Isolation and identification of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 10(-6) M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivatization, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23,25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1 alpha,25-dihydroxy[1 beta-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.     

Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway

About a decade ago calcitroic acid was isolated as a major side chain cleaved water-soluble metabolite of 1,25-dihydroxyvitamin D3 [Esvelt, R. P., Schnoes, H. K., & Decula, H. F. (1979) Biochemistry 18, 3977]. Presently, calcitroic acid is being considered as the major excretory form of 1,25-dihydroxyvitamin D3. However, the exact site or sites of calcitroic acid production and the possible side chain modified intermediary metabolites that may be formed during the conversion of 1,25-dihydroxyvitamin D3 into calcitroic acid are not fully understood. In the mean time there have been many advances in our understanding of the side-chain metabolism of 1,25-dihydroxyvitamin D3. It is now well established that both the kidney and the intestine metabolize 1,25-dihydroxyvitamin D3 through the C-24 oxidation pathway according to the following steps: 1,25-dihydroxyvitamin D3----1,24,25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3-----1,23,25-trihydroxy-24-oxovitamin D3. Recently, we identified 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-23 alcohol) as a major side chain cleaved lipid-soluble metabolite of 1,25-dihydroxyvitamin D3 and further extended the aforementioned C-24 oxidation pathway in the kidney by demonstrating 1,23,25-trihydroxy-24-oxovitamin D3 as the precursor of C-23 alcohol [Reddy, G. S., Tserng, K. Y., Thomas, B. R., Dayal, R., & Norman, A. W. (1987) Biochemistry 26, 324]. In this present study, we investigated the metabolic fate of 1,25-dihydroxyvitamin D3 (3 X 10(-10) M) in the perfused rat kidney and identified calcitroic acid as the major water-soluble metabolite of 1,25-dihydroxyvitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)     

C(24)- and C(23)-oxidation, converging pathways of intestinal 1,25-dihydroxyvitamin D3 metabolism: identification of 24-keto-1,23,25-trihydroxyvitamin D3

24-Keto-1,23,25-trihydroxyvitamin D3 has been identified as a major 1,25-dihydroxyvitamin D3 metabolite, produced by intestinal mucosa cells isolated from rats dosed chronically with 1,25-dihydroxyvitamin D3. The identification was based on ultraviolet absorbance spectroscopy, mass spectroscopy, and chemical derivatization. The pathway of biosynthesis proceeded through 1,24,25-trihydroxyvitamin D3 and 24-keto-1,25-dihydroxyvitamin D3, which are physiological metabolites of 1,25-dihydroxyvitamin D3. Previous work [Napoli, J. L., Pramanik, B. C., Royal, P. M., Reinhardt, T. A., & Horst, R. L. (1983) J. Biol. Chem. 258, 9100-9107] had shown that the amount of 24-keto-1,23,25-trihydroxyvitamin D3 in intestine in vivo, relative to its C(24)-oxidized precursors, is enhanced by chronically dosing rats with 1,25-dihydroxyvitamin D3. These results establish the C(24)-oxidation pathway as a predominant route of intestinal 1,25-dihydroxyvitamin D3 metabolism under physiological conditions and indicate that treatment of the rat with exogenous 1,25-dihydroxyvitamin D3 causes expression of C(23)-hydroxylase activity, which uses C(24)-oxidized 1,25-dihydroxyvitamin D3 metabolites as substrates.     

Intestinal synthesis of 24-keto-1,25-dihydroxyvitamin D3. A metabolite formed in vivo with high affinity for the vitamin D cytosolic receptor

24-Keto-1,25-dihydroxyvitamin D3 has been identified as an intestinal metabolite of 1,25-dihydroxyvitamin D3 by ultraviolet absorbance, mass spectroscopy, and chemical reactivity. The metabolite was produced from 1,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3 in rat intestinal mucosa homogenates. 24-Keto-1,25-dihydroxyvitamin D3 is present in vivo in the plasma and small intestinal mucosa of rats fed a stock diet, receiving no exogenous 1,25-dihydroxyvitamin D3, and in the plasma and small intestinal mucosa of rats dosed chronically with 1,25-dihydroxyvitamin D3. 24-Keto-1,25-dihydroxyvitamin D3 has affinity equivalent to 1,24R,25-trihydroxyvitamin D3 for the 3.7 S cytosolic receptor specific for 1,25-dihydroxyvitamin D3 in the intestine and thymus. In cytosolic preparations contaminated with the 5 S vitamin D-binding protein, both metabolites are about 7-fold less potent than 1,25-dihydroxyvitamin D3. In contrast, in cytosolic preparations largely free of the 5 S binding protein, both metabolites are equipotent with the parent compound. No evidence was obtained supporting a substantial presence of 23-keto-1,25-dihydroxyvitamin D3 in vivo; nor was the latter compound generated in detectable amounts from 1,25-dihydroxyvitamin D3 by intestinal homogenates. Thus, C-24 oxidation is a significant pathway of intestinal 1,25-dihydroxyvitamin D3 metabolism that produces metabolites with high affinity for the cytosolic receptor which mediates vitamin D action.     

