Circadian rhythms of blood minerals in humans.

Circadian rhythms of ionized calcium and phosphate concentrations have been demonstrated in human blood. A computer-derived model curve representing the 24-hour fluctuations in ionized calcium cannot be correlated consistently with curves for total calcium or phosphate. Knowledge of these circadian rhythms provides a physiological basis for further understanding the interactions between blood minerals and calcium-regulating hormones.

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8).

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) on the intracellular free calcium-ion concentration ([Ca2+]i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca2+]i at doses of 1 to 10 nM 1,25-(OH)2D3. At 10 nM, 1,25-(OH)2D3 elevated the [Ca2+]i from a control level of 118 +/- 4 nM to a peak value of 237 +/- 8 nM within 40 min. 1,25-(OH)2D3 also increased the initial rate of Ca2+ influx into ROS 17/2.8 cells, measured by 45Ca uptake, with a dose-response relationship which paralleled its effect on [Ca2+]i. Treatment of ROS 17/2.8 cells with Pb2+ at 1 and 5 microM significantly increased [Ca2+]i but significantly reduced the 1,25-(OH)2D3-induced elevation of [Ca2+]i. Simultaneous treatment of naive cells with 1,25-(OH)2D3 and Pb2+ produce little reduction of 1,25-(OH)2D3-induced 45Ca uptake while 40 min treatment with Pb2+ before addition of 1,25-(OH)2D3 significantly reduced the 1,25-(OH)2D3-induced increase in 45Ca influx. These findings suggest that Pb2+ acts by inhibiting 1,25-(OH)2D3-activation of Ca2+ channels and interferes with 1,25-(OH)2D3 regulation of Ca2+ metabolism in osteoblastic bone cells.

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+.

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.

Effect of lead on parathyroid hormone-induced responses in rat osteoblastic osteosarcoma cells (ROS 17/2.8) using 19F-NMR.

Using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, we have recently demonstrated that Pb2+ treatment elevates the intracellular free calcium ion concentration ([Ca2+]i) of rat osteoblastic osteosarcoma cells (ROS 17/2.8) (Proc. Natl. Acad. Sci. USA (1989) 86, 5133-5135). In this study, we have examined the effects of Pb2+ on the basal and parathyroid hormone (PTH)-stimulated levels of [Ca2+]i and cAMP in cultured ROS 17/2.8 cells. PTH treatment (400 ng/ml) stimulated a 150% elevation in [Ca2+]i from a control level of 105 +/- 25 nM to a concentration of 260 +/- 24 nM. Treatment of ROS 17/2.8 cells with Pb2+ (5 microM) alone produced a 50% elevation in the [Ca2+]i to 155 +/- 23 nM. Pb2+ treatment diminished subsequent elevation in [Ca2+]i in response to PTH administration thereby limiting the peak increase in [Ca2+]i to only 25% or 193 +/- 22 nM. In contrast to the dampening effect of Pb2+ on the peak rise in [Ca2+]i produced by PTH, Pb2+ (1 to 25 microM) had no effect on PTH-induced increments in intracellular cAMP levels. Hence, Pb2+ dissociated the PTH stimulation of adenylate cyclase from PTH effects on [Ca2+]i and shifted the regulation of [Ca2+]i beyond the control of PTH modulation. These observations further extend the hypothesis that an early toxic effect of Pb2+ at the cellular level is perturbation of [Ca2+]i homeostasis.

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.

Depressed excretion of 6 beta-hydroxycortisol in lead-toxic children.

6 beta-Hydroxycortisol (6 beta OHF) is a highly polar metabolite of cortisol, probably formed in the endoplasmic reticulum of hepatocytes by cytochrome P-450-dependent microsomal monoxygenases. Lead decreases the activity of cytochrome P-450-dependent microsomal hydroxylases in vivo and in vitro. To examine possible inhibitory effects of lead on 6 beta OHF metabolism, urinary 6 beta OHF excretion was measured in 26 children with mild to moderate increases in blood lead concentrations. Children were divided into 2 groups on the basis of their response to the EDTA provocative test. This test was used to assess the size of chelatable and potentially toxic lead stores in such children. Children with elevated urinary lead excretion after an EDTA provocative test, i.e. elevated tissue lead stores, had markedly decreased urinary excretion of 6 beta OHF (178 +/- 15 micrograms/m2 X 24 h) compared to children who had negative tests (333 +/- 40 micrograms/m2 X 24 h; P less than 0.01); their urinary cortisol excretion was not different from that of age-matched controls. These findings suggest that lead, at relatively low concentrations, may interfere with hepatic microsomal formation of a cortisol metabolite.

