The role of 1,25-dihydroxyvitamin D in the mechanism of acquired vitamin D deficiency.

OBJECTIVE: We wished to assess the effect of changes in the plasma concentration of 1,25-dihydroxyvitamin D on the plasma elimination half-time for 25-hydroxyvitamin D in man. DESIGN: The turnover of 25-hydroxyvitamin D in plasma was investigated after intravenous doses of the radioactively labelled metabolite had been given to a group of patients (n = 17) with disorders of bone and mineral metabolism before and after oral treatment with calcium or 1,25-dihydroxyvitamin D. PATIENTS: Seven patients with post-menopausal osteoporosis, five with hypoparathyroidism, three with hypophosphataemic osteomalacia, one with renal osteodystrophy and one patient with coeliac disease were studied. MEASUREMENTS: Intravenous injections of 3H-labelled 25-hydroxyvitamin D were given and plasma elimination half-time assessed over periods of 4-14 days during which frequent measurements of plasma calcium, phosphate, parathyroid hormone, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were made. Changes in the plasma elimination half-time for 3H-25-hydroxyvitamin D before and after treatment with calcium and 1,25-dihydroxyvitamin D were evaluated by non-parametric statistical analysis. RESULTS: The elimination half-time for 3H-25-hydroxyvitamin D in plasma was significantly shortened by raising the circulating concentration of 1,25-dihydroxyvitamin D. Conversely, in a patient with intestinal malabsorption of calcium, the metabolic clearance of 3H-25-hydroxyvitamin D was prolonged when the concentration of 1,25-dihydroxyvitamin D in plasma was decreased by suppressing secondary hyperparathyroidism with large calcium supplements. In the longer-term studies (n = 10) there was a highly significant inverse relation (r = -0.88, P < 0.001) between the change in the plasma concentration of 1,25-dihydroxyvitamin D and the induced change in the elimination half-time of 3H-25-hydroxyvitamin D. There was also a significant correlation (r = 0.66, p < 0.0025) between the observed fall in the plasma concentration of unlabelled 25-hydroxyvitamin D and the predicted fall calculated from the measured value for the half-time of the 3H-labelled metabolite. In acute studies in patients with post-menopausal osteoporosis (n = 7), enhanced metabolic inactivation of 3H-25-hydroxyvitamin D was detectable within 24 hours of oral administration of 1,25-dihydroxyvitamin D. CONCLUSIONS: The effect of 1,25-dihydroxyvitamin D on the catabolism of 25-hydroxyvitamin D can contribute to the development of vitamin D deficiency in many clinical disorders. When the natural supply of vitamin D is limited by sunlight deprivation, a sustained increase in the plasma concentration of 1,25-dihydroxyvitamin D due to primary or secondary hyperparathyroidism will lead to accelerated depletion of vitamin D stores.

Determinants of the serum concentration of 1,25-dihydroxyvitamin D in primary hyperparathyroidism.

1. The serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured in 44 patients with primary hyperparathyroidism. 2. In 14 patients the serum concentration of 1,25-dihydroxyvitamin D was greater than normal (142-337 pmol/l). One patient had a subnormal concentration of 1,25-dihydroxyvitamin D (36 pmol/l) but no other evidence of vitamin D deficiency. 3. The possible biological determinants of the serum concentration of 1,25-dihydroxyvitamin D were sought by multivariate analysis of relevant variables. The serum concentration of 1,25-dihydroxyvitamin D was found to be significantly and positively correlated with the serum concentrations of 25-hydroxyvitamin D (P less than 0.001) and parathyroid hormone (P less than 0.003), and with the glomerular filtration rate (P less than 0.03), and negatively correlated with the serum concentrations of calcium (P less than 0.02) and phosphate (P = 0.055) (multiple R = 0.638, P less than 0.002). 4. In primary hyperparathyroidism the major determinant of serum 1,25-dihydroxyvitamin D is the availability of precursor 25-hydroxyvitamin D. 5. The finding that serum 1,25-dihydroxyvitamin D is commonly normal in patients with primary hyperparathyroidism despite an adequate state of vitamin D nutrition, can be explained in terms of the constraining influences of hypercalcaemia and variable degrees of renal dysfunction on the biosynthesis of 1,25-dihydroxyvitamin D.

