Caren M. Gundberg

Osteocalcin: isolation, characterization, and detection.

Publisher Summary This chapter discusses the isolation, characterization, and detection of osteocalcin. Osteocalcin is a small protein (5800 daltons) comprising 10-20% of the noncollagenous protein in the bone. Because osteocalcin is tightly adsorbed to the hydroxyapatite mineral phase of the bone, thorough extraction is best achieved by dissolving the mineral of finely pulverized bone. Hydroxyapatite is soluble in neutral 0.5M ethylenediaminetetraacetic acid (EDTA), as well as in a variety of mineral and organic acids. Uniformly high yields of 1-2 mg of osteocalcin per gram of dry bone are obtained with EDTA, whereas acid procedures often leave 10-25% of the protein behind, presumably because of precipitation during excursions through the osteocalcin isoelectric point. The use of radioimmunoassay for the measurement of osteocalcin offers the advantages of specificity, sensitivity, and technical simplicity. The method can easily detect nanogram quantities of the protein in bone extracts, cell cultures, and serum. The assay is based on the competition of the radioactively labeled antigen and an identical nonlabeled antigen for binding to a specific antibody. The amount of labeled antigen bound to the antibody is inversely proportional to the amount of unlabeled antigen present in the system.

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 osteocalcins 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.

Clinical Use of Biochemical Markers of Bone Remodeling: Current Status and Future Directions

Abstract: Biochemical markers of bone turnover provide a means of evaluating skeletal dynamics that complements static measurements of bone mineral density (BMD). This review evaluates the use of commercially available bone turnover markers as aids in diagnosis and monitoring response to treatment in patients with osteoporosis. High within-person variability complicates but does not preclude their use. Elevated bone resorption markers appear to be associated with increased fracture risk in elderly women, but there is less evidence of a relationship between bone formation markers and fracture risk. The critical question of predicting fracture efficacy with treatment has not been answered. Changes in bone markers as currently determined do not predict BMD response to either bisphosphonates or hormone replacement therapy. Single measurements of markers do not predict BMD cross-sectionally (except possibly in the very elderly), or change in BMD in individual patients, either treated or untreated. On the other hand, research applications of bone turnover markers are of value in investigating the pathogenesis and treatment of bone diseases. Markers have potential in the clinical management of osteoporosis, but their use in this regard is not established. Additional studies with fracture endpoints and information on negative and positive predictive value are needed to evaluate fully the utility of bone turnover markers in individual patients.

Treatment of Congenital Osteopetrosis with High-Dose Calcitriol

We administered high doses of calcitriol (up to 32 micrograms per day) to an infant with malignant osteopetrosis, in an attempt to stimulate bone resorption. The patient was placed on a low-calcium diet to prevent hypercalcemia. Measures of bone turnover increased during calcitriol therapy; hydroxyproline excretion rose from 140 to 1358 micrograms per milligram of creatinine per 24 hours, with parallel increases in the ratio of calcium to creatinine in the urine, urinary gamma-carboxyglutamic acid, serum osteocalcin, and serum alkaline phosphatase. A pretreatment bone-biopsy specimen contained no osteoclasts with ruffled borders, a feature of active osteoclasts. After 11 days of calcitriol, ruffled borders were noted. After three months, numerous osteoclasts with ruffled borders and associated bony disruption were evident. Before therapy, the patients monocytes were incapable of in vitro bone resorption, but after calcitriol, their resorptive capacity was increased to 3.3 times control levels. These data demonstrate that calcitriol increased bone mineral and matrix turnover in our patient. However, during the three months of calcitriol therapy there was only slight clinical improvement in her severe disease. Early and sustained treatment with calcitriol may be useful in osteopetrosis.

Effect of Vitamin K Supplementation on Bone Loss in Elderly Men and Women

CONTEXT Vitamin K has been implicated in bone health, primarily in observational studies. However, little is known about the role of phylloquinone supplementation on prevention of bone loss in men and women. OBJECTIVE The objective of this study was to determine the effect of 3-yr phylloquinone supplementation on change in bone mineral density (BMD) of the femoral neck bone in older men and women who were calcium and vitamin D replete. DESIGN, PARTICIPANTS, AND INTERVENTION In this 3-yr, double-blind, controlled trial, 452 men and women (60-80 yr) were randomized equally to receive a multivitamin that contained either 500 mug/d or no phylloquinone plus a daily calcium (600 mg elemental calcium) and vitamin D (400 IU) supplement. MAIN OUTCOME MEASURES Measurements of the femoral neck, spine (L2-L4), and total-body BMD, bone turnover, and vitamins K and D status were measured every 6-12 months. Intent-to-treat analysis was used to compare change in measures in 401 participants who completed the trial. RESULTS There were no differences in changes in BMD measurements at any of the anatomical sites measured between the two groups. The group that received the phylloquinone supplement had significantly higher phylloquinone and significantly lower percent undercarboxylated osteocalcin concentrations compared with the group that did not receive phylloquinone. No other biochemical measures differed between the two groups. CONCLUSIONS Phylloquinone supplementation in a dose attainable in the diet does not confer any additional benefit for bone health at the spine or hip when taken with recommended amounts of calcium and vitamin D.

The role of osteocalcin in human glucose metabolism: marker or mediator?

