The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity.

The involvement of sodium/potassium-ATPase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the α-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested to be linked to increased hormone secretion. In this study, we used an in vivo rat model to determine the importance of sodium/potassium-ATPase in PTH secretion. Glands were exposed and treated in situ with vehicle or ouabain, a specific inhibitor of sodium/potassium-ATPase. PTH secretion was significantly increased in response to ethylene glycol tetraacetic acid-induced acute hypocalcemia and to the same extent in both vehicle and ouabain groups. The glands were removed, and inhibition of the ATPase was measured by (86)rubidium uptake, which was found to be significantly decreased in ouabain-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also examined but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium-ATPase activity in vivo had no effect on the secretory response to acute hypocalcemia. Hence, the suggested importance of this ATPase in the regulation of PTH secretion could not be confirmed in this in vivo model.

Three novel activating mutations in the calcium-sensing receptor responsible for autosomal dominant hypocalcemia.

We report three novel activating mutations in the calcium-sensing receptor (CASR) that are responsible for autosomal dominant hypocalcemia (ADH) in three unrelated families. Each mutation involves a missense substitution resulting in a nonconservative amino acid alteration, P221L, E228Q, and Q245R. These mutations were observed in affected family members, but not in unaffected family members or in unrelated control samples. All three mutations are clustered in the extracellular domain of the CASR in a region dominated by negatively charged amino acids. Each mutant and wild-type receptor was expressed in Cos-1 cells. A luciferase reporter gene assay was utilized to detect the level of receptor activity by utilizing a protein kinase C-activated promoter to drive the production of luciferin, the reporter gene product. All three mutant receptors exhibited an increased sensitivity to calcium at all concentrations tested when compared to the wild-type receptor, supporting the hypothesis that these are activating mutations and are responsible for the ADH phenotype in these families. The data presented in this study suggest the importance of this highly negatively charged region of the extracellular domain in normal CASR function.

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: variable expressivity of maternal illness during pregnancy and unusual presentation with infantile cholestasis and hypocalcaemia.

Patients with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency present with a Reye-like syndrome, cardiomyopathy, or sudden unexpected death. We describe an unusual presentation in a patient with unsuspected LCHAD deficiency. The proband presented at 2 months of age with an acute infantile hypocalcaemia and vitamin D deficiency associated with occult, unexplained cholestatic liver disease. Sudden, unexpected death occurred at 8 months. Molecular analysis revealed homozygosity for the prevalent LCHAD (1528G > C, E474Q) mutation. The mother had pre-eclampsia during the third trimester of her pregnancy. In a subsequent pregnancy, she developed severe acute fatty liver of pregnancy (AFLP) and intrauterine fetal death at 33 weeks of gestation. In conclusion, infantile hypocalcaemia is an unusual phenotype associated with LCHAD deficiency. The maternal pregnancy history documents that fetal LCHAD deficiency is associated with a spectrum of maternal illnesses ranging from pre-eclampsia to life-threatening AFLP.

Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D3-1alpha-hydroxylase gene in normocalcemic rats.

Regulation of vitamin D metabolism has long been examined by using vitamin D-deficient hypocalcemic animals. We previously reported that, in a rat model of chronic hyperparathyroidism, expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (CYP27B1) mRNA was markedly increased in renal proximal convoluted tubules. It is believed that the major regulator for the expression of renal CYP27B1 is parathyroid hormone (PTH). However, in the normocalcemic state, the mechanism to regulate the renal CYP27B1 gene could be different, since plasma levels of PTH are very low. In the present study, the effect of PTH and calcitonin (CT) on the expression of renal CYP27B1 mRNA was investigated in normocalcemic sham-operated rats and normocalcemic thyroparathyroidectomized (TPTX) rats generated by either PTH or CaCl2 infusion. A single injection of CT dose-dependently decreased the expression of vitamin D receptor mRNA in the kidney of normocalcemic sham-TPTX rats. Concomitantly, CT greatly increased the expression of CYP27B1 mRNA in the kidney of normocalcemic sham-TPTX rats. CT also increased the expression of CYP27B1 mRNA in the kidney of normocalcemic TPTX rats. Conversion of serum [3H]1alpha,25(OH)2D3 from 25-hydroxy[3H]vitamin D3 in vivo was also greatly increased by the injection of CT into sham-TPTX rats and normocalcemic TPTX rats, but not into hypocalcemic TPTX rats. In contrast, administration of PTH did not induce the expression of CYP27B1 mRNA in the kidney of vitamin D-replete sham-TPTX rats and hypocalcemic TPTX rats. PTH increased the expression of renal CYP27B1 mRNA only in vitamin D-deficient hypocalcemic TPTX rats. These results suggest that CT plays an important role in the maintenance of serum 1alpha,25(OH)2D3 under normocalcemic physiological conditions, at least in rats.

