Osteocalcin In Plasma Research Articles

Spironolactone reduces biochemical markers of bone turnover in postmenopausal women with primary aldosteronism

ContextPrimary aldosteronism (PA) is the most frequent form of endocrine hypertension. Besides its deleterious impact on cardiovascular target organ damage, PA is considered to cause osteoporosis.Patients and methodsWe assessed bone turnover in a subset of 36 postmenopausal women with PA. 18 patients had unilateral PA and were treated by adrenalectomy, whereas 18 patients had bilateral PA and received mineralocorticoid receptor antagonist (MRA) therapy respectively. 18 age- and BMI-matched females served as controls. To estimate bone remodeling, we measured the bone turnover markers intact procollagen 1 N-terminal propeptide, bone alkaline phosphatase, osteocalcin and tartrate resistant acid phosphatase 5b in plasma by chemiluminescent immunoassays at time of diagnosis and one year after initiation of treatment.Study designObservational longitudinal cohort study.SettingTertiary care hospital.ResultsCompared with controls, patients with PA had mildly elevated osteocalcin at baseline (p = 0.013), while the other bone markers were comparable between both groups. There were no differences between the unilateral and the bilateral PA subgroup. One year after initiation of MRA treatment with spironolactone bone resorption and bone formation markers had significantly decreased in patients with bilateral PA. In contrast, patients adrenalectomized because of unilateral PA showed no significant change of bone turnover markers.ConclusionThis study shows that aldosterone excess in postmenopausal women with PA is not associated with a relevant increase of bone turnover markers at baseline. However, we observed a significant decrease of bone markers in patients treated with spironolactone, but not in patients treated by adrenalectomy.

Soybean-Hop Alleviates Estrogen Deficiency-Related Bone Loss and Metabolic Dysfunction in Ovariectomized Rats Fed a High-Fat Diet.

Soybeans and hops have been traditionally used as a natural estrogen replacement therapy and their major active ingredients, isoflavones and prenylflavanones, are known to have estrogenic/antiestrogenic effects depending on the target organ. However, their potential benefits are still subject to controversies. The present study investigated the dual effect of soy isoflavones plus hop prenylflavanones (Soy-Hop) on bone loss and metabolic dysfunction under estrogen deficient condition. Rats were sham-operated (n = 10) or ovariectomized (OVX; n = 40) and then fed a high-fat diet (HFD) to develop hyperlipidemia in OVX rats within the experimental period of 8 weeks. The OVX/HFD rats were assigned to four groups to receive different doses of Soy-Hop (0, 30, 100, and 300 mg/kg) by oral gavage for 8 weeks. High-dose Soy-Hop significantly suppressed OVX/HFD-induced increases in food intake, body weight gain, fat mass, and circulating levels of leptin, adiponectin, LDL-cholesterol, total cholesterol, triglycerides, glucose, and insulin. High-dose Soy-Hop also attenuated OVX/HFD-induced elevation of osteocalcin, alkaline phosphatase, and CTX in plasma and RANKL/OPG gene expression ratio in femur. These findings were confirmed visually by confocal analysis of GLUT4 translocation in soleus muscle cells and micro-computed tomography scanning of the distal femoral epiphysis, respectively. These results suggest that Soy-Hop may have potential to ameliorate estrogen deficiency-related alterations in both metabolism and bone quality, at least in part, by hormonal factors secreted by adipocytes.

Altered Ethanol Consumption in Osteocalcin Null Mutant Mice.

Osteocalcin (OC) is an abundant extracellular calcium-binding protein synthesized by osteoblasts. Although most OC is bound to hydroxyapatite mineral during bone formation, a consistent amount is released directly to circulation. Plasma OC (pOC) levels are highly sensitive to stressful stimuli that alter stress-responsive hormones, such as glucocorticoids (cortisol or corticosterone) and the catecholamines norepinephrine and epinephrine. To gain a better understanding of the apparent relationship of OC to the effects of ethanol (EtOH) and the stress responses, we compared mice that have OC (WT [OC+/+] and HET [OC+/-]) with OC null mutants (KO [OC-/-]), which have no OC in either plasma or in bone. One experiment included chronic unpredictable stress, a second was conducted in the absence of any known stressors other than EtOH, while a third imposed a more severe acute immobilization stress in addition to EtOH consumption. The data obtained confirmed significant differences in EtOH consumption in mice that previously experienced various stressful stimuli. We also determined that adrenal tyrosine-hydroxylase expression was inversely proportional to EtOH consumption and tended to be lower in KO than in WT. Data suggest that OC possesses the ability to modulate the adrenal gene expression of the catecholamine synthetic pathway. This modulation may be responsible for differences in EtOH consumption under stress.

