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Regulation of vitamin D metabolism
Life Sciences, Vol . 17, pp . 1351-1358 Printed in D.S .A .
Pargamon Presa
MINIRE91~99 REGDLATION OF VITAMIN D METABOLISM Hector F . DeLuca Department of Biochemistry, College of Agricultural sad Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
Vitamin D can be regarded as a prohormone and its moat potent metabolite, 1,25-dihydroxyvitamin D3, a hormone xhich mobilizes calcite and phosphate from bone and intestine. Ia true horswaal fashion, the biosynthesis of 1,25-dihydroryvitamin D bq kidney mitochondria is feed-back regulated by serum calcium an~ serum phoephorw .levels . The lack of calcium brings about a secretion of parathyroid hormone xhich stimulates 1,25-dihydroayvitamin D3 ayatheaie xhile lox blood phosphorus stimulates 1,25-dihydroxyvitamia D ayathesie even in the absence of the parathyroid glands . For such regulation to occur, vitamin D moat be present probably because 1,25-dihydrozyvitamia D3 it~self is needed for the regulation . The molecular and cellular mechaniss~s xhereby 1,25-dihydroxyvitasiin Dg synthesis is regulated era uakaoxn despite many recent reports . Likely the elucidation of thpe nschaaisms must axait a detailed investigation of the enzymology of the seal 25-hydroryvitamin D -la-hydrozylase . In addition to the regulation at the 25-hydro:~vitasin D -larhydrorylase stop, vitamin D metabolis: is regulated at the he~atie vitamin D-25-hydrozylase level. This regulation is a suppression of the hydrozylase by the hepatic level of 25-hydroxyvitamin D itself by as unkaowa mechanism. Much remains to be leased concerning the regulation of this newly discounted endocrine system but already the findings are not only relevant to calcium homeostasis but also to as understanding of a variety of metabolic bone diseases . There is now little doubt that vitamin D must first be setabolised in the liver to 25-hydroxyvitamin D (25-OH-D ) and subsequently in the kidney to 1,2i-dihydrozyvitsmia D3 (1,~5-(OH)2D3~ before it can carry out its xell known functions on intestine and bone (1) . This concept couplsd xith the recognition that vitamin D is not normally required is the diet xhen sufficient ultraviolet irradiation is incident upon the skin hoe led to the belief that 1,25-(OH) 2D~ must be regarded as a hormone (1-3) . To be considered a hormone it is logical that the biogenesis or secretion must be feed-back regulated by the products of its metabolic efforts . That such is the case has nox been clearlq established at the physiologic level and it will be the put~+ose of this review to bring together those aaliant points establishing this regulation . Reaulation of 2S-Hydroxylstion So far there is no clear demonstration 25-OH-D acts directly is any of the target Accordiâgly serum calcium concentration and not appear to regulate this hydrorylation. 1351
that at physiologic concentrations tissues of vitamin D function . serum phosphate concentration do There is,~hoxever, evidence that
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the hepatic level of 25-OH-D3 does provide some degree of regulation of this initial entry of vitamin D into its metabolic sequence . Earlq efforts demonatrated that it was necessary to increase the dosage of vitamin D to hogs from 100 unite to 250,000 units per day to increase the circulating le~el of 25-OHDq from 1 unit to 12 units of antirachitic activity per milliliter (4) . Thus iL is clear that there is no direct relationship between the circulating levels of the metabolite sad the dosage of vitamin D given. Similar results have bean reported in man bq Haddad and his colleagues (5) . More directly it has been shown in both rata and chicks that the prior administration of vitamin D3 to rachitic or vitamin D-deficient animals will markedly depress hepatic 25hydrozglatioa of vitamin D3 meaaurnd in vitro (6, 7) . Furthermore this depression could bn demonstrated in vivo as wall and could be related to the hepatic level of this metabolite . Ît ie, therefore, apparent that the hepatic level of 25-OH-D in some way suppresses the 25-hydroxylation of vitamin D . Thn amount of vita~in D required for the suppression depends upon the apecie~ iaaamuch as Tucker et al . (B) could find no reduction in the 25-hydroxylase when .25 ug of vitamin 3 was given to chicks whereas Bhattacharyya and DaLuca (7) showed that in thin spacles dosages above .5 ug will bring about a reduction in the 25hydroxylase. Ia the rat, the reduction occurs at dosages of .25 ug or above. It ie of soma interest, hwevar, that the 25-hydroxylation of the Gloss analog of vitamin D3, namely dihydrotachysterol 3 , is not regulated hither by the prior administration of vitamin Dg or by the prior administration of dihydrotachysterol g itself (9) . Additionally it is important to realise that the adminiatration of 25-OH-D intravenously dons not exert a marked inhibition of the 25-hydrozylaee sinca3lt does not readily penetrate the liver (10) . It is uncertain whether the enzyme system which hydrozylatas vitamin D3 in the hepatic microsomes sad the anzyma that hgdrozylatas dihydrotachysterol g and cholesterol is the 25 position am one and the same . Instead thard is evidence to suspect that there is a non-specific 25-hydroxylasa which is sot regulated and which carries out 25-hydroxylation in a non-specific meaner at high substrata concentrations (11) . It moat also be borne in mind that the regulation of the vitamin D -25-hydroxylase is not absolute and largo amounts of vitamin D can overcome his regulation . Howâvar, the 25-hydroxylation stags must be r~gardad as a bottleneck reaction which is regulated at physiologic levels of vitamin D3 .
