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PubMed1992

Avidin-biotin enzyme immunoassay of osteocalcin in serum or plasma.

We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).

Adolescent↗
PubMed1992

Homologous radioimmunoassay of human osteocalcin.

Osteocalcin or bone gamma-glutamic acid-containing protein (GLA protein) was isolated from human bone and used to develop a homologous radioimmunoassay of human osteocalcin. The effect of age on serum osteocalcin was studied in 380 normal children and adolescents and 330 normal adults. The mean (+/- SD) values in adults were higher in men [25 +/- 5 micrograms/L (4.3 +/- 0.8 nmol/L)] than in premenopausal women [20 +/- 6 micrograms/L (3.4 +/- 1.0 nmol/L); P < 0.01], but both were lower than in postmenopausal women [29 +/- 2 micrograms/L (5.0 nmol/L)]. The highest concentrations were seen in girls [ages 10-12 years: 99 +/- 38 micrograms/L (17.0 nmol/L)] and boys [ages 14-16 years: 107 +/- 57 micrograms/L (18.4 nmol/L)]. These mean values were substantially higher than those previously reported for results of heterologous osteocalcin radioimmunoassays but the correlation (r = 0.87, n = 77, P < 0.001) between both sets of results was excellent. In patients with metabolic bone diseases characterized by high or low bone turnover, the increase or decrease in serum osteocalcin observed was as expected. This homologous radioimmunoassay of human osteocalcin thus reflects bone turnover but reports serum concentrations higher than previously suspected.

Adolescent↗
PubMed1992

Changes in expression of alpha 1 type 1 collagen and osteocalcin mRNA in osteoblasts and odontoblasts at different stages of maturity as shown by in situ hybridization.

We investigated whether the expressed phenotype of osteoblasts and odontoblasts is changing with increasing maturity of the cells. Thus we determined, using in situ hybridization techniques, whether the expression of mRNA's for osteocalcin and the alpha 1 type 1 collagen chain was different in newly developed and more mature cuboidal osteoblasts of the primary and secondary spongiosa of radii of 8 day old rats, in mature cuboidal and older flat osteoblasts in the metatarsals of pig embryos, and in apical and coronal odontoblasts of the developing unerupted molars of pig embryos. The results indicate that newly differentiated osteoblasts in the primary spongiosa of the 8 day rat radius contained approximately the same amount of type 1 collagen message as more mature osteoblasts in the secondary spongiosa. Osteocalcin mRNA, on the other hand, was undetectable in the newly differentiated osteoblasts but clearly detectable in the mature osteoblasts of the secondary spongiosa. When we compared expression of osteocalcin and collagen type 1 mRNA in mature cuboidal and older flat osteoblasts, we found that the amount of osteocalcin mRNA relative to collagen type 1 mRNA was higher in flat osteoblasts than in cuboidal osteoblasts. In odontoblasts, however, the steady state level of collagen type 1 mRNA was higher in the older coronal odontoblasts, and the level of osteocalcin message lower, when compared to the younger apical odontoblasts. The results indicate that relative levels of osteocalcin and collagen mRNA in osteoblasts and odontoblasts vary depending on the stage in their secretory lifetime. This heterogeneity of the osteoblast and odontoblast population suggests that the composition of the matrix produced by these cells also differs.

Animals↗
PubMed1992

Evolution of osteocalcin, alkaline phosphatase and parathyroid hormone after parathyroidectomy in patients receiving chronic maintenance hemodialysis.

Parathyroid hormone (PTH), osteocalcin and alkaline phosphatase (AP) were investigated before and after parathyroidectomy in 12 patients receiving hemodialysis. Early post-parathyroidectomy, PTH decreased (p less than 0.001), AP increased (p less than 0.05), but osteocalcin remained unchanged. At 3 months, osteocalcin and AP declined. A negative correlation was observed between aluminum staining and post-parathyroidectomy osteocalcin. In conclusion, early post-parathyroidectomy, osteocalcin and AP reflect persistent osteoblastic activity, which declined after 3 months. In patients receiving hemodialysis both variables may represent different aspects of osteoblastic activity and osteocalcin allows mixed uremic osteodystrophy after parathyroidectomy.

Adult↗
PubMed2005

Induction of type I collagen and osteocalcin in human dental pulp cells by retinoic acid.

