The synthesis and biological activity of 25,26-dihydroxy-cholecalciferol, a polar metabolite of vitamin D3.
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The uptake of Ca(2+) by isolated small-intestinal mucosa from vitamin D-depleted and -repleted rats was analysed for the effects of vitamin D on initial kinetics. The rapid association of Ca(2+) with the tissue, which is complete within 1min, was unaffected by the vitamin, whereas the subsequent, linear, uptake was significantly increased. Neither the tissue space accessible to inulin nor the permeability to thiourea was influenced by vitamin D treatment.
The cholecalcins are intracellular vitamin D-dependent calcium-binding proteins. High concentrations of 28 kDa cholecalcins have been found in the kidneys and cerebella of birds and mammals. However, whereas the synthesis of the renal protein is vitamin D-dependent, that of the cholecalcin in the cerebella of young growing chicks and rats is apparently not. In the present study a range of immunological, physicochemical and structural characteristics of renal and cerebellar cholecalcins isolated from a single species, the rat, has been examined to ascertain what, if any, differences there are between them, other than the vitamin D-dependence. Both proteins behaved in exactly the same way during purification and showed complete immunocross-reactivity in Ouchterlony double immunodiffusion and radioimmunoassay. No difference could be detected between them on electrophoresis under denaturing conditions or on two-dimensional isoelectric focusing/electrophoresis. Their amino acid compositions were very similar, as were their u.v.-absorption spectra and their chymotryptic and tryptic peptide maps. The N-terminal sequence was found to be Gly-Gly-Val-Ser... for both cholecalcins. We have thus been unable to show any significant difference between the two proteins and suggest that the 28 kDa cholecalcins of the rat cerebellum and kidney are extremely similar, if not identical.
Intestinal absorption of 47Ca was measured by a double-isotope technique in goats treated with 1, 5 or 25 micrograms of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). The effects of giving 1,25(OH)2D3 by intravenous (iv) infusion for 30-36 h were compared at each dose level with the effects obtained by oral administration of 1,25(OH)2D3 either in ethanol or protected against rumen degradation in fatty acid pellets. Dose-dependent increments in absorption followed the treatments, with a doubling of absorption at the 1 microgram dose and three- to fivefold increases with the 5 and 25 micrograms doses. 47Ca absorption was equally stimulated 2 and 6 d after treatment but had returned to pretreatment levels 12-14 d after treatment. Intravenous and protected oral administration of 1,25(OH)2D3 stimulated 47Ca absorption to the same extent, in spite of two- to fivefold higher plasma concentrations of 1,25(OH)2D3 after iv treatment. Somewhat lower increments in 47Ca absorption were seen using ethanol as the vehicle for oral administration. The naturally occurring metabolites 1,24(R),25-trihydroxycholecalciferol and 1,25(S),26-trihydroxycholecalciferol had only one-tenth to one-fifteenth the potency of 1,25(OH)2D3 in stimulating 47Ca absorption, while synthetic 1 alpha-hydroxycholecalciferol appeared to be twice as effective as 1,25(OH)2D3 when tested at a high (10 micrograms) dose.
Metabolic balance studies were undertaken to determine whether sodium bicarbonate (NaHCO3) supplements (4.5 mmol/day) altered 7-day cumulative calcium (Ca) phosphorus (P) balances in growing rats consuming either a basal diet providing 0.6% Ca and 0.3% P, or this diet plus 1,25-dihydroxycholecalciferol [40 ng 1,25(OH)2D3/day]. Feeding bicarbonate lowered urinary Ca but raised fecal Ca so that Ca balance became less positive. However, 1,25(OH)2D3 increased net absorption of Ca and P to the same degree when given to control rats and rats consuming bicarbonate. Nevertheless, bicarbonate-fed rats had lower net Ca absorption than controls, even when treated with high doses of 1,25(OH)2D3. Changes in net Ca absorption induced by bicarbonate may occur at a point in the gut distal to the duodenum since duodenal 45Ca absorption was decreased by bicarbonate feeding. The present results show that bicarbonate consumption depressed net Ca absorption in the rat. The effect appears to be independent of changes in 1,25(OH)2D3 metabolism because it is manifest in animals receiving high doses of 1,25(OH)2D3, which stimulate alimentary Ca absorption maximally, and because bicarbonate-fed rats are able to respond normally to exogenous 1,25(OH)2D3 by increasing their net absorption of Ca and P. In view of this demonstration that NaHCO3 supplements elevate fecal Ca loss in the rat, it is suggested that studies should be undertaken to determine whether bicarbonate exerts similar adverse effects on Ca balance in humans.
