25-Hydroxycholecalciferol, the Probable Metabolically Active Form of Vitamin D

Transcription

1 THE AMERIcAN JORNAL OF LINIcAL XuTiuTIox Vol. 22, No. 4, April, 1969, pp Printed in.s.a. 25-Hydroxycholecalciferol, the Probable Metabolically Active Form of Vitamin D Isolation, Identification, and Subcellular Location H. F. DELucA JT HAS BEEN REOGNIZED for many years that a lag exists between the time of vitamin D administration and the onset of its physiological action (1, 2). ntil recently no evidence had been developed to support one of several possible explanations. Then, it was shown in this laboratory that the effect of vitamin D on both the active transport of calcium across the intestine and the mobilization of bone mineral is blocked by actinomycin D (3, 4). A similar observation was reported for intestinal absorption of calcium in chicks (5). Through a series of critical experiments, it was shown (4) that actinomycin D was acting in the expected fashion, i.e., it blocked the transcription of DNA into mrna, indicating that the expression of vitamin D action involves the transcription of DNA and protein synthesis. This was confirmed by Stohs and others (6, 7), who dlemonstrated that vitamin D stimulated the incorporation of 3H orotic acid into nuclear RNA of intestine. Hence, the lag in vitamin D action must be partly due to the production of mrna and the subsequent synthesis of a protein component of the calcium transport system of intestine and probably also of bone (2). This by itself, however, was not sufficient to account for the entire lag in vitamin D action. This led to further studies aimed at elucidating what happens to the vitamin D molecule between the time of vitamin D administration and 1 From the Department of Biochemistry, niversi ty of \isconsin, Madison, Wisconsin. the initiation of its action. To examine these points, various radioactive vitamin D preparations were synthesized in this laboratory with high specific activity to permit experiments with truly physiological doses of the vitamin. Thus, Neville and DeLuca (8) prepared 1,2-3H-vitamin D3 (26, dpm/i). DeLuca et al. (9) prepared 22,23-3H-vitamin D4 (1, dpm/i) and Imrie et al. (1) random 14-vitamin D2 (3, dpm/i). Figure 1 demonstrates the lag in vitamin D action when 1 I of vitamin D3 are given either intravenously or orally. Obviously, 8-1 hr are required for the vitamin to express its characteristic stimulation of calcium transport in the intestine after an intravenous dose and! 2-22 hr after an oral dose. The first question to be answered is if there might be a lag in the transport of vitamin D to the targets of vitamin D action, i.e., intestine and bone. With the aid of 1, 2-3H-vitamin D3 it was shown that within an hour after administration of the vitamin the label was found in almost maximal concentration in intestine and bone (Figs. 2 and 3). The 3H concentration continued to increase for three more hours, but had reached at least 8% of its maximum within the 1st hr. It was thus apparent that the lag could not be attributed to the time required for the vitamin to be transported to the targets of vitamin D action (8). The next question centered around the possibility that vitamin D might be changed 412 Downloaded from ajcn.nutrition.org by guest on April 8, 216

2 25-Hydroxycholecalciferol 413 to a metabolically active form before it could function. Although this possibility had been suggested before, convincing cvidence was lacking (11). More recently, Kodicek and co-workers (12) as well as Haussler and Norman (13) have argued against the possibility of a metabolically active form of vitamin D. It remained for Lund and Dc- Luca (14) to demonstrate a major metabolite of vitamin D that is at least as active biologically as vitamin D itself. Silicic acid chromatographic profiles of tissue extracts of D-deficient rats given 1 I of 1, 2-3Hvitamin D3 intravenously 12 hr previously are shown in Fig. 4. There are four radioactive components in this profile. The first 4 I-) -J 4 l) Li -J 3 b VII. D5IV.. - -o 2,-S VII D ORAL J. - NO VII D TIME IN HORS Fic. 1. Intestinal calciuln transport in response to.25 g vitamin D, given either orally or intravenously. Rats were fed a.47% a,.3% P diet for 4 weeks. alcium transport was measured by the cv cited-sac method (3). _ I 8 246S FIG. 3. Time course of skeleton of rats after a 2 3 TIME (HORS) vitamin D, (8). J 211-,/M.oHI 3H TSED3 + EXTRAT LIVER BON( appearance of H in the O 1...L_A I.f%/\I I ER I LJ\L L_ ZO - t4testine 5. 3: o 5.( 5. BLOOD 5 BONE 3 I A j I LJ\il FRATION NMBER eocr Oi25-,.g dose of 1,2- Ha I-,- 4Oj z 2O a: Downloaded from ajcn.nutrition.org by guest on April 8, SMALL INTESTINE FIG. 4. Silicic acid column profiles of tissue extracts of rats 12 hr after they had received.25 g I,2-3H-vitamin D, intravenously. I5 1. a TIME (HORS) FI(;. 2. Time course of appearance of H in the intestine of rats after a O.25-g dose of 1,2- H-vitaliHu D, (8). component has been identified as an ester of vitamin D and long-chain fatty acids (15), while peak II represents a biologically active metabolite of vitamin D that is as yet unidentified. Both of these compounds appear to be of minor significance since their concentrations remain low regardliess of dose or time after dios. Peak III has been identified as unaltered vitamin D3 (16). Of major interest is the peak JV metabolite fraction that appears to be the principal

