ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 197, No. 1, October 1, pp. 119-125, 1979

Synthesis of Vitamin D,: Its Biological Activity Relative to Vitamins DB and D,’ JOSEPH

L. NAPOLLZ

MARY A. FIVIZZANI, HEINRICH AND HECTOR F. DELUCA3

Department of Biochemistry, College University of Wisconsin-Madison,

of Agricultural Madison,

K. SCHNOES,

and Life Sciences, Wisconsin 53706

Received October 24, 1978; revised May 23, 1979 The chemical synthesis, spectral characterization, and biological activity of vitamin D, in vitamin D-deficient rats is reported. Vitamin D, is about 180-fold less active than vitamin D, in calcification of rachitic cartilage and about lOO- to 200-fold less active in induction of bonecalcium mobilization. In stimulation of intestinal-calcium transport, vitamin D, is about 80. fold less active than vitamin DI. Vitamins D, and D, appear to be equiactive in all three responses when low doses are administered.

The D vitamins are a family of 9,10secosteroids which differ only in side-chain structure. Vitamin D, is the most physiologically important member of the family since it is the only form generated in viwo. Until recently, however, vitamin D, was the most important form commercially, since it was the D vitamin given to man and all domestic animals, except birds (1). Both vitamins D, and D, are converted in vivo to la,25-dihydroxyvitamin D &~,25-(0H),D)~ which is the hormonal form that directly regulates calcium and phosphorus metabolism in bone and intestine (2-5). With the exception of vitamin D, (6-8), other members of the D group, such as vitamin D, (9, 10) and vitamin D, (9, 11, 12), have not ’ Supported by Program-Project Grant No. AM14881 and Postdoctoral Training Grant No. DE-07031 from the National Institutes of Health and the Harry Steenbock Research Fund. * Present address: Department of Biochemistry, University of Texas Health Sciences Center, Dallas, Tex. 75235. 3 To whom all correspondence should be addressed. 4 Abbreviations used: 1,25-(OH),D, 1,25dihydroxyvitamin D; la-OH-D,, lo-hydroxyvitamin D,; 25 OH-D,, 25-hydroxyvitamin D,; NMR, nuclear magnetic resonance; glc, gas-liquid chromatography; hplc, high-pressure liquid chromatography.

been rigorously characterized either chemically or biochemically. Because of the current interest in D vitamins as potential candidates for treatment of renaI osteodystrophy and osteoporosis, we decided to compare vitamins D$, D3, and D, for efficacy in mediating calcium and phosphorus metabolism. This study was prompted in part by a report that excessive doses of vitamin D, are not as hypercalcemic as excessive doses of vitamin D, (13); and by a recent report that la-hydroxyvitamin D, (la-OH-D*) (14) may not stimulate bone-calcium mobilization to the same extent that it stimulates intestinal-calcium transport, relative to 25-hydroxyvitamin D3 (a&OH-D,) . (15).. This paper will demonstrate that vitamms Dz, DB, and D, have qualitatively, but not quantitatively, similar effects on calcium homeostasis when low, nonsaturating doses are administered. MATERIALS

AND METHODS

General Nuclear magnetic resonance (NMR) spectra were taken in CDCl, with a Bruker WH-270 FT spectrometer. High-resolution mass spectra were obtained at 120°C above ambient at 70 eV with an AEI MS-9 spectrometer coupled to a DS-50 Data System. Ultraviolet absorbanee spectra (u-v) were taken in ethanol 119

0003-9861/79/110119-07$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

120

NAPOLI

ET AL. D2 was purchased from Sigma Chemical Company, St. Louis, Missouri. The structure of vitamin D, was verified by NMR spectroscopy.

Synthesis

FIG. 1. Side-chain structures of D vitamins. The alkyl substituents in vitamins D, and D, are 24E. Those in vitamins D, and D, are 24s. Although in D, the alkyl group at C-24 is in the same absolute configuration as it is in D, the R designation changes to S in the absence of a 22-ene according to the Sequence Rules. The same is true for D, and D,. with a Beckman Model 24 recording spectrophotometer. Gas-liquid chromatography (glc) was done on a Packard Model 417 Becker gas chromatograph fitted with a 3% OV-101 on Chromasorb 30 column (2 mm x 6 ft) run at a helium flow rate of 30 mUmin. High-performance liquid chromatography (hplc) was done with a Waters Associates Model ALUGPC 204 using a microparticulate silica gel column (0.7 x 25 cm, 5-pm particles) developed with 1% 2propanophexane. Radioactivity was determined with a Packard Model 3375 Iiquid scintillation spectrometer. Scintillant was made by dissolving 2 g of 2,5-diphenyloxazole and 0.1 g of 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene in 1 liter of toluene.

