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Synthesis of 1α-[2-3H]Hydroxyvitamin D3
ANALYTICAL
BIOCHEMISTRY
77, 495-502 (1977)
Synthesis of 1~[2-3H]Hydroxyvitamin
D,
YASUO TOHIRA, KIYOSHIGE OCHI, ISAO MATSUNAGA, MASAFUMI FUKUSHIMA, SHIGERU TAKANASHI, KAZUHITO HATA,’ CHIKARA KANEKO,~ AND TATSUO SUDAN Research Laboratory of Chugai Pharmaceutical Co. Ltd., Takada, Toshima-ku, Tokyo, Japan 171, ‘Technical Department, Sinloihi Co. Ltd., Dai, Kamakura, Japan 247, ZDepartment of Pharmacy, University of Kanazawa, Takara-rho, Kanazawa, Japan 920, and 3Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ka, Tokyo, Japan 113
Received July 29. 1976; accepted October I. 1976 lo-[2-3H]Hydroxyvitamin D3 was synthesized chemically. The preparation was radiochemically pure and had a high enough specific activity (4.2 Wmmol) to permit experiments using 62.5 pmolkat. This preparation had as much effect as synthetic la-hydroxyvitamin D, in increasing the serum Ca level of vitamin D-deficient rats.
It is known that la-hydroxyvitamin D, (la-OH-D,)4 can replace the metabolically active form of vitamin D,, la,25dihydroxyvitamin D, [h~,25-(0H),-D,]~ in the treatment of various vitamin D-refractory syndromes (l-3). This synthetic analog appears to be of great value for clinical use because it is easier and less expensive to prepare than la,25(OH),-D3 (4-6). In addition, Zerwekh et al. (7) isolated a fraction containing la,25(OH),-D, from intestinal chromatin of rachitic chicks after administration of la-OH-D,. These findings suggest that the unnatural form of the D vitamin, la-OH-D,, is metabolized to the natural hormone of vitamin D,. Final proof of this, however, requires metabolic studies with radioactive la-OH-D,. Very recently, Holick et al. (8-10) synthesized Iu-[~-~H]OH-D~ and demonstrated that it was metabolized very rapidly to lc~,25-[~H](OH),D3 in viva and in vitro. Almost simultaneously we synthesized 1~[2-~H]OH-D3 and reached the same conclusion from studies on perfused rat liver (11). This paper reports the synthesis of ~cx-[~-~H]OH-D, with a specific activity of 4.2 Ci/mmol. Preliminary studies with this isotope on the metabolism of la-OH-D, in rats have been reported (11,12). 4 la-OH-D,, la-hydroxyvitamin 4. 5 la,25-(OH),-D,, lcl,2Sdihydroxyvitamin
4. 495
Copyright All rights
0 1977 by Academic Press, Inc. of reproductmn in any form reserved.
ISSN
0003-2697
496
TOHIRA ET AL.
