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From the Clinical Research Centers |
Department of Orthopaedics and Rehabilitation (C.M.G., S.D.N.), Yale University School of Medicine, New Haven, Connecticut 06510; Department of Pediatrics (S.A.), Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas 77030; and Department of Geriatrics (H.R.), Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Caren M. Gundberg, Department of Orthopaedics, Yale University School of Medicine, New Haven, Connecticut 06510. E-mail: caren.gundberg{at}yale.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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In a kit specific for uncarboxylated osteocalcin, we found good discrimination between carboxylated and uncarboxylated intact osteocalcin. However, the assay detected large osteocalcin fragments and overestimated their concentration by up to 350%. Values for uncarboxylated osteocalcin were not different in patients on coumadin compared with normal adults with this kit, but when normalized to the total intact osteocalcin, percent uncarboxylated osteocalcin was greater in patients on coumadin than in controls, as would be expected. Kit values for uncarboxylated osteocalcin in normal children were higher than intact values in the same subject, because of the increased reactivity of the kit toward circulating fragments that were elevated in children.
Thus, for estimation of undercarboxylated osteocalcin, care must be taken to standardize the hydroxyapatite or barium sulfite used for binding, to correct for the basal level of osteocalcin in the sample, to use immunoassays that do not detect small fragments, and to express the results as the percent of the total osteocalcin in the sample. Without these precautions, the assessment of undercarboxylated osteocalcin is not reliable.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent reports have found that assessment of undercarboxylated osteocalcin can be a sensitive measure of overall vitamin K nutritional status (8). Several studies have attempted to assess the degree of undercarboxylated osteocalcin in serum. Methods have been developed with the recognition that affinity of osteocalcin for hydroxyapatite (9) or barium sulfate (10) depends on the number of gla residues in the protein. The portion of immunoassayable osteocalcin that does not bind to hydroxyapatite or barium sulfate has been taken to be undercarboxylated osteocalcin. Furthermore, using these methods, studies have found that the amount of nonbound osteocalcin (and by inference undercarboxylated osteocalcin) is higher in postmenopausal women with osteoporosis than in premenopausal women (11, 12). Recently, in healthy elderly women, nonbound osteocalcin was shown to be associated with fracture risk when measured by hydroxyapatite binding assay and by an enzyme-linked immunosorbent assay (ELISA) specific for uncarboxylated osteocalcin (13).
We sought to determine the optimum conditions for measuring undercarboxylated osteocalcin. We report that undercarboxylated osteocalcin should be expressed as the percent of the total osteocalcin in the sample, and further, that even the percent undercarboxylated must be corrected for the total amount of immunoassayable osteocalcin in a particular serum sample. Furthermore, we find that for meaningful results, assays specific for intact osteocalcin must be used. Finally, we tested a new commercial kit that has been reported to be specific for uncarboxylated osteocalcin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteocalcin was purified from human bone as previously described (14). Uncarboxylated osteocalcin was produced by freeze-drying metal-free osteocalcin from a 50 mM HCl solution and heating the sample for 5 h at 110 C in vacuo. The uncarboxylated protein was then repurified by reverse phase high performance liquid chromatography (HPLC) using a C18 column (Waters, Milford, MA) with an 0–80% acetonitrile gradient in 0.1% trifluoroacetic acid. Human osteocalcin (glu 17, 21, and 24) and peptides 4–19, 11–29, 41–49, and 37–49 of the human sequence were synthesized by the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Human osteocalcin (gla 17, 21, and 24) was synthesized in the Department of Biochemistry, Albert Einstein College of Medicine. All synthetic proteins and peptides were repurified by preparative reverse- phase HPLC (RP-HPLC) as above. To facilitate intramolecular disulfide bonds in peptides containing cysteines, peptides were stirred in dilute solution (1 mg/mL) at ph 8.0 overnight.
