Biomedical subjects

D J Baylink

Publications and source records attributed to D J Baylink.

At least 271 records · Page 15Linked to original sources

PubMed1986

Skeletal growth factor is produced by human osteoblast-like cells in culture.

Human bone cells isolated from femoral heads were cultured in BGJb medium containing bovine serum albumin (100 micrograms/ml), insulin (1 microgram/ml) and epidermal growth factor (10 ng/ml), and the conditioned medium collected. The medium was concentrated, chromatographed using HPLC gel filtration (TSK 2000 SW), and assayed for mitogenic activity using [3H]thymidine incorporation into embryonic chick calvarial cells. The conditioned medium contained mitogenic activity which eluted with a different elution time than insulin or epidermal growth factor. Characterization of this activity suggests that it was due to human skeletal growth factor (SGF), a mitogen which had been previously isolated from human bone matrix. Common properties include: stimulation of DNA synthesis in cultured embryonic chick calvarial cells, competition with human SGF for binding to anti-SGF antibodies, elution from HPLC gel filtration as a large factor (Mr 100,000) under native conditions but as a small factor (Mr 10,000) under dissociative conditions (4 M guanidine HCl), elution time on HPLC reverse-phase chromatography (small SGF), inactivation by dithiothreitol, stability to heat, acidic or alkaline conditions and inactivation by trypsin and chymotrypsin. These observations provide evidence that human bone cells produce SGF. Conditioned medium from human skin cell cultures also contained mitogenic activity. However, the activity was less than that from bone cells and did not cross-react with the rat anti-SGF antibodies.

Animals↗

PubMed1986

Isolation and purification of a low-molecular-weight skeletal growth factor from human bones.

A low-molecular-weight potent bone cell mitogen termed human skeletal growth factor (human SGF) was purified to homogeneity from human bone matrix. Extraction and initial purification steps were done under dissociative conditions to separate human SGF from high-molecular-weight complexes of bone matrix proteins. SGF activity was extracted from human femoral heads by demineralization with 10% EDTA in the presence of 4 M guanidine-HCl and proteinase inhibitors and was purified by hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was homogeneous by HPLC reverse-phase chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of human SGF purified under dissociative conditions was 11,000. Human SGF stimulated bone cell proliferation ([3H]thymidine incorporation and cell number) at picomolar concentrations, with half maximum activity at 2-3 ng/ml (180-270 pM). Human SGF constitutes 0.00024% of organic bone matrix by weight.

Bone and Bones↗

PubMed1986

Embryonic bone matrix formation is increased after exposure to a low-amplitude capacitively coupled electric field, in vitro.

In order to investigate the mechanism(s) of electric field-stimulated osteogenesis, we have developed an in vitro model in which embryonic chick tibiae have consistently demonstrated increased bone matrix formation in response to a low amplitude (estimated 10(-5) V/m in the serum-free culture medium), capacitively coupled, 10 Hz sinusoidal electric field. Initial applications of this model revealed that 72 h of continuous exposure to the electric field increased tibial collagen production by 29% compared to untreated controls, P less than 0.01. Additional studies further revealed: (a) that when electric field exposure was limited to 30 min/day during the 72 h in vitro incubation, embryonic bone matrix formation was increased by 83%, compared to non-treated controls (P less than 0.001), suggesting an inductive mechanism; (b) that the osteogenic response to electric field exposure in vitro was not unique to embryonic chick tibiae, since a similar response was also seen with newborn mouse calvaria (+133%, P less than 0.02); (c) that electric field-exposure-stimulated chick bone matrix formation was associated with increased bone cell proliferation; and (d) that this mitogenic response to in vitro electric field exposure could also be observed with embryonic chick calvarial cells in monolayer, serum-free cultures.

Animals↗

PubMed1986

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1).

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.

Animals↗

PubMed1986

The role of skeletal calcium deficiency in postmenopausal osteoporosis.