OP0088 Discovery of a novel anti-bone resorption compound, AS2690168, which inhibits osteoclastogenesis induced by receptor activator of nuclear factor-kappa B ligand (RANKL)

BackgroundRANK/RANKL signaling is the pathway of osteoclast differentiation and activation. In many bone diseases, the balance of bone formation and bone resorption is impaired. In osteoporosis, bone resorption is predominant...

Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome

Soft-tissue mineralization is a tightly regulated process relying on the activity of systemic and tissue-specific inhibitors and promoters of calcium precipitation. Many of these, such as matrix gla protein (MGP) and osteocalcin (OC), need to undergo carboxylation to become active. This post-translational modification is catalyzed by the gammaglutamyl carboxylase GGCX and requires vitamin K (VK) as an essential co-factor. Recently, we described a novel phenotype characterized by aberrant mineralization of the elastic fibers resulting from mutations in GGCX. Because of the resemblance with pseudoxanthoma elasticum (PXE), a prototype disorder of elastic fiber mineralization, it was coined the PXE-like syndrome. As mutations in GGCX negatively affect protein carboxylation, it is likely that inactive inhibitors of calcification contribute to ectopic mineralization in PXE-like syndrome. Because of the remarkable similarities with PXE, we performed a comparative study of various forms of VK-dependent proteins in serum, plasma (using ELISA), and dermal tissues (using immunohistochemistry) of PXE-like and PXE patients using innovative, conformation-specific antibodies. Furthermore, we measured VK serum concentrations (using HPLC) in PXE-like and PXE samples to evaluate the VK status. In PXE-like patients, we noted an accumulation of uncarboxylated Gla proteins, MGP, and OC in plasma, serum, and in the dermis. Serum levels of VK were normal in these patients. In PXE patients, we found similar, although not identical results for the Gla proteins in the circulation and dermal tissue. However, the VK serum concentration in PXE patients was significantly decreased compared with controls. Our findings allow us to conclude that ectopic mineralization in the PXE-like syndrome and in PXE results from a deficient protein carboxylation of VK-dependent inhibitors of calcification. Although in PXE-like patients this is due to mutations in the GGCX gene, a deficiency of the carboxylation co-factor VK is at the basis of the decreased activity of calcification inhibitors in PXE.

Relationships between IGF-I, cortisol, and osteocalcin in peripheral plasma of growing pigs

Osteocalcin is a bone specific protein which is secreted by mature osteoblasts and is measurable in peripheral blood plasma. In growing female pigs (n = 6) osteocalcin was measured in daily blood samples between an age of 125 to 238 days and the values were compared to IGF-I and cortisol. Mean concentrations of osteocalcin were 158 +/- 2.7 ng/ml and the concentrations were significantly correlated to those of IGF-I (r = 0.12 p < or = 0.01 n = 581) and cortisol (r = 0.13 p < or = 0.01 n = 503). Both hormones and less pronounced osteocalcin revealed a rhythm-like pattern with a period of 10-14 days. In addition, rhythm-like variations with a period of about 5 weeks are visible. The highest correlation was found between IGF-I and cortisol both within the animals (r = 0.28, n = 508, p < or = 0.001) and between the individuals (r = 0.94, n = 6, p < or = 0.01). Thus a dialog between IGF-I and cortisol may be assumed. A possible physiological role is discussed.

