Genetic analysis of serum osteocalcin and bone mineral in multigenerational Afro-Caribbean families.

UNLABELLED: Osteocalcin is a major component of bone matrix. Concentrations of total, carboxylated, and uncarboxylated osteocalcin, are highly heritable and genetically correlated with bone mineral content (BMC) within African ancestry families. INTRODUCTION: Osteocalcin (OC) is a protein constituent of bone matrix and a marker of bone formation. We characterized the heritability of serum OC measures and identified genomic regions potentially involved in the regulation of OC via high-density genome-wide linkage analysis in African ancestry individuals. METHODS: African ancestry individuals (n = 459) were recruited, without regard to health status, from seven probands (mean family size = 66; 4,373 relative pairs). Residual heritability of serum OC measures was estimated and multipoint quantitative trait linkage analysis was performed using pedigree-based maximum likelihood methods. RESULTS: Residual heritabilities of total OC, uncarboxylated OC, carboxylated OC and percent uncarboxylated OC were 0.74 ± 0.10, 0.89 ± 0.08, 0.46 ± 0.10 and 0.41 ± 0.09, respectively. All OC measures were genetically correlated with whole body BMC. We obtained strong evidence of bivariate linkage for percent uncarboxylated OC and whole body BMC on chromosome 17 (logarithm of the odds [LOD] = 3.15, 99 cM). CONCLUSIONS: All forms of OC were highly heritable and genetically correlated with total body BMC in these African ancestry families. The identified linkage region contains several candidate genes for bone and energy metabolism including COL1A1 and TNFRSF11A. Further studies of this genomic region may reveal novel insight into the genetic regulation of OC and bone mineralization.

The effect of lead on bone mineral properties from female adult C57/BL6 mice.

Lead toxicity is a significant problem in the U.S. with elevated blood lead levels being highest among very young children and older adults >50 years old. Bone is the major reservoir of body lead, accounting for 75% in children and 90% in adults. Very little is known about the effect of lead on bone mineral properties in adults. We investigated the effect of lead on the femora from adult, 6 month old female C57/BL6 mice who were administered lead in the drinking water (250 ppm, blood lead 33 µg/dL) for 4 months. Bone mineral properties were examined using Fourier Transform Infrared Microscopy (FTIRM), quantitative microcomputed tomography (microCT) and whole bone mechanical testing. Lead significantly decreased the bone mineral density in the cortical and proximal cancellous bone and increased the marrow area in the cortical bone with microCT. Whole bone three-point bending showed a trend of decreased maximum and failure moments in the lead treated bones compared to controls. Lead significantly decreased the mineral/matrix ratio, collagen maturity and crystallinity in the trabecular bone as measured by FTIRM. In the cortical bone lead significantly decreased collagen maturity and bone crystal size by FTIRM. In contrast to cell culture studies, lead significantly increased serum osteocalcin levels. Lead also significantly increased the bone formation and resorption markers suggesting increased bone turnover. These data show that lead increases bone turnover resulting in weaker cortical bone in adult female mice and suggest that lead may exacerbate bone loss and osteoporosis in the elderly.

The (1)H NMR structure of bovine Pb(2+)-osteocalcin and implications for lead toxicity.

Structural information on the effect of Pb(2+) on proteins under physiologically relevant conditions is largely unknown. We have previously shown that low levels of lead increased the amount of osteocalcin bound to hydroxyapatite (BBA 1535:153). This suggested that lead induced a more compact structure in the protein. We have determined the 3D structure of Pb(2+)-osteocalcin (49 amino acids), a bone protein from a target tissue, using (1)H 2D NMR techniques. Lead, at a stoichiometry of only 1:1, induced a similar fold in the protein as that induced by Ca(2+) at a stoichiometry of 3:1. The structure consisted of an unstructured N-terminus and an ordered C-terminal consisting of a hydrophobic core (residues 16-49). The genetic algorithm-molecular dynamics simulation predicted the lead ion was coordinated by the Gla 24 and Gla 21 residues. It is proposed that mineral binding occurs via uncoordinated Gla oxygen ions binding to calcium in hydroxyapatite. A comparison of Pb(2+)- and Ca(2+)-osteocalcin suggests Pb(2+), at a lower stoichiometry, may induce similar conformational changes in proteins and subsequent molecular processes normally controlled by calcium alone. This may contribute to a molecular mechanism of lead toxicity for calcium binding proteins. Lead exposure may alter the amount of mineral bound osteocalcin and contribute to abnormal bone remodeling.

