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24R,25-Dihydroxyvitamin D₃: An Essential Vitamin D₃ Metabolite for Both Normal Bone Integrity and Healing of Tibial Fracture in Chicks¹

Department of Biochemistry (E.-G.S., A.W.N.) and the Division of Biomedical Sciences (A.W.N.), University of California, Riverside, California 92521; and the Department of Orthopedics, Mount Sinai School of Medicine (T.A.E.), New York, New York 10029

Address all correspondence and requests for reprints to: Prof. Anthony W. Norman, Department of Biochemistry, University of California, Riverside, California 92521. E-mail: norman{at}ucrac1.ucr.edu

	Abstract

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We tested the hypothesis that 24R,25-dihydroxyvitamin D₃ [24R,25-(OH)₂D₃] is an essential vitamin D metabolite for the development of normal bone integrity and the healing of fractures. The natural 24R,25-(OH)₂D₃ and its synthetic epimer 24S,25-dihydroxyvitamin D₃ [24S,25-(OH)₂D₃] were tested alone or in combination with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃], on normal bone development and other related variables of the Ca²⁺ homeostasis system [serum Ca²⁺, 25-hydroxyvitamin D₃ (25OHD₃), 24,25-(OH)₂D₃, and 1,25-(OH)₂D₃ levels] in chicks. Mechanical testing of torsional strength was carried out on the femur. 24R,25-(OH)₂D₃ (80 nmol/kg diet) alone was sufficient for normal bone growth and integrity similar to that achieved by the vitamin D₃-replete controls. Next, chicks were fed a 25OHD₃-replete diet (75 nmol/kg diet) for 8 days after hatching, and then 25OHD₃ was withdrawn to minimize any residual circulating metabolites before the imposition of standardized tibial fractures 14 days later. Vitamin D metabolites were administered for 2 weeks to determine their effects on the mechanical properties of healed tibia. 24S,25-(OH)₂D₃ combined with 1,25-(OH)₂D₃ or 1,25-(OH)₂D₃ alone resulted in poor healing [strength values of 0.158 ± 0.011 and 0.123 ± 0.009 Nm (Newton · meter), respectively] compared with that in the 25OHD₃-treated control group (0.374 ± 0.029 Nm). In contrast, the fractured tibia of the birds fed 24R,25-(OH)₂D₃ in combination with 1,25-(OH)₂D₃ showed healing equivalent to that in the control group, with strength values of 0.296 ± 0.043 Nm. These results suggest that when 24R,25-(OH)₂D₃ is present at normal physiological concentrations, it is an essential vitamin D₃ metabolite for both normal bone integrity and healing of fracture in chicks.

	Introduction

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IT IS WELL understood that the seco steroid vitamin D₃ undergoes metabolic conversion before it exerts its biological effects. The two metabolites that have attracted the most attention are 1,25-(OH)₂-vitamin D₃ [1,25-(OH)₂D₃] and 24R,25-(OH)₂-vitamin D₃ [24R,25-(OH)₂D₃]. The kidney is the principal site of production of both 1,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ (1, 2). The most active form of vitamin D₃ in terms of various classical actions, such as stimulating intestinal calcium absorption and maintaining calcium homeostasis, has been shown to be 1,25-(OH)₂D₃ (3). Despite the high plasma circulating level of 24R,25-(OH)₂D₃ (4), many aspects of its possible biological effects, including the identity of a receptor for 24R,25-(OH)₂D₃ or its mechanism(s) of action have not yet been clearly demonstrated (5, 6).

There are two viewpoints concerning the biological importance of 24R,25-(OH)₂D₃. Some have suggested that 24R,25-(OH)₂D₃ is a degradation metabolite that is not essential for embryo development in either the rat (7) or chick (8). Another study using 24,24-difluoro-25-hydroxyvitamin D₃ (24,25-difluoro-25OHD₃), which was postulated to block 24-hydroxylation, also concluded that 24R,25-(OH)₂D₃ was an unimportant metabolite (9).

