In vivo and in vitro effects of a pulsed electromagnetic field on net calcium flux in rat calvarial bone.

Although PEMF's have been found to promote fracture healing and to modulate the activity of bone cells in vitro, effects on bone metabolism are largely unexplored. A bioassay using neonatal rat calvarial bone was used to determine the early effects of a pulsing electromagnetic field (PEMF) exposure in vivo and in vitro on bone metabolic calcium exchange. Bone discs taken from whole body exposed animals (0-4 hours) show a log exposure time-dependent average increase in net Ca uptake in the 0-50% range (r2 = 0.83). This increase could be detected immediately after exposure and also after 24 hours, but not 48 hours later. Animals given whole body PEMF exposure also showed a decrease in serum calcium and did not elevate serum Ca after administration of exogenous parathyroid hormone (PTH). Bone discs from untreated rats, exposed to PEMF for 15 minutes in vitro and then assayed, showed net Ca uptake increases of a similar magnitude and also were refractory to the Ca-releasing effect of PTH. Unexposed discs responded normally to PTH by decreasing net Ca uptake. Treatment of calvarial discs with calcitonin or acetazolamide, both of which inactivate osteoclasts, made the bone refractory to further increases in Ca uptake by PEMF. These results suggest that PEMF exposure produces PTH-refractory osteoclastics and has a relatively rapid effect on increasing net bone Ca uptake, putatively due to a decrease in PTH/paracrine-mediated bone resorption.

Conjunctive effects of fibroblast growth factor and glycosaminoglycan on bone metabolism in neonatal bartter syndrome.

The calciotropic activity of urine from a subject with neonatal Bartter syndrome (NBS) has been partially purified using ion-exchange and gel chromatographic techniques. A bioassay using bone disks from rat calvaria was used to estimate calciotropic activity, which in the urine of the subject with NBS appears to be due to basic fibroblast growth factor (bFGF) bound to a glycosaminoglycan susceptible to heparitinase digestion. The calciotropic activity is eluted from DEAE-Sephacel and Sepharose CL-6B in a narrow band in association with metachromatic material and is destroyed by heparitinase and blocked by an antibody to bFGF. After treatment of purified preparations with heparitinase, a component that is inactive alone but develops calciotropic activity in association with heparin can be isolated by affinity chromatography on heparin-Sepharose columns. This component is recovered from the column at NaCl concentrations expected to elute bFGF and is inactivated by antibodies to bFGF. No calciotropic activity can be shown in glycosaminoglycan-containing fractions from urine from a normal boy or a normal man, but such fractions exhibit calciotropic activity if bFGF is added to the assay system. When bFGF is added to urine from either normal subject followed by ion-exchange chromatography on DEAE-Sephacel, calciotropic activity is eluted at NaCl concentrations closely similar to those found to elute calciotropic activity from the urine of the NBS subject. It appears that the abnormal findings in NBS urine are due to excess bFGF, although they could be due to some abnormality of the glycosaminoglycan component.

Melatonin: the dark force.

Although the pineal gland was described 2,300 years ago, its functions remained obscure and productive research was limited until 1958, when Lerner and associates defined melatonin. In 1965 Wurtman and Axelrod advanced the "melatonin hypothesis," according to which the pineal gland acts as a transducer responding to changes in circumambient light by changing its rates of melatonin output. Sites and mechanisms of melatonin action are still poorly understood. Two consistent effects are the induction of sleep and an antigonadotropic influence on reproductive structure and behavior. The former is demonstrable and clinically useful in human subjects; the latter has been shown in birds, rodents, and sheep. Alteration of skin color by the contraction of melanophores was effected by pineal extracts before the discovery of melatonin. This phenomenon, seen in reptiles, amphibians, and fish, has received little recent attention. Areas of greater interest and potential importance include the antimitotic effects of melatonin on some types of tumor cells in culture and the apparent in vivo protection of immunocompetent lymphocytes during chronic stress, which reduces the functional capacity of lymphocytes in control rodents. Clinical application of the antimitotic and immunosupportive properties of melatonin seems likely in the near future. Unfortunately, this innocent molecule has been touted in two recent books and many advertisements as an aphrodisiac, rejuvenator, protector against disease, and general wonder-worker. Because interest in melatonin is high, all physicians can expect questions and may have use for the information provided in this review.

Humoral factor in children with neonatal Bartter syndrome reduces bone calcium uptake in vitro.

