ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 188, No. 2, June, pp. 282-286, 1978

Vitamin D3 from Rat Skins Irradiated in Vitro with Ultraviolet Light 1 R. P. ESVELT, H. K. SCHNOES, AND H. F. DELUCA Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin--Madison, Madison, Wisconsin 53706 Received October 4, 1977; revised February 2, 1978 Vitamin D:3 was isolated and identified from intact rat skins subjected to ultraviolet irradiation. The addition of [1,2-:ill]vitamin D:~ (sp act 19 Ci/mmol) allowed us a means of following the vitamin through chromatography and of computing recoveries. Structural identification of vitamin D:3 was provided by the ultraviolet absorption spectrum, by mass spectrometry and by comigration of the ultraviolet absorbing peak with [1,2-:~H]vitamin D~ in several chromatographic systems. After correcting for recoveries, a yield of 320 ng of vitamin D~ per gram of rat skin was computed. No vitamin D2 could be detected. In a control isolation from skins receiving no ultraviolet light, no vitamin D could be detected. However, the ultraviolet absorption of the control sample at 264 nm indicated that less than 40 ng of vitamin D per gram of skin could have been present. It has therefore been unequivocally demonstrated that vitamin D:~ is produced upon ultraviolet irradiation of skin.

In 1924 Steenbock and Black (1, 2) discovered that ultraviolet (uv) irradiation of foods rendered them antirachitic and localized the activated substance to the sterol fraction. This discovery led to the identification of vitamin D2 from solutions of irradiated ergosterol by Askew et al. (3) and Windaus et al. (4) and, later, of vitamin D3 from solutions of irradiated 7-dehydrocholesterol in extracts from pig skin, thus leading to the inference that 7-dehydrocholesterol is the natural provitamin in vivo, yielding vitamin D3 in response to uv exposure. Knudso'n and Benford (7) studied the effectiveness of various Wavelengths of uv light in curing rickets in vivo. They found that 280.4-nm light was the most effective of the wavelengths tested, requiring 747,000 ergs for a 2+ score in the line test. Bunker et al. (8) demonstrated that the same wavelengths that were effective in vivo were also the most effective in activating 7-dehydrocholesterol in vitro. Thus, it has long been inferred that vi~This work was from the National NAS2-8752 from the Administration, and Fund.

supported by Grant AM-14881 Institutes of Health, Contract National Aeronautics and Space the Harry Steenbock Research

tamin D3 is produced in the skin in the same manner as it is produced from 7-dehydrocholesterol in organic solvents (9). Recently Petrova et al. (10) provided chromatographic evidence that previtamin D is produced upon uv irradiation of rat skin. Okano et al. (11) also invoked chromatographic evidence indicating that vitamin D can be isolated from saponified rat skins following uv irradiation either in vitro or in vivo. While the present report was in progress, Holick et al. (12) provided physical as well as chromatographic evidence that previtamin D3 and vitamin D3 are produced upon uv irradiation in vivo. In this study the above reports are confirmed with the isolation and characterization by physical measurements of vitamin D3 from rat skins irradiated in vitro with uv light. MATERIALS AND METHODS

Animals. Male rats (120 to 150 g) were obtained from the Holtzman Company (Madison, Wise.) and had been maintained on a low vitamin D stock diet containing 41% ground yellow corn, 11% soybean meal, 15% linseed meal, 5% alfalfa meal, 1% salt (iodized), 1% calcium phosphate, 20% skim milk, and 6% butter and shielded from uv light. Upon receipt from the breeder, the animals were killed by decapitation and the skins were removed from the backs and sides of 282

0003-9861/78/1882-0282502.00/0 Copyright 9 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

283

VITAMIN D:~ FROM U L T R A V I O L E T - I R R A D I A T E D R A T SKIN

control sample was chromatographed on the same column equilibrated in 5% H20 in CH3OH using a Dupont Model 830 high-pressure apparatus with a flow rate of 1.2 m l / m i n at 800 psi. In all hplc the uvabsorbing peaks were detected using a 254-nm uv monitor. Ultraviolet spectra were recorded with a Beckman Model 25 spectrophotometer. Mass spectra were obtained with an A.E.I. MS-9 mass spectrometer using a direct probe inlet at 90 ~ above the ambient temperature. [1,2-3H]Vitamin D3 was synthesized by the method of Neville and DeLuca (13). Radioactivity measurements were carried out with a Packard Model 3375 scintillation counter using a toluene counting solution containing 0.4% 2,5-diphenyloxazole and 0.03% dimethyl-l,4-his[2(5-phenytoxazolyi)] benzene. Counting efficiencies were routinely 30 to 35% as determined by [all]toluene internal standards.