A previous study of iliac crest composition identified skeletal calcium deficiency in 25% of 56 postmenopausal osteoporotic patients evaluated prior to the use of stanozolol or calcitonin. This report is a follow-up of biopsy data after 2 years of treatment with drug or placebo in 31 patients, 11 of whom had skeletal calcium deficiency. The study diet, consisting of 1 g elemental calcium plus 400 U Vitamin D, repaired the skeletal calcium deficiency in all patients, treated and untreated alike. Total body calcium (TBC) results were influenced by separation into calcium deficient and normal mineral groups, apparent treatment response being observed largely in patients with calcium deficiency. It is suggested that if all postmenopausal women ingested 1 g elemental calcium plus 400 U Vitamin D daily, skeletal calcium deficiency in osteoporosis would disappear as a problem. Meanwhile, it is important to recognize that repair of calcium deficiency is an important variable capable of influencing bone response to therapy and the evaluation thereof.

Bone and Bones↗

PubMed1986

Skeletal alkaline phosphatase activity as a bone formation index in vitro.

These studies were intended to examine the relationship between skeletal collagen formation and skeletal alkaline phosphatase (ALP) activity in vitro. Embryonic chick calvaria were exposed to skeletal effectors (including high and low pH, a range of [pi] and [Ca], PTH, NaF, etc), and collagen formation was assessed by the incorporation of 3[H]-proline as 3[H]-hydroxyproline (3[H]-hyp). ALP activity was measured in the serum-free conditioned medium and in 20% butanol extracts of the bones. We found that ALP activity and 3[H]-hyp incorporation were coordinately increased from pH 5.5 to pH 7.2 (r = .99, P less than 0.001). Calvarial ALP was not increased in response to low [Pi], but low [Ca] increased ALP and coordinately decreased collagen formation (r = -.81, P less than 0.05). Although calvarial ALP and 3[H]-hyp incorporation were coordinately increased by NaF, vanadate, PGE2, calcitonin, and insulin, the slopes of the correlations were not the same for all effectors (eg, NaF: r = .97, P less than 0.01, slope = 0.90; vanadate, r = .95, P less than 0.005, slope = 0.20), indicating differential actions on ALP and 3[H]-hyp incorporation. When a variety of effectors, including low [Ca], were used to treat different groups of calvaria, ALP activity was not correlated with 3[H]-hyp incorporation (r = .35), but when the exposure to effectors was limited to a preincubation, or when the low [Ca] data were excluded, a correlation was observed (r = .87, P less than 0.001, and r = .64, P less than 0.02, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1986

Characterization of mitogenic activities extracted from bovine bone matrix.

The mitogenic activity in the unfractionated mixture of proteins released from adult bovine bone matrix during demineralization with ethylenediaminetetraacetate (EDTA) has been examined. Bovine bone extract (BBE) from 1 to 25 micrograms protein per ml stimulated proliferation of chick embryo calvaria bone cells, newborn mouse bone cells, and osteoblastlike cell lines MMB-1 and ROS 17/2.8. BBE also stimulated DNA synthesis in cells from chick embryo cartilage, skin and skeletal muscle tissues and fibroblastlike BALB/c 3T3 and NRK cells. BBE contained beta transforming growth factor (TGF) activity (NRK cell colony formation in soft agar in the presence of epidermal growth factor EGF). The cell specificity results suggest that BBE contains more than one growth factor, including a beta TGF and a factor that is not specific for bone cells, and all of the bone derived growth factor activities that have been described previously, including SGF, are apparently present in BBE. Maximal stimulation of chick embryo calvarial cell DNA synthesis by BBE was equal to or exceeded maximal stimulation by nonosseous growth factors that have been reported to stimulate DNA synthesis in bone organ cultures (EGF, fibroblast growth factor, platelet-derived growth factor, insulinlike growth factor I, and multiplication stimulating activity). Combinations of BBE with maximally stimulatory concentrations of each growth factor stimulated DNA synthesis to a greater magnitude than did each growth factor alone. These results suggest that combinations of bone derived and systemic factors can coordinately stimulate bone cell proliferation.

Animals↗

PubMed1986

Characterization of a rapidly responding animal model for fluoride-stimulated bone formation.