In contrast, much evidence has been presented suggesting that 24R,25-(OH)₂D₃ has several important biological actions (10, 11) in tissues, including the parathyroid gland (12, 13) and the skeletal system (14, 15, 16, 17). The rachitic lesion present in a chick epiphyseal growth plate was shown to be healed by local administration of 24R,25-(OH)₂D₃ (18). Also, 24R,25-(OH)₂D₃ was shown to enhance the healing of chick tibial fractures (19, 20). A receptor for 24R,25-(OH)₂D₃ has been reported, but not confirmed, in the long bone of rats (21). Recently, Schwartz et al. (22, 23, 24) have shown that resting cartilage in the growth plate primarily responds to 24R,25-(OH)₂D₃. These findings suggest that tissues that undergo endochondral ossification (e.g. resting cartilage, growth cartilage, and fracture callus) may be specific targets for the actions of 24R,25-(OH)₂D₃.

The present study was undertaken to assess the physiological significance of long term administration of the naturally occurring 24R,25-(OH)₂D₃ or the unnatural 24S,25-(OH)₂D₃ compared to that of various other vitamin D₃ metabolites on bone integrity and fracture healing in chicks. Our results strongly suggest that 24R,25-(OH)₂D₃, present at physiological concentrations, is an essential vitamin D₃ metabolite for both normal bone development and fracture healing.

	Materials and Methods

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General description of experimental protocols
To study the consequences of administration of vitamin D₃ metabolites on normal bone growth and the fracture-healing process, a total of three experiments were carried out. In the first study (Exp I), newly hatched chicks were chronically fed vitamin D₃ metabolites alone or in combination for 4 weeks, and the intact femurs were tested for mechanical properties and ash content. The second study (Exp II) was used to develop and validate a chick tibial fracture-healing model. For this study, newly hatched chicks were raised on a vitamin D₃- or 25OHD₃-replete diet for 8 days, and then the vitamin D₃ or 25OHD₃ was withdrawn for 2 weeks, the birds were killed, and the bone ash was determined.

For the fracture-healing experiment (Exp III), newly hatched chicks were given 25OHD₃ for 8 days, and then the metabolite was withdrawn for 2 weeks in accordance with the model set up in Exp II. After the depletion period, a fracture of the right tibia was imposed under anesthesia. After 2 weeks of administration of one of several formulations of vitamin D₃ metabolites, the mechanical properties of the healing tibia fractures and the ash content of the intact femurs were determined. In addition, for Exp I, II, and III, the chick growth curves were recorded, and serum levels of Ca²⁺ and vitamin D₃ metabolites were measured.

In all three experiments, the chicks were raised in chick brooders in a room illuminated with incandescent light (16 h on, 8 h off). The chicks were normally fed ad libitum a vitamin D-deficient diet (25) that had been supplemented with the desired level of vitamin D₃ or its metabolites (see details below).

Design for Exp I: evaluation of normal bone growth and integrity
Male White Leghorn chicks were obtained at the time of hatching (Hyline International, Lakeview, CA) and fed a standard vitamin D-deficient diet containing 1.2% calcium and 1.2% phosphate for the duration of the experiment (25). The vitamin D₃ metabolites were mixed into the standard diet so that the birds orally ingested the metabolites provided. Each group consisted of at least 10–12 birds, and the levels of the metabolites fed were as follows: group A, standard vitamin D-deficient diet; group B, vitamin D₃ (50 nanomoles/kg diet); group C, 1,25-(OH)₂D₃ (1.2 nmol/kg diet); group D, 24R,25-(OH)₂D₃ (80 nmol/kg diet); group E, 24S,25-(OH)₂D₃ (80 nmol/kg diet); group F, 24R,25-(OH)₂D₃ (80 nmol/kg diet) plus 1,25-(OH)₂D₃ (1.2 nmol/kg diet); group G, 24S,25-(OH)₂D₃ (80 nmol/kg diet) plus 1,25-(OH)₂D₃ (1.2 nmol/kg diet); group H, 24R,25-(OH)₂D₃ (15 nmol/kg diet) plus 1,25-(OH)₂D₃ (1.2 nmol/kg diet); and group I, 24S,25-(OH)₂D₃ (15 nmol/kg diet) plus 1,25-(OH)₂D₃ (1.2 nmol/kg diet).

