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We evaluated circulating levels of biologically active and immunoreactive intact parathyroid hormone [iPTH-(1-84)] in 47 newborns at birth and eight hypocalcemic preterm infants during the first 10 d of life. Use of two sensitive detection systems, the cytochemical bioassay and an immunoradiometric assay specific for intact parathyroid hormone, enabled us to compare plasma concentrations of PTH-like bioactivity (bioPTH) and iPTH-(1-84). Mean umbilical venous plasma bioPTH was elevated in nondiabetic term and preterm newborns [22.5 +/- 3.1 (+/- SEM) and 15.8 +/- 2.5 ng-equiv/L, respectively] compared with normal adult subjects (9.8 +/- 2.6 ng-equiv/L; p less than 0.01). Umbilical bioPTH was suppressed in five term infants of diabetic mothers (2.6 +/- 0.4 ng-equiv/L). In contrast, iPTH-(1-84) was low in term and preterm nondiabetic infants' and term infants of diabetic mothers' umbilical samples (5.4 +/- 1.5, 4.3 +/- 1.5, and 2.4 +/- 1.0 ng/L, respectively). Umbilical venous bioPTH was highly correlated with the magnitude of the transplacental calcium gradient (r = 0.90; p less than 0.05). In eight preterm infants studied longitudinally, by 24-36 h of life, declining plasma total and ionized calcium (1.71 +/- 0.04 and 0.78 +/- 0.03 mmol/L, respectively) were accompanied by a significant rise in both bioPTH (41.2 +/- 6.3 ng-equiv/L) and iPTH-(1-84) (56.3 +/- 11.6 ng/L). These data indicate that the 3rd trimester fetoplacental circulation contains levels of bioPTH several-fold higher than those of immunoreactive intact hormone. We also conclude that even hypocalcemic preterm newborn infants can significantly elevate circulating levels of PTH.

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Fluoride ion (F-) alone or in conjunction with aluminum (Al3+) has been shown to stimulate the activity of guanine nucleotide-binding proteins (G proteins) in cell membrane preparations from a variety of cell types and in intact hepatic cells. Several studies have indicated that G proteins are involved in the regulation of parathyroid hormone (PTH) secretion. Intracellular second messengers which modulate PTH secretion (e.g., cAMP) have also been found to be regulated by G proteins. We have, therefore, employed F- as a probe to investigate the possible role of G proteins in the modulation of PTH release and the intracellular second messengers that have been implicated in the control of PTH secretion. F- produces a dose-dependent inhibition of PTH release with a maximal inhibitory effect (67%) at 5 mM. F- exerts its inhibitory effect within 5 min and the degree of suppression of PTH secretion gradually increases over 1 hr. F- (5 mM) inhibits PTH secretion at 0.5 mM Ca2+ to the level observed with 2 mM Ca2+ alone; moreover, the effects of F- and high Ca2+ are not additive. While 1 mM F- suppresses PTH secretion by only 21%, and 10 microM Al3+ has virtually no effect at all, together they inhibit PTH release approximately to the level (63% inhibition) observed with 5 mM NaF alone. In the presence of 10(-5) M dopamine, F- produces a concentration-dependent inhibition of cAMP accumulation (0.684 +/- 0.033 pmoles/10(5) cells at 0 mM F- vs. 0.256 +/- 0.048 at 5 mM F-). However, the F- -induced decrease in cAMP cannot account for the inhibition of PTH release by this agent, since addition of methylisobutylxanthine (10(-4) M) by F- -treated cells raises intracellular cAMP content above that of control cells but fails to reverse the inhibition of PTH release. The cytosolic calcium concentration in Fura-2-loaded cells increases from 210 +/- 20 nM to 340 +/- 44 nM after 5 mM F- was added to incubation media. Prior removal of extracellular Ca2+ by EGTA totally blocks the F- -induced rise in cytosolic Ca2+ without preventing the inhibition of PTH release by NaF. F- also produces a time- and dose-dependent increase in the accumulation of IP, IP2, and IP3 in cells prelabeled with [3H]inositol and incubated with 10 mM Li+, consistent with activation of phospholipase C. We conclude that F- is a potent inhibitor of PTH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

