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.

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.

Clinician follow-up of children screened for lead poisoning.

OBJECTIVES: This study assessed clinicians' compliance with Centers for Disease Control and Prevention recommendations for follow-up of children with blood lead (BPb) levels of 0.48 mumol/L (10 micrograms/dL) or higher. METHODS: Clinicians' success at follow-up was determined for 3 BPb ranges: > or = 0.97 mumol/L, 0.73 through 0.92 mumol/L, and 0.48 through 0.68 mumol/L (> or = 20 micrograms/dL, 15-19 micrograms/dL, and 10-14 micrograms/dL, respectively). RESULTS: A total of 410 children with elevated BPb levels were followed over a 12-month period; within 4 months, 71% of those with initial levels of 0.97 mumol/L or greater were retested and 57% and 34% of children with initial BPb levels of 0.73 through 0.92 mumol/L and 0.48 through 0.68 mumol/L, respectively, were retested. CONCLUSIONS: Follow-up of children with elevated BPb levels is inadequate within an urban ambulatory care network.

Low-level lead-induced neurotoxicity in children: an update on central nervous system effects.

The neurotoxicity of low-level long-term exposure to lead has a special relevance in children. An extensive database has provided a direct link between low-level lead exposure and deficits in the neurobehavioral-cognitive performance evidenced in childhood through adolescence. Electrophysiological studies showed that neurosensory processing may be affected by lead, with consequent decrease in auditory sensitivity and visuomotor performance. Lead disrupts the main structural components of the blood-brain barrier by primary injury to astrocytes with a secondary damage to the endothelial microvasculature. Within the brain, lead-induced damage occurs preferentially in the prefrontal cerebral cortex, hippocampus and cerebellum. Some characteristic clinical features of lead poisoning may be attributed to this specific anatomical pattern. The cellular, intracellular and molecular mechanisms of lead neurotoxicity are numerous, as lead impacts many biological activities at different levels of control: at the voltage-gated channels and on the first, second and third messenger systems. These effects could be related to lead's ability to interfere with the regulatory action of calcium in cell functions. Consequently, it may be assumed that lead acts as a chemical stressor and causes breakdown of the homeostatic cellular mechanisms. This is expressed in both the anatomical site and the neurotransmitter systems which are crucial in modulating emotional response, memory and learning. There is no threshold below which lead remains without effect on the central nervous system; thus, symptoms could simply be a clinical reflection of the brain regions preferentially involved. In integrating these physiological and clinical data, it may be suggested that the different mechanisms of low level lead neurotoxicity have a final common functional pathway.

Children with moderately elevated blood lead levels: a role for other diagnostic tests?

In this study we examined potential limitations of relying exclusively on blood lead (BPb) levels to evaluate children with moderately elevated BPb levels (1.21-2.12 micromol/l, or 25-44 microg/dl). We tested the following hypotheses: 1) such children without elevated erythrocyte protoporphyrin (EP) levels (>=0.62 micromol/l or >/= 35 microg/dl) are unlikely to respond to a chelating agent with a brisk urinary Pb diuresis; 2) those with elevated EP levels, but low hematologic indices consistent with iron deficiency, are also unlikely to respond to a chelating agent with a robust urinary Pb diuresis; and 3) those with elevated EP levels and iron sufficiency are more likely to respond to a chelating agent. To test these hypotheses, we performed retrospective analyses of the relationships between EP concentrations, hematologic indices, and urinary Pb excretion ratios (uPbr) in moderately Pb-poisoned children undergoing the CaNa2EDTA lead mobilization test (Pb-MT). Data from 122 children were available. Urinary Pb excretion was limited in children with an EP <0.62 micromol/l (<35 microg/dl); only 5% (1/21) of Pb-MTs were positive (uPbr >=0.6). In children with an EP >=0.62 micromol/l, low hematologic indices, such as a mean corpuscular hemoglobin (MCH) <23 pg, were associated with relatively little Pb excretion (0/14 positive Pb-MTs). In contrast, 32% (28/87) of Pb-MTs were positive in children with an EP >/= 0.62 micromol/l and iron sufficiency (p<0.01 by chi-square comparison between groups with EP >/= 0.62 micromol/l and either MCH <23 pg or MCH >/= 23 pg). We conclude that only a minority of moderately Pb-poisoned children will demonstrate enhanced urinary Pb excretion in response to chelation therapy. Some of the predicted nonresponders can be readily identified by adding the EP and complete blood count to the panel of tests performed.