All birds were killed 30 days after hatching except those in group A, which were killed at 26 days. The right femurs were carefully disarticulated at the hip and knee, and frozen immediately in liquid nitrogen for storage until mechanical testing. The left femurs were used for bone ash determination (25). Serum was collected from individual birds, and the serum Ca²⁺ level (26) as well as the levels of circulating 25OHD₃, 24,25-(OH)₂D₃, and 1,25-(OH)₂D₃ were measured.

Design for experiments on fracture healing (Exp II and III)
Exp II.
To determine the relative contributions of specific vitamin D₃ metabolites to the fracture-healing process, a standard chick tibial fracture-healing model was developed. A principal objective was that the animals should survive the fracture surgery and, at the same time, have very low circulating levels of vitamin D₃ metabolites. In this pilot experiment, birds were fed different levels of 25OHD₃ (25 or 75 nmol/kg diet) for 8 days; then the metabolites were withdrawn for about 2 weeks by the feeding of a vitamin D-deficient diet. Additional groups of birds fed the same level of 25OHD₃ or vitamin D₃ (150 nmol/kg diet) continuously served as positive controls. After the depletion period, bone ash, serum calcium, and levels of vitamin D metabolites were measured to identify the appropriate initial dose level of 25OHD₃, which, after withdrawal, resulted in a rapid depletion of the important vitamin D metabolites; the results are summarized in Table 3.

For the first 4 days after fracture, the birds were maintained in small cages in groups of two birds and treated with the appropriate metabolite(s) orally in a 1:1 dilution of ethanol-propandiol once a day to ensure the uptake of the metabolite(s). When all birds were ambulatory and eating ad libitum, they were moved into a larger cage, and the vitamin D₃ metabolite doses were again mixed into the vitamin D-deficient diet. Groups D and G were killed 14 days after fracture treatment, whereas groups A, C, E, and F were killed 16 days after the treatment. Each group had 8–10 birds with a healed tibia fracture. The fractured right tibiae were disarticulated at the knee and ankle, and frozen immediately in liquid nitrogen for storage until mechanical testing. The right femurs were collected for bone ash determination. Sera were collected for the measurements of the levels of calcium and vitamin D₃ metabolites.

Fracture surgery
The surgical procedure was approved by the chancellor’s committee on laboratory animal care (A-9306339–1). The chick was anesthetized with Metofane inhalation, and a skin incision (1 cm) was made over the right tibia. Muscle tissue overlying the tibia was longitudinally separated to expose the anterior surface of the bone. Using a dental burr, a hole approximately 2 mm in diameter was drilled at the middiaphysis to standardize the location of the fracture in the bones. The skin was then closed, and a fracture of the tibia was effected using the drill hole as a locus for stress concentration by applying light manual pressure. The fractures were not treated with any form of fixation.