Peptides 1–19, 21–43, and 45–49 were produced by tryptic digestion of the fully carboxylated or uncarboxylated human osteocalcin (HOC) as previously described (14). The 1–43 major peptide containing gla was generated by subjecting the fully carboxylated osteocalcin to limited tryptic digest as above, except that the duration of incubation was for only 30 min. The 1–43 peptide without gla (glu form) was generated by incubating uncarboxylated osteocalcin with cathepsin B (Calbiochem, La Jolla, CA)) at 37 C for 10 min as previously described by Price and co-workers (15). The resultant peptides from all enzymatic digestions were separated by RP-HPLC, which resolved the mixtures of peptides. Structures and concentrations of all peptides were determined by amino acid analysis and matrix-assisted laser desorption/ionization or electrospray mass spectrometry for gla-containing peptides.
Antibodies
Monoclonal antibodies were produced to peptides 1–19 and 37–49 by the method of Kohler and Milstein (16). IgG was purified on a protein-G Sepharose column. Purified monoclonal antibodies were biotinylated with sulfo-N-hydroxysuccinimido-biotin (Pierce, Rockford, IL). Polyclonal antibodies to synthetic human osteocalcin were produced in New Zealand white rabbits.
Immunoassays
RIAs were performed as described previously (14). The assay uses authentic human osteocalcin as standard and tracer. For two-site ELISA, medium binding polystyrene plates were coated overnight at 4 C with 0.5 µg/well of monoclonal antibody 640.1E (made to the 1–19 peptide) in PBS, pH 9.6. Plates were washed and then blocked with 1% BSA (RIA grade, Sigma Chemical, St. Louis, MO) at 37 C for 1 h. For assay, 10 µL control or standard were added to the wells, followed immediately with 100 µL (0.55 µg/mL) biotinylated 635–1K antibody, which was specific for the 37–49 peptide. Plates were incubated at 23 C for 1 h, then washed three times in PBS/0.01% Tween. One hundred microliters streptavidin horseradish peroxidase (Pierce) was added to each well and incubated for 30 min at 23 C. Plates were washed as above, and peroxidase substrate added to each well. After 15 min, the reaction was stopped with 100 µL of 2 M sulfuric acid, and the plates were read at 450 nm.
A commercially available kit for uncarboxylated osteocalcin (Takara Shuzo Co., Ltd. Japan) was used according to the manufacturer’s specifications.
Hydroxyapatite binding assays
Initial comparisons between synthetic and purified carboxylated or uncarboxylated human osteocalcin demonstrated no differences in hydroxyapatite binding characteristics of the comparable preparations. Therefore, synthetic preparations of the proteins were used throughout. For hydroxyapatite binding characterization studies, varying amounts of human fully carboxylated osteocalcin (3 gla) and/or uncarboxylated osteocalcin in 100 µL osteocalcin-free serum was added to 100 of a 2–40 mg/mL suspension of hydroxyapatite. The tubes were shaken vigorously for 1 h at room temperature and then centrifuged for 2 min in a microfuge (10,000 x g). The supernatant was removed, and the amount of unbound osteocalcin measured in the individual assays and compared with concentrations before hydroxyapatite binding. Two sources of hydroxyapatite were employed: Calbiochem hydroxyapatite and Sigma Chemical tricalcium phosphate type IV.
Barium sulfate binding assays
For barium sulfate binding assays, varying amounts of human fully carboxlyated osteocalcin and/or uncarboxylated osteocalcin in 100 µL osteocalcin-free serum was added to 100 µL of a 100 mg/mL barium sulfate solution (Sigma Chemical). The tubes were mixed end over end for 30 min at 4 C and then centrifuged for 2 min in a microfuge (10,000 x g) as previously described by Sokoll et al. (10). The supernatant was removed, and the amount of unbound osteocalcin measured in the individual assays and compared with assayed concentrations before barium sulfate binding.
Subjects
Serum was obtained from subjects who were recruited from a pool of patients from Yale-New Haven Hospital or Beth Israel Deaconess Medical Center, Boston, and who had been maintained on warfarin therapy for more than 1 yr. Reasons for anticoagulation included prosthetic heart valve, chronic atrial fibrillation, valvular heart disease without valve replacement, history of embolic stroke, or recurrent venous thromboembolic disease. Sera from healthy children were taken at 0800 h after an overnight fast. Children were part of ongoing studies of calcium metabolism in children of different ethnicities. Serum was obtained from normal laboratory volunteers or from archived samples from normal adults that were kept frozen at -70 C. All samples had been stored for less than 1 yr. Subjects were excluded who had any condition that affects bone density or who took medication that adversely affects bone density. All subjects and/or their parents or legal guardians gave informed consent. The protocol was approved by the Yale University Human Investigation Committee, The Committee on Clinical Investigation of the Beth Israel Deaconess Medical Center, and the Institutional Review Board of Baylor College of Medicine.