Early parathyroidectomy increases bone mineral density in patients with mild primary hyperparathyroidism: A prospective and randomized study

There is an ongoing controversy regarding how to take care of patients with mild primary hyperparathyroidism (PHPT) and how to grade their disease activity in terms of bone parameters. This prospective and randomized study was undertaken to evaluate skeletal effects of delayed surgical treatment in such patients. Fifty patients with mild PHPT (serum calcium, 2.55 to 2.95 mmol/L; 10.2 to 11.8 mg/dL) were randomized to parathyroidectomy either at diagnosis or 1 year later. Hip and spine bone mineral density (BMD, determined by dual energy x-ray absorptiometry), bone alkaline phosphatase in serum, osteocalcin and beta-CrossLaps in plasma, and calcium in urine were measured in all patients at inclusion in the study and 1 and 2 years later. The skeletal effects of mild PHPT varied with anatomy and time of exposure. Parathyroidectomy decreased all biochemical bone markers ( P < .0001) and increased lumbar spine BMD ( P < .05) equally in both groups, even in patients without overt osteoporosis, whereas hip BMD was increased ( P < .05) in the early intervention group only. Prolonged exposure to mild and seemingly stable PHPT is a risk factor for hip fractures, which adds to other reasons for surgical treatment of this condition without delay regardless of serum calcium levels.

Comparison of biochemical markers of bone remodelling in the assessment of the effects of alendronate on bone in postmenopausal osteoporosis

The effects of alendronate treatment on biochemical markers of bone remodelling and bone mineral density (BMD) were studied in 30 Caucasian women (postmenopausal for at least 3 years, age 42–76 years, with BMD of the lumbar spine at least 2 S.D. below the mean for mature, premenopausal women). The patients were randomly assigned to receive alendronate (10 mg/day) or placebo for 12 months (double blind). The study was subsequently extended to a second year of open alendronate treatment. The treatment with alendronate resulted in a significant and progressive increase in BMD of the lumbar spine and femoral neck. Under the treatment, the maximal decrease of biochemical markers of bone remodelling (osteocalcin in plasma, bone-specific alkaline phosphatase, N-terminal propeptide of type I procollagen and C-terminal telopeptide of type I collagen in serum, and cross-linked amino-terminal N-telopeptide and total hydroxyproline in urine) was observed at 6 months with no further change during the 2-year period. There were no significant differences in discriminating between patients treated for 1 year with alendronate or placebo using either the percentage change in spine BMD at month 12, or a single measurement of the marker at month 6, or log (percent of baseline at month 6 of value of the marker). In this respect, the power of all the biochemical markers were comparable. The markers are a valuable adjunct to the measurements of BMD, especially in the patients not showing an increase of 3% or more at the lumbar spine BMD after 1 year of treatment.

Production and characterisation of monoclonal and polyclonal amtobpdoes agaomst bovine osteocalcin

Monoclonal and polyclonal antibodies were produced against osteocalcin purified from bovine bone. The antibodies were used for the development of a two-site ELISA. A monoclonal antibody recognising residues 15-49 of bovine osteocatcin generated by cleavage with 70% formic acid was used as capture antibody and a biotinylated polyclonal antibody recognising the same peptide was used for detection. The two-site assay did not detect any tryptic peptides of bovine osteocalcin (residues 1-19, 20-43, and 45-49) nor did it detect the peptide 1-14 or the synthetic peptide 37-49. Cross-reaction was observed for osteocalcin in plasma from sheep, goat, chicken and human but not for osteocalcin from rat and pig.

Two-site enzyme immunoassay for measuring intact human osteocalcin in serum.

We developed a sensitive two-site sandwich ELISA for quantitative analysis of human osteocalcin in serum or plasma. Our method is based on two different highly specific antibodies recognizing epitopes at different ends of the protein so that only intact osteocalcin is detected. The method is fast (total analysis time less than 6 h/96 wells), precise (intraassay variation less than 2.3% at four different levels; n = 10, and interassay variation less than 2.5%, n = 5, respectively), and accurate, with a mean recovery of 105%. The detection limit in serum is approximately 0.1 micrograms/liter. The mean concentration of osteocalcin in normal serum with this assay is 3.3 micrograms/liter (SD 3.7 micrograms/liter; range 0.1-13.1 micrograms/liter; n = 41), and the reference range is 0.28-10.1 micrograms/liter (10 and 90% confidence limits). The method shows a reasonable positive linear correlation with other osteocalcin assays (Incstar, r = 0.55, p < 0.05, n = 13; Henning Oscatest, r = 0.52, p < 0.005, n = 34). A good correlation (r = 0.70, p < 0.001) between individual osteocalcin and bone-specific alkaline phosphatase serum concentrations was observed in normal subjects. We found a low or undetectable concentration of intact osteocalcin in serum of all four of our patients with acute primary hyperparathyroidism, and in all five patients with hypocalcemic secondary hyperparathyroidism, which suggests that PTH effectively inhibited the synthesis of osteocalcin in osteoblasts. The serum concentration of intact osteocalcin was elevated in two of three patients with chronic primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