3 414 DeLuca VIT H3VIT VIT VIT D3 D Do D4 AQEOS VITD3 I 1ff BIOLOGIAL ATIVITY OF VITAMIN D METABOLITES...:#{149}::: #{149}...:::.. 1 I I I I 1 o 3 4 i.v.41q Fic. 5. Relative ability of vitamin D and its metabolite fraction to cure rickets in rats. The assay was performed by the line-test method (22). 12 SOOiu. Lii QQ (21%) 4()(). I\ A c I --#{234}!!! IOOIu. (62%) 12- BOO - 4- J I.. I k... Z lolu. 1(83%) FRATION NMBER (IOmD Fic. 6. Effect of dose of 1,2- H-vitamin D, on the proportion of radioactivity found in the peak IV metabolite fraction of rat blood (14). -J l) 3 I TIME (HORS) Fic. 7. Response of intestinal calcium transport to an oral 1-l dose of peak IV metabolite fraction as compared to an equivalent dose of H-vitamin D, (17). I form in which vitamin D is found in the tissues after physiologic doses. Furthermore, this fraction is biologically active in the cure of rickets in rats (Fig. 5). Great care was taken to make sure that the peak IV metabolite was not an artifact of the methods used (14) and it could be shown that the proportion of this compound relative to unchanged vitamin D was highly dose dependent (Fig. 6). Moreover, Morii et al. (17) were able to show that the peak IV metabolite fraction mimicked the action of vitamin D in stimulating both calcium transport by intestine and mobilization of bone. Finally, of particular interest was the fact that oral administration of the metabolite to D-deficient rats produced a much more rapid intestinal response than did an equivalent dose of vitamin D3 (Fig. 7). These very interesting properties of the peak IV metabolite suggested that it might well be the metabolically active form of the vitamin and prompted our every effort to isolate and identify the metabolite. Dr. P. F. Neville in my laboratory had shown by both chemical and chromatographic means that peak IV was actually composed of at least five radioactive substances. Dr. G. Ponchon was then able to modify the silicic acid gradient systems to achieve the separation of various metabolites (Ponchon and DeLuca, ms in preparation). An example of the chromatographic profile of a plasma extract is shown in Fig. 8. The peak IV metabolite fraction has now been resolved into eight radioactive components. Of these, only fraction IV is biologically active in the cure of rickets in rats. sing this information, it was then possible to design a method for the isolation of the peak IV metabolite in sufficient quantity for chemical identification. Plasma from pigs fed large doses of vitamin D was used as the source material because it was shown by preliminary experiments that concentrations as high as 12-2 I of metabolite/ml plasma could be achieved by feeding 25, I D3/day to pigs (18). The preparation of the extract from which the metabolite was isolated in pure form is Downloaded from ajcn.nutrition.org by guest on April 8, 216

