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

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

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

7. First Generation PTH Asssay 184 1 H2NCOOH PTH COOH C-PTH Mid region PTH M, C Assay 1963-1983

8. First Generation PTH Asssay 184 1 H2NCOOH PTH N assay 1963-1983 H2N

9. Capture AbSignal Ab 184 H2NCOOH PTH Intact PTH Assays 1st Generation Sandwich Immunometric Assays 184 1983-1993

10. Lepage R et al. Clin Chem 1998:44:805-9. Nichols …… Incstar DSL 7-84 non(1-84) 1-84 Retention Time (min) 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 HPLC Fractionation of PTH forms in Serum

11. Capture AbSignal Ab 184 H2NCOOH PTH COOH Large C-PTH non (1-84)-PTH Total PTH Intact PTH Assays 1st Generation Sandwich Immunometric Assays 184 1983-1993 Further studies: non-(1-84) PTH = 10-20% of I-PTH in healthy, 40-60% of I-PTH in patients with terminal renal disease. 12-34

12. 2nd Generation Sandwich Immunometric Assays 184 Capture Ab Signal Ab 184 H2NCOOH PTH Whole PTH Assay of Whole PTH 1999 4-5

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

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

15. Utility in Clinical Diagnosis

16. Primary Hyperparathyroidism Secondary Hyperparathyroidism 1-84PTH Role of 1-84PTH Measurement in Intraoperative PTH Assay Yamashita et al., Annals of surgery 2002, 236(1):105-111. 29 patients 7 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

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

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

19. 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. Measurement of (1-84)PTH/ Non (1-84)PTH Ratio

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

21. BSAP: bone-specific alkaline phosphatase OC: osteocalcin Areas under ROC curve P<0.05

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

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

25. Lychee

26. No. LabsMethods Signal Capture 2nd generation Bayer ACS:18015CLIA (12-34) (39-84) Bayer ADVIA Centaur171CLIA (12-34) (39-84) Diasorin4IRMA (12-34) (39-84) DPC Immulite 100081CLIA AP(12-34)p (44-84)m DPC Immulite 2000123CLIA AP(12-34)p (44-84)m DPC Imulite 1000 Turbo84CLIA AP(12-34)p (44-84)m Nichols Advantage55CLIA Nichols Chemi4CLIA Nichols Isotopic18CLIA Nichols Quick-Intraoperative54CLIA AE(12-34)p (39-84)p Roches Elecsys/E17037ECLIA (26-32)m Ru(55-64)m Roche Elecsys 1010/2010122ECLIA (26-32)m Ru(55-64)m 3rd generation Nichols Advantage Bio-intact38 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 125 I (1-4) (39-84) Methods in Use (CAP, 2004C) AP(alkaline phosphatase) AE (acridinium ester) Ru (Ruthenium) m(monoclonal) p(polyclonal)