The neonatal Bartter syndrome (NBS) is associated with a complex disorder of mineral metabolism in children, including hypercalciuria, nephrocalcinosis, and diminished bone mineral density. Although cyclooxygenase inhibition usually brings about improvement in these findings, there is a variable component which is resistant to such therapy in many children. The factor mediating this disorder has not been identified. Blood and urine from 12 children with NBS were examined. When compared with samples from normal children and adults, all (NBS) sera reduced bone calcium uptake in a bone disc bioassay. This effect persisted in the presence of parathyroid hormone (PTH) antibody and PTH receptor blockade, indicating that neither PTH nor PTH related peptide was responsible. It was eliminated by indomethacin, suggesting that prostanoid generation was essential. Protamine was also inhibitory, as was the addition of ecteola, an anion binder. Activity could be recovered from ecteola by elution with hypertonic buffer. Urine samples from children with NBS had the same calcitropic effect. The agent was removed by ecteola and recovered by hypertonic elution. Activity was eliminated by protamine and by heparinase, but not by trypsin digestion. Size exclusion centrifugation showed that the activity was associated with a material between 10 and 30 kilodaltons. Finally, urine ecteola eluates from NBS patients raised serum concentrations of calcium after intraperitoneal injection in rats. These data suggest that children with NBS have a calcitropic substance in their serum and urine which is not found in normal individuals. The substance is heparin like, and mediates its effects through prostanoid production. These studies provide additional evidence against a direct renal cause of the urinary calcium disturbance characteristic of the disorder.

Interleukin 1beta mediated calciotropic activity in serum of children with juvenile rheumatoid arthritis.

OBJECTIVE: To detect the presence and source of calciotropic activity in the serum of children with juvenile rheumatoid arthritis (JRA). METHODS: Metabolic evaluation of an adolescent with polyarticular JRA and hypercalcemia/hypercalciuria included testing with a bone disc bioassay. The bioassay detects calciotropic activity (increased bone resorption or reduced bone formation) in serum. Interleukin 1 receptor antagonist (IL-1RA) was added to patient sera to test the role of IL-1beta. The results in this index case prompted additional study in 9 children with JRA. Correlation of calciotropic activity with disease activity score, erythrocyte sedimentation rate (ESR), and urinary calcium excretion was by Spearman rank correlation. RESULTS: Calciotropic activity was found in 2 consecutive samples from the index patient. This activity was eliminated by addition of IL-1RA (p < 0.001 compared to serum alone). Testing of the other 9 children showed calciotropic activity at least once in 7/9 and 10/15 samples studied. Addition of IL-1RA completely (6/8) or partially (2/8) neutralized calciotropic activity (p < 0.001 compared to serum alone) in the specimens available for testing. Calciotropic activity did not significantly correlate with disease activity score, ESR, or urine calcium. CONCLUSION: Our data indicate the presence of IL-1beta mediated calciotropic activity in the sera of children with JRA, and suggest a role for IL-1beta in JRA associated osteopenia.

Protamine and acute depletion of magnesium limit bone response to parathyroid hormone.

The effect of protamine on calcium homeostasis was studied in nine pediatric patients undergoing cardiopulmonary bypass. Total serum calcium decreased from 8.44 mg/dL to 7.49 mg/dL (P < 0.05) after protamine. Ionized calcium decreased from 1.39 to 1.31 mmol/L (P < 0.05). A bioassay determined the etiology of this response. Bone disks were placed in sera, protamine, parathyroid hormone, parathyroid hormone antibody, or magnesium-depleted solutions, then were incubated in solutions with known calcium content. The change in the media's calcium concentration reflects the bone's response to the initial stimulus. Calcium change is expressed as Experimental delta/Control delta (E/C). Normal bone responds to parathyroid hormone, E/C = 0.59 (P < 0.001). Protamine-treated bone loses this response, E/C = 0.9 (P = not significant [NS]). A parathyroid-hormone-induced osteoblast messenger was found. Protamine-treated bone continued to respond to this messenger, E/C = 0.42 (P < 0.001). Bone showed reversible loss of response to parathyroid hormone after incubation in magnesium-free solution, E/C = 0.93 (P = NS). With reincubation in magnesium, E/C = 0.69 (P < 0.01). Since protamine blocks parathyroid receptors, and magnesium depletion limits the bone's response to parathyroid hormone, this may explain the persistent hypocalcemia seen in some patients undergoing cardiopulmonary bypass.

Calcium kinetics in the hyperprostaglandin E syndrome.