Physiological significance of C-28 hydroxylation in the metabolism of 1alpha,25-dihydroxyvitamin D(2).

In our previous study, we indicated for the first time that C-28 hydroxylation plays a significant role in the metabolism of 1alpha, 25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] by identifying 1alpha,24(S),25,28-tetrahydroxyvitamin D(2) [1alpha,24(S),25, 28(OH)(4)D(2)] as a major renal metabolite of 1alpha,25(OH)(2)D(2) [G. S. Reddy and K-Y. Tserng Biochemistry 25, 5328-5336, 1986]. The present study was performed to establish the physiological significance of C-28 hydroxylation in the metabolism of 1alpha, 25(OH)(2)D(2). We perfused rat kidneys in vitro with 1alpha, 25(OH)(2)[26,27-(3)H]D(2) (5 x 10(-10)M) and demonstrated that 1alpha,24(R),25-trihydroxyvitamin D(2) [1alpha,24(R),25(OH)(3)D(2)] and 1alpha,24(S),25,28(OH)(4)D(2) are the only two major physiological metabolites of 1alpha,25(OH)(2)D(2). In the same perfusion experiments, we also noted that there is no conversion of 1alpha,25(OH)(2)D(2) into 1alpha,25,28-trihydroxyvitamin D(2 )[1alpha,25,28(OH)(3)D(2)]. Moreover, 1alpha,24(S),25,28(OH)(4)D(2) is not formed in the perfused rat kidney when synthetic 1alpha,25, 28(OH)(3)D(2) is used as the starting substrate. This finding indicates that C-28 hydroxylation of 1alpha,25(OH)(2)D(2) occurs only after 1alpha,25(OH)(2)D(2) is hydroxylated at C-24 position. At present the enzyme responsible for the C-28 hydroxylation of 1alpha, 24(R),25(OH)(3)D(2) in rat kidney is not known. Recently, it was found that 1alpha,25(OH)(2)D(3)-24-hydroxylase (CYP24) can hydroxylate carbons 23, 24, and 26 of various vitamin D(3) compounds. Thus, it may be speculated that CYP24 may also be responsible for the C-28 hydroxylation of 1alpha,24(R),25(OH)(3)D(2) to form 1alpha, 24(S),25,28(OH)(4)D(2). The biological activity of 1alpha,24(S),25, 28(OH)(4)D(2), determined by its ability to induce intestinal calcium transport and bone calcium resorption in the rat, was found to be almost negligible. Also, 1alpha,24(S),25,28(OH)(4)D(2) exhibited very low binding affinity toward bovine thymus vitamin D receptor. These studies firmly establish that C-28 hydroxylation is an important enzymatic reaction involved in the inactivation of 1alpha,25(OH)(2)D(2) in kidney under physiological conditions.     

1 Alpha-25,26-trihydroxyvitamin D3: an in vivo and in vitro metabolite of vitamin D3

A new metabolite of vitamin D3 has been isolated from the plasma of vitamin D3 treated cows and has been generated from 25(S),26-dihydroxyvitamin D3 with homogenates of vitamin D deficient chick kidney. This metabolite has been identified as 1,25,26-trihydroxyvitamin D3 by comigration with synthetic 1,25(S),26-trihydroxyvitamin D3 in four chromatographic systems, ultraviolet spectroscopy, mass spectrometry, and high-pressure liquid chromatography and mass spectrometry of derivatives. 1,25(S),26-Trihydroxyvitamin D3 is one-tenth as effective as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol 1,25-dihydroxyvitamin D receptor. Either 25(S),26-dihydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 can serve as precursor for in vitro production of 1,25,26-trihydroxyvitamin D3 by chick kidney tissue.     

