Production of C-24- and C-23-oxidized metabolites of 1,25-dihydroxycholecalciferol by cultured kidney cells (LLC PK1) and their presence in kidney in vivo.

1,25-Dihydroxy[3H]cholecalciferol was converted into several more-polar metabolites by a cultured pig kidney cell line (LLC PK1). The production of metabolites was stimulated by pretreating the cells with unlabelled 1,25-dihydroxycholecalciferol. A similar profile of metabolites was observed on high-pressure-liquid-chromatographic analysis of an extract from the kidneys of rats dosed intravenously with 1,25-dihydroxy[3H]cholecalciferol. Among the metabolites detected were 1,24,25-trihydroxycholecalciferol, 1,25-dihydroxy-24-oxocholecalciferol, 1,23,25-trihydroxy-24-oxocholecalciferol and 1,25-dihydroxycholecalciferol-26,23-lactone. The results are in accord with data reported for intestinal 1,25-dihydroxycholecalciferol metabolism [Napoli, Pramanik, Royal, Reinhardt & Horst (1983) J. Biol. Chem. 258, 9100-9107]. These data indicate that C-23- and C-24-oxidation of 1,25-dihydroxycholecalciferol are phenomena common to calciferol target tissues, and that regulation of 1,25-dihydroxycholecalciferol homoeostasis is dependent on the rate of its metabolism in addition to the rate of its synthesis.     

Comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites

The comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites were investigated to evaluate: (1) optimization of sensitivity (2) crossreactivity of these compounds in their respective assays and (3) side chain steric requirements of the vitamin D molecule for optimum binding to the calciferol binding protein or bovine thymus receptor. Disequilibrium assay conditions improved assay sensitivity 30-fold for the calciferol assay and approx 3-fold for metabolites in the 25-hydroxycalciferol and 1,25-dihydroxycalciferol assays. Ergocalciferol compounds were uniformly less efficient in their association with the proteins tested than were their cholecalciferol counterparts, with one exception. In the calciferol assay, cholecalciferol had greater affinity for the the calciferol binding protein than did ergocalciferol. In the 25-hydroxycalciferol assay affinity for the calciferol binding protein was 25-hydroxycholecalciferol = 24,25-dihydroxycholecalciferol greater than 25-hydroxyergocalciferol greater than 25S,26-dihydroxycholecalciferol greater than 24,25-dihydroxyergocalciferol greater than 25,26-dihydroxyergocalciferol. In the assay for 1,25-dihydroxycalciferol, bovine thymus receptor recognized 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol equally. From the forthcoming data it appears that hydroxyl and/or methyl groups on the calciferol side chain alter the ability of these physiological compounds to associate with the calciferol binding protein.     

Maternal-perinatal interrelationships of vitamins D metabolism in rats

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite that ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1.Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet.Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 5/25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected.Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol.Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

Isolation and identification of 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2: new metabolites of 1,25-dihydroxyvitamin D2 produced in rat kidney

Three new metabolites of vitamin D2 were produced in vitro by perfusing isolated rat kidneys with 1,25-dihydroxyvitamin D2. They were isolated and purified from the kidney perfusate by the techniques of methanol-methylene chloride lipid extraction and high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, and specific chemical reactions, the metabolites were identified as 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2. Both 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2 were also produced when a kidney was perfused with 1,24,25-trihydroxyvitamin D2. Thus, it becomes clear that 1,25-dihydroxyvitamin D2 is first hydroxylated at C-24 to form 1,24,25-trihydroxyvitamin D2, which is then further hydroxylated at C-28 and C-26 to form 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2, respectively. From several recent studies, it has been well established that 1,25-dihydroxyvitamin D3 is converted into various further metabolites in the kidney as a result of chemical reactions such as C-23, C-24, and C-26 hydroxylations, C-24 ketonization, and C-23:C-26 lactonization. From our study it is obvious that 1,25-dihydroxyvitamin D2 does not undergo all of the aforementioned chemical reactions except C-24 and C-26 hydroxylations. Also, our study indicates that C-28 hydroxylation plays a significant role in the further metabolism of 1,25-dihydroxyvitamin D2. Thus, for the first time, we describe a novel further metabolic pathway for 1,25-dihydroxyvitamin D2 in a mammalian kidney.     

Diastereoselective synthesis, binding affinity for vitamin D receptor, and chiral stationary phase chromatography of hydroxy analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol.

