CD ■D O D O C T O R A L D IS S E R T A T IO N SERIES Q . C g Q . The^Sffeds ■D CD C/) C/) 8 TITLE V ilam in Û fY a r iû U S Sources Of 3)ihvârohch^^}ero lJhi ■D T h e i i l o o i C â l e i n m a t o J T h o ^ j^ h o m s _________ 3. 3" CD _______________________________________________________________________2 h _________________________________________________________________ CD ■D O Q . C aO 3 "O o CD Q . ■D CD AU TH O R iB ^ tn x J x ix y x UNIVERSITY C/) CO DEG REE % L Z % g l< XCjSL^ pX 37) 'i JZ ' 4a *____ U DATE ^ PUBLIC ATIO N NO. C / ' T A L JL________ UNIVERSITY MICROFILMS ANN ARBOR - 7 MICHIGAN THE EFFECTS OF VARIOUS SOURCES OF VITAMIN D AND DIHÏDROTACHYSTEROL ON THE BLOOD CALCIUM AND PHOSPHORUS LEVELS IN COWS, DOGS, CHICKENS, AND RATS by Benjamin H a rtle y P r in g le A THESIS S ubm itted t o th e School o f G raduate S tu d ie s o f M ichigan S ta te C ollege o f A g ric u ltu re and A pplied S cience i n p a r t i a l f u lf i l l m e n t o f th e re q u ire m e n ts f o r th e degree o f DOCTOR OF PHILOSOPHY Departm ent o f C hem istry 19U7 R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. ACKNOV/LEDGaCENT The a u th o r w ishes to ex p ress h is a p p r e c ia tio n to D octor G. A. H oppert, f o r h is e n lig h te n in g under­ s ta n d in g of th e d i f f i c u l t i e s enco u n tered i n work­ in g w ith b io lo g ic a l m a te r ia l, and f o r h is i n t e r ­ e s t and k in d ly c r i ti c i s m th ro u g h o u t th e s e s tu d ie s and i n th e p r e p a ra tio n o f t h i s m a n u sc rip t. R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE OF CONTENTS Page I n tr o d u c tio n ................................................... H i s t o r i c a l ................ *........................... A. G e n e r a l . . . . . . . . . . ............ E. A n tir a c h itic Compounds and Side P ro d u c ts .................... 0 . E f f e c ts o f A n tir a c h itic Compounds and Side Prod­ u c ts on M in eral M etab o lism . .......... D. E f f e c ts o f A n tir a c h itic Compounds and S id e Prod­ u c ts on Blood C o n s titu e n ts .............................. E. T o x ic ity o f A n tir a c h itic Compounds and S ide Prod­ u c ts ................... C hem istry o f th e P ro v itam in s D and o f th e I r r a d i a t i o n P ro c e s s ................. P h y sio lo g y o f th e A n ti r a c h itic Compounds...................................... S ig n if ic a n c e .................................................... .... ......................................... A. Calcium l e v e l s ................... B. Phosphorus l e v e l s ................. ............................... G. P hosphatase l e v e l s . D e s c rip tio n o f M ethods............................................................................ A. C alcium . ................... B. P hosphorus................................................................................ C. P h o s p h a ta s e . ....................... D e s c rip tio n o f R a tio n s , P ro d u c ts, and Animals Used............... A. R a t s . . . . ........................... 6 . D o g s . . . . . . . ................................ C. C hickens..................... D. Costs..................................................................... E xp erim en tal P ro c e d u re s ................................................ A. R a ts ........................................ ... B. Dogs............................ C. C h i c k e n s . . . . . . . . . ........................................................ D. C o w s . . . . . . . . ................. D ata...................................................................................................................... A. C alciu m ,................... 1 . R a ts ............... 2 . Dogs....................................................................................... 3 . C hickens .......... U. Cows..................................................................... B. P hosphorus .................................... 1 . R a ts ......................... 2 . Dogs....................................................................................... 3 . C h i c k e n s ................................................. 1|. Cows........................................... C. P h o s p h a t a s e . . . . . . . ................... 1 . R a t s . . . . ................................................................................. 2 . Cows................... (G o n t'd n e x t page) R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 3 3 18 20 22 25 30 I4I4 U8 W 50 5l 53 53 5U 57 66 66 66 66 6? 69 69 69 69 70 71 71 71 78 60 82 83 83 85 88 91 93 93 95 TABLE OF CONTENTS (Gont'd.) Page D is c u ss io n ............................................... A. C a l c i u m .... .......... 1 . R a ts ..................... 2 . D o g s........................ 3 . C hickens .......... li. C o n 'S ........................ ..................... B. Phosphorus 1 . R a t s . . . ............................................. 2 . D o g s............................................................... 3 . C hickens ................. U* COTATS ................... C. P h o sp h a tase......................................................... 1 . R a ts ........... c . C o w s . . . . . . . . . ....................... ............................................. C o n clu sio n s B ib lio g ra p h y ............................................................ R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 96 96 96 98 99 100 101 101 102 103 103 lOU 10l| 10$ 106 1 INTRODUCTION S in ce i t s d isc o v e ry and i s o l a t i o n , v ita m in D has been s tu d ie d frcsn numerous v iew p o in ts w ith o u t r e g a rd f o r th e s id e p ro d u c ts produced i n th e a c tiv a tio n p ro c e s s ’. The a c t i v a t i o n o f e r g o s te r o l in v o lv e s th e fo rm atio n o f c e r t a i n in te rm e d ia te s such a s lu m is te r o l and t a c i ^ s t e r o l and upon c o n tin u ed i r r a d i a t i o n scane o f th e v ita m in D i s converted, t o to x ic compounds such a s t o x i s t e r o l and th e s u p r a s te r o ls . The p re se n c e o f th e s e to x ic compounds i n th e e a r ly v ita m in D p r e p a r a tio n s , l e d t o th e developm ent o f p u re v ita m in D p ro d u c ts w hich have been i n use f o r a number o f y e a r s . There has however been r e c e n tly developed an id e a t h a t th e n a tu r a l so u rc e s o f v ita m in D m ight c o