Isolation and identification of 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3. New metabolites of vitamin D3 produced by a C-24 oxidation pathway of metabolism for 1,25-dihydroxyvitamin D3 present in intestine and kidney

Two new vitamin D metabolites were isolated in pure form from separate incubations of homogenates of chick small intestinal mucosa or rat kidney employing either 1 alpha,25-dihydroxyvitamin D3 (28 microM) or 1 alpha,24R,25-trihydroxyvitamin D3 as substrate (0.17-1.3 microM). The newly characterized compounds and the amounts isolated in pure form from separate isolations are, respectively: 1 alpha,25-dihydroxy-24-oxo-vitamin D3 (1,25(OH)2-24-oxo-D3), 147 micrograms from kidney and 4.2 and 40 micrograms from intestine; 1 alpha,23,25-trihydroxy-24-oxo-vitamin D3 (1,23,25(OH)3-24-oxo-D3), 155 micrograms from kidney and 5.9 and 34 micrograms from intestine. Their structures were identified after extensive high pressure liquid chromatography by means of ultraviolet absorption spectrometry, mass spectrometry of the free compounds and their trimethylsilylated derivatives, proton nuclear magnetic resonance spectrometry, specific chemical reduction of the 24-oxo functionality with sodium borohydride, as well as direct comparison with synthetic 1,25(OH)2-24-oxo-D3. These structural assignments for both compounds correct previous determinations which had been proposed (Ohnuma, N., Kruse, J. R., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). The activity of the C-24 oxidation pathway used for the production of the 1,25(OH)2-24-oxo-D3 and 1,23,25(OH)3-24-oxo-D3 can be enhanced 10-fold by prior priming of the chicks or rats with a single intravenous dose of 1,25(OH)2D3 (1-12 nmol/100 g body weight); the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels within 12 h. Further, 1,25(OH)2D3, 1,24,25(OH)3D3, and 1,25(OH)2-24-oxo-D3 all were found to be capable of serving as a precursor with chick intestine and rat kidney homogenates of 1,23,25(OH)3-24-oxo-D3. Collectively these results suggest the existence of a C-24 oxidation pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney to 1,23,25(OH)3-24-oxo-D3. The pathway may play an important role in controlling the tissue levels of this hormonally active form of vitamin D3.     

Effect of dietary calcium and phosphorus on intestinal calcium absorption and vitamin D metabolism.

To understand better dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin D₃ and diets differing in calcium and phosphorus content was made. All known vitamin D metabolites were ultimately identified by high-pressure liquid chromatography. In addition to the known metabolites (25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃, and 1,24,25-trihydroxyvitamin D₃), several new and unidentified metabolites were found. In addition to 1,25-dihydroxyvitamin D₃ and 1,24,25-trihydroxyvitamin D₃, the levels of some of the unknown metabolites could be correlated with intestinal calcium transport. However, whether or not any of these metabolites plays a role in the stimulation of intestinal calcium absorption by low dietary calcium or low dietary phosphorus remains unknown.     

24,26-Dihydroxyvitamin D2: a unique physiological metabolite of vitamin D2

A new vitamin D2 metabolite, 24,26-dihydroxyvitamin D2, has been detected in the plasma of rats fed physiologic amounts of vitamin D2. The identity of the new metabolite (isolated from cow plasma) was established by ultraviolet absorbance, mass spectroscopy, chemical reactivity, and NMR spectroscopy. Among these, the mass spectrum was unique for the presence of a peak at M-48 that was attributed to an intramolecular rearrangement involving both the C-24 and C-26 hydroxyl groups. A 300-MHz 1H NMR spectrum of 40 micrograms of metabolite indicated a downfield shift of the C-28 methyl group signal to delta 1.30 and a multiplet at delta 3.66 corresponding to the hydroxylated C-26 methyl group. We determined that the formation of 24,26-dihydroxyvitamin D2 represented a major pathway for further metabolism of 24-hydroxyvitamin D2 in rats, exceeding the formation of 24,25-dihydroxyvitamin D2. Standard bioassays revealed that 24,26-dihydroxyvitamin D2 possessed very little biological activity and most likely represents a deactivation pathway for 24-hydroxyvitamin D2.     