1,25-dihydroxyvitamin D3-treated hypoparathyroidism: 35 patients years in 10 children.

Ten hormone-deficient hypoparathyroid children have been successfully treated with orally administered 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] within a dose range of 0.01--0.10 microgram/kg . day for a total of 35 patient yr. Resistance to 1,25-(OH)2D3 therapy was not observed. By monitoring the blood ionized calcium level (normal range, 4.05--5.14 mg/dl) at monthly intervals, 8 hypercalcemic and 10 hypocalcemic episodes were recognized during the entire treatment experience, though the patients were asymptomatic. In the former cases, cessation of treatment for 3 days resulted in normocalcemia; in the latter, half of the episodes were transient and related to intercurrent febrile illnesses. The remaining hypocalcemic occurrences responded to an increase in the dosage of 1,25-(OH)2D3. During 1,25-(OH)2D3 treatment, the mean ionized calcium level for individual patients ranged from 4.04--4.39 mg/dl. Urinary Ca excretion, expressed as a ratio of calcium to creatinine, ranged from 0.177--0.244. Twenty-four-hour mean levels of serum 1,25-(OH)2D did not correlate with the blood ionized calcium concentration in 4 patients who underwent sequential blood sampling every 1--2 h for a total of 59 time points (r = 0.05; P greater tha 0.10). To elucidate the temporal relationship between hormonal and mineral responses to exogenous PTH, we administered bovine PTH to four untreated hypoparathyroid children. Serum concentrations of PTH increased before the rise in ionized calcium, which in turn preceded the elevation in circulating 1,25-(OH)2D. This order was followed also in the fall in serum concentrations of these blood constituents to pre-PTH infusion values. These results indicate responsiveness of the l alpha-hydroxylase enzyme to exogenous PTH in hormone-deficient hypoparathyroid children.

Osteocalcin in human serum: a circadian rhythm.

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.

Temporal interrelationships between the circadian rhythms of serum parathyroid hormone and calcium concentrations.

The temporal relationships between the circadian rhythms of serum PTH, total calcium (Cat), and phosphate (Pi) and plasma ionized calcium (Cai) concentrations were determined in 9 normal men. Blood samples were collected every half hour for 24 h. Serum PTH was measured by an RIA specific for the midregion of the molecule. The mean circadian pattern for each variable was derived by calculating the average value across all men at concurrent time points. After the data were smoothed by the method of running means, the correlations between PTH and mineral values from concurrent time points were calculated, as were cross-correlations to 12 lag periods (6 h). Spectral and cross-spectral analyses were performed on the same data set. Both statistical methods yielded consistent results: 1) at concurrent time points (0 lag), high correlations were found between serum PTH and Cat (r = -0.74), PTH and Pi (r = 0.79), and PTH and Cai (r = -0.53); and 2) when the PTH series was lagged by 2 h, the PTH/Cai correlation improved to -0.70. A direct PTH/Cai correlation of 0.50 was found when the Cai series was lagged about 4.5 h. No improvement in the correlations were found when the other series were lagged. Spectral analyses also detected significant interrelations between PTH and Cai at 2 and 3.5 h. These data describe the timing of the bidirectional interaction between serum PTH and plasma Cai under steady state conditions in normal adult men; changes in Cai concentrations precede inverse changes in PTH levels by 2 h, whereas changes in PTH precede similar directional alterations in Cai by about 4 h.

Hereditary resistance to 1,25-dihydroxyvitamin D: defective function of receptors for 1,25-dihydroxyvitamin D in cells cultured from bone.

