room temperature for 2 h. This block solution was then discarded, and 100 µL of standard (human Glu-OC protein derived from recombinant DNA) or unknown samples (4-fold diluted in BlockAce; 25% in PBS) were added. After 1 h of incubation under slight agitation at room temperature, wells were washed three times with PBS, and 100 µL monoclonal antibody OCG3 (3000-fold diluted) labeled with horseradish peroxidase were added to each well. After 1 h of incubation under slight agitation at room temperature, unbound labeled antibody was removed by washing three times with PBS and 100 µL substrate solution (1 mg/mL OPD in 50 mmol/L citrate buffer, pH 5.0, and 0.01% H2O2) were added to each well. After 15 min of incubation in the dark, the reaction was stopped with 100 µL HCl (1 mol/L), and absorbance was read at 492 nm with an automated plate reader.
All serum samples were stored at -80 C until assayed, and biochemical measurements were performed in a blinded manner, i.e. without knowledge of the presence or absence of fracture.
Statistical analysis
The correlation coefficient for comparison between the indirect HAP assay for ucOC and the ELISA was calculated using linear regression analysis.
Clinical and biochemical characteristics between the two groups were assessed by ANOVA, in which the hip fracture patients were compared pairwise to the age-matched controls. The relative risks of hip fracture for increased ucOC level or total OC were estimated using the odds ratio (OR) before and after adjustments for confounding factors such as femoral neck BMD and gait speed, which were considered as continuous variables. The ORs were obtained from conditional logistic regression on matched sets to take into account the nested case-control design of the study. All analyses were performed using Statistical Analysis Software (SAS Institute, Cary, NC).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Specificity of the capture antibody for ucOC. To determine the
specificity of OC 4–5 for ucOC, competitive inhibition of
125I-labeled recombinant human Glu-OC binding by
recombinant human Glu-OC and by osteocalcin purified from human bone
that is mainly carboxylated was tested. As shown in Fig. 1
, the concentration that inhibits 50% of the binding
of 125I-labeled recombinant human Glu-OC was about 100-fold
lower for recombinant human Glu-OC (IC50 = 7 x
10-7 mol/L) than for human bone osteocalcin
(IC50 = 5 x 10-5 mol/L).
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, the estimated cross-reactivity of ELISA for human
bone OC was about 5%.
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. The detection limit of the method,
defined as being the minimum detectable concentration equivalent to
twice the SD of the zero standard, was 0.2 ng/mL. The
interassay variation evaluated by repeated measurements (n = 10)
of three sera (mean ucOC levels, 0.46, 1.7, and 2.7 ng/mL) was less
than 11.4%. The intraassay variation assessed by 10 measurements of
two sera (mean ucOC, 0.46 and 2.7 ng/mL) was less than 9%. The
recovery test was performed by adding known amounts of recombinant
human Glu-OC (0.5, 1, 2, and 4 ng/mL) to 2 serum samples. The recovery
ranged from 77–103%. The dose dilution curves of various normal sera
(samples A, B, and C) were parallel to the standard curve (Fig. 2).
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); ucOC levels measured by HAP
binding assay explained about 67% of those determined by ELISA.
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Baseline characteristics of elderly controls and patients with hip
fracture are shown in Table 2. There was no significant
difference in weight, serum calcium, creatinine, 25OHD, or total
alkaline phosphatase activity between the two age-matched groups.
Femoral neck BMD measured with dual energy x-ray absorptiometry using a
Lunar DPX+ device (Lunar, Madison, WI) was significantly decreased in
patients who subsequently sustained a hip fracture. Gait speed, a
fall-related factor, was significantly lower in the group of hip
fracture subjects. Albumin levels were also significantly decreased in
hip fracture patients compared to those in age-matched controls.
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). When ucOC levels were measured by
HAP and expressed as a percentage of the total OC (%ucOC), mean values
were higher in patients who will sustain a hip fracture than in
age-matched controls (Table 3).
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, elderly women with
baseline ucOC measured by ELISA in the highest quartile, who
represented 29% of the population, were at increased risk of hip
fracture, with an OR of 1.9. Similarly, %ucOC levels in the highest
quartile were associated with an increased hip fracture risk, with an
OR of 2. In contrast, increased levels of ucOC measured with the
conventional HAP method were not significantly associated with
increased hip fracture risk.
