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.

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.

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.

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.

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.

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.

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.

Vitamin K status and bone health: an analysis of methods for determination of undercarboxylated osteocalcin.

Recent studies suggest that fracture risk is associated with increased undercarboxylated osteocalcin. Methods use differences in binding of undercarboxylated and fully carboxylated osteocalcin to hydroxyapatite or barium sulfate. We evaluated these methods and found that results varied with the amount and preparation of the salts. Furthermore, patient samples with differing amounts of total osteocalcin could not be directly compared. Errors in the determination of undercarboxylated osteocalcin were minimized by expressing data as the percent of the total osteocalcin in the sample, and correcting for the basal level of osteocalcin using a polynomial equation derived from multiple binding curves. Errors from 5-15% in estimation of undercarboxylated osteocalcin were observed without both of these corrections. When differing types of assays were employed (RIA, intact, N-terminal), results also were affected. In normal adults and children and in patients on long-term warfarin therapy, the percent osteocalcin not bound to hydroxyapatite was lower when measured with an intact assay than by a polyclonal RIA. Differences were related to the amount of N-terminal osteocalcin fragments, which had low affinity for hydroxyapatite and resulted in variable overestimation of undercarboxylated osteocalcin. In a kit specific for uncarboxylated osteocalcin, we found good discrimination between carboxylated and uncarboxylated intact osteocalcin. However, the assay detected large osteocalcin fragments and overestimated their concentration by up to 350%. Values for uncarboxylated osteocalcin were not different in patients on coumadin compared with normal adults with this kit, but when normalized to the total intact osteocalcin, percent uncarboxylated osteocalcin was greater in patients on coumadin than in controls, as would be expected. Kit values for uncarboxylated osteocalcin in normal children were higher than intact values in the same subject, because of the increased reactivity of the kit toward circulating fragments that were elevated in children. Thus, for estimation of undercarboxylated osteocalcin, care must be taken to standardize the hydroxyapatite or barium sulfite used for binding, to correct for the basal level of osteocalcin in the sample, to use immunoassays that do not detect small fragments, and to express the results as the percent of the total osteocalcin in the sample. Without these precautions, the assessment of undercarboxylated osteocalcin is not reliable.

Osteocalcin production in primary osteoblast cultures derived from normal and Hyp mice.

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17-25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes.

A line of transgenic mice expressing a human osteocalcin genomic fragment (hOClocus) and a murine MC3T3-E1 cell line containing a stably integrated human osteocalcin promoter construct have been developed to characterize the osteogenic and hormonal regulation of human osteocalcin in vivo and in vitro. In this study, we used these models to demonstrate a species difference in the regulation of the mouse and human osteocalcin genes by vitamin D. Repeated administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to mice carrying the hOClocus transgene resulted in striking increases in serum human osteocalcin, whereas serum mouse osteocalcin levels were unchanged after 24 h and only modestly increased 48 h after the second dose of hormone. 1,25(OH)2D3 increased human calvarial mRNA expression by 1.8-fold and slightly decreased mouse osteocalcin mRNA levels by approximately 1.2-fold. Furthermore, treatment of primary calvarial osteoblasts from these mice with 1,25(OH)2D3 increased human osteocalcin production but inhibited mouse osteocalcin protein accumulation. To investigate further the mechanism for the apparent species difference in vitamin D3 induction of mouse and human osteocalcin, we examined the effect of 1,25(OH)2D3 in an MC3T3-E1 cell line (MC4) containing a stably integrated 3900 bp osteocalcin promoter-luciferase construct. Treatment of MC4 cells with ascorbic acid resulted in parallel increases of the endogenous mouse osteocalcin protein and luciferase reporter activity over a 12-day period. Continuous exposure of MC4 cells to 1,25(OH)2D3 resulted in time-and dose-dependent increases in the activity of the phOC3900 luciferase construct. By contrast, the hormone had no effect on mouse osteocalcin protein concentrations and inhibited its induction by ascorbic acid. However, when cells were treated acutely with 1,25(OH)2D3 at later times during growth in ascorbic acid, the induction of mouse osteocalcin protein was only partially inhibited. In conclusion, our results indicate that common osteogenic signals regulate both mouse and human osteocalcin gene expression, but the mouse gene is resistant to induction by vitamin D. This species difference in vitamin D regulation of osteocalcin appears to result from the failure of 1,25(OH)2D3 to transcriptionally activate the mouse osteocalcin gene.

Osteocalcin abnormalities in Hyp mice reflect altered genetic expression and are not due to altered clearance, affinity for mineral, or ambient phosphorus levels.

The Hyp mouse manifests rickets and renal wasting of phosphorus. We previously reported elevated circulating osteocalcin in Hyp mice, and a paradoxical decrease in response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. To investigate these abnormalities further, we characterized in detail the response of circulating osteocalcin to 1,25-(OH)2D3 and compared skeletal osteocalcin in normal and Hyp mice. The affinity of osteocalcin for hydroxyapatite and the protein's clearance were compared in Hyp and normal animals. Finally, the response of osteocalcin messenger RNA (mRNA) to 1,25-(OH)2D3 was examined in normal mice, Hyp mice, and normal mice subjected to dietary phosphorus deprivation. Multiple (n = 3-6) daily doses of 1,25-(OH)2D3 are required to increase serum osteocalcin levels in normal C57 BL/6 mice; the effect is apparent by 6 h and persists for at least 24 h after injection. Single doses of up to 50 ng have no significant effect. In contrast, an approximately 50% decrement in circulating osteocalcin occurs after a single dose of 1,25-(OH)2D3 in Hyp mice. Osteocalcin clearance in Hyp mice appears to be normal. Bone osteocalcin per U calcium or phosphorus is normal in Hyp mice, suggesting that its affinity for hydroxyapatite is normal. Osteocalcin mRNA from Hyp mice is expressed in greater abundance than that from normal animals, reflecting the differences in circulating levels of the protein. Similarly, osteocalcin mRNA from Hyp mice decreases in response to 1,25-(OH)2D3, whereas an increase in osteocalcin message is seen in normal animals. These studies indicate that normal mice are relatively resistant to 1,25-(OH)2D3 stimulation of osteocalcin production. Furthermore, the differences between Hyp and normal mice in circulating osteocalcin reflect differences in the regulation of gene expression.

Molecular basis and clinical application of biological markers of bone turnover.

An ideal battery of tests would include indices of bone resorption and formation. They should be unique to bone, reflect total skeletal activity, and should correlate with traditional measures of bone remodeling activity, such as radiocalcium kinetics, histomorphometry, or changes in bone mass. Factors that confound their measurement, such as circadian rhythms, diet, age, sex, bone mass, liver function, and kidney clearance rates, should be clearly defined (Fig. 9). To date, no bone marker has been established to meet all these criteria, and each marker may have its own specific advantages and limitations. There are still questions that must be answered before there can be complete confidence in the information gained from measurement of any of the bone markers. Furthermore, it should be emphasized that none of the markers are diagnostic for any particular bone disease and cannot be used for this purpose in individual patients. Nevertheless, recent advances in research and development have provided assays with increased specificity, sensitivity, and availability. Because of this, bone markers can be used for a variety of important purposes: as tools for basic bone biology research, for defining general physiological phenomenon in clinical studies or drug trials, and for following individual patients.