A series of analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol were obtained with an additional hydroxyl in the aliphatic side chain at carbon atom C-24. These analogs were synthesized by direct and diastereo-selective alpha-hydroxylation of enolates derived from respective vitamin D esters using Davies chiral oxaziridines. The use of (+)-(2R,8aS)-(8, 8-dichlorocamphoryl)sulfonyl oxaziridine resulted in (R) stereochemistry of the new asymmetric center for both series of analogs. Similarly, (-)-(2S,8aR) oxaziridine gave (S) analogs. The diastereomeric purity of hydroxy analogs was determined by high-performance liquid chromatography on a chiral stationary phase. High diastereopurity of hydroxylation of vitamin D esters was obtained without the use of any chiral auxiliary. The binding affinity of (24R)-1,24,25-trihydroxycholecalciferol for the calf thymus intracellular vitamin D receptor was one order of magnitude higher than that of the respective (24S)-diastereomer.     

Maternal-perinatal interrelationships of vitamin D metabolism in rats.

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite the ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1. Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet. Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected. Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol. Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

(23S)-1,23,25-Trihydroxycholecalciferol, an intestinal metabolite of 1,25-dihydroxycholecalciferol.

(23S)-23,25-Dihydroxycholecalciferol was converted into a polar metabolite in a calciferol-deficient chick kidney homogenate. The metabolite was identified as (23S)-1,23,25-trihydroxycholecalciferol by absorbance spectroscopy and mass spectrometry, and by formation of derivatives. (23S)-1,23,25-Trihydroxycholecalciferol was also observed as a 1,25-dihydroxycholecalciferol metabolite in intestinal cells isolated from 1,25-dihydroxycholecalciferol-treated rat. The trihydroxy metabolite was 50-fold less potent than 1,25-dihydroxycholecalciferol in the chick intestinal 1,25-dihydroxycholecalciferol receptor assay.     

23, 25, 26-trihydroxycholecalciferol. A 25-hydroxycholecalciferol-26,23-lactone precursor.

(23S)-23,25-Dihydroxycholecalciferol was converted into at least five metabolites in kidney homogenates prepared from 1,25-dihydroxycholecalciferol-treated chickens. One of these has been positively identified as 23,25,26-trihydroxycholecalciferol by u.v.-absorbance analysis, mass spectrometry and chemical formation of derivatives. 23,25,26-Trihydroxycholecaciferol produces 25-hydroxycholecalciferol-26,23-lactone when incubated in chick kidney homogenates.     

A simple method for generation of antibodies with specificity for 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol

A simple method for production of antisera with high affinity and selectivity for 1 alpha, 25-dihydroxyergocalciferol and 1 alpha, 25-dihydroxychole-calciferol is described. 1 alpha-Hydroxy-25,26,27-trisnorcholecalciferol-24-oic acid was coupled directly to bovine serum albumin. Rabbits immunized with this conjugate rapidly produced antibodies that bound 3H-1 alpha,-25-dihydroxycholecalciferol with high affinity and demonstrated nearly equal reactivity with 1 alpha, 25-dihydroxyergocalciferol and poor reactivity with 25-hydroxycholecalciferol; 24,25-dihydroxycholecalciferol; 25,26-dihydroxycholecalciferol; and 1 beta,25-dihydroxycholecalciferol. The use of one of these antisera has led to the development of a specific assay for 1 alpha,25-dihydroxyergocalciferol and 1 alpha,25-dihydroxycholecalciferol in human serum.     

The functional metabolism of vitamin D in chicks fed low-calcium and low-phosphorus diets

Radioactively labelled cholecalciferol was administered continuously to chicks that were fed normal, low-calcium and low-phosphorus diets. It has been possible to show that under such steady state conditions with regard to cholecalciferol, and mineral restriction, the animal reacts by increased accumulation of 1,25-dihydroxycholecalciferol in the intestinal and the kidney cell, which was associated in the intestine with an increased calcium-binding activity. A similar accumulation of 1,25-dihydroxycholecalciferol in bone was not noticed.It is proposed that the intestine and the kidney, but not bone, are the main target organs for cholecalciferol in the maintenance of calcium homeostasis, and that both calcium and phosphorus play a role in the regulation of the formation and subsequent function of 1,25-dihydroxycholecalciferol.     

The metabolism of cholecalciferol in the liver of Japanese quail (Coturnix coturnix japonica) with particular reference to the effects of oestrogen.