Statistical analyses
Correlations between groups were determined by Pearson’s product moment correlation. Differences between means were determined by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specificity of the antibodies used are given in Table 1
. All in-house assays are indifferent to
the presence or absence of gla residues. Monoclonal antibodies 640–1E
and 635–1K were used in a two-site enzyme-linked solid-phase assay
that recognized the intact protein. Monoclonal antibody 640–1E was
also used in a liquid-phase RIA (N-terminal). This assay displays equal
affinity for the intact molecule and smaller fragments derived from the
N-terminus (e.g. 1–19 or 1–43). The polyclonal HOC
reactivities suggest a nonlinear (conformational) epitope,
i.e. stretches of amino acids that are separated in the
primary structure have limited reactivity; but maximal reactivity when
brought into proximity when the protein is in its native form. This
antibody recognizes primarily the intact protein and the large 1–43
fragment, but also other C- and N-terminal peptides with low
affinity.
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Binding capacity of two commercial sources of hydroxyapatite and
one commercial source of barium sulfate were compared at three
different concentrations of carboxylated or uncarboxylated osteocalcin.
The HOC RIA assay was used for these initial studies. Figures 1 and 2
show the results for the two preparations of hydroxyapatite and for
barium sulfate, respectively. All three reagents bound carboxylated
osteocalcin to a greater extent than uncarboxylated osteocalcin and
could discriminate between the two. However, the amount of
hydroxyapatite needed to efficiently bind osteocalcin was dependent on
the source of the hydroxyapatite. Most striking, however, was the
difference in the absolute amount of osteocalcin bound or not bound
when the concentrations of the proteins were varied. For example, when
4, 7, or 15 ng/mL uncarboxylated osteocalcin were mixed with 10 mg
Calbiochem hydroxyapatite, 2.2, 4.2, and 10.3 ng/mL,
respectively, were not bound. The same trends were observed at all
concentration of the other binders to varying degrees. In an attempt to
minimize this effect, we expressed this data in terms of percent of the
total. Even with this correction, there were differences in the percent
not bound that were dependent on the amount of osteocalcin in the
sample.
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shows that for all
three binders, the percent unbound osteocalcin increased with the
proportion of uncarboxylated osteocalcin. However, the degree of
binding was not linearly related to the amount of carboxylated
osteocalcin in the sample. Furthermore, the binding varied with the
amount of total osteocalcin in the sample. When expressed as absolute
amount of osteocalcin not bound rather than percent of the total, this
discrepancy was even more apparent. For example, in a 1:1 mixture of 6,
10.6, 18.5, or 32.4 ng/mL carboxylated and uncarboxylated osteocalcin,
2.9, 5.6, 10.7, and 11.6 ng/mL, respectively, were not bound by Sigma
hydroxyapatite (49%, 53%, 58%, and 63%, respectively).
Therefore, data for subsequent studies are expressed as percent not
bound. To estimate the data according to the amount of total
osteocalcin in a given sample, we fit the following general equation to
Calbiochem curves similar to those shown in Fig. 3: % Uncarboxylated
osteocalcin = ((1.303 x % not bound) - (0.0261 x %
not bound2) + (0.000352 x % not
bound3)) + ((18.511) - (6.393 x total osteocalcin
in ng/mL) + (0.321 x total osteocalcin in ng/mL2) -
(0.000516 x total osteocalcin in ng/mL3).
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shows that the percent
osteocalcin not bound to hydroxyapatite generally decreased when the
amount of endogenous osteocalcin was reduced. This is consistent with
the above findings that binding varied (in absolute terms and in
percent of the total) with the amount of total osteocalcin in the
sample. The fact that the percent change was not similar in all samples
can be attributed to the contribution of two factors, i.e.
the basal levels of osteocalcin and the actual degree of carboxylation
in each sample.