4 25-Hydroxycholecalciferol 415 shown in Fig. 9. An extract of a small pig, injected with 1 mg 3H-vitamin D3 24 hr before, was used to label the peak IV metabolite. Preliminary purification was obtained by precipitating the plasma proteins with 7% saturation with (NH4)9S4 because Rikkers and DeLuca (19) had shown both vitamin D and its major metabolite were bound to an globulin of plasma. The z In I- z FRATION NMBER Fic. 8. hromatogram of a plasma extract of rats given.25 g H-vitamin D, 4 hr before by the gradient system of Lund and DeLuca (14) A, B, and by the new gradient system of Ponchon and DeLuca (in preparation) A, B, , Diethyl ether in Skelly B gradient , Methanol gradient. [ bo#{128}] L!I lb. bog 1 126day, 12.Omg.3H 25, 1.u. Dy dar J,2)hr. ril JProteinl eoojl plaemn J?lHSOk 1 ppt L I Hot extract 12:1)4WcHcl3. Fic. 9. Steps in the preparation of the extract from swine plasma for the isolation of 25-hydroxycholecalciferol. Silicic elite partition Hot acid Peak IV column G.L hydroxycholecalciferol e--_i 4 n.m.r.!.ss T.L.. Bloasseys spec. FIG. 1. Isolation of 25-hydroxycholecalciferol from swine plasma extract. FIG. 11. Silicic acid column profile of swine plasma extract (18). A 4 / \ -2 3 :: FRATION NMBER FIG. 12. Partition chromatography of 25-hydroxycholecalciferol isolated from swine plasma by silicic acid chromatography (peak IV in Fig. 11) (18). extract from the pigs was then subjected to silicic acid chromatography and finally to partition chromatography. This yielded 1.3 mg of a pure substance on which the indicated measurements were carried out (Fig. 1). The silicic acid column profile of the hog extract is shown in Fig. 11. Again, peak IV was very active biologically while peaks IVa and V were inactive in curing rickets by the line-test assay method. After peak IV had been subjected to a partition column (Fig. 12) it was found to be pure and gave the indicated ultraviolet spectrum (Fig. 13). Note in Fig. 12 that the absorbance at 265 rn/a exactly coincides with the radioactivity profile. Examination of this substance on gas-liquid chromatography (GL) (Fig. 14) revealed it to be a pure substance that has Downloaded from ajcn.nutrition.org by guest on April 8, 216

5 416 DeLuca I I I Ftc. 13. ltraviolet hydroxycholecalciferol ethyl ether) (18) rn,p spectrum of isolated 25- from swine plasma (in di- I I I I MINTES FIG. 14. Gas-liquid chromatography of isolated 25-hvdroxvcholccalciferol (18). a longer retention time than does vitamin D (18). The fact that the ultraviolet spectrum is identical to that of vitamin D and that GL produces a double peak similar to pyro- and i sopyrocholecalciferol demonstrates that the triene portion of the metaholite remains unchanged from the original vitamin D3. Mass spectrum (Fig. 15) of the substance showed a molecular weight of 4 or a hydroxylated vitamin D3. The fact that both vitamin D3 and the metabolite gave identical fragments at 271 m/e2 established that the extra hydroxyl function is on the side-chain of the molecule. A mass fragment at 59 m/e from the metabolite but not from the parent vitamin D3 strongly suggested the hydroxyl to be on the 25-position. Nuclear magnetic resonance spectra (NMR) established the structure as 25-hydroxycholecalciferol with a singlet at 61.2 ppm (Fig. l6a) identical to that in 25-hydroxycholesterol (Fig. 16B) and an absence of a doublet at 6.87 ppm due to the interaction of the 26, 27 methyl groups with tertiary hydrogen on the 25-position of cholecalciferol (Fig. l6). Thus, the structure was unequivocally demonstrated to be 25-hydroxycholecalciferol (Fig. 17) (18, 2). Table i (21) shows the biopotency of the 25-hydroxycholecalciferol as compared to vitamin D3 in the cure of rickets in rats by the line-test assay method (22). Repeated tests by members of our laboratory and of the Wisconsin Alumni Research Foundation show it to be 1.4 times as effective as vitamin D. Table ii shows also that it is about 1.4 times more effective than vitamin D3 in the prevention of rickets in chicks. Thus, in the two most widely studied animal species the metabolite has been shown to be much more effective than the parent vitamin D in the cure of rickets. Figure 18 demonstrates that administration of 25-OH vitamin D3 intravenously is effective in initiating calcium transport by everted sacs of rat small intestine. Most surprising is that the 25-OH vitamin D3 given at the.25-/ag (1 I) dosage level initiated calcium transport within 3 hr after injection while a similar dose of vitamin D3 required 8-1 hr before calcium transport was initiated (21). When bone mobilization experiments are carried out with rats on a low calcium diet, the 25-OH vitamin D3 is at least as effective as the parent vitamin (21) (Fig. 19). Of great interest is the fact that the rise in serum 2 Mass/charge. Downloaded from ajcn.nutrition.org by guest on April 8, 216