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Wilson's disease results in excess tissue accumulation of copper and is often complicated by skeletal and mineral abnormalities. We investigated vitamin D metabolism in rats fed a copper-laden diet rendering hepatic copper content comparable with that found in Wilson's disease. Injection of 25-hydroxyvitamin D3 [25(OH)D3] resulted in reduced 1,25-dihydroxyvitamin D [1,25(OH)2D] levels in copper-intoxicated rats. In vitro 25(OH)D-1 alpha-hydroxylase activity was impaired in renal mitochondria from copper-intoxicated animals. Activity was also inhibited in mitochondria from controls when copper was added to incubation media. Impaired conversion of 25(OH)D to 1,25(OH)2D occurs in copper intoxication and suggests that altered vitamin D metabolism is a potential factor in the development of bone and mineral abnormalities in Wilson's disease.

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A 2-year-old boy had signs and symptoms of chronic hypervitaminosis A. A course of increasing severity led to eventual death. A younger brother later had similar clinical features. Chicken liver spread containing up to 420 IU/g vitamin A was the likely source of intoxication. Markedly elevated circulating retinyl ester levels have persisted in the surviving sibling for 3 subsequent years despite severe restriction of vitamin A intake. A therapeutic trial of the carbohydrate-derived complexing agent 2-hydroxypropyl-beta-cyclodextrin was initiated. Circulating retinyl esters transiently increased during the infusion (from 407 to 4791 micrograms/dL), and urinary total vitamin A excretion, undetectable before infusion, increased to 23 micrograms/dL after infusion. The frequency of hypervitaminotic episodes has decreased somewhat in the 2 years since the infusion, probably related to dietary vitamin A restriction. The occurrence of this syndrome in two brothers, while a sister ingesting the same diet remains completely healthy, suggests an inherited variance in tolerance to vitamin A intake.

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We have examined possible mechanisms by which osmolality might modulate PTH secretion in dispersed bovine parathyroid cells. Increasing medium osmolality by adding sodium chloride causes a marked, dose-dependent increase in PTH release. The maximum effect (4-fold increase) is observed when osmolality is around 650 mosM, with half-maximal stimulation at about 400 mosM. When osmolality is increased to a similar extent with either sucrose or sodium chloride, PTH secretion is enhanced to a comparable degree, suggesting that osmolality itself, rather than ionic strength, is responsible for the increase in PTH secretion. Time course experiments show that the increased secretion of PTH with high osmolality occurs very rapidly (in less than 5 min). In contrast to the suppressive effects of high Ca2+ on PTH release, increasing calcium concentration in the incubation media does not inhibit the stimulation of PTH secretion by high osmolality. Moreover, the effects of dopamine (10(-5) M) and high osmolality on PTH release are additive, further suggesting that high osmolality and cAMP modulate PTH release by different mechanisms. In fact, direct measurement of cellular cAMP in cells exposed to high osmolality shows no change relative to control cells incubated with normal osmolality, 127 +/- 20 vs. 146 +/- 21 fmol/10(5) cells, respectively. Cytosolic Ca2+ increases from 257 +/- 29 nM to 703 +/- 50 nM after 200 mM NaCl is added to the incubation medium at low Ca2+ (0.5mM). Prior removal of extracellular calcium abolished this effect. Increasing the osmolality to a similar level by adding sucrose to the medium does not demonstrate any increase in cytosolic calcium. We conclude that high osmolality is a potent secretogogue in dispersed bovine parathyroid cells. Unlike dopamine and isoproterenol, high osmolality does not act through changes in intracellular cAMP. It also prevents the normal inhibitory effect of high Ca2+ on PTH release. Change of cytosolic Ca2+ is variable and suggests that the effect of high osmolality on PTH release cannot be explained by cytosolic Ca2+ alone. Further understanding of the mechanisms by which osmolality affects PTH release, therefore, may provide clues to the unusual inverse relationship between extracellular and cytosolic calcium and PTH release.