Retinoic acid has been known to play a key role in the regulation of bone cell differentiation and function. The effects of retinoic acid on human dental pulp cells, which contain several characteristics similar to those of bone cells, has yet to be elucidated extensively. The effects of retinoic acid on human dental pulp cells in terms of type I collagen and osteocalcin induction were investigated in vitro. Dental pulp cells obtained from the teeth of young patients (age between 18-22 years) were cultured and subsequently treated with various concentrations of retinoic acid (0, 10(-7), 10(-6), 10(-5) M) in serum-free DMEM. At different time intervals (8, 12 and 24 hours), the levels of type I collagen and osteocalcin secreted were determined using Type I Procollagen C-Peptide and Gla-type Osteocalcin EIA kits, respectively. Induction effects were evaluated using analysis of variance and the Duncan's multiple rank test. Retinoic acid at concentrations of 10(-5), 10(-6), 10(-7) M was able to induce type I collagen and osteocalcin production in human dental pulp cells within 12 hours of exposure. Dose-dependent induction was observed only after 24 hours. A two-fold increase in osteocalcin level was detected after exposed to 10(-5) M retinoic acid within 24 hours. Our data suggest that retinoic acid at concentrations of 10(-5), 10(-6), 10(-7) M has the ability to induce type I collagen and osteocalcin secretions in human dental pulp cells in vitro.

Adolescent↗
PubMed1991

[Clinical evaluation on serum osteocalcin in advanced prostate cancer patients].

The clinical significance of osteocalcin as a marker for advanced prostate cancer was examined. Osteocalcin is produced by osteoblasts and is also detected in the blood. Its change is a good index of osteometabolic diseases and especially of the osteoblastic activity. In the present study, we examined the serum osteocalcin concentration of those patients with urogenital tumor, especially prostate cancer, who had been confirmed for multiple bone-metastasis by clinical examination. These patients comprised an untreated group (15 cases) of patients with prostate cancer presenting confirmed bone-metastasis, and a group of patients without bone-metastasis. The respective serum osteocalcin concentrations of these two groups were compared with 51 cases of prostate hypertrophy used as the control group. The findings revealed that the serum osteocalcin concentration demonstrated high values in the first group with a tendency toward lowering during treatment. Neither the latter group nor the control group showed high values. On the other hand, false-positive cases (8%), and false-negative cases (20%) were found. In the case of bone-metastasis, these results suggest that measurement of serum osteocalcin concentration is useful for clinical periodical observation about the activity of the bone metastatic focus.

Adenocarcinoma↗
PubMedDate not supplied

Osteocalcin and bone alkaline phosphatase in the serum of women with liver disease.

To identify the types of liver disease in which osteopenia is a prominent feature and to understand the mechanisms of bone loss, bone mineral density was measured in the lumbar spine and hip, bone alkaline phosphatase, osteocalcin, and biochemical markers of calcium homeostasis were measured in 42 women, aged 33 to 52, with chronic liver disease and in 299 healthy women of similar age. In control women, bone alkaline phosphatase and osteocalcin correlated negatively with bone density at all sites (p less than 0.05). In women with liver disease, osteocalcin correlated negatively with bone density in the lumbar spine (p less than 0.007), whereas bone alkaline phosphatase did not correlate with bone density at any site. Bone alkaline phosphatase correlated positively with osteocalcin in control women (p = 0.001) and negatively with osteocalcin in women with liver disease (p = 0.03). Serum bone alkaline phosphatase in women with liver disease was increased significantly over serum bone alkaline phosphatase of control women, probably because of decreased clearance owing to defective function or decreased numbers of hepatic asialoglycoprotein receptors. Bone density was lower in the lumbar spines and hips of women with primary sclerosing cholangitis, primary biliary cirrhosis, and chronic active hepatitis or fibrosis without cirrhosis than in the lumbar spine and hips of control women. However, the differences were not significant, possibly because of the small sample size. It is concluded that, in liver disease, osteocalcin is a more reliable marker of osteoblastic function than bone alkaline phosphatase. Although our results show that bone density may decrease in women with cholestatic liver disease, larger studies are needed to determine the degree of osteopenia.

Adult↗
PubMedDate not supplied

Tc-99m HMDP bone scintigraphic findings in association with concurrent serum osteocalcin measurements.

