Metabolism of Vitamin D

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METABOLISM AND MECHANISM OF ACTION

Annu. Rev. Biochem. 1976.45:631-666. Downloaded from www.annualreviews.org by Chulalongkorn University on 09/25/12. For personal use only.
+929
OF VITAMIN Dl
Hector F. DeLuca and Heinrich K. Schnoes
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
CONTENTS
PERSPECTIVES AND SUMMARY INTRODUCTION METABOLISM OF VITAMIN DJ
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632 633 634
ENZYMES THAT CATALYZE VITAMIN DJ METABOLISM
Vitamin Dr25-Hydroxylase . .. . .... .. .... . ... . . . .. .. .. . .. .... ... . .. . .. ... ... .... . . ... .. ....... . . ..... 25-Hydroxyvitamin D-/a -Hydroxylase ......... ........................... ................................ Regulation of the Renal 10.- and 24-Hydroxylases ................. .. .......... ................. .. Regulation of the Renal Hydroxylases by Other Faclors ........ .......... ......................
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639
639 640
641 644
MECHANISM OF ACTION OF 1.25-(OH),DJ
. .. .................... . ................................... .. ...
645
. 645 649 649 650
Mechanism of Action in the Intestine .......... ............................................................ The Role of 1. 25-(OH)lD] in Bone ................ .... ....................... .............. . ................ The Function of 1. 25-(OH)}DJ in Other Tissues . ........... ..... ........ ............................ Summary of the Mechanism of Action of Vitamin D and Its Metabolites ............
CHEMISTRY OF VITAMIN D METABOLITES
Metabolite Characterization and Quantitation .. ................ ...... . ............... ............... Synthesis of Metabolites ............................................................................................ Structural Analogs of the Vitamin D Metabolites . .. . .. ... .. .. .. . .. ..... . ... .. . . ..... .... .. . . .. . .. . Side-chain analogs of 25-0H-D, ................... ............................................................
Side-chain-modified derivatives of 1a.25-(OH)lD,
.
.......... ................. .... ................ ..........
................. ....................................
Analogs modified in ring A ...................................................................................... Some generalizations on structure/activity relationships ... ................................... ......... .
Plant-Derived Factors Simulating la.25-(OH)}DJ Activity......................................

1
650 650 65 2 653 653 654 657 658 659
Abbreviations used are: 2S-0H-D 3. 2S-hydroxyvitamin 03; la.2S-(OH)2D3. 1a.25-dihy
droxyvitamin OJ; 24.25-(OHhDJ 24,25-dihydroxyvitamin OJ; 1.24,25-(OHhDJ, 1,24,25-
trihydroxyvitamin D3; 24-0HD3, 24hydroxyvitamin D3; 2S.26(OH)2D3' 2S.26dihydroxy vitamin D3; 2S-0H-D2 25-hydroxyvitamin Dz; 1.25-(OH)zDz 1.2S-dihydroxyvitamin 0z;
24.25-(OH)zD2 24,25-dihydroxyvitamin D2; ethane-l-hydroxy-l,l-diphosphonate; (OHhDJ la.24-dihydroxyvitamin OJ; 24-0H-Dz. 24-hydroxyvitamin Dz; EHDP. la-hydroxypregnaca\Ciferol; 1a-hydroxyvitamin OJ; 10..241 a-OH-PC, la-OH-DJ
I a-OH- 02 631
I a-hydroxyvitamin Oz.
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DELUCA & SCHNOES
PERSPECTIVES AND SUMMARY
Vitamin D, like the three other fat-soluble vitamins, has defied definition of its mechanism of action for over SO years. However, during the past several years great progress has been made, which has brought forth the demonstration that vitamin D must now be regarded as a prohormone giving rise to at least one hormone that plays a central role in metabolism of calcium and phosphorus, a situation unique among the known vitamins. It has been clearly established that vitamin D3 must first be converted to 2S-hydroxyvitamin D3 (2S-0H-DJ) in the liver. This compound, which can be regarded as the major circulating metabolite of vitamin D, does not act directly at physiologic concentrations, but must undergo at least one other alteration before it can carry out the functions of vitamin D. This conversion takes place in the kidney, where the mitochondria hydroxylate 25-0H-D3 on carbon- I to yield l a,2S-dihydroxyvitamin D3 [l a,2S-(OH)2D3]. It is this form of vitamin D which is then transferred to the intestine, bone, and kidney, where it or a further metabolite stimulates intestinal calcium transport, intestinal phosphate transport, the mobilization of calcium from bone, and renal reabsorption of calcium. Because I a,2S-(OHhD3 is formed exclusively in the kidney and has its functions in targets other than the kidney, it must be regarded as a hormone. In true hormonal fashion, production and accumulation of l a,25-(OH)2DJ is regulated either direct ly or indirectly by serum inorganic phosphorus and serum calcium concentration. A drop in serum calcium concentration signals the secretion of parathyroid hor mone, which then stimulates production of l a,25-(OH)2DJ. The l a,25-(OHhDJ in turn stimulates intestinal calcium absorption and together with parathyroid hormone stimulates the mobilization of calcium from bone and the renal reabsorp tion of calcium. These functions then return serum calcium to normal, suppressing parathyroid hormone secretion. Low plasma phosphorus concentration, even in the absence of parathyroid hormone, stimulates production and accumulation of la,2S-(OH)2D3, which functions to stimulate intestine and bone. Because of a complex interrelationship with the parathyroid hormone, l a,2S-(OH)2D3 can func tion both as a calcium-mobilizing hormone and as a phosphate transport hormone. Suppression of the I a-hydroxylase by high levels of circulating calcium or phos phate simultaneously stimulates another hydroxylase that converts 25-0H-DJ to 24,2S-dihydroxyvitamin D3 [24,25-(OHhD3]. This metabolite represents a probable excretory form of the vitamin in birds but may be functionally important in mam mals. Its 24-hydroxyl group has been shown to have the R configuration. The compound is further hydroxylated to form 1 ,24,25-trihydroxyvitamin DJ [ 1 ,24,25(OH)3D3]. The 1 ,24,25-(OH)3D3 can also be formed from l a,25-(OH)2D3 by the 24-hydroxylase. The 1,24,2S-(OH)3D3 may represent in mammals an important alternate hormone to l a,25-(OH)2D3, but thus far it has not received sufficient attention to delineate its role, if any, in vitamin D function. A great deal of effort has been expended on studies of the vitamin D hydroxylases of the kidney. Much progress has been made on the renal 25-0H-DJ-l a-hydroxy lase system, which has been shown to be composed of a flavoprotein, an iron-sulfur protein (renal ferredoxin), and cytochrome P-4S0. This system has been successfully
VITAMIN D METABOLISM AND MECHANISM OF ACTION
633
solubilized, and the components have been purified and recombined to produce an active l -hydroxylating system. The availability of labeled and unlabeled 1 a,2S-(OH}zD3 has permitted new advances in our understanding of both the vitamin D-initiated intestinal calcium transport system and the physiology of vitamin D function. In particular, evidence has been presented for the existence of a receptor protein in intestinal mucosa that binds specifically I a,2S-(OH)2D3 and that may function in the nucleus to initiate transcription of genomes that code for proteins involved in the transport phenome non. However, alternative mechanisms of action of l a,2S-(OH)2D3 are also being considered. Additionally, the existence of calcium-binding proteins and calcium binding complexes has been demonstrated in the intestine and in other organs that transport calcium in response to vitamin D, although it is not clear exactly how or if these proteins function in the calcium transport process or what their relationship to the transport process might be. It is in the area of mechanism of action of l a , 2S-(OH)2D3 that much effort can be expected i n the next few years. The discovery of this new endocrine system has brought with it many implications for the understanding and treatment of metabolic bone disease. These implications are beyond the scope of this review, but they have given great impetus to the use of the vitamin D metabolites or their analogs in the treatment of specific metabolic bone diseases such as renal osteodystrophy, hypoparathyroidism, pseudo-hypopara thyroidism, vitamin-D-dependency disease, and drug-induced osteomalacia. This has led to a marked resurgence of interest in the organic chemistry of the D vitamins and their analogs. All known vitamin D metabolites and a considerable number of structural analogs have been prepared by chemical synthesis. Among the latter, 1 a-hydroxyvitamin D3 ( 1 a-OH-D3)' the 2S-deoxy analog of 1 a,2S-(OH)2D3, repre sents perhaps the most important derivative because of its potential clinical use as a substitute for l a,2S-(OHhDJ. The future for vitamin D research is an extremely bright one for biochemical investigations at the level of mechanism of action, at the level of continued investiga tion into the metabolism of vitamin D to its excretory products, and in terms of the regulation of functional vitamin D metabolism. In addition, a fruitful area of analog synthesis has opened up which may permit the synthesis of compounds that can carry out specific functions of the vitamin. Finally, the medical and agricultural applications of the metabolites and their analogs may prove to be an important practical reward for the basic biochemical work of the past few years.
INTRODUCTION
For almost 30 years the area of vitamin D investigation had remained in a relative state of dormancy. The brilliant chemical work of Windaus and his collaborators on vitamin D ended with the identification of vitamin D3 in 1 937 ( 1 , 2), and the definitive work of Nicolaysen & Eeg-Larsen on the role of vitamin D in the intestinal absorption of calcium was completed by 1940 (3). Although the role of vitamin D in intestine and bone was further clarified during the next two decades, it was not until 1 967 that the breakthrough in vitamin D metabolism was registered, revealing
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DELUCA & SCHNOES
the concept that vitamin 0 is converted to metabolically active forms. This discov ery has led to the realization that vitamin 0 is a prohormone giving rise to at least one hormone that is produced in the kidney and functions in intestine and bone (4). This review brings together the features of the metabolism of vitamin 0 and its regulation as well as recent work on the function of vitamin O. Because of the intense interest in chemical synthesis of the metabolites and their analogs, a section on chemistry is included. Because of the massive and widespread nature of the recent literature available in this area, no attempt at a comprehensive list of contributions is made. Reviews covering different aspects of the field or other viewpoints have appeared in the past few years. Interested readers are directed to references 5-14.
METABOLISM OF VITAMIN D3
The clear demonstration that physiologic amounts of vitamin 03 are metabolized in large proportions to metabolites possessing biological activity equal to or greater than vitamin 03 (15) itself came following completion of chemical synthesis of [1,23H]vitamin 03 of high specific activity (16). The major circulating metabolite of vitamin D3 was then isolated from plasma of pigs and identified chemically as 25-0H-D3 (17) (Figure I). The liver was shown to be the site of this conversion (18-20), although evidence has been presented that some 25-hydroxylation occurs in kidney and intestine of chicks (21). A reexamination of this point has revealed that certainly in the rat and probably in the chick the liver is the major, if not sole, site of 25-hydroxylation ( 1 9; E. B. Olson & H. F. DeLuca, unpublished results). Even during the isolation of 25-0H-03 prior to its identification, the existence of metabolites more polar than 25-0H-03 was noted (17), but they were not further characterized, nor was their biological activity examined. Haussler, Myrtle & Nor man first detected a metabolite of vitamin 03 more polar than 25-0H-03 in the nuclear fraction of small intestine (22). Shortly thereafter Lawson, Wilson & Kodi cek (23) also located this metabolite of vitamin 0 and found that during its biosyn 3 3 thesis the 1 a- H from [ I a- H]vitamin 03 was lost. They used this observation to suggest that this metabolite possessed a modification on carbon-I, although other explanations were possible. Independently, Ponchon & DeLuca studied extensively the metabolites of vitamin D in a variety of tissues in both rats and chicks, demon strating the existence of what was termed peak V as well as other more polar metabolites (24). Synthesis of radioactive 25-0H-D3 (25) allowed a pursuit of its metabolites, and it was soon demonstrated that the polar metabolite of the small intestine, designated as peak V in the Wisconsin laboratories, peak p in the Cambridge laboratories, and 4B in the Riverside laboratories, was formed from 25-0H-03 (26, 27). Although Haussler, Myrtle, & Norman (22) first reported marked biological activity of the intestinal metabolite, this finding could not be uniformly reproduced. Haussler et al (28) discovered that the intestinal metabolite initiated intestinal calcium transport very rapidly and that its activity decreased very rapidly, accounting for the diver gency of reports on biological activity. This very rapid activity was confirmed in two
635
OH
Live r
OH
r
CH2 HO" HO" 25, 26-(OH)2 0 3 CH2
OH
CH2
03
2 5-0H-0
() a. ,<:l , l" o *-'

