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The Assessment of Circulating 25OHD and 1,25OH2D: Emergence as Clinically Important Diagnostic Tools Bruce W. Hollis, P

Vitamin D25(OH)D25(OH)D3-26,23-lactone24,25(OH)2D25,26(OH)2D1,25(OH)2D1,24,25(OH)3D1,25,26(OH)3D. Vitamin D2 and Vitamin D3 Metabolites that we could measure in a single plasma sample using hplc to separate. . Plasma (3-5 ml) 3H Metabolites. Diethylether (2X). Methanol: Methylene Chloride (1:3).

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The Assessment of Circulating 25OHD and 1,25OH2D: Emergence as Clinically Important Diagnostic Tools Bruce W. Hollis, P

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    4. Circulating 25(OH)D is the best indicator of nutritional vitamin D status. Circulating 1,25(OH)2D confirmatory test for disorders involving the vitamin D endocrine system.

    5. Indication for 25(OH)D Measurement When nutritional deficiency of vitamin D is suspected Intestinal malabsorption syndromes Limited exposure to the sun Limited intakes of oral vitamin D supplements Aged, homebound patients Skin pigmentation EVERYBODY should know their 25(OH)D status

    6. Indication for 1,25(OH)2D Measurement Monitor therapy Disease State Kidney disease Hyperparathyroidism Sarcoidosis Research

    7. Early 25(OH)D assays J.G. Haddad & K.J. Chyu, Competitive protein-binding radioassay for 25-hydroxycholecalciferol., Journal of Clinical Endocrinology and Metabolism 33 (1971) 992-995. R.E. Belsey, H.F. DeLuca & J.T. Potts, Jr., A rapid assay for 25-OH-vitamin D3 without preparative chromatography, J Clin Endocrinol Metab 38 (6) (1974) 1046-1051.

    8. Various Methods Competitive-Protein Binding Assays (CPBA) Advantages Inexpensive Small sample size Co-specific for 25(OH)D2 and 25(OH)D3 Disadvantages Subject to nonspecific interference Requires organic extraction and solvent evaporation Requires preparative chromatography Is limited to the use of 3H-tracer and thus liquid scintillation counting

    9. Physician’s View A lot of ergocalciferol 50,000 U (Drisdol and Calciferol) is used. Don't have easy access to large doses of vitamin D3 at this point is main reason to use vitamin D2. Works for me more often than not.

    10. UV Quantitation following HPLC (Gold Standard) J.A. Eisman, R.M. Shepard & H.F. DeLuca, Determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma using high pressure liquid chromatography., Analytical Biochemistry 80 (1977) 298-305. G. Jones, Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography., Clinical Chemistry 24 (1978) 287-298.

    11. UV Quantitation following HPLC (Gold Standard) Advantages Separate quantitation of 25(OH)D2 and 25(OH)D3 Very stable and repeatable Disadvantages Larger sample size required Very costly equipment required Internal standard [3H]-25(OH)D3, laurophenone Need preparative chromatography Sometimes assay is subject to interfering UV compounds High level of technical expertise required Slower sample turn around time

    12. B.W. Hollis & J.L. Napoli, Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status, Clin Chem 31 (11) (1985) 1815-1819. B.W. Hollis, J.Q. Kamerud, S.R. Selvaag, J.D. Lorenz & J.L. Napoli, Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer., Clinical Chemistry 39 (1993) 529-533. Radioimmunoassay (RIA)

    13. Radioimmunoassay (RIA) Advantages Small sample size Incorporation of 125I into tracer Quantitative extraction so no internal tracer required Not subject to nonspecific interference Rapid, inexpensive and accurate Disadvantages Still requires the use of radionuclides Some RIAs discriminate between 25(OH)D2 and 25(OH)D3

    14. LC-MS/MS Quantitation Same limitations as HPLC quantitation although sample size is quite small (200 ul) Difficult to resolve 3-epi-25(OH)D3 An inactive metabolite with respect to calcium metabolism

