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)