6 25-Hydroxycholecalciferol 417 NOIIIZINI slol 1N35d NOILSZINOI ejil LN33S3d o o - - -I e - -, 4 I -.) o.o ) Li) ) )...) I.....) ) - (.4 LI)... (.4 ) - (.4 ) If). N -) -) > x I.. - l -) I.. I.). Downloaded from ajcn.nutrition.org by guest on April 8, 216.) WI Li).), I -o Li,. If, ALISPL3LNI (ILe1. ALSN3LNI 3(TLI1)5

7 418 DeLuca HYDROXYHOL.EALIFEROL R Rt-t: R=OH HOLEALIFEROL : 25-HYDROXY- HOLEALIFEROL 2..5 I. 6. PPM.5 2. HOLEALIFEROL Fic. 17. Structure of 25-hydroxycholecalciferol (19, 21). TABLE I omparative effectiveness of 25-hydroxycholecalciferol and vitamin D3 in the cure of rickets in rats 25-OH-vitam in Da Vitamin Dz Type of Dosage I/g I//fg Oral (8) 58 ± 5a 4 ± 4a Oral(7) 52± 3 38± 5 Intravenous 56 ± 2 41 ± 3 Standard line-test assay for vitamin D activity was carried out as described in the. S. Pharmacopeia (23). Figures in parentheses represent number of rats per group. Standard deviation. Downloaded from ajcn.nutrition.org by guest on April 8, PPM FIG. 16A. NMR spectrum of 25-hydroxycholecalciferol (18). B. NMR spectrum of 25-hydroxycholesterol (18).. NMR spectrum of cholecalciferol (18). calcium of these rats occurs within 8 hr after 2.5 /Ag of the 25-OH vitamin D3, whereas more than 12 hr are required before 2.5 /Lg of vitamin D3 could give a like response (21). Furthermore, together with Dr. L. Raisz at the niversity of Rochester, it was possible to demonstrate that.9 I of the 25-OH vitamin D3/ml induced marked bone resorption in organ cultures, while vitamin D3 has a questionable effect at concentrations of 5-1, I/ml. The evidence is, therefore, strong that the 25-hydroxy- vitamin D3 is the metabolically active form of the vitamin. Experiments in-

8 25-Hydroxycholecalciferol 419 tended to provide further evidence are currently in progress in this laboratory. On the assumption that 25-hydroxycholecalciferol is the metabolically active form of the vitamin, approximately 5-6 hr of the 1-hr lag in vitamin D action (1 I) must be due to the time required for the conversion of vitamin D3 to the 25-hydroxy- vitamin D3. Antirachitic bone ash assay was carried out according to official Association of Official Agricultural hemists methods (195) using 2 chicks/ assay It has been possible to synthesize 25-hydroxycholecalciferol by two different routes (23), one of which is shown in Fig. 2. The synthetic material is identical in all respects with the 25-hydroxycholecalciferol, including physical constants and biological activity; this proves the structure beyond a doubt. The subcellular location of the 25-hydroxy- vitamin D3 or of vitamin D3 itself, or both, is of major importance to an understanding of the mechanism of vitamin D action. A meaningful analysis of this has been made possible only by the availability of radioactive vitamin D of high specific activity. Experiments carried out with vita- group. TABLE Biological activity of 25-hydroxycholecalciferol by chick bone ash assay Supplement, I/lO-g diet None 4.5vitiarninD vitamin D3 9. vitamind, vitamin D, 18. vitamin D, OH-vitamin D, 9.25-OH-vitamin D3 Ratio 25-OHvitamin OHvitamin D, Bone II ash, increase D vitamin Ds activity a E r-6 D w OH D3 I I I I I I I I TIME IN HORS Ftc. 18. Response of calcium transport system of intestine to either.25 g 25-hydroxycholecalciferol or to.25 g cholecalciferol (vitamin D3) (21). TIME (HORS) Fic. 19. Serum calcium response of vitamin D- deficient rats fed a low calcium diet to either 2.5 g 25-hydroxycholecalciferol or 2.5 zg cholecalciferol (vitamin D3) (21). mm D whose specific activity is too low may not lead to reliable conclusions. This is illustrated by the study of Haussler and Norman (13) who, using low specific activity 3H-vitamin D3, concluded that the intestine of chicks accumulates 4% of a 1-l dose of the vitamin. In repeated studies with 26,-16, dpm/i 3H-vitamin D, we detected only 2% of a 1-unit dose in the intestine of rats or chicks (8-1, 24). More recently, Haussler et al. (25) have reported that the chromatin fraction of the intestinal cell accumulates most of the radioactivity from 3H-vitamin D3 - sing very high specific activity preparations we have been unable, in spite of repeated attempts, to find any significant radioactivity from radioac- Downloaded from ajcn.nutrition.org by guest on April 8, 216