METHODS AND RESULTS
General Procedure Ultraviolet absorption spectra were determined in ethanol with a Hitachi 124 spectrophotometer. The following molar extinction coefficients were used: la-OH-proD,,6 hZBZnrn,E = 11,000; la-OH-preD,,’ h260nm, E = 9000; and la-OH-D,, hZ64n,,,, E = 18,100. Radioactivity was measured in a Nuclear Chicago, Mark I, liquid scintillation spectrometer. Samples were counted in 10 ml of scintillator solution consisting of 7 g of PPO and 90 mg of dimethyl-POPOP/liter of toluene. Thin-layer chromatography was carried out on Merck silica gel 60 containing FZs4, with 100% ether as soIvent. Radioscannograms were recorded with an Aloka JTC201 instrument. Column chromatography was carried out on 1.0 x 35-cm glass columns packed with 10 g of Sephadex LH-20 (Pharmacia) with 65% chloroform-35% n-hexane as solvent, according to the method of Holick and DeLuca (13). All solvents were distilled immediately before use. Preparation
of Tritiated
Lithium
Alumnium
Hydride
Metallic Li (12 mg) was heated in tritium gas for 135 min from 60 (initial) to 550°C (final) under a pressure of from 276 (initial) to 174 (final) mm Hg. A total volume of 12 cm3 (31 Ci) of tritium gas reacted on the metal. The yield, calculated from the amount of tritium gas which reacted, was 61%. To a portion (13 mg) of the reaction product, 24 mg of LiH as carrier and 1.3 ml of anhydrous ether were added; the mixture was stirred to obtain a homogeneous suspension. Then AlBr, (290 mg) in 1 ml of anhydrous ether was added to the suspension, and the mixture was refluxed for 200 min with stirring. The reaction mixture was filtered under suction, and the ether was evaporated. One hundred and nine milligrams of reaction product were obtained. This [3H]LiAlH4 was used for the following reaction without separation from LiBr or unreacted AlBr, because preliminary experiments indicated that these bromides did not affect the subsequent reaction. Preparation
of 1 LX-[2-3H]Hydroxy-7-dehydrocholesterol
(la-OH-proDJ
The 1,Cadduct of la,2a-epoxycholesta-5,7-dien-3P-ol with 4-phenyl1,2,4,-triazoline-3,5-dione [Fig. 1 (I)] was prepared by the method of Kaneko et al. (14). Thirty milligrams of (I) were added slowly to 3 ml of anhydrous tetrahydrofuran containing [3H]LiAlH,, and the mixture was refluxed gently for 3 hr. After destroying the excess 13HlLiAlH4 with a 6 la-OH-proD3, ’ la-OH-preD,,
la-hydroxyprovitamin la-hydroxyprevitamin
Da. DS.
ICY-[2-3H]HYDROXYVITAMIN
FIG.
497
D3
I. Synthesis of 1w[2-3H]hydroxyvitamin
D,.
saturated aqueous solution of NazS04, the organic layer was separated and dried over MgSO,. The solvent was evaporated under reduced pressure. The residue was subjected to thin-layer chromatography and successive column chromatography on Sephadex LH-20. Iuz-[~-~H]OHproD, [Fig. I (II)] was obtained from fractions 70-89 (l-ml fractions). The ultraviolet absorption spectrum of the material indicated a yield of 5.23 mg of (II), with uv maxima at 272, 282, and 294 nm. Irradiation
of 1 m [2-3H]OH-proD3
Tritiated la-OH-proD, (5.23 mg) in 350 ml of distilled ether was irradiated for 50 set with a 400-W Toshiba photochemical Hg lamp through a Pyrex filter under an argon atmosphere. The solvent was removed in vacua at room temperature. The residue was chromatographed on a Sephadex LH-20 column with chloroform-n-hexane (65:35, v/v) as solvent, and l-ml fractions of eluate were collected. la-OH-preD, [Fig. 1 (III)] was eluted in fractions 51-64 and la-OHproD, in fractions 79-87. These fractions yielded 1.43 mg of la-OHpreD, (12.08 mCi) and 1.27 mg of la-OH-proD, (11.21 mCi), respectively. la-OH-preD, showed an absorption maximum at 260 nm in ethanol. Conversion
of I (I-[3H]OH-preD,
to I LU-[~K~OH-D,
Storage of la-[3H]OH-preD, in ether (50 ml) under argon in the dark for 12 days afforded I(-w-[~H]OH-D~ [Fig. 1 (IV)]. As shown in Fig. 2 the conversion of (III) to (IV) was almost complete 9 days after irradiation. The ether was evaporated off in vacua, and the residue was chromato-
498
TOHIRA ET AL.
4
2
0
FIG. 2. Radioscannograms showing conversion of la-[2JH]hydroxyvitamin 4. Two (A), nine (B), and Samples of the solution were applied to silica gel ether. Radioactivity on the plates was recorded with a
1(Y-[2-3H]hydroxyprevitamin D3 to twelve days (C) after irradiation. plates and developed with ethyl radioscanner.