D
Regulatioa of the iCidnay 25-0H-D,-la-Hgdroxglaae By far the most important known regulation of vitamin D metabolism occurs at the 1-hydroxylation stage. In 1971 the first clear evidence that the 1hydroxylation mechanism is regulated was provided by Boyle et al . (12) . Boyle studied the in vivo accumilation of the 1,25-(OH) 2D3 metabolite in the plasma and target tiasuas following as intravenous done 3f 25-OH-D . They clearly demonstrated that rata on a low calcium diet accumulate lays amounts of 1,25-(OH) 2D in the plasma and target tissues whereas those animale given high calcium dins accumulate very little of this metabolite . In contrast the animals on a high calcium diet accumulated another metabolite which was aubsaquaatly identified ae 24,25-dihgdroxyvitamin D3 (24,25-(OH) 2D~) (14) . Of major importance is the realization that vitamin D-deficient animals did not show this degree of regulation . Vitamin D-deficient animals regardless of dietary treatment accumulatéd large amounts of 1,25-(OH) 2D3 in response to an injected dose of 3H-25-OH-D , illustrating the importance of some form of vitamin D in this regulation phenomenon . In another report, Boyle et al . (13) further demonstrated that the regulation in the rats given vitamin D is related to the resultant serum calcium concentration produced by the dietary manipulation (Figure 1) . SJhen the concentration of calcium in the blood is The normal, equal amounts of 1,25-(OH) 2D and 24,25-(OH) D3 appear in blood . hypocalcemic animals accumulated 1$r~e amounts of 1, 7 25-(OH) 2D3 . On the other
Vol . 17, No . 9
Regulation of Vitamin D Metabolism
W 20 i
FQ w î
135 3
1,25-(OH~ D3
10
N~
W N Z
24,25-(OH)2D3
3
4
!~______ l~ s __~__ __r_ v ~
5 6 7 8 9 SERUM CALCIUM mg/100 r~l
~ 10
FIG. 1 The elationship of serum lcium concentration to the plasma levels of [~)-24,25-(OH) D and ~]-1,25-(OH) D . Bats on a variety of diets for two wea~ i~aceived an injectio~ ~f 25-OH-[26,27- 3H)D and 1Z hours later setabolite levels xere determined in the plasma after estraction a~ chraa~atography (13) .
3
hand the hypercalcemic aaimaL accumulated little or ao 1,25-(OH) D . Boyle et al . (13) inferred from these etudiss that plasma calciun conce~t~ation plays âa important regulatory role in the renal 1-hydroxylation of 25-0H-D either directly or indirectly . Direct demonstration by in vitro measurements that such a regulation of the 1-hydroxylâse had actually taken place vas provided by studies in the chick by Omdahl et _al. working with high and lw calcium diets (15) . These investigators demoastratsd that chicks on a low calcium diet have a high level of 25-OH-D3 la-hydroxylase activity is their isolated mitochoadrie whereas these on a high calcium diet have very little of this enzyse and instead shoo large anouats of 25-OH-D3 24-hydrozylase . Similarly strontium, which ie known to shut down intestinal calcium absorption (16, 17), suppresses ZS-OA-D la-hydroxylase and stimulates the 25-OH-D -24-hydroxylase activities (18, 19)3 There vas, therefore, no doubt that the serum calcium conceatratioa vas e:arting its control on the renal hydroxylase enaymatic activities . These basic observations have recently been confirmed (20) .
3
g
Of great interest is that the administration of exogenous 1,25-(OH) D eliminates the repression of intestinal calcium transport by dietary cal~i~ (21) and strontium (18, 19) . It is, therefore, evidnat that the need for calcium as shown by even slight hypocalcemia in some way briaga about the increased activity of the 25-OH-D -lar-hydrozylase in the kidneys . This results in an iacreaaad produc .tioa of 1,25-(OH) D a hormone whose fuactioa it ie to mobilize calcium from both intestine an3 bone . this position has been largely confirmed in a number of laboratories (20, 22, 23) .
g
2 3,
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Regulation of Vitamin D Metabolism
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The fact that dietary calcium and serum calcium concentration in some way directs the activation of the renal 25-OH-D3-la-hydroxglasa lad to the idea that this regulation might be mediated by tRe parathyroid glands which are knows to secrete parathyroid hormone in response to hypocalcemia (24) . Because thyroparathyroidectamy is very difficult in chicks, experiments warn carried out in the rat and the accumulation of 1,25-(OH) D3 in the plasma and target tissues was meaeurad .~ In vitamin D-deficient ra~s given 1,25-(OH) Dg and a diet low in calcium, there is a predominant accumulation of 1,25-(~H72D 9 in the intestine and serum in response to a single dose of radioactive 25-OH-D g (24) . Following thyroparathyroidectamy there is a progressive diminution of the 3H-1,25-(OH) D3 in these tlsauna from 3H-25-OH-D3 until 48 hours poet surgery when no ~,25-(OH) D3 accumulates in these tissues . Iaatead 24,25(OH) 2D3 appears. These a~imals ware then given an exogenous source of parathyrbid hormone which restores the accumulation of 1,25-(OH) 2Dg in the target tiasuas. Fraser and Kodicek (23) have meseurad directly the 25-OH-D3-lahydrozylase of chick kidney in response to parathyroidectomy and parathyroid hormone injections illustrating that parathyroid hormone is soma unknown way stimulates the activity of the renal 25-OH-D -la-hydroaylase . Erperimenta carried out by Galaate et al . (22), although3of some interest, do not bear on the question of whathar physiological amounts of parathyroid hormone stimulate she production of 1,25-(OH) 2D3 in vivo . Other laboratories have also reported the stimulatory role of parathyroid hormone in production of 1,25-(OH) 2D3 (22, 2S, 26) . Less well astabliahed is the role of inorganic phosphorus in the control of 1,25-(OH) D3 synthesis . Based on the observation that low phosphorus dints stimilatn in~eitinal calcium absorption (27), evidence has bean provided that low phos hate diets result in an accumulation of large amounts of 3 H-1,25-(OH)2 D from ~-25-OH-D in the intestine (28) . Furthermore, is thyroparathgroidectomizad animals induction of serum phosphate concentration by dietary deprivation of phosphorus or by glucose feeding together with calcium gluconate, stimulates accumulation of 1,25-(OH) D in rat tissues (29) . Bacausa 1,25(OH) 2D3 stimulates an indapandeat ph~s~hate transport mechanism in the intestine (30) it seems reasonable that the biogenesis of 1,25-(OH) 2 D might be regulated by serum inorganic phosphate. Recently a report has a~peared suggaating that low phosphorus dints do not stimulate 1,25-(OH) 2D3 production in the chicken (20) . These authors claimed a "OS" phosphorus dZa[ made of corn and other dietary components which moat result in a dietary phosphorus level of at heat 0.35. In addition, dietary calcium was raised to extremely high lavala of 35 which couplicatea interpretation . Seruot phosphorus values in their case era not very low, which also suggests at best minimal phosphate daplation . In contrast, experi.nenta in our laboratory show that low phosphorus (0 .25) diets fed to chicks for 2 weeks show a 3-5 fold stimulation The in the renal 25-OH-Dg-la-hydroxylaee (Baxter and DeLuca, in preparation) . stimulation of 1,25-`OH)2D3 appearance in blood and tissue has been independently confirmed by three laboratories (30-32) . Hence a demand for phosphorus as rnvnalad by low serum phosphorus will stimulate the production of 1,25-(OH) 2D3 which does play a role in the mobilization of phosphate at least from intestine and possibly from other sources. It moat be borne in mind, however, in contrast to the stimulation of calcium absorption by low calcium diets, the stimulation by lov phosphorus dicta is not solely duo to a stimulation of 1,25-(OH) 2D3 synthesis since exogenous 1,25-(OH),D3 cannot raise calcium transport to levels achieved by phosphate depletion (33) . Tanaka and DeLuca have suggested that renal call levels of inorganic phosphate may be an important regulator based on measurement of renal cortex inorganic phosphate (29) . Thus parathyroid hormone administration and phosphata deprivation may lower cellular phosphate levels thereby stimulating 1-hydrorylation . Although of interest, much work is naedad before this hypothesis can
