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PubMed1988

The quantification of gammacarboxyglutamic acid residues in plasma-osteocalcin.

In this paper we describe an assay procedure for determining the amount of gammacarboxyglutamic acid (Gla) residues in serum- or plasma-osteocalcin. The test includes removing by ethanol precipitation the majority of the proteins from the plasma (e.g., the Gla-containing coagulation factors) and the specific extraction of osteocalcin with the aid of immobilized immunopurified antibodies. It is demonstrated that the Gla-content of circulating osteocalcin from normal cows is similar to that of osteocalcin obtained from bone. Hence, the fact that part of the newly synthesized osteocalcin does not bind to the hydroxyapatite matrix in bone cannot be explained by an undercarboxylation of the molecule.

1-Carboxyglutamic Acid↗
PubMed1993

Serum osteocalcin in metabolic bone diseases: what is its real significance?

This study has been carried out in order to elucidate the clinical significance of serum osteocalcin measurement. The changes of this marker paralleled those of serum total alkaline phosphatase activity (a marker of bone formation) following parathyroidectomy in hyperparathyroid patients with skeletal involvement. Furthermore, the percentage decrease of serum osteocalcin levels in respect to basal values (85 +/- 12), and the percentage decrease of serum alkaline phosphatase activity levels (82 +/- 7) were significantly lower (p < 0.001) in respect to that of the 24-h hydroxyproline/creatinine ratio (42 +/- 14) one week after parathyroid surgery. Instead, changes of serum osteocalcin levels were similar to those of serum free hydroxyproline (considered to be a marker of bone resorption) following acute calcitonin infusion in normal subjects. These results imply that the antibody used in our assay might recognize not only the entire osteocalcin molecule, but also small epitopes released during the process of bone matrix resorption. Alternatively, if we consider serum osteocalcin only as a marker related to some processes of bone formation, the experiment carried out on normal subjects strongly supports the evidence of calcitonin receptors in osteoblastic surfaces.

Adult↗
PubMedDate not supplied

Effect of insulin treatment on osteocalcin levels in diabetic children and adolescents.

To investigate bone mineral metabolism in insulin-dependent diabetes mellitus, serum osteocalcin, a marker of bone formation, was measured in 31 diabetic children at onset of disease and 15 days after metabolic improvement by insulin therapy. As a control group for osteocalcin levels we studied 31 healthy sex- and age-matched children. Mean values of serum osteocalcin at onset of diabetes were significantly lower than in control group (p < 0.001), but we did not find any difference after 15 days of insulin therapy. Osteocalcin and parathyroid hormone concentrations were significantly greater after 15 days of insulin treatment than at onset of disease (p < 0.001 and p < 0.01, respectively). The osteocalcin levels were negatively correlated both with fructosamine and with glycosylated hemoglobin (p < 0.01 and p < 0.001, respectively), and positively correlated with the degree of metabolic acidosis at onset (p < 0.05). Therefore we postulate that during glycometabolic imbalance there is a decrease in bone turnover that could be one of the etiological factors of diabetic osteopenia.

Child↗
PubMed2000

Intake of fermented soybean (natto) increases circulating vitamin K2 (menaquinone-7) and gamma-carboxylated osteocalcin concentration in normal individuals.

Changes in circulating vitamin K2 (menaquinone-7, MK-7) and gamma-carboxylated osteocalcin concentrations in normal individuals with the intake of fermented soybeans (natto) were investigated. Eight male volunteers were given sequentially fermented soybeans (natto) containing three different contents of MK-7 at an interval of 7 days as follows: regular natto including 775 micrograms/100 g (MK-7 x 1) or reinforced natto containing 1298 micrograms/100 g (MK-7 x 1.5) or 1765 micrograms/100 g (MK-7 x 2). Subsequently, it was found that serum MK-7 and gamma-carboxylated osteocalcin concentrations were significantly elevated following the start of dietary intake of MK-7 (1298 or 1765 micrograms/100 g). Serum undercarboxylated osteocalcin concentrations were significantly decreased by dietary MK-7 (1765 micrograms/100 g) supplementation. Moreover, the changes in serum MK-7 level with the frequency of dietary natto intake were examined in 134 healthy adults (85 men and 39 women) without and with occasional (a few times per month), and frequent (a few times per week) dietary intake of regular natto including MK-7 (775 micrograms/100 g). Serum MK-7 and gamma-carboxylated osteocalcin concentrations in men with the occasional or frequent dietary intake of natto were significantly higher than those without any intake. The present study suggests that intake of fermented soybean (natto) increases serum levels of MK-7 and gamma-carboxylated osteocalcin in normal individuals.