UNLABELLED: The syndrome of rickets, alopecia, hypocalcemia, and high circulating levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D) apparently is caused by resistance of target tissues to 1,25-(OH)2D. To evaluate this, we cultured cells from explants of long bone of one patient with this syndrome and from a control without any preexisting disorder of mineral metabolism. The cultured cells showed morphological features of fibroblasts but contained alkaline phosphatase activity without detectable acid phosphatase activity, indicating an osteoblastic origin for some or all of the cultured cells. Receptors for 1,25-(OH)2D were assessed by three methods: high affinity uptake of hormone in nuclei of dispersed cells, high affinity binding in hypertonic extracts (herein termed cytosol) from cells, and sedimentation velocity of bound [3H]1,25-(OH)2D3 in extracts of cell nuclei. With cells cultured from bone of the normal control, receptors for 1,25-(OH)2D exhibited properties indistinguishable from those found with cultured skin fibroblasts. With cells cultured from bone of the patient with resistance to 1,25-(OH)2D, high affinity uptake of 1,25-(OH)2D into nuclei was unmeasurable, but high affinity binding of hormone with cytosol was normal; these abnormal findings also were indistinguishable from abnormal findings obtained with fibroblasts cultured from skin of that patient. IN CONCLUSION: 1) Cells cultured from explants of human bone showed morphological features of fibroblasts but retained a marker enzyme characteristic of osteoblasts. Significant admixture of osteoblast-like cells with fibroblasts was possible. 2) Cells cultured from bone of a patient with familial resistance to 1,25-(OH)2D exhibit a defect in vitamin D metabolism, indistinguishable from the defect observed with cells cultured from skin of the same patient.

Absence of seasonal variation in serum concentrations of 1,25-dihydroxyvitamin D despite a rise in 25-hydroxyvitamin D in summer.

The serum concentrations of the vitamin D metabolites 25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D (calcitriol) have been measured in normal subjects whose ages varied from 18 months to 35 yr. Samples were obtained in all months of the year in order to assess the effects of season on serum concentration. During the months of April to September, 25OHD3 levels are higher than in the winter months. No seasonal variation in the 25OHD2 or calcitriol serum concentration was observed. Age-related differences in 25OHD2 and D3 concentrations did not exist. The levels of calcitriol are higher in adolescence and increase from 35 +/- 19 pg/ml (SD) at 1.5-10 yr of age to 54 +/- 21 pg/ml at 10-20 yr of age. In young adults, the levels fall again to 28 +/- 16 pg/ml. Accordingly, despite a seasonal variation in the precursor of calcitriol, the levels of this most active metabolite of vitamin D do not change in relation to sunlight exposure. This lack of seasonal variation is further evidence of the tight feedback regulation of calcitriol.

Effects of growth hormone therapy on circadian osteocalcin rhythms in idiopathic short stature.

The effects of GH administration on the circadian osteocalcin (Oc) rhythm were determined in four prepubertal children with idiopathic short stature (height, less than 5th percentile; growth velocity, less than 50th percentile for age). Each child underwent 24-h sequential blood sampling on three occasions: immediately before the initiation of GH treatment, 6 months later, and at the end of 12 months of treatment. The growth rate increased more than 50% over baseline in three of the four children during at least one of the 6-month periods. Insulin-like growth factor-I levels increased during treatment in all of the children. Twenty-four-hour Oc levels increased on 7 of the 8 treatment days evaluated. When mean 24-h Oc patterns for each of the 3 study days were derived by averaging across individual subjects at each time point and then compared, we noted an upward shift in the entire pattern during treatment (t = 13.2 at P less than 0.001 and t = 5.9 at P less than 0.001 for 6 and 12 month comparisons vs. the pretreatment day, respectively). This was more easily appreciated after the data were smoothed using the method of running means. There was, in addition, a progressive improvement in the shape of the Oc pattern compared to a normative model derived from a study of healthy adult men. The correlation between the model and the pre-GH day was 0.46, that between the model and the 6 months of GH day was 0.77, and that between the model and the 12 months of GH day was 0.96. Cross-correlation analyses showed that the peak correlation between the 2 treatment days and the model occurred at zero lag. In contrast, the peak correlation between the pre-GH day and the model or the pre-GH day and either of the 2 treatment days occurred when the pre-GH series was lagged by 2-3 h. Thus, an additional finding is the synchronization of the Oc series that occurred during treatment. We conclude that GH treatment increases Oc concentrations in children with idiopathic short stature by affecting its circadian rhythm. This rise in Oc values may not necessarily reflect an increase in growth velocity.