Metabolic investigations, including the use of stable isotopes of calcium, were used to study calcium kinetics in three children with the hyperprostaglandin E syndrome. The studies were performed both during indomethacin treatment and in the absence of therapy. Off therapy, each child had hypercalciuria (mean urinary calcium excretion 0.478 mM/kg/d), hyperprostaglandinuria, and elevated serum calcitriol concentration. All had diminished bone density and were euparathyroid. Indomethacin treatment was associated with a marked reduction in serum calcitriol concentration, as well as decreased prostaglandin E excretion. Mean urinary calcium excretion fell to 0.135 mM/kg/d. The stable isotope studies defined two components to the hypercalciuria of this disease: an indomethacin-sensitive dietary contribution and a relatively indomethacin-resistant bone resorptive element. Bone densitometry confirmed the presence of the resorptive element by demonstrating skeletal demineralization.

Pineal and adrenal effects on calcium homeostasis in the rat.

In human infants and newborn rats, white light at the intensity used to treat hyperbilirubinemia lowers serum calcium concentration. Occipital shielding or (in newborn rats) exogenous melatonin prevents this effect. Propranolol, by inhibiting melatonin synthesis, also causes hypocalcemia, which is preventable by melatonin. Metyrapone or adrenalectomy prevents hypocalcemia after light exposure or propranolol. Exogenous corticosterone lowers serum calcium; this is prevented by supplementary melatonin. In adult rats, the change in calcium after light, propranolol, or corticosterone is minimal. After parathyroidectomy or a diet with a high calcium/low phosphorus ratio, the hypocalcemic effect of these three agents is restored. Bone samples removed after light exposure or corticosterone administration show increased calcium uptake; this is blocked by supplementary melatonin in vivo or by addition of melatonin to the incubation medium. We postulated that the hypocalcemic effect of light or propranolol was due to an acute increase in corticosterone-mediated bone calcium uptake when circulating melatonin was decreased by reduction of the rate of melatonin synthesis. In our study, pinealectomized rats showed no change in serum calcium after light or propranolol; their hypocalcemic response to corticosterone was greater than that of sham-operated controls. Exogenous parathyroid hormone prevented light-induced hypocalcemia in newborn rats.

Hypercalciuria with Bartter syndrome: evidence for an abnormality of vitamin D metabolism.

Some children with Bartter syndrome have hypercalciuria. To determine the mechanism for this phenomenon, we studied tubular function and calcium metabolism in six such children. All patients had hypokalemic alkalosis, normotension, hyperreninemia, growth retardation, low fractional distal chloride reabsorption (4/5), and elevated urinary prostaglandin E2 excretion (5/6). In addition, all had hypercalciuria (urinary calcium 6.5 to 25.0 mg/kg/day), with evidence of nephrocalcinosis in five. None, however, had evidence of rickets or hyperparathyroidism. There was a marked elevation in the serum concentration of 1,25-dihydroxyvitamin D in all, and four patients had a response to oral calcium loading suggestive of absorptive hypercalciuria. Five children have had long-term therapy with indomethacin. They have had improvement in hypokalemia and reduced urinary prostaglandin E2 excretion as well as reductions in the serum concentration of 1,25-dihydroxyvitamin D and in urinary calcium excretion. These data suggest that hypercalciuria in some children with Bartter syndrome is associated with an excess of 1,25-dihydroxyvitamin D. The improvement in hypercalciuria with prostaglandin synthesis inhibition may result in part from correction of this vitamin D abnormality.

The cardiopulmonary effects of calcium infusion in infants with persistent pulmonary hypertension of the newborn.

Low blood ionized calcium levels have been reported previously in association with alkalosis in infants undergoing hyperventilation for persistent pulmonary hypertension. We investigated the effect of acute calcium infusions on the cardiopulmonary status of 10 hypocalcemic, hyperventilated infants with persistent pulmonary hypertension. Acid-base status, arterial partial pressure of oxygen, vital signs, and echocardiographically determined right and left systolic time interval ratios and left ventricular shortening fractions were obtained before and after rapid infusions of calcium gluconate or saline. At 5 and 15 min after calcium infusion, but not after saline infusion, there were significant decreases in both right and left ventricular systolic time interval ratios, and an increase in transcutaneous PO2 and PaO2 that coincided with peak levels of ionized calcium. These findings provide evidence that high levels of ionized calcium in newborns with persistent pulmonary hypertension transiently improve myocardial performance and oxygenation.

Calcemic responses to photic and pharmacologic manipulation of serum melatonin.