Hypocalcemic induced increase in creatine kinase in rats.

Calcium plays an important role in various myopathies. We report on an animal model with increased plasma creatine kinase (CK) resulting from hypocalcemia that will provide clues for studying human hypocalcemic myopathy. Male Wistar rats were pair-fed either a control or a calcium- and vitamin D3-deficient diet for 1, 2, 3, 4, or 5-6 weeks after weaning (3 weeks old). In the deficient diet-fed rats, plasma creatine kinase was increased and was accompanied by marked hypocalcemia. The omission of calcium and vitamin D3 from the diet for 1 or 2 weeks was enough to cause increased plasma creatine kinase; the creatine kinase ratio of deficient diet-fed rats to controls was 4.84 (1,777 IU L(-1)/367 IU L(-1)), and the calcium ion ratio was 0.41 (1.8 mg dL(-1)/4.4 mg dL(-1)) after 2 weeks. These values returned to control levels on treatment of the rats with the control diet and 1alpha-OH-vitamin D3 for 1 week.

Experimental production of hypocalcemia by EDTA infusion in calves: a critical appraisal assessed from the profile of blood chemicals and enzymes.

Physiological studies of the effects of Ca2+ withdrawal using Na2EDTA have been conducted with the prior basic assumption that Na2EDTA-specific direct or indirect effects on the functions under study were negligible. The present study aimed at providing unequivocal confirmation of such assumption by establishing the pattern of response of blood constituents to intravenous infusions of Na2EDTA in calves. Na2EDTA infusion in calves allowed effective chelation of blood Ca2+, leading to a progressive hypocalcemia. Magnesium levels remained constant and concentrations of other ions (Na+, K+, Pi, H+, HCO3-), although significantly altered (P < or = 0.0001), remained within the normal range. Comparison of enzymes, urea, and creatinine changes precluded renal, hepatic, or muscular parenchymatous damages as being the cause of dysfunctions in the context of Na2EDTA-induced hypocalcemia. It was not possible, however, to standardize the Na2EDTA infusion characteristics (flow, volume) to obtain previsible Ca2+ decay in different animals. Conversely, monitoring of systemic arterial pressure (SAP) offered a precious tool to estimate the degree of hypocalcemia reached. Infusion rate must, therefore, be manipulated using careful on-line monitoring of SAP to obtain an experimental range of Ca2+ as large as possible. It was concluded that physiological data collected during Na2EDTA perfusions can be reliably discussed in terms of Ca2+ dependence rather than in terms of either Na2EDTA toxicity, electrolytes maladjustment, acid-base imbalance, impaired blood oxygenation, or hepatic, renal, myocardic, or skeletal muscle damages.

[Ca(2+)-ATPase activity in the hypocalcemic cataract].

We used an animal model of hypocalcemic cataract to investigate the changes of the cation levels and the Ca2+ pump (Ca(2+)-ATPase) function in the lens. Wistar rats (4 weeks old) were fed with a low calcium and no vitamin D3 diet. After 4 weeks on this diet, anterior subcapsular cataract was recognized, when calcium concentration in the aqueous humor and serum had significantly decreased. Calcium content in the lens decreased and sodium content increased. Ca(2+)-ATPase activity detected by [gamma-32P] ATP assay did not show significant change. We concluded that cataract during the early stage of hypocalcemia is caused by membrane damage with low calcium level in the aqueous humor and sodium content increase in the lens. We also studied the ultracytochemical localization of Ca(2+)-ATPase activity and found it in the plasma membrane of the lens epithelium and cortex and also in the epithelium organelles.

