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Extract: This investigation confirms the high level of biologic activity and the similarity of the effects of small doses of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) and of its analog 1α-hydroxyvitamin D3(1α-OH-D3) on children with nutritional rickets, “pseudodeficiency” rickets (PDR), hereditary hypophosphatemia, chronic idiopathic hypoparathyroidism, and chronic renal failure. It also shows that cystinotic patients may develop, at the end stage of the disease, a certain degree of resistance to 1,25-(OH)2-D3. The comparison of the therapeutic effects of long term oral administration of 1,25-(OH)2-D3or 1α-OH-D3to two D-deficient children and two sibs with PDR demonstrates differences in sensitivity. In the patients with nutritional rickets, 0.5/μg/24 hr of either drug corrects the biochemical abnormalities, initiates healing of skeletal lesions in 28 days, and cures the metaphyseal lesions in 60 days of therapy. In contrast, it appears that doses of either drug that are curative in D deficiency rickets are only partly active in PDR. These observations indicate that the hypothesis of a deficit in 25-hydroxycholecalciferol 1α-hydroxylase in patients with PDR must await for confirmation more direct evidences, and that such a deficit, even if proven, may not account for all of the biochemical and skeletal alterations seen in patients with this inherited disorder.Speculation: The similarity of action of 1,25-(OH)2-D3and 1α-OH-D3observed in the present study presents additional evidence that 1α-OH-D3can be considered a valuable substitute for the hormonal form of cholecalciferol, especially for the management of children with chronic renal failure, hypoparathyroidism, or pseudo-deficiency rickets. In this latter disease, the active therapeutic doses of either drug appear to be greater than the amount required for the treatment of simple nutritional rickets. This observation indicates that investigations on the pathogenesis of pseudodeficiency rickets must be continued in order to confirm or invalidate the hypothesis suggested by us and others that this inherited disorder might be related to a deficit in 25-hydroxycholecalciferol 1α-hy-droxylase.

The calcaemic response of thyroidectomized parathyroid transplanted rats to a single dose of biosynthetic 1,25-dihydroxycholecalciferol (50 ng) injected into a jugular vein, was evaluated. The animals were fed a vitamin D-free, low calcium diet. Compared to sham-operated and to thyroidintact parathyroid transplanted rats thyroidectomized animals had a significantly reduced calcaemic response to 1,25-dihydroxycholecalciferol, Daily supplementation with d,l-thyroxine (100 μg/rat) during the experimental period restored a normal response. The increase in serum calcium concentration after 1,25-dihydroxycholecalciferol injection was similar in thyroidectomized bilaterally nephrectomized animals, and in thyroidectomized kidney-intact rats. The results suggest that in thyroxine depleted rats, the sensitivity of bone to the calcaemic effect of 1,25-dihydroxycholecalciferol is decreased.

Summary: The in vitro effects of vitamin D3 metabolites, parathyroid extract (PTE), purified parathyroid hormone (bPTH), vitamin A, and heparin on acid and alkaline phosphatases in rat or mouse calvaria in culture were investigated. Results show that: (a) when compared to values found in half calvaria incubated for 24 h in control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with PTE (1 USP/ml), bPTH (4×10−8M), heparin (5 USP/ml), vitamin A (23 USP/ml), 25-(OH)D3 (2.5×10−11 to 2.5×10−8M), 24,25-(OH)2D3, and 1,25(OH)2D3 (2.5×10−12 to 2.5×10−7M); (b) the presence of 24,25-(OH)2D3 at low concentrations in the incubation medium decreases significantly the PTE, bPTH, vitamin A, or heparin induced stimulation of the phosphatase activities. This interaction is also observed when measuringβ glucuronidase and glucose-6-phosphatase activities and45Ca release from previously labeled mouse calvaria; (c) a similar activity could not be found with 1,25-(OH)2D3 suggesting that 24,25-(OH)2D3 may have a specific role in bone metabolism.

Summary The metabolism of vitamin D3 was studied in 3-week-old, vitamin D deficient chicks, fed since hatching with a diet containing 3% ammonium chloride, 1% calcium, and 0.7% phosphorus. When kidney homogenates were incubated in vitro with [3H]25-(OH)D3, the production of 1,25-(OH)2D3 was reduced by 40% in acidotic birds. During in vivo experiments, after injection of [3H]D3 (1220 pM/bird), the level of 1,25-(OH)2D3 was also reduced in blood plasma, intestine, and tibiae in acidotic chicks as compared with the controls. As a large increase in plasma phosphate was found during acidosis, these results are discussed in relation to the possible role of phosphorus in the control of 1,25-(OH)2D3 synthesis.