Lead induced rise in intracellular free calcium is mediated through activation of protein kinase C in osteoblastic bone cells.

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.

Clinical applications of L-line X-ray fluorescence to estimate bone lead values in lead-poisoned young children and in children, teenagers, and adults from lead-exposed and non-lead-exposed suburban communities in the United States.

In summary, LXRF estimates of Pb in tibial cortical bone have yielded highly relevant clinical data relating to the efficacy of chelation therapy with CaNa2EDTA in lead poisoned children; diagnostic approach(es) to childhood lead poisoning; and evaluations of exposure in children, teenagers, and adults in lead-exposed and non-lead-exposed suburban communities. It is anticipated that KXRF and LXRF estimates of Pb in bone will yield new information concerning the epidemiology of hypertension, osteoporosis, and the contribution of maternal Pb to the developing fetus. It would be premature to delineate the age-developmental time boundaries over which these two systems [KXRF, LXRF] can be viewed as operated optimally. Consequently, it is appropriate to view the two approaches as providing complementary information. All such information might, in fact, be required to obtain a complete exposure profile and a comprehensive framework for assessment of health risks.

Moderate lead poisoning: trends in blood lead levels in unchelated children.

The appropriate clinical management of children who are moderately poisoned with lead (Pb) is under active investigation. To determine the pattern of change in blood Pb (BPb) levels in the absence of chelation therapy, we followed moderately Pb-poisoned children (initial blood Pb levels 1.21-2.66 mumol/l or 25-55 micrograms/dl) for 6 months with repeated BPb level measurements. Chelation therapy was not administered because all the children had negative lead mobilization tests indicating limited response to the chelating agent, calcium disodium edetate (CaNa2EDTA). Eligible children received the following interventions: notification of the health department to remediate lead hazards; reinforced educational efforts about the toxicity sources and treatment of Pb during 10 clinic and 3 home visits; and iron therapy for children with ferritin levels less than 16 micrograms/l. To quantify the lead paint hazards in the home, we combined a visual rating of the surfaces (intact to peeling) with an X-ray fluorescence (XRF) measurement of the lead content of the painted surface. The sum of these assessments is termed the home environmental score (HES). Data were analyzed from 79 children. BPb levels declined by 27%, on average, over 6 months. HES was correlated with BPb at enrollment, but neither the initial nor later HES measurements predicted BPb at other time points. The HES was highest at enrollment and declined by 50% and 75% at the second and third home visits, respectively. However, only a minority of the children (20%) achieved an HES of 0, indicating no lead paint hazards at home. Despite some ongoing Pb exposure, a parallel fall in BPb levels was observed in subgroups of children with either initially low or high HES (above or below the median HES of 37). Iron status did not account for the change in BPb levels. These data provide evidence that our measure, the HES, is quantifiably related to BPb levels in children, that this correlation is significant only prior to intervention; and that BPb levels decline in children who are moderately poisoned with Pb after they are enrolled in a comprehensive intervention program, even in the absence of chelation therapy and in the presence of ongoing lead paint exposure and Fe deficiency.

Childhood lead poisoning in China.