n ta in in te rm e d ia te s w hich m ight have p h y sio ­ l o g i c a l v a lu e . From ta c h y s te r o l th e r e has been produced a compound c a lle d d ih y d ro ta c h y s te r o l, known i n th e e a r ly h i s t o r y o f th e compound as A .T. 10 w hich has found c o n s id e ra b le u se i n r a i s i n g th e b lo o d calcium l e v e l in d e f ic ie n c ie s o f th e p a r a th y r o id . The p h y s io lo g ic a l e f f e c t s o f th e s id e -p ro d u c ts have n o t been s tu d ie d t o any e x te n t, ex c ep t t h a t i n th e case o f some o f them , t h e i r a n t i - r a c h i t i c v a lu e s have been d eterm in e d . C e rta in s tu d ie s have a ls o been made on t h e i r t o x i c i t y and on th e e f f e c t o f th e mode o f a d m in is tr a tio n on t h e i r p h y s io lo g ic a l a c t i v i t y . Because o f t h i s la c k o f in fo rm a tio n i t was c o n sid e re d t h a t a com parison betw een p u re v ita m in D and c e r t a i n n a tu r a l - 1 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. v ita m in D p ro d u c ts such a s f i s h o i l s and i r r a d i a t e d y e a s t m ig h t y ie ld seme i n t e r e s t i n g r e s u lt s * c e rn t o d ete rm in e w hether th e r e i s l o g i c a l b e h a v io r I t was o f c h ie f con­ a d if f e r e n c e i n p h y sio ­ between th e n a tu r a l and p u re p ro d u cts* A lthough m ost o f th e work on v ita m in D p ro d u c ts and on th e v a rio u s s id e -p ro d u c ts has been p erform ed on r a t s and c h ic k e n s , dogs and cows w ere a ls o used i n th e s e s tu d ie s * The b lo o d p i c t u r e was s tu d ie d t o d eterm in e th e e f f e c t s o f th e v a rio u s p ro d u c ts used* Because o f th e f a c t t h a t v ita m in D i s c h i e f l y concerned w ith c alciu m and phosphorus m etab o lism , a d e t a i le d s tu d y was made o f th e calcium and phosphorus changes o f th e b lo o d accompanying th e a d m in is tra ­ t i o n o f th e v a r io u s p ro d u cts* In a d d itio n some o f t h e e x p e ri­ m ents in v o lv e d th e d e te rm in a tio n o f th e a c id and a l k a l i n e p h o sp h ata se l e v e l i n th e b lo o d . -2- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. HISTQRIGAL A» G eneral In 1906 Hopkins su g g ested t h a t r i c k e t s WBS due t o some d e f ic ie n c y o f n u tr itio n * A v a ila b le ev id en ce a t t h a t tim e p o in te d t o th e c o n c lu sio n t h a t th e supposedly ad eq u ate d i e t needed some supplem entary c o n s titu e n t i n o rd e r t o p re v e n t r ic k e ts * E x h a u stiv e in v e s tig a tio n s have confirm ed th e views t h a t r i c k e t s h as t o be c o n sid e re d a s a d e f ic ie n c y d isea se* The names o f M ellanby, McCallum, Sherman, and Pappenheimer a re prcaninently connected w ith t h i s work* I t was found t h a t ex p erim en tal r i c k e t s could be induced i n dogs by g iv in g them a d i e t from w hich th e a n t i r a c h i t i c p r in c ip l e was m iss in g , and on th e o th e r hand t h a t ex p erim en tal r i c k e t s as w e ll a s th e r i c k e t s o c c u rrin g sp o n tan eo u sly could be c u red by supplem enting th e d i e t w ith c e r t a i n o i l s c o n ta in in g th e a n t i r o c h i t i c s u b sta n c e . Of th e s e , cod l i v e r o i l proved t o be th e m ost e f f e c t i v e . The symptoms o f r i c k e t s c o n s is t m ain ly in changes in th e developm ent o f th e s k e le to n and a r e u s u a lly accompanied by a g e n e ra l lo w erin g o f th e v i t a l i t y and by a r e t a r d a t i o n o f growth* The developm ent o f m ethods f o r prod u cin g ex p erim en tal r i c k e t s made i t p o s s ib le t o t e s t d i f f e r e n t food s t u f f s t h e i r a n t i r o c h i t i c p ro p e rtie s * fo r As ex p erim en tal r i c k e t s can be cu red by su p p ly in g th e a n t i r a c h i t i c su b sta n c e s i t i s p o s s ib le - 3- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. t o work o u t th e doses n e c e ssa ry to c u re r i c k e t s . T his i s c a ll e d I th e " c u r a tiv e t e s t " . The second method o f t e s t i n g c o n s is ts i n d eterm in in g th e amount o f a a n t i r a c h i t i c su b sta n c e n e c e s sa ry to p re v e n t th e developm ent o f r i c k e t s . tiv e te s t" . T his i s c a lle d th e "p rev en ­ I t has been found t h a t doses n e c e s s a ry t o cu re r i c k e t s a r e alw ays g r e a te r th a n th e doses p re v e n tin g r i c k e t s . More r e c e n tly chem ical methods have been used f o r th e d eterm in a­ tio n o f v ita m in D. The d isc o v e ry o f methods o f producing ex p erim en tal r i c k e t s fo llo w ed by th e developm ent o f t e s t s f o r m easuring th e a n t i ­ r a c h i t i c v a lu e was th e f i r s t g r e a t advance i n e lu c id a tin g th e n a tu r e o f r i c k e t s . The second g r e a t d isc o v e ry was due to a German p h y s ic ia n H uldschinsky, who i n 1919 found t h a t cod l i v e r o i l was n o t th e only c u r a tiv e a g e n t f o r r i c k e t s , and proved by a la r g e number o f c l i n i c a l o b s e rv a tio n s t h a t l i g h t , and e s p e c ia lly u l t r a ­ v i o l e t l i g h t , had e x c e lle n t c u r a tiv e a c tio n f o r human r i c k e t s . L a te r , 1922, Hess and Lindhagen, observed t h a t th e b lo o d c o n te n t o f in o rg a n ic phosphate d e c re a se s and in c r e a s e s w ith th e s e a s o n a l changes o f u l t r a - v i o l e t l i g h t from th e sun. R a ts p la c e d on a r ic k e ts - p ro d u c in g d i e t c o u ld be p r o te c te d a g a in s t r i c k e t s by s u n lig h t. U l t r a - v i o l e t l i g h t th e r e f o r e had th e same e f f e c t as th e su b stan ce v ita m in D. The co n n ectin g l i n k between th e s e a p p a re n tly u n re la te d o b s e rv a tio n s was th e t h i r d g re a t d isc o v e ry i n t h i s f i e l d . — R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. I n I 92U, Hess and Steenbock found sim u lta n e o u sly t h a t i t was by no means n e c e s sa ry t o expose th e r a c h i t i c anim al t o u l t r a - v i o l e t l i g h t p ro v id e d th e food was i r r a d i a t e d . t h i s d isc o v e ry was soon found. The i n t e r p r e t a t i o n o f The f o o d s tu f f s a s w e ll a s th e s k in c o n ta in su b stan ces w hich on i r r a d i a t i o n w ith u l t r a - v i o l e t l i g