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Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay

¹ Helsinki University Central Hospital, Laboratory, Haartmaninkatu 2, 00290 Helsinki, Finland

^aauthor for correspondence: fax 358-9-4717-4945, e-mail ursula.turpeinen@hus.fi

The first 300 words of the full text of this article appear below.

Vitamin D status is usually assessed by measuring the serum concentration of 25-hydroxyvitamin D [25(OH)D]. Its measurement is important as a clinical indicator of nutritional vitamin D deficiency, which is one of the causes of osteoporosis (1). Vitamin D exists in two forms: cholecalciferol (vitamin D₃) and ergocalciferol (vitamin D₂). Vitamin D₂ is further metabolized to 25(OH)D₂. Vitamin D₃ is formed in the skin from its precursor 7-dehydrocholesterol after ultraviolet irradiation or is absorbed from the diet (2). It is further hydroxylated in the liver to 25(OH)D₃ as the first step of its conversion in the kidney to 1,25-dihydroxyvitamin D₃, which is the biologically active form. 25(OH)D₃ is the main circulating form of vitamin D. Clinically it is important to measure both forms of 25-hydroxyvitamin D to monitor the effect of vitamin D₂ supplementation on total vitamin D status.

The first routine methods for measurement of 25(OH)D concentrations in human plasma were based on competitive protein binding and used vitamin D-binding protein and a tritium-labeled tracer (3). These methods were replaced by a simpler, rapid RIA (4), and a radioiodinated tracer was incorporated into the RIA in 1993 (5). This assay principle is the basis of several commercially available methods. Quantitative HPLC assays have been developed based on ultraviolet detection and normal-phase separation (6), combined use of normal- and reversed-phase separations (7), or reversed-phase separation alone(8). Recently, reversed-phase HPLC methods for 25(OH)D₃ in human plasma have been developed with normal-phase prepurification of the sample (9) or liquid extraction only (10).

Earlier HPLC methods for 25(OH)D₃ in serum were designed mainly for research purposes and were therefore too complicated for routine use. The present method was designed . . . [Full Text of this Article]

The following articles in journals at HighWire Press have cited this article:

				A. Leino, U. Turpeinen, and P. Koskinen Automated Measurement of 25-OH Vitamin D3 on the Roche Modular E170 Analyzer Clin. Chem., December 1, 2008; 54(12): 2059 - 2062. [Abstract] [Full Text] [PDF]

				M. Bjorkman, A. Sorva, J. Risteli, and R. Tilvis Vitamin D supplementation has minor effects on parathyroid hormone and bone turnover markers in vitamin D deficient bedridden older patients Age Ageing, January 1, 2008; 37(1): 25 - 31. [Abstract] [Full Text] [PDF]

				F. Ibrahim, C. Parmentier, and P. Boudou Divergence in Classification of 25-Hydroxyvitamin D Status with Respect to Immunoassays Clin. Chem., February 1, 2007; 53(2): 363 - 364. [Full Text] [PDF]

				J. A. Schmidt Measurement of 25-Hydroxyvitamin D Revisited Clin. Chem., December 1, 2006; 52(12): 2304 - 2305. [Full Text] [PDF]

				G. Lensmeyer, D. Wiebe, N. Binkley, and M. Drezner The authors of the article cited above respond: Clin. Chem., December 1, 2006; 52(12): 2305 - 2306. [Full Text] [PDF]

				G. L. Lensmeyer, D. A. Wiebe, N. Binkley, and M. K. Drezner HPLC Method for 25-Hydroxyvitamin D Measurement: Comparison with Contemporary Assays Clin. Chem., June 1, 2006; 52(6): 1120 - 1126. [Abstract] [Full Text] [PDF]

				M. Hedman, T. Matikainen, A. Fohr, M. Lappi, S. Piippo, M. Nuutinen, and M. Antikainen Efficacy and Safety of Pravastatin in Children and Adolescents with Heterozygous Familial Hypercholesterolemia: A Prospective Clinical Follow-Up Study J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 1942 - 1952. [Abstract] [Full Text] [PDF]

				M. Vogeser, A. Kyriatsoulis, E. Huber, and U. Kobold Candidate Reference Method for the Quantification of Circulating 25-Hydroxyvitamin D3 by Liquid Chromatography-Tandem Mass Spectrometry Clin. Chem., August 1, 2004; 50(8): 1415 - 1417. [Full Text] [PDF]

				N. Binkley, D. Krueger, C. S. Cowgill, L. Plum, E. Lake, K. E. Hansen, H. F. DeLuca, and M. K. Drezner Assay Variation Confounds the Diagnosis of Hypovitaminosis D: A Call for Standardization J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3152 - 3157. [Abstract] [Full Text] [PDF]