Biological Weanling male rats (21 days old), from the Holtzman Company, Madison, Wisconsin, were individually housed in overhanging wire cages and fed vitamin D-deficient diets of either low calcium (0.02%) and adequate phosphorus (0.3%) or high calcium (1.2%) and low phosphorus (0.1%) for 2-3 weeks prior to use (16). Animals were dosed intraperitoneally consecutively for 7 days with vehicle alone (control groups) or a test compound in 0.05 ml 1,Zpropanediol. All animals were sacrificed 24 h after the last dose. Duodena from rats on the low-calcium diet were used to measure intestinal-calcium transport by the everted intestinal sac technique of Martin and DeLuca (17). Sera (0.1 ml) from the same rats were diluted with 0.1% lanthanum chloride (1.9 ml) and measured for calcium content with a Perkin-Elmer Model 403 atomic absorption spectrometer. The degree of endochondral calcification was measured by the line-test method (18) with the radii and ulnae from rats on the lowphosphorus (rachitogenic) diet.

Chemical Vitamin D3 was purchased from the PhilipsDuphar Company, Weesp, The Netherlands. Vitamin

i-Sitosterol methyl ether (2). i-Stigmasteryl methyl ether (I, 5.0 g) in ethyl acetate (200 ml) over 5% Pd/C (2.0 g) was hydrogenated for 5.5 h at 65°C under 45 psi. Gas-liquid chromatographic analysis of the reaction mixture indicated that 97% reduction had occurred. Sitosterol (4). Sitosterol 3P-acetate, 3, was prepared by heating 2 with glacial acetic acid to give 3 (19, 20). An ethereal solution of 3 (4.8 g/40 ml) was treated with 0.1 M methanolic KOH (20 ml) at room temperature for 1.5 h to provide sitosterol 4, which was recrystallized from ethanol: mp 137-138°C [lit. (21) 137.5-139”C]; NMR and mass spectra were as expected. Alternatively, 4 was obtained by heating 2 (1.0 g) in 80% dioxaneiwater (24 ml) with p-toluenesulfonic acid (0.1 g) at 80°C for 2 h (19, 20). 7DehydrositosteroZ (5). A mixture of 3 (0.25 g, 0.55 mmol), sodium bicarbonate (0.27 g), and 1,3dibromo-5,5-dimethylhydantoin (0.11 g, 0.38 mmol) in hexane (8 ml) and benzene (5 ml) was heated at 70°C under nitrogen for 20 min. The mixture was cooled and filtered. The residue obtained upon evaporation of the filtrate was immediately dissolved in xyIene (8 ml) and s-collidine (2 ml) and heated at reflux under nitrogen for 90 min. Benzene was added and the organic phase washed with 1 N HCI (twice), water, dilute sodium bicarbonate, and saturated sodium chloride. The dried product was heated with p-toluene sulfonic acid (0.035 g) at 70°C under nitrogen in dry dioxane (7 ml) for 30 min. The mixture was diluted with ether; and washed with dilute sodium bicarbonate (twice), water, and brine. The residue obtained upon evaporation of solvent was allowed to stand in ether (2 ml) and 0.9 M methanolic KOH for 2.5 h. Water and ether were added. The phases were separated and the aqueous phase was washed with ether (thrice). The combined ether phases were washed with water (twice), brine, and dried (Na,SO,). The residue recovered after evaporation was chromatographed on a silica gel column (14 g, 1 x 30 cm) developed with ethyl acetate/chloroform (25175). The product 5 (95 mg), which eluted in 30 to 50 ml, was recrystallized once from ethanol: uv A n,al 262, 271, 281,291 nm; NMR 6 0.62 (s, 18-methyl), 0.82 (d, J = 6 Hz, 26,27-methyls), 0.85 (t, J = 6 Hz, 29-methyI), 0.95 (s, 1Bmethyl), 0.95 (d, J = 6 Hz, 21-methyl), 3.64 (broad m, 3cY-proton), 5.39, 5.57 (multiple& 6 and 7 protons); mass spectrum m/e (relative intensity, composition, m/e calcd.) 412.3719 (1.00, C,,H,,O, 412.3704), 394.3598 (0.09, C,,H,,, 394.3608), 379.3360 (0.62, CZ8H43,380.3365), 353.3205 (0.29, C,,H,,, 353.3208), 271.2055 (0.07, C&Hs,O, 271.2062), 253.1953 (0.13, C,,H,,, 253.1956), 211.1486