h t produce v ita m in D. v ita m in s D. These su b sta n c e s a re c a lle d p ro ­ T h e ir p re se n c e i s e a s i ly d em o n strated . V eg etab le o i l s in a c tiv e b e fo re i r r a d i a t i o n become a c tiv e a f t e r i r r a d i a t i o n . The n a tu r a lly - o c c u r r in g v ita m in D seems to be formed by photochem ical p ro c e ss e s from a p ro -v ita m in D. r i c h e r i n v ita m in D th a n w in te r m ilk . Summer m ilk i s T h is i s due la r g e ly t o th e p ro d u c tio n o f v ita m in D i n th e s k in o f th e an im a ls. The o r ig in o f v ita m in D i n f i s h e s i s n o t y e t c l e a r ly under­ s to o d . The cod f i s h l i v e s a t a d e p th where th e n e c e ssa ry amount o f u l t r a - v i o l e t l i g h t i s n o t c o n sid e red s u f f i c i e n t f o r th e form a­ t i o n of v ita m in D. Drummond (1) assum es t h a t th e cod p o s s e s s e s an e x c e p tio n a l power o f s to r in g v ita m in D. Coppens and Metz (2) found t h a t th e t i s s u e s o f th e lu n g s o f r a t s d e s tro y s v ita m in D. The absence o f lu n g s i n f is h e s m ight account f o r t h e i r la r g e s to r in g c a p a c ity . s y n th e s is o f A nother p o s s i b i l i t y would be a b io ch em ical v ita m in D i n f i s h e s . The in v e s tig a tio n o f i r r a d i a t e d v e g e ta b le o i l s gave e v i­ dence t h a t th e a c tiv e p r in c ip l e i s to be found in th e u n sa p o n if i a b l e p a r t o f th e o i l . examined. The p h y to s te r o ls , t h e r e f o r e , were A f te r i r r a d i a t i o n t h ^ became a c t iv e ; th e same - 5- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. a c t i v a t i o n was observed a f t e r th e i r r a d i a t i o n o f cru d e c h o le s­ te ro l. Thus i n 192$ i t was f i r s t b e lie v e d t h a t th e p ro -v ita m in D was a s te r o l* I n 1927 Pohl (1 )* in v e s tig a te d th e u l t r a - v i o l e t a b s o rp tio n o f c h o le s te ro l* The maximum was found a t 280 mu. D uring i r r a ­ d i a t i o n th e maximum i s s h if te d t o th e u l t r a - v i o l e t * A f te r a s h o r t i r r a d i a t i o n t h i s s h i f t was so c o n s id e ra b le t h a t a la r g e q u a n tity o f c h o le s te r o l was supposed t o have undergone a photo­ chem ical tra n sfo rm a tio n * The a n a ly a is o f th e i r r a d i a t e d m a te r ia l in d ic a te d i t t o be m o stly c h o le s te r o l , and a ls o seme a n t i r a c h i t i c su bstance* l i z a t i o n from a lco h o l* T his a c t i v i t y was l o s t by r e c r y s t a l ­ *Rosenheim and W ebster (3) o b ta in e d th e same r e s u l t , by p u r ify in g i r r a d i a t e d c h o le s te r o l by form a­ t i o n o f th e dibrom ide o r d ic h lo rid e * o r i g i n a l a c t i v i t y was l o s t . In so doing a l l o f th e C onsequently th e v ita m in D must be a s s o c ia te d w ith c h o le s te r o l o r th e phytostO T ols* of The study th e a b s o rp tio n s p e c tra o f p u re and c ru d e c h o le s te r o l i n d i ­ c a te d t h a t th e cru d e c h o le s te r o l had a maximum a t 280 mu w hereas th e p u r i f i e d c h o le s te r o l had a maximum a t 2I4.O mu. U" In 1927 Windeus (U) compared th e a b s o rp tio n s p e c tr a o f a l l th e known s t e r o l s w ith t h a t o f p r o -v ita m in D* They d i s ­ covered t h a t th e s t e r o l of e r g o t, e r g o s te r o l, had th e same a b s o rp tio n spectrum as p ro v ita m in D* They i r r a d i a t e d e r g o s te r o l and g o t a p ro d u c t 1000 tim es more a c tiv e th a n i r r a d i a t e d c h o le s te ro l* T h is proved e r g o s te r o l to b e p ro -v ita m in D* R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. I t was now assumed t h a t e r g o s te r o l i s a s s o c ia te d w ith th e known s t e r o l s , e s p e c ia lly th o se o f th e s k in . I t s p rese n c e i n s k in was supposed t o account f o r th e fo rm atio n o f v ita m in D upon exposure t o some so u rce of u l t r a v i o l e t lig h t* The re a s o n fear th e lo n g d ela y i n i s o l a t i n g th e n a tu r a lly - o c c u r r ­ in g p ro -v ita m in D i s e a s i ly se e n . I t s s o l u b i l i t y i s so much l i k e t h a t o f c h o le s te r o l t h a t i t was d i f f i c u l t t o s e p a ra te i t from c h o le s te r o l. A A«A A j. U d JL M W iC changed i n t o a ^3 j ^ v iia V a.1. ama#a I0m lA* tP x 'g U D o ^ j. wo. ^ ^n w w u -i.\x s tr o n g ly a n t i r a c h i t i c su b sta n c e th e i s o l a t i o n o f p ro -v ita m in D and o f v ita m in D from n a tu r a l so u rc e s was p o stp o n ed , and re s e a r c h was c o n c e n tra te d on th e a c t i v e sub­ s ta n c e s formed by i r r a d i a t i o n o f e r g o s te r o l . troublescane and slow . T his work was L i t t l e p ro g re s s was made u n t i l 1931 when WindauB i n Germar^, and Askew, B o u rd illo n and t h e i r c o l­ la b o r a to r s in England r e p o rte d th e i s o l a t i o n o f p u re c r y s t a l ­ l i n e v ita m in D from i r r a d i a t e d e r g o s te r o l. Windaus and h i s c o lle a g u e s r e a liz e d frcm th e b eg in n in g t h a t th e i r r a d i a t i o n o f e r g o s te r o l produced a m ix tu re o f com­ pounds. The f i r s t a n t i r a c h i t i c p ro d u ct is o l a t e d from i r r a ­ d ia te d e r g o s te r o l was a m ix tu re o f eq u a l m o lecu lar amounts o f v ita m in D w ith a second i r r a d i a t i o n compound c a l l e d lum i­ s te ro l. T his m ix tu re was d e s ig n a te d a s V itam in D^. A y e a r l a t e r W indaus, L in s e r t (^) and t h e i r c o lla b o r a to r s succeeded i n i s o l a t i n g p u re c r y s t a l l i n e v ita m in D. T his was now -7 — R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. d e s ig n a te d a s V itam in Ü2 « Thé name c a l c i f e r o l , used i n th e E n g lis h l i t e r a t u r e , was o r ig i n a l l y a p p lie d t o V itam in but was l a t e r t r a n s f e r r e d t o V itam in D2 » V itam in D2 and c a l c i ­ f e r o l a r e used synonymously. In th e fo llo w in g y e a r s o th e r p ro d u c ts o f i r r a d i a t i o n o f e r g o s te r o l w ere found by Windaus. They a r e arra n g e d in th e fo llo w in g p h o to -ch em ical s e r i e s : e r g o s te r o l lu m is te r o l > ta c h y s te r o l *■ S u p ra s te ro ls An e la a e n ta r y a n a ly s is o f th e s e p ro d u c ts gave th e s