6r
FIG.
3. Radioscannogram
of la-[2-3H]hydroxyvitamin
D,.
ICY-[2-3H]HYDROXYVITAMIN
Wavelength FIG. 4. Ultraviolet
499
D3
( nm)
absorption spectrum of 1(u-[2-3H]hydroxyvitamin
Dt.
graphed on a Sephadex LH-20 column. la-OH-D, was eluted in fractions 101-125 (OS-ml fractions), and these fractions yielded 972 pg of (IV) with a specific activity of 4.2 Ci/mmol. Figure 3 is a scannogram of the preparation with a radiochemical purity of 97.5%. Figure 4 shows the ultraviolet absorption spectrum of the compound with a maximum at 264 nm and minimum at 228 nm. When 0.02 ,ug (461,000 dpm) of laj3H]OH-D, was cochromatographed with 220 pg of authentic laOH-D3 (synthesized in our laboratory) on a Sephadex LH-20 column, its peak of ultraviolet absorption at 264 nm coincided exactly with the peak of radioactivity (Fig. 5). The radiochemical purity of the preparation was calculated to be over 9%.
500
TOHIRA ET AL.
8-
o-a-
IO
T 20 Fraction
30
number
40
-_
--50
60
70
( I ml 1
FIG. 5. Cochromatography of la-[2-3H]hydroxyvitamin D, with authentic Icu-hydroxyvitamin D, on a Sephadex LH-20 column. About 0.02 pg (461,000 dpm) of Icr[2-3H]OH-D3 and 220 pg of crystalline lo-OH-D3 were applied to a 1.0 x 35cm column packed with 10 g of Sephadex LH-20. The column was developed with chloroform:nhexane (65:35, v/v). Ultraviolet absorbance at 264 nm (- - - 0 ---); radioactivity (- 0 -).
Biological Activity of Ie[2-3H]OH-D3 Weanling male rats (Sprague-Dawley) were maintained for 6 weeks on a vitamin D-deficient diet (15). Then groups of six rats were injected intrajugularly with 0.25 pg of either 1cu-[2-3H]OH-D3 or authentic la-OH-Da, respectively, while six rats were injected with 0.05 ml of the vehicle, ethanol. The animals were decapitated 6 hr later, and blood samples were collected. The serum calcium concentration was determined with a Perkin-Elmer, Model 403, atomic absorption spectrometer. Results showed that 1a-[3H]OH-D3 was as active as authentic la-OH-D, in increasing the serum Ca concentration in vitamin D-deficient rats (Table 1). DISCUSSION
Recently, labeled preparations of vitamin D and its metabolites with high specific activities have been made by several investigators. DeLuca et al. (16) synthesized [22,23-3H]vitamin D4 with a specific activity of 750 mCilmmo1 by catalytic reduction with tritium gas, whereas Suda et al. (17) introduced tritiated methylene into an intermediate by a Grignard reaction to give 25-[26,27-3H]hydroxyvitamin
la-[2-3H]HYDROXYVITAMIN TABLE BIOLOGICAL
ACTIVITY
501
D,
I
OF ~c?[~-~H]HYDROXYVITAMIN
D3
Compound injected
Serum Ca (mg/dl)
95% Ethanol la-[2-3H]OH-D, (0.25 /.LgLg) la-OH-D3 to.25 ILg)
4.78 + 0.30 (6)” 6.36 ? 0.33 (6) 6.53 t 0.25 (6)