Hypocalcemic hyper-CK-emia in hypoparathyroidism.

A 15-year-old boy with increased serum creatine kinase (hyper-CK-emia) due to hypocalcemia in turn caused by idiopathic hypoparathyroidism (HP) is presented. Hyper-CK-emia was incidentally noted while managing a patient, aged nine, with mental retardation, epilepsy and mild hypocalcemia. Neurological examination showed normal deep tendon reflexes and no muscle weakness; electromyogram was normal. The hyper-CK-emia normalized during treatment for the hypocalcemia. Previously reported patients with hypocalcemic hyper-CK-emia or myopathy together with HP are discussed, as well as the degree of hypocalcemia and the wide spectrum of the muscle dysfunction.

Increased serum creatine kinase due to hypocalcemia in vitamin D deficiency.

An 8-year-old boy with vitamin D-deficiency rickets, increased serum creatine kinase (CK), and hypocalcemia is described. At 5 years of age, he was evaluated because of recurrent tonic seizures. He had growth retardation, microcephaly, quadriplegia, mental retardation, and epilepsy. Muscle strength was difficult to assess because of multiple joint contractures. Deep tendon reflexes were hyperactive. Laboratory data indicated rachitic changes on x-ray, hypocalcemia, low serum 25-hydroxyvitamin D3 (25-OH-D) and 1-alpha-25-dihydroxyvitamin D3 (1-alpha-25-[OH]2-D) levels, a normal response to the Ellsworth-Howard test, and markedly increased CK. Electromyography and nerve conduction velocities were normal. The patient responded to 1-alpha-OH-D treatment with increased serum calcium and normal CK activity; a significant correlation (p less than 0.01) was observed between calcium and log CK. The clinical course and laboratory findings supported the hypothesis that the increased CK was due to hypocalcemia, which in turn was due to the vitamin D deficiency in the severely handicapped child with malnutrition.

Calcium homeostasis in mentally handicapped epileptic patients.

Three groups of subjects have been studied to evaluate the role of long term hospitalization and chronic anti-epileptic therapy on calcium metabolism. The first group consisted of 32 epileptic patients, randomly selected from a population of inpatients in a hospital for the mentally handicapped, receiving various combinations of anti-epileptic drugs for at least 3 years. The second group was made up of 32 non-epileptic residents of the same hospital, individually matched for age and sex with epileptic patients and who had not received any anti-epileptic drugs in the last 3 years. The third group of 22 normal subjects was randomly selected from the staff of the University Hospital of Wales, matched for age and sex against the epileptic group, who had not received any anti-epileptic medication within the last 3 years. None of the subjects received any drugs (except anti-epileptic drugs) known to have effect on calcium metabolism. Significant differences in the serum levels of the total calcium, ionised calcium, total alkaline phosphatase and its liver iso-enzyme were seen. Serum total alkaline phosphatase and its liver iso-enzyme were significantly elevated in the epileptic group, showing the effect of anti-epileptic drugs. On the other hand serum total calcium was significantly lower in both residential groups compared to the normal population, epileptics being lower than non-epileptics showing the combined effect of hospitalization and anti-epileptic drugs. No significant difference was detected among the groups in the serum concentration of the bone alkaline phosphatase iso-enzyme.

Vitamin D-dependent rickets type I in pigs.

We have bred a strain of pigs with an inherited condition of hypocalcaemic rickets, transmitted by an autosomal-recessive mechanism. Homozygous (affected) piglets grew at half the rate of their heterozygous (clinically normal) littermates, and developed profound hypocalcaemia with severe secondary hyperparathyroidism and hypophosphataemia by 8 weeks of age. In the hypocalcaemic piglets, plasma 1,25-dihydroxyvitamin D levels were low or undetectable, and 24,25-dihydroxyvitamin D3 levels were also reduced despite 25-hydroxyvitamin D3 levels being 2-fold higher. There was no detectable 25-hydroxyvitamin D3-1- or -24-hydroxylase enzyme activity in renal homogenates prepared from affected animals. Plasma and intestinal calcium-binding protein levels were reduced in the hypocalcaemic piglets. Sucrose density gradient analysis of intestinal cytosol, prepared in high-salt buffer, revealed the presence of a similar amount of a specific less than 4.2S 1,25-dihydroxyvitamin D3 binder in both groups of piglets. Administration of pharmacological doses of vitamin D3 to affected animals reversed the hypocalcaemia. We conclude that this strain of pigs has vitamin D-dependent rickets type I.