VITAMIN

Dj ACTIVITY

RELATIVE

TO VITAMINS

121

D, AND D,

&F-+4? OCHl OCH, ..&-

RoL&+ 2, R=Ac

4,

R=H

h”

FIG. 3. Gas-liquid chromatogram of synthetic vitamin D,. The two peaks result from thermal isomerization of 7 to pyro- and isopyro-7 with retention times of 17.5 and 19.5 min (24), respectively.

FIG. 2. Synthesis of vitamin D,. i-Stigmasterol methyl ether, 1, was hydrogenated to give i-sitosterol methyl ether, 2. Compound 2 was converted to sitosterol acetate, 3, which was used to synthesize the provitamin 7-dehydrositosterol, 5. Irradiation of 5, gave previtamin 6 and thermal isomerization of 6 gave vitamin D,, 7. (0.12, C,,H,,, 199.1486).

211.1486),

199.1489 (0.11,

synthesized. 7-Dehydrositosterol would serve as provitamin for ?‘, but commercial sitosterol is only about 65% pure. Campesterol represents most of the remaining substances. Because of the similarities between the structures of sitosterol and campesterol, which differ only in that one possesses an ethyl group at the 24R-position, whereas

C,,H,,,

Vitumin D, (7). A cooled (ice bath) solution of 5 (22 mg) in ether (150 ml) under nitrogen was irradiated for 3 min with a mercury arc lamp (Hanau TQ 150 Zz) fitted with a Vycor filter. The residue obtained after evaporating the ether was purified by hplc. Pure 6 (13 mg) eluted in 32 ml: uv A,,, 260, A,,,in235nm. The previtamin 6 in ethanol (2.5 ml) was heated at 75°C for 2 h. Chromatography (hplc) of the residue yielded 7 which eluted in 48 ml: uv A,,, 265, ,&, 228 nm; NMR 6 0.54 (s, l&methyl), 0.82 (d, J = 6 Hz, 26,27-methyls), 0.85 (t, J = 6 Hz, 29-methyl), 0.90 (d, J = 6 Hz, 21-methyl), 3.95 (broad m, 3a-proton), 4.83, 5.05 (19-protons), 6.03, 6.24 (doublets, J = 12 Hz, 6- and i’-protons); mass spectrum m/e (relative intensity), composition, m/e calcd. 412.3732 (0.22, C,,H,,O, 412.3705), 379.3387(1.04, C28H430,379.3365), 271.2069 (0.03, C,,H,,O, 271.2062), 176.1218 (0.06, C,,H,,O, 176.1201), 158.1087 (0.13, f&H,,, 153.1096), 136.0874 (1.00, CsH,,O, 136.0888), 118.0734 (0.69, C,H,,, 118.0783). The vitamin 7 was homogeneous on glc with retention times of 17.5 and 19.5 min (pyroand isopyro forms; oven temperature 240°C). RESULTS

Synthesis

Pure vitamins D, and D, are commercially available, but vitamin D, (Fig. 1) had to be

0.01,

’

’

’

250

Wavelength

’

’

’

’

’

300

fi

( nm)

FIG. 4. Ultraviolet (uv) absorbance spectrum of vitamin D, in ethanol. The ratio of Amax 265 to Amin ~8 nm was 2.0.

122

NAPOLI

ET AL.