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be accepted . However, attempts to examine this question moat employ mild phosphate measuring techniques and the measurement must be oa renal cortex sad Obviously only those cells carrynot whole kidney as has been reported (20) . ing out the hydroxylation are of interest, not the entire kidaey . In the work of Boyle et al . (12, 13), it was demonstrated that vitamin Ddeficient animals do not show a regulation of 3H-1,25-(OH) D accumulation in the target tissues following a single injection of 3ü-25-0~-I~ 3. Tanaka et al . provided evidence that 1,25-(OH) 2D playa an important role iß the _in vivo regulation of 1,25-(OH) 2D accumulation (34) . It appears that 1,25-(OH) D3 given to vitamin D-defici~nt animals will bring about the appearance of ~tté 25-OH-D3 24-hydroxylase system and a repression of 25-OH-D3 la-hydroxylase (35) (Figure 2) . Furthermore, the 1,25-(OH) 2D3 must be present before regulation
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6 12 18 24 HOURS AFTER DOSE OF 1,25-i0H) Q D 3 FIC. 2
The suppression of 25-OH-D3 lar-hydrozylase sad the stimulation of 25-OH-D -24-hydrozylase by 1,25-(OH) D administered in vivo . RachitiL~ chicks on a lx calcium, 0 .5~ ~hoephorus diet were gives 650 pmoles 1,25-(OH) D3 intravenously and at various times thereafter, the chicks wee killed, their kidaey mitochondria isolated and their hydrozylase activity determined in vitro as described by Ghazarian et al . (35) . by parathyroid hormone and inorganic phosphate takes place . MacIntyre and his colleagues have presented in vitro evidence that 1,25-(OH) D suppresses production of 1,25-(OH) D3~y isolated renal tubules (36, ~7~ although it is not yet clear if this represents what happens in vivo .
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Because of the regulation of the 25-OH-D -la-hydroxylase by serum calcium and phosphorus in vivo , there have been numerus experiments oa the effects of calcium and phosphate, parathyroid hormone and calcitonin added in vitro to homogenates, isolated mitochondria and isolated renal tubules in vitro . Thus calcium can stimulate (38-40), inhibit (20, 35, 38, 39, 41) ; p~arathgroid hormone can stimulate (25) or do nothing (42) ; calcitonin can inhibit (25) while phosphate can both inhibit and stimulate 1,25-(OH) 2Dg synthesis (35, 43) . Many of these effects are likely not related to the phyeioiogical control which led to the etudq in the first place . The changeover in the renal la-hydroxylass activity requires many hours in response to a change in serum calcium (44), serum phosphorus (29), parathyroid hormone (24), or 1,25-(oH) 2D administraIf there is a minute to minute regulation oï ~he hydroaylasa tion (Figure 2) . in vivo by ionic activation or inhibition, it is yet evident . Esperimenta on surviving tissue preparations have serious limitations when long-term regulation is being studied and thus interpretation of the in vitro data in these systems at the verq least requirna great caution. Of some interest is the recent idea that 25-OH-D9 specificallq plays a role in the suppression of parathyroid hormone secretion. Thin interesting idea was put forth bq Oldham et al . and has been an attractive hypothesis (45) . Although not disprrnred, thin concept does not appear to have gained support. In fact, recent evidence obtained is pigs (46) and in rats (William and DeLuca, u~ublishad reaulta) have shown that the circulating parathyroid hormone levels are regulated by serum calcium concentration whether vitamin D is Furthermore, no evidence could be obtained that 1,25-(OH) 2D3 given or not . itself represses circulating parathyroid hormone levala until serum calcium rises. Recently a report of specific localization of 1,25-(OH) D in the parathyroid glands of chicks (47) has appeared, but is not yet convincing . Therefore, this interesting idea must have more convincing evidence before it can be taken seriously . It must be emphasized that the molecular and cellular mechanism of regulation of the 25-OH-D3-la-hydroxylases am not understood . It is, however, clear that 1,25-(OH) D 3 itself, serum inorganic phosphate and the parathgroid hormone physiologi~ailq play important regulatory roles in the production of 1,25-(OH) 2D9 by mechanism which are not immediate and which may well involve the regulation of enzyme synthesis or degradation. During the next several yearn the molecular mech .*,t~.,~~ of this regulation will undoubtedly come under close scrutiny . However, elucidation of the molecular mechanism of this regulation will probably require a detailed knowledge of the enzymology of the la-hydroxylase and the 24-hydroxylase . Progress has been verq rapid in this regard in which Ghazariaa et al . (48) have demonstrated clearly that the la-hydroxglaee contains and requires a cytochrome P-450 . This protein has bean partially purified, and when combined with beef adrenadoxin and beef adrenal flévoprotein can hydrozglate 25-OH-D in the la position (48) . Many exciting results at the biochemical level can nor be expected in this area is the next several years which will hopafullq cast light on the mechanism of regulation . The regulation of vitamin D metabolism by plasma calcium concentration and by plasma phosphate concentration has great significance phgaiologically and in medicine . For example, it may well be that hypoparathyroid patients Thus treatment produce very little 1,25-(OH) D in response to hypocalcemia . of these patients with 1,25-(~~ D plus dietary calcium may ba indicated. In fact, such treatment has resulta~ ~n marked success in the elevation of serum calcium concentration and in the management of this disease . The regulation phenomenon may also have major implications in the development of the renal ostaodgstrophic syndrome in which there is a high degree of osteomalacia and secondary hyperparathyroidlsm. The lack of 1,25-(OH) 2D3 may well bring about
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poor intestinal calcium absorption and poor mobilisation of calcium from bone resulting is a compensatory increased parathyroid hormone secretion and hypertrophy . Stimulation of 1,25-(OH~) 2D 3 synthesis by hypophosphatemia may well be as important factor is the well ia~own skeletal improvement in renal failure patients by phosphate restriction . Certainly the idea that 1,25-(OH) 2D3 synthesis is regulated provides a new dimension to an understandinY of calcium and phosphorus metabolism and its consequent implications in the management of metabolic boas disease . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 .
