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)

PTH

Osteoclasts

Resorptionbone turnover

Ca++

Kidneys

Ca++ reabsorption

Pi excretion

Hydroxylation of 25VD3

Osteoblasts

PTH1R

RANKL M-CSF IL-6

Parathyroid glandsCaSR

Collagenase-3Collagen I

Intestinal absorption

Proteolysis of 1-84 PTH

1

84

NH2

COOH

39-6844-68

NH2

COOH

24-8428-8434-8437-84b43-84b

C-PTH

N-PTH

M-PTHT1/2 (2-5 mins)

Parathyroide glandsliverkidneysbone

First Generation PTH Asssay

Radioimmunoassay (RIA) methods

1 84

Isotope-PTH/fragments

Antibody

PTH

1963-1983

First Generation PTH Asssay

1 84

1 84H2N COOH PTH

COOH C-PTH

Mid region PTH

M, C Assay

1963-1983

First Generation PTH Asssay

1 84

1 84H2N COOH PTH

N assay

1963-1983

H2N

Capture AbSignal Ab

1 84H2N COOH PTH

Intact PTH Assays1st Generation Sandwich Immunometric Assays

1 84

1983-1993

Lepage R et al. Clin Chem 1998:44:805-9.

Nichols

…… Incstar

DSL

7-84non(1-84) 1-84

Retention Time (min)

Pooled uremic serum samples

C18 u-Bondspak column (Waters)

Acetonitrile in 1g/L trifluoroacetic acid1.5 ml/min

1.5 ml/fraction for I-PTH checking

HPLC Fractionation of PTH forms in Serum

Capture AbSignal Ab

1 84H2N COOH PTH

COOH Large C-PTHnon (1-84)-PTH

Total PTH

Intact PTH Assays1st Generation Sandwich Immunometric Assays

1 84

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

2nd Generation Sandwich Immunometric Assays

1 84

Capture AbSignal Ab

1 84H2N COOH PTH

Whole PTH

Assay of Whole PTH

1999

4-5

Whole PTH assay (1-4)

Intact PTH Assay (12-23)

C18 u-Bondspak column (Waters)

Acetonitrile in 1g/L trifluoroacetic acid1.5 ml/min

1.5 ml/fraction for I-PTH checking

HPLC Fractionation of PTH forms in Serum and Parathyroid Cell Supernatant

D’Amour et al., Kidney International 2005,68:998-1007

Localization of the metabolically labeled residues wasdetermined by automated amino-terminal Edman degradation

D’Amour et al., Kidney International 2005,68:998-1007

Utility in Clinical Diagnosis

Primary Hyperparathyroidism

Secondary Hyperparathyroidism

1-84PTH

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

Non(1-84)PTH

PTH(1-84) Whole 1-84 PTH (1-4)Scantibodies Lab Inc.

Intact PTHIRMA intact iPTHNichols Institute Diagnostics

Parathyroid glands

PTH

Osteoclasts

Resorptionbone turnover

Ca++Kidneys

Ca++ reabsorption

Pi excretion

Hydroxylation of 25VD3

7-84PTH

Osteoblasts

PTHCR

Non(1-84) PTH = “Intact” PTH -(1-84)PTH

“Intact”PTH =(1-84)+non(1-84)1st Generation Sandwich Immunometric Assays

(1-84) PTH2nd Generation Sandwich Immunometric Assays

(1-84)PTH/ Non (1-84) PTH

Bone turnover Bone turnover

(1-84)PTH/ Non (1-84) PTH Ratio

Low bone turnover28 chronically dialyzed patients

High or normal bone turnover23 chronically dialyzed patients

Non(1-84)PTH

PT

H(1

-84)

/non

(1-8

4) P

TH

Monier-Faugere et al., Kidney International 2001,60:1460-1468.

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

A) :

PTH-(1-84)/non(1-84) PTH fragment ratio

PTH-(1-84)

“intact”PTH

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.

Monier-Faugere et al., Kidney International 2001,60:1460-1468.

BSAP: bone-specific alkaline phosphatase

OC: osteocalcin

Areas under ROC curve

P<0.05

P<0.05

Low bone turnover9 chronically dialyzed patients

High or normal bone turnover26 chronically dialyzed patients

Coen et al., American Journal of Kidney Diseases 2002,40:348-354.

Area under the ROC curve“intact”PTH 0.859PTH(1-84) 0.842(1-84)/non(1-84) 0.538 P<0.01

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)

Analytical assays

Clinical studies

Basic scientific research

what are we measuring?

Does it matter?Is it relevant?

Physiology, signaling of PTH and fragments

Lychee

No. Labs Methods Signal Capture2nd generationBayer 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)mDPC Immulite 2000 123 CLIA AP(12-34)p (44-84)mDPC Imulite 1000 Turbo 84 CLIA AP(12-34)p (44-84)mNichols Advantage 55 CLIANichols Chemi 4 CLIANichols Isotopic 18 CLIANichols Quick-Intraoperative 54 CLIA AE(12-34)p (39-84)pRoches Elecsys/E170 37 ECLIA (26-32)m Ru(55-64)mRoche Elecsys 1010/2010 122 ECLIA (26-32)m Ru(55-64)m

3rd generationNichols Advantage Bio-intact 38 CLIA AE(1-5)p (57-62)pNichols Advantage Quick IO Bio-intact 3 CLIA AE(1-5)p (57-62)pScantibodies 3 IRMA 125I (1-4) (39-84)

Methods in Use (CAP, 2004C)

AP(alkaline phosphatase) AE (acridinium ester) Ru (Ruthenium)m(monoclonal) p(polyclonal)