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Because fragments of osteocalcin are known to circulate, we
further sought to determine whether the assay used to measure
osteocalcin would affect the results. Therefore we performed
hydroxyapatite binding experiments using Calbiochem hydroxyapatite at 2
mg/mL. We assayed the total and nonbound osteocalcin by RIA, the intact
assay, and the N-terminal assay. To control for variations in total
immunoassayable osteocalcin, we performed these experiments in normal
adults and children, selected and grouped according to equivalent total
osteocalcin previously determined in the RIA. We also evaluated
unselected subjects on long-term coumadin therapy. Table 2 shows that in normal subjects, both the
percent osteocalcin not bound to hydroxyapatite and the calculated
degree of undercarboxylation is lower when measured with the intact
assay than when measured by RIA. In patients on long-term coumadin, the
calculated degree of undercarboxylation of osteocalcin was high, as
would be expected because of the inhibition of 
-carboxylation by the
drug. This was true when assessed by either the RIA or intact
assay.
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In the two groups of adults, the ratio of N-terminal/intact osteocalcin
was lower than in children. When we separated the adults into groups,
those with either equivalent N-terminal and intact osteocalcin or
with greater N-terminal than intact osteocalcin (N/I 
1.2 or 
1.2,
respectively), those with the greater amount of circulating fragments
had a significantly greater amount of unbound osteocalcin in the RIA
than in the intact assay. However those with low circulating fragments
(N = I) had equivalent amounts of nonbound osteocalcin in the two
assays (Table 3).
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Given the evident complexities of measuring carboxylation status
of osteocalcin using hydroxyapatite or barium sulfate binding assays,
we evaluated a commercial kit that has been recently introduced for
measurement of uncarboxylated osteocalcin. We first sought to determine
the reactivity of the antibodies to our in house proteins and peptides.
Table 4 shows that as stated by the
manufacturer, the kit was very effective in distinguishing carboxylated
from uncarboxylated intact osteocalcin. Furthermore, based on data from
three concentrations of our uncarboxylated osteocalcin, the standard
concentrations were equivalent to our in-house preparation. The
specificity of the kit appeared to be in the mid-molecular region
containing the gla residues, consistent with the published report that
the two antibodies used in these assay recognize residues 14–30 and
21–31 sequences, respectively (7). However, the large 1–43 and 4–43
fragments were overestimated by the kit. To ensure no error in our
preparations, these fragments were requantitated by amino acid analysis
and then reassayed in a second kit, and identical results were
obtained.
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shows that when determined by the
Takara kit, values for uncarboxylated osteocalcin were not different in
patients on long-term coumadin compared with normal adults. However,
the ratio of uncarboxylated osteocalcin (as determined by the Takara
kit) to the total intact osteocalcin measured by our intact assay was
greater in patients on coumadin than in controls (Takara/intact =
0.67 ± 0.36 and 0.40 ± 0.17, respectively,
P < 0.01). When the same samples were evaluated by
hydroxyapatite binding and intact assays, the degree of
undercarboxylation was significantly higher in the patients on
coumadin, as would be expected. These findings illustrate the need to
independently determine total osteocalcin when using this kit for
measuring relative undercarboxylation of osteocalcin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sensitive methods have long been sought both to determine nutritional vitamin K status and to predict risk of fracture. The measurement of undercarboxylated osteocalcin in serum has recently attracted much attention as a possible solution for both of these seemingly divergent problems. Our study documents the need to carefully standardize hydroxyapatite or barium sulfate binding assays for estimation of undercarboxylated osteocalcin. Some studies have expressed their data as the absolute amount of undercarboxylated osteocalcin, whereas others calculated the percent undercarboxylated. None have controlled for total osteocalcin in the sample. Furthermore, most use RIAs that are based on polyclonal antibodies to bovine osteocalcin. Characterization of such assays have revealed sensitivity to osteocalcin fragments (17, 18, 19, 20, 21). Therefore, several points must be taken into consideration when interpreting data.
First, reproducibility and homogeneity of different preparations of hydroxyapatite may vary. Our studies show that different preparations have different binding characteristics, and that each batch must be independently evaluated and characterized before use.