0 Mean k standard error (number of animals).
D3 (1.3 Ciimmol). These have been considered to be the two best methods for obtaining highly radioactive preparations. Very recently, Holick et al. (8,9) synthesized la-[6-3H]OH-D, with a specific activity of 4 Ci/mmol by reduction with [3H]NaBH, (20-40 Ci/ mmol). We also planned to prepare ~cx-[~H]OH-D~ by reduction with tritiated metal hydride. However, rH]NaBH, was not suitable for our synthetic method, but [3H]LiAlH4 was. The specific activity of the tritiated product depends on that of the [3H]LiAlH4. The specific activity of commercial [3H]LiAlH4 is only 50- 150 mCilmmo1, which is too low for our purpose. Therefore, we developed a method for preparing [3H]LiAlH, with a specific activity of more than 10 Ci/mmol. Reduction of Li with tritium gas at high temperature followed by addition of AlBr, in ether gave [3H]LiAlH,, whose specific activity, calculated from the amount of the tritium gas consumed, was 26 Ci/mmol. The intermediate to be reduced, the 1,6adduct of the la,2cy-epoxide with the triazoline derivative, was synthesized from cholesterol by the method of Kaneko et al. (14). The opening of the epoxide ring and cleavage of the triazoline group of this a-epoxidated cycloadduct with [3H]LiAlH, followed by irradiation and spontaneous conversion afforded ~cx-[~-~H]OH-D,. Reductive opening of such epoxidated steroids with LiAlD, gives 2p-deuteric products (18-21). Thus, the tritium atom on the 2-position of la-[3H]OH-D3 is considered to be entirely in the P-configuration. The specific activity of the Icx-[~H]OH-D~ synthesized can be calculated from that of the rH]LiAlH, used. Since only one hydrogen atom contributes to the reduction, the specific activity of the reaction product should be one-fourth of that of the [3H]LiAlH4. The specific activity of [3H]LiAlH, was calculated to be 26 Ci/mmol. Thus, the specific activity of ~cP[~H]OH-D, was expected to be 6.5 Ci/mmol. However, the actual specific activity of the la-[3H]0H-D, preparation, determined from its radioactivity and ultraviolet absorption, was 4.2 Ci/mmol.
502
TOHIRA ET AL.
This discrepancy may be because Li3H prepared from metallic Li and tritium gas is partly covered with unreacted melted metallic Li. Thus Li3H did not react quantitatively with AlBr,, so that the actual specific activity of the [3H]LiAIH, was lower than expected. Preliminary reports with this isotope on the metabolism of la-OH-D, in rats have been made (11,12). REFERENCES 1. Chalmers, T. M., Davie, M. W., Hunter, J. O., Szaz, K. F., Pelt, B., and Kodicek, E. (1973) Lance? 2, 696-699. 2. Chan, J. C. M., Oldham. S. B., Holick, M. F., and DeLuca, H. F. (1975) J. Amer.
3. Hirooka, J. Nutr.
Med.
Ass.
M., Wako, Sci.
234: 47-52.
H., Kaneko, C., Ishikawa, M., Sasaki, S., and Suda, T. (1975)
Vitaminol.
21, 277-285.
4. Holick, M. F., Semmler, E. J., Schnoes, H. K., and DeLuca, H. F. (1973) Science 180, 190- 191.
5. Barton, D. H. R., Hesse, R. H., Pechet, M. M., and Rizzardo, Chem.
Sot.
E. (1973) J. Amer.
95, 2748-2749.
6. Kaneko, C., Yamada, S., Sugimoto, A., Eguchi, Y., Ishikawa, M., Suda, T., Suzuki, M., Kakuta, S., and Sasaki, S. (1974) Steroids 23, 75-92. 7. Zerwekh, J. E., Brumbaugh, P. F., Haussler, D. H., Cork, D. J., and Haussler, M. R. (1974) Biochemistry 13, 4097-4102. 8. Holick, M. F., Holick, S. A., Tavela, T., Gallagher, B., Schnoes, H. K., and DeLuca, H. F. (1975) Science 190, 576-578. 9. Holick, M. F., Tavela, T. E., Holick, S. A., Schnoes, H. K., DeLuca, H. F., and Gallagher, B. M. (1976) J. Biol. Chem. 251, 1020-1024. IO. Holick, S. A., Holick, M. F., Tavela, T. E., Schnoes, H. K., and DeLuca, H. F. (1976) J. Biol.
Chem.
251,
1025-1028.