OHH OH
0,0;+"
(0 'Y
".&
(0 .. '",Y "-?;.
OH
OH
I) PTH 2) Low
CH2
Pi
I)Normal 2) Normol
Kidney
Co Pi CH2 OH 1,25-(OH)20 3
Kidney
HO" (24R)-24, 25-(OH)2 03 (24
R)-I, 24,25- (OH)3 03
Figure 1
Diagrammatic representation of vitamin D metabolism and its regulatory features.
laboratories (29,,30) and led to an intensive approach to the isolation and identifica tion. In early 1 97 1 this metabolite was isolated from the intestines of 1500 rachitic chickens given a single dose of radioactive vitamin D3 (31, 32). Isolation involved approximately ten chromatographic procedures through which the lipid extract of the intestines was processed, However, this led to a metabolite fraction con taminated with another trihydroxy sterol, which could then be separated upon derivatization as the 25-hydroxy-trimethylsilyl ether and subsequent chromatogra phy on Sephadex LH-20. Isolation of the metabolite from the intestine involved primarily the use of liquid-gel partition chromatography, which was introduced as a major chromatographic technique for the study of vitamin D metabolism (33). From the 1500 rachitic chickens given the radioactive vitamin D3, two micrograms of 25-trimethylsilyl ether derivative were isolated in pure form and the structure of the metabolite was unequivocally identified by chemical means as 1,25-(OH)2D3 (32). While this isolation was being carried out, Fraser & Kodicek (34) discovered that production of the intestinal metabolite took place in kidney tissue, and they were able to produce the metabolite in vitro by incubation of chick kidney homogen ates and isolated mitochondria. From in vitro incubations, Lawson et al (35) were able to obtain several micrograms of metabolite in approximately 30% purity, From
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DELUCA & SCHNOES
this preparation, independent evidence for the I ,25-(OH)zD3 structure was obtained (35). Therefore, it became clear that 25-0H-D3, which was produced predominantly if not exclusively in the liver, is hydroxylated to 1 ,25-(OH)zD3 in the kidney. The exclusive synthesis of 1 ,25-(OH)zD3 in the kidney could easily be demonstrated in nephrectomized rats given radioactive 25-0H-D3 intravenously (36). These animals failed to produce 1 ,25-(OH)zD3, while sham-operated controls were clearly able to perform the conversion. The ability of chick kidney homogenates and mitochondria to carry out the conversion of 25-0H-D3 to the 1,25-(OH)2D3 was confirmed in two different laboratories (36, 37). With the nephrectomized animal it was possible to demonstrate quite clearly that 1 ,25-(OH)2D3 is the active form of vitamin D3 in the initiation of intestinal calci um transport (38, 39), bone calcium mobilization (40), and intestinal phosphate transport (41). Briefly, nephrectomized animals do not show these responses to 25-0H-D3 when it is given in physiologic amounts, whereas 1 ,25-(OH)2D3 produces these responses whether the kidneys are present or not. Thus it is quite evident that 25-0H-D3 and vitamin D) itself cannot be considered metabolically active forms at physiologic concentrations. However, it is well known that vitamin D or 25-0H-D3 can produce responses in nephrectomized animals if given in large amounts (42, 43). In addition, in isolated cultures of bone (44, 45), in vascularly perfused intestine (46), and in isolated intestinal cultures (47), large amounts of 25-0H-D3 cause the mobilization of calcium from bone, the transport of calcium across intestinal mem brane, and the production of calcium-binding protein. The exact reason for re sponses to pharmacological amounts of these compounds is not known. Two possibilities are now considered: one that the receptor molecules or systems will respond to the unhydroxylated forms of vitamin D if present in high concentrations, or alternatively, that small amounts of trans-vitamin D or isotachysterol contami nants, which have their 3-hydroxyl in a pseudo 1 position, might produce a 1 ,25(OH)zDrlike response (48-50). In any case, from a physiologic point of view it is quite clear that conversion of 25-0H-D3 to 1 ,25-(OHhD3 is obligatory for vitamin D to carry out its functional role. Following the demonstration that J,25-(OHhD) or a further metabolite is the final metabolically active form of the vitamin, work has been carried out with radioactive 1 ,25-(OH)zD3 in an effort to learn whether it is further metabolized (5 1 -54). No other metabolites have been found during the time required for intestine and bone to respond to this compound, leaving the impression that 1 ,2S-(OH)2D3 is not further metabolized before it functions. Recent work in our laboratory has shown that in fact 1 ,25-(OH)zD3 is further metabolized even before the intestine responds (R. Kumar, D. Harnden & H. F. DeLuca, in preparation), but it is uncertain whether the resulting metabolite is of functional importance. Undoubt edly this area will be considerably clarified in the next few years, which will aid greatly in the final elucidation of the mechanism of action of 1 ,25-(OH)zD3. During the isolation and identification of 1 ,25-(OH)zD3, other metabolites of vitamin D3 were detected, isolated, and identified. In particular, from the peak V region described by Blunt, DeLuca & Schnoes ( 1 7), two metabolites were isolated and tentatively identified. The first, which was termed peak Va, was initially identi-
637
fied as 21 ,25-dihydroxyvitamin D3 (55), but upon more thorough characterization, the correct structure was deduced to be 24,25-(OHhD3 (56). This compound has proved to be a major metabolite of vitamin D3 whose exact function is still under examination. Chemical synthesis of this compound has been completed (57), and the two possible stereoisomers have been prepared independently by Ikekawa and his group (58) and by the Hoffmann-LaRoche group (5 9). Cochromatography of the natural radioactive product with the synthetic isomers as their silyl ether derivatives proved that the natural form was (24R)-24,2S-(OHhD3 (60). The natural product cochromatographs exactly with the 24R compound; no radioactivity migrated with the 24S compound. In the rat, the 24R compound has biological activity in intestinal calcium transport, calcification of bone, and mobilization of calcium from bone approaching that observed with 25-0H-DJ itself (60). Furthermore, the administra tion of radioactive 24,25-(OHhD3 to vitamin D-deficient rats reveals that it is not rapidly excreted, but is converted to another polar metabolite (61), which has been isolated in pure form and identified as 1 ,24,25-(OHhD3 (62). Presumably the config uration of the C-24-hydroxyl in this compound is also R. The 1 ,24,25-(OH)3D3 also possesses very potent biological activity in vitamin D-deficient rats (62); further more, both of these compounds persist in those animals (6 1 , 63), which argues against their representing an excretory pathway. The 1 ,24,25-(OH)3D3 is also syn thesized in vivo from 1,25-(OHhD3 (64), which may be the more important pathway since 1,25-(OH)2D3 actually stimulates or induces the 24-hydroxylase, which can use 1,25-(OHhD3 as a substrate (Y. Tanaka & H. F. DeLuca, unpublished results). Of great interest is that the (24S)-24, 25-(OH)2D3 is relatively inactive in almost every system with the possible exception of intestinal calcium transport (60). This marked discrimination against the 24S configuration could argue strongly for the biological importance of the 24R configuration (at least in mammals) or, alterna tively, for discrimination against the 24S configuration and tolerance for the 24R configuration. Only additional investigation will permit a resolution of these two possibilities. In any case; 24,25-(OH)zDJ must be considered a major metabolite in mammals with the possibility that the 24-hydroxylation reaction represents an important functional reaction that is not yet understood. In agreement with this, (24R)- and (24S)-24-hydroxyvitamin D have been prepared and their biological activity assessed in the vitamin D-deficient rat (65, 66). As might be expected, the (24R)-24-hydroxyvitamin D) (24-0H-D3) is approximately equal to 2S-0H-D) in biological activity in all respects, whereas the 24S compound is almost devoid of activity except in intestinal calcium transport. In contrast to rats, chicks show quite a different response to the 24-hydroxy compounds: both the (24R)- and the (24S)-24,25-dihydroxy compounds have much lower biological activity than 25-0H-D) (63, 67, 68). In this species as well the 24R configuration gives more biological activity than the 24S configuration (63, 67, 68). However, since both show only about one fifth the activity seen with 25-0H-DJ, it is strongly suspected that the 24-hydroxylated compounds are not of functional importance in the chick (63). Radioactive (24R)-24,25-(OH)2D3 is rapidly metabol ized by the chick and excreted into the feces (63). The (4R)-1 ,24,25-(OH)JD3 also shows weak biological activity in the chick (63) while demonstrating high biological
638
DELUCA & SCHNOES
activity in the rat (62). Because of the substitution on the 24 carbon and because chicks and other fowl discriminate against other vitamin D compounds that have a methyl group on the 24 carbon (69-71; G. Jones, L. Baxter & H. F. DeLuca, unpublished results), it is possible that substitution at this position constitutes a signal for elimination in the chicken or other birds (63). Thus, 24-hydroxylations in these species may represent the first reaction in the metabolic elimination of potentially toxic vitamin D compounds. It is possible, therefore, that birds discrimi nate against vitamin Dz compounds simply because they are analogs of the normal excretion product [(24R)-24,25-(OH)zD3J of vitamin D), the natural form of vitamin D (63). In agreement with this idea, it has been shown that vitamin Dz and its metabolites are rapidly metabolized and excreted via the bile into the feces (72). 25,26-Dihydroxyvitamin PJ [25,26-(OH)zD3J has also been isolated, identified (73), and chemically synthesized (74, 75). This compound is only weakly active in stimulating intestinal calcium transport and has little or no activity in the other systems known to be responsive to the vitamin. It appears in quantitatively smaller amounts than 24,25-(OH)zD3 and its role, if any, is not yet known. Paralleling the work on vitamin D3 metabolism have been occasional studies in the vitamin D2 series. 25-Hydroxyvitamin D2 (25-0H-D2) has been isolated and chemically identified (76), and 1 ,25-dihydroxyvitamin D2 [ 1 ,25-(OH)2D2] has been prepared in vitro with chick kidney mitochondria and its structure unequivocally elucidated (77). 24,25-Dihydroxyvitamin D2 [24,25-(OH)zDzJ has also been isolated and identified, as has been 24-hydroxyvitamin D2 (24-0H-Dz) (G. Jones, H. K. Schnoes & H. F. DeLuca, unpublished results). The biological activity of the vita min Dz compounds has been assessed in the rat and found to be identical in every respect to that of the vitamin D3 compounds. In the bird, however, as described above, the vitamin D2 compounds including 1,25-(OH)zDz are approximately one fifth to one tenth as active as their vitamin D3 counterparts (69-7 1 ; G. Jones, L. Baxter & H . F. DeLuca, unpublished results). Upjohn chemists have successfully synthesized 25-0H-D2 and 25-0H-24-epiD2; the latter is much less active than the former (J. A. Campbell, L. Reeve & H. F. DeLuca, unpublished results). New World monkeys also discriminate against vitamin Dz in terms of their biological response (78). Although the mechanism of this discrimination has not yet been determined, it might well be related to that described for the chick. In this regard it should be noted that the chick possesses the enzymes necessary for the synthesis of the active forms of vitamin D2 (79). That is, the 25-hydroxylase of chick liver does not discriminate against vitamin Dz and the I -hydroxylase and 24hydroxylase systems of chick kidney do not discriminate against 25-0H-D2 (80). However, following injection of radioactive vitamin Dz, little 25-0H-D2 is found in the circulation (7 1 ) and little 1 ,25-(OH}zDz is found in the intestine (79). Since chick intestine also responds poorly to. 1 ,25-(OHhDz compared with 1 ,25-(OH)zD3, it seems, as previously stated, that the bird discriminates against all forms of vitamin D substituted at the 24 position, probably as a consequence of their rapid metabo lism and excretion. A binding protein for 1 , 25-(OHhD3 from the cytosol of chick intestine is considered as a receptor, and binds 1 ,25-(OH)zDz as well as it binds \,25-(OH)zD3 (80; J. Eisman &.H. F. DeLuca, unpublished results). Thus discrimi nation does not take place at the receptor level.
639
Little is known about further metabolism of vitamin D metabolites to excretion products. Vitamin D sulfates have been described (8 1 -83), but have not yet been adequately demonstrated as products of the normal metabolic scheme or as normal excretion products. The primary route of excretion appears to occur via the bile, with less than 4% of the total radioactivity of vitamin D3 appearing in the urine (84-86). No biliary metabolites have yet been identified, although claims of glucuro nides or sulfates have appeared without adequate chemical identification (87, 88).
ENZYMES THAT CATALYZE VITAMIN D3 METABOLISM
Vitamin Dr25-H ydroxylase
There is little doubt that the liver is a site of 25-hydroxylation of vitamin D3 ( 1 8-20). There is some question whether this conversion occurs to any significant degree in tissues other than the liver. Tucker, Gagnon & Haussler (21) have reported that chick kidney and intestine also possess 2S-hydroxylating ability. The conversion in chick but not in rat intestine has been confirmed in our laboratory; confirmation of this activity in kidney has not been possible. Hepatectomy either markedly reduces or eliminates the ability of rats to produce 2S-0H-DJ. Although some of this metabolite has been detected in hepatectomized rats, the amount is very small in the face of very high concentrations of circulating vitamin D3 (E. B. Olson & H. F. DeLuca, unpublished results). Ordinarily vitamin D3 is rapidly taken up in large amounts in the liver and does not circulate in high concentrations (16, 18, 84, 86). Thus the contribution of extra-hepatic tissue to 2S-hydroxylation is likely small and perhaps absent under physiologic circumstances. In the bird this hydroxylation may occur in part in the intestine especially following oral administration of the vitamin. Liver homogenates carry out the 2S-hydroxylation when supported by added glucose-6-phosphate and magnesium ions utilizing the endogenous glucose-6-phos phate dehydrogenase (20, 89). However, the activity can be increased by the addition of an NADPH-generating system (2 1). Two cell components, the microsomal frac tion and the cytosolic fraction, are necessary for maximal rates of hydroxylation (89). The cytosol contains a heat-labile factor, which both protects the substrate and stimulates its conversion to 2S-0H-D3 (89), and may well be analogous to the systems described for cholesterol biosynthesis and for bile acid biosynthesis. The maximal extent of conversion, only 7% of the substrate, is below that which one might expect. However, there is sufficient enzymatic activity in the hepatic microso mal fraction to account for the observed in vivo 2S-hydroxylation. In addition, some 2S-hydroxylation can be obtained in the mitochondrial fraction, especially at higher substrate concentrations (90; M. H. Bhattacharyya & H. F. DeLuca, unpublished results). The mitochondrial fraction will also hydroxylate cholesterol to 2S-hydroxy cholesterol and dihydrotachysteroh to 2S-hydroxydihydrotachysteroh . The microsomal 2S-hydroxylation reaction is markedly suppressed by the prior administration of vitamin D3 (89, 9 1). The degree and duration of this suppression are dependent upon the dose of vitamin D and appear to be related to the hepatic level of 2S-0H-D3. External 25-0H-DJ does not enter the liver (92) and hence does not suppress the 25-hydroxylase. It is not known whether this suppression occurs as a result of product inhibition by 2S-0H-DJ' or whether the 2S-hydroxylase is
