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Biomedical subjects

W A Ratcliffe

Publications and source records attributed to W A Ratcliffe.

At least 37 records · Page 2Linked to original sources
PubMed1994

Parathyroid hormone and parathyroid hormone-related protein in the investigation of hypercalcaemia in two hospital populations.

BACKGROUND AND OBJECTIVE: Parathyroid hormone-related protein (PTHrP) produced by cancers has a central role as a humoral mediator of hypercalcaemia in patients with malignancy. Since the prevalences of hypercalcaemia of malignancy and parathyroid disease reflect the population studied, hypercalcaemic patients from a district general hospital (DGH) and an oncology centre (OC) were studied in order to assess the diagnostic role of assays for serum PTH and plasma PTHrP in different clinical settings. DESIGN: A prospective study of consecutive patients presenting with their first episode of hypercalcaemia during an 18-month period. PATIENTS: A total of 123 patients (DGH, n = 69; OC, n = 54) had corrected serum calcium concentrations > 2.65 mmol/l. MEASUREMENTS: PTH, PTHrP, calcium and albumin were measured together with urine calcium and creatinine, enabling fractional calcium excretion to be assessed. Urine cyclic adenosine monophosphate was also measured. RESULTS: Hypercalcaemia was attributed to malignancy alone in 72 patients (DGH, n = 20; OC, n = 52), benign causes in 42 (DGH, n = 42) and parathyroid disease coexisting with malignancy in 9 (DGH, n = 7; OC, n = 2). Plasma PTHrP levels were increased in 59/72 (82%) patients with hypercalcaemia due to malignancy. Measurements of both analytes contributed to a change in the final diagnosis in 12 patients (10%). Thus serum PTH was increased in seven patients with parathyroid disease coexisting with malignancy, and plasma PTHrP was increased in five patients with previous undiagnosed malignancy. Median survival for patients with parathyroid disease and coexisting malignancy was 13 months compared with 3 months for those with hypercalcaemia due to malignancy alone (P < 0.02). CONCLUSIONS: Since hypercalcaemia was attributable to parathyroid disease in 10% of all patients with malignancy we advise measurement of serum PTH at the initial presentation of hypercalcaemia in all patients, while plasma PTHrP may be useful to identify those patients in whom malignancy may not be clinically apparent, or coexist with primary hyperparathyroidism.

Calcium↗
PubMed1994

Expression and processing of parathyroid hormone-related protein in a pancreatic endocrine cell tumour associated with hypercalcaemia.

We describe a patient with a neuroendocrine tumour of the pancreas associated with hypercalcaemia which was attributed to production of parathyroid hormone-related protein (PTHrP) by the tumour. Plasma PTHrP 1-86 was significantly raised, and fell following surgical resection of the tumour. PTHrP mRNA and peptide were identified in tumour tissue by in-situ hybridization and immunohistochemistry respectively. PTHrP was quantitated in an extract of tumour tissue by three region-specific immunoassays (PTHrP 1-34 45.2 pmol/g, PTHrP 37-67 81.7 pmol/g, PTHrP 1-86 27.3 pmol/g) and suggested the presence of excess of amino-terminal and mid-region immunoreactivity. On chromatography of the tumour extract the first peak eluted as 22 kDa and comprised approximately equimolar 1-34, 37-67 and 1-86 activities. The second and major peak of 16 kDa contained only 37-67 activity, while the third peak of 6 kDa contained only 1-34 activity. This suggested that the tumour contained a native or intact form of PTHrP together with two major subfragments containing 37-67 and 1-34 activity respectively. Thus chromatographic separation and quantitation of PTHrP by region-specific immunoassays have provided new information on in-vivo proteolytic processing by tumour tissue by indicating that a site of cleavage is located between residues 17 and 61. Our findings are compatible with cleavage at residue 37, a site previously indicated from in-vitro studies.

Adult↗
PubMed1994

The role of parathyroid hormone-related protein in calcium homeostasis in the fetal pig.

The concentrations of plasma parathyroid hormone-like bioactivity and parathyroid hormone-related protein (1-86) (PTHrP) immunoreactivity were both higher in fetal pigs than in their mothers during the last 3 weeks of gestation. Both activities changed inversely with alterations in the plasma ionized calcium concentration. The data suggest that PTHrP may have a role in calcium homeostasis in the fetal pig, similar to its postulated role in sheep in the stimulation of calcium transport across the placenta.

Animals↗
PubMed1994

In vitro stability of endogenous parathyroid hormone-related protein in blood and plasma.