Determination of serum vitamin D₃metabolites levels
From a total of 12 birds, 6 samples of 2 ml serum pooled from 2 birds were used for the serum analyses of vitamin D₃ metabolites. Before the lipids were extracted from the serum, 9000 dpm each of [26,27-³H]25OHD₃ (15 Ci/mmol), [6,9,19-³H]24,25-(OH)₂D₃ (51.1 Ci/mmol; Kureha Chemical Industry Co., Tokyo, Japan), and [23,24-N-³H]1,25-(OH)₂D₃ (120 Ci/mmol; Amersham, Arlington Heights, IL) were added as tracers to permit determination of the percent recovery of each metabolite before the assay. Extracted lipids were dried under nitrogen, and the vitamin D₃ metabolites were separated by HPLC; the samples were chromatographed on a Radial Pak silica column (Waters Associates, Milford, MA), and the vitamin D₃ metabolites were eluted using a 5–20% isopropanol gradient in hexane. The regions for the 25OHD₃, 24,25-(OH)₂D₃, and 1,25-(OH)₂D₃ were pooled separately for their assay. Steroid competition assays were used to measure the serum levels of 25OHD₃ and 24,25-(OH)₂D₃ using vitamin D₃-binding protein, whereas a hydroxylapatite binding assay was used to measure 1,25-(OH)₂D₃ using a lyophilized preparation of chick intestinal cytosol receptor (27).

Biomechanical testing
The intact femora or fractured tibiae were thawed and dissected free of soft tissue attachments, and their ends were potted in Cerrobend (Jackson-Walter, King of Prussia, PA). Changes in length and diaphyseal diameter were determined with a micrometer. The potted bones were mounted in the grips of a Servo-actuated (direct current motor) torsion-testing apparatus (custom made, Department of Biomechanics, Hospital for Special Surgery, New York, NY) and loaded to failure. This apparatus uses a loading rate of 1 rps (360°/sec) and is a motorized modification of the Burstein-Frankel system. The data were digitized, displayed, and stored in an IBM PS-2 computer. Values were obtained for torsional strength, stiffness, angular deformation, and energy absorption.

Vitamin D₃ metabolites
Crystalline vitamin D₃ was obtained from Sigma Chemical Co. (St. Louis, MO), and 25OHD₃ and 1,25-(OH)₂D₃ were provided by Dr. M. R. Uskokovic (Hoffman LaRoche, Nutley, NJ). 24R,25-(OH)₂D₃ and 24S,25-(OH)₂D₃ were provided by Dr. N. Taniguchi of the Biomedical Laboratory of Kureha Chemical Industry Co. (Tokyo, Japan).

Statistical analysis
All data reporting the circulating levels of the vitamin D₃ metabolites (Tables 2 and 3) were analyzed by Tukey’s multiple comparison test (28). P 0.05 was used to define a significant difference between groups.

View this table: [in this window] [in a new window]   Table 2. Serum levels of calcium and circulating levels of 25OHD3, 24R,25-(OH)2D3, and 1,25-(OH)2D3 in the experiment on the normal bone development (Exp I)

	Results

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View larger version (23K): [in this window] [in a new window]   Figure 1. Growth curves of chicks in Exp I concerning normal bone development. A, Body weights of the birds fed one vitamin D3 metabolite. B, Body weights of the birds fed 24R,25-(OH)2D3 or 24S,25-(OH)2D3 in combination with 1,25-(OH)2D3. The levels of the metabolites given are indicated in brackets as nanomoles per kg diet. The doses were based on historical food consumption and growth curves recorded in our laboratory (data not shown) and were set at levels so that each bird could consume certain specified nanomoles of vitamin D3 metabolite per kg BW/day. For example, birds in group D consumed approximately 16 nmol 24R,25-(OH)2D3/kg BW·day. The vitamin D-deficient birds (group A) were killed on day 26, which is 4 days earlier than all other groups, because their continued survival was in question as a consequence of their severe hypocalcemia. Each group had at least 11 birds, and the data represent the mean ± SEM.

The circulating levels of the major vitamin D₃ metabolites in Exp I were measured to correlate their serum concentration to their observed biological effects on bone (see Table 2). In general, the circulating levels of the vitamin D metabolites were in accordance with those previously reported for chicks (4, 32). Interestingly, all the birds in group C [provided only 1,25-(OH)₂D₃] had plasma levels of 1,25-(OH)₂D₃ that fell within the lower boundary of the normal range [compare with group B]; however, their mechanical properties were distinctly suboptimal (Table 1). Normal serum Ca²⁺ levels were achieved by group B (vitamin D₃-replete group), group D (24R,25-(OH)₂D₃), and group F [24R,25-(OH)₂D₃ in combination with 1,25-(OH)₂D₃]. All birds fed 1,25-(OH)₂D₃ and/or 24S,25-(OH)₂D₃ were hypocalcemic, as were the vitamin D-deficient birds in group A.