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Prostate cancer continues to be a significant source of morbidity and mortality among older men. One possible means of reducing its impact on overall health and vitality is via cancer chemoprevention, both in the population that is unaffected but at some risk and in those who have undergone some form of curative therapy after the onset of the disease. Chemoprevention holds significant promise, but large phase III clinical trials evaluating chemopreventive agents in prostate cancer can require vast numbers of enrollees and require the commitment of significant financial resources and time before any therapeutic benefit may become apparent. One technique to shorten the time required for chemoprevention clinical trials is to use surrogate endpoint biomarkers in place of the currently used actual endpoints of cancer incidence or survival. The validation of such surrogate endpoint biomarkers will require small, well-designed phase I and/or II trials to accumulate data on the modulation of the surrogate biomarkers and the endpoints of cancer incidence or survival by the chemopreventive agent. Careful statistical correlation and clinical validation of the data will then allow us to justify the use such surrogates in place of the actual endpoint in large, randomized trials, potentially shortening trial duration, improving financial efficiency, and accelerating approval of the chemopreventive agent. To that end, we first review the theoretical construct of cancer chemoprevention trials with particular reference to prostate cancer. We thereafter describe the design of a small, randomized, double-blinded, placebo-controlled phase I/II clinical trial of an analogue of vitamin D, vitamin D5, which we believe could serve as a model for data accumulation on surrogate biomarkers and correlation with other clinical endpoints.
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The plasma concentrations of 25-hydroxycholecalciferol (25-OH-CC), immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) were measured at the age of 30 and 66 days in thirteen preterm neonates (birthweight: 970 to 1300 g). At the age of 30 days when all infants were fed only with breast milk (BM) serum iCT and iPTH levels were normal. During the second month 7 infants were fed with BM only (control group) and 6 infants were supplemented with formula (supplemented group). At the age of 66 days, mean +/- S.D. serum iPTH concentration was higher in the supplemented group than in the control group: 169 +/- 79 vs. 60 +/- 33 microliterEq/ml (p less than 0.01). Serum iCT levels remained undetectable (less than 150 pg/ml) in both groups. Plasma 25-OH-CC concentrations were normal and similar in both groups. Serum iPTH concentrations were positively correlated with phosphorus intake and negatively correlated with calcium intake from BM only. The results suggest that secondary hyperparathyroidism can be detected in very low birthweight infants supplemented with a formula, probably because of a phosphorus load or decreased intestinal absorption of calcium.
Eleven uremic children with osteodystrophy aged 3 to 17 years were studied during administration of 1,25-(OH)2D3 for periods up to 21 months. Nine children presented with pure hyperparathyroidism, one with osteomalacia and one with mixed bone disease. Bone biopsies were performed before initiation of therapy and after 6 to 21 months of treatment following double tetracycline labeling. Skeletal lesions were improved but not cured in 5 of 9 children with hyperparathyroidism. In three instances lesions remained unchanged and worsened in one. No significant change was observed in the child with osteomalacia. Moderate improvement was noted in the patient with mixed bone disease. The propensity to develop hypercalcemia was the major factor associated with treatment failure since it precluded administration of adequate amounts of medication. Therapy with 1,25-(OH)2D3 was associated with a spectacular improvement in growth velocity in two of six children under age twelve.
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