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.

Abnormal renal mitochondrial 25-hydroxyvitamin D3-1-hydroxylase activity in the vitamin D and calcium deficient X-linked Hyp mouse.

Despite severe hypophosphatemia, plasma levels of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) are not significantly elevated in the Hyp mouse, a murine homologue of X-linked hypophosphatemia in man. To examine the effect of the Hyp mutation on vitamin D hormone biosynthesis, the metabolism of 25-hydroxyvitamin D3 (25-OH-D3) by isolated renal mitochondria was studied. The ability of a vitamin D deficient, low calcium diet to stimulate renal mitochondrial 25-hydroxyvitamin D3-1-hydroxylase activity (1-OHase) in normal mice (n = 22) and Hyp littermates (n = 20) was examined. Both genotypes responded to the diet with an increase in 1-OHase activity and a decrease in 25-hydroxyvitamin D3-24-hydroxylase activity (24-OHase). The increase in 1-OHase activity, however, was significantly lower in Hyp mice (8-fold) than in normal littermates (13-fold, P less than 0.001). In spite of depressed 1-OHase in the mutant strain, enzyme activity was significantly correlated with serum calcium concentration in both normal and Hyp mice. The present results provide direct evidence for an abnormal 1-OHase response in renal mitochondria of Hyp mouse.

Pathophysiology behind anticonvulsant osteomalacia.

Anticonvulsant drug-induced disorders in mineral and bone metabolism are discussed. 'Anticonvulsant osteomalacia' includes hypocalcaemia, elevated serum alkaline phosphatase, decreased serum 25-hydroxycholecalciferol (25 OHD3), radiological and histological signs of osteomalacia and a lower bone mineral content (BMC) than normal. The pathophysiological mechanism behind anticonvulsant osteomalacia is thought be an induction of the microsomal enzyme system in the liver, leading to a disturbance in the metabolism of vitamin D and a resulting vitamin D deficiency. It has been shown that treatment with vitamin D2 increase BMC whereas serum calcium was unchanged. Treatment with vitamin D3 or 25 OHD3 increases serum calcium whereas BMC was unchanged. These findings suggest that vitamin D2 and D3 are metabolized differently in anticonvulsant treated patients. With the present knowledge, preventive treatment of this relative mild pathological condition is not generally indicated.

Pseudohypoparathyroidism and hypocalcemic "myopathy". A case report.

A patient with the clinical features of pseudohypoparathyroidism and elevated concentrations of serum CK and LDH, which normalized after successful therapy, is described. Clinical signs of myopathy did not exist. The bioptical material from the m. tibialis anterior was microscopically normal. The biochemical analysis revealed a reduced phosphorylase-a-activity with the total phosphorylase-activity (a and b) being within the normal range. The significance of these findings as well as possible pathogenetic mechanisms are discussed.

Hypoparathyroidism and elevated muscle enzymes.

We studied a patient with hypocalcemia and increased serum activity of sarcoplasmic enzymes. Idiopathic hypoparathyroidism was established by history, low serum parathyroid hormone content, and marked responsiveness of urinary cyclic AMP and phosphate to parathyroid hormone. Muscle-associated isoenzymes of creatine phosphokinase and lactic dehydrogenase were increased, but there was no concomitant muscle weakness. Muscle biopsy was normal by morphologic and histochemical examination. The patient was treated with calcium and vitamin D. As the calcium rose, there were corresponding decreases in the serum activities of creatine phosphokinase and lactic dehydrogenase, with correlation coefficients of -0.88 and -0.64, respectively (p less than 0.01).