Second, the optimal amount of hydroxyapatite or barium sulfate to be used in binding studies must effectively discriminate between carboxylated and undercarboxylated osteocalcin. However, at high concentrations of these binders a significant portion of the undercarboxylated protein will be adsorbed. Furthermore if hydroxyapatite or barium sulfate is used in excess, the amount of unbound osteocalcin will be at or below the detection limit of most immunoassays, and differences in carboxylation will be difficult to evaluate. On the other hand, serum proteins also bind to hydroxyapatite and will displace osteocalcin when concentrations of the binders are too low.
Third, estimation of undercarboxylated osteocalcin must be based on the total amount of osteocalcin in the sample. Other investigators have also observed that at a fixed amount of the binder there is a high correlation between total osteocalcin and the amount of undercarboxylated osteocalcin, and because of this, expressed their data as percent of the total (10, 22). Our studies show that even with this correction, there is still an error based on the total amount of osteocalcin in the sample. Because of the curvilinear nature of the binding curves, these differences can range from 5–15%, well within the reported differences between subjects and controls in some clinical studies. Others have observed the concentration dependence of osteocalcin for these types of studies and have used the average of two concentrations of hydroxyapatite in a single subject to estimate the amount of uncarboxylated osteocalcin (23), but this does not eliminate the error. We have attempted to better approximate the level of undercarboxylated osteocalcin in our samples by using a regression equation based on multiple binding curves. It should be stressed, however, that this equation is specific for our particular reagents and conditions and is not necessarily applicable to others.
Osteocalcin purified from human bone is incompletely -carboxylated
at the first potential gla residue (residue 17). Gamma-carboxylation at
this site in adult bone has been shown to range from 55–89%, whereas
residues 21 and 24 were greater than 90% carboxylated. Data suggest
that this is because of partial carboxylation of the protein during
synthesis (24). Circulating osteocalcin would be expected to be
similarly undercarboxylated, although this has not been tested. Serum
samples with mixtures of two and three gla species may behave
differently than mixtures of fully carboxylated and decarboxylated
protein. However, circular dichroism measurements show that the gla
residue at position 17 in osteocalcin is essential for the
conformational transition to an 
helix. (25). Because the helical
structure facilitates the selective binding of osteocalcin to
hydroxyapatite, partially carboxylated osteocalcin may have reduced
binding to hydroxyapatite. Studies with chicken osteocalcin demonstrate
that loss of two gla residues results in intermediate binding (26).
Furthermore, we have no information on the reactivity of the Takara kit
to partially carboxylated osteocalcin.
Fourth, the relative intactness of the osteocalcin in the sample will influence the apparent binding of osteocalcin to hydroxyapatite. In normal adults when only intact osteocalcin was considered, the nonbound fraction was reduced, especially in those serum samples in which N-terminal fragments were high. There is growing evidence that various fragments of osteocalcin circulate. Garnero et al. (27) has suggested that a major catabolic fragment present in the circulation is that spanning residues 1–43. This fragment binds to hydroxyapatite with the same characteristics as does the intact molecule. Therefore, assays that are known to be specific only for the intact molecule or intact plus the 1–43 fragment should be used for studies of osteocalcin carboxylation status.
It has been suggested that circulating osteocalcin fragments are generated by proteolysis in the circulation or during sample processing and storage (27). In our study, no attempt was made to standardize sample collection or processing times, and it is unclear whether the variation in N-terminal fragments in adults was because of sample handling. However, the N-terminal osteocalcin was consistently higher than intact osteocalcin in the children in this study. We have also observed this in a larger set of normal children (S. Abrams, C. Gundberg, unpublished observations). Two other studies also found increased circulating N-terminal immunoreactivity in individuals with high turnover states (28, 29). Because our assay will measure fragments smaller than the 1–43 species, it is possible that there are additional circulating fragments that may be clinically relevant.
Clinical utility
The effect of aging on total and carboxylated osteocalcin has been examined in both men and women. In adults, total osteocalcin levels are relatively stable but start to rise in men after the age of 60. In women, osteocalcin increases with menopause, and levels are correlated to an increase in the rate of bone turnover (30). Osteocalcin can remain elevated up to 40 yr after the menopause and is inversely related to bone mineral density (31). When undercarboxylated osteocalcin was evaluated, the percent of total osteocalcin not bound to hydroxyapatite was elevated in elderly institutionalized women compared with healthy premenopausal and postmenopausal ones (32). Whether undercarboxylated osteocalcin may have been an indicator of generalized poor total nutritional status in these institutionalized women is unknown. However in another study of healthy free-living subjects, both total and nonbound osteocalcin increased with age, but when expressed as percent of the total, there were no effects of aging on unbound osteocalcin (8).