We describe a systematic comparison of the effects of anticoagulants, protease inhibitors and conditions of sample handling on the in vitro stability of endogenous parathyroid hormone-related protein (PTHrP) in blood from patients with hypercalcaemia of malignancy (HM). When blood was separated within 15 min of collection, PTHrP1-86 levels measured by two-site immunoradiometric assay in serum and heparinized plasma were significantly lower than in ethylenediaminetetraacetic acid (EDTA) plasma (P < 0.02). PTHrP was unstable in blood kept at 20 degrees C for 4 h and inclusion of protease inhibitors reduced, but failed to abolish, this instability. In blood collected in the presence of EDTA, inclusion of leupeptin either alone or in combination with pepstatin and aprotinin increased the mean half-time of disappearance from 3.9 to 10.1 and 11.2 h, respectively (P < 0.05). In contrast, when blood containing EDTA was separated within 15 min, PTHrP was stable in plasma at 20 degrees C for at least 4 h. As a result of the instability of PTHrP1-86 immunoreactivity in whole blood at ambient temperatures we advise that for our immunoradiometric assay (IRMA) blood collected in EDTA should be separated within 15 min, and the plasma frozen until assay.

Anticoagulants↗
PubMed1994

Parathyroid hormone-related protein in human term placenta and membranes.

Parathyroid hormone-related protein (PTHrP), the hypercalcaemia of malignancy factor, is expressed in the tissues of the human uteroplacental unit, including the placenta, amnion and chorion. We have used three region-specific immunoassays to quantitate and compare the distribution of PTHrP in tissues obtained at term following spontaneous labour and vaginal delivery or elective Caesarean section. In non-labouring women highest PTHrP(1-86) and (37-67) immunoreactivity was found in amnion covering the placenta, rather than the decidua parietalis of the uterus (reflected amnion) (median 1020 vs 451 fmol/g; 2181 vs 1444 fmol/g respectively). In labouring women, the PTHrP(1-86) concentration in reflected amnion was inversely correlated with the interval between rupture of the membranes and delivery. Tissue PTHrP(1-86) concentrations were lower in placenta than in chorion and amnion (medians 12, 109 and 664 fmol/g respectively) and, in all tissues, PTHrP(1-34) and (37-67) concentrations were significantly higher than that of PTHrP(1-86). Bioactive PTHrP(1-34) was detected in placenta, chorion and amnion using the ROS cell bioassay. The PTHrP(1-86) concentration (mean +/- S.E.M. = 41.4 +/- 4.5 pmol/l) was high in amniotic fluid at term, although in maternal and cord plasma levels were only modestly increased. The molecular forms of PTHrP present in tissues and amniotic fluid were investigated by column chromatography which confirmed its molecular heterogeneity and suggested that processing is tissue-specific and occurs at both amino- and carboxy-terminals of the peptide.

Amnion↗
PubMed1993

Parathyroid hormone-related protein (PTHrP) gene expression in solid tumours associated with normocalcaemia and hypercalcaemia.

Parathyroid hormone-related protein (PTHrP) is an important humoral factor in the syndrome of humoral hypercalcaemia of malignancy (HHM) and its importance is evident by the many studies examining either PTHrP mRNA expression, intracellular peptide, or circulating PTHrP levels in patients with malignancy. However, the relationship between PTHrP mRNA expression, intracellular localization of peptide, and circulating PTHrP levels in the same group of patients with malignancy has not been examined. This study was carried out to explore this relationship in a group of patients with solid tumours associated with either normocalcaemia or hypercalcaemia. PTHrP mRNA and peptide were localized by in situ hybridization and immunohistochemistry respectively in 26 squamous carcinomas and 15 adenocarcinomas from patients who were either hypercalcaemic or normocalcaemic. Plasma PTHrP1-86 and serum PTH1-84 concentrations were measured by two-site immunoradiometric assays. PTHrP mRNA and peptide were localized in 11 (100 per cent) and 10 (91 per cent) of 11 squamous tumours from hypercalcaemic patients, all of whom had detectable circulating PTHrP levels, and in 14 (97 per cent) and 11 (73 per cent) respectively of 15 squamous tumours from normocalcaemic patients. PTHrP mRNA and peptide were localized in only two (28 per cent) and four (57 per cent) respectively of seven adenocarcinomas associated with hypercalcaemia. Since the majority of squamous tumours synthesized PTHrP irrespective of the calcium status of the patient, this suggests that the clinical expression of tumour-derived PTH-like bioactivity may depend on the rate of secretion of PTHrP rather than gene expression, and that the bioactivity of secreted PTHrP may be modulated by post-translational processing.

