A new, highly sensitive assay for 1,25-dihydroxyvitamin D not requiring high-performance liquid chromatography: application of monoclonal antibody against vitamin D receptor to radioreceptor assay.

A new, highly sensitive radioreceptor assay, which does not require high-performance liquid chromatography, has been developed for the determination of 1,25-dihydroxyvitamin D3 (1,25-(OH2)D3) in serum. The assay involves rapid extraction of serum, Sep Pak silica purification, and addition of 1,25-dihydroxyvitamin D3 receptor, radiolabeled 1,25-dihydroxyvitamin D3, bovine serum albumin, and monoclonal antibody to specifically precipitate the receptor. This method is sensitive to 0.3-0.6 pg/tube, with B50 occurring at 5.8 pg/tube. This sensitivity combined with overall recovery of 1,25-dihydroxyvitamin D3 (81.5 +/- 5.2%, n = 50, mean +/- SD) allows the measurement of serum 1,25-(OH)2D3 in duplicates with only 0.5 ml of serum. Intra- and interassay coefficient of variation were 9.5 and 14.6%, respectively. Dilution analysis, analytical recovery of added 1,25-dihydroxyvitamin D3, and comparison with a standard method using HPLC have been used to validate the assay. Serum 1,25-dihydroxyvitamin D3 level was for normal adults, 36.6 +/- 10.5 pg/ml (n = 14); in primary hyperparathyroidism, 98.9 +/- 19.9 pg/ml (n = 16); in chronic renal failure, 17.8 +/- 5.1 pg/ml (n = 12). This method allows large numbers of samples to be processed at once. Further, the method is rapid and provides an accurate assay using small amounts of serum.

Normalization of serum calcium restores fertility in vitamin D-deficient male rats.

We performed experiments to determine whether treatment with vitamin D or 1,25-dihydroxycholecalciferol could reverse male infertility caused by vitamin D deficiency. Additionally, an attempt was made to distinguish between a direct and an indirect effect of 1,25-dihydroxycholecalciferol on reproductive tissue. Vitamin D-deficient male rats with impaired fertility were treated with vitamin D and 1,25-dihydroxycholecalciferol for 3 wk, then mated. Secondly, vitamin D-deficient male rats were made normocalcemic by increasing dietary calcium, and their fertility was compared with that of vitamin D-deficient, hypocalcemic rats. The fertility of male rats was restored by treatment with either vitamin D or 1,25-dihydroxycholecalciferol. However, fertility was also restored in vitamin D-deficient animals by feeding them a diet supplemented with high levels of calcium. These results indicate that the influence of vitamin D and its active metabolite, 1,25-dihydroxycholecalciferol, on male fertility is indirect. Vitamin D and 1,25-dihydroxycholecalciferol seemed to influence male fertility by acting on classic target tissues and regulating levels of calcium in reproductive tissues.