Sodium fluoride (NaF) is the single most effective agent for increasing bone volume in the osteoporotic skeleton. However, the mechanism of fluoride-stimulated bone formation is not known, and investigation has been hampered by the lack of a suitable animal model. Young chicks show a rapid skeletal response to NaF that resembles the human skeletal response. This occurs at serum fluoride concentrations comparable to those obtained in humans. Fourteen-day-old chicks treated with NaF (4.2 mM NaF in the drinking water) for 2 weeks showed increases in bone-forming surface in the tibial metaphysis (130% of untreated controls, P less than 0.002), with no change in the number of osteoblasts per length of forming surface (104% of control). The NaF dose dependence of the change in bone-forming surface was biphasic, being optimal at 23 microM fluoride. Linear correlations were observed between dietary NaF and serum fluoride (r = 0.996, P less than 0.001), and serum fluoride and bone fluoride concentrations (r = 0.98, P less than 0.001). Correlations were also observed between the amount of alkaline phosphatase activity in the tibia and the serum fluoride concentration (r = 0.88, P less than 0.03), the serum fluoride concentration and the tibial ash weight (r = 0.93, P less than 0.01), and the bone fluoride concentration and tibial ash weight (r = 0.95, P less than 0.01). Preliminary studies of the time dependence of the skeletal fluoride response in young chicks revealed no difference between 2 weeks and 4 weeks of treatment (bone-forming surface increased to 124% and 139% of controls in separate studies, P less than 0.01 for each).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1986

Osteoporosis: radiographic detection of fluoride-induced extra-axial bone formation.

New bone formation in the peripheral skeleton was detected radiographically in 67% (22/33) of patients studied for new periarticular pain among 72 patients with osteoporosis being treated with fluoride, 66-88 mg/d, for 6 or more months. Changes included periosteal and endosteal new bone formation as well as trabecular thickening and were localized in areas of high mechanical stress. Sufficient mineral deposition for radiographic detection required 6 or more months of fluoride treatment. Radiologists must differentiate fluoride-induced new bone formation from other-processes, such as fractures and tumor, and elicit information on fluoride intake in suspicious cases.

Bone Development↗

PubMed1986

Alterations in calcium, phosphorus and C-terminal parathyroid hormone levels in equine acute renal disease.

The changes in serum and urinary levels of calcium and phosphorus and serum parathyroid hormone (PTH) were studied during controlled, chemically induced, acute renal disease in 4 ponies. There was an initial rise in daily urinary calcium and hydroxyproline excretion in 2 ponies which may have indicated increased bone resorption. Mild hypercalcemia, hypophosphatemia and elevated C-terminal PTH levels were associated with oliguria. Total daily urinary excretion of calcium and phosphorus decreased as oliguria developed. The levels of C-terminal PTH were increased in all four animals. This appears to have been related to their diminished renal function in that C-terminal fragments of intact PTH (which require functioning kidneys in order to be cleared) were probably being accumulated. Although there was no evidence with regard to an actual increase in PTH secretion, prolongation of intact PTH half-life due to renal tubular damage may have contributed to the development of hypercalcemia.

Acute Kidney Injury↗

PubMed1986

Osteoblast and osteoclast cell number and cell activity in postmenopausal osteoporosis.

This study evaluates osteoblast and osteoclast cell numbers and cell activities determined from quantitative histology of trabecular bone in human iliac crest bone biopsies. Subjects studied were postmenopausal osteoporotic patients (aged 53-81) for whom total body calcium and urine and serum clinical data were also available. Only 39% of the osteoclasts identified by acid phosphatase content were multinucleate; however, a significant correlation between multinucleate osteoclasts and the total number of osteoclasts was found (r = 0.87; p less than 0.001). The mean number of osteoclasts per square millimeter of total tissue area was 0.96 (+/- 0.14, SEM, n = 36); the mean number of osteoblasts was 6.8 (+/- 1.5, n = 14)/mm2 tissue area. Computed osteoclast activity (square millimeter bone resorbed per osteoclast nucleus per day) was 2.6 X 10(-4) +/- 1.0 X 10(-4) (n = 13). Computed osteoblast activity (square millimeter bone formed per osteoblast per day) was 2.9 X 10(-5) +/- 8.8 X 10(-6) (n = 13). Calculated mean rate of bone resorption was 1.6 X 10(-4) +/- 3.4 X 10(-5) mm2 bone resorbed per mm2 total tissue area per day (n = 19). These data indicate that although osteoclasts are not numerous in the iliac crest of postmenopausal osteoporotics, the individual osteoclast activity (in square millimeter bone resorbed osteoclast cell unit per day) is significantly greater (p less than 0.001) than the osteoblast activity (in square millimeter matrix deposited per cell per day). These data also point out that greater consideration should be given to the role that osteoclast cell activity may play in human postmenopausal osteoporosis.

Aged↗

PubMed1985

A mouse tumor-derived osteolytic factor stimulates bone resorption by a mechanism involving local prostaglandins production in bone.