640
DELUCA & SCHNOES
altered. Large amounts of vitamin D3 overcome this suppression (91, 93), and it is uncertain whether this is a result of competition with liver 2S-0H-D3' releasing the microsomal 2S-hydroxylation activity, or whether it results from extra-microsomal 2S-hydroxylation at high substrate concentrations. Thus regulation is observed only at physiologic doses of vitamin D and large amounts of the vitamin can increase the levels of circulating 2S-0H-D3 ( 1 7, 94, 9S). Little is known about the mechanism of the 2S-hydroxylation. It is not blocked by carbon monoxide or other cytochrome P-4S0 inhibitors, nor is it induced by phenobarbital (20, 89). Although some reports of increased activity with phenobar bital have appeared (96, 97), the increases are not remarkable and can be accounted for by increased liver size. Work has been carried out, however, on the possible interference with vitamin D metabolism of anti-epileptic drugs such as phenobarbital and dilantin. Patients on long-term therapy show reduced levels of circulating 2S-0H-D3 (98, 99) and evidence of bone disease. There is considerable disagreement regarding the extent of the drug-induced osteomalacia, but there is no doubt that it does appear. Hahn and collaborators have suggested that phenobarbital and dilantin cause a rapid degradation of vitamin D and its metabolites to inactive products ( 1 00). This inter esting idea has not yet received sufficient biochemical support to warrant its accep tance. Alternatively it has been suggested that phenobarbital and dilantin treatment interfere with 25-hydroxylation (99). The possibility of an increased metabolism of vitamin D under conditions of drug-induction is an idea that merits further investi gation.
25-Hydroxyvitamin Dla-Hydroxylase
Perhaps the most important hydroxylation reaction from a physiologic point of view occurs exclusively in the kidney, and is catalyzed by 2S-0H-Dr I a-hydroxylase (34, 36). This enzyme occurs exclusively in mitochondria (34, 1 0 1 ), and is found in a variety of animal species (102). The I -hydroxylase may be located in the inner mitochondrial membrane, but this has not yet been firmly established. 180 experi ments have shown that the I-hydroxylase is a mixed-function oxidase; molecular oxygen is incorporated into the substrate, 2S-0H-D3 (103). The hydroxylation in intact mitochondria is supported by succinate, malate, or almost any Krebs cycle substrate (34, 1 0 1 , 1 04), and requires the electron-transport chain and oxidative phosphorylation for maximal activity ( 1 04). Antimycin A blocks the reaction sup ported by these substrates completely, whereas cyanide, rotenone, and dinitrophenol reduce it to about SO% of maximum. Although a report has appeared that antimycin A does not block the renal I -hydroxylase ( l OS), great care must be taken in interpre tation of these results; antimycin A is relatively unstable and if it fails to inhibit, its effectiveness in blocking the respiratory chain must be demonstrated. When the mitochondria are swollen with calcium ions, NADPH but not NADH can supply the reducing equivalents (104). Thus the correct electron donor is NADPH. The I-hydroxylase is blocked by carbon monoxide-oxygen mixtures as demonstrated originally by Fraser & Kodicek (34) and subsequently in our laboratory ( 1 0 1 , 1 04). This inhibition is alleviated by white light (104), and more specifically by light of 4S0-nm wavelength (10S). Furthermore, glutethimide and metyrapone both inhibit
641
the reaction, which suggests that the reaction is indeed dependent upon cytochrome P-4S0 (106). Therefore, the reaction appears analogous to the steroidogenesis sys tems of the adrenal gland. The existence of a cytochrome P-4S0 in isolated chick mitochondria that is reducible by malate was clearly demonstrated by spectral means (106). Since its spectrum appeared only when malate or other mitochondrial substrates were added, the P-4S0 spectrum is clearly of mitochondrial origin and not due to microsomal contamination. This finding was followed by solubilization of the chick kidney la-hydroxylase system and the isolation of a P-450 fraction (106). When recombined with beef adrenal ferredoxin and beef adrenal flavoprotein together with NADPH, this P-450 fraction carries out the hydroxylation of 25-0H D3 to 1,2S-(OH)2D3 (106). Recently the P-4S0 preparation has been considerably improved and the chick renal ferredoxin has been isolated in pure form (J. Pedersen, J. Ghazarian, N. Orme-Johnson & H. F. DeLuca, in preparation). When these are combined with beef adrenal flavoprotein and NADPH, very substantial hydroxyla tion occurs with the appearance of the single product, J,25-(OH)zD3' These experi ments demonstrate clearly that the I -hydroxylation reaction involves the sequential reduction of flavoprotein, renal ferredoxin, and cytochrome P-4S0 (Figure 2). 2S-0H-D3-24-Hydroxylase also occurs in the kidney (61, 107), but probably not exclusively so (42). The reaction occurs in the mitochondria and in general is very similar to the 25-0H-Drla-hydroxylase (107). In intact mitochondria it is sup ported by Krebs cycle substrates, utilizes molecular oxygen, and is blocked by inhibitors of oxidative phosphorylation. In calcium-swollen mitochondria, NADPH will support the reaction, and under these circumstances the reaction is independent of the electron-transport scheme and oxidative phosphorylation. However, this reaction is not sensitive to carbon monoxide and does not appear to be dependent upon cytochrome P-4S0. It has not been further studied, nor has the responsible enzyme system been successfully solubilized at the present time.
Regulation of the Renal la- and 24-Hydroxylases
The reaction that is controlled physiologically and that makes the vitamin D system an endocrine system is 25-0H-Drl a-hydroxylase. Since this reaction occurs exclu sively in the kidney and since its product has a target of action in intestine and bone,
NADPH ----------
FP
FERREDOXIN
RENAL
Cytochrome
2 P450/C0 '" H 0'" 2 3
':
25-0H-D
Figure 2
Components of 25-0H-D)"1 u-hydroxyJase
of chick
kidney.
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DELUCA & SCHNOES
it appears to fit the criteria for a hormone. In true hormonal fashion the biosynthesis of this metabolite is strongly regulated physiologically by the need for calcium (l08, 109) and phosphorus (110). In 1971 it was clearly demonstrated that the production and appearance of 1,25-(OH)2D3 in the rat is markedly stimulated by the feeding of low-calcium diets and is suppressed in animals given a high-calcium diet (108). This suppression or stimulation takes place only in animals given a source of vitamin D. Animals maintained in the vitamin D-deficient state possess I -hydroxylase independent of dietary calcium (108). In animals given vitamin D there is a strict inverse relationship between the serum calcium level and the ability of the animals to produce 1,25-(OHhD3. As animals become hypocalcemic or illustrate the need for calcium, the synthesis of 1,25-(OH)2DJ is stimulated. When calcium is not needed, the production of 1,25-(OHhD3 is suppressed. In virtually all situations wherein 1,25-(OHhD3 synthesis is suppressed, 24,25-(OH)zD3 synthesis is stimu lated. Removal of the parathyroid glands eliminates the stimulation of 1 ,25-(OH)zD3 production by hypocalcemia; instead, the hypocalcemic animals make 24,25(OH)2D3 (III). The administration of parathyroid hormone restores their ability to make 1,25-(OH)2D3 and suppresses the production of 24,25 (OH)zD3 Experiments in chicks carried out by Fraser & Kodicek have confirmed that the parathyroid hormone is the primary controlling factor in determining synthesis of 1 ,25(OH)zD3 in response to the need for calcium (112). Attempts to show direct effects of parathyroid hormone on isolated chick renal tubules have been largely unsuccessful ( 1 1 3-11 5). This is probably related to the fact that in vivo the change over of the hydroxylases in response to parathyroid hormone or a change in serum calcium is a slow process involving many hours ( I I I , 112, 116). Undoubtedly isolated renal tubules do not survive for the periods of time necessary to permit expression of the in vivo stimulation of l a-hydroxylase by parathyroid hormone. Excessive amounts of parathyroid hormone are reported actually to suppress 1,25(OH)2D3 production, whereas large amounts of calcitonin, another calcium-related hormone, increase 1 ,25-(OH)2D3 production (117, 118). However, these experi ments were carried out in intact animals, which could have brought about secondary responses of the parathyroid gland or "c" cells of the thyroid, complicating interpre tation of the results; they have not been pursued further and their physiologic meaning remains in doubt. In any case it has been demonstrated that isolated mitochondria from chicks given high-calcium diets produce only 24,25-(OH)2D3, whereas those from chicks given a low-calcium diet produce exclusively 1,25(OH)2D3 (119). These enzymatic studies, which confirm the control by calcium, have been considerably expanded recently (120). In an attempt to understand the mechanism by which calcium and the para thyroid complex controls the I -hydroxylase system, studies involving the addition of calcium and/or parathyroid hormone and other ions to isolated mitochondria, isolated renal tubules, and other in vitro systems have been carried out ( 1 2 1 - 1 24). Added parathyroid hormone has no effect on the I -hydroxylase system of isolated renal mitochondria, while calcium can either stimulate or suppress, depending upon the nature of the medium used for isolation. When the mitochondria are isolated from EDTA media, calcium will stimulate ( 1 24), and when isolated from normal
643
media, the calcium will inhibit (122, 124). When the components of the I-hydroxy lase system are solubilized, isolated as described above, and recombined, the re constructed system is totally insensitive to calcium additions. Furthermore, calci um-swollen mitochondria carry out the I-hydroxylation even in the presence of 10 mM calcium. Thus the I-hydroxylase system itself is not inhibited by calcium ions. It seems likely that the inhibition by calcium in vitro is related to altered mitochon drial structure and to the inhibition of NADPH production (125). Whether or not this represents a true physiological control mechanism is in doubt. Many hours are required for the hydroxylases to respond to the parathyroid hormone or to changes in serum calcium concentration. If this were, the result of ionic inhibition or activa tion, one would expect the hydroxylases to change very rapidly. Thus the physi ologic meaning of the inhibition of I-hydroxylation in intact mitochondria by calcium is unclear. Experiments showing that the hydroxylases of the kidney have half-lives of approximately 2 hr (126, 127) suggest that the ionic environment of the cell may well regulate the biosynthesis or degradation of the enzymes or a control ling protein rather than exert a direct ionic inhibition or activation of the hydroxy lases. Because the I-hydroxylase of vitamin D--deficient rats or chicks does not show regulation by calcium or parathyroid hormone, it appears that some form of vita min D must be present for the regulation to occur. The administration of 1,25(OHhDJ to vitamin D--deficient rats brings about a suppression of the I-hydroxylase and a concomitant stimulation of the 24hydroxylase (128-130). In fact, current work suggests that 1,25-(OH)2D3 induces the formation or appearance of the 24hydroxylase system (129). It is only after the appearance of 1,2S-(OH)2D3 and the induction of the 24-hydroxylase system that parathyroid hormone and calcium can further modulate the production of the metabolites of vitamin D. It has been clearly shown. that 1,25-(OHhD3 stimulates RNA synthesis in kidney tissue (131). This effect may well be related to the induction of the 24-hydroxylase, but that remains unknown. Besides calcium and I,2S-(OHhD3 itself, phosphorus plays an important role in the regulation of 1,25-(OH)2D3 production. Maintaining rats on a low-phosphorus diet results in a marked accumulation of I,2S-(OHhD3 in blood and tissue even in the absence of the parathyroid gland (110). Thus in thyroparathyroidectomized rats a clear inverse relationship between serum levels of inorganic phosphorus and 1,25-(OH)2D3 production has been demonstrated (110). Under conditions of low blood phosphorus, 1,25(OHhD3 accumulation is stimulated, whereas with normal or high levels, 24,25-(OH)2D3 is accumulated. Because parathyroid hormone blocks renal reabsorption of phosphate, this may be the mechanism by which parathyroid hormone stimulates 1,25-(OH)2DJ production. Appropriate measurements show a correlation between the renal cortical level of inorganic phosphorus and the ability to produce 1,25-(OHhD3. Thus phosphate deprivation or administration of para thyroid hormone reduces renal cortical levels of inorganic phosphorus, whereas conditions favoring 24,25-(OH)zD3 synthesis cause an increase. A recent report suggests that renal 25-0H-D3- l a-hydroxylase is not stimulated by phosphate deprivation (120). An examination of those results reveals that the diet was not low in phosphorus nor was the length of time to which the animals were
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exposed to the low-phosphorus diet sufficient to stimulate the I-hydroxylase. Low phosphorus diets do, however, stimulate chick renal 2S-0H-Drl a-hydroxylase activity ( 1 32 ; L. A. Baxter & H. F. DeLuca, in preparation). It seems likely that phosphate deprivation also stimulates the intestine directly, inasmuch as rats main tained on exogenous 1 ,2 S-(OHhD3 as their sole source of vitamin D still show a marked elevation of intestinal calcium transport under conditions of phosphate deprivation (133). A similar conclusion was reached from studies using an analog of 1,25-(OHhD3, namely dihydrotachysterol3 ( 1 34). The biochemical mechanisms by which vitamin D hydroxylases of the kidney are regulated are thus not entirely clear. It is clear, however, that the need for calcium or phosphorus will stimulate production of 1 ,25-(OH)2D3' a hormone that functions in mobilization of both calcium and phosphate. Additionally, calcium regulation is mediated by parathyroid hormone, and 1 ,25-(OH)2D3 itself plays an important role in this regulation by inducing the 24-hydroxylase and by permitting modulation of the I -hydroxylase. It may seem disturbing that 1 ,25-(OH)2D3 can be considered a hormone with two signals (low calcium and low phosphorus) and two functions (calcium mobilization and phosphate mobilization). It would appear that a specific correction of the signal is not possible. However, the calcium signal causes parathyroid secretion; thus parathyroid hormone accompanies 1,25-(OH)2D3 in this circumstance, permitting mobilization of bone calcium and renal reabsorption of calcium. The effect of 1 ,25-(OHhD3 on serum phosphate is negated by the parathyroid hormone-induced loss of phosphate in urine (6). The composite effect of the low-calcium signal is to elevate serum calcium but not serum phosphate (6). The low-serum-phosphate signal causes 1 ,25-(OH)2D3 to appear without para thyroid hormone. Bone calcium is not readily mobilized without parathyroid hor mone, and calcium is not completely reabsorbed in the kidney. However, phosphate absorbed in intestine in response to 1,2S-(OH)2D3 is totally reabsorbed in the kidney without parathyroid hormone to cause phosphate excretion. The net effect is to increase serum phosphorus but not calcium (6). Thus 1 ,25-(OH)2D3 can be a dual purpose hormone with two different signals.
Regulation of the Renal Hydroxylases by Other Factors