11. Fukushima, M., Suzuki, Y.. Tohira, Y., Matsunaga, I., Ochi, Nishii, Y., and Suda, T. (1975) Biochem. Biophys. Res. Commun. 12. Fukushima. M., Suzuki, Y., Tohira, Y., Nishii, Y., Suzuki, and Suda, T. (1976) FEBS Lett. 65, 211-214. 13. Holick, M. F., and DeLuca, H. F. (1971) J. Lipid Res. 12, 460-465. 14. Kaneko, C., Sugimoto, Y., Eguchi, Y., Yamada, S., and Ishikawa, hedron
K., Nagano, H., 66, 632-638. M., Sasaki,
M. (1974) Tetra-
30, 2701-2705.
15. Suda, T., DeLuca, H. F., and Tanaka. Y. (1970) J. Nutr. 100, 1049-1052. 16. DeLuca, H. F., Weller, M., Blunt, J. W., and Neville, P. F. (1968) Arch. Biophys.
Biochem.
124, 122-128.
17. Suda, T., DeLuca, H. F., and Hallick, R. B. (1971)Anal. Biochem. 43, 18. Nambara, T., Hosoda, H., Anjyo, T., and Ikegawa, S. (1972) Chem. 20, 2256-2261. 19. Cookson, R. C., Hamon, D. P. G., and Parker, R. E. (1952) J. Chem. 5024. 20. Djerassi, C.. Shapiro, R. H., and Vandewalle, M. (196.5) J. Amer. 87, 4892-4902. 21. Corey, E. J., Howell, M. G., Boston, A., Young, R. L., and Sneen, J. Amer.
S.,
Chem.
Sot.
78, 5036-5040.
139-146. Pharm. Sot. Gem.
Bull.
5014Sot.
R. A. (1956)
BIOCHEMISTRY
77, 495-502 (1977)
Synthesis of 1~[2-3H]Hydroxyvitamin
D,
YASUO TOHIRA, KIYOSHIGE OCHI, ISAO MATSUNAGA, MASAFUMI FUKUSHIMA, SHIGERU TAKANASHI, KAZUHITO HATA,’ CHIKARA KANEKO,~ AND TATSUO SUDAN Research Laboratory of Chugai Pharmaceutical Co. Ltd., Takada, Toshima-ku, Tokyo, Japan 171, ‘Technical Department, Sinloihi Co. Ltd., Dai, Kamakura, Japan 247, ZDepartment of Pharmacy, University of Kanazawa, Takara-rho, Kanazawa, Japan 920, and 3Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ka, Tokyo, Japan 113
Received July 29. 1976; accepted October I. 1976 lo-[2-3H]Hydroxyvitamin D3 was synthesized chemically. The preparation was radiochemically pure and had a high enough specific activity (4.2 Wmmol) to permit experiments using 62.5 pmolkat. This preparation had as much effect as synthetic la-hydroxyvitamin D, in increasing the serum Ca level of vitamin D-deficient rats.
It is known that la-hydroxyvitamin D, (la-OH-D,)4 can replace the metabolically active form of vitamin D,, la,25dihydroxyvitamin D, [h~,25-(0H),-D,]~ in the treatment of various vitamin D-refractory syndromes (l-3). This synthetic analog appears to be of great value for clinical use because it is easier and less expensive to prepare than la,25(OH),-D3 (4-6). In addition, Zerwekh et al. (7) isolated a fraction containing la,25(OH),-D, from intestinal chromatin of rachitic chicks after administration of la-OH-D,. These findings suggest that the unnatural form of the D vitamin, la-OH-D,, is metabolized to the natural hormone of vitamin D,. Final proof of this, however, requires metabolic studies with radioactive la-OH-D,. Very recently, Holick et al. (8-10) synthesized Iu-[~-~H]OH-D~ and demonstrated that it was metabolized very rapidly to lc~,25-[~H](OH),D3 in viva and in vitro. Almost simultaneously we synthesized 1~[2-~H]OH-D3 and reached the same conclusion from studies on perfused rat liver (11). This paper reports the synthesis of ~cx-[~-~H]OH-D, with a specific activity of 4.2 Ci/mmol. Preliminary studies with this isotope on the metabolism of la-OH-D, in rats have been reported (11,12). 4 la-OH-D,, la-hydroxyvitamin 4. 5 la,25-(OH),-D,, lcl,2Sdihydroxyvitamin
4. 495
Copyright All rights
0 1977 by Academic Press, Inc. of reproductmn in any form reserved.