the animals. T h e skins were kept on ice and scraped free of hair and adipose tissue with a dull scalpel. T h e uv group of skins was spread onto an aluminum foil surface moistened with buffer containing 0.25 M sucrose, 50 mM Tris, 25 mM KC1, and 5 mM MgC[2 at pH 7.4. T h e y were irradiated at room temperature for 15 min, 20 cm from a Hanau TQ 150Z2 high-intensity mercury arc lamp cooled with a water jacket. Wavelengths shorter than 250 n m were filtered using a Vycor filter. T h e skins t h u s received a m a x i m u m of 630 J of radiant energy, as calculated from the lamp specifications. Control skins were similarly treated but were not irradiated. Extraction ~and chromatography. T h e skins were cut up into 5% (w/v) KOH in 95% methanol, and 21 ng of [ 1,2-:~H]vitamin D:3 (19 Ci/mmol) was added. T h e mixture was refluxed for 1 h with nitrogen gas bubbling through the solution. An equal volume of water was added to the saponified mixture and the lipide were removed by extracting twice with benzene and once with diethyl ether. T h e organic phases were washed with saturated NaC1 and the solvents were removed on a rotary evaporator. Chromatography was carried out as listed in Table I. All solvents were distilled before use. Lipidex 5000 (Packard Instrument Co., Inc., Downers Grove, Ill.) is a hydroxyalkyl-substituted Sephadex LH-20. Straightphase high-pressure liquid chromatography (hplc) was carried out on a Waters Model A L C / G P C 204 liquid chromatograph equipped with a 25 • 0.79-cm column of Zorbax SIL (Dupont) operating at a pressure of 800 psi and a flow rate of 2.0 ml/min. Reversed-phase hplc was carried out on the Waters instrument for the uvexposed sample using 0.4 • 30-cm micro-C18 column (Waters Associates) using 7% H20 in the CH3OH with a pressure of 1200 psi and flow rate of 2.0 ml/min. T h e

RESULTS

Skin tissue (414 g) f r o m 26 rats, was exposed to uv irradiation as described under Materials and Methods. T h e control group consisted of 96 g of skin tissue f r o m 10 rats. T h i s group received no uv light, b u t was otherwise t r e a t e d in the s a m e manner. E a c h group of skins was saponified after the addition of [1,2-~H]vitamin D3 and ext r a c t e d with benzene and diethyl ether. T h e recoveries of added [1,2-3H]vitamin D3 after saponification and extraction were 72.6% for the uv-exposed extract and 58.6% for the control skin extract. T a b l e I lists the

TABLE I CHROMATOGRAPHIC Colu~

Size

3 x 55cm

Columm Bed

L i p i d e x 5000

SYSTEMS

USED

FOR THE EXTRACTS

Solvent System

1:9

Peak Elution Volume (ml)

FROM RAT SKINS a Separation

577

S t r a i g h t Phase

87.7

205

Reversed Phase

82.6

CH3OH

39

Gel Filtration

80.6

27

Straight Phase HPLC

85.7

18.5

Reversed Phase HPLC

CHCI3 : S k e i l y B i x 60cm

Lipidex 5000

20 : 70 : I0 CHCI 3 : CH3OH : H20

i x 60cm

LH-20

7.9m~ x 25 cm

Dupont Zorbaxsil

1.5% isopropanol in hexane

4mm x 30 cm

~C18 Wat~ts

7.0% H20 in methanol

" T h e chromatographic systems used to isolate vitamin D.~ from rat skins are listed in order of use. T h e elution volumes and recoveries are reported for the sample from uv-exposed skins. T h e values for the control sample did not differ markedly except where noted in the text.