It is well known that strontium brings about a vitamin D-resistant form of rickets and markedly reduces intestinal calcium absorption ( 1 35). It is now clear that the feeding of strontium represses the renal 25-0H-D3- l a-hydroxylase and that the administration of 1 ,25-(OH)2D3 to strontium-fed animals will restore their ability to absorb calcium ( 1 36, 1 37). Thus the metabolic basis for strontium-induced rickets has been solved, at least partially. The administration of large amounts of ethane- I -hydroxy-I,l -diphosphonate (EHDP) also causes vitamin D-resistant rickets ( 1 38) and a markedly repressed intestinal calcium absorption. This result is also due to repressed biosynthesis of 1 ,25-(OH)2D3 ( 1 3 9, 140). The repressed intestinal absorption brought about by EHDP is reversed by administration of exogenous 1,25-(OH)2D3. It is unknown how the EHDP blocks the I -hydroxylase.
645
Although nephrectomized rats cannot form 1,25-(OHhDJ, homogenates of rat kidneys cannot carry out significant amounts of the 25-0H-DJ-I-hydroxylation (141). This finding contrasts sharply with the fact that renal homogenates and isolated mitochondria from chicks clearly carry out the hydroxylation step. The basis for this difference lies in the presence of a heat-labile inhibitor in rat tissues, which when added to chick kidney preparations also represses their ability to carry out the I-hydroxylation. This inhibitor has been found in large amounts in rat blood, intestine, and kidney fractions and to a much lesser extent in chick blood. It has been isolated from rat blood and shown to be a protein with a molecular weight of approximately 52,000 (K. Botham, J. G. Ghazarian & H. F. DeLuca, in prepara tion). It contains iron and can bind 25-0H-DJ. The physiological role of this inhibitor and the nature of its action remain unknown. Because of its presence, meaningful measurement of in vitro renal I -hydroxylation cannot be made in mam malian tissues. This inhibitor also blocks hydroxylation in calcium-swollen mito chondria and in the reconstructed system made from isolated P-450, renal fer redoxin, and flavoprotein. Prior to eggshell formation there is a marked stimulation of renal 25-0H-Dr l a-hydroxylation in Japanese quail (142). These results suggest that some humoral factor, released as the egg progresses down the oviduct to the shell gland, must stimulate 1,25-(OHhD3 production, which may in turn facilitate the transfer of calcium in the shell gland for deposition as eggshell. This observation implies the existence of yet an additional controlling factor and mechanism. Glucocorticoids also affect vitamin D metabolism (143). These compounds are known to repress intestinal calcium transport, but they appear not to interfere with production of 25-0H-D3 or 1,25-(OH)2DJ (144); instead they may repress calcium absorption even in animals given 1,25-(OH)2DJ' Carre et al report that glucocor ticoids cause the conversion of 1,25-(OHhD3 to a more polar, inactive metabolite in the intestine (145). This phenomenon may provide a basis for understanding why glucocorticoids are successful in the treatment of vitamin D toxicosis as well as new insight regarding hormonal control of the vitamin D-derived calcium-mobilizing hormone.
MECHANISM OF ACTION OF 1,25-(OHhD3
There are two known sites of action of 1,25-(OHhD3: intestine and bone. There is additional evidence that I ,25-(OHhD3 is active on kidney and indirect evidence that a metabolite of vitamin D may be active on muscle. By far the most thoroughly studied is the role of vitamin D in the intestine; less information is available concern ing the mechanism of action of 1,25-(OH)2DJ in bone.
Mechanism of Action in the Intestine
It is well known that vitamin D stimulates calcium absorption against an electro chemical potential gradient in the small intestine (146-151). The most rapid rate of calcium transport occurs in the duodenum followed by the jejunum and ileum (146, 152), but vitamin D improves intestinal calcium transport even in the colon (152).
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DELUCA & SCHNOES
Because of the time during which calcium is subject to absorption. it seems evident that the distal portions of the small intestine are primarily responsible for the bulk of intestinal calcium absorption. The movement of calcium against an electrical and concentration gradient re quires metabolic energy ( 1 46- 1 5 1). The site of vitamin D activation of this process is not entirely clear. but there is uniform agreement that vitamin D in some way alters the characteristics of the brush border membrane to permit entry of calcium ( I SO, 1 53, 1 54). Inconclusive evidence indicates that vitamin D may also improve the transfer of calcium across the serosal surface of the intestinal epithelial cell ( 1 53). The response of intestinal calcium transport to vitamin D is sensitive to pretreat ment by actinomycin D ( 1 55-1 57). However, it is unclear whether actinomycin D blocks the intestinal response to 1 .25-(OH)2D3' There is some evidence that the lifetime of 25-0H-Dr l -hydroxylase and its messenger is sufficiently short that the apparent block in vitamin D action on intestinal calcium transport may be due to the decay of the messenger and of the enzyme for the I -hydroxylation reaction in the kidney ( 1 26). Once the I -hydroxylase is bypassed by the administration of 1 ,25-(OHhD3, actinomycin D does not block the transport process, at least in the rat (1 58). Tsai. Midgett & Norman have provided conflicting evidence that actino mycin D does block the intestinal response to 1 ,25-(OH)zD3 in the chick when the antibiotic is administered every two'hours ( 1 27). Whether this is a bona fide block or a toxic reaction to the antibiotic remains undetermined. Wasserman and his colleagues have discovered a calcium-binding protein that appears in the intestine in response to vitamin D (5, 1 59). This protein has a molecular weight of 24.000 in the chick and 8()()().... 1 2.000 in the rat and other mammalian species. It binds four calcium ions per mole. and its appearance and concentration correlate approximately with the rate of intestinal calcium transport under a number of physiological conditions. Inasmuch as this protein binds calcium, appears only after vitamin D administration, and correlates approximately with intestinal calcium transport, it has been surmised that it must play a role in the transport process. However, there are conditions under which the correlation be tween calcium-binding protein and intestinal transport is quite poor. For example, cortisone inhibits intestinal calcium transport while it stimulates production of calcium-binding protein ( 1 60). Intestinal calcium transport precedes the appearance of calcium-binding protein as measured by the Chelex method ( 1 6 1). The amount of calcium-binding protein in the intestine at any time in response to vitamin D does not correlate well with the calcium transport phenomenon ( 1 6 1). Finally, following 1 ,25-(OH)2D3 administration, intestinal calcium transport in chicks decays to prein jection level while the level of calcium-binding protein remains high ( 1 62). It is evident, therefore, that the relationship between calcium-binding protein and cal cium transport has not been established. Corradino has developed an intestinal organ culture system using embryonic chick intestine (47). Before hatching, chick embryonic intestine does not contain calcium-binding protein. When this intestine is cultured in the presence of vitamin D or its metabolites, calcium-binding protein appears (as measured by immunoas-
647
say), and the ability of intestine to bind calcium increases. In this system vitamin D is effective, although much less so than 25-0H-D3, which is in turn less effective than 1 ,25-(OHhDJ in stimulating the appearance of calcium-binding protein. In this system both actinomycin D and a-aminitin, the RNA polymerase II inhibitor, prevent the response of calcium-binding protein to vitamin D metabolites ( 1 63). There is little doubt that the appearance of calcium-binding protein in this system is an induction phenomenon, but whether it is related to the response of young growing rats or chicks to vitamin D metabolites is unresolved. In this connection, it is known that chick embryo can metabolize vitamin D to 25-0H-D3 and also to 1 ,25-(OHhD3 (1 64), but the intestine prior to hatching does not respond to 1 ,25(OH)2D3; more recent evidence suggests that this tissue lacks a 3.7S receptor for 1 ,25-(OHhD3. Thus the failure to induce calcium-binding protein prior to hatching may result from lack of the receptor machinery for 1 ,25-(OH}zD3. A fluorescent antibody study has suggested that calcium-binding protein is formed in the goblet cells and secreted along the surface of the columnar epithelial tissues ( 1 65). The nuclear fraction of cells contains as much as 80% of administered radioactive 1 ,2S-(OHhD: (22, 23, 166). However, it is not certain that all of this radioactivity is contained within pure nuclei, because methods that yield pure nuclei from other tissues have not been successfully applied to the intestine. Even a method developed specifically for this purpose gives a low yield (20-40%) of pure intestinal nuclei (1 67), and only 30% of the cellular radioactivity can be accounted for in that fraction. It is not clear whether this preparation selects against the desired nuclei, or whether much of the radioactive 1 ,25-(OH)2D3 is present in a fraction that sediments with the nuclei. Chromatin isolated from chicks by the method of Maru shige & Bonner (1 68) contains between 30% and 40% of the total cellular radioac tivity, .while chromatin prepared by the method of Haussler, Myrtle & Norman contains most of the cellular radioactivity (22). However, as originally discussed by Chen, Weber & DeLuca, and more recently by Lawson & Wilson, the latter chroma tin preparation is far from homogeneous and, therefore, the question of chromatin localization of the radioactivity has not been settled (1 66, 1 69, 1 70). If the nuclear location of 1 ,25-(OHhD3 is of functional importance, then 1 ,25(OH)2D3 in the intestine may function in a manner similar to the steroid hormones. It has been suggested that 1,25-(OHhD3 becomes bound to a cytosolic receptor protein, which undergoes change as it enters the nucleus, and the receptor-bound steroid activates the transcription of specific genomes that code for functional proteins. In mammalian species, Haddad and co-workers have studied cytosolic proteins that bind 1 ,25-(OHhD3 ( 1 7 1 , 1 72); a 6S protein that binds both 25-0H-D3 and 1 ,25-(OH)2D3 has been detected. Binding constants for these proteins were reported, but since crude mixtures were used with charcoal-coated dextran to clear the unbound sterol, there is considerable question regarding their meaning. It is clear that the 6S protein has a higher affinity for 25-0H-DJ than for 1 ,25(OH)2D3, but its function is not clear; it does not bear the characteristics of a receptor protein inasmuch as it is found in both target and nontarget tissues ( 1 72) and does not preferentially bind 1 ,25-(OHhD3 Brumbaugh & Haussler have studied
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a cytosolic and nuclear receptor protein from chick intestine ( 1 73-1 75) and report the existence of a 3.0--3. 5S protein that binds specifically 1 ,25-(OHhD3. This protein has characteristics indicative of a receptor protein, namely high affinity and low capacity for 1 ,25-(OH)zD3' It is found to a much lesser degree in nontarget tissues, and, most importantly, under conditions of incubation with nuclei or chromatin, radioactive 1 ,25-(OH)2D3 is transferred in a rather specific manner to that fraction. They consider these properties as indicative that 1 ,25-(OH)2D3 functions by induc ing the transcription of genetic information, which in turn codes for the calcium transport system. Occurrence of a 3.7S protein in chicks has been confirmed in our laboratory (B. Kream & H. F. DeLuca, unpublished results) and by Lawson & Wilson ( 1 69). However, this protein is not found in the intestines of rats and mice, which also respond to 1 ,25-(OHhD3 In fact, no protein other than the 6S protein has yet been found that binds 1 ,25-(OHhD3 in the intestinal cytosol of mammalian species. Brumbaugh et al have shown that their "chromatin binding" method can be used as an assay for 1 ,25-(OHhD3 and have successfully applied it in the study of blood levels of this important metabolite in a variety of diseases ( 1 76). It seems clear that 1 ,25-(OH)2D3 must be involved in some induction process, but whether or not this is an induction of a calcium transport substance remains to be established. Lawson & Emtage have studied the formation of calcium-binding pro tein in cell-free preparations from vitamin D-deficient chick intestine and from chicks given vitamin D ( 1 77). Polysomes from chicks given vitamin D can form a substance that is immunologically similar to calcium-binding protein, whereas prep arations from vitamin D--deficient controls do not. Although evidence for a specific messenger for the calcium-binding protein that is formed in response to 1 ,25 (OH)zD3 is still incomplete, there seems little doubt that calcium-binding protein is assembled in direct or indirect response to 1 , 25-(OH)2D3 and that readout of messenger RNA must be involved. How 1 ,25-(OHhD3 initiates this phenomenon and, indeed, the mechanism of action of 1 ,25-(OH)2DJ in the calcium transport process are still not known. This area of investigation is an extremely fertile one, and will undoubtedly yield important new results in the near future. It is unknown how calcium traverses the columnar epithelial cell during the transfer process. It is likely bound to the mitochondria, which act as a buffer controlling the level of ionized calcium in the cell ( 1 78). The calcium is probably released at the basement membrane surface; this release requires the presence of sodium ions ( 1 79, 1 80). Exchange of sodium for calcium may bring about the final expulsion of calcium from the columnar epithelial cells, causing the mitochondria to release their calcium. It is known that 1 ,25-(OH)2DJ also activates the transfer of inorganic phosphate across intestinal ileum and jejunum (4 1 , 1 8 1-1 83). This process is independent of calcium transport and thus represents an entirely different function of the vitamin; 1 ,2S-(OH)zD3 rather than 25-0H-D3 appears to function in this process (4 1). It has not been settled whether phosphate transfer occurs by a diffusion process, a carrier mediated process, or an active transport process. No phosphate-binding protein has yet been found.
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The Role of 1,25-(OHJ;D3 in Bone
It is well known that vitamin D administration alleviates rickets and osteomalacia by stimulating the mineralization of the organic matrix of bone. Evidence to support the suggestion that vitamin D participates directly in the process of mineralization is lacking (4), but this possibility must be left open until it can be tested thoroughly. The major defect in the mineralization of bone in vitamin D deficiency appears to be a lack of supply of calcium and phosphorus to the mineralization centers (4). Vitamin D functions in the process of calcium mobilization from previously formed bone, making calcium available to the extracellular fluid upon demand by the calcium homeostatic system as described in the section on the regulation of vitamin D metabolism. This process has been studied in vivo in animals (1 84) and in organ culture (44, 45, 1 85). From the experiments described earlier (see the section on metabolism) it is clear that 1 ,25-(OHhD) rather than 2S-0H-D) or vitamin D). functions in the mobilization of calcium from bone under physiologic conditions, but how this process is initiated is not understood. In contrast to the response of intestinal calcium transport to 1 . 2S-(OH)2D3' mobilization of calcium from bone is blocked by the prior administration of actinomycin D, suggesting that in fact a transcriptive event is involved in this activation ( 1 84). In vivo this process requires the presence of parathyroid hormone ( 1 86), whereas in organ cultures 1 ,2S-(OH}zD3 functions without addition of the parathyroid hormone. The discrep ancy between the in vitro and the in vivo systems has not yet been explained.
