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653 CHROMATOGRAPHIC SEPARATION OF VITAMIN D3 SULFATE AND VITAMIN D3. Y. SORGUEand L. MIRAVET Unit~ de Recherche INSERM n° 18 - Centre Andr~ Lichtwitz HOpital Lariboisi~re - 6, rue Guy Patin - 75010 PARIS - FRANCE

Received

11-21-77

ABSTRACT This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin DR sulfate from free v i t a min DR and i t s metabolites. This technique ha~ been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin DR sulfate (35~ or 35S and 3H) only the peak of vitamin D sulfoconjuga~e was found i n chromatographicelution of serum extracts. INTRODUCTION The formation of the sulfoconjugates is a normal pathway in steroid metabolism (1). Recent evidence has shown that vitamin D is metabolized s i m i l a r l y to steroid hormone ; i t undergoes hydroxylation reactions leading to b i o l o g i c a l l y active metabolites such as 25 OH D3

(4) and 1,25(OH)2D3 (5). Recently, the vitamin D3 sulfate has been found in normal milk and found to be the major form of vitamin D in milk (10). One technical approach to the study of the sulfoconjugate of vitamin D was to carry out chemically or enzymatically desulfatation and then determination of the vitamin D liberated (10). However, none of these techniques is appropriate to study subsequent metabolism of tracer doses of vitamin D sulfate injected in vivo. The purpose of t h i s paper is to describe a simple chromatographic technique, using a method developed for the separation of steroids

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mono and disulfates (11), for the separation and isolation of sulfated vitamin D metabolites. This technique has been used to study the metabolism of vitamin D3 sulfate in vitamin D deficient rats. TECHNIQUES AND EXPERIMENTS Products. For the development of a vitamin D~ sulfate separation technique, we used pure Synthetic products. The coTd vitamin DR sulfate used was chemically synthetized by Dr N. Le Boulch (9) as described previously by Higaki et al. for vitamin D~ sulfate (7). The pure crystalline vitamin DR (cholecalciferol) ahd 25 OH vitamin D3 (25 hydroxycholecalciferoT), were obtained from Roussel (France). The chromatographic assays were performed on Sephadex LH 20 (25-100 ~m) from Pharmacia France S.A. All the solvents used, were of analytical grade. Chromatographic technique. About 1 mg of each product was put on the column of Sephadex • LH20. The elution was performed with a mixture of chloroform/methanol (50/50, v/v) and 0.01M NaCl. The eluates were directly collected in counting vials and evaporated to dryness under a stream of air. Then, each fraction was taken up in absolute ethanol and U.V. absorption was measured at 264.5 nm in a Zeiss PMQIII Spectrometer. Biological experiments. This technique has been used in the study of vitamin D3 sulfate metabolism. For these assays, we used Sprague-Dawley rats (Charles River France) at weanling, fed during three or four weeks with a semi-synthetic diet without vitamin D (6). The radioactive products came from C.E.A. - France. We used vitamin D~ sulfate 35c of specific radioactivity, 32 mCi/mmol and vitamin D~ sulfate 3H~1 alpha, 2 alpha(n)3Hl of specific radioa c t i v i t y , 9.4 mCi/mmol. The animals were injected intravenously with 35S vitamin D3 sulfate (4.8 pCi/ra~) or with a mixture of vitamin D3 sulfate 35R (0.04 pCi/rat~ and JH vitamin DR sulfate (0.56 pCi/rat). All proBucts were injected in 50 ul of ethan61 into the jugular vein. Blood was collected by cardiac puncture seven hours later. Extraction method and chrematography. The method is outlined in diagrammatic form in figure 1. The ethanol extraction was used because extraction with chloroform/methanol (3), as used for lipids and sterols, showed us that the conjugate was distributed in two phases chloroform and methanol-water. When the extraction of serum was performed with ethanol/water (9/1, v/v), all the free vitamin D derivatives and vitamin D sulfoconjugates were extracted (2).

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We used 9 ml of ethanol for 1 ml of serum and after mixing, samples were cooled and centrifuged at 3 000 rpm. The supernatant was evaporated to dryness in a rotor evaporator and then, taken up in 1 ml of elution solvent (chloroform/methanol, 50/50, v/v, NaCl 0.01 M) before being chromatographied, according to the method shown in figure 1. The elution was performed with 250 ml of chloroform/methanol, 50/50, v/v and NaCl 0.01M. Fractions of 5 ml each, were d i r e c t l y collected in counting vials and evaporated to dryness under a stream of a i r . The resulting residues were dissolved in a counting solution (Instagel - Packard, France). All radioactive determinations were carried out in an Intertechnic SL 40 l i q u i d s c i n t i l l a t i o n counter with external standard.