284

ESVELT, SCHNOES, AND DELUCA

chromatographic systems used in both isolations and reports the peak elution volumes and recoveries for the uv-exposed sample. The two extracts were subjected to chromatography on two Lipidex 5000 columns, straight- and reversed-phase, followed by gel filtration on LH-20 then two hplc steps. In each chromatographic step the region of vitamin D elution was detected by counting the radioactivity of aliquots of the fractions, and appropriate fractions were recombined and rotary evaporated. The overall recovery of added radioactivity following the straight-phase hplc step came to 36.3% for the uv-exposed sample and 10.5% for the control sample. The decreased overall recovery for the control was primarily due to low recoveries during extraction and a 46.7% recovery from the reversed-phase Lipidex 5000 column. Figures 1 and 2 show the hplc traces for a portion of the uv-exposed sample, clearly demonstrating the large peak of uv-absorbing material comigrating with [1,2-3H]vi tamin D3. The reversed-phase hplc trace of the control sample (Fig. 3) shows only a small uv absorbance in the region of the [1,2-3H]vitamin D3 despite a much reduced absorbance scale. The uv absorption spectrum of the vitamin D region from the reversed-phase hplc of the sample from uv-exposed skins clearly shows the maximum at 264 nm and a minimum at 230 nm (Fig. 4). The maximum (264 nm) to minimum (230 nm) ab-

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FIG. 3. Chromatographic profile of the sample from nonirradiated skins on reversed-phase hplc. Reversed-phase hplc of the vitamin D region from straight-phase high-pressure liquid chromatography of the control sample was carried out on the Dupont instrument using a 4-ram x 30-cm Waters pC18 column equilibrated in 5% H20 in methanol. The flow rate was 1.2 ml/min at a pressure of 800 psi. The solid line represents optical density at 254 nm and the clear bars represent the radioactivities of the fractions.

30

Fro. ]. Chromatographic profile of the sample from uv-exposed skins on straight-phase hplc. The hplc of the vitamin D region of the extract from uvexposed skins from the Lipidex 5000 columns and the LH-20 column was carried out on the Waters instrument using a 7.9 x 25-cm Zorbax SIL column bed equilibrated in 1.5% isopropanol in hexane. The flow rate was 2.0 ml/min at a pressure of 800 psi. The solid line represents optical density at 254 nm and the clear bars represent the radioactivity of the fractions.

sorbance ratio of 1.8 attests to the purity of the preparation. Using the uv absorbance at 264 nm (extinction coefficient for vitamin D3 = 18,800 cm -1 M-1) and correcting for recoveries of radioactivity, the amount of vitamin D3 in the uv-irradiated rat skins was calculated to be 132.6 #g, corresponding to 320 ng/g of skin. The ultraviolet absorption spectrum of the vitamin D region of the control sample

VITAMIN D3 FROM ULTRAVIOLET-IRRADIATED RAT SKIN on r e v e r s e d - p h a s e hplc shows neither a m a x i m u m a t 264 n m nor a m i n i m u m at 230 n m (Fig. 5). T h u s the level of v i t a m i n D in r a t skins receiving no uv irradiation is below the detectable range. After corrections for recoveries, the uv a b s o r b a n c e at 264 n m indicated t h a t less t h a n 40 ng of v i t a m i n D was p r e s e n t per g r a m of r a t skin. A portion of the v i t a m i n D region f r o m the s t r a i g h t - p h a s e hplc of the sample f r o m uv-exposed skins was subjected to m a s s s p e c t r o s c o p y (Fig. 6). T h e molecular ion at

285

m/e 384 and characteristic f r a g m e n t a t i o n s at 368 {loss of methyl), 366 (loss of H20), 351 (loss of methyl, HeO), 271 (loss of the side chain), 253 (loss of side chain, H20), 136 {loss of rings C and D, base peak), and 118 (loss of rings C and D, and H20) clearly d e m o n s t r a t e d t h a t v i t a m i n D3 was produced u p o n uv irradiation of r a t skins. No v i t a m i n De was detected, as evidenced by the absence of a molecular ion at m/e 396

