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PubMed1987

Changing osteocalcin concentrations during pregnancy and lactation: implications for maternal mineral metabolism.

We measured serum osteocalcin concentrations in 82 pregnant and 21 nonpregnant women. Osteocalcin values declined in the second trimester, but returned to nonpregnant levels late in the third trimester. The mean serum osteocalcin concentration in 36 women during pregnancy (mean gestation, 26 weeks) of 2.8 ng/mL was significantly lower than that in nonpregnant women (6.4 ng/mL; P less than 0.001) or term pregnant women at delivery (6.1 ng/mL; n = 46). Serum immunoreactive PTH (iPTH) levels were significantly higher during pregnancy than in nonpregnant women [97 +/- 5 vs. 56 +/- 4 ng/L (mean +/- SE); P less than 0.001]. No significant correlations were found between maternal osteocalcin concentrations and serum phosphorus, alkaline phosphatase, or iPTH, but significant negative correlations were found between osteocalcin and total calcium or total protein. Osteocalcin concentrations in midtrimester amniotic fluid were very low (mean, 0.3 +/- 0.1 ng/mL; n = 11). In 29 lactating mothers, the mean serum osteocalcin level was 9.5 +/- 1.5 ng/mL, significantly higher than in any of the other groups (P less than 0.05), but their serum calcium and iPTH levels were normal. There was no correlation between serum osteocalcin and calcium or iPTH concentrations in lactating women. These changes are compatible with a sequence in which bone turnover is reduced during early pregnancy, rebounds in the third trimester, and increases in postpartum lactating women.

Adolescent↗

PubMed1992

Serum osteocalcin is increased in patients with osteomalacia: correlations with biochemical and histomorphometric findings.

The synthesis of osteocalcin or bone gla protein by osteoblasts is markedly stimulated by 1,25-(OH)2D, a key hormone in the regulation of bone mineralization. The circulating levels of osteocalcin have been shown to reflect both the osteoid matrix production and the formation rate of mineralized bone in several metabolic bone diseases (osteoporosis, thyrotoxicosis, primary hyperparathyroidism) in which both mechanisms are tightly coupled because of the absence of mineralization defect. In this study, we measured in 12 patients (7 women, 5 men, 56 +/- 15 yr old) with untreated osteomalacia serum osteocalcin and vitamin D metabolites (25OHD and 1,25-(OH)2D). The results were correlated with biochemical and histomorphometric assessment of bone remodeling. Osteomalacia was due to vitamin D deficiency (5 cases), to vitamin D malabsorption (6 cases), and to hypophosphataemia in 1 case. When compared to control values, serum osteocalcin was increased in patients with osteomalacia (7.4 +/- 4 vs. 3.7 +/- 1.3 ng/mL; P less than 0.001) and was positively correlated with serum alkaline phosphatase (r = 0.65; P = 0.03) and negatively with 25 OHD (r = -0.61; P = 0.04). Serum osteocalcin was not correlated with 1,25-(OH)2D [r = -0.45; not significant (NS)] even after exclusion of the patient with hypophosphataemia. Serum osteocalcin was positively correlated with the osteoid volume and osteoid perimeter (r = 0.71 and 0.69 respectively; P less than 0.01) but not with any of the tetracycline-based parameter of bone mineralization at the tissue level (r ranging from -0.41 to +0.42, NS). Serum 25 OHD, but not 1,25-(OH)2D, was positively correlated with the mineralization rate (r = 0.59; P less than 0.05 and r = 0.54; NS). We conclude that in patients with osteomalacia, a condition which is characterized by an increased osteoid accumulation due to a decreased mineralization rate, the increased level of serum osteocalcin reflects the increased osteoid synthesis but not the mineralization defect. In this disease, serum osteocalcin is inversely correlated to the severity of vitamin D deficiency reflected by serum 25 OHD, but not to the serum levels of 1,25-(OH)2D.

Adult↗

PubMed1997

Pharmacological and biochemical evidence for the regulation of osteocalcin secretion by potassium channels in human osteoblast-like MG-63 cells.

