A major vitamin D metabolite was isolated in pure form from the blood plasma of chicks either maintenance levels or large doses of vitamin D3. The isolation involved methanol-chloroform extraction and five column chromatographic procedures. The metabolite purification and elution position on these columns were followed by a competitive protein binding assay. The metabolite was identified, using high- and low-resolution mass spectrometry, 270-MHz proton nuclear magnetic resonance spectrometry, ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, and specific chemical reactions, as 3 beta,-25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-lactone. The trivial names 25-hydroxyvitamin D3 26,23-lactone or calcidiol 26,23-lactone are suggested for this compound.

Vitamin D supplemented rats produce a metabolite of 25-hydroxy[3 alpha-3H]vitamin D3 that is easily separated from known metabolites by using high-performance liquid chromatography. The production of this metabolite in vivo as well as 1,25-dihydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 26,23-lactone is largely if not totally eliminated by nephrectomy. Kidney homogenates from vitamin D supplemented chickens incubated with 25-hydroxyvitamin D3 produce significant quantities of the new, unknown metabolite. This metabolite was isolated in pure form from such incubation mixtures by using both straight-phase and reversed-phase high-performance liquid chromatography. This metabolite has been positively identified as 23,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry, mass spectrometry, and derivatization. This structure was confirmed by chemical synthesis of both C-23 stereoisomers. Although the natural product exactly comigrates with one of the synthetic isomers, the exact stereochemistry of the natural product remains unknown. It is possible that this new metabolite is an intermediate in the biosynthesis of 25-hydroxyvitamin D3 26,23-lactone.

Labeled 25-hydroxyvitamin D3-26,23-lactone was isolated from the serum of vitamin D-repleted rats given [3a-3H-25-hydroxyvitamin D3 24 hr prior to sacrifice. The metabolite was identified by cochromatography with the authentic lactone on straight-phase and reversed-hase high-performance liquid chromatography. Production of the lactone was abolished by nephrectomy indicating that the kidney is the site of synthesis. Homogenate of kidneys from chickens given large doses of vitamin D can carry out in vitro production of the lactone from 25-hydroxyvitamin D3. When la,25-dihydroxyvitamin D3 was used as substrate, this system produced only traces of a compound believed to be la,25-dihydroxyvitamin D3-26,23-lactone. However, incubation of rachitic chicken kidney homogenates with 25-hydroxyvitamin Dx26,23-lactone produced substantial amounts of a compound that has been identified by mass spectrometry as la,25-dihydroxyvitamin D3x2,23-lactone.Thus, the development of a functional group on C-26 and eventual lactone formation takes place in kidney by a system acting on 25-hydroxyvitamin D3.Since the discovery that 25-hydroxyvitamin D3 (25-OH-D3) is the major circulating form of vitamin D3 (1, 2), the further metabolism of vitamin D has been the subject of intensive investigation. Some of the metabolites have been structurally identified and their physiological functions are well defined, but others either remain unidentified or their functions are unknown (1, 2). The 25-hydroxylation of vitamin D3 takes place in liver; the further hydroxylation of 25-OH-D3 to la,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (3), 24,25-dihydroxyvitamin D3 [24,2D3] (46), and 25,26-dihydroxyvitamin D3 [25,2D3] (7) takes place in kidney and elsewhere. Recently, a new metabolite of the vitamin has been isolated from the plasma of chickens and identified as 25-hydroxyvitamin D3-26,23-lactone (25-OH-D3-26,23-lactone) (8). This metabolite was first observed as an interfering substance in the determination of plasma levels of 24,25-(OH)2D3 by the use of the competitive rat plasma protein radioassay method (9). We have investigated the site of biosynthesis of 25-OH-D3-26,23-lactone from 25-OH-D3 and its la-hydroxylated analog. Our results suggest that lactone formation occurs in kidney and that the 25-OH-D3-26,23-lactone can serve as a substrate for the production by the renal la-hydroxylase system of the corresponding la-hydroxylactone [la-dihydroxyvitamin D3-26,23- per day for 6 weeks Two days before the conclusion of the experiment, the rats were given 25-OH-D3 (5 ,ug in 0.1 ml of 1,2-propandiol per day) subeutaneously. Twenty hours before sacrifice, the rats were either nephrectomized or sham operated and then given intrajugularly 10,uCi [3a-3H]-25-OH-D3 (28 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) in 0.05 ml of 95% ethanol.For in vitro production of

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These results suggest that felbamate given post hoc is remarkably effective in preventing delayed apoptosis secondary to global ischemia but that effective neuroprotection requires doses higher than those used for anticonvulsant treatment.