Determination of Mono- and Dihydroxy-Vitamin D Metabolites in Normal Subjects and Patients with Different Calcium Metabolic Diseases

Abstract

Hummer L, Riis BJ, Christiansen C, Rickers H. Determination of mono- and dihydroxy-vitamin D metabolites in normal subjects and patients with different calcium metabolic diseases. Scand J Clin Lab Invest 1985; 45: 611–619.

Reliable assays to determine the vitamin D metabolites are useful aids in the study of disorders involving vitamin D metabolism, and in the evaluation of the response in patients receiving vitamin D treatment. We report here the establishment in our laboratory of a method capable of measuring 25(OH)D, (including 25(OH)D₂ and 25(OH)D₃), 1,25(OH)₂D and 24,25(OH)₂D in a single blood sample. The method involves methanol/dichloromethane extraction and Sephadex LH-20 chromatography. The monhydroxylated fraction was purified on Lipidex 5000 and separated in 25(OH)D₂ and 25(OH)D₃ on high pressure liquid chromatography (HPLC), followed by ultra violet absorbance (UV) detection. The dihydroxylated fraction was separated by HPLC and quantified by protein-binding assays. The method is precise and accurate. The vitamin D metabolites were measured in different groups of patients and in normal subjects.

Key Words:

• calcium metabolism
• vitamin D metabolism
• 24,25-dihydroxy-vitamin D
• 24,25-dihydroxy-vitamin D
• 25-hydroxy-vitamin D