Previous reports have suggested the involvement of voltage-activated calcium (Ca2+) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast-like cells. We now report that potassium (K+) channels can also modulate the secretion of osteocalcin by MG-63 cells, a human osteosarcoma cell line. When 1,25-dihydroxyvitamin D3(1,25(OH)2D3)-treated MG-63 cells were depolarized by step increases of the extracellular K+ concentration ([K+]out) from 5-30 mM, osteocalcin (OC) secretion increased from a control value of 218 +/- 13 to 369 +/- 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance). In contrast, in the absence of 1,25(OH)2D3, there is no osteocalcin secretion nor any effect of cell depolarization on this activity. The depolarization-induced increase in 1,25(OH)2D3-dependent osteocalcin secretion was totally inhibited in the presence of 10 microM Nitrendipine (a Ca2+ channel blocker, p < 0.005) without affecting cellular alkaline phosphatase nor cell growth. Charybdotoxin, a selective blocker of Ca2+-dependent K+ channels (maxi-K) present in MG-63 cells, stimulated 1,25(OH)2D3-induced osteocalcin synthesis about 2-fold (p < 0.005) after either 30, 60, or 120 minutes of treatment. However, Charybdotoxin was without effect on basal release of osteocalcin in the absence of 1,25(OH)2D3 pretreatment. Using patch clamp technique, we occasionally observed the presence of a small conductance K+ channel, compatible with an ATP-dependent K+ channel (GK[ATP]) in nonstimulated cells, whereas multiple channel openings were observed when cells were treated with Diazoxide, a sulfonamide derivative which opens GK(ATP). Western blot analysis revealed the presence of the N-terminal peptide of GK(ATP) in MG-63 cells, and its expression was regulated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle. Glipizide and Glybenclamide, selective sulfonylureas which can block GK(ATP), dose-dependently enhanced 1,25(OH)2D3-induced OC secretion (p < 0.005). Reducing the extracellular calcium concentration with EGTA (microM range) totally inhibited the effect of Glipizide and Glybenclamide on osteocalcin secretion (p < 0.005), which remained at the same levels as controls. Diazoxide totally prevented the effect of these sulfonylureas. These results suggest that voltage-activated Ca2+ channels triggered via cell depolarization can enhance 1,25(OH)2D3-induced OC release by MG-63 cells. In addition, OC secretion is increased by blocking two types of K+ channels: maxi-K channels, which normally hyperpolarize cells and close Ca2+ channels, and GK(ATP) channels. The role of these channels is closely linked to the extracellular Ca2+ concentration.

Adenosine Triphosphate↗

PubMed1991

Relations between diurnal variations in serum osteocalcin, cortisol, parathyroid hormone, and ionized calcium in normal individuals.

Serum osteocalcin varies in a diurnal rhythm, with peak values during the night and minimum levels before noon, but the factors controlling this rhythm are unknown. In this study, we evaluated the temporal relations between the osteocalcin rhythm and variations in serum concentrations of cortisol, intact parathyroid hormone (PTH(1-84)), and ionized calcium (Ca2+) in 15 normal volunteers, aged 22-46 years. Serum cortisol varied in a typical way preceding inverse changes in serum osteocalcin by about 4 h (r = 0.78, p less than 0.0001). Changes in serum osteocalcin following the early morning increase in serum cortisol were statistically indistinguishable from the changes seen after oral administration of 2.5 or 10 mg of prednisone. Serum PTH (1-84) showed a diurnal rhythm (p less than 0.01) with peak values (4.06 +/- 0.42 pmol/l) at 20.30 h and nadir (2.81 +/- 0.10 pmol/l) around 10.30 h, preceding changes in serum osteocalcin in the same direction by 5 h (r = 0.55, p less than 0.02). Prednisone at a dose of 10 mg did not change the time course significantly. Serum Ca2+ varied in an almost bi-phasic pattern (p less than 0.01) with maximal mean levels around 16.30 and 09.30 h and minimal levels around 05.30 and 14.30 h. Serum Ca2+ correlated inversely with PTH (1-84) (r = 0.53, p less than 0.01), and serum osteocalcin was inversely related to Ca2+ at concurrent time points (r = 0.59, p less than 0.005). Prednisone caused a 2-3 h lasting increase in serum Ca2+ 3-5 h after ingestion (p less than 0.001). In conclusion, our results suggest that cortisol is strongly associated to the diurnal rhythm in serum osteocalcin. The biological relevance of the reported relation between serum osteocalcin and PTH (1-84) and serum Ca2+ is uncertain.

Administration, Oral↗

PubMed2005

Serum, saliva, and gingival crevicular fluid osteocalcin: their relation to periodontal status and bone mineral density in postmenopausal women.

