what are we referring to when we state pth level?
TRANSCRIPT
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What Are We Referring to When We
State PTH Level?
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Evolution of PTH assays
Clinical utility of new development
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Synthesized in parathyroid glands
Pre-proPTH (115 amino acids)
ProPTH (90-amino acid)
PTH (84 amino acids)
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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
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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
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First Generation PTH Asssay
Radioimmunoassay (RIA) methods
1 84
Isotope-PTH/fragments
Antibody
PTH
1963-1983
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First Generation PTH Asssay
1 84
1 84H2N COOH PTH
COOH C-PTH
Mid region PTH
M, C Assay
1963-1983
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First Generation PTH Asssay
1 84
1 84H2N COOH PTH
N assay
1963-1983
H2N
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Capture AbSignal Ab
1 84H2N COOH PTH
Intact PTH Assays1st Generation Sandwich Immunometric Assays
1 84
1983-1993
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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
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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
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2nd Generation Sandwich Immunometric Assays
1 84
Capture AbSignal Ab
1 84H2N COOH PTH
Whole PTH
Assay of Whole PTH
1999
4-5
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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
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Localization of the metabolically labeled residues wasdetermined by automated amino-terminal Edman degradation
D’Amour et al., Kidney International 2005,68:998-1007
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Utility in Clinical Diagnosis
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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
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Parathyroid glands
PTH
Osteoclasts
Resorptionbone turnover
Ca++Kidneys
Ca++ reabsorption
Pi excretion
Hydroxylation of 25VD3
7-84PTH
Osteoblasts
PTHCR
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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
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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
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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.
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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
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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
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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)
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Analytical assays
Clinical studies
Basic scientific research
what are we measuring?
Does it matter?Is it relevant?
Physiology, signaling of PTH and fragments
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Lychee
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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)