Osteocalcin is a noncollagenous bone protein produced by osteoblasts. Increase in serum osteocalcin level reflects osteoblast activity with acceleration of bone formation. To evaluate the correlation of Tc-HDP bone scintigraphic findings and concurrent serum osteocalcin, serum osteocalcin values of 40 consecutive patients were measured by radioimmunoassay at the time of initial referral for bone studies. These patients, all males aged 23-93, were studied for various disease entities, including prostate carcinoma. The results of the assay were categorized into three groups (gp) as follows: 17 at normal (1.8 to 6.6 ng/ml) level (gp 1), 21 low (gp 2), and two high (gp 3). Only one bronchogenic carcinoma patient with a high level had a right iliac lesion suggesting metastasis. The bone scans of nine patients with normal or low levels showed positive metastases. Two patients with stress fractures as shown on bone scans had normal levels. The images of patients with disparity between serum osteocalcin and positive bone lesions may be interpreted as follows: Instead of osteoblastic activity resulting in the synthesis and/or release of osteocalcin, area(s) of increased uptake in the bone scan may be predominantly reflecting increased regional blood flow and decreased sympathetic tone, resulting in an increase in bone-agent deposition.

Adult↗
PubMed1990

Partial amino acid sequence of osteocalcin from an extinct species of ratite bird.

Osteocalcin the major gamma carboxyglutamic acid containing protein of vertebrate bone has been purified from the bones of a specimen of Pachyornis elephantopus, a species of the extinct class of New Zealand ratite birds, the moas. The sequence of the N-terminal region of moa osteocalcin was determined using gas phase N-terminal sequencing. The N-terminal sequences of the ostrich and rhea osteocalcins were also determined. Alignment of the N-terminal sequence of osteocalcin from the extinct moa against the osteocalcins of the extant ostrich, rhea and emu reveals the homology amongst the ratite species is greater than the homology with the chicken osteocalcin.

Amino Acid Sequence↗
PubMed1990

Serum osteocalcin or bone Gla-protein, a biochemical marker for bone metabolism in horses: differences in serum levels with age.

Levels of alkaline phosphatase and osteocalcin or bone Gla-protein, a new marker of bone metabolism, were analyzed in blood samples of 50 clinically normal female Standardbred horses between four months and twenty years of age. Samples were collected in the morning before exercise. Serum osteocalcin was measured by radioimmunoassay using bovine antibodies. There was a significant inverse correlation between alkaline phosphatase, osteocalcin and the age of the animals up to 48 months. The decrease in osteocalcin levels in serum was very marked during the first 30 months of life. The mean osteocalcin concentration was respectively 47.3, 35.7 and 6.7 ng/mL for animals less than one year, between 1.5 and 2.5 years of age and older than 3.5 years. Alkaline phosphatase serum activity was higher in foals less than one year of age (means = 856 U/L) than in the two older groups (meansII = 339, meansIII = 351 U/L). We believe that osteocalcin is a useful parameter for the evaluation of bone metabolism in growing animals and in adults and is probably more specific than alkaline phosphatase.

Aging↗
PubMed1989

Radioimmunoassay of osteocalcin with polyclonal and monoclonal antibodies.

We have developed and thoroughly validated radioimmunoassays (RIAs) for osteocalcin in plasma and serum, demonstrating independence of volume and determining the recovery of added standard and within- and between-assay precision. Antibodies were raised in rabbits and mice by immunization with either osteocalcin adsorbed to polyvinylpyrrolidone or osteocalcin conjugated to bovine serum albumin. In both species, use of the conjugated osteocalcin was more efficacious. Correlation of the results obtained when groups of plasma or serum samples were analyzed by RIAs with different antibodies (two polyclonal and one monoclonal) indicated that the normal range observed for osteocalcin concentrations in serum depends on the antibody used. These findings may account for the wide range of "normal" osteocalcin values reported by different groups.

Adult↗
PubMed1986

Serum osteocalcin concentrations in Paget's disease of bone.

Total body scintiscans, serum alkaline phosphatase estimations, and serum osteocalcin radioimmunoassays were performed in 49 consecutive patients with Paget's disease of bone. Eleven were receiving calcitonin (salmon synthetic) at the time of the study. The serum alkaline phosphatase activities were elevated in all but one patient, with the highest value almost 50 times the upper limit of the reference range. Serum osteocalcin concentrations were elevated in 53% of patients and normal in the rest. The highest serum osteocalcin value was 4.2 times the upper limit of the reference range. The correlation coefficient between the extent of skeletal involvement and serum osteocalcin level was .70, while that between skeletal involvement and serum alkaline phosphatase level was .55. In spite of the better correlation between bone scintiscans and serum osteocalcin level, osteocalcin measurements are diagnostically less useful than serum alkaline phosphatase estimations in patients with Paget's disease of bone.

Aged↗
PubMedDate not supplied

[Osteocalcin and bone metabolism in rheumatoid arthritis and osteoarthritis].