BACKGROUND: Periodontitis and osteoporosis are characterized by the loss of bone mass. Osteocalcin levels have been postulated as a marker of inhibition of bone formation. The aim of the present study was to assess plasma, saliva, and gingival crevicular fluid (GCF) levels of osteocalcin and correlate them with periodontitis and osteoporosis. METHODS: Seventy-three postmenopausal women, over 35 years old, were recruited for the study. Serum, saliva, and GCF osteocalcin were measured. Vertebral bone mineral density was measured by dual-energy x-ray absorptiometry. Differences between groups were assessed by analysis of variance (ANOVA), chi-square test, and non-parametric Kruskal-Wallis test. RESULTS: Thirty-four (46.6%) were classified in the normal healthy bone group, 11 women (15.1%) in the osteopenic group, and 28 women (38.4%) in the osteoporotic group. No statistically significant differences between these densitometric groups were observed in probing depth (P = 0.24); clinical attachment level (P = 0.11); or mean osteocalcin concentrations in serum, saliva, and GCF. Twenty-seven (37.0%) of the women were classified without periodontitis (NPG) and 63.0% (N = 46) with periodontal disease (PG). There were no statistical differences in serum and saliva osteocalcin concentrations between these two groups. GCF osteocalcin concentrations were significantly higher in the PG women than in the NPG group (P = 0.008). Mean probing depth correlated significantly with GCF osteocalcin concentrations (r = 0.35; P = 0.002). CONCLUSION: The results further support the concept that osteocalcin levels in GCF correlates with periodontal but not with osteoporosis status.

Absorptiometry, Photon↗

PubMed1996

The determination of serum concentrations of osteocalcin in growing pigs and its relationship to end-measures of bone mineralization.

Osteocalcin, a 49-amino acid, gamma-carboxyglutamic acid-containing protein produced by the osteoblast, has been shown in laboratory animals to be a better marker of bone turnover than alkaline phosphatase. To determine serum osteocalcin levels in growing pigs, we isolated pure porcine osteocalcin and developed a double-antibody RIA. To evaluate the effects of dietary Ca and P levels on serum osteocalcin, 36 individually penned crossbred pigs (19.5 kg initial BW) were fed fortified corn-soybean meal diets (.95% lysine) containing four levels of Ca (.42, .66, .90, 1.14%) and P (.35, .55, .75, .95%) in a 30-d test. Increasing dietary Ca and P improved body weight gain quadratically (P < .02). Most bone traits improved quadratically (P < .05) with increasing Ca and P. Pigs were bled on d 0, 10, 20, and 30 to determine serum levels of alkaline phosphatase, 1,25-dihydroxyvitamin D3, and osteocalcin. Osteocalcin decreased (P < .02) linearly with increasing Ca and P on d 10, 20, and 30. However, this effect was much more pronounced on d 20 and 30. Alkaline phosphatase decreased with the first incremental increase in dietary Ca and P, but was not affected by higher levels on any day measured. Osteocalcin was inversely correlated with growth rate (r = -.54, P < .01), bone strength (r = -.57, P < .01), metacarpal ash (r = -.29, P < .10), femur ash (r = -.60, P < .01), and femur ash weight (r = -.65, P < .01). Similar results were found for 1,25-dihydroxyvitamin D3. Alkaline phosphatase was not correlated with performance or most bone traits on d 30. Based on this model, these results suggest that serum osteocalcin and 1,25-dihydroxyvitamin D3 are better predictors of bone mineralization and(or) turnover in pigs than serum alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1991

Osteocalcin: diagnostic methods and clinical applications.

Osteocalcin is a small (Mr 5800), very interesting bone specific protein, synthesized by osteoblasts and measured in plasma as a biochemical indicator of bone formation. Many immunoassays for osteocalcin have been developed, including radio- and enzymoimmunoassays, with the use of monoclonal and polyclonal antibodies. These are used in many different clinical settings, including bone, kidney, and liver diseases. However, there is a wide range of published values for plasma osteocalcin concentrations in control and patient samples and this has hindered a more widespread adoption of osteocalcin measurement by clinicians. This review discusses how various immunoassays for osteocalcin may contribute to the wide variation of published values and suggests approaches for the development of standardized assays. For example, epitope specificity and immunoreactivity with multiple forms of osteocalcin and osteocalcin peptides in plasma are discussed. It also includes a recent update on interesting clinical applications of osteocalcin.

Aging↗

PubMed2000

Assessment of serum osteocalcine level in pre-dialysis patients with chronic renal failure.