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Because of a slower gait speed in the EPIDOS population, which is a
significant predictor of hip fracture risk with an OR of 3.2 (CI,
1.2–10), adjustment for this parameter was also performed. After
adjustment for gait speed, ucOC (ELISA method) and %ucOC levels in the
highest quartile remained significantly associated with an increased
risk of hip fracture, with ORs of 1.9 (CI, 1.1–3.1) and 1.7 (CI,
1.0–2.8), respectively. As shown in Fig. 4, elderly
women with both low femoral neck BMD, defined by the lowest quartile,
and either ucOC (ELISA method) or %ucOC (HAP method) levels in the
highest quartile were at higher risk of hip fracture, with ORs of 5.5
(CI, 2.7–11.2) and 4.4 (CI, 2.3–8.4), respectively, than those with
only one of these two independent risk factors, i.e. with
either low BMD or high ucOC levels.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The immunoassay for ucOC uses two monoclonal antibodies; one of these recognizes specifically the 14–30 sequence in which the three glutamic acid are not carboxylated and recombinant human Glu OC as a standard. A typical test of dilution and recovery gave adequate results, indicating that this assay can be used to measure undercarboxylated OC in all serum samples. In addition, adding various amount of purified human bone OC in the ELISA, we determined that this assay consistently cross-reacts by about 5% with carboxylated OC. This figure compares well with, for example, the liver alkaline phosphatase cross-reactivity (16%) of bone-specific immunoassay recently developed (18), bone and liver isoenzymes, and carboxylated and noncarboxylated OC, differing only in posttranslational modifications.
We observed a significant correlation between undercarboxylated OC measured by immunoassay and that obtained by the HAP assay in a large population of elderly women (r = 0.81). However, HAP levels explained only 67% of the variance in the levels measured by the ELISA, suggesting that a significant proportion of molecules detected by the HAP method are different from those recognized in the immunoassay. Circulating OC is not a single 49-amino acid peptide. Actually, we recently showed that in addition to the intact molecule, several fragments of OC circulate, and that all together could represent about 50% of the total OC levels (16). In addition, it has been shown that in bone, the degree of carboxylation of the three glutamic acid residues may be different (19). Thus, partly carboxylated OC molecules with one, two, or three Gla residues could also be present in serum, adding to the heterogeneity of circulating OC. It would not be surprising if the HAP assay and the ELISA measured different OC moieties, although this has not yet been investigated. We also found that levels measured by ELISA were about 30% higher, on the average, than those determined by HAP assay. Indeed, it has been shown that the HAP binding assay underestimates ucOC levels because a significant proportion of decarboxylated OC does not bind specifically to HAP (14).
We previously found that increased ucOC levels measured with the conventional HAP method predict the subsequent risk of hip fracture in elderly institutionalized women followed prospectively for 3 yr (12, 13). However, one could argue that the results of that study may not be valid in the general population, as institutionalized women are known to be in poor nutritional and overall health status and, therefore, are more likely to be vitamin K deficient. Interestingly, in this study, we were able to confirm that although the differences observed between the two groups were small, increased levels of serum ucOC measured by a direct ELISA are still predictive of hip fracture risk in a large cohort of healthy elderly women, most of them living at home. In addition, the predictive value was independent of bone mass and mobility status, two major hip fracture risks (20, 21, 22), suggesting that ucOC measurement could be used in combination with these tests to identify a subgroup of women at very high risk for hip fracture. Indeed, we found that women with both low femoral neck BMD and high levels of ucOC were at higher risk to sustain hip fracture than women with only one of these two independent risk factors. Similar findings were obtained with levels of ucOC measured by the HAP method and expressed as a percentage of the total OC. In contrast, increased levels of ucOC measured by the HAP method and expressed as an absolute value were not consistently associated with increased hip fracture risk. This suggests that actually the direct ELISA, with increased precision and specificity, would be more sensitive to predict hip fracture risk than the indirect HAP method when results are expressed as an absolute value. As this new immunoassay is rapid (5 h) and requires a small volume of serum (25 µL), it should certainly represent an improvement over the cumbersome and serum-consuming HAP or barium sulfate indirect assays.
The mechanisms underlying the association between increased serum ucOC and increased hip fracture risk are still unknown. We previously showed in the same prospective study that total OC and bone alkaline phosphatase were not predictive of hip fracture risk, indicating that an increase in the overall bone turnover rate would not be responsible for that association (23). One of the most obvious reasons would be a decrease in the bone matrix content of OC as shown in warfarin-treated rats (4). The effect of this modification on bone metabolism is unknown, as the function of OC is unclear, although some studies have suggested a role for OC in the initiation of bone resorption (24, 25, 26), in the maintenance of the remodeling of trabecular bone (27), or, recently, in the inhibition of bone formation (28). However, although the mechanism by which ucOC is linked to bone fragility is not yet understood, increased serum ucOC levels probably reflect the low levels of both vitamin D and vitamin K that characterized the elderly population not only living in institutions (29) but also living at home (30). Thus, ucOC could represent an integrated and bone-specific index of a deficiency of those vitamins.
In conclusion, we have developed a new direct, specific, and convenient immunoassay for serum ucOC with low cross-reactivity with carboxylated OC. Using this immunoassay in a large cohort of healthy women, we have shown that ucOC, but not total OC, predicts hip fracture risk independently of bone mass in elderly women taken from the general population.
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Acknowledgments |
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Footnotes |
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Received August 29, 1996.
Revised November 19, 1996.
Accepted November 26, 1996.
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References |
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and for human bone OC 
. Results are
expressed as percentage of maximal binding (B°)