Summary The effects of acute or chronic administration of small doses (130 pmol) of 25-hydroxycholecalciferol, 24,25-dihydroxycholecalciferol, and 1,25-dihydroxycholecalciferol on rat calvaria acid and alkaline phosphatase activities were investigated in weanling male albino Wistar rats raised on a vitamin D-deficient, low-calcium diet. The results indicate that each of these active metabolites has a different effect on calvarial phosphatase activities. 25-hydroxycholecalciferol causes a significant increase, and 24,25-dihydroxycholecalciferol a decrease in the enzymatic activity. In animals treated with 1,25-dihydroxycholecalciferol these activities are lower after one injection, but after seven daily doses they are not different from those of ethanol-injected control rats. The observed changes do not seem to be related to changes in serum calcium and/or phosphorus concentrations.

After the injection of radioactive 25-hydroxyvitamin D3into rats given a normal diet, two metabolites can be detected on Sephadex LH-20 chromatography modified to reveal polar metabolites. The first is designated peak Va, which by means of periodate cleavage has been identified as 24,25-dihydroxyvitamin D3. The second more polar metabolite has been designated peak X1and is only partially sensitive to periodate. This metabolite appears in serum, bone, intestine, and liver and does not chromatograph on any system like any of the previously identified metabolites. Nephrectomy, especially in the animals given large doses of 25-hydroxyvitamin D3, does not abolish production of either peak Va or X1, revealing that both metabolites can be made in extra-renal tissue. Peak X1has marked ability to elevate serum calcium of rats on a vitamin D-deficient, low calcium diet, but requires the presence of kidneys to carry out this function.

Summary: The present method for the measurement of 24,25-dihydroxyvitamin D (24,25–(OH)2D) in serum associates the highly sensitive competitive protein binding assay of Preece et al. and a double-step chromatography of the serum lipid extract (Sephadex LH 20 column and high pressure liquid chromatograph). With this assay, values observed in 16 normal children, 10 adolescents and 5 neonates, were not significantly different from those found in 14 normal adults (25–(OH)D: 20 ± 7.4 ng/ml, 24,25–(OH)2D: 1.4 ± 0.8 ng/ml). No correlation was found between 25–(OH)D and 24,25–(OH)2D serum concentrations. Measurements of 2S-(OH)D and 24,25–(OH)2D serum concentrations in some pathologic states demonstrated the existence of a 25–(OH)D-24-hydroxylase in children with three types of vitamin D resistant rickets (hereditary hypophosphatemia, pseudodeficiency rickets, and vitamin D resistance associated with Recklinghausen's neurofibromatosis). Finally, results observed during vitamin D administration suggest a regulation of the 24,25–(OH)2D concentration in human serum: 1) 24,25–(OH)2D serum concentration increased after vitamin D administration, yet it was not found higher in a 3-wk-old child with vitamin D2intoxication (25–(OH)D: 900 ng/ml, 24,25–(OH)2D: 6 ng/ml) than in three children with vitamin D deficiency rickets in the 2nd wk after administration of vitamin D2and calcium (25–(OH)D: 25.5 ± 9.2 ng/ml, 24,25–(OH)2D: 13.4 ± 3.7 ng/ml; 2) In a child with pseudodeficiency rickets, 24,25–(OH)2D concentrations were found elevated before treatment. They decreased to normal values during treatment with la-(OH) cholecalciferol, whereas 25–(OH)D concentrations were not significantly different before and after treatment.Speculation: Development of methods for the measurement of 24,25–(OH)2D concentration in serum should provide valuable informations for a better understanding of vitamin D metabolism in physiologic and pathologic states in children.

This investigation confirms the high level of biologic activity and the similarity of the effects of small doses of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) and of its analog 1α-hydroxyvitamin D3(1α-OH-D3) on children with nutritional rickets, “pseudodeficiency” rickets (PDR), hereditary hypophosphatemia, chronic idiopathic hypoparathyroidism, and chronic renal failure. It also shows that cystinotic patients may develop, at the end stage of the disease, a certain degree of resistance to 1,25-(OH)2-D3. The comparison of the therapeutic effects of long term oral administration of 1,25-(OH)2-D3or 1α-OH-D3to two D-deficient children and two sibs with PDR demonstrates differences in sensitivity. In the patients with nutritional rickets, 0.5 /μg/24 hr of either drug corrects the biochemical abnormalities, initiates healing of skeletal lesions in 28 days, and cures the metaphyseal lesions in 60 days of therapy. In contrast, it appears that doses of either drug that are curative in D deficiency rickets are only partly active in PDR. These observations indicate that the hypothesis of a deficit in 25-hydroxycholecalciferol 1α-hydroxylase in patients with PDR must await for confirmation more direct evidences, and that such a deficit, even if proven, may not account for all of the biochemical and skeletal alterations seen in patients with this inherited disorder.