Adult↗
PubMed1983

Measurements of gamma-carboxyglutamate and circulating osteocalcin in normal children and adults.

We have measured serum osteocalcin by radioimmunoassay, and urinary gamma-carboxyglutamic acid (Gla) by Dowex-1 chromatography in healthy adults and children. Circulating osteocalcin is 6.8 +/- 0.5 and 5.8 +/- 0.5 ng/ml in adult males (n = 25) and females (n = 26), respectively. Values are higher in children (n = 26), ranging from 25-30 ng/ml in a 1-year-old and declining to the adult level at puberty. The ratio of urinary Gla/creatinine as a function of age parallels the pattern of serum osteocalcin. Free Gla ranges from a high of 150 +/- 20 mumol Gla/g creatinine in infants (n = 17), decreasing until puberty at which time the excretion stabilizes at 44 +/- 4 mumol Gla/g creatinine (n = 27). The greater amounts of circulating osteocalcin and urinary Gla most likely reflect synthesis of osteocalcin during growth. With epiphyseal closure, levels decline and, in the adult, probably represent normal bone remodelling.

1-Carboxyglutamic Acid↗
PubMed1983

The identification of the vitamin K-dependent bone protein osteocalcin as one of the gamma-carboxyglutamic acid containing proteins present in calcified atherosclerotic plaque and mineralized heart valves.

Calcium binding proteins containing gamma-carboxyglutamic acid (Gla) have previously been demonstrated to occur in calcified atherosclerotic plaque and calcified cardiac valves. Experiments were carried out to determine if one of the Gla-containing proteins in human cardiovascular calcifications is the vitamin K-dependent bone protein, osteocalcin. A radio-immunoassay for human osteocalcin was employed, and EDTA extractions of calcified atheromata, and aortic valves as well as relevant noncalcified material were analyzed. Tissue calcium levels were also determined, as were Gla levels as a measure of total vitamin K-dependent protein content. Osteocalcin was present at low levels in all calcified cardiovascular tissues (4.5-175.7 ng osteocalcin/mg protein) with trace levels or nondetectable amounts present in noncalcified tissue. Osteocalcin accounted for a small proportion of the total protein-bound Gla (0.01-0.05%). The relationship of osteocalcin to the other Gla-containing proteins of atherosclerotic plaque including atherocalcin, the principal extractable Gla-containing protein of calcified plaque, is discussed.

1-Carboxyglutamic Acid↗
PubMed1990

Effect of 1,25(OH)2D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells.

Hormone-dependent accumulation of specific binding sites for 1,25(OH)2D3 and changes in human 1,25-dihydroxy-vitamin D receptor (hVDR) mRNA levels were examined in cell lines (MG-63, SaOs-2 and U2-Os) derived from human bone. Osteocalcin synthesis and secretion as well as alkaline phosphatase activity were also characterized as biochemical markers of the osteoblastic phenotype. Specific binding sites for 1,25(OH)2D3 were quantified by incubating cultured intact cells with [3H]1,25(OH)2D3 at 37 degrees C. Based on the uptake of 1,25(OH)2D3, there were about 3000 to 4000 receptor molecules per cell with apparent dissociation constants varying between 0.02 to 0.03 nM. The binding was saturated with 1,25(OH)2D3 in 3 to 6 h after the hormone addition and further exposure to the hormone resulted in an upregulation of the bindings sites. The levels were elevated by as little as 10 to 200 pM 1,25(OH)2D3, and maximal binding was achieved with 0.2-0.7 nM 1,25(OH)2D3. Treatment with 1,25(OH)2D3 also resulted in a clear increase (about 3-fold) in hVDR mRNA by 24 h in all three cell lines. The increase in hVDR mRNA level was time- and dose-dependent. MG-63 cells responded with 2- and 15-fold increases, respectively, in intracellular and secreted levels of osteocalcin after the 1,25(OH)2D3-treatment. In dot-blot hybridization assay, MG-63 cells expressed osteocalcin mRNA which was inducible with 1,25(OH)2D3 while, in SaOs-2 and U2-Os cells, osteocalcin mRNA was not detected under the same circumstances. Also, no secretion of osteocalcin was detected in SaOs-2 and U2-Os cells with or without addition of 1,25(OH)2D3.