    17. Commercial 25(OH)D Assay RIA/EIA Methods DiaSorin RIA, 125I-ligand -ACN extraction, primary and secondary Ab -Co-specific fo 25(OH)D2 and 25(OH)D3 IDS RIA, 125I-ligand -ACN extraction, primary and secondary ab -discriminates against 25(OH)D2 (0.75) IDS EIA -no extraction -biotin labeled ligand, avidin labeled HRP -Co-specific for 25(OH)D2 and 25(OH)D3

    18. Automated Instrumentation Methods DiaSorin Liason, chemilumenescence -whole serum, w/antibody coated particles -Recognizes 25(OH)D2 and 25(OH)D3 equally -180 samples/h Nichols Advantage, chemilumenescence -whole serum, DBP coated particles -problem recognizing 25(OH)D2 -170 samples/h IDS EIA on Grifols TRITURUS

    20. DiaSorin Liaison

    21. precision

    22. Direct Methods HPLC, [3H]-internal standard or laurophenone -extraction -pre-purification -HPLC/UV quantification LC-MS/MS, deuterated internal standard -ACN extraction -HPLC -mass detection

    23. Vitamin D External Quality Assessment Scheme (DEQAS) Currently 322 worldwide participants in this QC survey 5 samples sent quarterly for 25(OH)D and /or 1,25(OH)2D quantitation

    26. Mean deviation (% bias) of the method mean from the target value (ALTM), over the period January 2000 to January 2004 unless stated otherwise.

    30. CORRELATION OF SAMPLES containing significant amount of 25(OH)D2

    31. 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 !

    33. DEQAS: Comparison of Low, Medium and High Standards (n) 25(OH)D (nmol) % CV Liaison 29 19.4 29.8 LC-MS 13 18.5 32.3 Liaison 37 34.1 18.9 LC-MS 14 38.1 28.5 Liaison 38 96.6 16.1 LC-MS 14 104.7 21.9

    34. DEQAS Linearity Study (1/07) 25(OH)D nmol 1 2 3 4 5 DS RIA 19.4 34.1 52.5 70.1 90.9 (37.3) (55.2) (73.1) DS LS 21.3 39.6 57.4 75.3 91.0 (38.8) (56.2) (73.6) LC-MS 18.5 38.1 63.4 80.8 104.7 (40.1) (61.6) (83.2) ALL HAVE EXCELLENT LINEARITY

    37. Who Is Right? 25(OH)D2 25(OH)D3 25(OH)D LabCorp DS-LI 15.6 Quest LC-MS-MS <4 45.0 45.0 Hollis Lab DS-RIA 21.3 Mayo Labs LC-MS <1 20.9 20.9 All values in ng/ml

    38. Picking an assay platform-recognize limitations Assay recognizes vitamin D2 and vitamin D3 metabolites equally. Sigma sells standards!! Be sure amounts are correct and compounds are pure. HPLC or LC-MS/MS Low throughput Cleanup steps are crucial Sample volume can be an issue More expertise required RIA or automated platform Higher throughput, small sample volume Can be more variable Prefer methods that have been validated against gold standard methods and accuracy is continuously being evaluated in external quality assessment schemes

    39. 1,25(OH)2D

    40. No need to develop an in-house 1,25(OH)2D assay. Commercially available kits work fine Each laboratory should have own internal control sample Pool of serum or plasma run in each assay to evaluate intra and inter assay variability Need for Standard Reference Standard (NIST) Other Issues

    41. Conclusion There are several valid methods by which to determine circulating 25(OH)D and/or 1,25(OH)2D. BE SURE THAT WHOMEVER PERFORMS THEM KNOWS WHAT THEY ARE DOING, HAS VALIDATED THE METHOD AND PARTICIPATES IN DEQAS !

    42. THANK YOU

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