The Function of 1,25-(OHJ;D3 in Other Tissues
Gran suggested in early work that vitamin D increases renal tubular reabsorption of calcium ( 1 87) and retention of calcium by kidney. However, in these experiments the renal physiological functions were not rigorously controlled. More recent experi ments show that 1 ,2S-(OH)2D3 does improve renal reabsorption of calcium ( 1 88). However, since 99% of the filtered calcium is reabsorbed even in the absence of vitamin D. this influence on renal reabsorption of calcium may not be very impor tant quantitatively. In any case it appears that 1 .25-(OH)2D3 does influence renal function. In agreement with this. a calcium-binding protein in the kidney that is depressed in vitamin D deficiency and is increased upon administration of vitamin D has been demonstrated (1 89). Again the mechanism of 1 ,25-(OHhD3 function in the kidney is not known. Some attention has been focused on a possible role of vitamin D in renal reabsorp tion of phosphate. Although early work suggested an increased renal reabsorption of phosphate ( 1 90), this appeared to be the result of an adjustment of the parathyroid glands and not a direct effect on renal reabsorption of phosphate. More recent experiments utilizing the metabolites of vitamin D are also not convincing, since vitamin D-deficient animals were not used ( 1 9 1 , 192), and thus pharmacological effects of the metabolites were studied. Other recent reports with rats suggest, but do not prove, an effect of vitamin D on the reabsorption of phosphate (1 93). Vitamin D increases serum phosphate levels of rachitic rats. which is in tum responsible for the mineralization of bone ( 1 94). The source of the phosphate has
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been examined in rats on extremely low phosphorus diets so that intestinal absorp tion would be a minor factor ( 1 94). The response to 1,25-(OH)2D3 was the same in these animals as in those on a 0. 1 % phosphorus diet. Increased renal reabsorption of phosphate was excluded by direct examination ( 1 88). The animals avidly reabsorb all of the phosphorus presented to the kidney even without vitamin D. The source of this phosphate was proved to be bone by 4SCa and 32p experiments (195). Para thyroidectomy did not prevent the response (194). Thus in hypophosphatemic animals, 1 ,25-(OH)2D3 can mobilize calcium and phosphorus from bone without parathyroid hormone ( 195).
Summar yo f the Mechanism o f Action o fV itamin D and Its Metabolites
Much work has been expended in this area, but so far a clear mechanism for the function of vitamin D remains undetermined. Most work has been carried out in intestine in which the major subcellular localization of 1,25-(OH)2D3 is found in the nuclear-debris fraction. A calcium-binding protein appears following the adminis tration of vitamin D and its metabolites that may play a role in calcium transport. In this transport process, which makes its appearance following vitamin D adminis tration, calcium is actively transported against an electrical and concentration gradi ent. The primary site of vitamin D function is at the brush border membrane, and evidence for its functioning elsewhere is equivocal. In addition to calcium transport, 1 ,25-(OH)zD3 stimulates intestinal phosphate transport, mobilization of calcium from bone, and renal reabsorption of calcium. In addition. some form of vitamin D increases muscle strength by an as-yet-undetermined process. Intensive biochemi cal investigations are required to elucidate the cellular and molecular mechanism of action of this vitamin-derived hormone.
CHEMISTRY OF VITAMIN D METABOLITES Metabolite Characterization and Quantitation
The extremely low concentrations of vitamin metabolites in animal tissues severely restrict the physical and chemical techniques that can be applied to their structural characterization. The amount of pure material that can be obtained is, in practice, about 2-1 0 J.Lg. At this level mass spectrometry and ultraviolet spectrophotometry are the only techniques that can be applied with confidence to the elucidation of structures. Fortunately vitamin D and its metabolites exhibit both a fairly character istic UV absorption (defining the cis triene chromophore) and relatively simple mass spectra, with only few, but diagnostically very useful, sets of ionic fragments, namely, (a) a group of peaks comprising the molecular ion and peaks due to loss of simple substituents (e.g. M-H20, M-CH3' or combinations of both); (b) a group of peaks corresponding to the loss of the side chain followed by further elimination of H20; and (c) a set of ions representing the ring A moiety plus carbons 6 and 7 of the triene system. From these peaks gross structural modifications (i.e. introduc tion of OH-functions) in either the side chain or ring A (or by difference in rings C and D of the molecule) can be determined. Complete structural analysis requires the mass spectral analysis of at least several derivatives (e.g. trimethylsilyl ethers.
65 1
acetates) and, where possible and appropriate, confirmation by other chemical transformations (e.g. periodate cleavage, reduction, or ozonolysis), which can be carried out on the submicrogram scale. This approach has been used successfully and has proved adequate for the identification of metabolites encountered thus far. The characterization (3 1 , 32) of l a-25-(OH)2D3 is a useful example: the pure metabolite exhibits UV absorption with Am 265 nm and AmiD 228 nm, from which the presence of a cis triene chromophore can be deduced. The mass spectrum, exhibiting a molecular ion at mle 4 1 6, suggested a dihydroxyvitamin D3 structure and, from the typical fragmentation pattern, one of the additional hydroxy groups could be assigned to the side chain and one to ring A of the vitamin. Mass spectra of several derivatives, including fully and partially trimethylsilylated metabolite. conclusively established the presence of three hydroxy functions and localized one of them at C-25 of the side chain. The biosynthetic origin of the metabolite de manded the presence of a C-3-hydroxyl group. but proof of a 1 a-hydroxy substitu ent required further chemical transformations. i.e. a periodate test to establish that the ring A hydroxyls were not vicinal. and catalytic reduction of the compound. which resulted in hydrogenolysis of the C- I -OH substituent. These data lead to an unambiguous gross structure. although proof of stereochemistry at C-l had to await chemical synthesis. The chief experimental problem in characterization of the vitamin D metabolite is usually the isolation of material in a sufficiently pure state to avoid ambiguities and errors in the interpretation of mass spectral data (55). Although it is often easy to recognize non-vitamin contributions to a mass spectrum. there is a danger of interpreting peaks due to impurities in terms of the vitamin metabolite thought to be present (35). and simplification of the pattern by subtraction of presumed non vitamin contributions ( 196) is really safe only if the pattern for pure metabolite is already known or can be predicted with confidence. Nuclear magnetic resonance spectroscopy has been used only for the identifica tion of 25-0H-D3 and 25-0H-D2 0 7. 76). Rapid advances in design and methodol ogy should eventually lead to the routine application of this technique for metabolite characterization in the microgram scale. Recent proton magnetic resonance studies of vitamin Dz vitamin D3 l a.25-(OHhD3 and several structural analogs (1 97199), using high-field instruments. have established that ring A of the vitamins and of the 1 a-hydroxylated derivatives exists in solution as an equilibrium mixture of roughly equal populations of two chair conformers. Since the discovery of the vitamin D metabolites, the search for reliable. sensitive, and convenient analytical methods for their quantitation in animal fluids and tissues has been pursued vigorously. Efforts have concentrated in particular on the develop ment of competitive protein-binding assays. and considerable progress has been made in this area. Useful competitive protein-binding assays for vitamin D3 and 25-0H-D3 using either protein from rat serum (200) or protein from kidney cytosol (20 1 202), have been described. The general topic of blood and tissue binding proteins for the D vitamins and their metabolites and their application to vitamin metabolite assays have been reviewed by Edelstein (203). A more recent develop ment is the successful application of a competitive protein-binding assay for the
,
652
DELUCA & SCHNOES
quantitation of 1 a,25-(OH}ZD3 ( 1 76, 204), using a reconstituted cytosol-chromatin receptor system from chick intestinal 'mucosa. This method appears to be quite sensitive [detection limit of about 20 pg of I a,25-(OH)2D3] and specific for I a, 25-(OH)2D3, but perhaps somewhat laborious as four column-chromatographic steps are required. 25-0H-DJ at lOOO -fold higher concentration competes effectively for the protein-binding sites, requiring its removal by chromatography. The method has been applied to the quantitation of l a-,25-(OH)2D3 in human plasma. For normal individuals an average level of '" 4.0 ng/ I OO ml was determined, whereas patients with kidney disease give values ranging from undetectable to '" 1 . 5 ng/ I OO m ! . A successful radioimmunoassay for the D metabolites has not yet been devel oped, and newer physicochemical techniques have not been explored sufficiently to allow evaluation of their potential for metabolite analysis. Recently a binding assay using chick intestinal cytosol only has been developed. This assay has improved sensitivity and is simpler to perform. Normal values with this assay are 2.9 ng/I OO m l 0 . 2 (1. A. Eisman and H. F . DeLuca, i n preparation).
S ynthesis 0 / Metabolites
The isolation and characterization of active vitamin D metabolites has been followed by a dramatic resurgence of interest in the synthetic chemistry of the D vitamins. The need to confirm assigned structures, especially stereochemical details, and the need for larger quantities of material than could be provided from natural sources, were the chief factors prompting both academic and pharmaceutical laboratories to initiate synthetic programs. The chemical work, providing vitamin metabolites and analogs in experimentally useful amounts, has in turn led to more rapid advances in the biochemical and biomedical research areas, and has made it possible to bridge the gap between laboratory discovery and clinical application in a remarkably short time. A detailed discussion of the chemical aspects of the published synthesis would go far beyond the scope of a review in this series. We restrict ourselves here to a summary of the compounds that have been prepared. With one exception (205), all published syntheses of vitamin metabolites (or their analogs) are based on the photochemical conversion of the appropriately hydroxy-substituted provitamin-a steroidal 5,7-diene intermediate-to the previtamin derivative, and the thermal isomerization of the latter to the desired vitamin metabolite or analog. The chief synthetic problem then involves the introduction of the required hydroxy sub stituent(s) into a preformed steroid skeleton. This problem has been solved in a number of ways, with the result that currently all known vitamin D3 metabolites have been prepared. In the preceding discussion of vitamin metabolism, several synthetic metabolites have been mentioned. The listing below is, therefore, a recapitulation of the compounds prepared in the vitamin D3 series: 25-0H-D3 (206-2 1 2); I a,25-(OH}zD3 (2 1 3-2 1 7); 24t,25-dihydroxyvitamin D3, 24,25-(OH)2D3 (57, 2 1 8, 2 1 9); (24R) and (24S)-24,25-(OH)2D3 (58, 59); 25c,26-dihydroxyvitamin D3, 25,26-(OH)2D3 (74, 75, 2 1 8); and (24R) and (24S)-1 ,24,25-(OH)3D3 (220; M. R. Uskokovic, personal communication). The references cited include in each case procedures for the preparation of the vitamin metabolite and of the appropriately hydroxy-substituted cholesterol precur-
653
sors, which can be converted to the respective vitamin metabolites by well-estab lished methods. We note once more that synthetic (24R)-24,25-(OH)2D3 is iden tical with the natural metabolite (60) (see Figure I); its 24-epimer, (24S)-24,25(OH)2D3' is not a natural product. Since 1 ,24,25-(OHhD3 arises biosynthetically both by C-24 hydroxylation of l a,25-(OH)2D3 and by Co l hydroxylation of natural 24,25-(OH)2D) (i.e. the 24R-epimer), it follows that natural l,24,25-(OH)D) should be represented as (24R)-la,24,25-(OH)D3 as shown in Figure 1. With the exception of 25,26-(OH)2D3' all vitamin D3 metabolites are, therefore, completely defined structurally. Syntheses of 25,26-(OHhD3 have thus far yielded mixtures of C-25 epimers, from which the pure 25R and 25S isomers have not yet been obtained. In the vitamin D2 series there has been relatively little synthetic activity. Thus far only 25-0H-Dz and its 24-epimer, 25-0H-24-epiD2, have been prepared syn thetically (J. A. Campbell, personal communication). The more recent demonstra tion of other metabolites, i.e. 1 ,25-(OH)2D2 (77), 24,25-(OH)2D2' and 24-0H-D2 (G. Jones, H. K. Schnoes & H. F. DeLuca, unpublished results), is likely to lead to further intensive efforts in this area.
Structural Analogs 0 / the V itamin D Metabolites
The requirement for metabolic "activation" of vitamin D3 by a two-step hydroxyla tion sequence suggests a logical explanation for the observation that certain simple analogs of vitamin D3, prepared many years ago, exhibited either no (e.g. 3-bromo, 3-chloro, 3-thio derivatives and others) or very little (e.g. 3-epivitamin D3) antirachitic activity (for summaries see 221-223). It also raises the question of the relative functional importance of distinct structural elements of the vitamin D molecule, in particular of the functional significance of the hydroxy groups introduced during metabolism. Since the vitamin acts in at least two distinct organs (intestine and bone) and promotes the independent intestinal transport of both calcium and phos phate, these diverse functions might be differentially sensitive to structural modifica tions. The general problem of structure/activity relationships in the vitamin D series has been approached by the synthesis of various structural analogs. This work has contributed some important insights into the structural units essential for activity and has also furnished synthetic derivatives of very promising clinical potential. We discuss here only those compounds on which relatively extensive biological data are available. Table I offers a summary of compounds prepared to date, and includes references to purely chemical work designed to furnish the appropriately modified steroid skeleton from which the respective vitamin analog can be derived by known methods. Figure 3 presents the structures of analogs discussed in the text. The biological activity of some recently prepared members of this series, e.g. (24R)- and (24S)-24-0H-D3 and (24S)- 24,25(OH)zD3 (see Table I and Figure 3), have already been discussed in connection with the naturally occurring metabolite (24R)-24,25-(OH)2D3' Other simple side-chain analogs include 27-nor-25-0H-D3 and 26,27-bisnor-25-0H-D3 (cf Figure 3) (225). These compounds retain partial, though quite low, activity (compared to 25-0H-D3) as measured by in vivo assays of calcium transport and bone calcium mobilization, but exhibit little antirachitic activity. Bontekoe et al (226) have prepared the same
SIDE-CHAIN ANALOGS OF 25-0H-D,
654
Table 1
DELUCA & SCHNOES Synthetic analogs of vitamin D metabolites