Alkaline Phosphatase↗
PubMed1995

Transcriptional control of the osteocalcin gene by 1,25-dihydroxyvitamin D-2 and its 24-epimer in rat osteosarcoma cells.

The effects of two vitamin D analogs, 1,25-dihydroxyvitamin D-2 and 24-epi-1,25-dihydroxyvitamin D-2, were examined on osteocalcin gene expression in the rat osteosarcoma cell line ROS 17/28. Our results indicate that these analogs are more transcriptionally active than 1,25-dihydroxyvitamin D-3, particularly the 24-epimer. Assessment of reporter gene chloramphenicol acetyltransferase (CAT) activity, using the vitamin D responsive element (VDRE) derived from the human osteocalcin gene promoter. revealed that both analogs stimulated CAT activity 5- to 10-fold. 1,25-Dihydroxyvitamin D-2 was slightly more active than 1,25-dihydroxyvitamin D-3, while the 24-epimer was twice as effective. 1,25-Dihydroxyvitamin D-3 also stimulated osteocalcin mRNA accumulation by 2-fold over vehicle-treated cells, 1,25-dihydroxyvitamin D-2 by 2.5-fold, and 24-epi-1,25-dihydroxyvitamin D-2 by 4-fold. Electrophoretic mobility shift assays using the osteocalcin vitamin D responsive element revealed no increase in DNA binding with either analog when compared to 1,25-(OH)2D3. Examination of CAT activity using the rat 24-hydroxylase VDRE indicated no significant difference in transcription with these compounds, suggesting that the vitamin D-2 analogs preferentially activate osteocalcin gene expression.

Animals↗
PubMed1984

Production of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids.

Cells have been cultured from human bone that possess several characteristics of osteoblasts, including the capacity to produce osteocalcin (bone Gla protein). In these cultures the production of osteocalcin is dependent on 1,25(OH)2D3 but is not affected by 24,25(OH)2D3 either alone or in combination with 1,25(OH)2D3. Two glucocorticoids, prednisolone and deflazacort, reverse the stimulation of osteocalcin synthesis by 1,25(OH)2D3 in a dose-dependent manner (10(-9) - 10(-6)M). Parathyroid hormone also inhibits osteocalcin production in a dose-dependent fashion (0.2-5 IU/ml). These results demonstrate that human bone cell cultures may be of considerable value in investigating the hormonal and pharmacologic regulation of the production of osteocalcin and other bone proteins in vitro.

24,25-Dihydroxyvitamin D 3↗
PubMed1996

Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination.

Age at death determination based on aspartic acid racemization in dentin has been applied successfully in forensic odontology for several years now. An age-dependent accumulation of D-aspartic acid has also recently been demonstrated in bone osteocalcin, one of the most abundant noncollagenous proteins of the organic bone matrix. Evaluation of these initial data on in vivo racemization of aspartic acid in bone osteocalcin was taken a step further. After purification of osteocalcin from 53 skull bone specimens, the extent of aspartic acid racemization in this peptide was determined. The D-aspartic acid content of purified bone osteocalcin exhibited a very close relationship to age at death. This confirmed identification of bone osteocalcin as a permanent, 'aging' peptide of the organic bone matrix. Its D-aspartic acid content may be used as a measure of its age and hence that of the entire organism. The new biochemical approach to determination of age at death by analyzing bone is complex and demanding from a methodologic point of view, but appears to be superior in precision and reproducibility to most other methods applicable to bone.

Adult↗
PubMed1992

Serum osteocalcin in the management of myeloma.

We have measured serum osteocalcin, a vitamin K-dependent glycoprotein synthesised by osteoblasts in 62 patients, 49 with myeloma, 26 at presentation and 23 previously treated, 7 with Waldenstrom's macroglobulinaemia (WM), and 6 with monoclonal gammopathy of uncertain significance (MGUS). Osteocalcin levels were normal in WM and MGUS. High values were found in 5/26 (19%) patients with myeloma at presentation. There was no relationship between serum osteocalcin and stage of disease. Osteocalcin was normal in all patients in plateau phase, falling to low levels in relapsed patients who failed to respond to further treatment. Serum osteocalcin may be a useful indicator of bone metabolism in myeloma.

