Challenges in vitamin D analysis

J Med Biochem 2012; 31 (4) DOI: 10.2478/v10011-012-0016-z UDK 577.1 : 61 ISSN 1452-8258 J Med Biochem 31: 326–332, 2012 Review article Pregledni ~lanak CHALLENGES IN VITAMIN D ANALYSIS IZAZOVI U ANALIZI VITAMINA D Mustafa Serteser, Abdurrahman Coskun, Tamer C. Inal, Ibrahim Unsal Acibadem University, School of Medicine, Department of Medical Biochemistr y, Acibadem Labmed Clinical Laboratories, Istanbul, Turkey Summary: Vitamin D is an important deter minant for the regulation of calcium and phosphorus levels and mineralization of the bone. The most reliable indicator of vitamin D status is the measur ement of plasma or ser um 25OH-D concentration. Several studies r eported discrepancies between the results of assays. These high variabilities in 25OH-D measurements are due to used assay technologies and lack of standardization against the r eference materials. Different assays have been employed for the measur ement of 25OHD levels: Competitive Protein Binding Assays, immunoassays, direct detection methods. Choosing an assay platfor m is important both for clinical laborator y professionals and researchers, and several factors affect this process. Recently, liquid chromatography and tandem mass spectrometry is an alternative method to traditional assays and pr ovides higher specificity and sensitivity than many assays; ther efore, it has been suggested as a candidate r eference method for circulating 25OH-D3. Standardization of methods for the quantification of 25OH-D by using the human-based samples would reduce the inter-method variability. The best way for laboratories to demonstrate the accuracy of their results is by participating in the exter nal quality assessment scheme. Standardization of the assays is also r equired to provide clinicians with the accurate tools to diagnose hypovitaminosis. In addition, assay -specific decision limits ar e needed to define appropriate thresholds of treatment. Keywords: 25 Hydroxy Vitamin D, assay, standardization, external quality control, LC-MSMS Kratak sadr`aj: Vitamin D je va`na deter minanta u regulaciji nivoa kalcijuma i fosfora i mineralizaciji kostiju. Naj pouzdaniji indikator statusa vitamina D je odr e|ivanje koncentracije 25OH-D u plazmi ili ser umu. Nekoliko studija je pokazalo neslaganja izme|u r ezultata razli~itih testova. Ovako velike varijacije u odr e|ivanjima 25OH-D su posledica kori{}enih tehnologija testova i nepostojanja standar dizacije u odnosu na referentne materijale. Razli~iti testovi se koriste za odr e|ivanje koncentracija 25OH-D: testovi kompetitivnog vezivanja za pr oteine, imunoodre|ivanja, metode direktnog odre|ivanja. Izbor platforme odre|ivanja je va`an i za klini~ko-laboratorijsku praksu i za istra`ivanja i nekoliko faktora uti~e na ovaj proces. Odnedavno, te~na hromatografija i tandem masena spektr ometrija predstavlja alternativu tradicionalnim testovima, ima ve}u osetljivost i specifi~nost od mnogih testova i pr edlo`ena je za kandidata za r eferentnu metodu odr e|ivanja 25OH-D3. Standar dizacija metoda za kvantifikaciju 25OH-D upotr ebom uzoraka humanog se ruma bi smanjila varijacije izme|u metoda. Najbolji na~in za potvrdu ta~nosti odre|ivanja rezultata u laboratoriji je u~e{}e u programu spolja{nje kontr ole. Standardizacija testova je tako|e potrebna da bi se klini~arima obezbedile ta~ne informacije za dijagnozu hipovitaminoze. T ako|e, neophodni su nivoi odluke specifi~ni za test da bi se definisale odgovaraju}e vrednosti praga za terapiju. Klju~ne re~i: 25 hidroksi vitamin D, test, standar dizacija, spolja{nja kontrola kvaliteta, LC-MSMS Introduction Address for correspondence: Mustafa Serteser, M.D. Acibadem University School of Medicine Department of Medical Biochemistr y Acibadem Labmed Clinical Laboratories Fahrettin Kerim Gokay Cad. No. 49 Altunizade, Uskudar, Istanbul, Turkey Tel: +90 216 544 39 45 Fax: +90 216 544 39 40 e-mail: mustafa.serteserªacibademlabmed.com.tr mustafa.serteserªacibadem.edu.tr Vitamin D is a pr o-hormone, known for its im portant role in the r egulation of calcium and phosphorus levels and mineralization of the bone. Hypo vitaminosis D is known to contribute to osteopor osis through decreased calcium absorption, subsequent secondary hyperparathyroidism and incr eased bone resorption. For this reason, decreased vitamin D levels are usually associated with the incr eased parathyroid hormone (PTH) levels. R ecent studies have shown that the Vitamin D r eceptors are present in a variety J Med Biochem 2012; 31 (4) of cells and have biological effects which ar e far beyond the mineral metabolism (1). Low Vitamin D levels have been found to be associated with the asthma in childr en (2), endothelial dysfunction (3, 4), harmful immunomodulatory effects (5), car diovascular risk (6), cognitive impair ment (7), and lost antitumoral activity potentiating a number of cytotoxic anti-cancer agents (8 ). In oncology patients, it has been shown that low ser um vitamin D levels pr edict an advanced stage of disease (9). It has been estimated that globally mor e than one billion people ar e vitamin D deficient and in the States, more than 75% of the adult population is vitamin D insufficient (1, 10). Increases in vitamin D testing is attributed to gr owing global deficiency due to blockage in sun exposur e and incr eased number of evidence between vitamin D deficiency and health conditions. Vitamin D is metabolized in the liver to pr oduce 25-OH-Vitamin D (25OH-D) and 1,25 (OH)2- Vitamin D is produced in kidneys (11). 25OH-D is a pr edominant form in the circulation and generally accepted as the best single marker of vitamin D status (1, 12). There are two types of 25OH-D found in the circulation: 25-OH-Vitamin D2 (25OH-D2) is also cal led ergocalciferol and derives mainly from plants and fish (13). 25-OH-Vitamin D3 (25OH-D3) or cholecalciferol accounts for appr oximately 95% of the cir culating 25OH-D pool, wher eas 25OH-D2 r epresents only a minor fraction unless vitamin D2-containing medication is taken by the individual (13). 1,25 (OH)2-Vitamin D is closely r egulated by PTH and intestinal calcium. It cir culates at extremely low concentrations what makes it mor e difficult to be measured accurately. Since vitamin D itself is tightly bound to vitamin D binding pr otein, it is the most highly lipid soluble form of the vitamin D (14). 25OH-D is better indicator of the patient’s vitamin D status than the vitamin itself . This is because the hydroxyl group makes 25OH-D less fat soluble and makes it have lower affinity to vitamin D binding protein than the actual vitamin. These factors make the circulating concentrations of 25OH-D about 1,000 times more concentrated than the steroid hormone form of vitamin D. 25OH-D levels also cor relate well with the clinical signs of vitamin D deficiency (15). Measurement of 25-Hydroxyvitamin D Competitive Protein Binding Assays The history of developing a sensitive method for the estimation of 25OH-D levels dated back to ne (...truncated)