ISSN
0003-2697
496
TOHIRA ET AL.
METHODS AND RESULTS
General Procedure Ultraviolet absorption spectra were determined in ethanol with a Hitachi 124 spectrophotometer. The following molar extinction coefficients were used: la-OH-proD,,6 hZBZnrn,E = 11,000; la-OH-preD,,’ h260nm, E = 9000; and la-OH-D,, hZ64n,,,, E = 18,100. Radioactivity was measured in a Nuclear Chicago, Mark I, liquid scintillation spectrometer. Samples were counted in 10 ml of scintillator solution consisting of 7 g of PPO and 90 mg of dimethyl-POPOP/liter of toluene. Thin-layer chromatography was carried out on Merck silica gel 60 containing FZs4, with 100% ether as soIvent. Radioscannograms were recorded with an Aloka JTC201 instrument. Column chromatography was carried out on 1.0 x 35-cm glass columns packed with 10 g of Sephadex LH-20 (Pharmacia) with 65% chloroform-35% n-hexane as solvent, according to the method of Holick and DeLuca (13). All solvents were distilled immediately before use. Preparation
of Tritiated
Lithium
Alumnium
Hydride
Metallic Li (12 mg) was heated in tritium gas for 135 min from 60 (initial) to 550°C (final) under a pressure of from 276 (initial) to 174 (final) mm Hg. A total volume of 12 cm3 (31 Ci) of tritium gas reacted on the metal. The yield, calculated from the amount of tritium gas which reacted, was 61%. To a portion (13 mg) of the reaction product, 24 mg of LiH as carrier and 1.3 ml of anhydrous ether were added; the mixture was stirred to obtain a homogeneous suspension. Then AlBr, (290 mg) in 1 ml of anhydrous ether was added to the suspension, and the mixture was refluxed for 200 min with stirring. The reaction mixture was filtered under suction, and the ether was evaporated. One hundred and nine milligrams of reaction product were obtained. This [3H]LiAlH4 was used for the following reaction without separation from LiBr or unreacted AlBr, because preliminary experiments indicated that these bromides did not affect the subsequent reaction. Preparation
of 1 LX-[2-3H]Hydroxy-7-dehydrocholesterol
(la-OH-proDJ
The 1,Cadduct of la,2a-epoxycholesta-5,7-dien-3P-ol with 4-phenyl1,2,4,-triazoline-3,5-dione [Fig. 1 (I)] was prepared by the method of Kaneko et al. (14). Thirty milligrams of (I) were added slowly to 3 ml of anhydrous tetrahydrofuran containing [3H]LiAlH,, and the mixture was refluxed gently for 3 hr. After destroying the excess 13HlLiAlH4 with a 6 la-OH-proD3, ’ la-OH-preD,,
la-hydroxyprovitamin la-hydroxyprevitamin
Da. DS.
ICY-[2-3H]HYDROXYVITAMIN
FIG.
497
D3
I. Synthesis of 1w[2-3H]hydroxyvitamin
D,.