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A newly developed calcium-sensitive dye, Fura-2, was employed in dispersed bovine parathyroid cells to study the effects of extracellular calcium and magnesium on cytosolic calcium concentration and parathyroid hormone (PTH) release. In comparison with control cells, Fura-2-loaded parathyroid cells showed the same maximal rate of PTH release, set-point for extracellular Ca++ (the calcium concentration producing half of the maximal inhibition of PTH release), and maximal inhibition of PTH release (71.6%) by high extracellular Ca++. At an extracellular Mg++ concentration of 0.5 mM, raising extracellular Ca++ in a stepwise fashion from 0.5 mM to 2.0 mM produced a dose-dependent, statistically significant (p less than 0.01) increase in cytosolic Ca++ from 198 +/- 24 nM (0.5 mM Ca++) to 411 +/- 21 nM (2.0 mM Ca++) which closely paralleled the concomitant decrease in PTH release. An elevation of extracellular Mg++ from 0.5 mM to 5 mM, at an extracellular Ca++ of 0.5 mM, resulted in a transient spike of cytosolic Ca++ which lasted for approximately 30 seconds, followed by a small but stable increase in the cytosolic Ca++ concentration (174 +/- 7 nM vs. 237 +/- 10 nM, n = 4, p less than 0.01). Prior removal of extracellular calcium by addition of an excess of EGTA did not abolish the transient spike induced by high extracellular magnesium concentrations in Fura-2-loaded cells, suggesting that this rapid increase in cytosolic Ca++ arises, at least in part, from intracellular stores of Ca++. This is supported by the observation that pretreating cells with ionomycin resulted in disappearance of the magnesium-induced spike.(ABSTRACT TRUNCATED AT 250 WORDS)

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Resistance to vitamin D in magnesium depletion has been observed in humans and in animal studies. Variable levels of 1,25-dihydroxyvitamin D [1,25(OH)2D] have been reported in patients with magnesium depletion, and studies of vitamin D metabolism in states of magnesium depletion have not yielded consistent results. We examined effects of magnesium deprivation on circulating 1,25(OH)2D levels before and after a loading dose of 25-hydroxyvitamin D3 [25(OH)D3], on in vivo conversion of small doses of radiolabeled 25(OH)D3 to 1,25(OH)2D3 in intact rats, and on in vitro 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity in rat renal mitochondria. The effects of magnesium-free media on mitochondrial 1 alpha-hydroxylase activity was examined. Magnesium depletion did not affect in vivo conversion of 25(OH)D to 1,25(OH)2D. In vitro 1 alpha-hydroxylase activity was comparable in magnesium-replete and -deplete animals and was evident in the absence of added magnesium in incubation media. Our in vivo and in vitro studies are consistent with one another and demonstrate that in the rat conversion of 25(OH)D to 1,25(OH)2D is unimpaired in magnesium deficiency. Resistance to vitamin D in magnesium depletion is likely due to the impaired skeletal responsivity to 1,25(OH)2D, as demonstrated in earlier studies.

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Evidence to date has failed to show a consistent effect of vitamin D metabolites on PTH secretion. This study was undertaken to assess the possible direct, acute effects of vitamin D metabolites on PTH secretion in vitro. Ethanol has been used in several published studies as the vehicle for vitamin D metabolites. We found that 0.2-1.0% ethanol inhibited PTH release from dispersed bovine parathyroid cells (PTC). Our experiments with vitamin D metabolites used ethanol as a vehicle at a concentration less than 0.1%. When compared to ethanol treatment, 10-100 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 25 and 100 nM 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 100 nM 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3) had no effect on PTH release from PTC incubated for up to 4 h. A combination of 1,25(OH)2D3 and 24,25(OH)2D3 (each 25 or 100 nM) was without effect. Also, 100 nM 1,25(OH)2D3 had no effect on PTH release from either bovine parathyroid gland slices or from parathyroid glands from either vitamin D-replete (+D) or vitamin D-deficient (-D) rats incubated for up to 4 h. The i.v. injection of 1 microgram 1.25(OH)2D3 into -D rats had no effect on either serum PTH or calcium (Ca), either 0.5 or 1.0 h after treatment. Parathyroid glands from -D rats incubated with 0.75 mM Ca secreted more PTH than glands of similar weight from rats given 25 micrograms vitamin D3 3 days earlier, suggesting that vitamin D or a metabolite of vitamin D may modulate the sensitivity of the parathyroid gland to medium Ca. In summary, we found no evidence for a direct, acute effect of vitamin D metabolites on PTH secretion under diverse experimental conditions.

