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What Are We Referring to When We State PTH Level?

What Are We Referring to When We State PTH Level?. Evolution of PTH assays. Clinical utility of new development. Synthesized in parathyroid glands. Pre-proPTH (115 amino acids). ProPTH (90-amino acid) . PTH (84 amino acids). Parathyroid glands. CaSR. PTH1R. Osteoblasts. PTH.

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What Are We Referring to When We State PTH Level?

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  1. What Are We Referring to When We State PTH Level?

  2. Evolution of PTH assays Clinical utility of new development

  3. Synthesized in parathyroid glands Pre-proPTH (115 amino acids) ProPTH (90-amino acid) PTH (84 amino acids)

  4. Parathyroid glands CaSR PTH1R Osteoblasts PTH Kidneys Ca++ Ca++ reabsorption RANKL  M-CSF  IL-6 Pi excretion  Collagenase-3 Collagen I Hydroxylation of 25VD3 Osteoclasts Intestinal absorption Resorption bone turnover

  5. Proteolysis of 1-84 PTH NH2 NH2 1 N-PTH T1/2 (2-5 mins) M-PTH 39-68 44-68 Parathyroide glands liver kidneys bone 24-84 28-84 34-84 37-84b 43-84b 84 COOH COOH C-PTH

  6. First Generation PTH Asssay Radioimmunoassay (RIA) methods 84 1 PTH Antibody Isotope-PTH/fragments 1963-1983

  7. 1 84 PTH H2N COOH C-PTH COOH Mid region PTH First Generation PTH Asssay 1 84 M, C Assay 1963-1983

  8. 1 84 PTH H2N COOH H2N First Generation PTH Asssay 1 84 N assay 1963-1983

  9. 1 84 PTH H2N COOH Intact PTH Assays 1st Generation Sandwich Immunometric Assays 1 84 Signal Ab Capture Ab 1983-1993

  10. HPLC Fractionation of PTH forms in Serum 7-84 non(1-84) 1-84 Pooled uremic serum samples C18 u-Bondspak column (Waters) Acetonitrile in 1g/L trifluoroacetic acid 1.5 ml/min 1.5 ml/fraction for I-PTH checking Retention Time (min) Nichols …… Incstar DSL Lepage R et al. Clin Chem 1998:44:805-9.

  11. 1 84 PTH H2N COOH Large C-PTH non (1-84)-PTH COOH Intact PTH Assays 1st Generation Sandwich Immunometric Assays 12-34 1 84 Signal Ab Capture Ab Total PTH Further studies: non-(1-84) PTH = 10-20% of I-PTH in healthy, 40-60% of I-PTH in patients with terminal renal disease. 1983-1993

  12. 1 84 PTH H2N COOH Assay of Whole PTH 2nd Generation Sandwich Immunometric Assays 4-5 1 84 Capture Ab Signal Ab Whole PTH 1999

  13. HPLC Fractionation of PTH forms in Serum and Parathyroid Cell Supernatant C18 u-Bondspak column (Waters) Acetonitrile in 1g/L trifluoroacetic acid 1.5 ml/min 1.5 ml/fraction for I-PTH checking Whole PTH assay (1-4) Intact PTH Assay (12-23) D’Amour et al., Kidney International 2005,68:998-1007

  14. Localization of the metabolically labeled residues was determined by automated amino-terminal Edman degradation D’Amour et al., Kidney International 2005,68:998-1007

  15. Utility in Clinical Diagnosis

  16. 1-84PTH 1-84PTH Role of 1-84PTH Measurement in Intraoperative PTH Assay Primary Hyperparathyroidism 29 patients Non(1-84)PTH PTH(1-84) Whole 1-84 PTH (1-4) Scantibodies Lab Inc. Intact PTH IRMA intact iPTH Nichols Institute Diagnostics Non(1-84)PTH Secondary Hyperparathyroidism 7 patients Yamashita et al., Annals of surgery 2002, 236(1):105-111.