H . F . DSLUCA, Fed . Proc . 33, 2211-2219 (1974) . A . W . NORMAN, Am. J . Med . 57, 21-27 (1974) . E . BODICE&, Lancet_1, 325-329 (1974) . J . W . BLUNT, R . F . DELUCA, and H . & . SCHNOES, Biochemistry _7, 3317-3322 (1967j . J . G .~SADDAD and T . C . B . STAMP, Am . J . Med . 57, 57-62 (1974) . . Chem . 248, 2969-2973 (1973) . M . H : BHATTACHARYYA and H . F . DELDCA,_J .Biol M . H . BHATTACHARYYA and H . F . DELUCA, Hiochem . Biophye . Ras . Commun . 59, 734-741 (1974) . G . T[JCRER, III, R . E . GAGNON, and M . R . HAUSSLER, Arch . Biochem . Hiophgs . 155, 47-57 (1973) . M . H . HHATTACRARYYA and H . F . DELUCA, J . Biol . Chem . 248, 2974-2977 (1973) . G . PONCHON, H . F . DELUCA, and T . SUDA, Arch . Biochem .Bi s . 141, 397408 (1970) . M . B . BHATTACHARYYA and H . F . DSLUCA, Arch . Biochem . Biophye . 160, 58-62 (1474) . I . T . BOYLE, R . W . GRAY, and H . F . DELIICA . Proc . Nat . Acad . Sçi . IISA 68, 2131-2134 (1971) . I . T . BOYLE, R . W . GRAY, J . L . OMDAHL, sad H . F . DELUCA, Endocriaolo~ 1971 , pp . 468-476, Wen . Heinemann Medical Books Ltd ., London (1972) . M . F . HOLICR, H . H . SCHNOES, H . F . DSLUCA, R . W . GRAY, I . T . BOYLE, and T . SUDA, Biochemistry _11, 4251-4255 (1972) . J . L . ülmAHL, R . W . GRAY, I . T . BOYLE, J . 1CNIITSON, and H . F . DELIICA, Nature _New Biol . 237, 63-64 (1972) . J . EHEL, P . H . CRAIG, A . N . TAYLOR, sad R . H . WASSERR . A . COR$ADINO, .G MAN, Calc . Ties . _Res . _7, 93-102 (1971) . R . A . CORRADINO, J . G . EBEL, P . H . CRAIG, A . N . TAYLOR, and R . H . WASSERMAN, Calc . Tise . Res . 7, 81-92 (1971) . H F . DELUCA, Scleace 174, 949-951 (1971) . J . L . OMDAHL and . J . L . OMDAHL and H . F . DELUCA, J . Biol .Chem . 247, 5520-5526 (1972) . $ . L . HENRY, R . J . MIDGETT, andA . W . NORMAN, _J .Biol . Chem . 249, 75847592 (1974) . J . L . OMDAHL and H . F . DELUCA, Physiol . _Rev . 53, 327-372 (1973) . L . GALANTE, S . J . MACAULEY, R . W . COLSTON, and I . MACINTYRE, Lancet 1, 985-988 (1972) . D . R . FRASER sad E . BODICE&, Nature New Biol . 241, 163-166 (1973) . M . GARAHEDIAN, M . F . HOLICR, H . F . DELUCA, andÎ. T . BOYLE, Proc . Nat . Acad . Sçi . US A _69, 1673-1676 (1972) . H . RASMUSSEN, M . WONG, D . BIBLE, and D . B . P . GOODMAN, J . Clin . Imrest . S1, 2502-2504 (1972) . L . F . HILL and E . B . MAWER, Annual General Meeting of the Medical Research Society of London, December (1972), abstract . R, L . MORRISSEY and R . H . WASSERMAN, _Am . _J . Ph aiol . _220, 1509-1515 (1971) . Y . TAMARA, H . FRANK, and H . F . DELUCA, Science 181, 564-566 (1973) . Y . TAMARA and H . F . DELUCA, Arch . Biochem . Biophye 154, 566-574 (1973) . S . EDELSTEIIQ, A . HARELL, A . BAR, and S . HURWITZ, Biochim . Biophye . Acts 385, 438-442 (1975) .