UNLABELLED: The application of serum osteocalcine as a marker of osseous synthesis in patients with renal osteodystrophy is still disputable because of its predominantly renal excretion. The aim of the present study was to investigate the level of serum osteocalcine in pre-dialysis patients with chronic renal failure (CRF). MATERIAL AND METHODS: 47 patients aged 22-60 years (26 males and 22 females) with chronic renal failure were studied. 23 of them were stage I CRF patients (creatinine up to 353.6 mumol/l) and 24 were stage II and III CRF patients (creatinine up to 800 mumol/l). 35 healthy subjects (15 males and 20 females) were used as controls. Serum osteocalcine was measured by a radioimmunologic assay (ELSA-OSTEO-CIS, France). Serum creatinine, calcium, phosphorus and alkaline phosphatase were detected on a biochemical analyzer "Optima" (Kone Instruments, Finland) using the standard techniques recommended by IFCC. RESULTS: Serum osteocalcine was significantly elevated in patients with stage I CRF (45.61 +/- 7.75 ng/ml), compared to the control group (14.61 +/- 1.02, p < 0.001; u = 3.96). A significant increase was also found in patients with stage II and III CRF (120.48 +/- 15.96 ng/ml, p < 0.001; u = 4.22). No significant difference in osteocalcine level was found between male and female patients (83.77 +/- 15.09 vs. 94.52 +/- 16.88). 32 (68%) patients of the entire sample had osteocalcine above the reference values. These included 11 out of 23 patients with stage I CRF (47%) and 21 out of 24 patients with stage II and III CRF (87%). A moderately positive correlation was established between osteocalcine level and the duration of CRF (0.57), as well as between serum creatinine (0.39) and phosphorus (0.34). A moderately negative correlation was discovered between creatinine clearance (-0.42) and total serum calcium (-0.37). CONCLUSIONS: Serum osteocalcine could be used as a marker for bone synthesis in pre-dialysis patients with CRF. Our results indicate that more than 50% of the patients show evidence for renal osteodystrophy.

Adult↗

PubMed2002

Validation of a new automated immunoassay for measurement of intact osteocalcin.

Bone turnover is assessed indirectly by measurement of biochemical markers of bone turnover. Osteocalcin, a 49-amino-acid protein is a major noncollagenous protein of bone matrix, synthesized by osteoblasts and odontoblasts. Various assays exist for assessment of osteocalcin and concentrations in the same serum or plasma sample may vary enormously. The used antibodies may recognize intact osteocalcin and/or circulating fragments of osteocalcin. We here describe and validate a new automated immunoassay system for measurement of intact osteocalcin (DPC IMMULITE assay) using monoclonal antibodies (mouse) against the C-terminus of osteocalcin (AA 44-49). For detection polyclonal antibodies (goat) directed against the N-terminus (AA 1-17) conjugated with alkaline phosphatase are used. While different laboratory assays show marked clinical discordance, we evaluated our results comparatively to an established IRMA method (Nichols). We observed a highly significant correlation between both assays (r = 0.9352, p < 0.0001, n = 286) for healthy persons and also for patient samples (osteoporosis, diabetes type 1, rheumatoid arthritis). Very low inter- and intraassay covariance as well as highly significant linearity (analytical recovery near 100%) tested by serial dilutions were demonstrated for the DPC IMMULITE intact osteocalcin assay. We conclude that the IMMULITE assay is a useful method for assessment of intact osteocalcin giving valuable results in comparison to an established non-automated assay.

Antibodies, Monoclonal↗

PubMed1992

Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin gene promoter.