la,25-(Ofl ) 2 D J A nalogs
25-0H-D3 Analogs 24S,25-(O H ) 2 D 3 24R-OH-D3 (65 , 2 2 4 ) 24S-0H-D3 (65 , 2 24 ) 27-nor-25-0H-D3
Side-chain modified
1a-OH-D3 (205, 1 a-OB-D 2 (25 2 ) la-oH-pe ( 2 5 3 ) 24-nor- la,25-(OH ) 2 D 3 1<>,24R-(OH)2D3 (224, 254) 1<>,24S-(OH ) 2 D 3 ( 2 24 , 254 ) la,24S,25-(OH)3D3 (220)
Ring A-modified
Ring A - and side-chain 3-deoxy- 1 a-OIl-D 3 (255-257) 1a-oH-D3 3-mcthy1cthcr (2 2 3 ) la-OH-3-cpiD3 ( 2 6 4 ) 4a-OH-D3 (265 ) 2<>-OB-D3 (266) 5 ,6-tran,-1) 3 (49, 26 2 ) D I I T 3 ( 2 5 9 , 260)
modified
(5 8, 59)
228-233)
3-dcoxy- 1 a , 25 -(QfI )2 D3 (25 1 ) 25-0H-5 ,6-lrall,-D3 (49 , 5 9 ) 25-0H-DHT 3 ( 26 1 )
( 2 25 , 2 2 6) 26,2 7-bisnor-25-0H-D3 ( 2 2 5 , 2 2 6) 2U,25-(OH)21)3 (226) 27-nor-20,25-(OH)2 D 3 (226) 22S-0H-D 20-0H-PC (225) 4
(223)
(227)
compounds, as well as several others (see Table I ), and found all of them devoid of antirachitic potency_ Since the efficacy of these analogs depends on prior C- I hydroxylation (225), their low activity i n vivo is not unexpected and can be rational ized as a manifestation of poor affinity for (a) the l a-hydroxylase, (b) transport proteins, or (c) the target tissue receptor proteins_ Accordingly, drastic alteration of side-chain structure gives compounds [e_g_ 20-hydroxypregnacalciferol (225)] exhibiting no activity in vivo_
SIDE-CHAIN-MODIFIED DERIVATIVES OF 1 a,25-(OH),D,
la -H ydroxyvitamin D3 The first close analog of l a,25-(OH)2D3 to be prepared (228) may also prove to be the one of greatest practical utility_ The compound is accessible conveniently from cholesterol (228-233) and has also been prepared by total synthesis (205). It is distinguished by a biological potency approaching that of l a,25-(OH)2D3 (228, 234), a fact that has prompted its extensive biochemical study (43, 235, 236), It has already been exploited for clinical application (237-239). Extensive bioassay results obtained for the rat, measuring bone calcification, mainte nance of serum calcium, and stimulation of calcium absorption, indicate that on a weight basis the material has about 50% of the potency of l a,25-(OH)2DJ (235). Studies with hypoparathyroid patients gave a similar estimate of potency (240). Of practical interest is the fact that in the rat, the biopotency of l a -OHDJ is unaffected by route of administration. Oral or intraperitoneal doses give very similar activity patterns, a finding that differs strikingly from earlier experiences with I a, 2S-(OH)2DJ' which is much less effective when given orally in rats (24 1)_ In chicks (234, 237), the efficacy of l a -OH-D3 in stimulating intestinal calcium absorption, in mobilizing bone mineral, and in calcifying bone is about equal to that of I a, 2S (OH)2D 3 which may indicate a species difference in the utilization of this analog_ More recently Toffolon, Pechet & Isselbacher have reported (242) intravenous administration of I a-OH-DJ to rachitic rats to produce extremely rapid onset of a biological response. Peak activity for intestinal calcium transport was observed 1 hr
'
655
after administration of the analog [or of l a,25-(OHhDJ1, a result that is difficult to reconcile with all previous data (228, 236, 239), which rather consistently show maximal stimulation of intestinal calcium transport (in either rat or chick) between 6 and 1 2 hr. Based on their results, Toffolin, Pechet & Isselbacher have suggested that l a-OH-DJ might act directly on mucosal membranes rather than via new RNA or protein synthesis (242), and that the compound might be active without undergo ing further metabolism to the natural hormone, 1 a,25-(OH)2DJ. This latter pro posal is weakened by the recent unambiguous demonstration that l a -OH-D, is indeed very rapidly metabolized to l a,25-(OH)2DJ (see below). Before the sugges tion of a direct membrane action of 1 a-OH-DJ or 1 a,25-(OH)2DJ can be entertained seriously, additional confirmatory experiments demonstrating their rapid action are essential. The first studies (228, 234) on the biological activity of 1 a-OH-DJ suggested that its action might require prior metabolism to 1 a,25-(OH)2DJ, and more recently Zerwekh et al (236) obtained evidence based on a chromatin receptor assay to support this possibility. C-2S Hydroxylation of 1 a-OH-DJ. which based on the known transformation of dihydrotachysterolJ to 25-hydroxydihydrotachysterolJ
,' ' .or ,('