Osteocalcin is the most abundant noncollagenous protein of bone and is regarded as the best biochemical marker for bone formation. The synthesis of this protein in osteoblasts is stimulated by 1.25-Dihydroxyvitamin D3 (1.25(OH)2D3). The aim of this study was to examine the rate of bone formation measured by osteocalcin in patients (pts) with rheumatoid arthritis (RA) (n = 58) and osteoarthrosis (OA) (n = 14) and its dependence on various parameters of calcium and phosphate metabolism, especially vitamin D metabolites. Furthermore we compared the significance of alkaline phosphatase and osteocalcin as a parameter for bone turnover in these patients. According to treatment pts with RA were divided into four groups: one receiving gold salts (n = 14), one glucocorticoids (n = 13), one chloroquine (n = 14), and one nonsteroidal antiinflammatory drugs (NSAID) (n = 17). Pts with OA and RA treated with NSAID showed significantly lower values of osteocalcin than pts with RA treated with glucocorticoids or gold. In contrast to osteocalcin, alkaline phosphatase was significantly higher in all pts with RA than in pts with OA. 1.25(OH)2D, which was significantly (p less than 0.05) elevated in pts with RA treated with glucocorticoids, correlated significantly with parathyroid hormone (PTH). These data indicate that bone metabolism, at least in pts with OA and RA treated with NSAID is characterized by a decreased bone formation which is probably compensated in part in pts treated with glucocorticoids, as 1.25(OH)2D and PTH are significantly (p less than 0.05) elevated. Furthermore, the osteocalcin values were closely correlated with 1.25(OH)2D in pts with OA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1995

Effect of glucocorticoids on serum osteocalcin concentration in horses.

The effects of dexamethasone (0.2 mg/kg of body weight; IV, IM, and PO) and methylprednisolone acetate (120 mg given intra-articularly) on serum osteocalcin and cortisol concentrations were studied in 6 horses. Serum osteocalcin and cortisol concentrations were serially monitored after each treatment. A significant (P < 0.05) decrease in serum osteocalcin and cortisol concentrations was observed from 12 to 24 and 2 to 48 hours, respectively, after IV and IM administrations of dexamethasone. Serum osteocalcin and cortisol concentrations were significantly decreased from 6 to 48 and 3 to 72 hours, respectively, after oral administration. In contrast, a change in serum osteocalcin concentration was not detected after intra-articular administration of methylprednisolone. Oral, IV, or IM treatment with 0.2 mg of dexamethasone/kg caused a decrease in serum osteocalcin concentration in horses.

Animals↗
PubMed1995

Myeloma cells upregulate interleukin-6 secretion in osteoblastic cells through cell-to-cell contact but downregulate osteocalcin.

Previous studies have shown that bone marrow, especially the bone microenvironment, may play an important role in the pathogenesis of multiple myeloma (MM). To elucidate the relationship between myeloma cells and bone cells, mainly osteoblasts, we have established a coculture system between two interleukin-6 (IL-6)-dependent myeloma cell lines, XG1 and XG6, and the osteosarcoma cell lines Saos-2 and MG63. Both osteosarcoma cell lines have retained major functions of normal osteoblasts; principally, the capacity to produce hematopoietic growth factors (including IL-6) and osteocalcin, a noncollagenic protein essential in the bone formation process. Because IL-6 is a critical growth factor in MM, we have examined the IL-6 osteoblastic cell production in our coculture system. XG1 cells strongly upregulate IL-6 production by MG63 and Saos-2 cells. Of major interest, the triggering of IL-6 is totally dependent on the physical contact between myeloma cells and osteoblastic cells, contact that is partly mediated by CD44, CD56, and fibronectin interactions. Osteocalcin production by MG63 and Saos-2 cells has previously been shown to be dependent on 1,25-(OH)2D3. We demonstrate that XG1 and XG6 cells reduced the amount of osteocalcin in MG63 coculture cell supernatants, a reduction that is partly mediated by a soluble factor and by cell-to-cell contact. Notably, whereas one of the myeloma cell lines, XG6, has lost its capacity to stimulate IL-6 production by osteoblastic cell lines, both XG1 and XG6 cell lines remain able to reduce the osteocalcin amount, indicating that IL-6 and osteocalcin levels are regulated by two different pathways. In conclusion, these data strongly support the concept that the bone microenvironment is directly modified by contact with myeloma cells and are consistent with the characteristics observed in vivo in patients with MM patients, ie, abnormally high IL-6 and low osteocalcin levels, respectively.