t r i k ­ in g r e s u l t t h a t a l l have th e same form ula a s e r g o s te r o l3 nam ely, C2fi^l|l|Q* s te ro ls . L ike e r g o s te r o l, th e y a l l a r e u n s a tu ra te d The f i r s t assum ption was t h a t th e a c tio n o f l i g h t on e r g o s te r o l produced m ain ly s t e r i c rearran g em en ts and s h i f t ­ in g o f th e double b o nds. F u rth e r in v e s tig a tio n s by I ,e t tr e * ( 6 ) showed t h a t th e p ro c e ss was f a r more co m p licated th a n had been th o u g h t i n th e b e g in n in g . A ll p ro o f t o d a te h as shown t h a t th e a n t i r a c h i t i c p r o p e rty depends on th e p re sen ce o f a f r e e hydroxyl group p r e s e n t i n th e 3 r in g s k e le to n , p ro v id e d w ith t h r e e co n ju g ated double bonds i n c lo s e ly lim ite d p o s itio n s . The to x i c e f f e c t i s produced by su b stan ces w hich c o n ta in two or th r e e co n ju g ated double bonds i n th e 3 r in g s k e le to n . — 8— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. T h is i s svnnmed up by th e fo llo w in g ta b l e ta k e n from F riedm ann's book, " S te r o ls and R e la te d Compounds", p g . 60 (7)* 'I Rings E rg o s te ro l L u m istero l T a c h y ste ro l V itam in Dg T o x is te ro l S u p ra s te ro l I S u p ra s te ro l I I h k 3 3 3 (? ) U k (3) Double Bonds C onjugated Double Bonds 3 3 h h h (? ) 3 3 (U) I s o la t e d A n tira c h itic Double Bonds 2 2 3 3 2 (?) — — 1 1 1 1 2 (?) — 3 (W Toxic — “ “ + + i — — — The s u c c e s s fu l i s o l a t i o n o f a su b sta n c e w ith h ig h ly a n t i ­ r a c h i t i c p r o p e r tie s from i r r a d i a t e d e r g o s te r o l b r in g s th e r e ­ s e a rc h back t o i t s s t a r t i n g p o in t—th e i s o l a t i o n o f v ita m in frcan n a tu r a l sources* Was th e v ita m in D i n cod l i v e r o i l s im ila r t o o r i d e n t i c a l w ith th e v ita m in from i r r a d i a t i o n o f e r g o s te r o l? Much evidence showed t h i s t o be t r u e . The chem ical co m p o sitio n , s o l u b i l i t y and r e a c tio n s tow ards d i f f e r e n t re a g e n ts was s im ila r ; i t was to x ic i n h ig h d o se s, and o f fe re d th e same r a tio ;b e tw e e n to x ic and a n t i r a c h i t i c doses a s v ita m in Dg* However, i t s p h y sio ­ l o g i c a l a c t i v i t y in d ic a te d t h a t th e r e was a d iffe re n c e * A ccm- p a ra so n o f t h e i r a n t i r a c h i t i c a c t i v i t y showed no d if f e r e n c e i n r a t s w ith v ita m in Dg, b u t i n th e case o f c h ic k s th e v ita m in I) from f i s h o i l so u rc e s was much more p o w e rfu l. Windaus su b se q u e n tly d isc o v e re d th e e x is te n c e o f a n o th e r p ro -v ita m in D; nam ely, 7- d e h y d ro c h o le s te ro l. T his compound b e a rs th e same r e l a t io n s h i p t o th e v ita m in D i n f i s h l i v e r o i l s R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. a s e r g o s te r o l does t o V itam in T>2 * I n 1936 W erder and Windaus ( 8) i s o l a t e d v ita m in from th e i r r a d i a t i o n p ro d u c ts o f 7-d e h y d ro o h o le s te ro l * A .T. 10 o r d ih y d ro ta c h y s te ro l a d e r r i v a t i v e o f an i r r a ­ d i a t i o n p ro d u ct o f e r g o s te r o l, i s known t o have im p o rta n t e f f e c t s upon calciu m and phosphorous m etabolism . T h is has been amply shown n o t only by H oltz (9) and o th e r s , i t s d is c o v e r e r s , who showed i t s a b i l i t y to r a i s e serum calcium c o n c e n tra tio n a f t e r rem oval o f th e p a r a th y r o id s , b u t a ls o by th e s tu d ie s o f A lb rig h t (1 0 ,1 1 ) and c o lla b o r a to r s on th e m etabolism o f calcium and phosphorus i n id io p a th ic hyp o p arath y ra id is m and i n r i c k e t s . They compared th e e f f e c t s o f A .T. 10 w ith th o s e o f v ita m in D2 and p a r a th y r o id e x t r a c t . I t i s t h e i r t h e s i s t h a t v ita m in D has two p rim ary a c tio n s j t o in c r e a s e p h o sp h ate e x c re tio n in th e u r in e , and t o in c r e a s e calciu m a b s o rp tio n from th e g a s t r o i n t e s t i n a l t r a c t . P a ra th y ­ r o id hormone does n o t in c re a s e a b s o rp tio n o f calcium b u t does in c r e a s e phosphate i n th e u r in e m arkedly. A ccording t o th e s e a u th o rs A .T. 10 has th e same two a c tio n s a s v ita m in D, b u t in c r e a s e s calcium a b s o rp tio n l e s s and p h o s p h a te .e x c re tio n m ore. I n c o n firm a tio n o f t h i s h y p o th e sis i t has been shown t h a t A .T. 10 w i l l p re v e n t o r cu re r i c k e t s i n r a t s produced by a low calcium -low phosphorus d i e t in th e same dosage t h a t i s w ith o u t e f f e c t on th e r i c k e t s produced by th e c l a s s i c a l hig h calcium -low phosphorous d i e t s by S hohl, F a r r and F o rb e r(1 2 ). - 10- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. I t has been r e p o r te d by Hornapp (13)» t h a t A.T. 10 has l/UOO t o / 1 /6 0 0 th e a n t i r a c h e t i c p o ten cy o f v ita m in Dg* Shohl and F orber (11+) have shown t h a t A.T. 10 i n amounts w hich approxim ate to x ic d o sag e, i s e f f e c t i v e in p re v e n tin g e x p e rim e n ta l r i c k e t s i n r a t s f e d a h ig h -c alciu m -lo w phosphorus r a e h ito g e n ic d i e t . I t was n o te d by Hess and Suppléé (1$) t h a t i r r a d i a t e d e r g o s te r o l i s much l e s s e f f e c t i v e th a n v ita m in D frcm n a tu r a l so u rc e s in prom oting norm al c a l c i f i c a t i o n i n th e c h ic k . These o b s e rv a tio n s have been amply confirm ed, b u t no s a t i s f a c t o r y e x p la n a tio n o f th e phenomenon has been fo u n d . R e c e n tly , C c r r e ll and Wise (16) have shown t h a t one a n t i r a c h i t i c r a t u n i t o f d e h y d ro ta c h y s te ro l i s e q u iv a le n t i n th e c h ic k u n i t s o f th e U .S .P . R eferen ce O il. t o betw een i+ and $ A s im ila r r e la t io n s h ip o f dose l e v e l t o r a t i o o f e f f e c tiv e n e s s f o r th e c h ick has been shown t o e x i s t betw een v ita m in D2 and n a tu r a l v ita m in D by M assengole and B e lls ( 1 7 ). Summarizing th e r e e x i s t s t h i s p e c u lia r s it u a t i o n : V itam in D2 i s e q u iv a le n t i n th e r a t t o 1+0,000 U .S .P . U n its p e r m i l l i ­ gram (Cawood 18) b u t i n th e c h ic k to on ly ab o u t 