The Isolation and Identification of 5,6-trrmns-25-Hydroxyvitamin D, from the Plasma of Rats Dosed With Vitamin D3 EVIDENCE FOR A NOVEL MECHANISM IN THE METABOLISM OF VITAMIN D3*

We isolated 5,6-trans-25-hydroxyvitamin D3 from the plasma of vitamin D-deficient rats which had received 5,6-cis-vitamin D3 orally. The relative amounts of 5,6-tmmd5-hydroxyvitamin D3 and 5,6-cis-25-hydroxyvi- tamin D3 in plasma were 1:s. Control experiments in which 5,6-cis-25-hydroxyvitamin D3 was added to plasma or water showed that less than 0.5% of the added 25-hydroxyvitamin DS was converted to 5,6- trans-25-hydroxyvitamin D3 during the isolation procedure ( p < 0.001, control versu8 experimental). We synthesized 5,6-tr~m~-25-hydroxyvitamin D3 from 25-hydroxyvitamin D3 and demonstrated that authentic 5,6-tnuzs-25-hydroxyvitamin D3 co-eluted with the iso- lated putative 5,6-trans-25-hydroxyvitamin D3 on a high performance liquid chromatography system. In addition, the isolated metabolite demonstrated mass spectral, ultraviolet absorption, and protein-binding properties similar to those of synthetic 5,6-tmns-25- hydroxyvitamin D3. As 5,6-tmm-25-hydroxyvitamin D3 binds more efficiently to intestinal cytosol-binding protein than 25-hydroxyvitamin D3, this observation could explain,

Vitamin D3 plays an important role in normal calcium and phosphorus physiology (1). D : is metabolized in the liver to This work was supported by Grants AM 25409 and AM 26808 from the National Institutes of Health, and by grants from the R. K. Mellon Foundation and the Grainger Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

MATERIALS AND METHODS
General-All solvents were distilled prior to use. UV spectra of D3 analogs were recorded in ethanol with a Beckman model 35 spectrophotometer (Beckman Instruments, Palo Alto, CA). Mass spectra were obtained at 70 eV on a Kratos MS-50/DS-55 high resolution mass spectrometer/computer system (Kratos Instruments, United Kingdom) at a source temperature of 200 "C. Sephadex LH-20 was obtained from Pharmacia (Piscataway, NJ). HPLC was performed on a Beckman Isocratic liquid chromatograph model 330 with a UV detector set at 254 nm.
Animals-Fifty-to sixty-g male, albino, weanling rats were obtained from the Holtnan Company (Madison, WI) and maintained in individual overhanging wire cages for 4 weeks. The rats were fed a 0.02% calcium, 0.30% phosphorus, D-deficient diet ad libitum. Vitamin D Analogs-D, was obtained from the Aldrich Chemical Co. (Milwaukee, WI). 25(OH)D3 was a gift of the Upjohn Co., Kalamazoo, MI. 1,25(OH)~D3 was a slft from Hoffman-La Roche, Inc., Nutley, NJ. All of the D compounds were pure based upon UV spectroscopy, HPLC, and mass spectrometry.

RESULTS
The HPLC profile of the plasma from experimental animals is shown in Fig. 3. The UV spectrum of the peak eluting between 38 and 42 min shows a X, , , at 272-273 nm (Fig. 4). Comparison UV spectra of authentic cis and trans metabolites are plotted on the same figure. Following HPLC on a reversedphase column, the UV spectrum displayed the same features as shown in Fig. 4. The mass spectrum of the material isolated after reversed-phase HPLC (Fig. 1A) Table I).
The isolated peak eluted in a manner similar to synthetic 5,6-trans-25(OH)D3 (Fig. 3). We examined the binding of 5,6- We repeated the entire isolation procedure a second time with identical results.

DISCUSSION
We have isolated a new metabolite from the plasma of rats dosed with D3. The data show that 1) the metabolite has a molecular weight of 400 (C27Hu02), suggesting the presence of one additional hydroxyl group in the D3 molecule. 2) The diMe3Si ether derivative has a molecular weight of 544 (CsHmOzSiz), suggesting the presence of two hydroxyl groups For control 1 versus 2, p > 0.05; for control 1 or 2 versus experimental, p < 0.001. , confirming that the extra hydroxyl group is not in the A ring. The mass spectral data are compatible with the structure of a 9,10-secocholesta-5,7,10(19)triene-3P,25-diol. No information relative to the nature of the 5,6 double bond can be obtained from the mass spectrum. 5,6cis and trans isomers behave in a similar fashion on mass spectrometry. 6) A X,, at 272-273 nm appears on UV spectroscopy, that is characteristic of a 5,6-trans or 5E configura. tion. The metabolite also has 7) chromatographic and mass spectral characteristics that are identical with synthetic 5,6trans-25(0H)D3, and 8) a similar Bm value when compared with that of synthetic 5,6-trans-25(OH)D3. On the basis of the above observations, we conclude that the new metabolite is 5,6-trans-25(OH)D~ (9,10-secocholesta-5,7,10(19)-triene-3P,25-dio1,5E isomer). It is unlikely that the compound is formed during the isolation procedure, because appropriate controls show only trace amounts of the 5,6-trans isomer.
The formation of 5,6-trans-25(OH)D3 may explain, in part, why large doses of 25(OH)D3 or D3 are effective in anephric or hypoparathyroid individuals. Holick et al. (1 1) , Lawson and Bell (lZ), and Kraft et al. (13) have previously shown thht the 5,6-trans isomers of vitamin D analogs are biologically active as trans isomers because rotation of the "A" ring of the secosteroid by 180' provides the correct stereochemical features necessary for binding to the intestinal cytosol receptor. We speculate that when other analogs of D3 (such as 24,25dihydroxyvitamin D3) are administered in large doses, they may undergo similar isomerization in vivo and thus become active. The site of formation of this new metabolite and the mechanism by which it is formed are unknown at present.
Our observations strongly support the existence of a heretofore unknown metabolic route in vitamin DS metabolism, namely, the conversion of 5,6-cis compounds to 5,6-trans compounds. The mechanism of this transformation is not clear at present; it could be enzymatic or nonenzymatic. The site of formation of 5,6-trans-25-hydroxyvitamin DS is unknown.
In summary, when rats are administered large doses of vitamin DS, 5,6-trans-25(OH)D3 is found in plasma. This unique process of 5,6-cis-trans isomerization may be of biological significance, particularly when la-hydroxylase activity is absent or greatly reduced.