1. A radioimmunoassay for 25,26-dihydroxycholecalciferol has been developed with an anti-serum raised in a sheep, tritiated 1,25-dihydroxycholecalciferol as tracer and synthetic 25,26-dihydroxycholecalciferol as standard. The metabolite was purified from serum extracts by Sephadex LH 20 and high-pressure liquid chromatography; recovery was monitored with biologically generated, tritiated 25,26-dihydroxycholecalciferol.

2. The mean ± sem concentration of 25,26-dihydroxycholecalciferol in serum from 18 healthy subjects was 587 ± 65 pmol/l. Seven Asian patients with osteomalacia due to vitamin D deficiency had very low or undetectable (< 96–231 pmol/l) circulating 25,26-dihydroxycholecalciferol concentrations.

3. The metabolite was detectable in the sera from seven anephric patients (mean 262 ± 43 pmol/l), indicating that extrarenal sites for the 26-hydroxylation of 25-hydroxycholecalciferol exist in man.

4. A strong positive correlation between the concentrations of 25-hydroxycholecalciferol and those of 25,26-dihydroxycholecalciferol in serum was obtained. Thus it appears that in man the production of this dihydroxy metabolite of vitamin D depends on the concentration of its precursor, 25-hydroxycholecalciferol.

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