In China, comprehensive epidemiological data relating to the prevalence of childhood lead poisoning are not available. However, existing data suggest that this disease may be widely pervasive as a result of rapid industrialization and the use of leaded gasoline. Seventeen publications have reported elevated blood lead levels in children from different areas of the country. Children residing in industrial and busy traffic areas had average blood lead levels(BPb) of 21.8-67.9 mu g/dl. The percentages of BPb values above 10 mu g/dl, which is the definition of lead poisoning in children, ranged from 64.9% to 99.5%. Even for 'unexposed' children, about 50% of them had BPb values above 10 mu g/dl. Furthermore, several retrospective pilot studies were conducted in Shanghai, Shenyang, Fuzhou and Beijing to evaluate the health effects of lead at current degree of exposure. The link between low-level lead exposure and deficits in IQ, neurobehavioral development and physical growth is remarkably consistent without exception. In summary, the harmful health effects of childhood lead poisoning in limited studies of exposed and 'unexposed' children demonstrate that this totally preventable disease warrants considerable public health attention in China.

Relationships among blood lead levels, iron deficiency, and cognitive development in two-year-old children.

The goals of this study were to explore the relationship of declining blood lead levels and cognitive development in 42 moderately lead poisoned children around 2 years of age and to investigate the potential interaction between iron and lead levels in the course of development. The cognitive functioning of children was assessed upon enrollment into a comprehensive intervention and 6 months later. The intervention consisted of chelation treatment, if appropriate, iron supplementation, if needed, and steps to eliminate the source of lead in the home environment. The children were referred because of blood lead levels between 25 and 55 mu g/dl; they were also selected on the basis of age between 18 and 30 months. The outcome measures were the global score on a standardized test of cognitive development and subscale scores for perceptual-motor and language functioning. Cognitive change over 6 months was related to an interaction between change in blood lead and initial iron status. Specifically, the change in standardized score (particularly change in perceptual-motor performance) was strongly related to change in blood lead in children who were iron sufficient at the outset: there was an increase of 1.2 points for every 1 mu g/dl decrease in blood lead. There was no such relationship in iron-deficient children. Secondary analyses suggested that 1) the change in cognitive functioning of iron-deficient children was related to change in hemoglobin, and 2) the decline in blood lead was less in iron-deficient than in iron-sufficient children. Thus, when iron is sufficient, changes in blood lead and changes in cognition are inversely related. When iron is deficient, other processes affect the outcome.

Adverse health effects of lead at low exposure levels: trends in the management of childhood lead poisoning.

An extensive database has provided a direct link between low-level lead exposure during early development and deficits in neurobehavioral-cognitive performance evident late in childhood through adolescence. These consistent studies have demonstrated the presence of a constellation of neurotoxic and other adverse effects of lead at blood lead (BPb) levels at least as low as 10 micrograms/dl). Federal agencies and advisory groups have redefined childhood lead poisoning as a BPb level of 10 micrograms/dl. Before discussing some of these studies in greater detail, the pervasiveness of this entirely preventable disease today in millions of American children must be recognized.

The effects of succimer on the absorption of lead in adults determined by using the stable isotope 204Pb.

The chelating agent succimer (meso-2,3-dimercaptosuccinic acid) is orally effective at inducing a urinary lead diuresis and a decrease in blood lead levels in lead poisoned children and adults. However, there are concerns that succimer may increase the absorption of lead from the gastrointestinal (GI) tract during treatment, particularly in cases of continuing lead exposure, which would compromise its effectiveness in reducing whole body lead stores. This preliminary study investigated the effects of succimer on the absorption of lead in adults using a stable lead isotopic tracer (204Pb). Twelve male subjects were divided into control (no succimer), 10, and 30 mg succimer/kg body wt treatment groups of 4 individuals each. All subjects ingested a single tracer dose (200 micrograms) of 204Pb, followed by a single oral dose of placebo (control) or succimer. Whole blood was collected at intervals of 0, 2, 4, and approximately 26 hr following ingestion of the 204Pb tracer, and composite urine and feces samples were collected over the duration of the study (approximately 26 hr). Mean intestinal excretion of 204Pb was reduced in the succimer-treated groups compared to the control (placebo), whereas urinary diuresis of 204Pb was higher in the succimer groups. The amount of lead 204Pb tracer accounted for at the end of the study was lower in the succimer-treated groups. These results suggest that GI lead absorption was enhanced by succimer and that succimer mediated the redistribution of lead from the circulation to other tissues. However, none of the differences between treatment groups were statistically significant (P < 0.05, t test) because of the relatively large within-group variability. This study demonstrates the utility of microgram doses of a stable lead isotopic tracer to assess the efficacy of clinical chelating agents in humans. Future studies are necessary to further clarify the effects of succimer on the absorption and retention of lead in adults and children. Although, based upon these preliminary data, it appears advisable that patients be maintained in a lead-safe environment while being treated with succimer.

