A highly sensitive method for large-scale measurements of 1,25-dihydroxyvitamin D.
A quantitative method for measuring 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was developed utilizing a luciferase reporter gene under the control of the highly inducible 25-hydroxyvitamin D3 24-hydroxylase promoter in a stably transfected cell line. Transient transfections with constructs containing the 24-hydroxylase gene promoter 5' to a luciferase reporter were first performed in cell lines with high levels of vitamin D receptor, i.e., the rat osteosarcoma (ROS 17/2.8) and human breast cancer (T-47D) cell lines. ROS 17/2.8 cells, stably transfected with the plasmid, gave a 60-fold stimulation with 10(-10) M 1,25-(OH)2D3. A standard curve was constructed showing a large range of response to 1,25-(OH)2D3 (1 pg to 1 ng). The assay was adapted to microtiter plates, which permits a large number of samples to be assayed simultaneously. Other metabolites of vitamin D and analogs such as 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 1 alpha-hydroxyvitamin D3 have negligible effects on the detection of 1,25-(OH)2D3, thus eliminating the need for purification of sample. The sensitivity of the method permitted the use of 100 microliters of serum with excellent results. Comparison of this method with a commercially available assay demonstrates that it gives higher sensitivity, simpler manipulations, and comparable results.
Arbour
N C
NC
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Ross
T K
TK
Zierold
C
C
Prahl
J M
JM
DeLuca
H F
HF
D015650:24,25-Dihydroxyvitamin D 3 / Q000008:administration & dosage / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D053493:Cholestanetriol 26-Monooxygenase; D004274:DNA, Recombinant; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006887:Hydroxycholecalciferols / Q000008:administration & dosage / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008156:Luciferases / Q000032:analysis / Q000187:drug effects / Q000235:genetics; D008722:Methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013250:Steroid Hydroxylases / Q000235:genetics; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D006887:Hydroxycholecalciferols; D007457:Iodine Radioisotopes; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins; D014807:Vitamin D; D015650:24,25-Dihydroxyvitamin D 3; C097949:1,25-dihydroxyvitamin D; D003907:Dexamethasone; D008156:Luciferases; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; C008088:alfacalcidol