Metabolic inactivation of vitamin D is enhanced in primary hyperparathyroidism.

1. The elimination half-time of 25-hydroxyvitamin D in plasma was estimated after intravenous injection of the radioactively labelled metabolite in seven patients with primary hyperparathyroidism before and after excision of a parathyroid adenoma. 2. The elimination half-time of 25-hydroxyvitamin D was significantly shortened in primary hyperparathyroidism and reverted towards normal after parathyroidectomy. 3. The increased metabolic clearance of 25-hydroxyvitamin D in primary hyperparathyroidism was accounted for by an increased excretion of vitamin D-derived inactivation products in the faeces. 4. Enhanced hepatic inactivation of 25-hydroxyvitamin D may be important in the development of vitamin D deficiency in primary hyperparathyroidism.

Bone and mineral metabolism and chronic alcohol abuse.

Studies of bone and mineral metabolism were made in 22 patients with chronic alcohol abuse and varying degrees of liver damage. None of the patients had clinical evidence of metabolic bone disease, but quantitative bone histology showed that six had osteoporosis, three osteomalacia, and two osteoporosis and osteomalacia combined. Trabecular bone volume (TBV) tended to be reduced in relation to age, and there was histological evidence of reduced bone formation particularly among the patients with osteoporosis. Multivariate analysis of the relevant variables showed that the major determinants of age-adjusted trabecular bone volume were the serum concentration of albumin and the dietary calcium. The presence of osteoporosis was related to the state of liver function and the type of alcohol habitually consumed, and was a particular feature of patients with severe liver disease and those who only drank spirits. Six patients (five with osteoporosis) had biochemical evidence of hyperparathyroidism, but none showed histological evidence of increased bone resorption or of osteitis fibrosa. In four patients the development of hyperparathyroidism was probably related to underlying magnesium deficiency. Serum calcidiol tended to be reduced and was directly related to the state of liver function; four patients had reduced or low normal serum concentrations of calcitriol. In only three patients could the development of osteomalacia be related to vitamin D deficiency; in two patients the cause of the osteomalacia was obscure. Significant changes in bone structure and mass appear to be common among heavy drinkers even in the absence of clinical metabolic bone disease. These skeletal abnormalities are likely to be relevant to the increased fracture risk associated with heavy drinking.

Familial hypocalciuric hypercalcaemia: evidence for continued enhanced renal tubular reabsorption of calcium following total parathyroidectomy.

A patient with familial hypocalciuric hypercalcaemia (FHH) is reported. Seven years after total parathyroidectomy he remained hypocalcaemic, with biochemical evidence of hypoparathyroidism (enhanced renal tubular reabsorption of phosphate, low nephrogenic cyclic AMP excretion, and reduced serum concentration of 1,25-dihydroxycholecalciferol in the presence of normal renal function and normal serum 25-hydroxyvitamin D levels). Iv infusions of calcium were given before and 6 years after total parathyroidectomy. The renal tubular reabsorption of calcium was compared in these two situations. No difference was found. Before and after parathyroidectomy there was enhanced renal tubular reabsorption of calcium. It is concluded that the enhanced renal tubular reabsorption of calcium in FHH is independent of parathyroid hormone. Total parathyroidectomy corrects the hypercalcaemia in FHH by a reduction in the input of calcium into the extra-cellular fluid from gut and or bone perhaps as a result of reduced renal synthesis of 1,25-dihydroxycholecalciferol.

ADP binds similarly to rigor muscle myofibrils and to actomyosin-subfragment one.

The binding of Mg2+ ADP to both rabbit skeletal and bovine cardiac myofibrils has been studied at two different temperatures. In each case a single class of binding sites was observed with a binding constant very close to that reported for the analogous actomyosin-subfragment one but much weaker than that seen with the analogous myosin subfragment one alone. These findings are discussed in terms of the constraints on the myosin cross-bridges imposed by the regular array of thick and thin filaments found in myofibrils.

