Studies of the effects of cholecalciferol upon the metabolism of rachitic chick growth cartilage.

When a physiological dose of cholecalciferol was given to rachitic chicks, an early response was increased amino acid incorporation into growth cartilage protein, including collagen, but no evidence was found for induction of specific protein synthesis as in the intestine. Increased release into the medium of 45Ca and of hydroxyproline was observed when growth cartilage tissue was incubated in medium containing 1,25-dihydroxycholecalciferol. These studies suggest that the metabolism of cartilage tissue from the zones of proliferation and maturation was more affected than tissue from the zones of hypertrophy and calcification. Cholecalciferol metabolites may therefore have a direct effect upon chondrocytes as well as on bone cells during endochondral bone formation.

The effect of various cholecalciferol-related substances on the biosynthesis of the cholecalciferol-dependent calcium-binding protein in the small intestine of the rachitic chick and its relation to rickets.

Rachitic chicks were injected with different dose-levels of cholecalciferol and several cholecalciferol-related substances, i.e., dihydrotachysterol 3, ergocalciferol, 5,6-transcholecalciferol and 25-hydroxycholecalciferol. The response to treatment was assayed by the amount of cholecalciferol-dependent calcium-binding protein produced in the mucosa of the small intestine. The biological activity of these substances in the healing of rickets in the rat was also estimated except for ergocalciferol. The relative potencies in stimulating calcium-binding protein production were: 25-hydroxycholecalciferol > cholecalciferol > 5,6-trans-cholecalciferol > ergocalciferol > dihydrotachysterol 3.

Influence of plasma calcium and vitamin D on bone collagen. Effects on lysine hydroxylation and crosslink formation.

In chick bone collagen the degree of lysine hydroxylation and the magnitude of the crosslink ratio dihydroxylysinonorleucine/hydroxylysinonorleucine were both found to be inversely related to the concentration of plasma calcium. Lysine hydroxylation was also affected by a second factor related to vitamin D status.

Effect of vitamin D deficiency on bone formation in the chick.

1. The process of diaphyseal bone formation can be investigated by studying the rate of incorporation of radioactive precursors, administered in vivo into bone fractions of increasing density. 2. In the 4-week-old vitamin D-treated chick most of the osteoid becomes calcified within 12h and almost all within 2 days. The low-density calcified phase that is formed is converted into a higher density form and within 7 days the greater proportion of the calcified tissue is in the higher density form. 3. In the vitamin D-deficient chick of similar age the rate of calcification of osteoid is decreased, as is the rate of conversion into the higher density phase with the resultant accumulation of the lower density calcified form. 4. The higher density phase probably corresponds to hydroxyapatite and the lower density one to the ACP-pase described by Termine & Posner [(1967) Calcif. Tissue Res. 1, 8--23]. 5. The disorder in the process of calcification seems to be unrelated to the alteration in blood Ca2+ and phosphate concentrations, but related to the presence or absence of cholecalciferol.

1-alphahydroxycholecalciferol in chronic renal failure. Studies of the effect or oral doses.

Four patients with advanced chronic renal failure and osteodystrophy were treated with 1-alphahydroxycholecalciferol, a synthetic vitamin D analogue, in a daily oral dose of 1.5 to 2.0 mug, for periods up to 1 year. They showed increased calcium absorption, positive calcium and phosphorus balances, moderate increases in serum calcium levels, marked reductions in serum alkaline phosphatase levels, a decrease in serum immunoreactive parathyroid hormone levels, and radiologic and histologic improvement in bone disease. One patient with proximal myopathy showed improvement in muscular strength. 1-Alphahydroxycholecalciferol appears to be effective therapy for renal osteodystrophy.

The investigation and treatment of renal osteodystrophy.

Skeletal demineralisation was measured for two years in 16 patients on maintenance haemodialysis; the technique employed was neutron activation analysis of a hand. The magnesium concentration of the dialysate was increased in six of the patients; oral calcium carbonate was administered to seven patients and 1-alpha-hydroxycholecalciferol, a potent vitamin-D analogue was supplied to a further three patients. An increase in skeletal calcium content of a hand was noted in the groups of patients receiving supplementary calcium and 1-alpha-hydroxycholecalciferol. It is concluded that 1-alpha-hydroxycholecalciferol appears to be effective therapy for renal osteodystropy, that oral calcium supplements may promote skeletal remineralisation and that supplementary magnesium therapy may reduce the progressive losses of skeletal calcium from some patients on maintenance haemodialysis.

I alpha-hydroxycholecalciferol: a treatment of renal bone disease.

Three patients with chronic renal failure on maintenance haemodialysis have been treated with 1 alpha-hydroxycholecalciferol (1 alpha-OHCC), a synthetic vitamin D analogue. A daily dose of 2 mug by mouth produced a significant increase in both calcium absorption from the gastrointestinal tract and calcium content of bone. Treatment with 1 alpha-OHCC appears to be effective in cases of metabolic bone disease associated with chronic renal failure.

The response of the small intestine to vitamin D. Correlation between calcium-binding-protein production and increased calcium absorption.

1. The synthesis of calcium-binding protein, a protein produced in the small intestine in response to vitamin D, was investigated with a view to determining whether calcium-binding-protein production could be correlated with the stimulation of calcium absorption by vitamin D. 2. A radioimmunological assay, which can quantitatively estimate calcium-binding-protein concentrations as low as 1mug/g wet wt., was used to detect the synthesis of soluble calcium-binding protein. 3. When used on intestinal supernatants from chicks dosed with vitamin D, calcium-binding protein was not detectable at 8h but was present after 12h at a concentration of 8.6mug/g wet wt.; in agreement with this an increase in calcium absorption due to vitamin D was detected at 12h but not at 8h. 4. The synthesis of calcium-binding protein was also monitored directly by making use of the ability of the iodinated antiserum to bind specifically to nascent calcium-binding protein chains on intestinal polyribosomes; in this way calcium-binding-protein synthesis could be detected 8h after dosage with vitamin D. Further, the binding reaction indicated a near linear increase in the calcium-binding-protein-synthesizing capacity over a 16h period. 5. From the amount of calcium-binding protein present 12 and 24h after vitamin D administration it is calculated that calcium-binding-protein mRNA is produced at approx. 1mol/min per intestinal cell. 6. It is concluded that the high correlation between the initiation of calcium-binding-protein synthesis and the stimulation of calcium absorption by vitamin D strengthens the proposal that calcium-binding protein plays an important role in calcium transport.

The response of the small intestine to vitamin D. Isolation and properties of chick intestinal polyribosomes.

Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30-35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.

The transporting proteins of cholecalciferol and 25-hydroxycholecalciferol in serum of chicks and other species. Partial purification and characterization of the chick proteins.

Chick serum contains two cholecalciferol-binding proteins, one of which binds mainly cholecalciferol (cholecalciferol-binding protein) and the other binds 25-hydroxycholecalciferol (25-hydroxycholecalciferol-binding protein). By means of Cohn fractionation, (NH(4))(2)SO(4) precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephadex and an additional gel-filtration step on Sephadex G-100, these two binding proteins were purified. Both proteins possess beta-globulin mobility on analytical polyacrylamide-disc-gel electrophoresis, a sedimentation coefficient of 3.5S and approximate molecular weights of 60000 for the cholecalciferol-binding protein and 54000 for the 25-hydroxycholecalciferol-binding protein. Sera obtained from rat, pig, human and monkey were shown to contain a single binding protein that is responsible for the transport of both cholecalciferol and 25-hydroxycholecalciferol. In the toad the lipoproteins are used for the transport of these two steroids.