the other has a methyl group at the 24Rposition, the two compounds could not be conveniently separated by recrystallization, hplc, or argenation-layer chromatography. However, the compounds are sufficiently different so that their respective vitamins may have quite different biological activity. It is obvious that pure sitosterol was imperative for sound studies. It appeared that the simplest route to pure sitosterol, 4, would be by synthesis from the methyl ether of i-stigmasterol, 1. Thus, steroid 1 was hydrogenated to give methyl i-sitosterol, 2, in excellent yield in a reasonable time (Fig. 2). Milder reduction conditions, or platinum as catalyst, resulted in long reduction times and incomplete reductions that created problems in conveniently separating i-stigmasteryl methyl ether from i-sitosteryl methyl ether. Conversely, more stringent conditions caused reduction of the 3,5-bond. The hydrogenation product 2 was rearranged to sitosterol acetate, 3, with warm glacial acetic acid, or directly to sitosterol, 4, by treatment with p-toluenesulfonic acid. Sitosterol thus obtained was pure as determined by its mass and NMR spectra, and by comparison of its melting point with literature values (21,22). Sitosterol acetate, 3, was converted to 7dehydrositosterol (5, provitamin D5) by bromination and dehydrobromination5 followed by alkaline hydrolysis (25). The uv, NMR, and high-resolution mass spectra of 5 were as expected and clearly established the structure of 5 as pure provitamin D,. Irradiation of 5 gave the previtamin 6 which was separated from provitamin 5 and other irradiation products by hplc. The 6 obtained was homogeneous by hplc and its uv spectrum showed only one absorbance maximum at 260 nm which additionally demonstrated it was free of contaminating materials. Thermally induced rearrangement of 6 provided vitamin Dg, 7, which was homogeneous by glc analysis (Fig. 3). The two peaks in the glc trace of 7 result from the well-known thermal isomerization of vitamin D-like compounds to their pyroand isopyro forms (24). The uv spectrum of 7 (Fig. 4) displayed the typical vitamin 5 T. Narwid, Hoffmann-LaRoche N. J., personal communication.

Co.,

Nutley,

VITAMIN

D, ACTIVITY

RELATIVE TABLE

DOSE-RESPONSE

RELATIONSHIPS MOBILIZATION

TO VITAMINS

123

D, AND D,

I

OF VITAMINS D, AND D, IN PROMOTING AND INTESTINAL-CALCIUM TRANSPORTS

BONE-CALCIUM

Compound

Dose (rig/day)

Serum calcium (mg/lOO ml)

Intestinal transport (Wa serosal/45Ca mucosal)

1,2-Propanediol

-

4.3 r 0.1

2.1 + 0.2

Vitamin D,

2.5 12.5 25.0

4.4 t 0.1 4.8 k 0.2 5.1 + 0.1”

3.8 2 0.4’ 5.8 + 0.8c 4.8 iz 0.5c

Vitamin D,

2.5 12.5 25.0 50.0

4.4 4.7 4.9 5.0

3.3 2 0.4’ 4.5 2 0.7’ 4.2 2 0.6’ -

2 0.1 -c 0.1 2 0.1* rt 0.1”

4 Values are means 2 SEM of data from 5-6 animals. Animals were dosed daily for ‘7 days (see Materials and Methods). b.c Significantly different from controls; P < 0.005 and P < 0.01, respectively.

D absorbance resulting from the cis-triene chromophore. NMR spectroscopy confirmed the assignment as a vitamin D structure and established the presence of an ethyl substituent on the molecule. A highresolution mass spectrum (Fig. 5), and the knowledge that the provitamin was sitosterol, completed the structural characterization of 7.

tion and the mineralization of rachitic cartilage in vitamin D-deficient rats. Neither vitamin D, nor vitamin D, caused an increase in serum-calcium concentration after a dose of 2.5 rig/day for 1 week (Table I). An equivalent response was produced when 12.5 nglday of either vitamin was dosed. But at least 25 rig/day of either vitamin was necessary to give a rise in serum calcium significantly different from control at the P < 0.005 probability. Vitamin D, appeared to be slightly more active than vitamin D2 in stimulating bone-calcium mobilization; however, there is no statistical

Biological Assay

Vitamins DS, Dz, and D, were compared for ability to induce intestinalcalcium transport, bone-calcium mobilizaTABLE DOSE-RESPONSE

II

RELATIONSHIP OF VITAMIN D, IN STIMULATION OF BONE-CALCIUM MOBILIZATION AND INTESTINAL-CALCIUM TRANSPORT”

Compound

Dose @g/day)

Serum calcium (mg/lOO ml)

Intestinal transport (%a serosal/?a mucosal)

1,2-Propanediol

-

4.2 ‘- 0.1

1.4 k 0.1

4.9 k 0.2*

5.3 2 0.7’

4.4 4.4 4.8 4.9 5.5

2.9 3.5 5.9 3.9 4.1

Vitamin D,

25

Vitamin D,

500 1,000 2,500 5,000 10,000

2 + 5 + k

0.1 0.1 O.lb 0.1” 0.2b

2 + -t 2 ”

0.3’ 0.2” 0.7” 0.4’ 0.4c

a Data are expressed as means + SEM of values from 5-6 animals. Animals were dosed daily for 7 days (see Materials and Methods). b,c Significantly different from controls; P < 0.005 and P < 0.01, respectively.

