La determinazione della Vitamina D: utilità e problemi analitici

Transcription

1 La determinazione della Vitamina D: utilità e problemi analitici Antonio Fortunato Ospedale San Bortolo - Vicenza [email protected]

2 A reevaluation of the physiological and pharmacological actions of vitamin D produced evidence that vitamin D can regulate the immune system and thereby is implicated in several immune-mediated diseases. Single serum 25OHD measurements are of limited value because of the known seasonal variation and lack of consistency between methods Epidemiological studies provide only indirect evidence that vitamin D, resulting from exposure to UVB, is a protective agent. Therefore, vitamin D supplementation studies are important to establish whether vitamin D has a direct protective role. THE IMPLICATIONS OF NEW ROLES FOR VITAMIN D FOR CLINICAL CHEMISTRY The measurement of 25OHD in blood and other biological fluids is challenging. As we seek to understand the importance of newly discovered roles for vitamin D in health and disease, the accurate and precise measurement of 25OHD in blood is essential. 2

3 Early methods that relied on competitive protein binding or immunoassay to measure 25OHD included a solvent extraction step to remove the sterol from vitamin D binding protein. Manufacturers of 25OHD immunoassay methods have sought to replace this solvent-extraction step with blocking agents to facilitate the inclusion of 25OHD assays on automated platforms. Evidence in practice suggests that the success of this block-and-displace approach is limited. Clinical chemists are aware of spuriously high 25OHD results in individual patient samples and higher than expected imprecision. For example, the all-method mean CV for 20 recent specimens distributed through the Vitamin D External Quality Assessment Scheme was 18.7% (range 16.6% 20.2%), and the CV for some individual immunoassay methods was greater than this all-method mean. A second challenge to the measurement of 25OHD arises from the application of the assay to assess the adequacy of vitamin D replacement therapy. Vitamin D that is used for replacement purposes occurs in 2 major forms. Cholecalciferol (vitamin D3) is the natural animal form, and ergocalciferol (vitamin D2) is derived from plants. Increasing globalization of health and medicinal products means that both vitamin D3 and D2 are used by the public and by patients in all countries. Although vitamins D3 and D2 are structurally similar, the differences are sufficient to cause a variable response in the immunoassays used to measure the hydroxylated form of these preparations. Some immunoassays are claimed to be equipotent for the measurement of 25OHD3 and 25OHD2, whereas others preferentially detect one form. Some immunoassays in use may lead to serious clinical misclassification of individual patients. 3

4 La Vitamina D Forme principali che costituiscono il gruppo vitaminico D: vitamina D1 : composto costituito in parti 1:1 di ergocalciferolo e lumisterolo vitamina D2 : calciferolo vitamina D3 : colecalciferolo vitamina D4 : diidroergocalciferolo vitamina D5 : sitocalciferolo 4

5 La Vitamina D 7-Deidrocolesterolo Ergosterolo 5 Colecalciferolo (D3) Ergocalciferolo (D2) La vitamina D è un gruppo di pro-ormoni liposolubili; il gruppo si presenta sotto due forme principali dall'attività biologica molto simile: il colecalciferolo (D3), derivante dal colesterolo e sintetizzato negli organismi animali, e l'ergocalciferolo (D 2 ), di provenienza vegetale (ergosterolo).

6 Il Laboratorio e la misura della Vitamina D Raramente si misura Vitamina D sotto forma di pro-ormone I due metaboliti prevalentemente misurati sono: 25 idrossi Vitamina D 2+3 (25OH D) fornisce indicazioni sulle riserve di Vitamina di apportate dalla dieta, dall esposizione al sole, dalla supplementazione 1,25 di-idrossi Vitamina D (1,25-OH 2 D) è la forma attiva 6

7 7 Il Laboratorio e la misura della Vitamina D

8 Given the range of methodologies available for the measurement of 25OHD the choice of which is the best method for an individual laboratory is challenging especially given the dramatic increase, over the last few years, of both routine clinical and research use of the measurement. It is clear that there are advantages and limitations for each method and these are summarised in Table 4. 8

9 segnale Il Laboratorio e la misura della Vitamina D Cromatografia (HPLC) 9 tempo

