A sensitive method for the assay of calcitonin is described with a much lower detection limit than that reported hitherto. Calcitonin was injected intravenously into fasting Wistar rats and aortic blood was collected 30 min. later under anaesthesia. Equivalent hypocalcaemia was produced in 3-week-old rats by doses less than a third of those required in 5-week-old ones. Less than 0·1 m-u. calcitonin/3-week-old rat could be measured with an index of precision of about 0·2.
Thyroid hormone (TH) deficiency results in delayed proliferation and migration of cerebellar granule cells. Although extensive cell loss during the development of the cerebellum under hypothyroid conditions is known, its nature and its mechanism are poorly understood. Bcl-2 family gene expression is known to determine the fate of cells to undergo apoptosis. We evaluated the effect of hypothyroidism on Bcl-2 family gene expression in the developing rat cerebellum. Electrophoresis and Western blotting were used to analyze DNA fragmentation and expression of DNA fragmentation factor (DFF-45), Bcl-2, Bcl-xL and Bax genes respectively. In the hypothyroid condition, extensive DNA fragmentation and enhanced cleavage of DFF-45 were seen throughout development (postnatal day 0 to day 24) and adulthood whereas they were absent in the euthyroid state. The anti-apoptotic genes Bcl-2 and Bcl-xL were down-regulated and the pro-apoptotic gene Bax was expressed at higher levels compared with the euthyroid state. These results suggest that normal levels of TH prevent cerebellar apoptosis to a large extent, whereas hypothyroidism not only increases the extent but also the duration of apoptosis by down-regulating the anti-apoptotic genes and maintaining a high level of the pro-apoptotic gene Bax.
(1) Calcitonin preparations from acetone-dried thyroid were administered to rats by various routes.
(2) Intravenous administration, especially by infusion, produced a much greater fall in plasma calcium than s.c. or i.p. injection.
(3) The log dose-effect curves after i.v. injection or infusion showed no evidence of non-linearity over a 100-fold dose range and had highly significant slopes.
(4) The potency ratio of two preparations was estimated by means of a (2+2) assay design using both i.v. infusion and single i.v. injection. There was satisfactory agreement.
(5) The i.v. injection method is recommended for the routine assay of calcitonin. A simple assay schedule is given in the Appendix.
(6) A unit of calcitonin activity is defined in terms of a standard preparation.
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