9 42 DeLuca #{231}6 #{176} Aco,cl_HoAI OH FIG. 2. hemical synthesis of 25-hydroxycholecalciferol (23). TABLE Location of radioactivity in rat intestinal 8 hr after an intravenous dose of.25 g 1,2-3H-vitamin D3 ell fractionation methods are as described by Stohs and DeLuca (24).. jllaih. Fractions % Total radioact ivity in itric acid nuclei ± 3.8 rude nuclei 57.6 ± 3.4 Mitochondria 6.7 ±.4 Microsomes 1.3 ± 1.2 Ribosomes.6 ±.2 ytoplasm 22.8 ± 6.2 tive vitamin D3 or 25-hydroxy- vitamin D3 in chromatin prepared by the method of Bonner and associates (26). sing high specific activity vitamin D and ordinary cell fractionation techniques, Stohs and DeLuca (24) demonstrated that some 6% of the intestinal l radioactivity was found in the crude nuclear pellet and some 2% was found in the supernatant or cytoplasmic fraction (Table iii). However when pure nuclei were isolated from citric acid solutions by the method of Mirsky and Pollister (27) only 12% of the l radioactivity was found in this preparation. Examination of these nuclei under the electron microscope revealed intact nuclei including chromatin but with the outer nuclear membrane removed. This surprising finding prompted our preparing nuclei in 2.3 M sucrose by the method of haveau (28). These nuclei did retain the 3H from the 3H-vitamin D3 as shown in Table iv. If the nuclei are first prepared in 2.3 M sucrose III and then treated with 1% citric acid, most of the 3H is removed. In addition, treatment of the 2.3 M sucrose nuclei with 1% Triton X-l as described by Blobel and Potter (29) also removes the 3H from the nuclei. itric acid (3) and the Triton X-1 (29) treatments are known to remove the outer nuclear membrane, a fact which our electron micrographs (Figs. 21 and 22) confirm. These observations suggest that the nuclear membrane is a major site of binding for vitamin D3 or its metabolites. Rat Rat Rat Rat hick hick hick hick Prepared in 2.2 M sucrose, extraction and chromatography of pure intestinal nuclei from chicks given 1 I 3H-vitamin D3 8 hr earlier showed that at least 8% of the radioactivity was found in the 25-hydroxycholecalciferol fraction. This was not removed by DNA-ase treatment, was not found in deoxyribonucleoprotein isolated by the method of Zubay and Doty (31), nor in chromatin isolated by the method of Marushige and Bonner (26), nor in citric acid nuclei isolated by the method of Mirsky and Pollister (27). Thus, all evidence sug- Nuclear source Method of preparation liver l deoxyribonucleoprotein TABLE 3H in nuclei after a 1-l iv 1% itric acid 2.2 M Sucrose 2.3 M Sucrose EDT + NaI 1% itric acid 2.3 M Sucrose 2.2 M Sucrose + l/ citric acid 2.2 M Sucrose, washed 3 times with 1% Triton X-lOO Percent of total H in tissue#{176} 12.2 ± ± ± ± ± ± ± ± 7.4 O Each value represents the average of 4-8 animals. All animals were given 1 I I,2-3Hvitamin D3 or 22,23-3H-vitamin D4. Rats were sacrificed after 8 hr and chicks after 12 hr. IV dose of 3H-vitamin D3 Downloaded from ajcn.nutrition.org by guest on April 8, 216

10 25-Hydroxycholecalciferol 421 Downloaded from ajcn.nutrition.org by guest on April 8, 216 FIG. 21. hick intestinal nuclei isolated in 2.3 M sucrose (24). gests that a major location of 25-hydroxyvitamin D.1 is the nuclear membrane of the intestinal cell. When large doses of 3H-vitamin D are given, only a small fraction of the radioactivity is found in the nuclei (Table v). This is not so surprising since much of the vitamin of the tissues is probably in a nonfunctional storage form. Finally, if D-deficient chicks are first given 5, I of nonradioactive vitamin D followed by 1 I of 1,2-3H-vitamin D3, the amount of radioactivity

