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A two-site immunochemiluminometric assay (ICMA) for intact (1–84) parathyroid hormone (PTH)
Abstracts from the Bone and Tooth Society Meeting
48
PSEUDOHYPOPARATHYROIDISM TYPE I: DISPARITY BETWEEN bioPTH LEVELS MEASURED BY THE RENAL AND METATARSAL CYTOCHEMICAL BIOASSAYS J. Bradbeer,’ J. Dunham,’ J.A. Fischer,2 C. Nagant de Deuxchaisnes3 and N. Loveridge’ ‘Kennedy Institute of Rheumatology, London, UK, ‘University of Zurich, Zurich, Switzerland, and 3Louvain University, Brussels, Belgium. Patients with pseudohypoparathyroidism type I (PSPI) are resistant to the action of PTH on the kidney. We have postulated that this resistance is due to the presence of a circulating inhibrtor of PTH action. However, there have been reports that in PSPI the skeletal response to circulating PTH may be comparable to that seen in primary hyperparathyroidism. We have compared the levels of bioPTH in PSPI plasma as assayed in the renal and metatarsal cytochemical bioassays. In normal subjects the ratio of bioPTH measured by the metatarsal CBA to that assayed by the renal CBA ranged from 0.4 to 2.4 (mean 1.3, n = 7). In patients with PSPI the levels of bioPTH were within the normal range in the renal CBA (0.5-6.9, mean 1.5 pgiml, n = lo), whereas in the metatarsal CBA the level of bioPTH was higher (1.5-50, mean 20.7 pg/ml) although only four of these were outside the normal range. The ratio of bioPTH between the metatarsal and renal assays was significantly raised in the PSPI patients (mean 33.7, range 0.7-120, P < 0.01). As reported by us and others, there was a dissociation between bioPTH levels measured by the renal CBA and both N and C terminal IPTH. However, significant correlations were found between bioPTH as measured by the metatarsal CBA and the levels of iPTH (N terminal: r = 0.685, P < 0.02; C terminal: r = 0.69, P < 0.02). In conclusion, PTH bioactivity as measured by the metatarsal and renal bioassays is similar in normal subjects. In patients with PSPI, however, broPTH levels as measured by the metatarsal CBA are generally hrgher. This indicates that the inhibitor of PTH bioactivrty IS not as effective against bone, which probably accounts for the spectrum of skeletal responses seen in this disease.
A TWO-SITE IMMUNOCHEMILUMINOMETRIC (ICMA) FOR INTACT (l-84) PARATHYROID (PTH).
ASSAY HORMONE
R.C. Brown, J.P. Aston, I. Weeks, and J.S. Woodhead Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK. lmmunoassays for PTH suffer from problems associated with cross-reactivity, poor sensitivity, overlap between patient groups, long incubations, and reagent preparation. Most current assays measure fragments, and rnterpretation of data may be difficult Measurement of the Intact 1-84 PTH peptide would have the advantage in that circulating concentrations more closely reflect secretory activity of the glandular tissue. We have developed a two-site ICMA specific for the intact peptide. Serum (100 ~1) or standard is incubated with affinity purified sheep anti (l-34 h PTH)-antibody labeled with aryl acridinium ester (4 h). Separation of bound label is achieved by the addition of solid-phase mouse monoclonal anti (44-68 h PTH) antibody. After washing and centrifugation, the bound lumrnescence is counted in an automatic luminometer Luml-
nescence is directly proportional to the amount of PTH present. The working range of the assay is 0.8-200 pmolil 1-84 PTH. No interference has been observed from PTH fragments. Circulating concentrations in normals and patients with surgically proven primary hyperparathyroldism (PHP), chronic renal failure (CRF), malignancy associated hypercalcaemia (MHCA), and hypoparathyroidism (HYPOP) are given in the table. We conclude that this ICMA for intact PTH should be of considerable diagnostic potential in diseased states associated with disorders of extracellular Ca metabolism. PTH (pmoi/l) ”
Mean Range
Normal
PHP
CRF
HYPOP
MHCA
87 3.6
29 24 3
20 29 4
3
8 -
2 4-78
~08
09-79
11 5-51
THE EFFECT OF CONTINUOUS AND PULSED CALCITONIN ON BONE LOSS FOLLOWING IMMOBILISATION IN THE RABBIT S. Caine,’
I. Boyle,’
and 6.F Boyce2
‘Department of Medicine Glasgow Royal Infirmary, G31 2ER, UK.