In Exp II, which served to explore and validate the animal model for the subsequent tibial fracture-healing study (Exp III), the birds were divided into five different groups after hatching and fed one of two levels of 25OHD₃ (either 75 or 25 nmol/kg diet) for 8 days or continuously. In contrast to Exp I, 25OHD₃ was used as the positive normal control, as opposed to vitamin D₃. 25OHD₃ is the immediate precursor of both dihydroxylated metabolites, and importantly, when 25OHD₃ is withdrawn from the diet, the chicks become vitamin D metabolite depleted more rapidly. In a preliminary experiment (data not shown) we found that a continuous dietary supply of 25OHD₃ (75 nmol/kg diet) resulted in the same rate of growth as a generous dose of vitamin D₃ (150 nmol/kg diet). Further, it was found that the supply of 25OHD₃ (75 nmol/kg diet) for 8 days followed by a 12- to 13-day depletion was suitable for the study; the birds were physiologically robust enough to withstand the stress of the fracture treatment, and at the same time, there were no significant amounts of residual vitamin D₃ metabolites present after 12–13 days of depletion (Table 3).

As shown in Fig. 2, around day 20 when the fracture treatment would be performed in the subsequent experiment, the reduction of circulating levels of the vitamin D₃ metabolites became evident in the depleted groups A and C, as indicated indirectly by the presence of an inflection in the body weight curves. However, the overall body weight was not much lower than that in those birds given 25OHD₃ continuously (groups B and D). Depletion for 8 days greatly reduced the circulating level of 25OHD₃ without adversely affecting the total body growth or bone ash content (Table 3), indicating that these birds would probably survive the fracture surgery.

View larger version (23K): [in this window] [in a new window]   Figure 2. Growth curves of chicks in Exp II using the repletion-depletion model. Chicks were given 25OHD3 (25 or 75 nmol/kg diet) for 8 days or continuously for 29 days. Group E was given 150 nmol vitamin D3 for comparison. At the initiation of the experiment, there were approximately 16 birds in each group, and birds were killed on days 20 and 29 for analysis. Some of the data are presented in Table 3. The levels of the metabolites given are indicated in brackets as nanomoles per kg diet.

View larger version (29K): [in this window] [in a new window]   Figure 3. Growth curves of chicks in Exp III concerning fracture healing. One-day-old chicks were given 25OHD3 (75 nmol/kg diet) for 8 days, and 25OHD3 was withdrawn until the day of the fracture surgery for all groups except group A. After 12 days of depletion, group B was killed to serve as a vitamin D-deficient diet control and to document the levels of the vitamin D metabolites remaining before the repletion phase of the experiment. All remaining birds (except group A) experienced imposition of a right tibial fracture on day 22 and were randomly divided to create groups C, D, E, F, and G, which received the indicated (see figure) vitamin D metabolite either alone or in combination. Details are described in Materials and Methods. The levels of the metabolites given are indicated in brackets as nanomoles per kg diet. At least 10–12 birds were available from each group at the end of the experiment.

