Calcium‐binding protein synthesis on chick intestinal polysomes is induced within 2 h of injecting vitamin‐D‐deficient birds with 1,25‐dihydroxycholecalciferol. The induction is short‐lived: the maximum output of the binding protein is reached by 13 h after hormone injection, and declines rapidly thereafter. This induction of calcium‐binding protein synthesis occurs by the production of active mRNA for the protein. The sedimentation coefficient of this mRNA in denaturing conditions is 18 S, equivalent to a molecular weight of approximately 700000, and the molecule contains a tract of polyadenylate. Both polysomal and poly(A)‐containing RNA extracted from intestinal polysomes stimulate the synthesis of a range of proteins (up to 70000 molecular weight) by the wheat germ cell‐free system. Immunoprecipitable calcium‐binding protein is translated from RNA obtained from 1,25‐dihydroxycholecalciferol‐dosed birds but not from control birds. This calcium‐binding protein is the same size (27000 molecular weight) as authentic chick calcium‐binding protein. No other proteins are specifically precipitated by the antiserum. Thus in chickens 1,25‐dihydroxycholecalciferol‐induced calcium‐binding protein is not synthesised via any precursor molecule. The implications of this result are discussed.

911 Comment Increases in osteoid volume with sodium fluoride, calcium, and vitamin D treatment are well documented. Small decreases in mineralisation rate have also been reported.2 But osteomalacia in the presence of high plasma 25-OHD concentrations has not been described in patients treated with this regimen. The plasma 1,25-(OH)2D3 concentration in our patient was just below the lower limit of normal, but the total plasma 1,25-(OH)2D (1,25-(OH)2D2-+1,25-(OH)2D3) concentration was probably normal since she was taking vitamin D2 and the radioimmunoassay did not measure plasma 1,25-(OH)2D2. The mechanisms by which fluoride may produce osteomalacia despite high plasma 25-OHD concentrations require further investigation. Possibilities include fluoride-induced end-organ resistance in bone to active vitamin D metabolites or an effect of fluoride on processes of bone mineralisation that are unaffected by vitamin D metabolites. Alternatively, fluoride might affect the metabolism of 25-OHD to other metabolites. Although lack of calcium supplements was probably unimportant in our patient, since the plasma calcium concentration remained above 2 40 mmol,l throughout treatment and dietary calcium intake was adequate, we cannot exclude it as a factor in the development of osteomalacia. Our results indicate that vitamin D in doses that produce high plasma 25-OHD concentrations does not protect against fluoride-induced mineralisation defects and that patients treated with this regimen require careful supervision. Trans-iliac biopsy provides a sensitive method for diagnosing generalised bone disease such as osteomalacia and may be necessary to detect its development when, as in our patient, plasma biochemical changes are not diagnostic. We thank J Sainsbury Ltd and the Special Trustees, St Thomas's Hospital, for generous financial support, and Dr T L Clemens, the Middlesex Hospital, London, WI, for 1,25-(OH)2D3 assays. Jowsey J, Riggs BL, Kelly PJ, Hoffman DL. Effect of combined therapy with sodium fluoride, vitamin D and calcium in osteoporosis. AmJ Med 1972 ;53 :43-9. 2 Meunier PJ, Bressot C, Vignon E, et al. Radiological and histological evolution of post-menopausal osteoporosis treated with sodium fluoride, vitamin D, and calcium. Preliminary results. In: Courvoisier B, Donath A, Baud CA, eds. Fluoride and bone. Berne: Hans Huber, 1978:263-76. 3Cass RM, Croft JD, Perkins P, Nye W, Waterhouse C, Terry R. New bone formation in osteoporosis following treatment with sodium fluoride.

1. The rapid stimulation of intestinal Ca(2+) transport observed in vitamin D-deficient chicks after receiving 1,25-dihydroxycholecalciferol has necessitated a re-evaluation of the correlation hitherto observed between this stimulation and the induction of calcium-binding protein synthesis. By 1h after a dose of 125ng of 1,25-dihydroxycholecalciferol, Ca(2+) transport is increased. This is at least 2h before calcium-binding protein can be detected immunologically and 1h before synthesis of the protein begins on polyribosomes, and thus the hormone stimulates Ca(2+) transport before calcium-binding-protein biosynthesis is induced. 2. The maximum increase in Ca(2+) transport observed after this dose of 1,25-dihydroxycholecalciferol (attained by 8h) is similar to that observed after 1.25-25mug of cholecalciferol, but the stimulation is only short-lived, in contrast with the effect observed after the vitamin. At later times after the hormone, however, when Ca(2+) transport has declined to its basal rate, the cellular content of calcium-binding protein remains elevated. 3. Calcium-binding protein is synthesized on free rather than membrane-bound polyribosomes, which implies that it is an intracellular protein. 4. Rachitic chicks require the presence of dietary calcium for maximum stimulation of calcium-binding protein production by cholecalciferol. 5. These results suggest that calcium-binding protein is an intracellular protein, and that its synthesis may be a consequence of the raised intracellular calcium content of the intestinal epithelial cells resulting from 1,25-dihydroxycholecalciferol-stimulated Ca(2+) transport. We propose that calcium-binding-protein synthesis is necessary for maintaining the stimulated rate of Ca(2+) transport, which is initiated by other factors.

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Stimulation of intestinal calcium transport by the hormone 1,25-dihydroxycholecalciferol appears to involve RNA transcriptions and the synthesis of new proteins. Although one of these proteins has been identified as calcium-binding protein, no RNA molecules specifically induced by the hormone in the nucleus have been identified. Nuclear RNA from intestine of vitamin D-deficient chicks before and at various time intervals after treatment with the hormone or cholecalciferol was tested for its ability to code for calcium-binding protein in a cell-free system. Calcium-binding-protein mRNA could only just be detected in the intestinal nuclei 2h after dosing with these steroids which is the same time that it was first observed in the polyribosomes. Thus 1,25-dihydroxycholecalciferol induces the production of new calcium-binding protein by stimulating the formation and rapid release from the nucleus ofnew mRNA molecules for this protein. Polyribosomal translation of the mRNA continued only as long as it was being synthesized, and the maximum rate of synthesis following a pulse dose of 125 ng of the hormone was the same as that observed after prolonged stimulation with cholecalciferol. The possibility that other 1,25-dihydroxycholecalciferol-dependent events may be occurring in the nucleus in the lag period between accumulation of the hormone in the intestine and the appearance of active calcium-binding-protein mRNA, and that these may ultimately control the synthesis of that mRNA, is discussed.

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