Phototherapy of newborn rats (NBR) resulted in a decrease in serum calcium and melatonin levels. Transcranial light penetrance in NBR increased with wavelength. Below 640 nm (penetrance = 6.9%), no hypocalcemic effect could be demonstrated. Shielding the occiput of NBR prevented a decrease in serum calcium during phototherapy and substantially reduced the decrease in melatonin found in unshielded NBR. Intraperitoneal injection of propranolol, an inhibitor of melatonin synthesis, caused a decrease in serum calcium in shaded NBR. In contrast, when melatonin was injected with propranolol a decrease in serum calcium did not occur. Additionally, intraperitoneal isoproterenol before phototherapy protected against a decrease in serum calcium. These data are consistent with an hypothesis that a decrease in serum calcium during phototherapy results from transcranial photic inhibition of melatonin synthesis.

Stimulation of calcium uptake in rat calvaria by prostacyclin.

Treatment of newborn rat calvaria discs with a variety of unsaturated fatty acids led to a 50% enhancement of calcium uptake. Arachidonic acid was effective at lower concentrations than cis-vaccenic or oleic acid, while trans-vaccenic acid and saturated fatty acids did not enhance calcium uptake. Cyclooxygenase inhibitors indomethacin and acetylsalicylic acid abolished the enhancement of calcium uptake seen in response to cis-vaccenic acid and inhibited calcium uptake by otherwise untreated bones. Prostacyclin was found to produce up to 2 fold stimulation of calcium uptake with an EC50 of approximately 0.1 microM. No statistically significant stimulation of calcium uptake was seen in response to PGE2 or PGE1 alpha up to 25 microM, while slight stimulation was produced by 6-keto PGE1 alpha but only at concentrations of 10 microM. Prostacyclin production by calvaria was demonstrated and was stimulated over 50% by cis-vaccenic acid. These results suggest that not only is enhanced prostacyclin production responsible for elevation of calcium uptake in response to unsaturated fatty acids, but also that prostacyclin may be an important regulator of bone calcium homeostasis.

Effect of hyperventilation on total calcium, ionized calcium, and serum phosphorus in neonates.

The effect of alkalosis (pH greater than 7.55) on total calcium, ionized calcium, and serum phosphorus was studied in seven infants with persistent pulmonary hypertension (PPH) before, during, and after hyperventilation. Hyperventilation-induced alkalosis resulted in marked decreases in total calcium, phosphorus, and ionized calcium. There was an inverse correlation between plasma Ca+2 and pH; a 0.1-unit increase in blood pH decreased Ca+2 by 0.42 mg/dl. Two of the study infants had ionized calcium concentrations less than 2.5 mg/dl during hyperventilation. These disturbing changes in total calcium, ionized calcium, and serum phosphorus could have potentially detrimental effects on neonates with PPH.

Phototherapy-induced hypocalcemia in newborn rats: prevention by melatonin.

When young rats are exposed to white fluorescent light the concentration of calcium in their serum decreases. This effect is prevented by shielding the occiput, by inhibiting corticosterone synthesis, and by exogenous melatonin. Furthermore, the expected hypocalcemic response to cortisol injection is prevented by melatonin. Light-induced hypocalcemia may result from increased calcium uptake by bone when the blocking effect of melatonin decreases after pineal inhibition by transcranial illumination.

Glucagon-induced hypocalcemia in the rat: effects of maturation and insulin.

In adults of several species including man, a small transient decrease in serum calcium concentration follows glucagon administration in doses of 1 to 10 mg/kg. The effects of maturation and insulin on this phenomenon were assessed by comparing the response of newborn and adult rats to equivalent doses of glucagon with and without prior insulin administration. After injection of 1 microgram/g of glucagon, the decrease in serum calcium concentration at 60 min was significant in the newborn rats (-1.75 mg/dl; P less than 0.001) and not significant in the intact adults (-0.07 mg/dl; P greater than 0.1). In pancreatomized adults, the decrease in serum calcium after the same dose of glucagon became significant (-1.23 mg/dl; P less than or equal to 0.01). This hypocalcemic effect was prevented in the pancreatectomized adult rat if insulin in a dose of 0.01 micron/g was given 15 min before glucagon. In the newborn rats, the same dose of insulin decreased the hypocalcemic effect, but the change was still significant (-0.74 mg/dl; P less than 0.01). Glucagon decreased serum calcium at one hr in newborn rats but not in adults. After pancreatectomy, the adult response to glucagon was significant and similar to that of the newborn. Insulin cancelled this effect of glucagon in the pancreatectomized adults and reduced it in the newborns.