The displacement of calcium from osteocalcin at submicromolar concentrations of free lead.

Lead, an environmental toxin, is known to impair some of the functional properties of osteocalcin, a small protein (MW, 5700) active in bone mineralization and resorption. To investigate a possible mechanism of lead toxicity at the molecular level, we have studied the interaction of lead with osteocalcin using 43Ca and 1H NMR. The measured 43Ca NMR linewidth as well as longitudinal relaxation rate (1/T1) of 43CaCl2 progressively increased with increasing amounts of added osteocalcin. A titration measuring 43Ca linewidth as a function of [Ca2+]/[Osteocalcin] ratio could be fitted to a single metal binding site with a dissociation constant of 7 microM. The 43Ca 1/T1 of Ca-osteocalcin decreased in the presence of Pb2+ due to competitive displacement of Ca2+ by Pb2+. The magnitude of decrease in the effect of osteocalcin on 43Ca 1/T1 in the presence of Pb2+ was consistent with the existence of only one tight divalent cation binding site. An analysis of the NMR T1 data in osteocalcin solutions containing both Pb2+ and Ca2+ yielded a Pb-osteocalcin dissociation constant of about 2 nM. The 1H NMR spectra showed Pb-induced changes in the same aliphatic and aromatic resonances of osteocalcin that are also affected by Ca(2+)-binding, supporting interaction of Pb2+ at the Ca2+ site. However, the existence of significant differences between the Pb-osteocalcin and Ca-osteocalcin NMR spectra indicates some differences in the structures of the two complexes. Since Pb2+ inhibits the binding of osteocalcin to hydroxyapatite, the high affinity of Pb2+ for osteocalcin would indicate significant inactivation of osteocalcin even at submicromolar free lead levels. Pb(2+)-induced inactivation of osteocalcin could affect bone mineral dynamics and may be related to the observed inverse correlation between blood Pb(2+)-levels and stature and chest circumference observed in growing children.

Lead perturbs 1,25 dihydroxyvitamin D3 modulation of intracellular calcium metabolism in clonal rat osteoblastic (ROS 17/2.8) cells.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is known to modulate Ca2+ metabolism in several cell types. 1,25(OH)2D3 causes an increase in Ca2+ influx and probably exerts many of its effects via the Ca2+ messenger system. Lead (Pb2+) interacts with and perturbs normal Ca2+ signalling pathways; hence, the purpose of this work was to determine if Pb2+ perturbs 1,25(OH)2D3 modulation of Ca2+ metabolism in ROS 17/2.8 cells, which express receptors for and respond to 1,25(OH)2D3, and to determine the effect of 1,25(OH)2D3 on Pb2+ metabolism in these cells. In both cases three kinetic compartments described the intracellular metabolism of the isotope. These data show that 1 microM Pb2+ inhibits 1,25(OH)2D3 modulated increases in Ca2+ flux, whereas 5 microM Pb2+ increases membrane fluxes, all intracellular Ca2+ pools, and total cell Ca2+. In the Pb2+ metabolism studies it was found that 10 nM 1,25(OH)2D3 increases intracellular Pb2+. Pb2+ appears to disrupt the modulation of intracellular steady-state Ca2+ homeostasis by 1,25(OH)2D3 in a complex, biphasic manner and may therefore perturb functions that are modulated by 1,25(OH)2D3 via the Ca2+ messenger system.