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ESVELT, SCHNOES, AND DELUCA

286

and corresponding fragments. This is especially significant since none of the chromatographic steps prior to recording of the mass spectrum is able to resolve the two vitamins, and the rats would have received significant amounts of ergosterol in their diet. DISCUSSION T h e present report firmly establishes that vitamin D3 is produced upon uv irradiation of rat skins. This was demonstrated by the isolation of vitamin D3 from uvexposed rat skins and comparison to levels found in unexposed skins. Identification of vitamin D3 was provided by chromatographic comigration of a uv absorbing peak with [1,2-aHJvitamin D3, by the uv absorption spectrum, and by the characteristic mass spectrum. T h e mass spectrum did not show any peaks which would indicate the presence of vitamin D2 despite the presence of ergosterol in the rats' diet. It was calculated that approximately 320 ng of vitamin D:j was present per gram of uv-exposed skin without correcting for loss of previtamin D3 since the thermal equilibrium between the isomers allows recovery of only about 90% of the vitamin (9), whereas nonirradiated skin contained less than 40 ng of the vitamin per gram of tissue. This level of vitamin D:~ production is sufficient to explain the antirachitic activity of extracts from rat skins receiving comparable uv exposure (Esvelt and DeLuca, unpublished results). T h e demonstration of the uv light-dependent production of vitamin D.~ in rat skin directly implies that previtamin D3 is the initial photoproduct (9) which subsequently undergoes thermal isomerization to the vitamin and that 7-dehydrocholesterol is the natural provitamin in vivo. Indeed, 7-dehydrocholesterol attains higher concentrations in the skin than in most body tissues (14) and is localized mainly in the epidermis and epidermal appendages in the skin (15, 16), thus allowing accessibility of the provitamin to uv light. At the present time, it is unknown whether or not the uv-dependent formation of vitamin D3 in skin is a controlled process. Experiments in this laboratory using skin homogenates and heat-denatured controls have produced no evidence for the involve-

meEt of proteins in either the photochemical or thermal isomerizations leading to the vitamin (Esvelt and DeLuca, unpublished results). It is possible that 7-dehydrocholesterol levels could be varied to modulate the production of vitamin D. Wells and Baumann (17) were able to demonstrate increases in the 7-dehydrocholesterol content of skin in response to long-term uv light exposures. W h e t h e r or not this leads to increased vitamin D production is open to question. In any case it appears that the major regulated steps in vitamin D activity are at the 1- and 25-hydroxylation reactions (18). REFERENCES 1. STEENBOCK, H., AND BLACK, A. (1924) J. Biol. Chem. 61, 405-422. 2. STEENBOCK, H., AND BLACK, A. (1925) J. Biol. Chem. 64, 263-298. 3. ASKEW,F. A., BOtJRDILI.ION,R. B., BRUCE,H. M., 4.

5. 6. 7. 8. 9. 10. 11.

12.

JENKINS, R. G. C., AND WEBSTEn, T. A. (1930) Proc. Roy. Soc. (London) Ser. B 107, 76-90. WINDAUS, h., LINSEBT, O., LUTTRINGHAUS, i . , AND WEIDLICH, G. (1932) Ann. Org. Chem. 492, 226-241. SCHENK, F. (1957) Naturwissenenschaflen 25, 159-166. WINDAUS, A., AND BOCK, F. (1936) Hoppe-Seyler's Z. Physiol. Chem. 245, 168-174. KNUDSON. A., AND BENFORD, F. (1938) J. Biol. Chem. 124, 287-299. BUNKER. J. W. M., HnitnIS, R. S., AND MOSItER, L. M. (1940) J. Amer. Chem. Soc. 62, 508-511. HAVINGA, E. (1973) Experientia 29, 1181-1316. PETnOVA, E. Z., NIKULICHEVA, S. I., AND LAZAIiEVA, N. P. (1976) Vopr. Pitan. 5, 50-55. OKANO, T., YASUMURA, M., MIZITNO, K., AND KOBAYASHI, T. (1977) J. Nutr. SeN. Vitaminol. 23, 165-168. HOLICK, M. F., FROMMER, J., McNEILL, S., RICHT, N., HENLEY, J., ANDPOTTS,J. T., Jlr (1977) Biochem. Biophys. Res. Commun. 76,

107-114. 13. NEVILLE, P. F., AND DELUCA,H. F. (1966) Biochemistry 5, 2201-2207. 14. BILLS,E. C. (1954) in The Vitamins: Chemistry,

Physiology and Pathology, (Sebrell, W. H., and Harris, R. S., eds.), Vol. II, pp. 132-223, Academic Press, New York. 15. REINERTSON, R. P., AND WHEATLY, V. R. (1959) J. Invest. Dermatol. 32, 49-55. 16. GAYLOR, J. L., AND SAULT, F. U. (1964) J. Lipid Res. 5, 422-431. 17. WELLS, W. W., AND BAUMANN, C. A. (1954) Arch. Bioehem. Biophys. 53, 471-476. 18. DELwcA,H. F. (1975) Life SeN. 17, 1351-1358.