24-Hydroxylation of 1,25-dihydroxyergocalciferol. An unambiguous deactivation process

1,24,25-Trihydroxyergocalciferol was isolated from bovine kidney homogenates incubated with 1,25-dihydroxyergocalciferol and from chick kidney homogenates incubated with 24,25-dihydroxyergocalciferol. The identity was established by ultraviolet absorbance, sensitivity to periodate, nuclear magnetic resonance, and mass spectrometry. The new metabolite had an affinity equal to 1,24,25-trihydroxycholecalciferol for the bovine-thymus and chick-intestinal 1,25-dihydroxyvitamin D receptor and had an affinity twice that of 1,24,25-trihydroxycholecalciferol for the rat-intestinal receptor. It was 3- and 6-fold less competitive than either 1,25-dihydroxycholecalciferol or 1,24,25-trihydroxycholecalciferol, respectively, for the rat plasma vitamin D transport protein. 1,24,25-Trihydroxyergocalciferol was at least 10-fold less active than 1,25-dihydroxycholecalciferol, 1,25-dihydroxyergocalciferol, and 1,24,25-trihydroxycholecalciferol at stimulating intestinal-calcium transport and was also relatively ineffective at stimulating bone-calcium resorption in rats. Moreover, in rats, [3H]1,24,25-trihydroxyergocalciferol was cleared from plasma approximately 40% faster than [3H]1,24,25-trihydroxycholecalciferol. These data suggest that C-24 hydroxylation of 1,25-dihydroxyergocalciferol represents a significant in vivo deactivation step, whereas equivalent deactivation of 1,25-dihydroxycholecalciferol seems to involve metabolic steps subsequent to C-24 hydroxylation (C-24 ketonization). C-24 ketonization of 1,25-trihydroxyergocalciferol would not be anticipated due to the presence of the 24(S)-methyl group. These results reveal further dissimilarities between ergocalciferol and cholecalciferol metabolism in mammals and suggest a mechanism for the lesser tendency of ergocalciferol to cause hypercalcemia relative to cholecalciferol.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

Isolation and identification of vitamin D3, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 in Solanum malacoxylon incubated with ruminal fluid.

It has been shown that Solanum malacoxylon contains 1 alpha,25-dihydroxyvitamin D3-glycoside. The presence of vitamin D3 and 25-hydroxyvitamin D3 has also been suggested. In the present study vitamin D3 and three of its metabolites, including 1 alpha,25-dihydroxyvitamin D3, were detected in plant leaf extracts preincubated with ruminal fluid (SMRF). Extraction of SMRF with non-polar organic solvents and purification of the lipid extract by TLC followed by HPLC yielded nine ultraviolet-absorbing (264 nm) peaks. Four of them comigrated on a Zorbax-Sil HPLC column with synthetic standards of vitamin D3, 25-hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3, respectively. These compounds were unequivocally identified by means of mass spectrometry. The results confirm that Solanum malacoxylon contains, in addition to 1 alpha,25-dihydroxyvitamin D3, vitamin D3, 25-hydroxyvitamin D3 and possibly other as yet unidentified derivatives. As 1,24,25-trihydroxyvitamin D3 is absent in plant extracts not incubated with ruminal fluid, the data also indicate that rumen microbes may convert 1 alpha,25-dihydroxyvitamin D3 into 1,24,25-trihydroxyvitamin D3.     

Synthesis and biological activity of 1 alpha,23,25,26-tetrahydroxyvitamin D3

The metabolic pathway from 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone includes the formation of 1 alpha,23,25-26-tetrahydroxyvitamin D3 [1 alpha,23,25,26-(OH)4D3]. The aim of the current study was to explore the as yet unknown biological properties of this vitamin D3 sterol. The four diastereoisomers of 1 alpha,23,25,26-(OH)4D3 were chemically synthesized. They were compared to 1 alpha,25-(OH)2D3 in terms of their affinity for the chick intestinal 1 alpha,25-(OH)2D3 receptor and their biologic activity in vivo (stimulation of intestinal calcium absorption and mobilization of calcium from bone in vitamin D-deficient rats). The 1,25-(OH)2D3 receptor binding affinities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4 D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 were 11, 100, 216, and 443 times weaker than the binding affinity of 1 alpha,25-(OH)2D3, respectively. Compared to 1 alpha,25-(OH)2D3, the relative capacities of the 1 alpha,23,25,26-(OH)4D3 compounds to stimulate intestinal calcium absorption were 1/4 for 1 alpha,23(R)25(R)26-(OH)4D3; 1/19 for 1 alpha,23(S)25(S)26-(OH)4D3; 1/90 for 1 alpha,23(S)25(R)26-(OH)4D3; and 1/136 for 1 alpha,23(R)25(S)26-(OH)4D3. Maximal stimulation of intestinal calcium transport occurred 8 h after administration of vitamin D3 metabolites. Mobilization of calcium from bone was quantitated by serum calcium concentration measurements. The activities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 to increase serum calcium were estimated to be 4, 13, 43, and 69 times weaker than that of 1 alpha,25-(OH)2D3, respectively. These results illustrate the stereospecificity of the chicken intestine 1 alpha,25-(OH)2D3 receptor for binding of 1 alpha,23,25,26-(OH)4D3 and suggest that the 1 alpha,23,25,26-(OH)4D3 exerts its biological activity in the rat through an interaction with 1,25-(OH)2D3 receptors. In summary, the 1 alpha,23,25,26-(OH)4D3 had a markedly lower biological activity than 1 alpha,25-(OH)2D3.     