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M . R . HAIISSLSR, Clin . Endocrinol . (Proceedings of the Fifth International Conference of Endocrinology, London), (1976), is press . I . MACIIiTYRS, Clin . Endocriaol . (Proceedings of the Fifth Intaraational Conference of Endocrinology, London), (1976), in press . T . C . CHEM, L . CASTILLO, M . 1CORYC&A-DAHI., and H . F . DSLÜCA, J . Nutr . 104, 1056-1060 (1974), . Y . TAMAT,A and H . F . DSLIICA, Science 183, 1198-1200 (1974) . J . G . Gw~~~~~u , Y . TAMARA, and H . F. DELIICA, Calcium Regulating Hormonen , E:cerpta Medics, Amsterdam (1975), in press . R . G . LARiCINS, S . J . MACAULEY, A . RAPOPORT, T . J . MARTIM, B . R . TIILLOCH, P . G . H . BYFIELD, 8 . W . MATTHEWS, and I . MACINTYRE, Clin . Sçi . Mol . Med . 46, 569-582 (1974) . Î. MACINTYRB . L . S . GALANTE, & . W . COLSTON, I . M . A . EVANS, R . G . LAßRINS, 3 . J . MACAIILEY, C . J . HILLYARD, P . B . GREEMBERG, E . W . MATTHEWS, and P . G . fl . BYFIELD, Calcium Regulating Hormones , Ezcerpta Medics, Amsterdam (1975), in prass . N . HORIIICHI, T . SIJDA, S . 3ASAHI, I . EZAWA, Y . SANG, aad E . OGATA, FEBS Letters 43, 353-356 (1974) . K . W . COLSTOM, I . M . A . SVAMS, L . GALANTE, I . MACIMIRRE, sad D . W . MOSS, Biochem . J . 134, 817-820 (1973) . D . D . BIIR.S and H . RASMTSSEN, J . Clin . Imrent . 55, 292-298 (1975) . D . R . FRASER and E . BODICE[, Nature 228, 764-766 (1970) . 3 . A . SHAIM, J . Siol . Chan . 247, 4404-4413 (1972) . H . F . DSLIICA,Nm. J . timed . 57,1-12 (1974) . H . F . DELUCA, Biochan . _Soc . Spec . Publ . 3, 5-26 (1974) . S . B . OLDHAH, J . A . FISCHSR, L . H . SHEN, and C . D . ARMAUD, Bioch~i.stry 13, 4790-4796 (1974) . A . D . CARE, R . F . L . BATES, R. SWAMIMATHAM, C . G . SCAMES, M . PEACOC&, E . B . MAWER, C . M . TAYIAR, H . F . DELUCA, S . TOMLINSON, and J . L . H . O'RIORDAM, Calcins Regulating Hormones , E :cerpta Medico, Amsterdam (1975), is press . H . HENRY sad A . W . MOBMAN, Biochem . Biophss . Bes . Commam . 62, 781-783 (1975) . J . G . c-~e~~uteN , C . R . JEFCOATE, J . C . 1QiIJTSON, W . H . ORME-JOHNSOM, and H . F . DELUCA, J . Biol . Chain . 249, 3026-3033 (1974) .
Pargamon Presa
MINIRE91~99 REGDLATION OF VITAMIN D METABOLISM Hector F . DeLuca Department of Biochemistry, College of Agricultural sad Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
Vitamin D can be regarded as a prohormone and its moat potent metabolite, 1,25-dihydroxyvitamin D3, a hormone xhich mobilizes calcite and phosphate from bone and intestine. Ia true horswaal fashion, the biosynthesis of 1,25-dihydroryvitamin D bq kidney mitochondria is feed-back regulated by serum calcium an~ serum phoephorw .levels . The lack of calcium brings about a secretion of parathyroid hormone xhich stimulates 1,25-dihydroayvitamin D3 ayatheaie xhile lox blood phosphorus stimulates 1,25-dihydroxyvitamia D ayathesie even in the absence of the parathyroid glands . For such regulation to occur, vitamin D moat be present probably because 1,25-dihydrozyvitamia D3 it~self is needed for the regulation . The molecular and cellular mechaniss~s xhereby 1,25-dihydroxyvitasiin Dg synthesis is regulated era uakaoxn despite many recent reports . Likely the elucidation of thpe nschaaisms must axait a detailed investigation of the enzymology of the seal 25-hydroryvitamin D -la-hydrozylase . In addition to the regulation at the 25-hydro:~vitasin D -larhydrorylase stop, vitamin D metabolis: is regulated at the he~atie vitamin D-25-hydrozylase level. This regulation is a suppression of the hydrozylase by the hepatic level of 25-hydroxyvitamin D itself by as unkaowa mechanism. Much remains to be leased concerning the regulation of this newly discounted endocrine system but already the findings are not only relevant to calcium homeostasis but also to as understanding of a variety of metabolic bone diseases . There is now little doubt that vitamin D must first be setabolised in the liver to 25-hydroxyvitamin D (25-OH-D ) and subsequently in the kidney to 1,2i-dihydrozyvitsmia D3 (1,~5-(OH)2D3~ before it can carry out its xell known functions on intestine and bone (1) . This concept couplsd xith the recognition that vitamin D is not normally required is the diet xhen sufficient ultraviolet irradiation is incident upon the skin hoe led to the belief that 1,25-(OH) 2D~ must be regarded as a hormone (1-3) . To be considered a hormone it is logical that the biogenesis or secretion must be feed-back regulated by the products of its metabolic efforts . That such is the case has nox been clearlq established at the physiologic level and it will be the put~+ose of this review to bring together those aaliant points establishing this regulation . Reaulation of 2S-Hydroxylstion So far there is no clear demonstration 25-OH-D acts directly is any of the target Accordiâgly serum calcium concentration and not appear to regulate this hydrorylation. 1351
that at physiologic concentrations tissues of vitamin D function . serum phosphate concentration do There is,~hoxever, evidence that
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Regvlatioa of Vitamin D
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the hepatic level of 25-OH-D3 does provide some degree of regulation of this initial entry of vitamin D into its metabolic sequence . Earlq efforts demonatrated that it was necessary to increase the dosage of vitamin D to hogs from 100 unite to 250,000 units per day to increase the circulating le~el of 25-OHDq from 1 unit to 12 units of antirachitic activity per milliliter (4) . Thus iL is clear that there is no direct relationship between the circulating levels of the metabolite sad the dosage of vitamin D given. Similar results have bean reported in man bq Haddad and his colleagues (5) . More directly it has been shown in both rata and chicks that the prior administration of vitamin D3 to rachitic or vitamin D-deficient animals will markedly depress hepatic 25hydrozglatioa of vitamin D3 meaaurnd in vitro (6, 7) . Furthermore this depression could bn demonstrated in vivo as wall and could be related to the hepatic level of this metabolite . Ît ie, therefore, apparent that the hepatic level of 25-OH-D in some way suppresses the 25-hydroxylation of vitamin D . Thn amount of vita~in D required for the suppression depends upon the apecie~ iaaamuch as Tucker et al . (B) could find no reduction in the 25-hydroxylase when .25 ug of vitamin 3 was given to chicks whereas Bhattacharyya and DaLuca (7) showed that in thin spacles dosages above .5 ug will bring about a reduction in the 25hydroxylase. Ia the rat, the reduction occurs at dosages of .25 ug or above. It ie of soma interest, hwevar, that the 25-hydroxylation of the Gloss analog of vitamin D3, namely dihydrotachysterol 3 , is not regulated hither by the prior administration of vitamin Dg or by the prior administration of dihydrotachysterol g itself (9) . Additionally it is important to realise that the adminiatration of 25-OH-D intravenously dons not exert a marked inhibition of the 25-hydrozylaee sinca3lt does not readily penetrate the liver (10) . It is uncertain whether the enzyme system which hydrozylatas vitamin D3 in the hepatic microsomes sad the anzyma that hgdrozylatas dihydrotachysterol g and cholesterol is the 25 position am one and the same . Instead thard is evidence to suspect that there is a non-specific 25-hydroxylasa which is sot regulated and which carries out 25-hydroxylation in a non-specific meaner at high substrata concentrations (11) . It moat also be borne in mind that the regulation of the vitamin D -25-hydroxylase is not absolute and largo amounts of vitamin D can overcome his regulation . Howâvar, the 25-hydroxylation stags must be r~gardad as a bottleneck reaction which is regulated at physiologic levels of vitamin D3 .