The mechanical properties of the healing tibial fracture calluses for Exp III are presented in Tables 4 and 5. As it was not possible to determine bone ash on the healed tibia that was used for assessment of mechanical properties, the bone ash measurement was carried out on the ipsilateral tibia. It has been reported that most bone parameters are affected similarly due to the systemic effects of osteogenic factors produced after fracture treatment (33). The bone ash content and the torsional strength (Table 4) and size measurement (Table 5) of the fractured tibiae of group A (continuous 25OHD₃) and group C (intermittent 25OHD₃) were similar. Although group B, which served as the vitamin D-deficient diet control group, had a much higher ash content (27.5 ± 0.5%) than group D (19.6 ± 0.7%), the strength of the fracture callus was much lower due to the small size of the bone (Table 5), as these birds were killed at a younger age (16 days earlier). Statistical analysis of the linear regression between bone ash and strength measurements excluded group B, as no fractures were produced in this group. The rank order of the strength and stiffness was B < D < G < E F < C A and was highly correlated to the ash content (r² > 71%). The site of failure in the tibiae produced by these tests occurred through both the fracture calluses as well as the adjacent intact bone. Although the length of the tibiae in groups C, E, and F, all of which experienced both the depletion-repletion protocol as well as a fracture, were comparable (75–100%) to the length of the tibiae of the birds that received continuous 25OHD₃ but did not experience a fracture (group A), the antero-posterior and mediolateral lengths of the tibiae in groups D, E, and F were substantially less (40–50%) than those of the tibiae in the birds of group A (Table 5).

The serum levels of Ca²⁺ and the various vitamin D₃ metabolites are shown in Table 6. The serum calcium level was normal in all groups except group B (25OHD₃ for 8 days), group D [1,25-(OH)₂D₃], and group G [24S,25-(OH)₂D₃ plus 1,25-(OH)₂D₃], as expected. Despite the 12-day depletion period, the levels of the three vitamin D₃ metabolites measured in group C (the birds that received 25OHD₃ after fracture) at the end of the experiment were not different from those in group A (birds fed 25OHD₃ continuously), which was indirectly indicated by the similar growth curves of the two groups after the replacement period (see Fig. 3). With the exceptions of the two groups that received 25OHD₃ (groups A and C), the level of 25OHD₃ in other groups was negligible. This indicates that the 12- to 13-day interval during which no 25OHD₃ was administered effectively depleted the plasma concentration of this vitamin D metabolite. In terms of plasma 24,25-(OH)₂D₃, the groups fed 25OHD₃ (groups A and C), 24R,25-(OH)₂D₃ (group F), and 24S,25-(OH)₂D₃ (group G) were all within the normal range (3–5 nM), whereas the other groups (B, D, and E) had insignificant levels. Interestingly, in group E, which was fed 1,25-(OH)₂D₃ at a 3 times higher level than other groups, the serum level of 1,25-(OH)₂D₃ was not significantly different from those in groups D and G. This result is compatible with the suggestion that 1,25-(OH)₂D₃ is metabolized to 1,24R,25-(OH)₃D₃, which is an analog of 24R,25-(OH)₂D₃ (34, 35). The enhancing effect of the higher administered dietary level of 1,25-(OH)₂D₃ in group E vs. group F (3.6 vs. 1.2 nmol/kg diet) on bone ash and bone strength probably cannot be directly attributed to 1,25-(OH)₂D₃ because there was not an increase in the plasma concentration of 1,25-(OH)₂D₃.

	Discussion

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Despite very active research in the area of bone biology in recent years, the biological role of 24R,25-(OH)₂D₃ is not yet clearly understood. In that context, the present study was undertaken to evaluate the role of 24R,25-(OH)₂D₃ in both normal bone development and fracture healing. The primary approach was to use two chick models in which 24R,25-(OH)₂D₃, 24S,25-(OH)₂D₃, and 1,25-(OH)₂D₃, alone or in combination, were provided via diet supplementation over 2–4 weeks so as to achieve serum levels of 24,25-(OH)₂D₃ or 1,25-(OH)₂D₃ that are in the known physiological range and then to assess their relative contributions to body growth, serum Ca²⁺, and several bone strength parameters as described by Einhorn and colleagues (36).