Loss of the transcription factor p45 NF-E2 results in a developmental arrest of megakaryocyte differentiation and the onset of a high bone mass phenotype.

NF-E2 is a transcription factor required for megakaryocyte differentiation. The phenotype of mice deficient in p45 NF-E2 has been characterized by increased numbers of immature megakaryocytes and the absence of functional platelets. These mice also exhibited a high bone mass phenotype with up to a 6-fold increase in trabecular bone volume and a 3- to 5-fold increase in the bone formation rate. Our data indicated that both osteoblast and osteoclast numbers were increased in vivo with a 4- to 10-fold increase in osteoblast number/tissue area and approximately a 5-fold increase in osteoclast number/tissue area. Serum osteocalcin levels were also increased in NF-E2-deficient mice, corroborating the histomorphometric data and confirming that the osteoblasts were functional. Urinary cross-links levels were measured to confirm osteoclast activity. Interestingly, the increased bone was observed only in bony sites of hematopoiesis, and was not seen in flat bones such as calvariae. We showed that cells of the osteoblast lineage do not express NF-E2 mRNA. The increased bone phenotype was adoptively transferred into irradiated wild-type mice using spleen cells from NF-E2-deficient mice. These observations suggest that a megakaryocyte-osteoblast interaction occurs which is anabolic for bone.

The three-dimensional structure of bovine calcium ion-bound osteocalcin using 1H NMR spectroscopy.

Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using (1)H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and (1)H and (13)C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16-49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16-49 include type III turns (residues 20-25) and two alpha-helical regions (residues 27-35 and 41-44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm-molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 A in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.

Patterns of osteocalcin and bone specific alkaline phosphatase by age, gender, and race or ethnicity.

A variety of biochemical markers of bone turnover that assess bone formation or resorption are now available for research and clinical application. However, our understanding of the usual pattern of these measures over age in the general population is limited. Therefore, values of two bone formation markers, serum osteocalcin (Oc) and bone specific alkaline phosphatase (bone ALP), were compared by age, gender, and race or ethnicity using serum obtained from a subsample of blacks, whites, and Mexican Americans from the third National Health and Nutrition Examination Survey (NHANES). In all racial and ethnic groups, mean values of both serum Oc and bone ALP were lower in women than in men <50 years old. In individuals > or =50 years of age, Oc was significantly higher in women than in men. When analyzed in these two broad age groups, Oc was lower in older black men than in white or Mexican American men, but bone ALP was not different among the groups. In women, Oc levels tended to be lower in the black women than in white or Mexican American women. In contrast, bone ALP tended to be lower in white women than in black or Mexican American women. On the other hand, when analyzed by decade, patterns differed between the two markers in both men and women. In women, both Oc and bone ALP rose postmenopausally. However, bone ALP plateaued in the sixth through eighth decades, whereas Oc levels tended to increase further. In men, Oc was highest in the 20-29 year age group, declined and stabilized, then increased again in the seventh decade. In contrast, mean bone ALP did not differ by decade in men. Our data document differences in levels of circulating Oc and bone ALP by age, gender, and race/ethnicity. The age patterns reflected by the two markers are not concordant and distinctions are most evident in the latter decades. Our findings suggest that the specific osteoblast activity reflected by these markers responds differently to the physiologic changes that occur later in life.

Effects of a hydrogenated form of vitamin K on bone formation and resorption.

BACKGROUND: Hydrogenation of vegetable oils affects blood lipid and lipoprotein concentrations. However, little is known about the effects of hydrogenation on other components, such as vitamin K. Low phylloquinone (vitamin K1) intake is a potential risk factor for bone fracture, although the mechanisms of this are unknown. OBJECTIVE: The objective was to compare the biological effects of phylloquinone and its hydrogenated form, dihydrophylloquinone, on vitamin K status and markers of bone formation and resorption. DESIGN: In a randomized crossover study in a metabolic unit, 15 young adults were fed a phylloquinone-restricted diet (10 microg/d) for 15 d followed by 10 d of repletion (200 microg/d) with either phylloquinone or dihydrophylloquinone. RESULTS: There was an increase and subsequent decrease in measures of bone formation (P = 0.002) and resorption (P = 0.08) after dietary phylloquinone restriction and repletion, respectively. In comparison with phylloquinone, dihydrophylloquinone was less absorbed and had no measurable biological effect on measures of bone formation and resorption. CONCLUSION: Hydrogenation of plant oils appears to decrease the absorption and biological effect of vitamin K in bone.