Calcitriol↗
PubMed1995

Undercarboxylated osteocalcin and development of a method to determine vitamin K status.

We developed a RIA involving a polyclonal antibody against bovine osteocalcin, which has a carboxy-terminal epitope. Although the antibody recognizes both native and descarboxy osteocalcin, the two forms of osteocalcin were differentiated by adsorption to barium sulfate, taking advantage of the calcium-binding properties of the vitamin K-dependent gla domain. To test the clinical application of undercarboxylated osteocalcin, we examined the effect of minidose warfarin on this measure in nine healthy subjects, ages 60 to 80 years. The percentage of undercarboxylated osteocalcin increased by 170% +/- 36% (mean +/- SE) after 7 days of treatment with warfarin, 1 mg/day. The effectiveness of undercarboxylated osteocalcin as a sensitive measure of vitamin K nutritional status was further established when concentrations dropped to 17% +/- 14% below baseline with 2 days of repletion with vitamin K1, 5 mg/day, during which prothrombin times did not leave the normal range.

Adsorption↗
PubMed1994

Osteocalcin in patients with rheumatoid arthritis. A one-year followup study.

OBJECTIVE: To analyze clinical, radiological, and drug (disease modifying antirheumatic drug, DMARD) dependent factors influencing bone turnover in patients with rheumatoid arthritis (RA). METHODS: We investigated in a one-year double blind randomized study comparing intramuscular (im) gold with im methotrexate (MTX), whether the variation of inflammatory activity or functional capacity, the ascending anatomic stage, or DMARD treatments have an influence on bone formation (osteocalcin) in patients with RA. RESULTS: Patients (n = 48) enrolled at the beginning of our study had significantly increased osteocalcin levels (3.45 +/- 0.93-->4.42 +/- 1.39 ng/ml p < 0.02) after one year if inflammatory activity decreased (> or = 1 SD: erythrocyte sedimentation rate (ESR) 26.4 mm/h, C-reactive protein (CRP) 3.8 mg/dl). We found a significant negative correlation of the one-year CRP- (r = -0.44, p < 0.001) or ESR differences (r = -0.45, p < 0.001) with the corresponding osteocalcin differences. This was also evident if these patients were pooled with 15 patients excluded from the double blind study as already receiving DMARD treatment (n = 63; p < 0.01). Patients with impaired functional capacity also had significantly reduced osteocalcin levels (p < 0.01). In both cases, alkaline phosphatase showed no significant differences. CONCLUSIONS: Our data suggest that osteocalcin, a useful followup variable of bone turnover, is changed significantly (p < 0.02) in patients with RA regarding inflammatory activity and functional capacity. In contrast to alkaline phosphatase, a fall in inflammatory activity stimulated and impairment of functional capacity significantly decreased osteocalcin levels in patients with RA.

Adult↗
PubMed1993

Heterotopic ossification. The utility of osteocalcin in diagnosis and management.

The status of 12 patients after severe traumatic brain injury was investigated to determine the clinical significance of serum osteocalcin levels during the active phase of neurogenic heterotopic ossification. The diagnosis of heterotopic ossification was confirmed on the basis of radiologic findings. The mean interval between the initial injury and diagnosis of heterotopic ossification was 29 weeks. At the time of diagnosis, serum osteocalcin and total alkaline phosphatase levels were determined along with 24-hour urinary calcium and hydroxyproline excretion. The mean serum osteocalcin level was normal at 4.3 ng/ml (normal range 1.6-6.6 ng/ml) in contrast to the elevated mean values for serum alkaline phosphatase (mean = 173 units/liter; normal range 0-105 units/liter) and urinary hydroxyproline concentration (mean = 39.6 mg/24 h; normal range 7-25 mg/24 hr). Only 17% of the subjects demonstrated an elevated serum osteocalcin level, whereas 58% of the patients had an elevated serum alkaline phosphatase level. Urinary calcium excretion and hydroxyproline excretion were elevated in 33% and 70% of subjects, respectively. These data did not reveal a significant correlation between serum osteocalcin and serum alkaline phosphatase, urinary calcium excretion or urinary hydroxyproline at the time of diagnosis or in serial measurements. Therefore, serum osteocalcin is not a valuable adjunct in confirming the diagnosis of neurogenic heterotopic ossification once the diagnosis has been suggested on the basis of clinical findings and it does not appear to play a role in assessing the maturation of heterotopic ossification.

Adolescent↗