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RADIOIMMUNOASSAY OF 1,25‐DIHYDROXY VITAMIN D 2 : STUDIES ON THE METABOLISM OF VITAMIN D 2 IN MAN
Author(s) -
FRAHER L. J.,
ADAMI S.,
CLEMENS T. L.,
JONES G.,
O'RIORDAN J. L. H.
Publication year - 1983
Publication title -
clinical endocrinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.055
H-Index - 147
eISSN - 1365-2265
pISSN - 0300-0664
DOI - 10.1111/j.1365-2265.1983.tb02977.x
Subject(s) - vitamin , vitamin d and neurology , radioimmunoassay , chemistry , ergocalciferol , endocrinology , medicine , vitamin d binding protein , chromatography , high performance liquid chromatography , antiserum , cholecalciferol , antibody , biochemistry , immunology
SUMMARY A sensitive radioimmunoassay for 1,25‐dihydroxy vitamin D 2 was developed using a sheep antiserum which preferentially reacts with 1‐hydroxylated forms of vitamin D. An improved isolation procedure was also developed using acetonitrile for the initial extraction of serum followed by chromatography on cartridges of C 18 silica and high pressure liquid chromatography eluted with a ternary solvent system to separate 1,25‐dihydroxy vitamin D 2 and 1,25‐dihydroxy vitamin D 3. 25‐hydroxy vitamin D 2 and 25‐hydroxy vitamin D 3 were separated by further reverse phase high pressure liquid chromatography prior to competitive protein binding assay. The limits of detection were 4.3 pmol/l (2.0 pg/ml) for the 1,25‐dihydroxy metabolites and 1.25 nmol/l (0.5 ng/ml) for both 25‐hydroxy vitamin D 2 and 25‐hydroxy vitamin D 3 . 25‐hydroxy vitamin D 2 ranged from 2.0 to 11.3 nmol/l (0.8–4.5 ng/ml) with a mean of 4.75 nmol/l (1.9 ng/ml) in thirteen healthy British adults and this accounted for 9.0% of the mean total 25‐hydroxy vitamin D. 1,25‐dihydroxy vitamin D 2 was detected in the sera of only one of these subjects whereas 1,25‐dihydroxy vitamin D 3 was present in all ranging from 48 to 163 pmol/l (20–65 pg/ml) with a mean of 100 pmol/l (42 pg/ml). Both 1,25‐dihydroxy vitamin D 2 and 1,25‐dihydroxy vitamin D 3 were detected in the sera of hypoparathyroid patients treated with vitamin D 2 but the relationship between 25‐hydroxy vitamin D and 1,25‐dihydroxy vitamin D was complex. For example, when an excess of 25‐hydroxy vitamin D 2 was present the serum concentration of 1,25‐dihydroxy vitamin D 3 was disproportionately high. Conversely, in patients who had previously been treated with vitamin D 2 but were receiving only vitamin D 3 at the time of study, the major 25‐hydroxy metabolite was in the vitamin D 3 form and there was a disproportionately high amount of 1,25‐dihydroxy vitamin D 2. Total 1,25‐dihydroxy vitamin D ranged from 110 to 400 pmol/l (45–165 pg/ml) and was above the upper limit of normal for 1,25‐dihydroxy vitamin D 3 in half of these hypoparathyroid patients treated with pharmacological doses of vitamin D.