Parameters of high bone-turnover predict bone loss in renal transplant patients: a longitudinal study.

BACKGROUND: Osteoporosis is a serious complication of kidney transplantation. Various factors have been postulated to contribute to posttransplant bone loss, among them treatment with corticosteroids, the use of cyclosporine and cyclosporine-like agents, and persistent hyperparathyroidism. In a previous cross-sectional study of long-term renal transplant recipients, we observed that osteoporosis or osteopenia was present in 88% of patients. Because biochemical markers of bone formation (serum osteocalcin) and bone resorption (urine pyridinoline, PYD, and deoxypyridinoline, DPD) were elevated in the majority of study subjects, we hypothesized that elevated rates of bone-turnover contribute to posttransplant bone loss in long-term renal transplant patients. This study was performed to examine this hypothesis. METHODS: The study population was composed of 62 patients who were more than 1-year postrenal transplantation and who had preserved renal function. They were followed prospectively for 1 year. Biochemical markers of bone-turnover were measured at study entry, and patients were classified as having high bone-turnover based on elevated urinary levels of at least one marker of bone resorption (i.e., PYD or DPD) and/or serum osteocalcin (group 1). If none of these were present, they were classified as having normal bone-turnover (group 2). Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry (DEXA) at time of entry into the study and again after 1 year of follow-up. The changes in BMD at the lumbar spine, hip, and wrist over the period of the study were compared between the high and normal bone-turnover groups. RESULTS: Forty-three patients (69%) were classified as having high bone-turnover (Group 1), and 19 patients (31%) were classified as having normal bone-turnover (Group 2). There was a statistically significant difference in change in BMD between the two groups at the lumbar spine (-1.11+/-0.42%, high bone-turnover, vs. 0.64+/-0.54%, normal bone-turnover; P=0.02) and the hip (-0.69+/-0.38%, high bone-turnover, vs. 1.36+/-0.66%, normal bone-turnover; P=0.006). Whereas group 2 had stable bone mass, group 1 exhibited bone loss at these skeletal sites. CONCLUSIONS: Our results indicate that bone loss is greater in renal transplant recipients with elevated biochemical markers of bone-turnover, suggesting that these markers may be useful in identifying patients at risk for continued bone loss. These data support the hypothesis that continued bone loss in long-term renal transplant recipients is associated with high bone-turnover. If accelerated bone resorption does play a role in posttransplant bone loss, this would provide a strong rationale for use of antiresorptive therapy for the prevention and treatment of this complication.

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

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

Clinical use of biochemical markers of bone remodeling: current status and future directions.

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.

Biochemical markers of bone formation.

The procollagen peptides, ALP and osteocalcin evaluate a different osteoblastic function. With increased understanding of the precise functions of these markers and the factors that govern their biosynthesis and metabolic clearances, abnormalities in specific osteoblast activities may be discerned. It is hoped that further advances in research and development will provide assays with increased specificity, sensitivity, and availability. Clearly, measurement of bone markers does not substitute for bone mass measurement, the latter giving, at this time, the most useful information about fracture risk. The combined use of BMD and marker measurements, however, has the potential to be valuable in individual risk assessment. It is not clear whether a combination of markers or a single marker will gain the highest predictive results in this context.

Calcium absorption, bone mass accumulation, and kinetics increase during early pubertal development in girls.