A proinflammatory cytokine cascade, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8, is activated in response to infection or immunologic insult. Besides their immunologic effects, several of these mediators stimulate bone resorption and inhibit bone formation. Osteocalcin, the most abundant noncollagenous protein present in bone, is an osteoblast-specific product whose production closely correlates with bone formation, and which has also been implicated in control of bone resorption. IL-1 and TNF have previously been shown to down-regulate osteocalcin production in vitro and in vivo, although the mechanism of this inhibition is unknown. In the present studies, IL-1 beta and TNF-alpha both inhibited 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin protein and mRNA by ROS 17/2.8 osteosarcoma cells, whereas IL-6 had no effect on protein and only weakly inhibited mRNA. To determine if down-regulation was exerted at the transcriptional level, an osteocalcin promoter-chloramphenicol acetyltransferase (CAT) fusion gene was constructed (PHOC-CAT). After transient transfection of PHOC-CAT into ROS 17/2.8 osteosarcoma cells, reporter CAT activity was up-regulated by vitamin D at concentrations above 10(-12) M. In screening studies, TNF-alpha (-57%) and IL-6 (-37%) inhibited vitamin D-stimulated osteocalcin transcription, whereas IL-1 alpha, IL-1 beta, and IL-8 had no effect. Other immune cytokines and growth factors, including IL-2, IL-3, IL-7, and M-CSF, also failed to regulate osteocalcin transcription. Despite their lack of promoter regulation, IL-1 alpha and IL-1 beta also stimulated PGE2 production by ROS 17/2.8, further confirming the ability of the host cell to respond to these mediators. In dose-response experiments, down-regulation by TNF-alpha was significant at concentrations as low as 0.14 pM (0.1 U/ml), whereas approximately 10(4)-fold higher concentration of IL-6 was required to exert a similar effect. TNF-alpha-mediated down-regulation was unaffected by indomethacin. These data demonstrate that of these cytokines, TNF-alpha alone potently down-regulates osteocalcin promoter function, whereas IL-1 acts post-transcriptionally, possibly by reducing mRNA stability. Heterogeneity therefore exists among the proinflammatory cytokines with respect to the level at which control of osteocalcin expression is exerted.

Cell Line↗

PubMed1992

Serum and synovial fluid osteocalcin in rheumatic diseases.

The mechanisms involved in juxta-articular bone destruction are poorly understood. Osteocalcin or gamma-carboxyglutamic acid (GLA) protein is a small non-collagenous bone protein. It is a sensitive marker of osteoblastic bone formation. Its seric variations in the serum in such rheumatisms as rheumatoid arthritis remain unclear. Further information on local osteoblastic activity may be obtained by assaying the level of osteocalcin in the synovium. Its serum level can be evaluated by radioimmunoassay. The same method can be used in the synovial fluid. Paired serum and synovial fluid samples have been assayed from 63 patients, 33 patients with inflammatory arthritis (rheumatoid arthritis, psoriasis, chondrocalcinosis, pyogenic arthritis) and 30 patients with mechanical joint effusion (osteoarthritis, meniscal lesions). Serum levels of osteocalcin were the same in the inflammatory group (m: 8.69 +/- 0.68 ng/ml) and in the mechanical group (m: 10.2 +/- 0.67 ng/ml). In the synovial fluid, the levels of osteocalcin were significantly lower in the inflammatory group (m: 3.27 +/- 0.40 ng/ml) than in the mechanical group (m: 6.91 +/- 0.47 ng/ml). The same results were obtained with the ratio of synovial fluid osteocalcin on serum osteocalcin. There was a significant correlation between serum and synovial fluid osteocalcin and an inverse correlation between synovial fluid osteocalcin and the number of synovial fluid cells. The present study suggests that periarticular osteoblastic depression, among patients with inflammatory arthritis, is likely.

Adult↗

PubMed1988

Cyclic AMP-mediated stabilization of osteocalcin mRNA in rat osteoblast-like cells treated with parathyroid hormone.

Osteocalcin (bone gamma-carboxyglutamic acid-containing protein) is exclusively produced by osteoblasts, which are the major target cells of parathyroid hormone (PTH) in bone. This study examined the effect of human (h) PTH(1-34) on osteocalcin gene expression in the rat osteoblast-like osteosarcoma cells ROS17/2.8. hPTH(1-34) increased in a dose-dependent manner the steady state levels of osteocalcin mRNA 2- to 3-fold with an ED50 of about 5 X 10(-10) M. This effect was detectable at 12 h, peaked at 24 h, and lasted at least up to 48 h. Forskolin, cyclic 8-bromo-AMP, isobutylmethylxanthine, cholera toxin, and (-)-isoproterenol similarly elevated osteocalcin mRNA. hPTH(1-34) did not alter the transcriptional rate of the osteocalcin gene, estimated by nuclear run-on assays, but increased the stability of osteocalcin mRNA. hPTH(1-34) also increased 2- to 3-fold the osteocalcin level in the culture media determined by radioimmunoassay. PTH, thus, promoted osteocalcin gene expression in these cells at least in part through mRNA stabilization via cyclic AMP mediation, a mechanism known only in few systems.

1-Methyl-3-isobutylxanthine↗

PubMed1988

Osteocalcin. Biochemical considerations and clinical applications.

