Clinical Chemistry
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Clinical Chemistry 50: 1315-1321, 2004. First published May 20, 2004; 10.1373/clinchem.2004.033126
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(Clinical Chemistry. 2004;50:1315-1321.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Vitamin D Receptor mRNA Measured in Leukocytes with the TaqMan Fluorogenic Detection System: Effect of Calcitriol Administration

Laura Soldati1,a, Donatella Adamo1, Cristiana Bianchin2, Teresa Arcidiacono2, Annalisa Terranegra1, Maria Luisa Bianchi4, Stefano Mora3, Daniele Cusi1 and Giuseppe Vezzoli2

1 Department of Sciences and Biomedical Technologies, University of Milan, Milan, Italy. 2 Division of Nephrology Dialysis and Hypertension and 3 Laboratory of Pediatric Endocrinology, Istituto di Ricovero e Cura a Carattere Scientifico, San Raffaele Hospital, Milan, Italy. 4 Bone Metabolic Unit, Istituto di Ricovero e Cura a Carattere Scientifico, Istituto Auxologico Italiano, Milan, Italy.

aAddress correspondence to this author at: Department of Sciences and Biomedical Technologies, University of Milan, Via Fratelli Cervi 93, 20090 Milan, Italy. Fax 39-02-50330434; e-mail laura.soldati{at}unimi.it.

Background: The aim of the present study was to investigate the interactions between the circulating concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and the mRNA concentration of its specific nuclear receptor in human leukocytes.

Methods: We measured vitamin D receptor (VDR) mRNA extracted from leukocytes by use of TaqMan fluorescence analysis applied to the reverse transcription-PCR (RT-PCR) technique in 16 volunteers before and after calcitriol administration. VDR mRNA was also measured in leukocytes from calcium-stone-formers (37 hypercalciuric and 34 normocalciuric patients). The relationship between VDR mRNA concentrations and genetic VDR polymorphisms was analyzed in these patients.

Results: Imprecision (CV) of RT-PCR was 1.3% within assay (n = 10) and 1.7% between assays (n = 4). Oral 1,25(OH)2D3 increased mean (SE) serum 1,25(OH)2D3 1.6 (0.3)-fold and VDR mRNA 1.6 (0.1)-fold 8 h after administration. The maximum VDR mRNA was reached 3.6 (1.3) h after 1,25(OH)2D3 ingestion. No differences in leukocyte VDR mRNA concentrations were found between normocalciuric and hypercalciuric stone-formers in the absence of stimulation. Finally, no association was found between VDR mRNA concentrations and genetic VDR polymorphisms in stone-formers.

Conclusions: The TaqMan RT-PCR assay is a rapid and accurate method to measure VDR mRNA, and leukocytes are a useful model to study VDR and 1,25(OH)2D3 interactions. In humans, VDR mRNA is increased by agonist 1,25(OH)2D3, a finding resembling previously reported results obtained in cellular and animal models.






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