1000 U .S .P . U n its , Remp and M a rsh a ll (1 9 ). V itam in i s e q u iv a le n t t o 1+0,000 U .S .P . U n its i n b o th r a t and ch ick en and c r y s t a l l i n e d ih y d ro ta c h y s te ro l i s e q u iv a le n t t o 80 U .S .P . U n its p e r m i l l i ­ gram b u t t o about 360 u n i t s i n th e chicken* M ost r e c e n t work shows v ita m in D2 and Dj e q u iv a le n t t o about ^0,000 u n its p e r m illig ra m . - 11- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. R u s s e ll and co-w orkers (20,21) have atte m p te d t o e x p la in ^ th e low e f f e c tiv e n e s s i n th e ch ick by showing t h a t t h e amounts o f v ita m in D2 and n a tu r a l v ita m in D re c o v e ra b le from th e fe c e s o f th e c h ick a r e q u ite com parable (2 6 .5 and 3U*1^ o f th e dose a d m in iste re d r e s p e c tiv e ly ) * I t was n o t d e f i n i t e l y s e t t l e d w h eth er o r n o t a i ^ o f th e rem ainder i n th e d ig e s tiv e t r a c t . o f th e dose was d e s tro y e d They d id show t h a t th e v ita m in was e f f e c tiv e i n th e ch ick when given e i t h e r by mouth o r i n t r a p e r i to n e a l ly , w hereas s ix tim es a s many u n its o f v i t - ^ n D2 g iv en i n t r a p e r i t o n e a l l y i n o i l f a i l e d t o promote e q u iv a le n t c a lc ific a tio n . From th e s e s tu d ie s th e y concluded t h a t th e low e f f e c tiv e n e s s o f v ita m in D2 i n th e ch ick en could n o t b e a t t r i ­ b u te d t o f a i l u r e o f a b s o rp tio n frcm o r d e s tr u c tio n in th e d i ­ g e s tiv e tr a c t* McChesney and w orkers (2 2 ,2 3 ,2 L ), found t h a t th e r a t i o s o f o r a l e f f e c tiv e n e s s i n th e chick o f t o D2 was 35s i and o f d ih y d ro s ta c h y s te ro l t o D3 = U«5 s l confirm ing th e work of o th e r w o rk ers. Most w orkers have found v ita m in D3 , D2 , and DHT m ost e f f e c t i v e when g iv e n o r a l ly . These a u th o rs (25) have a ls o found i n stu d y in g o f th e o r a l and p a r e n te r a l u t i l i z a ­ t i o n o f v ita m in s D2 and D3 t h a t th e l a t t e r (D3 ) i s somewhat b e t t e r absorbed from th e d ig e s tiv e t r a c t . C o r r e ll and Wise (26) found A.T. 10 had an a n t i r a c h i t i c p o ten cy o f 300 u n i t s p e r mgm by r a t te c h n iq u e . They a ls o found t h a t th e a c tio n o f A .T. 10 resem b les t h a t o f v ita m in D 12 — — R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. r a t h e r th a n th e p a ra th y ro id hormone* R at u n it f o r r a t u n it th e y found A.T. 10 fo u r tim es more e f f e c t i v e a s an a n t i r a c h i t i c th a n v ita m in D from cod l i v e r o i l . On a r a t u n it b a s is th e y a ls o found A.T* 10 t o be n e a r ly 200 tim es more e f f e c tiv e on th e c h ic k th a n Dg* Jones and R apaport (2?) found t h a t i r r a d i a t e d e r g o s te r o l fe d t o dogs on b a s is o f 30,000 u n its o f v ita m in D p e r k ilo o f body w eight produced a l^ e r c a lc e m ia * S chneider and Steenback (28) found t h a t a s y n th e tic d i e t low in phosphorous and f r e e o f v ita m in D produced r i c k e t s , b u t when supplem ented w ith s u f f i c i e n t phosphorous and v ita m in D, i t s u f f ic e d as th e s o le so u rce of n u t r i e n t s f o r th r e e s u c c e ssiv e g e n e ra tio n o f r a t s . They found t h a t v ita m in D induced th e u t i l i z a t i o n o f P by bone, th e re b y d e p riv in g th e s o f t t i s s u e s o f t h e i r supply of P , which i n tu r n in h ib ite d growth* C o rre ll and Wise (29) found t h a t th e a n t i r a c h i t i c v ita m in from s e v e ra l so u rces e x e rte d v ary in g d eg re e s o f in flu e n c e on th e p h o sp h atase v a lu e s i n chicken serum* God l i v e r o i l was more e f f e c tiv e th a n i r r a d i a t e d e r g o s te r o l and th e tu n a l i v e r o i l s i n red u cin g serum p h o sp h atase a c t i v i t y in th e growing c h ic k . In 1938 R apaport and G uest (3 0 ), r e p o rte d t h a t in r a t s g iv en a h ig h Ga and low P d i e t (ra e h ito g e n ic ) th e development o f r i c k e t s was a s s o c ia te d w ith d e c re a se s f i r s t o f in o rg a n ic 4 P and o f a d e o s in e trip h o s p h a te (A*T*P*) and th e n o f diphosphog ly c e r ic a c id i n th e blo o d c e lls * In t h e h e a lin g o f r i c k e t s , —13— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited without p erm ission. t h e re v e rs e o f t h i s p ro c e ss "was found to ta k e p la c e . Th^ su g g e st th e fo llc w in g scheme: T rio se p h o sp h a te - p y ru v ic a c id p h o sp h o g ly crate H exosephosphate In o rg a n ic P A d en o sin etrip h o sp h ate Jo n es (31) , in 19UUj found sm all doses o f v ita m in D g iv en t o r a t s k e p t on a low p h o sp h orus-high calcium d i e t produce a iQ rpercalcem ia. I r r a d i a t e d e r g o s te r o l, p u re c a l c i f e r o l , o r i r r a d i a t e d 7 -d e h y d ro c h o le s te ro l w ere e q u a lly e f f e c t i v e , w hereas A.T. 10 was much l e s s e f f e c t i v e . The degree o f hypercalcem ia produced was dependent on th e amount o f v ita m in D a d m in is te re d . The deg ree o f growth i n h i b i t i o n was d i r e c t l y r e l a t e d t o th e degree o f l^ypercalcem ia. C a lc if ic a tio n a s judged by femur ash was g r e a t e r i n th o se c a se s i n w hich th e r e was a d e f i n i t i v e in ­ c re a s e i n calciu m . Thus t h i s d a ta gave a d d itio n a l su p p o rt i n fa v o r o f th e view t h a t v ita m in D in c r e a s e s t h e a b s o rp tio n o f calcium from th e i n t e s t i n e s and s e rv e s to i n d i c a t e th e im p o rt­ ance o f th e calcium -phosphorus p ro d u c t o f th e serum in c a l c i f i ­ c a tio n . W addell (32) has shown t h a t i r r a d i a t e d cru d e c h o le s te r o l i s e q u iv a le n t t o cod l i v e r o i l and s u p e rio r t o i r r a d i a t e d e r g o s te r o l i n p re v e n tin g th e developm ent o f le g weakness in c h ic k s . B ethke (33) has su g g ested t h a t p erh ap s two o r more f a c t o r s may be p r e s e n t i n i r r a d i a t e d e r g o s te r o l, one o f w hich -lU - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. i s e f f e c t i v e i n b o th th e chicken and th e r a t and th e o th e r only i n th e r a t . T his d o e s n 't seem t o h o ld b ecau se i t has been shown t h a t c r y