11 422 DeLuca Downloaded from ajcn.nutrition.org by guest on April 8, 216 Fic. 22. hick intestinal nuclei isolated in 1% citric acid (24). in the intestinal nuclei is greatly decreased. This suggests specific receptor sites for the functional form of vitamin D (Table vi). The question of how the 25-hydroxy- vitamin D3 is bound to the nuclei and how it initiates the transcription of a specific DNA into mrna remains open. Another question relates to the nature of the calcium transport protein that is synthesized in response to vitamin D. Although Wasserman and co-workers have described a calciumbinding protein that they consider to be the protein made in response to vitamin D (32-34), further studly is necessary to determine

12 25-Hydroxycholecalciferol 423 TABLE rude Subcellular distribution of 5, I Hvitamin D, in intestinal of vitamin D-deficient rats 8 hr after intrajugular administration Mitochondria Microsomes Supernatant Fraction Percent total H in nuclei V 71. ± ± ± ± 1.2 Each rat received 5, I generally labeled H-vitamin D, intrajugularly for 8 hr. Fractionation was accomplished by differential centrifugation in.25 M sucrose +.5 at tris chloride, ph 7.4. Each value represents the average of four animals. TABLE Prevention of H accumulation from 1, 2- H vitamin D, in chick l nuclei Treatment 1 I (.25 jig) H-vitamin D, 5, I (125 Mg) H-vitamin D3 5, I (125 jig) D, + 1 I H-vitamin D, 125 pg 7-Dehydrocholesterol + 1 I H-vitamin ID, 125 pg Dihydrotachysterol I H-vitamin D3 VI Percent of total radioactivity in ± ± ± ± ± 7.9 All nuclei were prepared in 2.2 or 2.3 M sucrose. a Sixty-two pg of nonradioactive vitamin D,, 7-dehydrocholesterol or dihydrotachysterol-2 was given 36 hr and 24 hr before sacrificing the chicks. The 1,2- H-vitamin D, was given 12 hr prior to sacrificing. Each value represents the average of four to six animals. its role if any in the vitamin D-induced calcium transport system. SMMARY The isolation, identification, and chemical synthesis of 25-hydroxycholecalciferol have been achieved, with the synthesized product identical in all respects to the isolated material. Evidence has been presented that strongly suggests this compound is the metabolically active form of vitamin D. As compared to vitamin D3, it is more active biologically in rats and chicks and acts much more rapidly to induce bone mobilization and intestinal transport of calcium. It also induces bone mobilization in vitro, whereas vitamin D3 is either not, or only minimally, effective in culture. Finally, the nucleus, more probably the nuclear membrane, is the primary subcellular site of localization of 25-hydroxycholecalciferol. REFERENES 1. ARLSSON, A. Tracer experiments on the effect of vitamin D on skeletal metabolism of calcium to phosphorus. Acta Physiol. Scand. 26: 212, DELA, H. F. Mechanism of action and metabolic fate of vitamin D. Vitamins Hormones 25: 315, Zuu., J. E., E. wtnowska-mlsrnu. and H. F. DELA. Actinomycin D inhibition of vitamin D action. Science 149: 182, Zuu, J. E., E. ziutnowska-miszm.i. AND H. F. DELA. On the relationship between vitamin D action and actinomycin-sensitive processes. Proc. Nati. Acad. Sci..S. 55: 177, NORMAN, A. W. Actinomycin D and the response to vitamin D. Science 149: 185, SToats, S. J., J. E. ZLL AND H. F. DELuA. Vitamin D stimulation of H orotic acid incorporation into RNA of rat intestinal. Biochemistry 6: 134, Zuu, J. E., S. J. STOHS AND H. F. DELA. The relationship between vitamin D action and actinomycin sensitive processes. Federation Proc. 25: 545, NEvILLE, P., AND H. F. DELA. The synthesis of [1,2 H] vitamin D, and the tissue localization of a.25 pg (1 i.u.) dose per rat. Biochemistry 5: 221, DELA, H. F., M. WELLER, J. W. BLNT AND P. F. NEvILii. Synthesis, biological activity and metabolism of 22,23- H vitamin D.. Arch. Biochem. Biophys. 124: 122, IMRIE, M. H., P. F. NEvILLE, A. W. SNELLGROVE AND H. F. DELA. The metabolism of vitamin D, and vitamin D, in the rachitic chick. Arch. Biochern. Biophys. 12: 525, SALLIs, J. D., AND E. S. HouswoRTH. alcium metabolism in relation to vitamin D, and adrenal function in the chick. Am. J. Physiol. 23: 56, ALLOW, R. K., E. KODIEK AND E. A. THOMPSON. Downloaded from ajcn.nutrition.org by guest on April 8, 216