D
Regulatioa of the iCidnay 25-0H-D,-la-Hgdroxglaae By far the most important known regulation of vitamin D metabolism occurs at the 1-hydroxylation stage. In 1971 the first clear evidence that the 1hydroxylation mechanism is regulated was provided by Boyle et al . (12) . Boyle studied the in vivo accumilation of the 1,25-(OH) 2D3 metabolite in the plasma and target tiasuas following as intravenous done 3f 25-OH-D . They clearly demonstrated that rata on a low calcium diet accumulate lays amounts of 1,25-(OH) 2D in the plasma and target tissues whereas those animale given high calcium dins accumulate very little of this metabolite . In contrast the animals on a high calcium diet accumulated another metabolite which was aubsaquaatly identified ae 24,25-dihgdroxyvitamin D3 (24,25-(OH) 2D~) (14) . Of major importance is the realization that vitamin D-deficient animals did not show this degree of regulation . Vitamin D-deficient animals regardless of dietary treatment accumulatéd large amounts of 1,25-(OH) 2D3 in response to an injected dose of 3H-25-OH-D , illustrating the importance of some form of vitamin D in this regulation phenomenon . In another report, Boyle et al . (13) further demonstrated that the regulation in the rats given vitamin D is related to the resultant serum calcium concentration produced by the dietary manipulation (Figure 1) . SJhen the concentration of calcium in the blood is The normal, equal amounts of 1,25-(OH) 2D and 24,25-(OH) D3 appear in blood . hypocalcemic animals accumulated 1$r~e amounts of 1, 7 25-(OH) 2D3 . On the other
Vol . 17, No . 9
Regulation of Vitamin D Metabolism
W 20 i
FQ w î
135 3
1,25-(OH~ D3
10
N~
W N Z
24,25-(OH)2D3
3
4
!~______ l~ s __~__ __r_ v ~
5 6 7 8 9 SERUM CALCIUM mg/100 r~l
~ 10
FIG. 1 The elationship of serum lcium concentration to the plasma levels of [~)-24,25-(OH) D and ~]-1,25-(OH) D . Bats on a variety of diets for two wea~ i~aceived an injectio~ ~f 25-OH-[26,27- 3H)D and 1Z hours later setabolite levels xere determined in the plasma after estraction a~ chraa~atography (13) .
3
hand the hypercalcemic aaimaL accumulated little or ao 1,25-(OH) D . Boyle et al . (13) inferred from these etudiss that plasma calciun conce~t~ation plays âa important regulatory role in the renal 1-hydroxylation of 25-0H-D either directly or indirectly . Direct demonstration by in vitro measurements that such a regulation of the 1-hydroxylâse had actually taken place vas provided by studies in the chick by Omdahl et _al. working with high and lw calcium diets (15) . These investigators demoastratsd that chicks on a low calcium diet have a high level of 25-OH-D3 la-hydroxylase activity is their isolated mitochoadrie whereas these on a high calcium diet have very little of this enzyse and instead shoo large anouats of 25-OH-D3 24-hydrozylase . Similarly strontium, which ie known to shut down intestinal calcium absorption (16, 17), suppresses ZS-OA-D la-hydroxylase and stimulates the 25-OH-D -24-hydroxylase activities (18, 19)3 There vas, therefore, no doubt that the serum calcium conceatratioa vas e:arting its control on the renal hydroxylase enaymatic activities . These basic observations have recently been confirmed (20) .
3
g
Of great interest is that the administration of exogenous 1,25-(OH) D eliminates the repression of intestinal calcium transport by dietary cal~i~ (21) and strontium (18, 19) . It is, therefore, evidnat that the need for calcium as shown by even slight hypocalcemia in some way briaga about the increased activity of the 25-OH-D -lar-hydrozylase in the kidneys . This results in an iacreaaad produc .tioa of 1,25-(OH) D a hormone whose fuactioa it ie to mobilize calcium from both intestine an3 bone . this position has been largely confirmed in a number of laboratories (20, 22, 23) .