The finding that has generated the most interest is the relationship between undercarboxylated osteocalcin and risk of hip fracture (11). As part of a large French study of vitamin D and calcium treatment on the incidence of hip fractures in elderly women, Szulc et al. (12) found that the absolute amount of osteocalcin not bound to hydroxyapatite was negatively correlated with BMD, but the correlation decreased when the data was expressed as a percent of the total. Furthermore, the correlation with BMD was greater with nonbound osteocalcin than with total osteocalcin. The study of Szulc employed a bovine-directed polyclonal assay, and it is intriguing to speculate that the greater correlation between BMD and nonbound osteocalcin as compared with total osteocalcin was because of the circulating fragments of osteocalcin that do not bind to hydroxyapatite rather than the degree of carboxylation.
In a follow-up study to the French series, the absolute amount of unbound osteocalcin was higher in 30 institutionalized patients who subsequently developed a hip fracture than in 153 subjects who did not. Total osteocalcin was equally increased in those with fractures (33). In a subsequent larger study in these subjects, Vergnaud et al. (13) determined undercarboxylated osteocalcin by the hydroxyapatite binding method using an assay specific for the intact plus 1–43 fragment. Although the subjects and controls were not matched for total osteocalcin, the means of two groups were equivalent. A significant difference in percent nonbound osteocalcin at baseline was found in 104 subjects who subsequently sustained hip fractures compared with 255 controls. In addition, the Takara kit was used to measure uncarboxylated osteocalcin in this study, but there was no significant difference between subjects with hip fractures and controls in this case. Whether these values would have reached significance if this parameter was expressed as a percent of the total is unknown. Nevertheless, this study provides evidence of an association between increased risk of hip fracture and undercarboxylated osteocalcin. Whether vitamin K nutrition per se, total nutritional status, or some other unknown factors are responsible for this association has not been defined.
In summary, if using hydroxyapatite or barium sulfate to measure differential binding of osteocalcin, the salts must be standardized before use. Percent binding rather than absolute amount of undercarboxylated osteocalcin should be reported. Results for individuals cannot be directly compared without correcting for the basal level of osteocalcin in the sample. Furthermore, studies should be performed with antibodies that do not detect osteocalcin fragments that have low affinity for these salts. Finally, immunoassays that are reportedly specific for either carboxylated or undercarboxylated osteocalcin must not detect fragments. These values must also be normalized to total intact osteocalcin in the sample. With these provisos in mind, future assessment of undercarboxylated osteocalcin may serve as a sensitive index of osteoblastic vitamin K status.
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Footnotes |
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Received March 4, 1998.
Revised May 28, 1998.
Accepted June 5, 1998.
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S. L Booth, I. Golly, J. M Sacheck, R. Roubenoff, G. E Dallal, K. Hamada, and J. B Blumberg Effect of vitamin E supplementation on vitamin K status in adults with normal coagulation status Am. J. Clinical Nutrition, July 1, 2004; 80(1): 143 - 148. [Abstract] [Full Text] [PDF]
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K. J. Schulze, K. O. O'Brien, E. L. Germain-Lee, S. L. Booth, A. Leonard, and B. J. Rosenstein Calcium Kinetics Are Altered in Clinically Stable Girls with Cystic Fibrosis J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3385 - 3391. [Abstract] [Full Text] [PDF]
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 S. L. Booth, L. Martini, J. W. Peterson, E. Saltzman, G. E. Dallal, and R. J. Wood Dietary Phylloquinone Depletion and Repletion in Older Women J. Nutr., August 1, 2003; 133(8): 2565 - 2569. [Abstract] [Full Text] [PDF]
