Transcript Parathyroid hormone

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
PTH
PTH1R
Kidneys
Osteoblasts
Ca++ reabsorption
RANKL
 M-CSF
 IL-6
 Collagenase-3
Collagen I
Osteoclasts
Pi excretion
Hydroxylation of 25VD3
Intestinal absorption
Resorption
bone turnover
Ca++
Proteolysis of 1-84 PTH
1
NH2
NH2
N-PTH
T1/2 (2-5 mins)
Parathyroide glands
liver
kidneys
bone
84
COOH
M-PTH
24-84
28-84
34-84
37-84b
43-84b
COOH
C-PTH
39-68
44-68
First Generation PTH Asssay
Radioimmunoassay (RIA) methods
84
1
PTH
Antibody
Isotope-PTH/fragments
1963-1983
First Generation PTH Asssay
1
84
M, C Assay
1
H2N
84
COOH
COOH
PTH
C-PTH
Mid region PTH
1963-1983
First Generation PTH Asssay
1
84
N assay
1
H2N
H2N
1963-1983
84
COOH
PTH
Intact PTH Assays
1st Generation Sandwich Immunometric Assays
1
84
Signal Ab
1
H2N
1983-1993
Capture Ab
84
COOH
PTH
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.
Intact PTH Assays
1st Generation Sandwich Immunometric Assays
12-34
1
84
Signal Ab
Capture Ab
1
84
H2N
COOH
PTH
COOH
Large C-PTH
non (1-84)-PTH
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
Assay of Whole PTH
2nd Generation Sandwich Immunometric Assays
4-5
1
84
Signal Ab
Capture Ab
1
84
H2N
COOH
Whole PTH
1999
PTH
HPLC Fractionation of PTH forms in Serum and Parathyroid Cell Supernatant
C18 u-Bondspak column (Waters)
Whole PTH assay (1-4)
Intact PTH Assay (12-23)
Acetonitrile in 1g/L trifluoroacetic acid
1.5 ml/min
1.5 ml/fraction for I-PTH checking
D’Amour et al., Kidney International 2005,68:998-1007
Localization of the metabolically labeled residues was
determined by automated amino-terminal Edman degradation
D’Amour et al., Kidney International 2005,68:998-1007
Utility in Clinical Diagnosis
Role of 1-84PTH Measurement in Intraoperative PTH Assay
Primary Hyperparathyroidism
29 patients
Non(1-84)PTH
PTH(1-84)
1-84PTH
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
1-84PTH
Yamashita et al., Annals of surgery 2002, 236(1):105-111.
Parathyroid glands
PTH
7-84PTH
PTHCR
Osteoblasts
Osteoclasts
Kidneys
Ca++ reabsorption
Pi excretion
Hydroxylation of 25VD3
Resorption
bone turnover
Ca++
(1-84)PTH/ Non (1-84) PTH Ratio
“Intact”PTH =(1-84)+non(1-84)
1st Generation Sandwich Immunometric Assays
2nd Generation Sandwich Immunometric Assays
(1-84) PTH
Non(1-84) PTH = “Intact” PTH -(1-84)PTH
(1-84)PTH/ Non (1-84) PTH
Bone turnover
Bone turnover
Measurement of (1-84)PTH/ Non (1-84)PTH Ratio
Low bone turnover
28 chronically dialyzed patients
Non(1-84)PTH
High or normal bone turnover
23 chronically dialyzed patients
Monier-Faugere et al., Kidney International 2001,60:1460-1468.
Receiver-operator characteristics (ROC) curves for the prediction of bone turnover
in chronically dialyzed patients.
A) :
(B)
PTH-(1-84)/non(1-84) PTH fragment
ratio
bone-specific alkaline phosphatase
PTH-(1-84)
osteocalcin
“intact”PTH
Monier-Faugere et al., Kidney International 2001,60:1460-1468.
Areas under ROC curve
P<0.05
P<0.05
BSAP: bone-specific alkaline phosphatase
OC: osteocalcin
Monier-Faugere et al., Kidney International 2001,60:1460-1468.
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.
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:
PTH(1-84):
IRMA Intact PTH, Nichols Institute Diagnostics
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
what are we measuring?
Clinical studies
Does it matter?
Is it relevant?
Basic scientific research
Physiology, signaling of PTH and fragments
Lychee
Methods in Use (CAP, 2004C)
No. Labs Methods
Signal
Capture
15
171
4
81
123
84
55
4
18
54
37
122
CLIA
CLIA
IRMA
CLIA
CLIA
CLIA
CLIA
CLIA
CLIA
CLIA
ECLIA
ECLIA
(12-34)
(12-34)
(12-34)
AP(12-34)p
AP(12-34)p
AP(12-34)p
(39-84)
(39-84)
(39-84)
(44-84)m
(44-84)m
(44-84)m
38
3
3
CLIA
CLIA
IRMA
AE(1-5)p
AE(1-5)p
125I (1-4)
2nd generation
Bayer ACS:180
Bayer ADVIA Centaur
Diasorin
DPC Immulite 1000
DPC Immulite 2000
DPC Imulite 1000 Turbo
Nichols Advantage
Nichols Chemi
Nichols Isotopic
Nichols Quick-Intraoperative
Roches Elecsys/E170
Roche Elecsys 1010/2010
AE(12-34)p
(39-84)p
(26-32)m
Ru(55-64)m
(26-32)m
Ru(55-64)m
3rd generation
Nichols Advantage Bio-intact
Nichols Advantage Quick IO Bio-intact
Scantibodies
AP(alkaline phosphatase) AE (acridinium ester) Ru (Ruthenium)
m(monoclonal) p(polyclonal)
(57-62)p
(57-62)p
(39-84)