Culture medium which was conditioned by tissue of a CE mouse breast tumor in vitro contained dose-dependent osteolytic activity. The osteolytic activity was not soluble in dichloromethane and ethylacetate, indicating that it was not attributable to vitamin D metabolites or prostaglandins. However, breast tumor-conditioned medium stimulated production and release of prostaglandin E2 from mouse calvaria in vitro, and the stimulation of bone resorption in vitro by breast tumor-conditioned medium was blocked by a dose of indomethacin that prevented stimulation of mouse calvarial prostaglandin E2 production and release. The resorptive activity of parathyroid hormone (PTH) was not affected by the same dose of indomethacin, suggesting that the osteolytic factor was not PTH. This was further supported by observation that mouse kidney cell cAMP production was stimulated by PTH, but not by the aqueous phase of ethylacetate-extracted breast tumor-conditioned medium. In addition to osteolytic activity, breast tumor-conditioned medium contained a dose-dependent bone cell mitogenic activity, demonstrated by the stimulation of [3H]thymidine incorporation into trichloroacetic acid-insoluble macromolecules and a corresponding increase in bone cell number in monolayer cultures of bone cells. Breast tumor-conditioned medium also contained a dose-dependent transforming growth factor-(TGF-) like activity as defined by its ability to transform anchorage-dependent growth of nontransformed cells to anchorage-independent growth. The TGF in breast tumor-conditioned medium did not compete with epidermal growth factor (EGF) for EGF receptor binding, but its transforming activity was greatly enhanced by EGF, indicating that it was a beta-type TGF. Both the osteolytic and mitogenic activities were nondialyzable, sensitive to reducing agent, and not removable by dichloromethane and ethylacetate extractions. Furthermore, the TGF activity was not removed by ethylacetate extraction. Thus, the possibility that these activities in breast tumor-conditioned medium might be mediated by the same molecule must be considered. In summary, our data suggest that the CE mouse mammary carcinoma cells produce and secrete into the culture medium an osteolytic factor which is neither PTH nor prostaglandin and which stimulates local synthesis in bone of prostaglandin E2 which in turn increases bone resorption in vitro.

Animals↗

PubMed1985

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase.

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.

4-Nitrophenylphosphatase↗

PubMed1985

Intestinal metabolism and portal venous transport of 1,25(OH)2D3, 25(OH)D3, and vitamin D3 in the rat.

We compared the intestinal absorption of three vitamin D3 sterols and tested the hypothesis that the intestine hydroxylates absorbed vitamin D and transports polar metabolites in portal venous blood. Micellar solutions containing 50 nmol of a radiolabeled vitamin D3 sterol (1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or vitamin D3) were placed in closed jejunal segments of rats prepared with lymphatic and mesenteric venous fistulas. Venous blood loss was replaced by infusion of donor rat blood into the saphenous vein. After 1-2 h the rats were killed, and intestinal homogenates, mesenteric blood, and lymph were analyzed. The average rate of absorption of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was two- and fivefold higher than that of 25-hydroxyvitamin D3 [25(OH)D3] and vitamin D3 (D3), respectively. Transport of hydroxylated vitamin D sterols was primarily via the venous route; the average rate of venous transport of 1,25(OH)2D3 was 18.3 X 10(2) nmol X min-1 X g-1 compared with 8.8 X 10(2) for 25(OH)D3 and 0.13 X 10(2) for D3. High-performance liquid chromatography of intestinal and plasma extracts revealed that there was 25-hydroxylation of absorbed D3, 24- and putative 1-hydroxylation of absorbed 25(OH)D3, and prompt portal venous transport of all hydroxylated metabolites. When 1,25(OH)2D3 was infused into the lumen, the composition of radiolabeled sterols found in intestinal homogenates and mesenteric venous plasma was virtually identical to that of the infusate. These studies provide in vivo evidence for the intestinal metabolism of pharmacological quantities of absorbed vitamin D3 sterols and the prompt portal venous transport of more polar metabolites.

Animals↗

PubMed1985

Calcium balance in uremia: longitudinal study of long-term survivors.