Antisperm antibodies in mouse vasectomy sera react with embryonal teratocarcinoma.

Sera from vasectomized BDF1 mice were tested by immunofluorescence and radioimmunoassay for antibodies reacting with sperm and the mouse teratocarcinoma cell lines F9 and PYS. Antibody titers to both sperm and F9 cells increased with time after vasectomy and were highest in long-term (15 to 30 mo) vasectomized animals. By immunofluorescence assay, 35% of long-term vs 10% of age-matched control mice had antisperm antibodies (p less than or equal to 0.01), 54% of vasectomized vs 21% of controls had antibodies reacting with F9 teratocarcinoma (p less than or equal to 0.01), and 5% of vasectomized vs no controls reacted to the PYS teratocarcinoma cell line. No sera from either group reacted with thymocytes or cultured fibroblasts from male 129/Sv mice (negative somatic cell controls). Kendall's rank order statistical correlation test showed a significant association (p less than or equal to 0.01) between individual responses to sperm and F9 cells. Absorption of positive sera with sperm removed both sperm and F9 reactivity, indicating that the F9 cell-reactive antibodies in vasectomy sera are directed against identical or immunologically cross-reactive antigens present on the surface of sperm and F9 embryonal teratocarcinoma. A combination of electrophoresis and nitrocellulose blotting techniques (Western blot analysis) revealed that sera from some vasectomized mice contain IgG antibodies that react with 50,000 and 150,000 dalton (approximately) antigen bands present in sperm and F9 extracts, but not in PYS or thymocyte extracts. It remains to be determined whether the teratocarcinoma antigens detected in this study are true embryonic antigens or germinal antigens (the F9 cell line may have originated from primordial germ cells in transplanted embryonic tissue). In either case, teratocarcinoma cell lines provide an abundant source of material for the further characterization of these autoantigenic molecules.

Familial hypocalciuric hypercalcaemia: observations on vitamin D metabolism and parathyroid function.

Serum vitamin D metabolites, the renal tubular maximum reabsorptive rate for phosphate (TMP/GFR) nephrogenic cyclic AMP (NcAMPI, and CaE (urinary calcium excretion per litre of glomerular filtrate) were measured in 14 adults with familial hypocalciuric hypercalcaemia (FHH). The findings were compared with analyses in 14 patients with surgically proven primary hyperparathyroidism matched for serum calcium, creatinine clearance and vitamin D status (assessed by serum concentrations of 25 hydroxyvitamin D). Vitamin D metabolites were also measured in 16 normocalcaemic relatives of patients with FHH. The serum concentration of 24,25 dihydroxycholecalciferol was appropriate for the prevailing 25 hydroxyvitamin D and no difference was found between groups. The serum concentration of 1,25 dihydroxycholecalciferol was significantly greater in primary hyperparathyroidism (P less than 0.0005) compared with patients with FHH and their normocalcaemic relatives. TMP/GFR was reduced in both primary hyperparathyroidism (0.53 +/- 0.12 mmol/l GF, mean +/- SEM) and FHH (0.86 +/- 0.14 mmol/l GF). Patients with primary hyperparathyroidism showed an increase in NcAMP output in the urine (38.5 +/- 16 mmol/l GF) which was significantly greater (P less than 0.0001) than the normal NcAMP (13.5 +/- 9.2 nmol/l GF) found in FHH. CaE was low in FHH indicating increased renal tubular reabsorption of calcium. It is concluded that there is no abnormality of vitamin D metabolism in FHH comparable with the changes observed in primary hyperparathyroidism. It is suggested that the biochemical abnormalities in FHH cannot be explained solely upon an increased sensitivity of the renal tubules to the effects of endogenous parathyroid hormone.

Measurement of trabecular bone mineral by dual energy computed tomography.