saturated aqueous solution of NazS04, the organic layer was separated and dried over MgSO,. The solvent was evaporated under reduced pressure. The residue was subjected to thin-layer chromatography and successive column chromatography on Sephadex LH-20. Iuz-[~-~H]OHproD, [Fig. I (II)] was obtained from fractions 70-89 (l-ml fractions). The ultraviolet absorption spectrum of the material indicated a yield of 5.23 mg of (II), with uv maxima at 272, 282, and 294 nm. Irradiation
of 1 m [2-3H]OH-proD3
Tritiated la-OH-proD, (5.23 mg) in 350 ml of distilled ether was irradiated for 50 set with a 400-W Toshiba photochemical Hg lamp through a Pyrex filter under an argon atmosphere. The solvent was removed in vacua at room temperature. The residue was chromatographed on a Sephadex LH-20 column with chloroform-n-hexane (65:35, v/v) as solvent, and l-ml fractions of eluate were collected. la-OH-preD, [Fig. 1 (III)] was eluted in fractions 51-64 and la-OHproD, in fractions 79-87. These fractions yielded 1.43 mg of la-OHpreD, (12.08 mCi) and 1.27 mg of la-OH-proD, (11.21 mCi), respectively. la-OH-preD, showed an absorption maximum at 260 nm in ethanol. Conversion
of I (I-[3H]OH-preD,
to I LU-[~K~OH-D,
Storage of la-[3H]OH-preD, in ether (50 ml) under argon in the dark for 12 days afforded I(-w-[~H]OH-D~ [Fig. 1 (IV)]. As shown in Fig. 2 the conversion of (III) to (IV) was almost complete 9 days after irradiation. The ether was evaporated off in vacua, and the residue was chromato-
498
TOHIRA ET AL.
4
2
0
FIG. 2. Radioscannograms showing conversion of la-[2JH]hydroxyvitamin 4. Two (A), nine (B), and Samples of the solution were applied to silica gel ether. Radioactivity on the plates was recorded with a
1(Y-[2-3H]hydroxyprevitamin D3 to twelve days (C) after irradiation. plates and developed with ethyl radioscanner.
6r
FIG.
3. Radioscannogram
of la-[2-3H]hydroxyvitamin
D,.
ICY-[2-3H]HYDROXYVITAMIN
Wavelength FIG. 4. Ultraviolet
499
D3
( nm)
absorption spectrum of 1(u-[2-3H]hydroxyvitamin
Dt.
graphed on a Sephadex LH-20 column. la-OH-D, was eluted in fractions 101-125 (OS-ml fractions), and these fractions yielded 972 pg of (IV) with a specific activity of 4.2 Ci/mmol. Figure 3 is a scannogram of the preparation with a radiochemical purity of 97.5%. Figure 4 shows the ultraviolet absorption spectrum of the compound with a maximum at 264 nm and minimum at 228 nm. When 0.02 ,ug (461,000 dpm) of laj3H]OH-D, was cochromatographed with 220 pg of authentic laOH-D3 (synthesized in our laboratory) on a Sephadex LH-20 column, its peak of ultraviolet absorption at 264 nm coincided exactly with the peak of radioactivity (Fig. 5). The radiochemical purity of the preparation was calculated to be over 9%.
500
TOHIRA ET AL.
8-
o-a-
IO
T 20 Fraction
30
number
40
-_
--50
60
70
( I ml 1
FIG. 5. Cochromatography of la-[2-3H]hydroxyvitamin D, with authentic Icu-hydroxyvitamin D, on a Sephadex LH-20 column. About 0.02 pg (461,000 dpm) of Icr[2-3H]OH-D3 and 220 pg of crystalline lo-OH-D3 were applied to a 1.0 x 35cm column packed with 10 g of Sephadex LH-20. The column was developed with chloroform:nhexane (65:35, v/v). Ultraviolet absorbance at 264 nm (- - - 0 ---); radioactivity (- 0 -).