Summary: The present study was undertaken to test the in vitro action of aluminum on bone phosphatase activities and the possible interaction of this metal with parathyroid hormone (bPTH) or vitamin D3 dihydroxymetabolites [1,25- and 24,25(OH)2D3). Three-day-old rat calvaria were incubated for 24 h with one of the following: bPTH at 510−8M, 1,25-or 24,25(OH)2D3 at 2.510−9M, Al at concentrations ranging from 310−11M to 610−6M, or their corresponding solvents. Al effects were also investigated when the medium phosphate or calcium concentrations were modified. In some experiments, Al was added simultaneously with bPTH or one of the vitamin D3 metabolites at the beginning of the 24 h incubation. At the end of all incubations, acid and alkaline phosphatase activities were measured in bone cytoplasmic extract. The results show that: (a) When compared to the value found in half calvaria incubated in a control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with Al (310−11M to 1.510−6M) as well as with bPTH, 1,25-, or 24,25(OH)2D3 and sharply decreased with higher Al concentrations (610−6M). (b) The Al effect on phosphatase activities is modified in a free phosphate or a free calcium medium. (c) The presence of Al at 1.510−6M or 610−6M significantly decreases the bPTH or 1,25(OH)2D3-induced stimulation of bone phosphatase activities. (d) A similar interaction could not be found between Al and 24,25-(OH)2D3.

Summary: Results from in vitro works suggest that 1,25- and 24,25-dihydroxyvitamin D3 (1,25-(OH)2D3 and 24,25-(OH)2D3) act on bone via different mechanisms. The present investigation was performed to study the effect of these two metabolites and of their precursor 25-hyxdroxyvitamin D3 (25-(OH)D3) on bone cAMP content in vitro. Rats' paired half calvaria were incubated under sterile conditions with one vitamin D3 derivative (10−13 to 10−9 M) or with ethanol (0.005 ml for 15 min to 24 h in 1 ml medium containing 0, 0.2, 1, 2, or 3 mM calcium. In some experiments: (a) cycloheximide (10−5M) was added simultaneously with the vitamin D3 metabolites; (b) 1–84 bPTH (5 10−8 M) was added for 5 or 15 min at the end of the 24 h incubation. Calvaria were immersed in 1 ml TCA 5% 4C and homogenized. The cAMP was extracted with diethylether and measured by a competitive protein binding assay. Results bring further evidence for a particular effect of low doses of 24,25-(OH)2D3 (10−9 to 10−12M) and of 25-(OH)D3 (10−9 to 10−11M) on bone, different from that of 1,25-(OH)2D3: cAMP content was higher in 24,25-(OH)2D3- or 25-(OH)D3-treated and lower in 1,25-(OH)2D3-treated calvaria than in ethanol-treated ones with 1 mM calcium. The 1,25-(OH)2D3 effect persisted in calcium-free medium whereas 25-(OH)D3 and 24,25-(OH)2D3 effects could not be observed with 0 mM nor with 3 mM calcium. The required duration of the preincubation (over 1 h) as well as the inhibitory action of cycloheximide may suggest an involvement of protein synthesis in the vitamin D3 metabolites effects. Neither 1,25-(OH)2D3 nor 24,25-(OH)2D3 affected the PTH-induced increase in bone cAMP content.

The metabolism of 25-hydroxycholecalciferol (25-(OH)D3) was investigated in rats fed a diet low in calcium and without vitamin D for 4 weeks after hypophysectomy. Compared with intact rats on the same diet these animals had a low serum phosphorus concentration, a less marked degree of hypocalcaemia and their parathyroid gland was not hypertrophied. Eighteen hours after i.p. injection of a single dose of tritiated 25-(OH)D3, chromatography of serum extracts on Sephadex LH-20 showed that the percentage of radioactivity corresponding to 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was lower in hypophysectomized rats than in control rats. High-pressure liquid chromatography demonstrated that 81% of this material had the same elution profile as synthetic 1,25-(OH)2D3. The percentage of 1,25-(OH)2D3in the serum of hypophysectomized rats could be increased to the level seen in the controls by chronic treatment with bovine growth hormone. This action of growth hormone was most probably independent of the parathyroid glands since the injection of parathyroid extract did not alter 25-(OH)D3metabolism in hypophysectomized animals. These results suggest that the decrease in the conversion of 25-(OH)D3to 1,25-(OH)2D3after hypophysectomy may be related to the lack of growth hormone.