and *Departmenf of Pathology, 70 Alexandra Parade, Glasgow
The effect of calcitonrn on disuse osteoporosis in the rabbit has been studied. Using adult male Dutch rabbits, the quadriceps and Achilles tendons of the left hind limb were sectioned, and the entire limb was then immobilized in flexion for 4 to 6 months. During this period, the diet was supplemented with either calcium or phosphate and salmon calcitonin (10 IUikg BW) given intramuscularly elther daily or for 4 days every 2 weeks. The intermittent therapy was based on the coherence concept as described by Frost.’ At the time of sacrifice, blood was collected by intracardiac puncture, and both rear limbs were x-rayed and excsed. The calcanei and trbrofibuiae were cleaned and weighed wet, and the tibiofibulae were weighed again after ashing. Histological analysis of the calcanei was carried out after plastrc embedding. Immobilisation resulted in a loss of some 20% in weight from the calcanei and 10% from the tibiofibulae as compared with the bones in the control right limbs. None of the various therapy regimens, including either pulsed or continuous salmon calcitonin, had any effect on this weight loss or on the histologically demonstrated loss of both trabecular and cortical bone in the calcanei. Serum analyses revealed a significant elevation of serum phosphate in those animals given phosphate supplements and a reduced serum 1.25 dihydroxyvttamrn D in animals treated with calcitonin. 1 Frost H M
Metab
Bone
DLS Rei Res 25 317-321.
1980
INCREASED RATES OF SPINAL TRABECULAR LOSS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE
BONE
J. Compston, R. Motley, E.O. Crawley, W.D. Evans, C. Evans, and J. Rhodes Departments of Pathology, Gastroenterology, RadIology,
48
PSEUDOHYPOPARATHYROIDISM TYPE I: DISPARITY BETWEEN bioPTH LEVELS MEASURED BY THE RENAL AND METATARSAL CYTOCHEMICAL BIOASSAYS J. Bradbeer,’ J. Dunham,’ J.A. Fischer,2 C. Nagant de Deuxchaisnes3 and N. Loveridge’ ‘Kennedy Institute of Rheumatology, London, UK, ‘University of Zurich, Zurich, Switzerland, and 3Louvain University, Brussels, Belgium. Patients with pseudohypoparathyroidism type I (PSPI) are resistant to the action of PTH on the kidney. We have postulated that this resistance is due to the presence of a circulating inhibrtor of PTH action. However, there have been reports that in PSPI the skeletal response to circulating PTH may be comparable to that seen in primary hyperparathyroidism. We have compared the levels of bioPTH in PSPI plasma as assayed in the renal and metatarsal cytochemical bioassays. In normal subjects the ratio of bioPTH measured by the metatarsal CBA to that assayed by the renal CBA ranged from 0.4 to 2.4 (mean 1.3, n = 7). In patients with PSPI the levels of bioPTH were within the normal range in the renal CBA (0.5-6.9, mean 1.5 pgiml, n = lo), whereas in the metatarsal CBA the level of bioPTH was higher (1.5-50, mean 20.7 pg/ml) although only four of these were outside the normal range. The ratio of bioPTH between the metatarsal and renal assays was significantly raised in the PSPI patients (mean 33.7, range 0.7-120, P < 0.01). As reported by us and others, there was a dissociation between bioPTH levels measured by the renal CBA and both N and C terminal IPTH. However, significant correlations were found between bioPTH as measured by the metatarsal CBA and the levels of iPTH (N terminal: r = 0.685, P < 0.02; C terminal: r = 0.69, P < 0.02). In conclusion, PTH bioactivity as measured by the metatarsal and renal bioassays is similar in normal subjects. In patients with PSPI, however, broPTH levels as measured by the metatarsal CBA are generally hrgher. This indicates that the inhibitor of PTH bioactivrty IS not as effective against bone, which probably accounts for the spectrum of skeletal responses seen in this disease.