s t a l l i n e v ita m in Dg and V io a te ro l w ere found t o have r e l a t i v e l y th e same potency when t e s t e d on r a t s as w e ll a s on chicks* Thus i n th e c h ick i t seems t o be a m a tte r o f th e d eg ree o f u t i l i z a t i o n o f th e s e v e ra l forms* McChesney (3i|) found t h a t d if f e r e n c e s o f a b s o rp tio n and o r d e s tr u c tio n can n o t a lo n e account f o r th e v e ry h ig h doses o f v ita m in Dg needed t o produce th e hypercalcém ie e f f e c t i n c h ic k s . I t r e q u ir e d 12$ mgms p e r kg o f v ita m in Dg t o r a i s e th e serum calcium t o a l e v e l o f 12 m g n ^ . T h is l e v e l was a ls o reac h e d in th e ch ick - w ith -a dose o f 10 mpi p e r - k g - o f v ita in in n o t a f t e r a dose o f 5 mgm p e r kg. Caward but (35 ) in a group o f s e p a ra te r e s e a r c h e r s e s ta b lis h e d t h a t v ita m in s Dg and have th e same a n t i r a c h i t i c p o ten cy i n r a t s ; i . e . , UOjOOO I .U . p e r mgm. R u s s e ll (36) i n a review concluded t h a t th e v ita m in was 30 tim e s as e f f e c t i v e as Dg and 6 tim es a s e f f e c t i v e a s A .T. 10 i n th e chick* Toxic e f f e c t s were observ ed when cod l i v e r o i l was fe d a t ab o u t 5000 tim e s , th e c a l c i f e r o l a t ab o u t 100 tim e s , and A.T. 10 a t about 10 tim es th e l e v e l s n e c e s sa ry f o r norm al m in e ra l m etabolism . Morgan and S h im ato ri (37) i n t h e i r work on dogs fe d v a rio u s so u rces o f v ita m in Dg and t h a t th e r e was produced a prolonged b lo o d calcium r i s e , which was g r e a t e s t i n th e case o f i r r a d i a t e d e r g o s te r o l fo llo w ed by D e is te r o l and Tuna l i v e r o i l . —1$— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. Sherman and Booker (38) found t h a t when th e d i e t was com­ p osed o f n a t u r a l food s t u f f s a rran g ed so a s t o make calcium th e s o le s ig n i f i c a n t v a r ia b le , t h a t a t each o f th e ages s tu d ie d , t h e amount o f calcium i n th e body o f th e growing anim al v a r ie d i n accordance w ith th e calcium c o n te n t o f th e d i e t . Lanford and Sherman (39) found on d i e t s o f d i f f e r e n t calcium le v e ls t h a t alth o u g h th e p e rc e n ta g e o f body calcium f i n a l l y reach ed on th e two h ig h e r calcium r a t io n s were th e same, th e r a t e o f c a l c i f i c a t i o n was somewhat g r e a te r on th e d i e t p ro v id in g .8 p e r c e n t th e n on t h a t w ith »6l\ p e rc e n t o f calciu m . Jo n e s, R ap ap o rt and Hades (UO), i n working w ith dogs, r e p o r t t h a t th e so u rce o f th e calcium i n th e hypercalcem ia produced by i r r a ­ d ia t e d e r g o s te r o l i s th e fo o d and n o t th e body t i s s u e . G reen- w ald (W.) o bserved t h a t i t was im p o ssib le t o e s t a b l i s h a d e f i n i t e r e l a t io n s h ip betw een th e c o n c e n tra tio n s o f calcium , p h o sp h ate , and p r o te in in th e s e ra o f d o g s. lELein and R u s s e ll (^2) found t h a t o f th e i r r a d i a t e d erg o s­ t e r o l fe d t o c h ic k s d u rin g th e f i r s t U weeks o f l i f e , 26.$ p e r­ c e n t was re c o v e re d in th e d ro p p in g s. When cod l i v e r o i l was u sed U3 .1 p e rc e n t was re c o v e re d . O uthouse, Sm ith, and Twomey (b3) found t h a t r a t i o n s con­ t a i n i n g l a c to s e and cod l i v e r o i l caused g r e a te r r e t e n t i o n o f ca lciu m , phosp h o ru s, and magnesium th a n r a t io n s c o n ta in in g s ta r c h o r s u c ro s e . - 16- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. McLean (14;) found t h a t d ih y d ro ta c h y s te ro l and c a l c i f e r o l a r e h ig h ly e f f e c t i v e in in c r e a s in g th e c o n c e n tra tio n o f c a l ­ cium i n th e b lo o d and i n r e l ie v in g th e symptoms r e s u l t i n g from hypocalcem ia i n c a s e s o f in s u f f ic ie n c y o f th e p a r a th y ro id s . He found no to x ic e f f e c t s nrhen a d m in iste re d over c o n s id e ra b le p e rio d s o f tim e . A lb rig h t and co-w orkers (U5) w orking w ith human s u b je c ts e s ta b lis h e d th e fo llo w in g r e s u l t s w ith v ita m in D, A.T. 1 0, and p a ra th y ro id hormone; Calcium A b so rp tio n V itam in D A .T. 10 P a ra th y ro id e x t r a c t + 4 + + 0 Phosphorus e x c re tio n i n th e u r in e + 4 4 4 4 4 4 4 Cohn and Greenberg (i;6 ) in t h e i r work w ith rad io p h o sp h o ru s an d v ita m in D concluded t h a t th e in flu e n c e o f v ita m in D on th e d e p o s itio n o f in o rg a n ic phosphorus i n th e bone o f r a c h i t i c r a t s m ust be a s c rib e d t o a more d i r e c t e f f e c t th a n one m erely r e s u l t ­ in g from an in c r e a s in g a b s o rp tio n . They th o u g h t t h a t v ita m in D m ust have a s p e c i f i c e f f e c t on o rg an ic bone phosphorus and a s p e c i f i c r o l e o f t h i s f r a c t i o n i n bone fo rm atio n i s in d ic a te d . McDonald and M assengole (U7) found t h a t m assive doses o f i r r a d i a t e d e r g o s te r o l a d m in iste re d t o p u l l e t s gave on ly 185 tim e s more v ita m in D p o ten cy i n th e eggs th a n d id one te n th o u sa n d th a s much v ita m in ( in th e form of cod l i v e r o i l ) . R u s s e ll, T a y lo r and W ilcox, i n work on c h ic k s (J48) o b ta in e d e s s e n t i a l l y th e same bone ash p e rc e n ta g e s when v ita m in D was a d m in iste re d by i n j e c t i o n o r by c a p s u le , - 17- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. B. A n t i r a c h i ti c Compounds and Side P ro d u cts V itam in D i s shomi t o be a g e n e tic name f o r a c la s s of su b sta n c e s p o s s e s s in g a n t i r a c h i t i c p r o p e r t i e s . C e rta in mem­ b e r s o f th e group have b een p u r if i e d , and t h e i r s tr u c tu r e s e lu c id a te d . The commonest p ro ced u re f o r th e fo rm a tio n o f an a n t i r a c h i t i c su b stan ce i s i r r a d i a t i o n w ith u l t r a - v i o l e t i n w hich e r g o s te r o l and 7 dehydro c h o le s te r o l a r e th e u su a l s t a r t i n g ccxapouhds. A n t i r a c h i ti c a ll y a c tin g p ro d u c ts may a ls o be produced from c h o le s te r o l by a v a r i e t y o f chem ical p ro c e d u re s . These p ro d u c ts have however n o t a t t a i n e d com aer- c i a l im p o rta n ce . (U9). The