The results of Exp I show that 24R,25-(OH)₂D₃ alone over 4 weeks is sufficient to achieve body growth and bone integrity equivalent to a vitamin D₃-replete group or the group given 24R,25-(OH)₂D₃ in combination with 1,25-(OH)₂D₃. These results extend the early observations of Henry et al. (37) by virtue of the comparison of 24R,25-(OH)₂D₃ and 24S,25-(OH)₂D₃ in the presence and absence of 1,25-(OH)₂D₃. Despite its presence in the serum within the normal range, the administration of 1,25-(OH)₂D₃ alone for 4 weeks failed to support normal growth and bone integrity in the chick. Animals treated with 1,25-(OH)₂D₃ alone were hypocalcemic in both Exp I and III (groups C and D), suggesting that 1,25-(OH)₂D₃ or its subsequent metabolites cannot replace the actions of 24R,25-(OH)₂D₃. However, the addition of 1,25-(OH)₂D₃ to 24R,25-(OH)₂D₃ improved the serum calcium level in Exp I and III (groups F and F). Previous studies have also indicated that a combination of 24R,25-(OH)₂D₃ and 1,25-(OH)₂D₃ was required to achieve completely normal responses; these include egg hatchability (10, 12), calcium and phosphate homeostasis (38), and treatment of rickets (39).

One of the complications of the present study is that both 1,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ can be metabolized to the same daughter metabolite, namely 1,24R,25-(OH)₃D₃; furthermore, it is a reasonable postulate that 1,24R,25-(OH)₃D₃ is a good analog of both 1,25-(OH)₂D₃ and 24R,25-(OH)₂D₃. Thus, a complication in designing the protocols that involved the administration of a single dihydroxylated metabolite was to avoid doses of either 24R,25-(OH)₂D₃ or 1,25-(OH)₂D₃ that were on the high side of the normal range so as to minimize the presence of 1,24R,25-(OH)₃D₃. There are only a limited number of reports describing the biological activities of 1,24R,25-(OH)₃D₃, and these studies were limited exclusively to short term, rather than chronic, administration of this trihydroxylated metabolite (34, 35, 40). Another complication raised by seco steroid metabolism is the possible conversion of 24R,25-(OH)₂D₃ to 25(OH)-24-oxo-D₃ and then to either 1,25-(OH)₂-24-oxo-D₃ or 23,25-(OH)₂-24-oxo-D₃ (41). Although these metabolites are known to be produced in vivo (42), they are not believed to circulate in high concentrations in the serum. It remains to future studies to define the extent of metabolism of 24R,25-(OH)₂D₃ and the serum concentrations of the metabolites that may be achieved.

Although there is some possibility that 24S,25-(OH)₂D₃ could be converted to 24R,25-(OH)₂D₃ in vivo (43), in the present study in Exp I and III, 24S,25-(OH)₂D₃ at the doses administered was not able to generate normal biological effects. 24R,25-(OH)₂D₃ has a chiral center at carbon 24. The naturally occurring form of 24,25-(OH)₂D₃ has its hydroxyl on carbon 24 in the R orientation. If the biological responses mediated by 24R,25-(OH)₂D₃ involve interaction of the ligand with a receptor, it seems reasonable that the putative receptor could distinguish between the naturally occurring metabolite, which has a 24R-hydroxyl, from the unnatural metabolite, in which the hydroxyl is 24S. Thus, although our results are similar to those reported by others (29, 44, 45), confirming our hypothesis that this unnatural compound is biologically inactive or that only a very small proportion of it is converted to the naturally occurring 24R,25-(OH)₂D₃, a new contribution reported here for the first time is that 24S,25-(OH)₂D₃ is not able to support the development of bone integrity with normal strength parameters (groups E and G).