g
2 3,
1354
Regulation of Vitamin D Metabolism
Vol. 17, No . 9
The fact that dietary calcium and serum calcium concentration in some way directs the activation of the renal 25-OH-D3-la-hydroxglasa lad to the idea that this regulation might be mediated by tRe parathyroid glands which are knows to secrete parathyroid hormone in response to hypocalcemia (24) . Because thyroparathyroidectamy is very difficult in chicks, experiments warn carried out in the rat and the accumulation of 1,25-(OH) D3 in the plasma and target tissues was meaeurad .~ In vitamin D-deficient ra~s given 1,25-(OH) Dg and a diet low in calcium, there is a predominant accumulation of 1,25-(~H72D 9 in the intestine and serum in response to a single dose of radioactive 25-OH-D g (24) . Following thyroparathyroidectamy there is a progressive diminution of the 3H-1,25-(OH) D3 in these tlsauna from 3H-25-OH-D3 until 48 hours poet surgery when no ~,25-(OH) D3 accumulates in these tissues . Iaatead 24,25(OH) 2D3 appears. These a~imals ware then given an exogenous source of parathyrbid hormone which restores the accumulation of 1,25-(OH) 2Dg in the target tiasuas. Fraser and Kodicek (23) have meseurad directly the 25-OH-D3-lahydrozylase of chick kidney in response to parathyroidectomy and parathyroid hormone injections illustrating that parathyroid hormone is soma unknown way stimulates the activity of the renal 25-OH-D -la-hydroaylase . Erperimenta carried out by Galaate et al . (22), although3of some interest, do not bear on the question of whathar physiological amounts of parathyroid hormone stimulate she production of 1,25-(OH) 2D3 in vivo . Other laboratories have also reported the stimulatory role of parathyroid hormone in production of 1,25-(OH) 2D3 (22, 2S, 26) . Less well astabliahed is the role of inorganic phosphorus in the control of 1,25-(OH) D3 synthesis . Based on the observation that low phosphorus dints stimilatn in~eitinal calcium absorption (27), evidence has bean provided that low phos hate diets result in an accumulation of large amounts of 3 H-1,25-(OH)2 D from ~-25-OH-D in the intestine (28) . Furthermore, is thyroparathgroidectomizad animals induction of serum phosphate concentration by dietary deprivation of phosphorus or by glucose feeding together with calcium gluconate, stimulates accumulation of 1,25-(OH) D in rat tissues (29) . Bacausa 1,25(OH) 2D3 stimulates an indapandeat ph~s~hate transport mechanism in the intestine (30) it seems reasonable that the biogenesis of 1,25-(OH) 2 D might be regulated by serum inorganic phosphate. Recently a report has a~peared suggaating that low phosphorus dints do not stimulate 1,25-(OH) 2D3 production in the chicken (20) . These authors claimed a "OS" phosphorus dZa[ made of corn and other dietary components which moat result in a dietary phosphorus level of at heat 0.35. In addition, dietary calcium was raised to extremely high lavala of 35 which couplicatea interpretation . Seruot phosphorus values in their case era not very low, which also suggests at best minimal phosphate daplation . In contrast, experi.nenta in our laboratory show that low phosphorus (0 .25) diets fed to chicks for 2 weeks show a 3-5 fold stimulation The in the renal 25-OH-Dg-la-hydroxylaee (Baxter and DeLuca, in preparation) . stimulation of 1,25-`OH)2D3 appearance in blood and tissue has been independently confirmed by three laboratories (30-32) . Hence a demand for phosphorus as rnvnalad by low serum phosphorus will stimulate the production of 1,25-(OH) 2D3 which does play a role in the mobilization of phosphate at least from intestine and possibly from other sources. It moat be borne in mind, however, in contrast to the stimulation of calcium absorption by low calcium diets, the stimulation by lov phosphorus dicta is not solely duo to a stimulation of 1,25-(OH) 2D3 synthesis since exogenous 1,25-(OH),D3 cannot raise calcium transport to levels achieved by phosphate depletion (33) . Tanaka and DeLuca have suggested that renal call levels of inorganic phosphate may be an important regulator based on measurement of renal cortex inorganic phosphate (29) . Thus parathyroid hormone administration and phosphata deprivation may lower cellular phosphate levels thereby stimulating 1-hydrorylation . Although of interest, much work is naedad before this hypothesis can
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Requlatton of vitamin D Metabolism
1355
be accepted . However, attempts to examine this question moat employ mild phosphate measuring techniques and the measurement must be oa renal cortex sad Obviously only those cells carrynot whole kidney as has been reported (20) . ing out the hydroxylation are of interest, not the entire kidaey . In the work of Boyle et al . (12, 13), it was demonstrated that vitamin Ddeficient animals do not show a regulation of 3H-1,25-(OH) D accumulation in the target tissues following a single injection of 3ü-25-0~-I~ 3. Tanaka et al . provided evidence that 1,25-(OH) 2D playa an important role iß the _in vivo regulation of 1,25-(OH) 2D accumulation (34) . It appears that 1,25-(OH) D3 given to vitamin D-defici~nt animals will bring about the appearance of ~tté 25-OH-D3 24-hydroxylase system and a repression of 25-OH-D3 la-hydroxylase (35) (Figure 2) . Furthermore, the 1,25-(OH) 2D3 must be present before regulation
~. 2 0 O
d M O
ac 10
d G 4 F O H 0
6 12 18 24 HOURS AFTER DOSE OF 1,25-i0H) Q D 3 FIC. 2
The suppression of 25-OH-D3 lar-hydrozylase sad the stimulation of 25-OH-D -24-hydrozylase by 1,25-(OH) D administered in vivo . RachitiL~ chicks on a lx calcium, 0 .5~ ~hoephorus diet were gives 650 pmoles 1,25-(OH) D3 intravenously and at various times thereafter, the chicks wee killed, their kidaey mitochondria isolated and their hydrozylase activity determined in vitro as described by Ghazarian et al . (35) . by parathyroid hormone and inorganic phosphate takes place . MacIntyre and his colleagues have presented in vitro evidence that 1,25-(OH) D suppresses production of 1,25-(OH) D3~y isolated renal tubules (36, ~7~ although it is not yet clear if this represents what happens in vivo .