An investigation into the possible effect of fat on the estimation of trabecular bone mineral concentration by computed tomography (CT) is described. Computed tomographic attenuation and effective atomic number were measured in models simulating trabecular bone, and in cadaveric femoral using a simultaneous dual energy technique. The composition of the area of bone scanned was measured by chemical analysis. In the bone models, CT attenuation value and effective atomic number were positively correlated to the calcium concentration; fat modified CT attenuation value but not atomic number. The calcium concentration of the trabecular bone samples was positively correlated with effective atomic number (r = 0.972; p less than 0.001) and to a lesser extent with CT attenuation value (r = 0.67; p less than 0.01), but both relations were independent of fat. Under the conditions of these experiments, the determination of effective atomic number by dual energy CT provides a more accurate measure of trabecular bone mineral concentration than does CT attenuation value by a single energy technique. There was a considerable spatial variation in the amount of bone tissue in the trabecular bone space, and CT attenuation values varied with the site and size of the area of interest. Accurate registration is essential for the precise measurement of trabecular bone mineral by CT.

Computed tomography and the localisation of parathyroid tumours.

The value of computed tomography (CT) in the pre-operative localisation of parathyroid tumour was assessed in 18 patients with primary hyperparathyroidism. A discrete tumour mass was identified in one patient. The factors which limit the usefulness of CT in the localisation of parathyroid tumours are discussed.

Familial hypocalciuric hypercalcaemia and acute pancreatitis.

Four families with familial hypocalciuric hypercalcaemia were studied. The probands presented with abdominal pain, which in three was due to acute pancreatitis; in two the condition was life threatening. Serum concentrations of calcium, magnesium, phosphate, and immunoassayable parathyroid hormone, urinary calcium excretion, and the rate of renal tubular reabsorption of phosphate were measured; the findings were compared with results in 10 patients with primary hyperparathyroidism matched for serum calcium concentration to establish differences between the diseases. Familial hypocalciuric hypercalcaemia should be suspected in patients with hypercalcaemia in whom daily urinary calcium excretion is below 5 mmol (200 mg) provided renal insufficiency, vitamin D deficiency, and ingestion of drugs that reduce calcium excretion have been excluded. Most cases appear to run a benign course, but some may suffer considerable morbidity. Surgical treatment should be reserved for patients with severe complications, when all parathyroid tissue should be removed.

Primary hyperparathyroidism and thiazide diuretics.

Six patients receiving thiazide diuretics were referred for evaluation of mild to moderate hypercalcaemia (serum calcium 2.65-2.98 mmol/l). All patients were considered to be suffering from primary hyperparathyroidism. Withdrawal of the diuretic was followed by a reduction in the serum calcium, one patient becoming normocalcaemia. The mechanisms responsible for these changes are discussed. In hypercalcaemic patients taking thiazides, it is recommended that the effects of withholding the diuretic should be observed before more radical measures are considered.

The continuing risk of vitamin-D intoxication.

Eight cases of vitamin-D poisoning are described. In six patients this therapy was unnecessary and in the remaining two patients inadequate supervision of the treatment resulted in overdosage. In five cases the history of vitamin-D therapy was either unknown or not appreciated by the referring clinician so that three surgical procedures of dubious value were performed on two patients. Large doses of vitamin D should only be used when strictly indicated and on the understanding that close biochemical and clinical supervision is necessary.

Vitamin D metabolism and the response to 1,25-dihydroxycholecalciferol in Osteoporosis.

The metabolism of isotopically-labelled cholecalciferol and the response to small doses of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in a group of women with osteoporosis presenting with crush vertebral fracture. No abnormality of vitamin D metabolism was detected. The administration of 1 microgram 1,25-(OH)2D3 for between 8 and 20 days was associated with an increased intestinal absorption and urinary excretion of calcium but caused no improvement in calcium balance. There was a small but significant rise in serum calcium and phosphorus and significant reduction in immunoassayable parathyroid hormone levels during treatment. It is concluded that 1,25-(OH)2D3 is unlikely to be of value in the management of osteoporosis.

Thermodynamics of cross links.

Comparison of the melting temperatures of native lysozyme and a cross-linked ester derivative of lysozyme (Imoto and Rupley, 1973) yielded a value of 5.5 kcal/mole for the free energy of stabilization developed through forming the cross link and a value of zero for the corresponding enthalpy. There is close agreement between experiment and calculation from the statistical theory of polymers.