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M. J. Horwitz, M. B. Tedesco, C. Gundberg, A. Garcia-Ocana, and A. F. Stewart Short-Term, High-Dose Parathyroid Hormone-Related Protein as a Skeletal Anabolic Agent for the Treatment of Postmenopausal Osteoporosis J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 569 - 575. [Abstract] [Full Text] [PDF]
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S. L. Booth, A. H. Lichtenstein, and G. E. Dallal Phylloquinone Absorption from Phylloquinone-Fortified Oil Is Greater than from a Vegetable in Younger and Older Men and Women , J. Nutr., September 1, 2002; 132(9): 2609 - 2612. [Abstract] [Full Text] [PDF]
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L. J. Schurgers, M. J. Shearer, B. A. M. Soute, I. Elmadfa, J. Harvey, K.-H. Wagner, R. Tomasch, and C. Vermeer Novel effects of diets enriched with corn oil or with an olive oil/sunflower oil mixture on vitamin K metabolism and vitamin K-dependent proteins in young men J. Lipid Res., June 1, 2002; 43(6): 878 - 884. [Abstract] [Full Text] [PDF]
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N. M. McKeown, P. F. Jacques, C. M. Gundberg, J. W. Peterson, K. L. Tucker, D. P. Kiel, P. W. F. Wilson, and S. L. Booth Dietary and Nondietary Determinants of Vitamin K Biochemical Measures in Men and Women J. Nutr., June 1, 2002; 132(6): 1329 - 1334. [Abstract] [Full Text] [PDF]
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S. L Booth, A. H Lichtenstein, M. O'Brien-Morse, N. M McKeown, R. J Wood, E. Saltzman, and C. M Gundberg Effects of a hydrogenated form of vitamin K on bone formation and resorption Am. J. Clinical Nutrition, December 1, 2001; 74(6): 783 - 790. [Abstract] [Full Text]
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R. Gopalakrishnan, H. Ouyang, M. J. Somerman, L. K. McCauley, and R. T. Franceschi Matrix {gamma}-Carboxyglutamic Acid Protein Is a Key Regulator of PTH-Mediated Inhibition of Mineralization in MC3T3-E1 Osteoblast-Like Cells Endocrinology, October 1, 2001; 142(10): 4379 - 4388. [Abstract] [Full Text] [PDF]
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 M. J. Seibel, M. Lang, and W.-J. Geilenkeuser Interlaboratory Variation of Biochemical Markers of Bone Turnover Clin. Chem., August 1, 2001; 47(8): 1443 - 1450. [Abstract] [Full Text] [PDF]
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S. A. Abrams, K. C. Copeland, S. K. Gunn, C. M. Gundberg, K. O. Klein, and K. J. Ellis Calcium Absorption, Bone Mass Accumulation, and Kinetics Increase during Early Pubertal Development in Girls J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1805 - 1809. [Abstract] [Full Text]
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S.-M. Kakonen, J. Hellman, M. Karp, P. Laaksonen, K. J. Obrant, H. K. Vaananen, T. Lovgren, and K. Pettersson Development and Evaluation of Three Immunofluorometric Assays That Measure Different Forms of Osteocalcin in Serum Clin. Chem., March 1, 2000; 46(3): 332 - 337. [Abstract] [Full Text] [PDF]
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S. L Booth, M. E O'Brien-Morse, G. E Dallal, K. W Davidson, and C. M Gundberg Response of vitamin K status to different intakes and sources of phylloquinone-rich foods: comparison of younger and older adults Am. J. Clinical Nutrition, September 1, 1999; 70(3): 368 - 377. [Abstract] [Full Text] [PDF]
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S. A. Abrams, K. C. Copeland, S. K. Gunn, J. E. Stuff, L. L. Clarke, and K. J. Ellis Calcium Absorption and Kinetics Are Similar in 7- and 8-Year-Old Mexican-American and Caucasian Girls Despite Hormonal Differences J. Nutr., March 1, 1999; 129(3): 666 - 671. [Abstract] [Full Text]
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) and uncarboxylated (•) osteocalcin in
osteocalcin-free serum after incubation with 2–40 mg/mL hydroxyapatite
purchased from Calbiochem (HA-C) or Sigma (HA-S). Results are expressed
as percent of total added remaining in supernatant.
; and 32.4 ng/mL, 
) were evaluated with 2
mg/mL HA-C (top), 20 mg/mL HA-S (middle),
and 50 mg/mL barium sulfate (bottom). Results are
expressed as percent of total added remaining in supernatant.
; 15.5
ng/mL, 
; 17.2 ng/mL,