Physiological and clinical studies of calcium-regulating hormones calcitonin and parathyroid hormone-related protein

Sammanfattning: The present studies were performed to elucidate several physiological and clinical questions of calcitonin (CT) and parathyroid hormone-related protein (PTHrP) such as: a) Does age influence the basal and calcium-stimulated levels and circulating molecular forms of CT in healthy females? b) Are osteoporotic patients lacking circulating monomeric CT? c) How is salmon CT (sCT) absorbed and cleared during treatment of diseases? d) Is there any effect of acute physical activity on circulating CT, PTH, PTHrP and bone metabolic markers? e) Is there an absorbance of PTHrP in milk by the neonate? and f) Are there any interactions between CT and PTHrP? In addition, by use of time-resolved dissociation and enhancement lanthanide fluorometry, the effort was put to develop two-site immunofluorometric assays (IFMA) for sCT and PTHrP as well as a quantitative competitive PCR (QC-PCR) for measuring PTHrP mRNA and use these methods in some of the investigations. Heterogeneity of CT forms in serum was disclosed after immunoextraction, fast protein liquid chromatography (FPLC) and radioimmunoassay (RIA) in healthy females and osteoporotic patients. All young women studied had considerable amounts of monomer-like CT whereas several elderly had undetectable or low levels. After calcium stimulation, young women had higher serum concentrations of CT than elderly women whereas the difference between their basal levels was not significant. Three different molecular sizes of immunoreactive CT was found in osteoporotic patients as well as in healthy subjects and none of the studied male and female patients lacked monomer-like CT. After extraction and FPLC slightly higher CT concentrations were found in osteoporotic women than in age-matched healthy women. A two-site IFMA for intact sCT has been developed using the same polyclonal antibodies both for solid phase immobilisation and after being Eu-labelled for signalling and the sensitivity was I . I pmol/L. The assay was improved by employing a biotinylated monoclonal antibody for "capturing" and the above mentioned polyclonal antibodies for signalling, and the detection limit was lowered to 0.3 pmol/L. The preliminary pharmacokinetics of sCT both in osteoporotic patients and in healthy men was studied by use of the IFMA. The results indicate that there are large interindividual variations in absorption and clearance of sCT. The influence of acute endurance and strength exercise on circulating levels of CT, PTH, PTHrP, osteocalcin and carboxyterminal cross-linked telopeptide of type I collagen (ICTP) was evaluated in healthy young males by use of various immunoassays. Strength exercise increased PTH levels but no significant effects on CT or PTHrP levels could be observed. ICTP levels showed more pronounced decrease following physical activity whereas osteocalcin followed the same pattern as the controls except for after prolonged endurance exercise when a decrease was abolished. An increase in PTH after strength exercise and a decrease in ICTP after all exercise together with a relative increase in osteocalcin after prolonged endurance exercise might reflect some mechanisms involved in the positive effect of physical activity on bone mass. A two-site IFMA for PTHrP has been developed by use of a biotinylated rabbit anti-PTHrP 38-67 and a Eu-labelled sheep anti-PTHrP 1-34, with a sensitivity of 0.3 pmol/L. PTHrP levels were determined in the plasma of neonatal, parturient and non-pregnant, non-lactating goats as well as in the goat milk. The circulating PTHrP levels were significantly in creased at day I and day 3 after birth in the male kids bottle-fed with milk from the dams. In the female kids fed with formula there was no such increase. This indicates that PTHrP in milk might be absorbed into the circulation of the neonates. In the parturient goats plasma PTHrP was higher during and after parturition in comparison with before. PTHrP levels in goat milk were in the nanomolar range and were highest in the concentrated colostrum. A QC-PCR assayed by time-resolved lanthanide fluorometry for PTHrP mRNA after reverse transcription has been developed. The effect of sCT on PTHrP gene expression in a breast cancer cell line, MCF-7, was investigated. After incubation with 10-10 to 10-8 moi/L of sCT, both PTHrP mRNA production and PTHrP levels in the conditioned medium were decreased by a dose-dependent pattern. Key Words: bone metabolism, breast cancer, calcitonin, calcium, exercise, lanthanide, milk, neonate, osteoporosis, parathyroid hormone-related protein, parturition, polymerase chain reaction, time-resolved fluorometry ISBN 91-628-2431-7

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