TheLC-MS/MSisourreference

ThequantificationofthephysiologicallyactiveVitaminDmetabolite1,25-dihydroxyVitaminDisanecessarydiagnostictoolforseveralpathologicalconditionssuchas:

• Kidneydiseases
• Kidneytransplantation
• Sarcoidosis
• Rickets
• Osteomalacia

Thequalityofthemeasuringmethod,whichourassayfulfillsahundredpercent,thereforeisindispensable.

Thelevelsof1,25-dihydroxyVitaminDinplasmaorserumis100to1000-foldlessthanthatof25-hydroxyVitaminD.Duetoitslowconcentrationsandthepresenceofmanysimilarmetabolites,theexactmeasurementof1,25-dihydroxyvitaminDstillrequirestodoanextractionandseparationstepwithorganicsolventsofthesampleprioranymeasurementsystem.Thisextractionwithorganicsolventsstilltodayisthegoldstandardextractionmethod.

"Thesuccessiveabandonmentofsampleextraction,chromatography,[…]inimmunoassayshasundoubtedlycontributedtotheinconsistenciesreportedbyFarrelletal,[…]."(GrahamCarter,DEQASorganizer,CarterGD.ClinChem.2012;58(3):486-8.)

Theextractionsuppressesthepotentialinterferencefromsamplematrix,heterophilicantibodiesandabnormalconcentrationsofVitaminDbindingproteins.AndtheadditionalseparationonsolidphasecartridgesfurthersuppressestheinterferencefromotherVitaminDmetabolitessuchas25-hydroxyVitaminD,24,25-dihydroxyVitaminD,3-epi-25-hydroxyVitaminDand25,26-dihydroxyVitaminD.

ThisleadstogoodcorrelationofourELISAwiththegoldstandardtechnologyLC-MS/MS.

FurThermoretheELISAwasalsocorrelatedtothestillwidelyusedrADIoimmunoassayfromDiasorin.AlsoheretheELISAshowedagoodfit.

KeyfeaturesofourELISAare:

• GoodcorrelationwithLC-MS/MS
• VerylowLimitofDetection(1.7pg/mL)
• Limitednumberofincubationsteps
• DEQASsamplesaccuratelymeasured
• 108%cross-reactivityagainst1,25(OH)₂VitaminD2
• GoldStandardextractionprotocolensuringnointerference

Generalinformation

1,25(OH)₂VitaminDistheactiveformofVitaminDwithregardtotheknownfunctionswhereas25OHVitaminDandVitaminDitselfcanbeexcludedasbeingphysiologicallyfunctional.1,25(OH)₂VitaminDstimulatestheintestinalabsorptionofbothcalciumandphosphorus.Italsostimulatesboneresorptionandmineralizationtherebypreventingthedevelopmentofricketsandosteomalacia.1,25(OH)₂VitaminDisalsoactiveinothertissuesresponsibleforCalciumtransport(placenta,kidney,mammarygland,...)andendocrineglandssuchasparathyroidglands.1,25(OH)₂VitaminDisrapidlymetabolizedanditshalflifeinplasmaisapproximately12h.Itsisfurthermetabolizedtoeithercalcitroicacid,essentiallywithoutanyBIOLOGicalactivity,orvia24-hydroxylationto1,24,25-trihydroxyvitaminD.Thiscompoundhaslessbiologicalactivitythanitsparentandthismetabolicrouteisconsideredasaminorpathway.Thelevelsof1,25(OH)₂VitaminDinplasmaorserumis100-to1000-foldlessthanthatof25OHVitaminD.Duetoitslowconcentrationsandthepresenceofmanysimilarmetabolites,themeasurementof1,25(OH)₂VitaminDrequiresextractionandseparationbychromatography.