1356
~
RegUlatibn oP vitamin D I4eetaboli~sm
Vol . 17, No . 9
Because of the regulation of the 25-OH-D -la-hydroxylase by serum calcium and phosphorus in vivo , there have been numerus experiments oa the effects of calcium and phosphate, parathyroid hormone and calcitonin added in vitro to homogenates, isolated mitochondria and isolated renal tubules in vitro . Thus calcium can stimulate (38-40), inhibit (20, 35, 38, 39, 41) ; p~arathgroid hormone can stimulate (25) or do nothing (42) ; calcitonin can inhibit (25) while phosphate can both inhibit and stimulate 1,25-(OH) 2Dg synthesis (35, 43) . Many of these effects are likely not related to the phyeioiogical control which led to the etudq in the first place . The changeover in the renal la-hydroxylass activity requires many hours in response to a change in serum calcium (44), serum phosphorus (29), parathyroid hormone (24), or 1,25-(oH) 2D administraIf there is a minute to minute regulation oï ~he hydroaylasa tion (Figure 2) . in vivo by ionic activation or inhibition, it is yet evident . Esperimenta on surviving tissue preparations have serious limitations when long-term regulation is being studied and thus interpretation of the in vitro data in these systems at the verq least requirna great caution. Of some interest is the recent idea that 25-OH-D9 specificallq plays a role in the suppression of parathyroid hormone secretion. Thin interesting idea was put forth bq Oldham et al . and has been an attractive hypothesis (45) . Although not disprrnred, thin concept does not appear to have gained support. In fact, recent evidence obtained is pigs (46) and in rats (William and DeLuca, u~ublishad reaulta) have shown that the circulating parathyroid hormone levels are regulated by serum calcium concentration whether vitamin D is Furthermore, no evidence could be obtained that 1,25-(OH) 2D3 given or not . itself represses circulating parathyroid hormone levala until serum calcium rises. Recently a report of specific localization of 1,25-(OH) D in the parathyroid glands of chicks (47) has appeared, but is not yet convincing . Therefore, this interesting idea must have more convincing evidence before it can be taken seriously . It must be emphasized that the molecular and cellular mechanism of regulation of the 25-OH-D3-la-hydroxylases am not understood . It is, however, clear that 1,25-(OH) D 3 itself, serum inorganic phosphate and the parathgroid hormone physiologi~ailq play important regulatory roles in the production of 1,25-(OH) 2D9 by mechanism which are not immediate and which may well involve the regulation of enzyme synthesis or degradation. During the next several yearn the molecular mech .*,t~.,~~ of this regulation will undoubtedly come under close scrutiny . However, elucidation of the molecular mechanism of this regulation will probably require a detailed knowledge of the enzymology of the la-hydroxylase and the 24-hydroxylase . Progress has been verq rapid in this regard in which Ghazariaa et al . (48) have demonstrated clearly that the la-hydroxglaee contains and requires a cytochrome P-450 . This protein has bean partially purified, and when combined with beef adrenadoxin and beef adrenal flévoprotein can hydrozglate 25-OH-D in the la position (48) . Many exciting results at the biochemical level can nor be expected in this area is the next several years which will hopafullq cast light on the mechanism of regulation . The regulation of vitamin D metabolism by plasma calcium concentration and by plasma phosphate concentration has great significance phgaiologically and in medicine . For example, it may well be that hypoparathyroid patients Thus treatment produce very little 1,25-(OH) D in response to hypocalcemia . of these patients with 1,25-(~~ D plus dietary calcium may ba indicated. In fact, such treatment has resulta~ ~n marked success in the elevation of serum calcium concentration and in the management of this disease . The regulation phenomenon may also have major implications in the development of the renal ostaodgstrophic syndrome in which there is a high degree of osteomalacia and secondary hyperparathyroidlsm. The lack of 1,25-(OH) 2D3 may well bring about
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Regulation of Vitamin D Mataboliam
135 7
poor intestinal calcium absorption and poor mobilisation of calcium from bone resulting is a compensatory increased parathyroid hormone secretion and hypertrophy . Stimulation of 1,25-(OH~) 2D 3 synthesis by hypophosphatemia may well be as important factor is the well ia~own skeletal improvement in renal failure patients by phosphate restriction . Certainly the idea that 1,25-(OH) 2D3 synthesis is regulated provides a new dimension to an understandinY of calcium and phosphorus metabolism and its consequent implications in the management of metabolic boas disease . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 .
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M . R . HAIISSLSR, Clin . Endocrinol . (Proceedings of the Fifth International Conference of Endocrinology, London), (1976), is press . I . MACIIiTYRS, Clin . Endocriaol . (Proceedings of the Fifth Intaraational Conference of Endocrinology, London), (1976), in press . T . C . CHEM, L . CASTILLO, M . 1CORYC&A-DAHI., and H . F . DSLÜCA, J . Nutr . 104, 1056-1060 (1974), . Y . TAMAT,A and H . F . DSLIICA, Science 183, 1198-1200 (1974) . J . G . Gw~~~~~u , Y . TAMARA, and H . F. DELIICA, Calcium Regulating Hormonen , E:cerpta Medics, Amsterdam (1975), in press . R . G . LARiCINS, S . J . MACAULEY, A . RAPOPORT, T . J . MARTIM, B . R . TIILLOCH, P . G . H . BYFIELD, 8 . W . MATTHEWS, and I . MACINTYRE, Clin . Sçi . Mol . Med . 46, 569-582 (1974) . Î. MACINTYRB . L . S . GALANTE, & . W . COLSTON, I . M . A . EVANS, R . G . LAßRINS, 3 . J . MACAIILEY, C . J . HILLYARD, P . B . GREEMBERG, E . W . MATTHEWS, and P . G . fl . BYFIELD, Calcium Regulating Hormones , Ezcerpta Medics, Amsterdam (1975), in prass . N . HORIIICHI, T . SIJDA, S . 3ASAHI, I . EZAWA, Y . SANG, aad E . OGATA, FEBS Letters 43, 353-356 (1974) . K . W . COLSTOM, I . M . A . SVAMS, L . GALANTE, I . MACIMIRRE, sad D . W . MOSS, Biochem . J . 134, 817-820 (1973) . D . D . BIIR.S and H . RASMTSSEN, J . Clin . Imrent . 55, 292-298 (1975) . D . R . FRASER and E . BODICE[, Nature 228, 764-766 (1970) . 3 . A . SHAIM, J . Siol . Chan . 247, 4404-4413 (1972) . H . F . DSLIICA,Nm. J . timed . 57,1-12 (1974) . H . F . DELUCA, Biochan . _Soc . Spec . Publ . 3, 5-26 (1974) . S . B . OLDHAH, J . A . FISCHSR, L . H . SHEN, and C . D . ARMAUD, Bioch~i.stry 13, 4790-4796 (1974) . A . D . CARE, R . F . L . BATES, R. SWAMIMATHAM, C . G . SCAMES, M . PEACOC&, E . B . MAWER, C . M . TAYIAR, H . F . DELUCA, S . TOMLINSON, and J . L . H . O'RIORDAM, Calcins Regulating Hormones , E :cerpta Medico, Amsterdam (1975), is press . H . HENRY sad A . W . MOBMAN, Biochem . Biophss . Bes . Commam . 62, 781-783 (1975) . J . G . c-~e~~uteN , C . R . JEFCOATE, J . C . 1QiIJTSON, W . H . ORME-JOHNSOM, and H . F . DELUCA, J . Biol . Chain . 249, 3026-3033 (1974) .