124

NAPOLI ET AL. TABLE III

ANTIRACHITIC

(CALCIFICATION) OF D VITAMINS” Dose

Compound 1,2-Propanediol Vitamin D3 Vitamin D, Vitamin D,

(rig/day) 20 21 1000 5000

PROPERTIES

Calcification score 0.0 3.6 3.9 0.9 4.1

2 0.0 2 0.7 +- 0.1 -t 0.5 + 0.3

1~/Pie 0.0 36.0 37.0 0.2 0.2

a Data are expressed as means f SEM of scores from 4-6 animals. b International units per microgram. Calculated by: calcification score x lOOO/(ng/day x 5).

difference between D, and D, at dosages of 12.5 or 25 rig/day. Both vitamins DZ and D, produced saturating intestinal-calcium transport responses when 12.5 rig/day or more was administered. The 2.5 rig/day dose of each produced a significant response which was not maximal. These data confirm previously published data on the biological activity of vitamin D3 (25, 26), and extend the observations to vitamin D,. Vitamin D, doses of 2500-5000 nglday compared well with a dose of 25 rig/day of vitamin D3 in producing bone-calcium mobilization (Table II). On the other hand, vitamin D, caused a saturated intestinalcalcium transport response when about 1000 rig/day was dosed. Therefore, vitamin D5 is about 80-fold less active than vitamin D, in the intestine and about lOO- to 200fold less active in bone; whereas, at low doses, vitamins D2 and D3 have similar activities. Likewise, vitamins D2 and D3 were equipotent in inducing new endochondral calcification in the radii and ulnae of vitamin D-deficient rats on the low-phosphorus diet (Table III). Their calculated biopotencies in IU/pg were comparable. However, about 180 times as much vitamin D, was required for a score comparable to those produced by vitamins D, and D,. DISCUSSION

It is evident from the results that vitamins D3 and D, have similar effects on calcium metabolism in bone and intestine

when low doses are chronically administered. Vitamin Dg, on the other hand, is much less active than either vitamin D, or DS, but like vitamins D, and Da, shows no conspicuous preferential activity in bone or intestine. Notably, for all three vitamins, doses from two- to five-fold greater than the doses that initiated intestinal-calcium transport were required to stimulate bonecalcium mobilization. Thus, in assaying vitamin D analogs for differential tissue activity, or for ability to selectively inhibit vitamin D-mediated calcium metabolism in specific tissues, the more sensitive nature of the vitamin D-mediated intestinal response, relative to the bone response, must be considered. Apparently, C-24 alkyl substitution does not have a strong effect on selectivity of vitamin D action in regulating blood calcium concentration. It is important to emphasize, however, that the assays used in this work primarily measure blood-calcium homeostasis effects and do not necessarily reflect bone remodeling modulation. Since bone remodeling and blood-calcium regulation are related, but distinct physiological events, it is conceivable that each of these vitamins could affect remodeling differently despite their apparent similarities in regulating blood-calcium concentration As such, the side-chain analogs of vitamin D3 remain candidates for evaluation in longterm vitamin D therapy where a minimum of bone resorption may be desirable. The similar activity of vitamins Dz and D3 indicates that substitution of both a 24S-methyl group and a 22,23-ene in the vitamin D, side-chain does not affect biopotency of vitamin D compounds. But introduction of only the methyl group, in the same configuration decreases activity as shown by vitamin D4, which is about twothirds as active as vitamins D, and D, (8). Substitution of an ethyl group in the opposite configuration further decreases bioactivity to the extent that vitamin D, becomes IOO- to 200-fold less active than vitamin DS. By analogy to vitamins D, and D4, vitamin D, should be about twice as active as vitamin D,. We, therefore, can summarize the order of activity in mammals as: D, = D, > D, s Ds > Dg. Itisprobable that the discrimination by birds (27-29) and

VITAMIN

D, ACTIVITY

RELATIVE

new world monkeys (30) against vitamin D, would also be suffered by the other sidechain substituted D vitamins. The differences among the D vitamins in mammals could be the result of affinity differences for: transport proteins; liver and/or kidney hydroxylases; or target-tissue receptors, to name a few. Synthesis and evaluation of la-hydroxylated vitamin D side-chain analogs could be of help in distinguishing among the possibilities. ACKNOWLEDGMENTS We help Sean NMR

wish to thank Mr. Melvin Micke for technical with the high-resolution mass spectra and Mr. Hehir for technical help with the 270-MHz spectra.