To evaluate the changes in calcium and bone mineral metabolism associated with early pubertal development, we performed longitudinal measurements of calcium absorption, calcium kinetics, bone mineral content, and hormonal markers related to puberty in a multiethnic group of girls beginning when they were 7 or 8 yr old. Girls were Tanner stage 1 (breast) at the start of the study. They were placed on a 1200 mg/day dietary calcium intake and studied at approximately 6-month intervals until they reached Tanner stage 2 (breast). Results at that time point (PUB) were compared to values obtained approximately 1 yr earlier (LatePRE) and those 1 yr before that (EarlyPRE). We found an increase in calcium absorption comparing PUB to LatePRE (n = 34; 36.6 +/- 8.7% vs. 30.7 +/- 9.9%; P = 0.002). Using whole body, dual energy, x-ray absorptiometry scanning, we found an increase in calcium gain during the LatePRE to PUB period compared with that during the EarlyPRE to LatePRE period (135 +/- 53 vs. 110 +/- 45 mg/day; P = 0.04). Calcium kinetic studies showed a significant increase in the bone calcium deposition rate (Vo+) during the PUB compared to the LatePRE period. Hormonal and biochemical markers of bone development were also significantly increased at PUB compared to LatePRE. Hormonal activity, as evidenced by the unstimulated LH level, was significantly correlated with calcium gain between the LatePRE and PUB studies and the bone calcium deposition rate in the PUB study. These data demonstrate, using multiple independent methods, an increase in calcium utilization associated with the earliest physical signs of puberty.

Diet- or warfarin-induced vitamin K insufficiency elevates circulating undercarboxylated osteocalcin without altering skeletal status in growing female rats.

To further characterize the skeletal role of vitamin K (K), markers of bone turnover, density, and strength were evaluated in rats with diet- or warfarin (W)-induced K insufficiency. One hundred two, 7-week-old, female rats were randomly assigned to low K (phylloquinone [K1], 20 microg/kg diet), control K (K1, 1300 microg/kg diet), low-dose W (W, 1.5 mg/kg control diet), or high-dose W plus K (W/K1, 10/100 mg/kg diet). Femur bone mineral content (BMC) and bone mineral density (BMD), plasma prothrombin time (PT) and prothrombin concentration (PC), and serum total alkaline phosphatase (ALP) and skeletal alkaline phosphatase (sALP) were measured at baseline and days 20, 40, 60, and 80. Serum total osteocalcin (OC) and undercarboxylated osteocalcin (ucOC) and femur length (FL) were measured at baseline and day 80. Left femur OC was measured and biomechanical testing of the right femur and third lumbar vertebral body was performed at day 80. Low dietary K elevated circulating ucOC (17% higher than control; p < 0.0001) at day 80. Furthermore, in both W groups, essentially all circulating OC was undercarboxylated and femur OC was lower than control (p < 0.0001). However, there was no change in femur percent ucOC, suggesting deposition of less newly synthesized OC. No between group differences were observed in PT, ALP, sALP, FL, BMC, BMD, or bone strength. In conclusion, skeletal K insufficiency can be induced by W or diet manipulation. This does not hinder peak bone mass attainment in female rats; however, W causes less newly synthesized OC to be deposited in bone.

Posttransplant bone disease: evidence for a high bone resorption state.

Loss of bone is a significant problem after renal transplant. Although bone loss in the first post transplant year has been well documented, conflicting data exist concerning bone loss after this time. It is equally unclear whether bone loss in long-term renal transplant recipients correlates with bone turnover as it does in postmenapausal osteoporosis. To examine these issues, we conducted a cross-sectional study to define the prevalence of osteoporosis in long-term (> 1 year) renal transplant recipients with preserved renal function (mean creatinine clearance 73 +/- 23 ml/min). Bone mineral density (BMD) was measured at the hip, spine and wrist by DEXA in 69 patients. Markers for bone formation (serum osteocalcin) and bone resorption [urinary levels of pyridinoline (PYD) and deoxypyridinoline (DPD)] were also measured as well as parameters of calcium metabolism. Correlations were made between these parameters and BMD at the various sites. The mean age of the patients was 45 +/- 11 years. Eighty eight percent of patients were on cyclosporine (12% on tacrolimus) and all but 2 were on prednisone [mean dose 9 +/- 2 mg/day)]. Osteoporosis (BMD more than 2.5 SD below peak adult BMD) at the spine or hip was diagnosed in 44% of patients and osteopenia was present in an additional 44%. Elevated levels of intact parathyroid hormone (i PTH) were observed in 81% of patients. Elevated urinary levels of PYD or DPD were present in 73% of patients and 38% had elevated serum levels of osteocalcin. Levels of calcium, and of 25(OH) and 1,25(OH)2 vitamin D were normal. In a stepwise multiple regression model that included osteocalcin, PYD, DPD, intact PTH, age, years posttransplant, duration of dialysis, cumulative prednisone dose, smoking, and diabetes: urinary PYD was the strongest predictor of bone mass. These results demonstrate that osteoporosis is common in long-term renal transplant recipients. The data also suggest that elevated rates of bone resorption contribute importantly to this process.