Twelve male patients surviving uremia of 9-15 years duration were studied sequentially over a mean period of 9.2 years. Radiographs, bone biopsies, bone mineral mass (BMM) measured by photon absorptiometry, and total body calcium (TBC) determined by in vivo neutron activation analysis (NAA) were used. Pathologic fractures correlated with TBC corrected for height: 4 of 5 patients without fractures had TBC above 80% predicted, whereas 5 of 7 patients with fractures fell below this value. Unlike previous studies, negative calcium balance was seen in 3 patients with prolonged hyperparathyroidism. Though soft tissue calcification occurred in 6 patients, only 1 with massive shoulder calcification had an increase in TBC; this drawback of NAA has been previously overemphasized. Moreover, radial BMM also increased in 2 patients due to soft tissue calcification. When viewed at a single point in time, nearly all patients fell within the wide normal limits of BMM vs TBC. However, 6 patients with variable bone histology studied sequentially over a mean of 8.5 years showed significant preferential loss of radial bone mass compared to TBC. Aluminium retention did not influence calcium balance in these subjects.

Adult↗

PubMed1985

Spaceflight results in formation of defective bone.

Growing rats were flown on 19 day spaceflights aboard Cosmos 782 and 936 biosatellites. Spaceflight resulted in a prominent skeletal defect at the periosteal surface of the tibia diaphysis. The defect, termed an arrest line, was approximately 3 micron across and separated the bone formed in space from that formed following spaceflight. The bone matrix at the arrest line region was abnormal in that collagen fibers were preferentially orientated parallel to the periosteal surface. In addition, the bone matrix was hypomineralized. The altered bone was inferior to normal bone in resistance to abrasion and may be partially responsible for the decrease in torsional strength observed after spaceflight.

Animals↗

PubMed1984

Differential response in alveolar bone osteoclasts residing at two different bone sites.

Using a histochemical method for demonstrating acid phosphatase activity, we have studied osteoclasts residing at two different bone sites in rat incisor alveolar bone, one at the endosteum and the other at the tooth socket, and compared the response of these osteoclasts to systemic changes. After 12 days of calcium (0%) or phosphorus (0.2%) deprivation, the number of osteoclasts/cross section at the endosteum increased 463% (P less than 0.001) and 103% (P less than 0.002), respectively. After 10 days of calcium or phosphorus replenishment, the number of osteoclasts at this bone site decreased to levels not significantly different from those in the control. In contrast, the number of osteoclasts at the incisor socket remained insignificantly changed throughout the experimental period. A similar osteoclast differential response was also observed in the alveolar bone surrounding the first molar tooth. After 12 days of calcium deprivation, the number of osteoclasts/mm bone surface increased 371% (P less than 0.001) at the endosteum but remained insignificantly changed at the first molar socket. These results suggest that an osteoclast differential response exists in alveolar bone and that the response may be of significance inasmuch as the major function of alveolar bone is to support the teeth. The work described here supports the concept of local as well as systemic regulation of bone metabolism to simultaneously perform the dual functions of mineral homeostasis and mechanical support.

Animals↗

PubMed1984

24,25(OH)2D3, bone formation, and bone resorption in vitamin D-deficient, azotemic rats.

Bone formation, mineralization, and resorption were measured in vitamin D-deficient, azotemic rats given two different dosages of 24,25(OH)2D3 daily and in vehicle-treated controls (C). The intraperitoneal administration of 65 pmol over a 10 day period corrected the hypocalcemia observed in C, whereas 130 pmol produced mild hypercalcemia. Both dosages reduced osteoid width, osteoid area, and mineralization front width from control values. The rates of bone and matrix formation were unaffected by treatment. In C, matrix formation exceeded bone formation and resulted in osteoid accumulation; both dosages of 24,25(OH)2D3 reversed this relationship such that bone formation exceeded matrix formation in each treatment group. The rates of osteoid maturation and initial mineralization increased during repletion with 24,25(OH)2D3 at both dosage levels. However, the serum calcium concentration was correlated with both osteoid maturation rate (r = 0.68, P less than 0.01) and initial mineralization rate (r = 0.63, P less than 0.01) when all three experimental groups were considered. Bone resorption was unchanged from control values during treatment with 24,25(OH)2D3. The results suggest that 24,25(OH)2D3 promotes the maturation and mineralization of osteoid, and that this metabolite differs in its effects on bone formation and resorption. It is not clear, however, that the changes in bone dynamics observed are independent of the calcemic response induced by metabolite repletion under the conditions of this experiment.

24,25-Dihydroxyvitamin D 3↗