Metabolism and pharmacokinetics of 24,25-dihydroxyvitamin D3 in the vitamin D3-replete rat

The time course of in vivo metabolism of 24,25-dihydroxyvitamin D3 in rats has been examined. Several tissues were surveyed in an effort to discover new metabolites of 24,25-dihydroxyvitamin D3 and to estimate the concentrations of previously identified metabolites. Rapidly growing male rats were dosed with 24,25-dihydroxyvitamin D3 orally until plasma concentrations of 24,25-dihydroxyvitamin D3 were at steady state. 24,25-Dihydroxyvitamin [3-3H]D3 was then administered. At 10 min and 1, 6, 15, 24, 96, and 192 h after dosing, the animals were killed, and plasma, liver, intestine, and bones were analyzed with a newly developed gradient straight-phase high performance liquid chromatography system. The high performance liquid chromatography system is capable of base-line resolution of most of the major vitamin D metabolites. 24,25-Dihydroxyvitamin D3 clearance from plasma, liver, and kidney but not intestine followed a two-compartment model. 24,25-Dihydroxyvitamin D3 disappeared from plasma with a half-life of 0.55 h (fast phase) and 73.8 h (slow phase). Only two lipid-soluble metabolites of 24,25-dihydroxyvitamin D3 were detected: 24-oxo-25-hydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3. These compounds circulate at very low concentrations in the plasma (50 pg/ml of plasma).     

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.     

Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney

1,25-Dihydroxyvitamin D3 induces both 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3- 24-hydroxylase activities. However, whether 24-hydroxylation of these substrates is catalyzed by a single enzyme is unknown. We have examined the substrate specificity of the enzyme using the solubilized and reconstituted chick renal mitochondrial 24-hydroxylase enzyme system. The soluble enzyme catalyzes 24-hydroxylation of both substrates. The apparent Km of the 24-hydroxylase for 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 were 1.47 and 0.14 microM, respectively. Kinetic studies demonstrated that 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 act as competitive inhibitors with respect to each other. 1,25-Dihydroxyvitamin D3 inhibited the production of 24,25-dihydroxyvitamin D3 with an apparent Ki of 0.09 microM and 25-hydroxyvitamin D3 inhibited the production of 1,24,25-trihydroxyvitamin D3 with an apparent Ki of 3.9 microM. These results indicate that chick 24-hydroxylase preferentially hydroxylates 1,25-dihydroxyvitamin D3 and support the idea that the 24-hydroxylation of these substrates is catalyzed by a single enzyme.     

24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2: novel metabolites of vitamin D2

Understanding of the inactivation pathways of 25-hydroxyvitamin D2 and 24-hydroxyvitamin D2, the two physiologically significant monohydroxylated metabolites of vitamin D2, is of importance, especially during hypervitaminosis D2. In a recent study, it has been demonstrated that the inactivation of 24-hydroxyvitamin D2 occurs through its conversion into 24,26-dihydroxyvitamin D2 [Koszewski, N.J., Reinhardt, T.A., Napoli, J.L., Beitz, C.D., & Horst, R.L. (1988) Biochemistry 27, 5785]. At present, little information is available regarding the inactivation pathway of 25-hydroxyvitamin D2 except its further metabolism into 24,25-dihydroxyvitamin D2 [Jones, G., Rosenthal, A., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Rabinovich, D., & Shakked, Z. (1979) Biochemistry 18, 1094]. In our present study, we investigated the metabolic fate of 25-hydroxyvitamin D2 in the isolated perfused rat kidney and demonstrated its conversion not only into 24,25-dihydroxyvitamin D2 but also into two other new metabolites, namely, 24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2. The structure identification of the new metabolites was established by the techniques of ultraviolet absorption spectrophotometry and mass spectrometry and by the characteristic nature of each new metabolite's susceptibility to sodium metaperiodate oxidation. In order to demonstrate the physiological significance of the two new trihydroxy metabolites of vitamin D2, we induced hypervitaminosis D2 in a rat using [3 alpha-3H]vitamin D2 and analyzed its plasma for the various [3 alpha-3H]vitamin D2 metabolites on two different high-pressure liquid chromatography systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] (0.43 microM). Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3). Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.