Biological Activity of Ie[2-3H]OH-D3 Weanling male rats (Sprague-Dawley) were maintained for 6 weeks on a vitamin D-deficient diet (15). Then groups of six rats were injected intrajugularly with 0.25 pg of either 1cu-[2-3H]OH-D3 or authentic la-OH-Da, respectively, while six rats were injected with 0.05 ml of the vehicle, ethanol. The animals were decapitated 6 hr later, and blood samples were collected. The serum calcium concentration was determined with a Perkin-Elmer, Model 403, atomic absorption spectrometer. Results showed that 1a-[3H]OH-D3 was as active as authentic la-OH-D, in increasing the serum Ca concentration in vitamin D-deficient rats (Table 1). DISCUSSION
Recently, labeled preparations of vitamin D and its metabolites with high specific activities have been made by several investigators. DeLuca et al. (16) synthesized [22,23-3H]vitamin D4 with a specific activity of 750 mCilmmo1 by catalytic reduction with tritium gas, whereas Suda et al. (17) introduced tritiated methylene into an intermediate by a Grignard reaction to give 25-[26,27-3H]hydroxyvitamin
la-[2-3H]HYDROXYVITAMIN TABLE BIOLOGICAL
ACTIVITY
501
D,
I
OF ~c?[~-~H]HYDROXYVITAMIN
D3
Compound injected
Serum Ca (mg/dl)
95% Ethanol la-[2-3H]OH-D, (0.25 /.LgLg) la-OH-D3 to.25 ILg)
4.78 + 0.30 (6)” 6.36 ? 0.33 (6) 6.53 t 0.25 (6)
0 Mean k standard error (number of animals).
D3 (1.3 Ciimmol). These have been considered to be the two best methods for obtaining highly radioactive preparations. Very recently, Holick et al. (8,9) synthesized la-[6-3H]OH-D, with a specific activity of 4 Ci/mmol by reduction with [3H]NaBH, (20-40 Ci/ mmol). We also planned to prepare ~cx-[~H]OH-D~ by reduction with tritiated metal hydride. However, rH]NaBH, was not suitable for our synthetic method, but [3H]LiAlH4 was. The specific activity of the tritiated product depends on that of the [3H]LiAlH4. The specific activity of commercial [3H]LiAlH4 is only 50- 150 mCilmmo1, which is too low for our purpose. Therefore, we developed a method for preparing [3H]LiAlH, with a specific activity of more than 10 Ci/mmol. Reduction of Li with tritium gas at high temperature followed by addition of AlBr, in ether gave [3H]LiAlH,, whose specific activity, calculated from the amount of the tritium gas consumed, was 26 Ci/mmol. The intermediate to be reduced, the 1,6adduct of the la,2cy-epoxide with the triazoline derivative, was synthesized from cholesterol by the method of Kaneko et al. (14). The opening of the epoxide ring and cleavage of the triazoline group of this a-epoxidated cycloadduct with [3H]LiAlH, followed by irradiation and spontaneous conversion afforded ~cx-[~-~H]OH-D,. Reductive opening of such epoxidated steroids with LiAlD, gives 2p-deuteric products (18-21). Thus, the tritium atom on the 2-position of la-[3H]OH-D3 is considered to be entirely in the P-configuration. The specific activity of the Icx-[~H]OH-D~ synthesized can be calculated from that of the rH]LiAlH, used. Since only one hydrogen atom contributes to the reduction, the specific activity of the reaction product should be one-fourth of that of the [3H]LiAlH4. The specific activity of [3H]LiAlH, was calculated to be 26 Ci/mmol. Thus, the specific activity of ~cP[~H]OH-D, was expected to be 6.5 Ci/mmol. However, the actual specific activity of the la-[3H]0H-D, preparation, determined from its radioactivity and ultraviolet absorption, was 4.2 Ci/mmol.
502
TOHIRA ET AL.
This discrepancy may be because Li3H prepared from metallic Li and tritium gas is partly covered with unreacted melted metallic Li. Thus Li3H did not react quantitatively with AlBr,, so that the actual specific activity of the [3H]LiAIH, was lower than expected. Preliminary reports with this isotope on the metabolism of la-OH-D, in rats have been made (11,12). REFERENCES 1. Chalmers, T. M., Davie, M. W., Hunter, J. O., Szaz, K. F., Pelt, B., and Kodicek, E. (1973) Lance? 2, 696-699. 2. Chan, J. C. M., Oldham. S. B., Holick, M. F., and DeLuca, H. F. (1975) J. Amer.
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