The vitamin K-dependent protein of bone, osteocalcin (bone Gla protein) is a specific product of the osteoblast. A small fraction of that synthesized does not accumulate in bone but is secreted directly into the circulation. Upon catabolism of osteocalcin, its characteristic amino acid, gamma-carboxyglutamic acid (Gla), is excreted into the urine. Both serum osteocalcin and urine Gla are currently being used for the clinical assessment of bone disease. The authors summarize the current understanding of the structure and function of osteocalcin in bone and evaluate the clinical studies done using serum osteocalcin and urinary Gla to monitor bone turnover. Factors that affect the measurement of osteocalcin concentrations in the blood are osteoblastic synthesis, content of Gla in the protein, drug-induced alterations in osteocalcin's affinity for bone, hormonal status, renal function, age, sex, timing of blood collection, and specificity of the radioimmunoassay. With these considerations, serum osteocalcin measurements provide a noninvasive specific marker of bone metabolism.

1-Carboxyglutamic Acid↗

PubMedDate not supplied

A heterologous radioimmunoassay system for human osteocalcin. Applications in performance sports girls.

Taking into account the involvement of the measurement of osteocalcin, a protein-containing gamma-carboxyglutamic acid (Gla), in investigating the physiopathology of bone and calcium metabolism, a radioimmunoassay (RIA) method useful in human clinics was developed. The reagents for the RIA of human osteocalcin namely, the radioiodination preparation, the reference preparation and the antiserum were obtained starting from bovine bone on account of the structure similarities between bovine and human osteocalcin. The reagents were incubated at 4 degrees C for 48 h followed by the polyethylene-glycol (PEG) separation technique. The sensitivity of the osteocalcin RIA method is 2.0 ng/ml, the intraassay precision is 3-5% and the interassay precision, 8-12% (coeficient of variation). Osteocalcin was measured in the sera of 3 groups of performance sports girls under basal conditions and being of more or less similar ages: rowing girls (n = 10, mean age 16 yr 6 mos), gymnast girls (n = 13, mean age 14 yr 8 mos) and table tennis girls (n = 9, mean age 16 yr). The same samples were used for the measurement of myoglobin. Osteocalcin quantification showed similar levels in the rowing and the table tennis girls, i.e. 11.91 +/- 3.39 ng/ml (X +/- SD) and 9.50 +/- 4.15 ng/ml, respectively. By contrast, in the gymnast girls, the osteocalcin levels were about 4 times higher (44.31 +/- 7.27 ng/ml) than in the other two groups. Myoglobin measurements showed lower levels in the gymnast group, than in the rowing and the table tennis groups, the latter two having similar levels. The multiplicative increase of osteocalcin in the gymnast girls is interpreted and commented in relation to their age and sexual development.

Adolescent↗

PubMed1987

Serum osteocalcin, bone alkaline phosphatase isoenzyme and plasma tartrate resistant acid phosphatase in patients on chronic maintenance hemodialysis.

Biochemical indices of bone formation (serum osteocalcin and bone alkaline phosphatase isoenzyme) and osteoclastic function (plasma tartrate resistant acid phosphatase) were measured in 43 patients undergoing chronic hemodialysis and in 27 patients with primary hyperparathyroidism. The mean values for bone alkaline phosphatase isoenzyme and plasma tartrate resistant acid phosphatase but not for osteocalcin were significantly higher in primary hyperparathyroidism as compared with dialyzed patients. A significant positive correlation was found between the biochemical indices of osteoblasts and osteoclasts both in primary hyperparathyroidism and in dialyzed patients, indicating biological coupling between bone resorption and formation under these conditions. The regressions of osteocalcin vs bone alkaline phosphatase isoenzyme and/or plasma tartrate resistant acid phosphatase in dialyzed patients paralleled those in primary hyperparathyroidism but their distance differed significantly. It is concluded that in patients with renal failure, an increase in circulating osteocalcin by a relatively constant portion reflects decreased renal clearance. Any additional increase in osteocalcin serum level indicates an increased skeletal production of osteocalcin. The clinical value of bone alkaline phosphatase isoenzyme and plasma tartrate resistant acid phosphatase appears to be comparable with that of serum osteocalcin in primary hyperparathyroidism, and more exact than osteocalcin in renal failure.

Acid Phosphatase↗

PubMed1983

Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid-containing proteins in developing bone.