more common a n t i r a c h i t i c compounds a r e l i s t e d below : (1) V itam in Dg, C a lif e r o l . e r g o s te r o l. The p ro d u c t o f i r r a d i a t i o n o f T his o ccu rs in many i r r a d i a t e d foods o f p l a n t o r ig i n . (2) V itam in Dj^, 2 2 - d ih y d r o o a lc if e ro l. The p ro d u ct o f i r r a d i a ­ t i o n o f 22- d ih y d r o e r g o s te r o l, (3 ) E r tr o n . T h is i s e l e c t r i c a l l y a c tiv a te d e r g o s te r o l and may p o s s ib ly be a d i s t i n c t e n t i t y . (li) The p ro d u c t from th e a c tio n o f n i t r i t e s on e r g o s te r o l . T h is su b sta n c e i s f e e b ly a c tiv e b u t i s c e r t a i n l y d i f f e r e n t from o th e r form s o f v ita m in D w hich a r e d e stro y e d by n itrite s . (^ ) The p ro d u c t o f i r r a d i a t i o n o f 22, 2 3 -o x id o - e r g o s te r o l. T h is su b sta n c e i s a ls o f e e b ly a n t i r a c h i t i c . —18— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. (6 ) V itam in B y i r r a d i a t e d 7 -d e h y d ro c h o le ste ro l* T h is has been shown t o be th e c h ie f farm o f th e v ita m in found in c e rta in f is h o ils . (7 ) The p ro d u c t o f i r r a d i a t i o n o f 7 - d e h y d r o s ito s te r o l. p ro b ab ly o ccu rs t o a sm all e x te n t in T his n a tu r e , a lth o u g h i t i s o nly f e e b ly a n t i r a c h i t i c . I t h as been shown, t h a t in a d d itio n t o th e s e compounds, th e r e i s a p o s s i b i l i t y t h a t s t i l l o th e r form s o f v ita m in D may e x i s t i n cod l i v e r o i l o r i n o th e r n a t u r a l p ro d u c ts . I t h as been shown t h a t a r e a l and s ig n if ic a n t d if f e r ­ e n ce, b o th q u a n tita tiv e and q u a l i t a t i v e , e x i s t s betw een th e c h ic k and th e r a t i n th e r e a c tio n o f th e v a rio u s form s of v ita m in D, and i t is concluded t h a t r e c e n t s tu d ie s o f th e a c tio n o f s e v e r a l form s o f v ita m in D show t h a t th e human r e ­ a c t s i n g e n e ra l i n th e same manner a s th e r a t t o th e v a rio u s v ita m in s D. I n th e i r r a d i a t i o n o f e r g o s te r o l th e r e a r e form ed a s p re v io u s ly m entioned s e v e ra l s id e o r r e l a t e d p ro d u c ts such a s lu m is te r o l, ta c h y s te r o l, t o x i s t e r o l , s u p r a s te r o l I , and Supra­ s te ro l I I . T h e ir a n t i r a c h i t i c a c t i v i t i e s a r e r e l a t i v e l y s l i g h t a lth o u g h c e r ta in w orkers have shown some of t h e i r d e r r i v a t i v e s such a s d ih y d ro ta c h y s te ro l, t o have a p p re c ia b le a n t i r a c h i t i c e f f e c t and a p a r t i c u l a r l y marked in flu e n c e on th e b lo o d calcium l e v e l . -19- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. G» E f f e c ts o f A n t i - r a c h i t i c Ccmpounds and S id e -p ro d u c ts on M ineral I^etatiolïsm The s tu d y o f th e in flu e n c e o f v ita m in D on m in era l m etabolism h as occupied th e a t t e n t i o n o f many in v e s tig a to r s ^ and i t should b e a p p re c ia te d a t once t h a t such in v e s tig a tio n s o f f e r maiy d i f f i c u l t i e s . As a conseque. s e , th e o b s e rv a tio n s have n o t been uniform , and th e c o n c lu sio n s have l e d , th e r e ­ f o r e , t o much c o n fu sio n . Eddy and D a lld o rf (^0) in t h e i r work on th e calcium in ta k e o f r a t s found t h a t norm ally th e r a t lo s e s 20 t o UO p e r c e n t o f th e calcium in ta k e and 15 p e r c e n t o f th e phosphorus i n th e f e c e s , w hereas i n r a c h i t i c anim als th e calcium lo s s amounts t o 90-100 p e r c e n t and phosphorus 60-70 p e r c e n t. When v ita m in D i s a d m in iste re d t o r a c h i t i c r a t s r e s t o r e s th e p ro ­ p o r tio n a te a b s o rp tio n . T h is makes i t appear t h a t th e f u n c tio n o f v ita m in D i s t o prom ote a b s o rp tio n o f calcium and phosp h o ru s. The p ro c e ss i s somewhat more co m p licated how ever. Kem and Montgomery ( 5 l) found t h a t on h ig h do ses o f v ita m in D, th e r e was an in c re a s e d i n t e s t i n a l a b s o rp tio n o f calcium and t o a l e s s e x te n t phosphorous. T ay lo r and Weld (52) i n t h e i r work on r a t s found t h a t v ita m in D h y p ercalcem ia was due to m o b iliz a tio n o f calcium f r o a th e bones and t h a t th e v ita m in d e p re sse d th e power o f th e i n ­ t e s t i n e t o e x c r e te calciu m . B auer, M arble, and G la f fin (53) found no e f f e c t on n itr o g e n m etabolism from th e in g e s tio n of v ita m in D over p e rio d s up t o a month o r m ore. - B lood calcium 20 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. and phOBphonis l e v e l s were lik e w is e u n a f fe c te d . They found th e m in e ra l b a la n c e s n o t a p p re c ia b ly a f f e c t e d . In an i n t e r e s t i n g stu d y o f th e e f f e c t s o f v ita m in D on c a lv e s , Duncan etnd Huffman (5U) found t h a t th e r e was b u t l i t t l e a l t e r a t i o n o f b lo o d calciu m , w hereas in o i'g an ic phos­ phorus was in c re a s e d d e f i n i t e l y and r e tu r n e d t o normal l e v e l s a f t e r f o u rte e n d a y s . There was d ecrease d f e c a l and in c r e a s e d r e n a l e x c re tio n o f b o th elem en ts. W a llis , P alm er, and G u llick so n (55) r e p o r te d t h a t a c a l cium -phosphorus r e t e n t io n r a t i o o f 2 was p r e s e n t i n b o th normal and r a c h i t i c c a lv e s and t h a t v ita m in D in c r e a s e d r e t e n t io n w ith ­ o u t a l t e r i n g th e r a t i o . P atro h a rd en and C h itr e (56) claim ed t h a t I1.500 u n i t s o f v ita m in D fe d d a i l y to a d u lt r a t s in c r e a s e d r e n a l e x c re tio n o f calciu m , phosphorus and n itr o g e n . Reed, S tru c k , and S teck (57) i n t h e i r m in e ra l b a la n c e stusfy on humans w ith a r t h r i t i s , could re a c h no d e f i n i t e c o n c lu sio n s b ecause o f such v a r i a t i o n s . N ico lay sen (5 8 , 59, 60, 6 1 , 62, and 63 ) i n a s e r i e s o f s tu d ie s on r a c h i t i c r a t s has shown t h a t th e r a t may a d a p t i t s e l f t o e x c e ss v ita m in D w hich would cause in c o n s is te n t r e s u l t s as i n th e c a se o f th e humans w ith a r t h r i t i s . He a ls o found t h a t w ith v a ry in g l e v e l s o