Development and evaluation of an osteocalcin chemiluminoimmunoassay

We developed a competitive chemiluminoimmunoassay of osteocalcin that is similar to radioimmunoassay but uses acridinium-ester-labeled antigen instead of 125I-labeled osteocalcin; a second antibody immobilized on plastic beads is used to separate free and bound fractions. There was good correlation of the new chemiluminoimmunoassay (y) with a polyclonal antiserum (R102) radioimmunoassay (x) used in many previous clinical studies (r = 0.96, y = 0.968x + 2.69, Sy/x = 0.029, n = 86). The new assay recognized both the intact and the small fragment of osteocalcin in plasma and detected decreases of them (total, approximately 47%) after a 24-h infusion of parathyroid hormone. Patients with primary hyperparathyroidism had increased concentrations of intact osteocalcin. Children had higher concentrations of osteocalcin than adults did. Healthy women had greater osteocalcin concentrations at ages 50-70 years than earlier. Inverse correlations of bone mineral density and osteocalcin were found in healthy women and in women with osteoporosis.

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).

Comparison of monoclonal and polyclonal antibody-based immunoassays for osteocalcin: A study of sources of variation in assay results

We have compared two assays for osteocalcin (BGP, bone gla protein), one based on a monoclonal antibody, the other based on a polyclonal serum, in an effort to better understand the factors that contribute to the wide variation reported for osteocalcin normal ranges. The two assays compared well using serum samples, with a correlation coefficient of 0.9071 (n = 48). However, the monoclonal antibody assay returns values increased over the polyclonal assay by approximately 1.5-fold. Gel permeation studies indicate that these larger values are due primarily to increased reactivity in the monoclonal assay with high- and low-molecular-weight forms of osteocalcin in plasma; both assays give similar reactions to intact osteocalcin. Analysis of samples from individuals with increased bone resorption due to parathyroid hormone administration reveals that the decrease seen in osteocalcin values after hormone infusion occurs primarily in the fraction that corresponds to intact osteocalcin. During the course of these studies, we re-evaluated the extinction coefficient for osteocalcin, arriving at the value E = 1.33 mg/ml-1, cm-1. We also observed a significant negative interference in both assays (and a commercial assay) caused by hemolysis. This interference is due to proteolysis of osteocalcin by enzymatic activity released from the lysed red cells.

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

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.

Bisphosphonates inhibit 1,25-dihydroxyvitamin D3-induced increase of osteocalcin in plasma of ratsin vivo and in culture medium of rat calvariain vitro

In order to test whether bisphosphonates, which are potent inhibitors of osteoclastic bone resorption, may also act upon osteoblasts, we studied the effect of dichloromethylenebisphosphonate (Cl2MBP) and 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP) on in vivo levels and in vitro release of osteocalcin, a bone-specific protein produced by osteoblasts. In rats, 161 mumol/kg of Cl2MBP or 1.61 mumol/kg AHBuBP strongly inhibited the increase of plasma osteocalcin induced by 1,25(OH)2D3. The inhibition was measurable within 24 hours after the administration of bisphosphonate and was independent of any change in bone resorption. The effect upon osteocalcin release was also present in calvaria cultures. 250 microM Cl2MBP strongly inhibited the osteocalcin release induced by 10(-8) M 1,25(OH)2D3. In the presence of 1,25(OH)2D3, protein synthesis and DNA synthesis were also decreased, whereas in the absence of 1,25(OH)2D3, protein synthesis was increased. Thus, bisphosphonates affect the production of a bone-specific protein by osteoblasts in addition to their inhibitory action on osteoclasts.