Purification of plasma vitamin D metabolites for radioimmunoassay.

The ability of four solvent systems to extract tritiated 25-hydroxy vitamin D3, 24,25-dihydroxy vitamin D3, 25,26-dihydroxy vitamin D3 and 1 alpha, 25-dihydroxy vitamin D3 from plasma was compared. Diethyl ether gave the highest yield of metabolites and lowest yield of "lipid" materials, the dihydroxylated metabolites were more readily extracted than 25-hydroxy vitamin D3. Following extraction with ether the vitamin D metabolites could be separated, without prior purification, on a 6.2 mm X 250 mm Zorbax-Sil high pressure liquid chromatography (HPLC) column, a simplification of previous methods. Plasma 1 alpha, 25-dihydroxy vitamin D levels measured after purification by this method were not significantly different from those obtained by an established, more complex method.

A radioimmunoassay for plasma androstenedione using 125I tracer.

A radioimmunoassay for plasma androst-4-en-3,17-dione is described using ether extracts of plasma and 125I radioligand. The reagent is easily prepared by an established radiochemical method from androst-4-en-3,17-dione-6 beta-(carboxymethyl) thioether and 125I-histamine.

The relation between plasma androstenedione, plasma oestrone and androstenedione to oestrone conversion rates in post-menopausal women with and without fractures.

1. Plasma androstenedione, plasma oestrone and the conversion of plasma androstenedione into oestrone were measured in 19 post-menopausal women without fractures (six of them oophorectomized) and 18 with vertebral or femoral neck fractures (four of them oophorectomized). 2. In the series as a whole, the main determinant of the plasma oestrone level was the plasma androstenedione concentration. Only in the small oophorectomized group did the variation in conversion rate make a significant contribution to the variation in plasma oestrone. 3. The conversion rates were not different as between non-fracture and fracture cases but the mean plasma androstenedione and oestrone concentrations were lower (though not significantly) in the latter.

Thermal transformation of cholecalciferol between 100--170 degrees C.

Cholecalciferol is transformed, irreversibly, to pyrocholecalciferol and isopyrocholecalciferol. The rate constant, as a function of temperature, for this transformation from the equilibrium mixture of cholecalciferol and precholecalciferol has been determined for temperatures between 100--170 degrees C and is slower than the precholecalciferol-cholecalciferol interconversion. The ratio of rates of production of pyro to isopyro derivatives is 2:1 throughout.

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.

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 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.

Synthesis of (1,2- 3 H 2 )cholecalciferol and metabolism of (4- 14 C,1,2- 3 H 2 )- and (4- 14 C,1- 3 H)-cholecalciferol in rachitic rats and chicks.

[1,2-(3)H(2)]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-(14)C,1-(3)H(2)]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-(14)C,1,2-(3)H(2)]cholecalciferol retained 36% of (3)H, the amount expected from its distribution between C-1 and C-2, the (3)H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The (3)H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal (3)H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its (3)H at C-1 is not a precursor of the intestinal (3)H-deficient substance P.