Circadian variations in serum zinc (Zn) concentrations: correlation with blood ionized calcium, serum total calcium and phosphate in humans.

Serum zinc (Zn) concentrations were determined in sequentially drawn blood samples from six healthy adult males. Each subject had blood sampling performed every 30 minutes for 24 hours. Mean Zn concentrations at each time point (n = 6, 48 time points) were plotted and fitted by a polynomial regression of the data against time. A "U" shaped curve was derived; we found peak Zn levels at 9:30 AM, a midtrough at 8 PM and a peak-trough difference of 19 micrograms/dl. Correlation with a similarly derived 24-h ionized calcium pattern was strikingly high, r = .923, p less than .001. An intermediate correlation between the Zn and phosphate patterns was observed, r = -.493, p less than .01; and no significant correlation occurred between Zn and total calcium rhythms, r = .167. These data conclusively demonstrate the presence of a circadian rhythm in serum Zn in healthy adult males. Furthermore, the high correlation between the Zn and ionized calcium patterns suggests a common regulator.

Association between age, blood lead concentration, and serum 1,25-dihydroxycholecalciferol levels in children.

Serum levels of 1,25-dihydroxycholecalciferol (1,25-CC), the form of vitamin D active in stimulating intestinal absorption of calcium, phosphorus, and lead, were determined in 177 human subjects ages 1 to 16 yr. Significant negative association (r = -0.88) was observed between serum 1.25-CC levels and blood lead concentrations over the entire range of blood lead levels, 12 to 120 micrograms/dl. Adolescents ages 11 to 16 yr had serum 1,25-CC levels higher than those observed among children 10 yr old or younger. No effect of sex or season on serum 1,25-CC level was observed. When the 1,25-CC values for children with blood lead concentrations greater than 30 micrograms/dl were excluded from the analysis, no significant effect of geographic location on 1,25-CC levels was observed.

Marrow transplantation for juvenile osteopetrosis.

Two children with the juvenile form of osteopetrosis were treated with marrow transplants from their HLA identical siblings. Following transplantation each child exhibited extensive bone reabsorption with a marked augmentation of osteoclastic function attributable to donor osteoclasts, including remodeling of bone with expansion of intramedullary hematopoiesis and correction of associated abnormalities of thymic factor and natural killer cells. Osteopetrosis ultimately recurred in one patient in whom engraftment of donor hematopoietic elements was not achieved. Our studies indicate that marrow transplantation will correct osteopetrosis but that permanent reconstitution necessitates sustained engraftment of marrow precursors of cells with osteoclastic activity.

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.

Significance of plasma lead levels in normal and lead-intoxicated children.

Plasma lead (Pb) levels have been measured in normal and lead-intoxicated children, newborns, and children with sickle cell disease. The results in all groups were contant over a wide range of red cell Pb concentration. These results support the thesis that the red cell represents a large repository for Pb, maintaining plasma Pb concentration within closely defined limits, and that methods other than measurements of plasma Pb will be necessary to uncover a presumably dynamic transport system between red cell and plasma. Indeed, we have demonstrated in vitro that ionized calcium (Ca(2+)) lowers red cell Pb content according to a linear dose-response curve. Ca(2+) may thereby control Pb transport from red cell to plasma, and fluctuations in the concentration of Ca(2+) in serum and extracellular fluid may influence the toxic activities of Pb. In bone organ culture, changes in the concentration of Ca(2+) and phosphate in the medium alter the release of previously incorporated (210)Pb from fetal rat bones in response to parathyroid hormone (PTH). Therefore, both PTH and the ionic milieu of the medium apparently regulate bone Pb metabolism.We would expect that understanding further the dynamics of Pb transport in plasma and bone may lead to a more exact definition of the real hazards of low level Pb toxicity in children.

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.

Cellular and molecular toxicity of lead in bone.

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.