10 segnale Il Laboratorio e la misura della Vitamina D concentrazione 10

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13 Supplementation of Vitamin D DRUGS D 3 based drugs are widely used in all the countries WW in US D 2 based drugs only are available D 2 based drugs are widely used in a no official (almost hidden) way even in D 3 utilizing Countries (e.g. France, UK, Belgium, Italy, Germany, Canada, etc) or sometimes in OTC supplementation or for vegan people Nowadays people are much more frequently travelling around 13 Consequently a proper and accurate monitoring of therapy MUST be performed with an analytical method that detect both D 2 and D 3 metabolites, even for one single subject

14 IL LABORATORIO E LA MISURA DELLA VITAMINA D Specificity D2 0.0 % D3 0.0 % 25 OH D % 25 OH D % 1,25D2 40 % 1,25D3 17 % 14

15 IL LABORATORIO E LA MISURA DELLA VITAMINA D Precision intra-run CV% 4.5 ng/ml 5 % 36.2 ng/ml 4 % 87.3 ng/ml 3 % Precision inter-run CV% 4.5 ng/ml 11% 36.2 ng/ml 6 % 87.3 ng/ml 6 % 15

16 % Recupero Vit D esogena 25OH D3 25OH D nm 25ODH D endogena Metodi

17 IL LABORATORIO E LA MISURA DELLA VITAMINA D Serum sample (100 µl) Pretreatment Denaturation w. methanol-propanol Vortex mixing Extraction in hexane Dried under nitrogen reconstituted Runtime 8 min Within CV: 9,5% (52 nmol/l) for D2 5,1% (55 nmol/l) for D3 100 samples/12,5 h = 8 samples/h 17

18 Despite the sophisticated principle of analyte detection in LC MS/MS it is important to consider issues which may compromise the reliability of such analyses 56 compounds were identified as potential interferers It also has to be considered that in LC MS/MS interference might arise from several charged compounds with a higher molecular mass than the target analyte if both share one mass-to-charge ratio. 18

19 IL LABORATORIO E LA MISURA DELLA VITAMINA D LC-MS is clearly a superior analytical technique by which to assess circulating 25(OH)D The data clearly do not support this statement! DEQAS results demonstrate that DiaSorin LIAISON and LC-MS are clinically equivalent IF LC-MS is properly performed. Further, a recent publication by Binkley et. al. (Clin Chem 2006) states that separate determination and reporting of 25(OH)D2 and 25(OH)D3 does nothing more than confuse the physician. It is TOTAL 25(OH)D that is important! 19

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21 21 D E Q A S Vitamin D External Quality Assessment Scheme Scheme Organiser: Graham Carter Administration: Julia Jones Software: Richard Carter Endocrine Laboratory - Charing Cross Hospital - Fulham Palace Road - London W6 8RF - UK Telephone: +44 (0) Facsimile: +44 (0) [email protected]

22 22 Laboratorio di Chimica clinica ed Ematologia Ospedale San Bortolo - Vicenza

23 ng/ml Laboratorio di Chimica clinica ed Ematologia Ospedale San Bortolo - Vicenza 25OH-Vitamina D vs decadi età ,1 0, Decade 2Decade 3Decade 4Decade 5Decade 6Decade 9Decade 10Decade 11Decade

24 Laboratorio di Chimica clinica ed Ematologia Ospedale San Bortolo - Vicenza anni N Media Mediana P.le ,7 24,3 16,8-36, ,4 24,2 16,7-32, ,2 23,1 15,6-31, ,5 20,4 12,9-29, ,5 20,7 12,9-29, ,0 21,9 14,1-30, ,2 21,4 13,1-30, ,3 17,9 9,3-27, ,7 13,3 6,0-24, ,1 10,8 5,0-24, ,7 17,8 6,3-29,5 2 4 Totale determinazioni

25 Laboratorio di Chimica clinica ed Ematologia Ospedale San Bortolo - Vicenza Donne N = 109 Età media = 38 min = 16 max = 46 Settembre ng/ml

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28 J.C. Souberbielle «La recherche et la correction d un déficit en calcium et/ou vitamine D non seulement constitue un préalable indispensable à la mise en route d un traitement anti-ostéoporotique, mais doivent être poursuivies au long cours.» 28