Lead activation of protein kinase C from rat brain. Determination of free calcium, lead, and zinc by 19F NMR.

Lead (Pb2+) has been reported to activate calcium/phospholipid-dependent protein kinase C (PKC) at subnanomolar concentrations (Markovac, J., and Goldstein, G. W. (1988) Nature 334, 732-734); however, others have failed to find any Pb(2+)-induced activation of PKC (Murakami, K., Feng, G., and Chen, S. G. (1993) J. Pharmacol. Exp. Ther. 264, 757-761). In neither of these studies was the actual free Pb2+ or Ca2+ concentration measured. In this study, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA) was used to buffer Pb2+ and Ca2+ concentrations in the PKC reaction mixture. The specific free ion concentrations of Pb2+ and Ca2+, as well as Zn2+ and other divalent cations contained in the PKC reaction mixtures, were determined by 19F NMR spectroscopy. Using this approach to set and confirm the free Pb2+ and Ca2+ concentrations, we measured the Pb(2+)-dependent and the Ca(2+)-dependent activation of phosphotydylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous acid precipitable proteins in the 100,000 x g supernatant from homogenized rat brain cortex. We found that free Pb2+ activates PKC in the range from 10(-11) to 10(-8) M, Kact = 5.5 x 10(-11) M, while Ca2+ activates PKC in the range from 10(-8) to 10(-5) M, Kact = 2.56 x 10(-7) M. These findings clearly resolve the activation of PKC by subnanomolar concentrations of free Pb2+ from activation induced by Ca2+ or other divalent cations. Furthermore, it documents the utility of 5F-BAPTA as buffer and indicator when resolving the contributions of multiple divalent cations in biochemical processes.

Effects of calcium disodium versenate (CaNa2EDTA) chelation in moderate childhood lead poisoning.

BACKGROUND: For children with asymptomatic moderate lead poisoning (Blood lead level [BPb] 25 to 55 micrograms/dL [1.21 to 2.66 mumol/L]), treatment with the chelating agent calcium disodium versenate (CaNa2EDTA) is recommended for all those children with a BPb level > 45 micrograms/dL (2.17 mumol/L) and for those with a BPb level of 25 to 44 micrograms/dL (1.21 to 2.13 mumol/L) who also have a positive lead mobilization test. However, controlled studies demonstrating its efficacy at inducing a sustained reduction in BPb level or lead-related toxicity have not been performed in children with moderate lead poisoning. This study assesses the relationship between CaNa2EDTA chelation and measures of lead burden and toxicity in children with moderate lead poisoning. METHODS: Two hundred one children with moderate lead poisoning were enrolled. Sequential changes in BPb concentrations, bone lead level as measured by L alpha-x-ray fluorescence, and lead-induced toxicity as assessed by erythrocyte protoporphyrin levels were determined over a 7-week period. From this group, children with a positive lead mobilization test received CaNa2EDTA chelation therapy. RESULTS: Children with positive lead mobilization tests had on average higher initial BPb, bone lead, and erythrocyte protoporphyrin concentrations. The chelated children decreased approximately 4.7 micrograms/dL (0.23 mumol/L), 41 corrected net counts, and 24 micrograms/dL (0.46 mumol/L) more than the unchelated children on BPb, bone lead, and erythrocyte protoporphyrin values, respectively. However, children with higher initial levels decreased the most, whereas children with lower initial levels showed the least decline, with or without treatment. When the initial values on the measures were controlled analytically, or when subgroups matched on initial levels were compared, there were no significant differences between the chelated and unchelated children. CONCLUSIONS: The apparent effectiveness of CaNa2EDTA at reducing lead burden and toxicity is no longer observed when the pretreatment levels are considered. The findings suggest that sufficient doubt about CaNa2EDTA efficacy now exists to warrant a randomized controlled trial of chelation therapy in moderately lead-poisoned children. However, until such studies are performed, it would be premature to withhold chelation treatment on the basis of this study alone.