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A neonate with severe primary hyperparathyroidism was successfully managed by parathyroidectomy and heterotopic autotransplantation (one third of one gland of the infant was implanted in the forearm). In vitro studies of parathyroid tissue from the infant revealed a severe defect in parathyroid suppressibility. Postoperatively, the infant had modest hypercalcemia, normal serum immunoreactive parathyroid hormone levels, hypermagnesemia, and relative hypocalciuria. The parents were related and both had asymptomatic hypercalcemia with mean serum immunoreactive parathyroid hormone levels that were within the normal range. Similar to the findings in the infant postoperatively, relative hypocalciuria in the presence of hypercalcemia was found in the mother; in contrast, the father had hypercalciuria. The presumed dominantly transmitted hypercalcemia in this kindred is consistent with familial hypocalciuric hypercalcemia with a confounding factor of ethanol possibly accounting for the hypercalciuria in the father.

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We examined the ability of the mononuclear phagocyte in vitro to degrade 45Ca-labeled bone particles to determine whether this assay allowed us to monitor disease activity in patients with juvenile rheumatoid arthritis. The monocytes from patients with juvenile rheumatoid arthritis receiving no anti-erosive therapy (n = 10) degraded significantly more bone than did cells obtained from normal controls (n = 10, P less than 0.001) or patients with juvenile rheumatoid arthritis receiving either gold thioglucose (n = 4, P less than 0.001) or D-penicillamine (n = 6, P less than 0.005). In two patients monitored for either 8 or 11 months, results of monocyte assays were found to parallel the clinical course. We conclude that in vitro monocyte bone degradation assays may provide a means of assessing joint activity in patients with juvenile rheumatoid arthritis. Further, this study and others indicate that mononuclear phagocytes are capable of causing erosive changes.

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Serum immunoreactive parathyroid hormone (iPTH), calcium, magnesium, and phosphorus levels were measured in 13 premature infants during the first 96 hours of life. Hypocalcemia at 12-24 hours of age was associated with a markedly elevated mean serum iPTH level. Six of the hypocalcemic infants received a continuous infusion of calcium while seven were not treated. In the untreated infants, the mean serum calcium remained in the hypocalcemic range while the serum iPTH progressively increased. By contrast, the mean serum calcium in the treated infants increased to 2.35 mmol/l at 96 hours of age and was accompanied by a decline in serum iPTH. At 72 and 96 hours, the mean serum iPTH was twofold greater in the untreated than in the treated infants. The results indicate that the parathyroid glands of premature infants respond to calcium signals and that a factor(s), other than parathyroid insufficiency, plays an etiologic role in the hypocalcemia of prematurity.

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Previous studies indicate that magnesium, like calcium, stimulates the release of calcitonin (CT) from the thyroid gland. On the other hand, C-cell hyperplasia has been noted in magnesium-deficient dogs and rats. To explore further possible interrelationships between magnesium and CT, 21-day-old Sprague-Dawley male rats fed a control diet (0.043% Mg and 0.47 Ca) were match-fed with rats given either a control low calcium diet (0.043% Mg and 0.15% Ca) or a low magnesium-low calcium diet (0.001% Mg and 0.15% Ca). The low calcium content in the magnesium-deficient diet prevented the development of hypercalcemia characteristic of the magnesium-deficient rat. After 17 days, animals were killed by decapitation. Blood was obtained from some animals in the basal state and in other animals 1 min postpentagastrin or 1 min postmagnesium chloride infusion. No significant difference was found in the serum calcium level in the three groups, while the mean serum immunoreactive CT (iCT) level was significantly higher in magnesium-deficient rats both before and after pentagastrin. An acute iv infusion of MgCl2 resulted in significant increases in serum iCT in both the control and magnesium-deficient animals. The results of this study demonstrate that basal serum iCT levels and their response to pentagastrin are increased in magnesium-deficient, normocalcemic animals. The further increase in serum iCT after magnesium infusion in magnesium-depleted animals appears paradoxical and indicates that the relationship between extracellular magnesium and iCT release is not a simple feedback mechanism. It is possible that the increase in circulating iCT may be a response to extracellular-intracellular differences in magnesium concentration. Alternatively, the increased C-cell activity may be secondary to some unknown metabolic alteration induced by magnesium deficiency, rather than to magnesium deficiency per se.