1. Studies were carried out in vitro with the livers of Japanese quail that had been fed from hatching on diets supplying their full requirements for vitamin D. 2. 25-Hydroxycholecalciferol was the major metabolite when liver homogenates of egg-laying female and oestrogen-treated quail of both sexes were incubated with [3H]cholecalciferol. 3. Very little 25-hydroxycholecalciferol was generated from liver homogenates of adult male and immature quail. Instead the cholecalciferol was converted into one or more compounds less polar than 25-hydroxycholecalciferol and into a number of highly polar metabolites, some of which were water-soluble. 4. Oestrogen not only stimulated the 25-hydroxylation of cholecalciferol but also protected both cholecalciferol and 25-hydroxycholecalciferol from degradation by the enzymic pathways active in immature and male birds. 5. These actions of oestrogen may be of physiological significance in relation to the high requirements of laying birds for 1,25-dihydroxycholecalciferol to support the intense metabolism of calcium associated with egg-shell calcification.     

Convergent synthesis, chiral HPLC, and vitamin D receptor affinity of analogs of 1,25-dihydroxycholecalciferol

A series of analogs of 1,25-dihydroxycholecalciferol was obtained with an additional chiral center at the terminus of the aliphatic side chain (C-25). The analogs were obtained from (+)-(R)- and (-)-(S)-2-methylglycidols, by opening of the oxirane ring with the carbanions derived from vitamin D C23a,24- or C22-sulfones. The diastereomeric purity of the analogs was determined by high-performance liquid chromatography on a chiral stationary phase. The binding affinity of analogs for the calf thymus intracellular vitamin D receptor (VDR) was two orders of magnitude lower than that of the lead compound of this group, 24a,24b-dihomo-1,25-dihydroxycholecalciferol, and it was comparable to the affinity of analogs of 24-nor-1,25-dihydroxycholecalciferol. However, a twofold difference was observed for analogs diastereomeric at C-25 in their affinity for VDR. The diastereodifferentiation of the binding affinity was found to be specific for vitamin D vicinal 25,26-diols as it disappears for analogs where 26-hydroxyl, neighboring the C-25 chiral center, is replaced with methyl.     

Nuclear and cytoplasmic receptors for 1,25-dihydroxycholecalciferol in intestinal mucosa

The apparent hormonal form of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-(OH)₂-CC), was incubated with intestinal mucosa homogenates and whole intestinal tissue, $\text{in}\text{vitro}$. After 40–70 min, 1,25-(OH)₂-CC was specifically associated with the nuclear chromatin fraction. This sterol remains bound to the cytosol fraction at 0°C and a dramatic movement to the nuclear chromatin occurs at 37°C indicating that the subcellular localization of the sterol is temperature dependent. Isolated intestinal cytosol, previously incubated with 1,25-(OH)₂-CC, is required for transportation of the hormone to the intestinal chromatin fraction; cytosol fractions from other tissues are ineffective mediators of this sterol migration. It is concluded that the intestinal cytosol contains a specific receptor that functions to transport 1,25-(OH)₂-CC to the nucleus, its probable site of action.     

Regulation of Metabolism of 25-Hydroxycholecalciferol by Kidney Tissue <Emphasis Type="Italic">in vitro</Emphasis> by Dietary Calcium

IT is now recognized that hydroxylated metabolites of vitamin D (that is, cholecalciferol) function as effectors of the physiological actions originally attributed to the unaltered vitamin¹. The activation of vitamin D by specific hydroxylation reactions and sequestration of the resultant metabolites by target tissues represents a hormonal control loop which is feed-back sensitive. 25-Hydroxycholecalciferol (25-HCC) and 1,25-dihydroxycholecalciferol (1,25-DHCC) have been shown to be participants in the control loop, vitamin D being first metabolized in the liver to 25-HCC² which in turn is hydroxylated in the C-1 position to 1,25-DHCC in the kidney^3,4. The metabolically active form in the intestine appears to be 1,25-DHCC^5,6.     

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.     

Formation of 1,24-24-trihydroxyvitamin D3 from 1,25-dihydroxyvitamin D3

Incubation of [26,27-³H₂]-25-hydroxyvitamin D₃ with kidney homogenates from rats fed a high (3%) calcium vitamin D-supplemented diet results in the production of a more polar metabolite which cochromatographs with 1,24,25-trihydroxyvitamin D₃. On the other hand, incubation with kidney homogenates from vitamin D-deficient or calcium-deficient rats did not produce the polar metabolite. Mitochondria but not microsomes carry out the reaction and evidence has been produced to demonstrate that the 1,24,25-trihydroxyvitamin D₃ can be produced in vivo from either 1,25-dihydroxyvitamin D₃ as previously reported.     

