Vitamin A metabolism: analysis of steady-state neutral metabolites in rat tissues.

High-performance liquid chromatography systems were developed to rapidly separate retinol from its esters, analyze the total spectrum of neutral vitamin A compounds, and purify retinyl esters to homogeneity. Chemical ionization mass spectrometric techniques were used to identify vitamin A compounds; these techniques are also applicable to quantification of tissue vitamin A compounds. These methods provide rapid and sensitive techniques for separation and quantification of neutral retinol metabolites.

Arachidonic acid epoxidation: epoxyeicosatrienoic acids are endogenous constituents of rat liver.

Epoxyeicosatrienoic acids have been isolated and purified from the livers of male rats. They were identified by gas chromatography-mass spectrometric techniques. These results expand the list of in vivo-produced eicosanoids.

Formation of 19-nor-10-keto-25-hydroxyvitamin D3 in cultured mammalian cells.

Cultured dog kidney cells convert 25-hydroxyvitamin D3 into more polar metabolites during in vitro incubations. One elutes with 1,25-dihydroxyvitamin D3 from high pressure liquid chromatographic silica columns with hexane:isopropanol (9:1), but can be separated from 1,25-dihydroxyvitamin D3 by elution with dichloromethane:isopropanol (95:5). This peak has been isolated, purified and identified by mass spectral analysis to be 19-nor-10-keto-25-hydroxyvitamin D3.

The isolation and identification of vitamin D2 and vitamin D3 from Medicago sativa (alfalfa plant).

Vitamin D2 and vitamin D3 were isolated from Medicago sativa (alfalfa) grown under field and laboratory conditions and then irradiated with ultraviolet light. The vitamins were identified by ultraviolet absorption, mass spectroscopy, and comparison with synthetic standards on several chromatographic systems. Sun-cured, field-grown alfalfa contained vitamin D2 at a concentration of 48 ng/g (1920 IU/kg) and vitamin D3 at 0.

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.

Vitamin A metabolism: alpha-tocopherol modulates tissue retinol levels in vivo, and retinyl palmitate hydrolysis in vitro.

The steady-state concentrations of retinol in rat tissues varied as a function of dietary alpha-tocopherol. The liver, kidney, and intestinal retinol concentrations increased in animals fed an alpha-tocopherol-deficient diet despite a decrease (liver) or no change (kidney and intestine) in the concentrations of total vitamin A. In contrast, in lung the concentrations of both retinol and total vitamin A decreased.

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.

Metabolism of orally administered [3H]ergocalciferol and [3H]cholecalciferol by dairy calves.

Concentrations of ergocalciferol, cholecalciferol, and their metabolites in plasma were determined after a single oral dose of [3H]ergocalciferol or [3H]cholecalciferol was given to 95- to 105-kg Jersey bull calves. One group (three calves) was given 365 muCi of [3H]ergocalciferol (1.2 Ci/mmol) per calf, and the other group (three calves) was given 365 muCi of [3H]cholecalciferol (1.

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.

13-cis-retinoic acid metabolism in vivo. The major tissue metabolites in the rat have the all-trans configuration.

The liver and intestinal metabolites of orally dosed 13-cis-[11-3H]retinoic acid were analyzed in normal and 13-cis-retinoic acid treated rats 3 h after administration of the radiolabeled retinoid. all-trans-Retinoic acid was identified as a liver and intestinal mucosa metabolite in normal rats given physiological doses of 13-cis-[3H]retinoic acid. all-trans-Retinoyl glucuronide was identified as the most abundant radiolabeled metabolite in mucosa and a prominent liver metabolite under the same conditions.

19-nor-10-ketovitamin D derivatives: unique metabolites of vitamin D3, vitamin D2, and 25-hydroxyvitamin D3.

Three metabolites were isolated after incubation of vitamin D3, vitamin D2, or 25-hydroxyvitamin D3 with bovine rumen microbes. They are identified as 5(E)-19-nor-10-ketovitamin D3, 5(E)-19-nor-10-ketovitamin D2, and 5(E)-19-nor-10-keto-25-hydroxyvitamin D3, respectively. The identifications were based on ultraviolet absorbance, mass spectroscopy, and chemical reactivity.