REFERENCES 1. DELUCA, H. F. (1976) in Handbook of Physiology (Aurbach, G. D., ed.), Vol. 7, Chap. 11, American Physiological Society, Washington, D. C. 2. DELUCA, H. F. (19’78) in Handbook of Lipid Research (DeLuca, H. F., ed.), Vol. 2, Chap. 2, pp. 69-132, Plenum, New York. 3. DELUCA, H. F., AND SCHNOES, H. K. (1976) Annu. Rev. Biochem. 45, 631-666. 4. KODICEK, E. (1974) Lancet 1, 325-329. 5. JONES, G., SCHNOES, H. K., AND DELUCA, H. F. (1976) J. Biol. Chem. 251, 24-28. 6. WINDAUS, A., AND TRAUTMAN, G. (1937) Z. Physiol. Chem. 247, 185-188. 7. WINDAUS, A., AND GONTZEL, B. (1939) Ann. Chem. 538, 120-127. 8. DELUCA, H. F., WELLER, M., BLUNT, J. W., AND NEVILLE, P. F. (1968)Arch. Biochem. Biophys. 124, 122- 138. 9. GRAB,

W. (1936)

2. Physiol.

Chem.

243, 63-89.

TO VITAMINS

D, AND

DZ

125

10. W~NDERLICH, W. (1936) Z. Physiol. Chem. 241, 116-124. 11. LINSERT, 0. (1936) 2. Physiol. Chem. 241, 125128. 12. HASSLEWOOD, G. A. D. (1939) Biochem. J. 33, 454-456. 13. ROBORGH, J. R., AND DEMAN, T. J. (1960) Biothem. Pharmacol. 3, 272-282. 14. LAM, H.-Y., SCHNOES, H. K., AND DELUCA, H. F. (1974) Science 186, 1038-1040. 15. REEVE, L. E., SCHNOES, H. K., AND DELUCA, H. F. (1978)Arch. Biochem. Biophys. 186,164167. 16. SUDA, T., DELUCA, H. F., AND TANAKA, Y. (1970) J. N&r. 100, 1049-1052. 17. MARTIN, D. L., ANDDELUCA, H. F. (1969)Amer. J. Physiol. 216, 1351-1359. 18. U. S. Pharmacopoeia (1955) 15th Revision, p. 889, Mack, Easton, Pa. 19. PARTRIDGE, J. J., FABER, S., AND USKOKOVIC. M. R. (1974) Helv. Chim. Acta 57, 764-771. 20. NARWID, T. A., COONEY, K. E., AND USKOKOVIC, M. R. (1974) Helv. Chim. Acta 57, 771-781. 21. STEELE, J. A., AND MOSETTIG, E. (1963) J. Org. Chem. 28, 571-572. 22. BERNSTEIN, S., AND WALLIS, E. S. (1937) J. Org. Chem. 2, 341-345. 23. HUNZIKER, F., AND MOLLNER, F. X. (1958) Helv. China. Acta 41, 70-73. 24. HAVINGA, E. (1973) Experientia 29, 1181-1316. 25. TANAKA, Y., FRANK, H., AND DELUCA, H. F. (1975) Endocrinology 92, 417-422. 26. HOLICK, M. F., KASTEN-SCHRAUFROGEL, P., TAVELA, T., AND DELUCA, H. F. (1975) Arch. Biochem. Biophys. 166, 63-66. 27. WINDAUS, A., AND TRAUTMAN, G. (1937) Z. Physiol. Chem. 247, 185-188. 28. CHEN, P. S., AND BOSMANN, H. B. (1964) J. Nutr. 83, 133-139. 29. DRESCHER, D., DELUCA, H. F., AND IMRIE, M. H. (1969)Arch. Biochem. Biophys. 130,657661. 30. HUNT, R. D., GARCIA, F. G., AND HEGSTED, D. M. (1967) Lab. Anim. Care 17, 222-234.