  17. Parathyroid glands PTH 7-84PTH PTHCR Ca++ Kidneys Osteoblasts Ca++ reabsorption Osteoclasts Pi excretion Hydroxylation of 25VD3 Resorption bone turnover

  18. 1st Generation Sandwich Immunometric Assays “Intact”PTH =(1-84)+non(1-84) 2nd Generation Sandwich Immunometric Assays (1-84) PTH Bone turnover Bone turnover (1-84)PTH/ Non (1-84) PTH Ratio Non(1-84) PTH = “Intact” PTH -(1-84)PTH (1-84)PTH/ Non (1-84) PTH

  19. Measurement of (1-84)PTH/ Non (1-84)PTH Ratio Low bone turnover 28 chronically dialyzed patients High or normal bone turnover 23 chronically dialyzed patients Non(1-84)PTH PTH(1-84)/non(1-84) PTH Monier-Faugere et al., Kidney International 2001,60:1460-1468.

  20. Receiver-operator characteristics (ROC) curves for the prediction of bone turnover in chronically dialyzed patients. A) : PTH-(1-84)/non(1-84) PTH fragment ratio PTH-(1-84) “intact”PTH (B) bone-specific alkaline phosphatase osteocalcin Monier-Faugere et al., Kidney International 2001,60:1460-1468.

  21. P<0.05 P<0.05 Areas under ROC curve BSAP: bone-specific alkaline phosphatase OC: osteocalcin Monier-Faugere et al., Kidney International 2001,60:1460-1468.

  22. Low bone turnover 9 chronically dialyzed patients High or normal bone turnover 26 chronically dialyzed patients Area under the ROC curve “intact”PTH 0.859 PTH(1-84) 0.842 (1-84)/non(1-84) 0.538 P<0.01 Coen et al., American Journal of Kidney Diseases 2002,40:348-354.

  23. Monier-Faugere et al Intact PTH: IRMA Intact PTH Assay, Nichols Institute Diagnostics PTH(1-84): Whole 1-84 PTH Assay, Scantibodies Lab Inc Non(1-84) = Intact(Nichols) - Whole(Scantibodies) Coen et al Intact PTH: IRMA Intact PTH, Nichols Institute Diagnostics PTH(1-84): Whole (1-84) PTH, Scantibodies Lab Inc. Total (1-84) PTH : (1-84)+non(1-84), Scantibodies Lab Inc. Non(1-84) = Total (1-84) PTH (Scantibodies) - Whole (Scantibodies)

  24. Analytical assays what are we measuring? Clinical studies Does it matter? Is it relevant? Basic scientific research Physiology, signaling of PTH and fragments

  25. Lychee

  26. Methods in Use (CAP, 2004C) No. Labs Methods Signal Capture 2nd generation Bayer ACS:180 15 CLIA (12-34) (39-84) Bayer ADVIA Centaur 171 CLIA (12-34) (39-84) Diasorin 4 IRMA (12-34) (39-84) DPC Immulite 1000 81 CLIA AP(12-34)p (44-84)m DPC Immulite 2000 123 CLIA AP(12-34)p (44-84)m DPC Imulite 1000 Turbo 84 CLIA AP(12-34)p (44-84)m Nichols Advantage 55 CLIA Nichols Chemi 4 CLIA Nichols Isotopic 18 CLIA Nichols Quick-Intraoperative 54 CLIA AE(12-34)p (39-84)p Roches Elecsys/E170 37 ECLIA (26-32)m Ru(55-64)m Roche Elecsys 1010/2010 122 ECLIA (26-32)m Ru(55-64)m 3rd generation Nichols Advantage Bio-intact 38 CLIA AE(1-5)p (57-62)p Nichols Advantage Quick IO Bio-intact 3 CLIA AE(1-5)p (57-62)p Scantibodies 3 IRMA 125I(1-4) (39-84) AP(alkaline phosphatase) AE (acridinium ester) Ru (Ruthenium) m(monoclonal) p(polyclonal)

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