(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.

Dietary phosphate deprivation increases 1,25-dihyroxyvitamin D3 synthesis in rat kidney in vitro.

A sensitive radioreceptor assay has been used to measure in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) synthesis in vitamin D-replete rats. Incubation of kidney cortical slices with 25-hydroxyvitamin D3 produced a product which co-migrated on high performance liquid chromatography with authentic 1,25(OH)2D3 in two different solvent systems and displaced 1,25(OH)2D3 from its intestinal receptor. In addition, mass spectral analysis of the product produced a mass fragmentation consistent with that of authentic 1,25(OH)2D3.

23-Keto-25-hydroxyvitamin D3: a vitamin D3 metabolite with high affinity for the 1,25-dihydroxyvitamin D specific cytosol receptor.

A new metabolite of 23,25-dihydroxyvitamin D3 has been generated with kidney homogenates prepared from vitamin D treated chicks. The metabolite was purified with three high-performance liquid chromatographic steps and was identified as 23-keto-25-hydroxyvitamin D3 by ultraviolet absorption spectroscopy, mass spectrometry, and chemical reactivity. The R stereoisomer of 23,25-dihydroxyvitamin D3 was 10-fold more effective as an in vitro precursor to 23-keto-25-hydroxyvitamin D3 than was the naturally occurring S stereoisomer.

23S,25-dihydroxyvitamin D3 as a circulating metabolite of vitamin D3. Its role in 25-hydroxyvitamin D3-26,23-lactone biosynthesis.

23,25-Dihydroxyvitamin D3 was isolated from vitamin D-toxic pig plasma by sequential chromatography through two gravity columns and three high performance liquid chromatography systems. Two of the high performance liquid chromatography systems separated the R and S diastereomers of 23,25-dihydroxyvitamin D3 and demonstrated that the metabolite has the 23S configuration. Ultraviolet absorbance and mass spectroscopy of the pure metabolite and mass spectroscopy of its trisilylated derivative confirmed the structural assignment.

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.

Discrimination in the metabolism of orally dosed ergocalciferol and cholecalciferol by the pig, rat and chick.

Vitamin D-deficient pigs, rats and chicks were repleted with four daily oral doses of crystalline ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) containing equal concentrations of each. At 24 h after the last dose, the plasma of each species was analysed for vitamin D and 25-hydroxyvitamin D by standard methods. The mean (+/- S.

Metabolism of 5,6-epoxyretinoic acid in vivo: isolation of a major intestinal metabolite.

The major metabolite in the small intestinal mucosa of vitamin A deficient rats dosed intrajugularly with 5,6-epoxy[3H]-retinoic acid has been identified as 5,6-epoxyretinoyl beta-glucuronide. The assignment was based on the metabolite's chemical, spectral, and chromatographic properties. Incubation of the metabolite with beta-glucuronidase released 5,6-epoxyretinoic acid.

Identification of 5,6-epoxyretinoic acid as an endogenous retinol metabolite.

Rats on a normal diet were administered physiological doses of [3H]retinyl acetate or [3H]retinol orally for 5 days to label endogenous retinoid pools. The kidney retinoids were extracted and separated by DEAE-Sephadex into neutral and acidic fractions. All-trans-retinoic acid and 5,6-epoxyretinoic acid were isolated and unequivocally identified by chromatographic analysis, chemical derivatization, and mass spectroscopy.

Tissue dependence of retinoic acid metabolism in vivo.

Metabolites formed in vivo from [3H]retinoic acid dosed intrajugularly to vitamin A-deficient rats and to vitamin A-deficient rats supplemented orally with unlabeled retinoic acid were investigated. Extracts of liver, small intestinal mucosa, kidney, testes and serum were separated into charged uncharged fractions by DEAE-Sephadex. This allowed the direct application of 20-40% of the combined charged extracts from up to six organs to be loaded onto a high-performance liquid chromatography column.