Longitudinal changes in bone density in hyperparathyroidism.

Primary hyperparathyroidism (HPTH) is a known risk factor for cortical bone loss. The primary objective of this study was to examine the time course and location of changes in bone mass within the first year after parathyroidectomy (PAX). The secondary goal was to evaluate the efficacy of combined estrogen therapy and parathyroidectomy in postmenopausal women. Thirty-two subjects with primary HPTH participated in a prospective, longitudinal study for at least 1 yr. Twenty-seven subjects underwent PTX, while five received no therapy (control). Among the PTX patients, 21 were postmenopausal women, and 8 of these women also received estrogen. Subjects had serial measurements of parathyroid hormone levels, serum chemistries, and bone density at multiple sites. Among all PTX patients, lumbar spine, hip, and whole body bone mineral content increased significantly (3.8-6%; p < 0.005) at 12 mo, with most of the increments observed by 3 mo. In postmenopausal women, estrogen treatment resulted in higher increments in the femoral neck (8.6 +/- 2% vs 4.9 +/- 1.2%, respectively; p = 0.07) and the whole body (6 +/- 2% vs 2.4 +/- 1.6%, respectively; p = 0.07). In HPTH, early and generalized increments in bone mass follow PTX, and the combination of surgery with estrogen therapy may be superior to surgery without estrogen treatment. A randomized, controlled trial including PTX, estrogen, and a combination of the two is needed to determine the optimal therapy in postmenopausal women.

Response of vitamin K status to different intakes and sources of phylloquinone-rich foods: comparison of younger and older adults.

BACKGROUND: Phylloquinone, found in dark-green vegetables and certain plant oils, is the primary dietary source of the fat-soluble vitamin K. Limited data suggest that the relative bioavailability of phylloquinone from vegetables is lower than that from a supplement. This finding is relevant to the maintenance of optimal vitamin K status. OBJECTIVE: The objective of this study was to compare, in younger and older adults, the relative bioavailability of phylloquinone from a vegetable with that of a fortified oil. DESIGN: In a crossover design with three 15-d residency periods in a metabolic unit, younger and older men and women (n = 36) consumed a mixed diet containing 100 microg phylloquinone/d. During 2 residency periods, the mixed diet was supplemented for 5 d with either broccoli (377 microg phylloquinone/d; broccoli diet) or phylloquinone-fortified oil (417 microg/d; oil diet). The relative bioavailability of phylloquinone was defined by the difference in plasma phylloquinone, percentage serum undercarboxylated osteocalcin (%ucOC), and urinary gamma-carboxyglutamic acid in response to 5 d of supplementation. RESULTS: For both younger and older adults, plasma phylloquinone concentrations were higher (P < 0.001) and %ucOC values were lower (P = 0.001) after the broccoli and oil diets than after the mixed diet only. Overall, the response to broccoli supplementation was not significantly different from the response to the fortified oil in either age group. Urinary gamma-carboxyglutamic acid did not change in response to supplementation. CONCLUSIONS: There was no significant difference in the relative bioavailability of phylloquinone, as evidenced by the lack of a significant difference in plasma phylloquinone and %ucOC between the 2 groups after either the broccoli or oil diets for younger and older adults.

Targeted expression of calcitonin gene-related peptide to osteoblasts increases bone density in mice.