OH
""
R' H : OHT
R = H ; 5,6- trons - 0
R H : l a .O H O
l a OH0
R OH : 25.0HDHT ,
R ' O H : 5,6. t r a n s . 2 50HD3
R ' C H : l a O H D, 3
3 - methy\ ether
,,' I
H : ( 245) .24, 2 5 . (OH)2 D
O H : ( 2 4 5 ) . la , 24 , 2 5 (OH ) D , ,
r,e r r' t
I , "" I I "" I I
""
, R .OH.R2 H : ( 24R).24 .0H.D , 3 R ' C H ' 27nor.25.0H.D , , la O H - PC R .H, R . OH : ( 24S) 240H - D
'
R H : 26 , 27 Bisnor-25.0H.D
I H : 3-deox y . l a . OH-D , OH : 3 . deoxY l a , 25.(OH) D 2 ,
R . H : l a . O H . 3 - epi D ,
R . OH " a , 25.(OH) 3 - epi D , 2
2 4 - nor . l a , 2 5 . ( O H)
D,
Figure 3
Vitamin D analogs.
656
DELUCA & SCHNOES
(243) and of 5.6- trans-vitamin 0) to 25-hydroxy-5.6- trans-vitamin 0) (244) cer. tainly appeared quite probable a priori. has now been demonstrated directly with tritiated I a-OH-03 (245-247). Within 2 hr after intravenous administration of l a 3 OH-[6- H]OJ to rats or chicks. tritiated I a, 25-(OH)20J appears in intestine and bone. and the appearance of the metabolite precedes the biological response by at least 2 hr. Although these data do not preclude a direct action by I a-OH-D3 (particularly at high levels). detailed studies of the time course of accumulation of l a .25-(OH)z-[6-3 H]D3 in the target tissues and the onset of a biological response after a dose of I a-OH-[6-3 H]D3 suggest strongly that the activity of I a-OH-D3 depends on prior hydroxylation at C-25. These studies have also shown that the route of administration of l a-OH-D3 affects its tissue distribution very markedly. 3 In the rat. both intestine and bone accumulate l a-OH-[6- H]03 and the metabolite 3 1 a.25-(OHh-]6- H]03 equally well after an intravenous dose of I a-OH-[6-3 H]D3 but after an oral dose much less of l a-OH-03 and l a.25-(OHh03 appears in blood and bone than in intestine (246). In the chick a similar distribution pattern depend ing on route of administration has been observed (247). There is also an interesting species difference in the metabolism of l a-OH-03. In the chick. homogenates of both liver and intestine convert I a-OH-D3 to 1 .25-(OHhD3. while in the rat the reaction can be demonstrated with liver but not with intestinal preparations (247). This observation might offer a partial explanation for the apparently greater efficacy of l a-OH-D3 in the chicken. l a-OH-D3 has also been tested extensively in two in vitro systems: the bone resorption assay (59. 248. 249) and the cytosol/chromatin receptor assay ( 1 75. 236. 250. 25 1 ). The former assay shows the compound to be about 2-3 orders of magni tude less potent than I a.25-(ORhD3 (52). and about equal to 25-0H-D3. a value that parallels the data from competition experiments using the intestinal receptor preparation where I a-OH-O) binds at least 500-fold less well than the natural hormone (2 36). It is clear from these qata that at physiologic dose levels. a 25-0H group is an essential functional parameter. Given at sufficiently high doses [i.e. WOO-fold greater abundance than l a.25-(OHh03] the analog will. of course. com pete effectively for intestinal receptor sites and thus induce the appropriate response. At physiologic dose levels, however. there is little doubt that 25-hydroxylation is an obligatory step.
Other Side-Chain Analogs I a-OH-02, prepared and tested recently (252). exhibits an activity pattern in the rat essentially indistinguishable from l a-OR-D3. Al though not yet demonstrated, this compound. like l a-OH-D3 undoubtedly un dergoes hydroxylation to 1 a.25-(OHhOz (77) in vivo. If the 25-hydroxy function represents an important element as far as expression of biological activity is concerned. then any alteration of the side chain that would prevent 25-hydroxylation should result in inactive (or poorly active) analogs. This has been confirmed by preparation of two analogs, I a-hydroxypregnacalciferol (1 a-OH-PC) and 24-nor- 1 a,25-(OH)203' The former is inactive when tested in vivo even at relatively high dose levels (6.25 nmoles), but the compound elicits a response in the in vitro bone resorption system, although again only at high concentrations
657
( 1Q-6M) (253). Presumably this analog would also exhibit some affinity for the intestinal receptor, and one may regard I a-OH-PC, therefore, as a vitamin of modest intrinsic functional capacity, which remains unexpressed in vivo (at least at the doses tested) as a result of other factors (impaired transport, more rapid excre tion, etc) that prevent target tissue accumulation to the relatively high levels re quired. Likewise, 24-nor- l a,25-(OHhDJ exhibits no activity in vivo, but elicits a fairly pronounced bone resorption response in vitro (H.- Y. Lam, H. K. Schnoes, P. H. Stern, A. F. Chen & H. F. DeLuca, unpublished). In Table 1 several other side-chain analogs are listed as having been synthesized: (24R)- and (24S)- 1 a,24-(OHhD3 (254) and (24S)- 1 a,24,25-(OHhD3, the C-24 epimer of the naturally occurring metabolite (220; M. R. Uskokovic, personal communication), but no conclusive biological data are available for these com pounds at present.
ANALOGS MODIFIED IN RING A The activity pattern of the natural vitamin metabolites and of analogs mentioned thus far provides clear evidence for the crucial importance of the C- I hydroxy group for activity at physiological levels. The relative significance of the C-3 hydroxy group has been assessed by the preparation of three analogs: 3-deoxy- I a-OH-DJ (255-257), l a-OH-DJ 3-methyl ether (223), and 3deoxy- I a,25-(OH)2D3 (25 1). All three analogs are active in vivo; in both rats (255) and chicks (256, 258), a pronounced stimulation of intestinal calcium transport is observed, with 3-deoxy- 1 a-OH-D) or I a-OH-D) methyl ether, although their effect on in vivo liberation of bone calcium in the rat is relatively modest. 3-Deoxy- 1 a OH-D3 elicits no response in the in vitro assay for bone resorption (249). The 1 ,25-dihydroxy analog, 3-deoxy- l a, 25-(OHhD), has been described as "at least as active as I a ,25-(OH)2D/' (25 1 ) in stimulating intestinal calcium transport in the chick, but it is difficult to find support for this view among the available experimental data. At the very high (and probably saturating) dose level of 6.5 nmo\es, 3-deoxy1 a,25-(OH)2D) indeed elicits an intestinal calcium transport response approaching that of I a,25-(OHhD), but considerable differences in potency are clearly evident (25 1 ) at lower doses. The compound is, however, the most effective analog yet known in competing with 1 a,2S-(OH)2D] for the cytosol/chromatin receptor sites, demonstrating once again the key importance of the I a and 2S-hydroxy groups in determining affinity for the intestinal receptor protein. It can be expected that 3-deoxy- 1 a,2 5-(OHhD) will prove very effective in promoting calcium release from bone in culture, since published data (248-25 1) indicate a fairly similar order of relative potency for the vitamin 0 metabolites and analogs in these two in vitro systems (cytOSOl/chromatin receptor and bone resorption assays). In view of the metabolism of I a-OH-D) to I a,25-(OH)2D), the speculation that the in vivo efficacy of 3-deoxyl- l a-OH-D3 and the 3-methyl ether derivative of l a-OH-D3 depends on prior conversion to the corresponding 25-0H analog appears warranted. Under the category of l a,25-(OH)2D) analogs, Table 1 also lists several more distantly related compounds: dihydrotachysterol 3 , 25-hydroxydihydrotachysteroh, 5,6- trans-vitamin D) (S,6- trans-DJ), and 5,6- trans-2S-0H-DJ. Since these biologi cally active analogs have been discussed fairly thoroughly in earlier reviews (4, 1 3,
658
DELUCA & SCHNOES
223), we forego here another summary of their biological properties. Their biological activity (in normal as well as nephrectomized animals) is explained by the presence of a hydroxy group at a pseudo Co l position (see Figure 3); in both the 5,6- trans and the dihydrotachysterol series the 25-hydroxy analog is the more potent com pound in agreement with the results presented in the preceding discussion, which identify the Co l and C-25-hydroxy functions as key parameters for expression of biological activity. It is highly likely that the in vivo activity of dihydrotachyste rol3 and 5,6- trans-D3 depends on prior metabolism to the respective 25-0H deriva tives, a conversion that has been demonstrated for both compounds (243, 244). The fact that neither compound is capable of calcium mobilization from bone in culture, whereas both 25-hydroxydihydrotachysterolJ and 5,6- trans-25-0H-DJ are active in this assay (59, 248, 263), is in accord with this assumption. Since both 3-deoxy- 1 a-OH-D3 and 3-deoxy- 1 a,25-(OH)2D3 have now been synthesized, detailed activity comparisons in vivo and in vitro (especially receptor binding and calcium release from bone) of compounds of the series 3-deoxy- 1 a-OH-D3, 5,6- trans-D3, dihydrota chysteroh and 3-deoxy- l a,25-(OHhD3, 25-0H-5,6- trans-D) and 25-hydroxydihy drotachysterol3' should yield necessary data for an assessment of the relative functional importance of the triene chromophore, which as yet has received little attention (compare structures in Figure 3). The effect of ring A hydroxy stereochemistry on biological efficacy also deserves additional investigation. Preparation of the C-3-epimer of l a-OH-DJ ( l a-OH-3epiDJ) has been announced (264), but no biological data are available. The C-3epimer of I a,25-(OH)2D) (l a,25-(OH}z-3-epiD3) was prepared a few years ago in connection with the first synthesis of l a,25-(OHhD) (2 1 3); it is far less potent than the natural hormone. The biological results on analogs synthesized thus far allow a few important generaliza tions: apparently all three hydroxy groups of I a,25-(OH)2DJ contribute to its biological potency and to ligand-receptor affinity. However, those at Co l and C-25 are most important for expression of activity in vivo and, based on a qualitative evaluation of results from competitive receptor-binding assays, both make about an equal contribution to the overall affinity of the molecule for the receptors. The assay data for in vitro bone resorption are roughly in agreement with this observation. Data from in vitro assay systems (either receptor binding or bone resorption in culture) suggest the following order of increasing activity: D3 < < 3-deoxy-25OH-DJ 3-deoxy- I a-OH-D3 < 25-0H-D) l a-OH-D) < 3-deoxy- l a,25(OH)2D3 < l a,25-(OH)2D3' In an in vivo system this order will not be maintained, of course, principally because of metabolic transformation. For example, although 3-deoxy- l a,25-(OHhD3 is a more active analog than 3-deoxy-l a-OH-D3 in any of the in vitro systems, both might be roughly equally potent in vivo, because of the likelihood of metabolic conversion of 3-deoxy- l a-OH-DJ to 3-deoxy- l a,25-(OH)2 D3 Similarly the known transformation of 1 a-OH-D3 to 1 a,25-(OH)2D3 is likely ' to make l a-OH-D3 the more effective analog in vivo compared with 3-deoxy- l a , 25-(OHhD3, which is the more potent compound as measured by in vitro assays
SOME GENERALIZATIONS ON STRUCTURE/ACTIVITY RELATIONSHIPS
659
[an in vivo conversion of 3-deoxy- l a,25-(OHhD3 to l a,25-(OHhD3 appears less likely). These generalizations hold only for those compounds differing in hydroxy substitution of C- I , C-3, and C-25 while retaining the normal vitamin skeleton. Introduction of additional hydroxyl groups [e.g. 24,25- and 25,26-(OH)2D3]' change of stereochemistry (e.g. 24R vs 24S, 3a or 3Jl-OH), and change of other structural parameters [i.e. trans-vitamins, I a-OH-PC, 24-nor- 1 a,25-(OHhD3] would further modulate this basic activity sequence. The equal potency of vitamins of the D3 and D2 series in the rat suggests that, at least in mammals, the exact structure of the hydrocarbon portion of the side chain is of minor biological consequence as long as a 25-0H function (or a site for its introduction in vivo) is available. On the other hand, stereochemistry of the hydroxyl group at C-24 has a notable effect on activity patterns. The observation that the various 24S hydroxy epimers prepared thus far can elicit an intestinal calcium transport response without affecting the bone mineral mobilization process or intestinal phosphate transport represents the first example of a structure-dependent discrimination between vitamin D responsive systems, which, although far from understood in molecular terms, may have interesting and useful practical consequences.
Plant-Derived Factors Simulatinf( Ja,25-(OH hDJ Activity
Within the past few years there has been increasing interest in the nature and biochemical action of various plant-derived factors that produce severe calcinosis, reminiscent of vitamin D-intoxication, in grazing animals. A disease of cattle, characterized by symptoms of wasting, stiffness, and excessive soft tissue calcifica tion and known as "Enteque seco" and "Espichamento" in Argentina and Brazil, respectively, has been shown to be due to ingestion of the leaves of Solanum malacoxylon (267, 268). A disease with similar symptoms reported from the alpine areas of Germany has been traced to the plant Trisetum flavescens (269), and in the United States, the shrub Cestrum diurnum, indigenous to areas of Florida, has been shown to cause calcinosis in animals grazing on it (270). Similar diseases in other parts of the world have been described (268, 270) without implicating a specific plant as the cause. The toxic action of S. malacoxylon has been studied most intensively; the dried leaves of the plant or an aqueous extract of them cause hypercalcemia and hyperphosphatemia in several animal species with consequent soft tissue calcifica tion (27 1-273). These effects are due to increased intestinal calcium absorption (27 1), and the demonstration of a marked stimulation of intestinal calcium transport . in vitamin D-deficient rats after administration of powdered leaf material (274) called attention to the strikingly vitamin D-like activity of the causative factor. The stimulating effect of S. malacoxylon factor on intestinal calcium transport has been demonstrated in a number of laboratories (274-279), but there is no general agree ment on the effect of the factor on bone mineral mobilization in vivo (272, 275, 276, 279-282). The factor stimulates synthesis of the intestinal calcium-binding protein and can overcome strontium inhibition of intestinal calcium transport and calcium binding protein synthesis (277), indicating an activity pattern remarkably like that of I a,25-(OH)2D3' The functional similarity between the unknown factor and I a , 25-(OHhDJ is demonstrated also by the ability of S. malacoxylon extract to stimu-
660
DELUCA & SCHNOES
late intestinal calcium transport in nephrectomized rats, and by its ability to com pete with I a,25-(OHhD3 for the cytosol/chromatin receptor (280). Although in these assays the factor behaves remarkably like I a,25-(OH)2D3' it must differ from the latter chemically since it is water-soluble and insoluble in organic solvents (283). In the absence of a pure preparation of S. malacoxylon factor, which precludes reasonably reliable quantitative evaluations of activity in different assays, and with out reliable chemical information, structural speculations are premature. The active principle of C diurnum exhibits similar biological activity; it stimu lates intestinal calcium absorption and can overcome strontium inhibition of intesti nal calcium-binding protein synthesis and intestinal calcium transport (270). Unlike the S. malacoxylon factor, the activity from C diurnum is soluble in CHCVMeOH mixtures. A number of laboratories are now engaged in the purification and identifi cation of these factors, and some concrete structural information and therefore perhaps some explanation of activity may become available in the near future. These compounds, especially if they are not simple vitamin derivatives, could represent an important addition to the arsenal of synthetic and natural structural variants with which to probe the molecular complexities of the diverse vitamin D-regulated processes.
ACKNOWLEDGMENTS
Some of the original research reported in this contribution was supported by grant no. AM- 1488 1 and contract no. 72-2226 from the National Institutes of Health, contract no. E( 1 I - I )- 1 668 from the US Energy Research and Development Admin istration, and funds from the Wisconsin Alumni Research Foundation.
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Metabolism of Vitamin D