Adenocarcinoma↗
PubMed1993

Molecular forms of parathyroid hormone-related protein in tumours and biological fluids.

OBJECTIVE: We investigated the content and compared the molecular forms of parathyroid hormone-related protein in tumour tissue, plasma and pleural fluid. DESIGN: Measurement of parathyroid hormone-related protein in tumour extracts and biological fluids and comparison of the elution profiles of parathyroid hormone-related protein (PTHRP) immunoreactivity following gel filtration chromatography on Bio-gel P100. PATIENTS: Tumours and plasma from patients with humoral hypercalcaemia of malignancy were studied, together with tumours and pleural fluids from patients who were normocalcaemic. MEASUREMENTS: Immunoreactivity in column fractions, plasma and tumour extracts was measured by a highly sensitive immunoradiometric assay for PTHRP 1-86 with specificity directed at the 17-61 region of PTHRP. RESULTS: Similar levels of PTHRP immunoreactivity were measured in tumours from normocalcaemic and hyper-calcaemic patients. PTHRP 1-86 (28-4630 fmol/g) was detected in eight of the nine tumours studied. Immunoreactivity in tumour extracts eluted as major peaks in the range 22-33 kDa with an additional peak of 15 kDa in three out of six tumours studied. In contrast, immunoreactivity in plasma and pleural fluid eluted within the range 7-14 kDa. CONCLUSIONS: The major species of parathyroid hormone-related protein in plasma and pleural fluids was consistently smaller than that in tumour tissue (22-33 kDa) suggesting that tumour-derived parathyroid hormone-related protein is processed at the COOH-terminus to form a species of approximately 10 kDa which circulates in patients with humoral hypercalcaemia of malignancy.

Adult↗
PubMed1993

Humoral hypercalcaemia in renal carcinoma due to parathyroid hormone related protein.

Parathyroid hormone related protein (PTHRP) has been implicated in humoral hypercalcaemia of malignancy (HHM). We describe a patient with a renal carcinoma and hypercalcaemia, and for the first time have demonstrated a 4-fold PTHRP concentration gradient across a renal tumour bed. This provides further strong evidence that the primary tumour was the source of the PTHRP. The PTHRP 1-86 content of the tumour tissue was 4.8 ng/g, similar to that previously found in tumours associated with HHM. In PTHRP-secreting tumours, PTHRP may be a useful oncological marker after surgical resection.

Biomarkers, Tumor↗
PubMed1993

Malignant phaeochromocytoma and hypercalcaemia.

We describe a case of hypercalcaemia secondary to recurrent malignant phaeochromocytoma. Parathyroid-related protein (PTHrp 1-86) immunoreactivity was identified in plasma and PTHrp was identified by immunocytochemistry in tumour tissue.

Adrenal Gland Neoplasms↗
PubMed1993

Parathyroid hormone-related protein as a tumour marker in humoral hypercalcaemia associated with occult malignancy.

The tumour-derived factor PTH-related protein (PTHRP) is the primary humoral factor responsible for hypercalcaemia in patients with solid tumours. In a woman presenting with anaemia and hypercalcaemia, the finding of raised plasma PTHRP and undetectable serum PTH concentrations led to further investigations and the subsequent identification of a uterine tumour. No evidence of tumour spread was found at operation, and removal of the tumour resulted in normalization of both serum calcium and plasma PTHRP. Expression of PTHRP by the tumour was shown by immunohistochemistry and in situ hybridization. We conclude that the identification of an occult tumour in a patient with hypercalcaemia and raised plasma PTHRP provides evidence of the diagnostic utility of PTHRP immunoassays in the investigation of patients with hypercalcaemia and suspected malignancy.

Biomarkers, Tumor↗
PubMed1993

Clearance of exogenous parathyroid hormone-related protein in pregnant, non-pregnant and fetal sheep, goats and pigs.

In the sheep, goat and pig, radiolabelled parathyroid hormone related protein (PTHRP) and immunoreactive PTHRP(1-34) and (1-86) were rapidly cleared from the circulation. Metabolic clearance rates (MCR) were in the range of 1.25-7.5 ml/min per kg and were slightly slower than that of intact PTH in man (10 ml/min per kg); while the mean MCR of labelled PTHRP(1-86) in fetal sheep and goats was significantly faster than that in their respective mothers (14.4 vs 4.0 ml/min per kg respectively). This may reflect increased metabolism of PTHRP by fetal tissues, e.g. the placenta. Similar rates of clearance of radiolabelled PTHRP(1-141), (1-86) and (1-34) suggest that clearance involves the amino terminus of the molecule.

Animals↗
PubMed1992

Immunochemical characterisation of parathyroid hormone-related protein from tumour and non-tumour cells.