Increasing evidence supports an association between the skeleton and energy metabolism. These interactions are mediated by a variety of hormones, cytokines and nutrients. Here, the evidence for a role of osteocalcin in the regulation of glucose metabolism in humans is reviewed. Osteocalcin is a bone matrix protein that regulates hydroxyapatite size and shape through its vitamin-K-dependent, γ-carboxylated form. The concentration of osteocalcin in the circulation is a measure of bone formation. The undercarboxylated form of osteocalcin is active in glucose metabolism in mice. Total serum osteocalcin concentrations in humans are inversely associated with measures of glucose metabolism; however, human data are inconclusive with regard to the role of uncarboxylated osteocalcin in glucose metabolism because most studies do not account for the influence of vitamin K on the proportion of undercarboxylated osteocalcin or differentiate between the total and uncarboxylated forms of osteocalcin. Furthermore, most human studies do not concomitantly measure other bone turnover markers to isolate the role of osteocalcin as a measure of bone formation from its effect on glucose metabolism. Carefully designed studies are required to define the role of osteocalcin and its carboxylated or undercarboxylated forms in the regulation of glucose metabolism in humans.

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism

Long-term glucocorticoid treatment is associated with numerous adverse outcomes, including weight gain, insulin resistance, and diabetes; however, the pathogenesis of these side effects remains obscure. Glucocorticoids also suppress osteoblast function, including osteocalcin synthesis. Osteocalcin is an osteoblast-specific peptide that is reported to be involved in normal murine fuel metabolism. We now demonstrate that osteoblasts play a pivotal role in the pathogenesis of glucocorticoid-induced dysmetabolism. Osteoblast-targeted disruption of glucocorticoid signaling significantly attenuated the suppression of osteocalcin synthesis and prevented the development of insulin resistance, glucose intolerance, and abnormal weight gain in corticosterone-treated mice. Nearly identical effects were observed in glucocorticoid-treated animals following heterotopic (hepatic) expression of both carboxylated and uncarboxylated osteocalcin through gene therapy, which additionally led to a reduction in hepatic lipid deposition and improved phosphorylation of the insulin receptor. These data suggest that the effects of exogenous high-dose glucocorticoids on insulin target tissues and systemic energy metabolism are mediated, at least in part, through the skeleton.

The presence of both an energy deficiency and estrogen deficiency exacerbate alterations of bone metabolism in exercising women

BACKGROUND Bone loss in amenorrheic athletes has been attributed to energy deficiency-related suppression of bone formation, but not increased resorption despite hypoestrogenism. OBJECTIVE To assess the independent and combined effects of energy deficiency and estrogen deficiency on bone turnover markers in exercising women. DESIGN PINP, osteocalcin, U-CTX-I, TT3, leptin, and ghrelin were measured repeatedly, and bone mineral density (BMD) was measured once in 44 exercising women. Resting energy expenditure (REE) was used to determine energy status (deficient or replete) and was corroborated with measures of metabolic hormones. Daily levels of urinary estrone and pregnanediol glucuronides (E1G, PdG), were assessed to determine menstrual and estrogen status. Volunteers were then retrospectively categorized into 4 groups: 1) Energy Replete+Estrogen Replete (EnR+E2R), (n=22), 2) Energy Replete+Estrogen Deficient (EnR+E2D), (n=7), 3) Energy Deficient+Estrogen Replete (EnD+E2R), (n=7), and 4) Energy Deficient+Estrogen Deficient (EnD+E2D), (n=8). RESULTS The groups were similar (p>0.05) with respect to age (24.05+/-1.75 yrs), weight (57.7+/-2.2 kg), and BMI (21.05+/-0.7 kg/m2). By design, REE/FFM (p=0.028) and REE:pREE (p<0.001) were lower in the EnD vs. EnR group, and the E2D group had a lower REE:pREE (p=0.005) compared to the E2R group. The EnD+E2D group had suppressed PINP (p=0.034), and elevated U-CTX-I (p=0.052) and ghrelin (p=0.028) levels compared to the other groups. These same women also had convincing evidence of energy conservation, including TT3 levels that were 29% lower (p=0.057) and ghrelin levels that were 44% higher (p=0.028) than that observed in the other groups. Energy deficiency was associated with suppressed osteocalcin, and TT3 (p<0.05), whereas estrogen deficiency was associated with decreased E1G (p<0.02), and lower L2-L4 BMD (p=0.033). Leptin was significant in predicting markers of bone formation, but not markers of bone resorption. CONCLUSIONS When the energy status of exercising women was adequate (replete), there were no apparent perturbations of bone formation or resorption, regardless of estrogen status. Estrogen deficiency in exercising women, in the presence of an energy deficiency, was associated with bone loss and involved suppressed bone formation and increased bone resorption. These findings underscore the importance of avoiding energy deficiency, which is associated with hypoestrogenism, to avoid bone health problems.

Dilatational band formation in bone

Toughening in hierarchically structured materials like bone arises from the arrangement of constituent material elements and their interactions. Unlike microcracking, which entails micrometer-level separation, there is no known evidence of fracture at the level of bone’s nanostructure. Here, we show that the initiation of fracture occurs in bone at the nanometer scale by dilatational bands. Through fatigue and indentation tests and laser confocal, scanning electron, and atomic force microscopies on human and bovine bone specimens, we established that dilatational bands of the order of 100 nm form as ellipsoidal voids in between fused mineral aggregates and two adjacent proteins, osteocalcin (OC) and osteopontin (OPN). Laser microdissection and ELISA of bone microdamage support our claim that OC and OPN colocalize with dilatational bands. Fracture tests on bones from OC and/or OPN knockout mice (OC−/−, OPN−/−, OC-OPN−/−;−/−) confirm that these two proteins regulate dilatational band formation and bone matrix toughness. On the basis of these observations, we propose molecular deformation and fracture mechanics models, illustrating the role of OC and OPN in dilatational band formation, and predict that the nanometer scale of tissue organization, associated with dilatational bands, affects fracture at higher scales and determines fracture toughness of bone.

Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2

Mice deficient in GATA‐1 or NF‐E2 have a 200–300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6‐fold when cultured with megakaryocytes. A megakaryocyte‐osteoblast interaction plays a role in the increased bone formation in these mice.