Isolation and Identification of 25-Hydroxyvitamin D3-26,23-peroxylactone A NOVEL IN VIVO METABOLITE OF VITAMIN D3*

A new vitamin D3 metabolite was isolated in pure form (18.2 pg) from the serum of rats given large doses (two doses of 26 pmol/rat) of vitamin D3. The new metabolite has been unequivocally identified as 3p,25-dihydroxy-9,10-seco-5,7,10(19)-cholestat~eno-26,23- peroxylactone by ultraviolet absorption spectropho-tometry, Fourier transform infrared spectrophotome-try, mass spectrometry, field desorption mass spec- trometry, and specific chemical reaction with triphenyl phosphine. The stereochemical configuration at the C- 23 and C-25 positions of the 25-hydroxyvitamin DB-26,23-peroxylactone was definitely determined to be the 23(S)25(R),25-hydroxyvitamin D3-26,23-peroxylac-tone. The trivial name 25-hydroxyvitamin D3-26,23-per-oxylactone is suggested for this metabolite. The isola- tion involved chloroform-methanol extraction and four column chromatographic procedures. The metabolite purification and elution position on these columns were followed by UV measurement at 264 nm. This metabo- lite was ultimately resolved from the previously known 25-hydroxyvitamin D3-26,234actone by high pressure liquid chromatography using a Zorbax Si1

7 To whom all inquiries should be addressed. It is widely accepted that vitamin DJ must undergo metabolism in vivo to express its physiological activities (1,Z). Since the discovery that 25(OH)D,' is the major circulating form of vitamin Ds (3), the further metabolism of vitamin D3 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 physiological functions are unknown. The two dihydroxylated metabolites, 1,25(OH)zD3 and 24,25(OH)tD3, are believed to be responsible for the spectrum of biological responses attributable to the parent vitamin D3 (4-6).
To date, 20 vitamin D metabolites have been isolated and chemically characterized   Table I.
To conserve space, the initial Sephadex LH-20 column chromatographic profie is not presented.

DISCUSSION
This report demonstrates that the 25(OH)D:,-26,23-peroxylactone is a major metabolite of 25(OH)D3 present in the serum of rats under the conditions of the isolation procedure employed. The structure assignment is based on UV absorption spectrometry, Fourier transform infrared spectrophotometry, mass spectrometry, field desorption mass spectrometry, as well as specific chemical reaction of the peroxylactonesecosteroid with triphenyl phosphine. The new metabolite was isolated in pure form by successive chromatography on Sephadex LH-20 and HPLC columns of Zorbax Sil.
An essential feature of the HPLC Zorbax S i l chromatographic purification was the use of the two different solvent systems, isopropyl alcoho1:n-hexane (9:91) and methano1:dichloromethane (1.5:98.5). The subtle structural differences of many of the vitamin D metabolites are such that even the high resolving power of HPLC cannot achieve with one solvent system an unequivocal separation of all the vitamin D metabolites that may be present in the same sample. Thus, the  (14). The structural confirmation of the 25(OH)D3-26,23-peroxylactone was carried out as follows: (i) the anomalous chromatographic behavior of the metabolite is similar to that of the 25(OH)D3-26,23-lactone (11)(12)(13)(14) (Fig. 2); (ii) the UV absorption spectrum of the metabolite displays the typical vitamin Dri cis-triene chromophore with X, , , = 264 nm, X, , , = 228 nm, and A2Cd/A228 = 1.76 (Fig. 3); (iii) the field desorption mass spectrum of the metabolite gives a parent ion M, = 444 (Fig. 5); (iv) the mass spectrum of the metabolite gives m/z 412,400,372,354,339,313,271,253,136, and 118 (Fig. 6). The fragments m/z 271 and 253 indicate that the secosteroid nucleus of vitamin DY has remained unchanged and that all the metabolic alterations have taken place on the side chain; (v) the Fourier transform infrared spectrum of the metabolite indicates the very intense absorbance at 1733 cm", indicative of a &lactone moiety (Fig. 7); (vi) the metabolite easily converts nonenzymatically to the 25(OH)D3-26,23-lactone at room temperature or -20 "C, because it is extremely unstable (Figs. 8, 9, and 10). It is well known that the peroxylactone derivatives such as y-methyl-y-peroxyvalerolactone and y-peroxy-y-butyrolactone are extremely unstable (24,25); (vii) the reaction of the metabolite with triphenylphosphine results in stoichiometric amounts of 25(OH)D3-26,23-lactone and triphenyl phosphine oxide (Fig. ll). A similar reaction was reported by Adam and Szendry (24). The reaction of the ymethyl-y-peroxyvalerolactone with triphenylphosphine resulted in a stoichiometric amount of y-methyl-y-valerolactone and triphenyl phosphine oxide; and (viii) the stereochemistry of the generated 25(OH)D3-26,23-lactone from the metabolite is definitely determined to be 23(S)25(R)25(OH)D3-26,23-lactone (Fig. 12). From these results, the structure of the new vitamin D metabolite was unequivocally determined to be