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RESULTS Chrgmato~raphic technique. The chromatographic technique resulted in a clear separation of vitamin D3 sulfate from vitamin D3 and of i t s major metabolite 25 OH vitamin D3 (Fig. 2). As shown in figure 2 - A, when a mixture of vitamin D3, 25 OH vitamin D3 and vitamin D3 sulfate was subjected to chromatography, the elution volumes for vitamin D3 and 25 OH D3 (30-50 ml) were similar, but that of vitamin D3 sulfate (100-150 ml) was considerably greater with an elution ratio about 3. This f i r s t peak containing both vitamin D3 and 25 OH D3 was collected and rechromatographied on a Sephadex LH 20 column (2 x 30 cm) with chloroform/hexane (65/35, v/v) (8). The two derivatives were correctly separated (Fig. 2 - B). Biological experiments. After injection of 35S vitamin D3 sulfate, the chromatogram of the serum extract prepared from serum obtained 7 hours after injection, showed only a peak corresponding to the vitamin D3 sulfate (Fig. 3 - A). This peak accounted for more than 90 % of the total radioactivity of the serum extract put on the column. The simultaneous injection of 3H vitamin D3 sulfate and 35S vitamin D3 sulfate confirmed this result and the v a l i d i t y of the technique. When an extract of serum was prepared from animals injected with both isotopically labelled compounds and then subjected to chromatography, the two isotopes appeared in the vitamin D3 sulfate fraction (Fig. 3 , B). There was no detectable peak of 3H vitamin D3 or 25 OH vitamin D3. The ratio 3H~5S of the vitamin D3 sulfate extracted from the

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serum seven hours after injection, was similar to that ofthei~ected material. These results provide strong evidence that the vitamin D3 sulfate did not undergo significant deconjugation during this period.

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CONCLUSION The chromatographic system developed can be used e f f i c i e n t l y to separate the vitamin D3 sulfate from the vitamin D3 and 25 OH vitamin D3 .

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In the serum extracts of rats which had received vitamin D3 sulfate, seven hours before bleeding, we found only the vitamin D3 sulfate. Thus, the r e l i a b i l i t y of this technique indicated that i t will be possible to measure the proportion of sulfoconjugate which remains after injection of 35S vitamin D3 sulfate.

ACKNOWLEDGMENTS We thank Mrs N. Le Boulch who provided the vitamin DR sulfate and the Roussel Laboratories (France) for the 25 hydroxycBolecalciferol. The authors wish to acknowled~ePr H. Rasmussen for his assistance in the preparation of the manuscript. This investigation was supported by a research grant, CL 75.5.0524 from the I.N.S.E.R.M. - Paris, France. REFERENCES 1 - Baulieu, E.E., Emiliozzi, R., Lebeau, M.C., Corpechot, C. and Robel, P., ADVANCES IN TRACERMETHODOLOGY, Plenum Press, 1966, 285. 2 - Belsey, R.E., De Luca, H.F. and Potts, J.T., J. CLIN. ENDOCR. 38, 1046 (1974). 3 - Bligh, E.G. and Dyer, W.J., CANAD. J. BIOCHEM. PHYSIOL. 3__77, 911 (1959). 4 - Blunt, J.W., De Luca, H.F. and Schnoes, H.K., BIOCHEM.Z, 3317 (1968). 5 - Fraser, D.R. and Kodicek, E., NATURE (LONDON) NEW BIOL. 228, 764 (1970). 6 - Guroff, G., De Luca, H.F. and Steenbock, H., AMER. J. PHYSIOL. 204, 833 (1963). 7 - Higaki, M., Takahashi, M. and Sahashi, Y., J. VITAMINOL. 11, 261 (1965). 8 - Holick, M.F. and De Luca, H.F., J. LIPID RES. 12, 460 (1971).

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9 - Ingelman-Sundberg, M. Rane, A. and Gustafsson, J.A., BIOCHEM. 14 (2), 429 (1975). 10 - Le Boulch, N. and Marnay-Gulat, C., BIOCHIMIE 53, 1219 (1971). 11

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Le Boulch, N., Marnay-Gulat, C. and Raoul, Y., INT. J. VITAM. NUTR. RES. 4_44, 167 (1974).