A TWO-SITE IMMUNOCHEMILUMINOMETRIC (ICMA) FOR INTACT (l-84) PARATHYROID (PTH).
ASSAY HORMONE
R.C. Brown, J.P. Aston, I. Weeks, and J.S. Woodhead Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK. lmmunoassays for PTH suffer from problems associated with cross-reactivity, poor sensitivity, overlap between patient groups, long incubations, and reagent preparation. Most current assays measure fragments, and rnterpretation of data may be difficult Measurement of the Intact 1-84 PTH peptide would have the advantage in that circulating concentrations more closely reflect secretory activity of the glandular tissue. We have developed a two-site ICMA specific for the intact peptide. Serum (100 ~1) or standard is incubated with affinity purified sheep anti (l-34 h PTH)-antibody labeled with aryl acridinium ester (4 h). Separation of bound label is achieved by the addition of solid-phase mouse monoclonal anti (44-68 h PTH) antibody. After washing and centrifugation, the bound lumrnescence is counted in an automatic luminometer Luml-
nescence is directly proportional to the amount of PTH present. The working range of the assay is 0.8-200 pmolil 1-84 PTH. No interference has been observed from PTH fragments. Circulating concentrations in normals and patients with surgically proven primary hyperparathyroldism (PHP), chronic renal failure (CRF), malignancy associated hypercalcaemia (MHCA), and hypoparathyroidism (HYPOP) are given in the table. We conclude that this ICMA for intact PTH should be of considerable diagnostic potential in diseased states associated with disorders of extracellular Ca metabolism. PTH (pmoi/l) ”
Mean Range
Normal
PHP
CRF
HYPOP
MHCA
87 3.6
29 24 3
20 29 4
3
8 -
2 4-78
~08
09-79
11 5-51
THE EFFECT OF CONTINUOUS AND PULSED CALCITONIN ON BONE LOSS FOLLOWING IMMOBILISATION IN THE RABBIT S. Caine,’
I. Boyle,’
and 6.F Boyce2
‘Department of Medicine Glasgow Royal Infirmary, G31 2ER, UK.
and *Departmenf of Pathology, 70 Alexandra Parade, Glasgow
The effect of calcitonrn on disuse osteoporosis in the rabbit has been studied. Using adult male Dutch rabbits, the quadriceps and Achilles tendons of the left hind limb were sectioned, and the entire limb was then immobilized in flexion for 4 to 6 months. During this period, the diet was supplemented with either calcium or phosphate and salmon calcitonin (10 IUikg BW) given intramuscularly elther daily or for 4 days every 2 weeks. The intermittent therapy was based on the coherence concept as described by Frost.’ At the time of sacrifice, blood was collected by intracardiac puncture, and both rear limbs were x-rayed and excsed. The calcanei and trbrofibuiae were cleaned and weighed wet, and the tibiofibulae were weighed again after ashing. Histological analysis of the calcanei was carried out after plastrc embedding. Immobilisation resulted in a loss of some 20% in weight from the calcanei and 10% from the tibiofibulae as compared with the bones in the control right limbs. None of the various therapy regimens, including either pulsed or continuous salmon calcitonin, had any effect on this weight loss or on the histologically demonstrated loss of both trabecular and cortical bone in the calcanei. Serum analyses revealed a significant elevation of serum phosphate in those animals given phosphate supplements and a reduced serum 1.25 dihydroxyvttamrn D in animals treated with calcitonin. 1 Frost H M
Metab
Bone
DLS Rei Res 25 317-321.
1980
INCREASED RATES OF SPINAL TRABECULAR LOSS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE
BONE
J. Compston, R. Motley, E.O. Crawley, W.D. Evans, C. Evans, and J. Rhodes Departments of Pathology, Gastroenterology, RadIology,