Development of a sensitive radioimmunoassay for the vitamin K-dependent bone protein osteocalcin in avian species has provided new information on the biosynthesis of this protein in bone. Chicken osteocalcin shares many structural features, including the sequence positions of its 3 gamma-carboxyglutamic acid (Gla) residues, with osteocalcins of human, monkey, cow, and rat, but is cryptic in the radioimmunoassays for these species. In the chicken assay system, the intact 50-residue (Mr = 5670) protein is required for immunoreactivity. Reduction and alkylation of the disulfide bond (Cys 23-Cys 29) or tryptic removal of the COOH-terminal pentapeptide abolish antibody binding activity. Decarboxylation of the 3 Gla residues enhances the affinity for antibody by 1.5- to 2-fold. Osteocalcin appears coincident with the very earliest detectable perichondral mineralization in developing long bone (tibiotarsus) of the 7- to 8-day-old chick embryo (stages 31-33). However, amino acid analysis demonstrates an excess of Gla in embryonic bone compared to the level of osteocalcin by radioimmunoassay. Two independent experimental approaches have partially resolved this paradox. First, extraction and gel filtration in 4 M guanidine hydrochloride of total bone proteins has revealed high molecular weight species which share antigenic determinants with osteocalcin, namely, 10,000 (+/- 1,000), 15,000 (+/- 2,000), 35,000 (+/- 5,000), and 85,000 (+/- 15,000), in addition to 5,670 osteocalcin. Second, a selective 3H exchange labeling procedure for Gla residues has revealed Gla-containing proteins in bone in the same molecular weight classes. One or more of these may represent precursors in the biosynthetic pathway for osteocalcin.

1-Carboxyglutamic Acid↗

PubMed1992

The osteocalcin propeptide is not secreted in vivo or in vitro.

Osteocalcin is initially synthesized as an 11 kD molecule consisting of a 23-residue translocation signal peptide that is cleaved during translation, a 26-residue propeptide that targets the protein for gamma-carboxylation, and the 49-residue mature protein. Although the majority of newly synthesized osteocalcin is deposited into bone matrix, a small amount can be detected in blood, and it is this characteristic that has led to its current clinical use as a specific index of osteoblastic activity. Nothing is known, however, about the fate of the propeptide. If osteocalcin and the propeptide are cosecreted, then the concentration of the propeptide could also be useful as a marker of osteoblastic function and, further, may be superior to osteocalcin because it would be unaffected by binding to bone. To test this hypothesis, we synthesized a peptide corresponding to 21 residues of the osteocalcin propeptide from humans and produced a polyclonal antibody to this peptide. Human sera were screened for the presence of the propeptide, and the human osteosarcoma cell line MG-63 was tested for secretion of the propeptide. We could not detect any osteocalcin propeptide in sera from normal adults or individuals with renal failure or primary hyperparathyroidism or those on long-term coumadin therapy. Likewise there was no propeptide present in media from cells grown in the presence of vitamin K, 1,25-(OH)2D3, warfarin, or warfarin plus 1,25-(OH)2D3. In contrast, the cell extract, characterized by high-performance liquid chromatography, contained mature osteocalcin, free propeptide, and the proosteocalcin precursor when cells were grown in the presence of 1,25-(OH)2D3 alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitriol↗

PubMed1996

Ectopic secretion of osteocalcin, the major non-collagenous bone protein, by the myeloma cell line NCI-H929.

In this study we provide the first evidence that human non-osteoblastic cells secrete the bone-specific protein osteocalcin. We show that the myeloma cell line NCI-H929 constitutively produces low amounts of osteocalcin. Furthermore, we demonstrate that this production is strongly enhanced after 1,25(OH)2D3 stimulation. An amplification or a deregulation of the osteocalcin gene could be responsible for the observed phenomenon because NCI-H929 has the unusual characteristic of having at least three chromosomes 1, where the osteocalcin gene was mapped (1q25-q31). It is of interest to note that the patient in whom the NCI-H929 cell line originated never developed osteolytic lesions despite extensive disease apparent at autopsy. Although lytic bone lesions with low osteocalcin serum levels are a common feature of multiple myeloma (MM), a small subset of patients does not develop lytic bone lesions or even demonstrate osteosclerotic MM. This occurrence is associated with high osteocalcin serum levels of unknown origin. In this context, osteocalcin production by immortalized tumor lacking any osteolytic potential could be relevant to the pathophysiology of this particular type of MM.

Blotting, Western↗