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To fully understand the significance of bone as a target tissue of lead toxicity, as well as a reservoir of systemic lead, it is necessary to define the effects of lead on the cellular components of bone. Skeletal development and the regulation of skeletal mass are ultimately determined by the four different types of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. These cells, which line and penetrate the mineralized matrix, are responsible for matrix formation, mineralization, and bone resorption, under the control of both systemic and local factors. Systemic components of regulation include parathyroid hormone, 1,25-dihydroxyvitamin D3, and calcitonin: local regulators include numerous cytokines and growth factors. Lead intoxication directly and indirectly alters many aspects of bone cell function. First, lead may indirectly alter bone cell function through changes in the circulating levels of those hormones, particularly 1,25-dihydroxyvitamin D3, which modulate bone cell function. These hormonal changes have been well established in clinical studies, although the functional significance remains to be established. Second, lead may directly alter bone cell function by perturbing the ability of bone cells to respond to hormonal regulation. For example, the 1,25-dihydroxyvitamin D3-stimulated synthesis of osteocalcin, a calcium-binding protein synthesized by osteoblastic bone cells, is inhibited by low levels of lead. Impaired osteocalcin production may inhibit new bone formation, as well as the functional coupling of osteoblasts and osteoclasts. Third, lead may impair the ability of cells to synthesize or secrete other components of the bone matrix, such as collagen or bone sialoproteins (osteopontin). Finally, lead may directly effect or substitute for calcium in the active sites of the calcium messenger system, resulting in loss of physiological regulation. The effects of lead on the recruitment and differentiation of bone cells remains to be established. Compartmental analysis indicates that the kinetic distribution and behavior of intracellular lead in osteoblasts and osteoclasts is similar to several other cell types. Many of the toxic effects of lead on bone cell function may be produced by perturbation of the calcium and cAMP messenger systems in these cells.

Osteocalcin, the vitamin K-dependent protein synthesized in bone, is found in blood. The level of circulating osteocalcin has recently been used as an indicator of the rate of bone turnover. We measured serum osteocalcin during 24-h periods in 6 normal 20- to 30-yr-old men and 4 women. Blood was sampled via an indwelling venous catheter every 30 or 60 min for 24 h. Circadian rhythmicity in circulating osteocalcin was found in 9 of the 10 individuals studied. Osteocalcin levels fell during the morning, rose in the afternoon and early evening, and reached a peak nocturnally. There were no consistent correlations between osteocalcin concentrations and circulating levels of ionized calcium, total calcium, or inorganic phosphate in the subjects tested. This study illustrates the importance of regulating the time of blood sampling for osteocalcin determinations in clinical investigations of metabolic bone disease.

An x-ray fluorescence system which utilizes polarized radiation to measure lead in vivo in human subjects is described. The minimum detection limit is approximately 6.4 ppm wet weight lead in the cortex of the tibia with 4 mm of overlying soft tissue. This appears to be adequate for assessing lead stores in lead-toxic preschool children. The measurement requires 16.5 min and is associated with an effective equivalent whole body dose to the subject of 2.5 muSv. The system, its calibration and its validation are described herein.

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 α-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 Å in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.The family of vitamin K-dependent γ-carboxylated calcium binding proteins is important in a variety of tissues and cellular functions. The formation of γ-carboxyglutamic acid (Gla) 1 in the liver derived blood clotting factors, for example, results in calcium-dependent conformational changes in the proteins and functionally important interactions with acidic phospholipid surfaces (1-3). The predominant Gla protein found in bone matrix is † Support for this project was provided by NIH Grant ES-02030 to T.D., support from the Division of Environmental Sciences,Children's Hospital at Montefiore (CHAM), at the Albert Einstein College of Medicine to T.D., and NIH Grant AR-38460 to C.G. *Corresponding author. Address: Montefiore Medical Center, Moses Bldg. Rm. 401, 111 East 210th Street, Bronx, NY, 10467. Phone: 718-920-2276. Fax: 718-920-4377. dowd@aecom.yu.edu. 1 Abbreviations: Gla, γ-carboxyglutamic acid; Fmoc, 9-flourenyl-methyloxycarbonyl; CD, circular dichroism; NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum correlation; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; rmsd, root-mean-square deviation; TOCSY, total correlation spectroscopy. HHS Public Access Author manuscriptBiochemistry. Author manuscript; available in PMC 2015 July 28. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript osteocalcin (4, 5), a small Ca 2+ binding protein containing three Gla residues which are thought to facil...

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.

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