Studies on calciferol metabolism. VII. The effects of actinomycin D and cycloheximide on the metabolism, tissue and subcellular localization, and action of vitamin D3

It was originally postulated, primarily on the basis of experiments employing actinomycin D, that calciferol (vitamin D) mediated its characteristic physiological responses in the intestine via the activation of information stored in the intestinal genome. A more recent alternative hypothesis suggested that actinomycin D blocked the biological response to calciferol by inhibiting the mandatory metabolism of cholecalciferol to 1,25-dihydroxycholecalciferol. Presented in this paper are the results of recent experiments studying the effects of both actinomycin D and cycloheximide on the metabolism, subcellular localization, and action of cholecalciferol or its metabolites, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Actinomycin D was found to inhibit calcium transport stimulated by cholecalciferol or its metabolites without inhibiting their metabolism or localization in the target tissue, the intestinal mucosa. However, actinomycin D had to be administered in four doses at 2-hr intervals to block the stimulation of calcium transport by 1,25-dihydroxycholecalciferol. Actinomycin D was also found not to lower the renal levels of 25-hydroxycholecalciferol-1-hydroxylase, which were measured in vitro. In contrast, cycloheximide was found to inhibit the localization of the sterols in the intestine. Also cycloheximide lowered the renal enzyme levels which were measured in vitro following administration of the antibiotic in vivo. From these data it can be calculated that the 25-hydroxycholecalciferol-1-hydroxylase appears to have a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approximately 3 hr. Thus, the inhibition of intestinal calcium transport by these two antibiotics may in fact occur at two different target organs; cycloheximide by a lowering of the kidney levels of 25-hydroxycholecalciferol-1-hydroxylase and actinomycin D by blocking the action of 1,25-dihydroxycholecalciferol in the intestine.     

Metabolism of dihydrotachysterol and 5,6-trans-cholecalciferol in the chick and the rat

Dihydrotachysterol and 5,6-trans-cholecalciferol are biologically active analogues of cholecalciferol (vitamin D) with a similarity in steric structure to 1,25-dihydroxycholecalciferol, the active form of the vitamin. The question arises as to the nature of the active form of these analogues. High specific radioactivity (14)C- and (3)H-labelled forms of dihydrotachysterol and 5,6-trans-cholecalciferol and its 25-hydroxy derivative were synthesized and their metabolism was studied in chicks and rats. All these steroids were very rapidly metabolized compared with cholecalciferol; 20% of the dihydrotachysterol dose was excreted in bile in the first 24h, about 50% as a carboxylic acid derivative. Although polar metabolites were detected in tissues, no 1-hydroxy form was observed. Larger proportions of the parent steroid and its 25-hydroxy metabolite were detected in tissues compared with cholecalciferol, but no single metabolite was detected at the intracellular site of action of cholecalciferol. It is suggested that analogues of cholecalciferol will be biologically active if they possess a hydroxyl group in the same steric position as that at C-1 of cholecalciferol, with the greatest activity shown by those that also have a C-25 hydroxyl group. The implication of these findings for the chemical features necessary for binding to receptor proteins are briefly discussed.     

Intranuclear localization and receptor proteins for 1,25-dihydroxycholecalciferol in chick intestine

1. The intranuclear distribution of cholecalciferol and its metabolites was studied in the intestine of rachitic chicks. 2. At high doses of cholecalciferol the nuclei contain the vitamin and its 25-hydroxy metabolite, but over 80% of this is localized on the nuclear membranes. The hormone, 1,25-dihydroxycholecalciferol, is found within the cell nuclei irrespective of the intake of cholecalciferol, but significant amounts could not be found with chromatin isolated free of nuclear membranes. 3. 1,25-Dihydroxycholecalciferol is associated in the nucleus with an acidic protein. Since one of the actions of 1,25-dihydroxycholecalciferol is to control the synthesis of mRNA for calcium-binding protein it was to be expected that the hormone would be bound to chromatin, as with the other steroid hormones. It is suggested that the hormone-receptor complex exists as part of an equilibrium mixture of the complex bound to the DNA and in a free form. 4. A protein extract of nuclei was obtained, which when incubated at 4 degrees C for 1h took up the 1,25-dihydroxycholecalciferol. The nature of this binding was studied. 5. There appear to be two nuclear proteins able to bind the hormone one of which is the intestinal nuclear receptor. The binding sites on this protein are saturable with the hormone, have an association constant of 2x10(9)m(-1) and show a high chemical specificity for the 1,25-dihydroxycholecalciferol. The number of nuclear binding sites for the hormone provided by this receptor is similar to the maximum intestinal hormone concentration so far observed. Its sedimentation coefficient is 3.5S, and is very close to that observed for the nuclear protein to which is attached the 1,25-dihydroxycholecalciferol formed in vivo from vitamin D. 6. The cytoplasmic protein has an association constant of 1x10(9)m(-1)and a sedimentation coefficient of 3.0S, but its relation with the nuclear receptor is not yet clear.     

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)