The neuropeptide calcitonin gene-related peptide (CGRP) is concentrated in fine sensory nerve endings innervating all tissues, including bone. CGRP inhibits osteoclasts, stimulates insulin-like growth factor I and inhibits tumor necrosis factor alpha production by osteoblasts in vitro. To investigate the role of CGRP in bone in vivo, mice were engineered to express CGRP in osteoblasts by placing the human CGRP gene under the control of the rat osteocalcin promoter (Ost-CGRP tg+ mice). Calvaria cultures from transgene positive (tg+), but not tg- mice, produced bioactive CGRP. Trabecular bone density and bone volume, determined by peripheral quantitative computed tomography and bone histomorphometry, respectively, were higher in tg+ than tg- littermates. This increase in bone volume was associated with an increased bone formation rate. Trabecular bone density decreased in tg+ mice as a result of ovariectomy, but remained higher than in sham tg- mice. Targeting CGRP to osteoblasts appears to favor the establishment of a higher trabecular bone mass in mice.

Changes in bone turnover in young women consuming different levels of dietary protein.

Although high protein diets are known to increase urinary calcium excretion and induce negative calcium balance, the impact of dietary protein on bone turnover and fractures is controversial. We therefore evaluated the effect of dietary protein on markers of bone turnover in 16 healthy young women. The experiment consisted of 2 weeks of a well balanced diet containing moderate amounts of calcium, sodium, and protein followed by 4 days of an experimental diet containing one of three levels of protein (low, medium, or high). On day 4, serum and urinary calcium, serum PTH, 1,25-dihydroxyvitamin D, serum osteocalcin, bone-specific alkaline phosphatase, and urinary N-telopeptide excretion were measured. Urinary calcium excretion was significantly higher on the high than on the low protein diet. Secondary hyperparathyroidism occurred on the low protein diet. Urinary N-telopeptide excretion was significantly greater during the high protein than during the low protein intake (48.2 +/- 7.2 vs. 32.7 +/- 5.3 nM bone collagen equivalents/mM creatinine; P < 0.05). There was no increase in osteocalcin or bone-specific alkaline phosphatase when comparing the low to the high diet, suggesting that bone resorption was increased without a compensatory increase in bone formation. Our data suggest that at high levels of dietary protein, at least a portion of the increase in urinary calcium reflects increased bone resorption.

Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone.

Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNAand accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low Mr bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP.

Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin.

Osteocalcin, the gamma-carboxyglutamic acid-containing protein, which in most species is the predominant noncollagenous protein of bone and dentin, has been postulated to play roles in bone formation and remodeling. Recently, genetic studies showed that osteocalcin acts as an inhibitor of osteoblast function. Based on von Kossa staining and measurement of mineral apposition rates in tetracycline-labeled bones, osteocalcin knockout animals were reported to have no detectable alterations in bone mineralization. To test the hypothesis that, in addition to regulating osteoblastic activity, osteocalcin is involved in regulating mineral properties, a more sensitive assay of mineralization, Fourier transform infrared microspectroscopy (FT-IRM) was used to study thin sections of femora of 4-week-, 6-month- (intact and ovariectomized), and 9-month-old wild-type and osteocalcin-knockout mice. FT-IRM spectra provided spatially resolved measures of relative mineral and carbonate contents, and parameters indicative of apatite crystal size and perfection. No differences were detected in the mineral properties of the 4-week-old knockout and wild-type mice indicating that the mineralization process was not altered at this time point. Six-month-old wild-type animals had higher mineral contents (mineral:matrix ratios) in cortical as compared with trabecular bones; mineral contents in knockout and wild-type bones were not different. At each age studied, carbonate:phosphate ratios tended to be greater in the wild-type as compared with knockout animals. Detailed analysis of the phosphate nu1,nu3 vibrations in the spectra from 6-month-old wild-type animals indicated that the crystals were larger/more perfect in the cortical as opposed to the trabecular bones. In contrast, in the knockout animals' bones at 6 months, there were no differences between trabecular and cortical bone in terms of carbonate content or crystallite size and perfection. Spectral parameters of the cortical and trabecular bone of the knockout animals resembled those in the wild-type trabecular bone and differed from wild-type cortical bone. In ovariectomized 6-month-old animals, the mineral content (mineral:matrix ratio) in the wild-type cortices increased from periosteum to endosteum, whereas, in the knockout animals' bones, the mineral:matrix ratio was constant. Ovariectomized knockout cortices had lower carbonate:phosphate ratios than wild-type, and crystallite size and perfection resembled that in wild-type trabeculae, and did not increase from periosteum to endosteum. These spatially resolved data provide evidence that osteocalcin is required to stimulate bone mineral maturation.