The present method for the measurement of 24,25-dihydroxyvitamin D (24,25–(OH)2D) in serum associates the highly sensitive competitive protein binding assay of Preece et al. and a double-step chromatography of the serum lipid extract (Sephadex LH 20 column and high pressure liquid chromatograph). With this assay, values observed in 16 normal children, 10 adolescents and 5 neonates, were not significantly different from those found in 14 normal adults (25–(OH)D: 20 ± 7.4 ng/ml, 24,25–(OH)2D: 1.4 ± 0.8 ng/ml). No correlation was found between 25–(OH)D and 24,25–(OH)2D serum concentrations. Measurements of 2S-(OH)D and 24,25–(OH)2D serum concentrations in some pathologic states demonstrated the existence of a 25–(OH)D-24-hydroxylase in children with three types of vitamin D resistant rickets (hereditary hypophosphatemia, pseudodeficiency rickets, and vitamin D resistance associated with Recklinghausen's neurofibromatosis). Finally, results observed during vitamin D administration suggest a regulation of the 24,25–(OH)2D concentration in human serum: 1) 24,25–(OH)2D serum concentration increased after vitamin D administration, yet it was not found higher in a 3-wk-old child with vitamin D2intoxication (25–(OH)D: 900 ng/ml, 24,25–(OH)2D: 6 ng/ml) than in three children with vitamin D deficiency rickets in the 2nd wk after administration of vitamin D2and calcium (25–(OH)D: 25.5 ± 9.2 ng/ml, 24,25–(OH)2D: 13.4 ± 3.7 ng/ml; 2) In a child with pseudodeficiency rickets, 24,25–(OH)2D concentrations were found elevated before treatment. They decreased to normal values during treatment with la-(OH) cholecalciferol, whereas 25–(OH)D concentrations were not significantly different before and after treatment.

The metabolism of 25-hydroxycholecalciferol (25-(OH)D3), plasma concentration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and the amount of calcium-binding protein (CaBP) in duodenal mucosa were determined in ovariectomized rats and were compared with data observed in normal age-matched cyclic rats. Sephadex LH-20 and high-pressure liquid chromatography were used for the study of the metabolism of 25-(OH)D3. The concentration of 1,25-(OH)2D3in plasma and prolactin in serum were measured by radioimmunoassay. Calcium-binding protein in duodenal mucosa was determined immunologically using electroimmunodiffusion. The results showed that the lack of ovarian hormones and low prolactin levels observed in ovariectomized rats did not promote a significant change in the metabolism of 25-(OH)D3, in the levels of 1,25-(OH)2D3in the circulation or in the amount of CaBP in duodenal mucosa. It is possible that the regulation of 25-(OH)D3by sex hormones is restricted to the state of calcium stress such as during egg-laying in birds or pregnancy and lactation in mammals.

The effect of thyroparathyroidectomy (TPTX) on the plasma concentrations of the vitamin D metabolites (25-(OH)D, 24,25-(OH)2D and 1,25-(OH)2D) has been studied in pregnant rats and their fetuses during the last quarter of gestation.Maternal and fetal vitamin D metabolites were not significantly affected by TPTX. A significant increase in plasma 1,25-(OH)2D concentrations was observed in both TPTX and control mothers and fetuses from days 19 to 21. Fetal and maternal plasma 25-(OH)D were positively correlated in both control and TPTX groups. Such a correlation was also found for 24,25-(OH)2D in the two groups. In contrast, a positive correlation between maternal and fetal plasma concentrations of 1,25-(OH)2D was found in TPTX but not in control rats.These data suggest that major alterations in calcium metabolism, such as that produced by maternal TPTX, are insufficient to affect the changes in maternal and fetal plasma 1,25-(OH)2D during late pregnancy significantly. They also suggest that parathyroid hormone, thyroxine, and/or calcitonin may control a possible placental transfer of 1,25-(OH)2D in the rat.J. Endocr.(1988) 116,381–385