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Metabolism of Vitamin D

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METABOLISM AND MECHANISM OF ACTION

632 633 634

ENZYMES THAT CATALYZE VITAMIN DJ METABOLISM

MECHANISM OF ACTION OF 1.25-(OH),DJ

. .. .................... . ................................... .. ...

.......... ................. .... ................ ..........

Plant-Derived Factors Simulating la.25-(OH)}DJ Activity......................................

650 650 65 2 653 653 654 657 658 659

Abbreviations used are: 2S-0H-D 3. 2S-hydroxyvitamin 03; la.2S-(OH)2D3. 1a.25-dihy

droxyvitamin OJ; 24.25-(OHhDJ 24,25-dihydroxyvitamin OJ; 1.24,25-(OHhDJ, 1,24,25-

DELUCA & SCHNOES

PERSPECTIVES AND SUMMARY

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

() a. ,<:l , l" o *-'

HO" (24R)-24, 25-(OH)2 03 (24

R)-I, 24,25- (OH)3 03

Diagrammatic representation of vitamin D metabolism and its regulatory features.

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

Vitamin Dr25-H ydroxylase

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

2 P450/C0 '" H 0'" 2 3

Components of 25-0H-D)"1 u-hydroxyJase

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

The Role of 1,25-(OHJ;D3 in Bone

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES Synthetic analogs of vitamin D metabolites

25-0H-D3 Analogs 24S,25-(O H ) 2 D 3 24R-OH-D3 (65 , 2 2 4 ) 24S-0H-D3 (65 , 2 24 ) 27-nor-25-0H-D3

3-dcoxy- 1 a , 25 -(QfI )2 D3 (25 1 ) 25-0H-5 ,6-lrall,-D3 (49 , 5 9 ) 25-0H-DHT 3 ( 26 1 )

( 2 25 , 2 2 6) 26,2 7-bisnor-25-0H-D3 ( 2 2 5 , 2 2 6) 2U,25-(OH)21)3 (226) 27-nor-20,25-(OH)2 D 3 (226) 22S-0H-D 20-0H-PC (225) 4

VITAMIN D METABOLISM AND MECHANISM OF ACTION

,' ' .or ,('

R ' O H : 5,6. t r a n s . 2 50HD3

H : ( 245) .24, 2 5 . (OH)2 D

I H : 3-deox y . l a . OH-D , OH : 3 . deoxY l a , 25.(OH) D 2 ,

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

VITAMIN D METABOLISM AND MECHANISM OF ACTION

DELUCA & SCHNOES

Schenck, F. 1 937. Naturwissenscha ften

\3. 14. 1 5. 1 6. 17. 1 8.

VITAMIN D METABOLISM AND MECHANISM OF ACTION

24. Ponchon, G., DeLuca, H. F. 1 969. Nutr. 99: 1 57-67

25. Suda, T., DeLuca, H. F., Hallick, R. B.