Advances and challenges in the measurement of 1,25-dihydroxyvitamin D: a comprehensive review

Vitamin D has received significant attention from clinical societies, researchers, and the general population in recent years. While 25-hydroxyvitamin D (25(OH)D) is the most commonly-used biomarker of vitamin D status, 1α,25-dihydroxyvitamin D (1,25(OH)₂D), its bioactive form, plays a critical role in regulating calcium and phosphorus homeostasis and is also involved in the immune system and cellular differentiation. Consequently, accurate measurements of 1,25(OH)₂D can aid in the differential diagnosis of calcium-related disorders such as hypocalcemia in vitamin D-dependent rickets and hypercalcemia due to inappropriate increase of serum 1,25(OH)₂D in granulomatous diseases. However, due to its lipophilicity and very low circulating concentration, the measurement of 1,25(OH)₂D is particularly challenging. Over the past several decades, numerous efforts have been made to develop sensitive, specific, and practical laboratory methods for measuring 1,25(OH)₂D. Methods using radioreceptor assay, radioimmunoassay, enzyme immunoassay, enzyme-linked immunosorbent assay, automated chemiluminescent immunoassay, and liquid chromatography-tandem mass spectrometry have been described. Each of these methods has unique advantages and limitations, and some are no longer used. Despite the sophisticated methods in use today, substantial variations between methods still exist. A concerted effort toward standardization of 1,25(OH)₂D measurement is needed to ensure accurate and reliable results across laboratories and methods.