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtration chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1-86 and 1-34) and bioactivity (1-34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19-22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10-16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1-34 or 37-67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24-25 kDa, consistently slightly larger than recombinant PTHRP(1-141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.

Animals↗
PubMed1992

Production and characterisation of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein.

The production and characterisation of monoclonal antibodies (MAb) to the mid-region sequence 37-67 of human parathyroid hormone-related protein (PTHRP) is described. In spite of the poor immunogenicity of this sub-fragment of PTHRP, a high percentage of specific hybrids were produced by boosting with conjugate and free peptide prior to cell fusion. Seven of the MAbs produced cross-reacted with PTHRP37-67, PTHRP1-86 and native forms of PTHRP. Inhibition studies with peptide sub-fragments of PTHRP37-67 indicated that the majority recognised the 45-59 region. In a RIA for PTHRP1-86, detection limits ranged from 0.17 to 0.9 ng PTHRP1-86/tube, and no cross-reaction was found with PTH1-84. Two MAbs 1D11 and 4B10 were shown to be of potential use in measuring PTHRP1-86 in a two-site immunoradiometric assay in combination with either a solid phase consisting of a MAb to PTHRP1-34, or iodinated affinity purified rabbit antibodies to PTHRP1-34. MAb 1D11 coupled to Sepharose was suitable for immunoextraction of PTHRP, and successfully localised PTHRP on immunoblots. Two additional MAbs were produced which recognised an epitope unique to PTHRP37-67 located in the 37-46 region of the peptide.

Animals↗
PubMed1992

Role of assays for parathyroid-hormone-related protein in investigation of hypercalcaemia.

Parathyroid-hormone-related protein (PTHrP) has been implicated as a humoral mediator of hypercalcaemia in malignant disease. We have investigated the contributions of PTHrP and parathyroid hormone (PTH) to the hypercalcaemia seen in routine clinical practice by means of highly sensitive immunoradiometric assays. PTHrP concentrations in plasma and PTH concentrations in serum were measured in 121 consecutive patients with hypercalcaemia (corrected serum calcium above 2.65 mmol/l) identified from routine biochemical profiles in a district general hospital. Hypercalcaemia was due to primary hyperparathyroidism in 63 (52%) patients and to malignant disease in 40 (49%). Plasma PTHrP was detectable in 35 (88%) of 40 patients with solid tumours and 3 of 9 patients with haematological malignant disease; it was undetectable in 92% of patients with primary hyperparathyroidism. 7 patients with malignant disease had PTH concentrations above 4.0 pmol/l, consistent with coexisting primary hyperparathyroidism. Measurement of both PTH and PTHrP in all patients led to a change in the diagnosis in 7% of patients. This study provides direct evidence for a humoral role of tumour-derived PTHrP in hypercalcaemia, and shows how PTHrP assays can be used appropriately, in conjunction with PTH assays, to investigate hypercalcaemia in routine clinical practice.

Biomarkers↗
PubMed1992

Parathyroid hormone related protein and skeletal morbidity in breast cancer.

The presence of parathyroid hormone related protein (PTHRP) in human breast cancers has been assessed by immunohistochemistry using a polyclonal antiserum specific for the mid-region sequence 37-67 in an immunoperoxidase technique. The primary tumours from 155 normocalcaemic, consecutive women with early breast cancer who had been followed up for a minimum of 5 years were assessed. Dewaxed paraffin sections of formalin fixed tissue was used throughout. Positive PTHRP staining was detected in 56% of the cancers and was unrelated to standard prognostic factors, recurrence or survival. However, PTHRP positivity was related to the development of bone metastases (P less than or equal to 0.03) and hypercalcaemic episodes. PTHRP is implicated as the humoral factor responsible for hypercalcaemia associated with breast cancer and tumour positivity may be a useful predictor of which women will develop bone metastases.

Aged↗
PubMed1992

Parathyroid hormone related protein in hypercalcaemia of Hodgkin's disease.

The role of parathyroid hormone related protein (PTHRP) as a humoral mediator of hypercalcaemia was investigated in a patient with lymphocyte depleted Hodgkin's disease during an episode of hypercalcaemia, using an immunohistochemical staining technique for PTHRP on the tumour tissue and an immunoradiometric (IRMA) assay for PTHRP1-86 on the patient's plasma. The plasma PTHRP was less than 0.23 pmol/l in the range found in normocalcaemic controls, and the immunohistochemical staining was not positive for protein. PTHRP did not have a role in the pathogenesis of hypercalcaemia in this patient.

Aged↗