13 424 DeLuca Metabolism of tritiated vitamin D. Proc. Roy. Soc. London, Ser. B. 164: 1, HASSLER, M. R., AND A. W. NORMAN. The subcellular distribution of physiological doses of vitamin D,. Arch. Biochem. Biophys. 118: 145, LND, J, AND H. F. DELA. Biologically active metabolite of vitamin D, from bone, liver, and blood serum. J. Lipid Res. 7: 739, LND, J., H. F. DELA AND M. HORSTING. The formation of vitamin D esters in vivo. Arch. Biochem. Biophys. 12: 513, NORMAN, A. W., J. LND AND H. F. DELA. Biologically active forms of vitamin D, in kidney and intestine. Arch. Biochem. Biophys. 18: 12, MoRn, H., J. LND, P. NEVILLE AND H. F. DE- LA. Biological activity of vitamin D metabolite. Arch. Biochem. Biophys. 12: 58, BLNT, J. W., H. F. DELA AND H. K. SHNOES. 25-Hydroxycholecalciferol: a biologically active metabolite of vitamin D,. Biochemistry 7: 3317, RIKKERS, H., AND H. F. DELA. An in vivo study of the carrier proteins of H-vitamin D, and D4 in rat serum. Am. J Physiol. 213: 38, BLNT, J. W., H. F. DELA AND H. K. SHNOES. 25-Hydroxycholecalciferol: a biologically active metabolite of cholecalciferol. hem. ommun. 14: 81, BLNT, J. W., Y. TANAKA AND H. F. DELA. Biological activity of 25-hydroxycholecalciferol. Proc. Nati. Acad. Sci.S. In press S. Pharmacopeia, XV. Easton, Pa.: Mack, 1955, p BLNT, J. W.. AND H. F. DELA. hemical synthesis of 25 hydroxycholecalciferol-a biologically active metabolite of vitamin D. Biochemistry. In press. 24. STOHS, S. J., AND H. F. DELA. Subcellular location of 1, 2-H -vitamin D, and its metabolites in intestinal. Biochemistry 6: 3338, HASSLER, M. R., J. F. MYRTLE AND A. W. NORMAN. The association of a metabolite of vitamin D, with intestinal chromatin in vivo. J. Biol. hem. 243: 455, MARSHIGE, K., AND J. BONNER. Template properties of liver chromatin. J. Mol. Biol. 15: 16, MIRSKY, A. E., AND A. W. POLLISTER. hromosin, a deoxyribase nucleoprotein complex of the cell nucleus. J. Gen. Physiol. 39: 117, HAVEA, J. Nouvelle methode d isolement des noyaux cellulaires. ompt. Rend. 235: 92, BLOBEL, G., AND V. R. POTTER. Nuclei from rat liver: isolation mcthod that combines purity with high yield. Science 154: 1662, GRR, M. I., J. B. FINEAN AND J. N. HAWTHORNE. Phospholipids of liver cell fractions. I. Phospholipid composition of the liver cell nucleus. Biochim. Biophys. Acta 7: 46, ZBAY, G., AND P. DOTY. The isolation and properties of deoxyribonucleoprotein particles containing single nucleic acid molecules. J. Mol. Biol. 1: 1, WASSERMAN, R. H., AND A. N. TAYLOR. Vitamin D3 induced calcium binding protein in chick intestinal. Science 152: 791, TAYLOR, A. N., AND R. H. WASSERMAN. Vitamin D3 induced calcium binding protein: partial purification, electrophoretic visualization and tissue distribution. Arch. Biochem. Biophys. 119: 536, KALLFELZ, F. A., A. N. TAYLOR AND R. H. WASSERMAN. Vitamin D-induced calcium binding factor in rat intestinal. Proc. Soc. Exptl. Biol. Med. 125: 54, Downloaded from ajcn.nutrition.org by guest on April 8, 216