Another intriguing observation concerns the level of 24,25-(OH)₂D₃ present in the serum of the groups given 24R,25-(OH)₂D₃ or 24S,25-(OH)₂D₃ in combination with 1,25-(OH)₂D₃ (groups F and G); the levels of 24,25-(OH)₂D₃ were higher in the groups fed combinations than in the groups fed 24R,25-(OH)₂D₃ or 24S,25-(OH)₂D₃ alone (groups D and E). Whether the addition of 1,25-(OH)₂D₃ retarded the further metabolism of 24R,25-(OH)₂D₃/24S,25-(OH)₂D₃ that is administered is not known. However, it is possible that in the group given 24R,25-(OH)₂D₃ or 24S,25-(OH)₂D₃ alone, a higher proportion of the administered metabolite could have been metabolized to metabolites with a 1-hydroxyl group, as some vitamin D₃ metabolites with 1-hydroxyl group [for example, 1,24R,25-(OH)₃D₃] can mimic biological actions of 1,25-(OH)₂D₃ by binding to its nuclear receptor with partial affinity (46).

The present study demonstrates that 24R,25-(OH)₂D₃ is indispensable for the healing process of a fracture. We have shown previously that both the renal 25OHD₃-24-hydroxylase activity and the circulating 24,25-(OH)₂D₃ levels are highly elevated 10–12 days after fracture treatment in chicks (47, 48). This suggests that more 24R,25-(OH)₂D₃ is required during this period. Even the high level of 1,25-(OH)₂D₃ (Exp III, group E) could not reach the strength value of the control group fed 25OHD₃, suggesting that 1,25-(OH)₂D₃ alone cannot mediate the complete spectrum of biological actions on bone development. There is also a possibility that we did not use a high enough level of 1,25-(OH)₂D₃ to achieve the best results, because even with the higher level of 1,25-(OH)₂D₃, the animals did not achieve as high a serum calcium level as that of the control group, and the circulating level of 1,25-(OH)₂D₃ was only half that in the control group. However, this might be equally true for 24R,25-(OH)₂D₃; for example, the strength of the fractured tibiae from the birds given 24R,25-(OH)₂D₃ plus 1,25-(OH)₂D₃ (Exp III, group F) was lower than the strength of those from control birds given 25OHD₃ (Exp III, group C). This may be simply because, as a control group, we administered a much higher level of 25OHD₃ (75 nmol/kg diet) in Exp III than of vitamin D₃ (50 nmol/kg diet) in Exp I that was metabolized to a lower amount of 25OHD₃. Furthermore, in separate experiments (data not shown), a higher level of 1,25-(OH)₂D₃ resulted in reduction of bone ash content and a further blunting of body growth.

It has been reported that 1,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ have selective effects on different stages of cartilage development (24, 49) and opposite actions on bone resorption (29) or PTH secretion (50). It seems obvious that these two metabolites have distinct actions on different cell types. The fracture-healing process is complex, involving many cellular reactions and interactions, including an inflammatory response and the sequential differentiation of mesenchymal cells to form fibroblasts, chondrocytes, and osteoblasts.

The results of the present study, which demonstrate the biological effectiveness of 24R,25-(OH)₂D₃ are consistent with a recent study that demonstrated that homozygous mice in whom the 24-hydroxylase gene had been knocked out had abnormalities in bone structure and intramembranous ossification that led to their death shortly after weaning (51). The results of this study suggest that in the absence of a functional 25OHD₃-24-hydroxylase, which would block the production of 24R,25-(OH)₂D₃, there is an absence of an essential vitamin D metabolite for normal bone development.

In summary, the present studies demonstrate that physiological concentrations of the naturally occurring vitamin D metabolite 24R,25-(OH)₂D₃ is essential for both normal bone development and fracture healing. Studies are currently in progress to identify the nature of the cellular interactions and the details of the involved signal transduction pathway.

	Acknowledgments

 
We thank Dr. Charles Huszar for his advice with the statistical analysis, Ms. Tiana Michel for her assistance with the bone ash determinations, and Prof. Helen Henry for her extensive advice and comments.

	Footnotes

 
¹ This work was supported in part by USPHS Grant DK-09012–032.

Received March 21, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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