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An 11-year-old girl with Wilson's disease presented with mild hypocalcemia (8.0 mg per deciliter), hypophosphatemia (2.7 mg per deciliter), hypercalciuria (569 mg per day), and hyperphosphaturia (tubular reabsorption of phosphate, 67 per cent). The hyperphosphaturia and hypercalciuria were attributed to the Fanconi syndrome, a known component of Wilson's disease. Circulating immunoreactive parathyroid hormone was usually undetectable or, occasionally, detectable at minimal levels in the presence of depressed blood levels of ionized calcium. Normal levels of ionized calcium were not maintained throughout a 24-hour monitoring period. The patient had tetany during a period of rapid reduction in ionized calcium levels, and an appropriate rise in circulating immunoreactive parathyroid levels was never demonstrated. Induced hypocalcemia during citrate infusion did not stimulate parathyroid secretion, nor did infusion of magnesium. We conclude that parathyroid insufficiency may be associated with Wilson's disease. We speculate that it is due to deposition of copper in the parathyroid glands.

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Hypocalcemia is characteristically observed in magnesium deficiency in a number of animal species. Previous studies demonstrated impaired release of PTH in magnesium-depleted hypocalcemic humans. However, an enigma remains in that, unlike in other animals, hypercalcemia, rather than hypocalcemia, accompanies magnesium deficiency in the rat. Because intact parathyroids are necessary for the development of this hypercalcemia, it has been postulated that magnesium depletion stimulates, rather than impairs, PTH secretion in the rat. In an effort to more directly evaluate this thesis, sequential measurements of circulating immunoreactive PTH (iPTH) were made over a 30-day period in rats maintained on a magnesium-deficient diet and in match-fed controls. In the control rats, serum calcium, magnesium, and iPTH remained relatively constant throughout the study. By contrast, during the first 4 days of a low magnesium diet, serum magnesium decreased to 1.0 mg/dl, serum calcium increased moderately, while serum iPTH increased to a mean level that was twice that in controls. After 5 days, when serum magnesium progressively fell to levels less than 0.6 mg/dl and serum calcium continued to rise, serum iPTH fell to levels significantly lower than the control value. In a second set of experiments, the effect of hypocalcemia on circulating iPTH in magnesium deficiency was evaluated. Circulating iPTH was greatly increased and not significantly different in magnesium-deficient and magnesium-replete animals who were rendered chronically hypocalcemic by diets deficient in either calcium or vitamin D. The results of this study indicate that: 1) in the rat, an increase in PTH secretion occurs early in the genesis of magnesium deficiency in the presence of a modest increase in serum calcium; however, the subsequent further increase in serum calcium counteracts the stimulatory effect of hypomagnesemia on PTH secretion; 2) unlike the human parathyroid gland, the rat parathyroid gland responds appropriately to both hypo- and hypercalcemia in magnesium deficiency; and 3) the hypercalcemia that occurs in the magnesium-deficient rat is not due to increased PTH secretion and must be accounted for by another mechanism.

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Resorption of devitalized bone particles by monocytes grown in culture was stimulated by platelet-derived growth factor (PDGF) in a dose-dependent manner. Bone resorption in response to PDGF was time-dependent with a significant increase over control cultures evident by 72 hours. These data are the first to demonstrate stimulation of bone resorption by PDGF in a specific cell type known to resorb bone in vivo and in vitro.

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Three experiments were carried out to test the time course of effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the ultrastructural morphometry of osteoclasts. The addition of lactose to a vitamin D-deficient diet with a high calcium and phosphate content, fed to weanling rats for 4 weeks, ensured normacalcemia and normophosphatemia and allowed thyroparathyroidectomy without ill effects. In these vitamin D-deficient thyroparathyroidectomized rats, iv injection of 50 ng 1,25-(OH)2D3 resulted in significant changes in the osteoclasts in the metaphysis of the tibiae compared to those in corresponding controls; the size of these cells, their nuclei, ruffled borders, and clear zones enlarged after 6 h and the number of osteoclasts increased after 48 h. Serum calcium and serum phosphate levels increased after 12 h in one experiment, but not in a second experiment. Serum 25-hydroxyvitamin D and serum immunoreactive parathyroid hormone levels were undetectable. Mineralization of metaphyseal bone matrix was normal, as quantified by histomorphometry. When, dependent on the mineral content in the diet, mineralization was impaired and the volume density of the osteoid seams was increased, activation of osteoclasts by 1,25(OH)2D3 was not seen until 12--24 h after injection. It is concluded that a physiological dose of 1,25-(OH)2D3 stimulates the activity of osteoclasts in the absence of parathyroid hormone.

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