Adult↗
PubMed1988

Effects of bone associated growth factors on DNA, collagen and osteocalcin synthesis in cultured fetal rat calvariae.

Studies in bone and bone cell cultures have shown that osteocalcin synthesis is dependent on the maturity of the osteoblast and the presence of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3. The bone matrix is a rich source of growth factors that play a role in bone formation, but their effects on osteocalcin synthesis and their interactions with 1,25(OH)2D3 have not been examined. Insulin-like growth factor I (IGF I), basic and acidic fibroblast growth factor (bFGF and aFGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta), are growth factors associated with the bone matrix. These factors were shown to stimulate [3H]thymidine incorporation into DNA in 24 h cultures of fetal rat calvariae, and their effect was not modified by 1,25(OH)2D3. IGF I and TGF beta stimulated [3H]proline incorporation into calvarial collagen while the other growth factors studied did not; 1,25(OH)2D3 inhibited collagen synthesis in control as well as in IGF I and TGF beta treated calvariae. IGF I, bFGF and aFGF stimulated osteocalcin synthesis 1.5 to 2.5 fold but only IGF I was synergistic with the stimulatory effect of 1,25(OH)2D3. PDGF and TGF beta had no effect on osteocalcin synthesis. In conclusion, bone matrix-associated factors have important mitogenic effects in bone cultures, but only IGF I and FGFs stimulate osteocalcin synthesis, an effect that is of small magnitude when compared to that of 1,25(OH)2D3.

Animals↗
PubMed1990

Phenytoin affects osteocalcin secretion from osteoblastic rat osteosarcoma 17/2.8 cells in culture.

5,5-diphenylhydantoin (Phenytoin, PHT), a widely used anticonvulsant, is also a vitamin K antagonist and disrupts bone metabolism, leading to osteomalacia. The vitamin K-dependently synthesized protein, osteocalcin, has been implicated as a key regulatory protein in bone resorption. The purpose of the present study was to determine whether PHT had an effect on osteocalcin secretion. Cells were grown to confluence in Ham's F-12 nutrient mixture, and treated with 1,25 (OH)2 vitamin D3 (2.6 microM to 2.6 pM) or PHT (5-100 micrograms/mL) for either 24 or 48 h of pretreatment. The media were then discarded, replaced with fresh media and test reagents, and quantitated for osteocalcin by radioimmunoassay at 0, 4, and 8 h secretion time points. Results were statistically analyzed by the Student's two-tailed t test. Controls showed a nearly linear secretion rate of osteocalcin, reaching 8-9 ng/10(6) cells by 8 h. Vitamin D3 (2.6 nM) maximally stimulated secretion nearly two-fold after 24 or 48 h of pretreatment in comparison to controls. PHT alone (25-100 micrograms/mL) exerted an inhibitory effect, which appeared dose-dependent and was most evident at 4 and 8 h. PHT (50 micrograms/mL) had a significant effect, in the presence of a range of vitamin D3 concentrations (2.6 microM to 2.6 pM), after 48 h of pretreatment. A maximal PHT dose of 100 micrograms/mL had no effect on either the viability or the numbers of cultured cells. These data indicate that PHT affects osteocalcin secretion from osteoblastic rat osteosarcoma (ROS 17/2.8) cells.

Animals↗
PubMed2004

Activation of p38 mitogen-activated protein kinase mediates thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

It is well known that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p44/p42 mitogen-activated protein (MAP) kinase, which limits T(3)-induced alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p44/p42 MAP kinase or p38 MAP kinase is involved in the thyroid hormone-stimulated osteocalcin synthesis in these cells. T(3) markedly induced the phosphorylation of p38 MAP kinase in addition to p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the T(3)-induced synthesis of osteocalcin. On the contrary, the T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 and PD169316, inhibitors of p38 MAP kinase. SB203580, PD169316 or PD98059 suppressed the T(3)-phosphorylation of myelin basic protein. T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 or PD169316 also in primary cultured mouse osteoblasts. These results strongly suggest that p38 MAP kinase but not p44/p42 MAP kinase takes part in the thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

3T3 Cells↗
PubMed2001

The effect of Pb(2+) on the structure and hydroxyapatite binding properties of osteocalcin.

Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb(2+) at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb(2+) on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb(2+) induces a similar structure in osteocalcin as Ca(2+) but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb(2+) to osteocalcin (K(d)=0.085 microM) than Ca(2+) (K(d)=1.25 mM). The hydroxyapatite binding assays show that Pb(2+) causes an increased adsorption to hydroxyapatite, similar to Ca(2+), but at 2-3 orders of magnitude lower concentration. Low Pb(2+) levels (1 microM) in addition to physiological Ca(2+) levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca(2+) alone. These results suggest a molecular mechanism of Pb(2+) toxicity where low Pb(2+) levels can inappropriately perturb Ca(2+) regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb(2+)-intoxicated animals.

Animals↗
PubMed1996

Identification of an osteoblastic silencer element in the first intron of the rat osteocalcin gene.

The osteocalcin gene has been used as a model for studying the regulation of gene expression by 1,25-dihydroxyvitamin D3, as well as for examining factors which contribute to osteoblast-specific regulation of gene expression. Most of these studies have focused on transactivation. We report the identification of a sequence in the first intron of the rat osteocalcin gene which suppresses the expression of osteocalcin-CAT fusion genes approximately 10-fold in ROS 17/2.8 and UMR 106 osteosarcoma cells. Mutation of a TTTCTTT motif in the first intron abolishes this suppression. The silencing effect of this motif is also observed after bone morphogenic protein-2 (BMP-2)-induced expression of the osteoblastic phenotype in the MLB13MYC clone 17 cell line. Mutation of the splice donor site does not affect suppression by these sequences in ROS 17/2.8 cells. When multimerized and placed upstream of the native osteocalcin promoter, these sequences retain their ability to mediate transcriptional repression. Electrophoresis mobility shift analysis demonstrates a specific protein-DNA interaction with the TTTCTTT motif in nuclear extracts from ROS 17/2.8, UMR 106, and MLB13MYC clone 17 cells but not those from COS-7 kidney cells. The mutation of this motif, which abolishes suppressing activity in the native context, also abolishes binding. The presence and activity of this suppressor in cells of the osteoblast lineage suggest that it is expressed with other cell-specific transcriptional regulators of the osteocalcin gene, coordinately regulating expression of this gene in bone cells.

Animals↗
PubMed1999

Gingival crevicular fluid osteocalcin in adult periodontitis.

This study aimed to detect the levels of osteocalcin in gingival crevicular fluid (GCF) from healthy (< or =3 mm sulcus depth and non-bleeding) and diseased sites (> or =6 mm probing depth and bleeding) in subjects with adult periodontitis, in order to further investigate its potential as a possible marker of the disease process. Periodontal probing depths, attachment levels and gingival indices were recorded from one healthy and one diseased site in each of 20 subjects with adult periodontitis. Both GCF accumulated in the periodontal pocket or sulci and GCF flowing into the periodontal pocket or sulci over a three-minute interval were sampled. The amounts of osteocalcin in each GCF sample was determined using immunoassays. A mean of 2.34 ng/site (2.7 microg/ml) osteocalcin was found at diseased sites and a mean of 2.47 ng/site (5.47 microg/ml) was found at healthy sites for the accumulated GCF collection method. A mean of 0.17 ng/ site (2.17 microg/ml) osteocalcin was found at diseased sites and a mean of 0.14 ng/ site (1.85 microg/ml) at healthy sites for the flow method of GCF collection. There were no statistically significant differences between osteocalcin levels in diseased and healthy sites in subjects with adult periodontitis.

Adult↗
PubMed2003

Bone recognition mechanism of porcine osteocalcin from crystal structure.

Osteocalcin is the most abundant noncollagenous protein in bone, and its concentration in serum is closely linked to bone metabolism and serves as a biological marker for the clinical assessment of bone disease. Although its precise mechanism of action is unclear, osteocalcin influences bone mineralization, in part through its ability to bind with high affinity to the mineral component of bone, hydroxyapatite. In addition to binding to hydroxyapatite, osteocalcin functions in cell signalling and the recruitment of osteoclasts and osteoblasts, which have active roles in bone resorption and deposition, respectively. Here we present the X-ray crystal structure of porcine osteocalcin at 2.0 A resolution, which reveals a negatively charged protein surface that coordinates five calcium ions in a spatial orientation that is complementary to calcium ions in a hydroxyapatite crystal lattice. On the basis of our findings, we propose a model of osteocalcin binding to hydroxyapatite and draw parallels with other proteins that engage crystal lattices.

Animals↗