f calcium added t o t h i s d i e t , th e o u tp u t o f endogenous phosphorus i n th e fe c e s was p r o g r e s s iv e ly in c r e a s e d , b u t t h e v ita m in g e n e r a lly i n h i b i t e d th e in c r e a s e t o scane e x te n t. W ith v a ry in g l e v e l s o f phosphorus in th e d i e t , th e f e c a l o u tp u t - 21 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. o f calciu m was lik e w is e p r o g re s s iv e ly in c r e a s e d and a g a in th e in c r e a s e was l e s s when v ita m in D was g iv e n . I t i s a p p a re n t t h a t th e r e i s no c o n v e n tio n a l p i c t u r e of m in e ra l m etabolism in a r t h r i t i s and t h a t th e r e i s no c h a r a c te r ­ i s t i c resp o n se t o v ita m in D t h a t can be c o n sid e re d s p e c i f i c in th is r e la tio n . A lso i t i s a p p a re n t t h a t th e v ita m in i s cap ab le o f p ro d u cin g o p p o s ite r e s u l t s under d i f f e r e n t c o n d itio n s . What f a c t o r s a re r e s p o n s ib le f o r e s ta b lis h in g th e p o in t o f re v e rsia l i s n o t e n t i r e l y c l e a r , b u t c e r t a i n l y th e s i z e o f th e dose i s n o t th e o nly f a c t o r in v o lv e d . The a b s o lu te amounts o f calcium a s w e ll a s th e calcium -phosphorus r a tio y th e s t a t e o f th e d ig e s tiv e t r a c t a s w e ll as o f th e l i v e r , a r e a l l co n cern ed . There may be many o th e r im p o rta n t in f lu e n c e s . D. E f f e c ts o f A n t i r a c h itic Compounds and S id e -p ro d u c ts on Blood C o n s titu e n ts S in ce th e b lo o d sta n d s a s an in te rm e d ia ry betw een th e a lim e n ta ry c a n a l, th e p e r ip h e r a l t i s s u e s , and th e k id n e y s, i t i s w here one would ex p ect to lo o k f o r some c lu e as t o th e n a tu re o f th e m e ta b o lic re sp o n s e s t o v ita m in D. A lte r a tio n s i n b lo o d com position a re n o t alw ays s p e c i f ic and o f te n cannot be i n t e r p r e t e d a lo n e , y e t c e r t a i n tre n d s a r e re c o g n iz a b le as more o r l e s s i n d i c a t iv e o f changes i n th e t i s s u e s . A lthough i t i s p ro b ab le t h a t to o much s t r e s s has been l a i d on c e r t a i n b lo o d changes in re sp o n se t o v ita m in D, y e t i n th e absence o f b e t t e r c r i t e r i a i t has been n e c e s sa ry t o make use o f such 22 — — R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. d a ta i n seek in g in fo rm a tio n o f i t s a ctio n * Schm idt and Greenberg (6U) i n t h e i r s tu d ie s on some o f th e e l e c t r o l y t e s i n body f l u i d s found th e p h y s io lo g ic a l ran g e f o r calcium i n th e b lo o d o f humans t o ran g e betw een 9 and 1 1 .^ . The norm al b lood calcium l e v e l i u most common la b o r a to r y anim als i s o f about th e same o rd e r as f o r man except i n c a se o f b ir d s d u rin g th e la y in g season* S p ecies d if f e r e n c e s a re n o t r e a d i l y e x p la in e d . Q u ite commonly one f in d s sta te m e n ts t o th e e f f e c t t h a t th e a c tio n o f v ita m in D i s to produce hypercalcem ia o r hyperphos­ phatem ia o r b o th , y e t th e r e i s abundant ev idence t h a t e i t h e r one may occur in d ep e n d en tly o r t h a t n e ith e r may fo llo w t r a t i o n o f la r g e amounts o f v ita m in D* th e adm inis­ Very o fte n th e e x p e ri­ m en tal c o n d itio n s a r e n o t s u f f i c i e n t l y s ta n d a rd iz e d to -e n a b le one t o d eterm in e j u s t wty a g iv en v a r i a t io n o ccu rs; i n f a c t , even when a l l c o n d itio n s a re as w e ll s ta n d a rd iz e d a s p o s s ib le , i t may s t i l l be d i f f i c u l t t o account f o r th e s e v a r ia tio n s * There i s now in c o n tr o v e r tib le ev idence t h a t a h ig h degree o f hyp ercalcem ia o f long d u r a tio n i s co m p atib le w ith l i f e . S u sta in e d hyperphosphatem ia i s much more l i k e l y t o d is tu r b body f u n c tio n s . Some y e a rs ago Reed and Leed su g g ested t h a t th e p rim ary g ro ss e f f e c t o f v ita m in D i s one o f s t a b i l i z i n g calcium -phosphorus m etabolism , r e g a rd le s s o f d i r e c t io n . Not many o b se rv a tio n s have been r e p o r te d on th e e f f e c t s o f v ita m in D on th e formed elem ents o f th e b lo o d , P h i l l i p s and -2 3 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. c o llo b o ra to r s (65) have shown v ita m in D t o in c re a s e th e throm bocytes o f th e blood and to d e c re a se c o a g u la tio n tim e in r a t s . Sure and Kik (66) in t h e i r s tu d y o f heanatopoisis in r a t s found t h a t anemia o f r i c k e t s i s n o t due to a v ita m in D d e f ic ie n c y . T a y lo r, Weld, and B ranion and Kay (6 ?) re p o rte d t h a t v ita m in D g r e a tly reduced th e phosphatase c o n te n t in th e r a t , r a b b i t , fo w l, and dog. L eenhardt and co-w orkers (68) i n t h e i r s tu d ie s o f r i c h i t i c c h ild re n found t h a t th e a c id s o lu b le phos­ phorous was in c re a s e d m arkedly w hereas th e p h o sp h atase a c t i v i t y was d e c re a se d . Dreyer and Reed (69) worked w ith a group o f a r t h r it ic p a tie n ts fed m assive doses o f vitam in D and found th a t th e serum phosphatase a c t iv it y was apparently o f l i t t l e use as an o b je c tiv e d ia g n o stic t e s t in a r t h r it i s , because o f th e wide v a r ia tio n in v a lu es found. In summary, s tu d ie s on b lo o d showed t h a t th e o n ly con­ s t i t u e n t s a f f e c te d i n any c o n s is te n t manner by m assive doses o f v ita m in D w ere t o t a l calcium (upw ard), c h o l e s te r o l, g lu ­ c o se , ph o sp h atase (g e n e ra l tr e n d downward), and phosphorus (upw ard). D ih y d ro ta c h y ste ro l has b een shown t o cause in c r e a s e s i n b o th phosphorus and calcium and a downward tre n d i n phos­ p h a ta s e a c t i v i t y . —2l|— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. E . T o x ic ity o f A n t i r a c h iti c Ccaapotmds and S id e -p ro d u c ts The volum inous d is c u s s io n o f th e to x ic e f f e c t s o f v ita m in D has been co m p licated by a number o f co n fu sin g f a c t o r s . th e f i r s t p la c e s ta n d a rd term in o lo g y In i s s t i l l la c k in g . "H y p erv itam in o sis D", and "V itam in D in to x ic a tio n " , have a l l been used lo o s e ly and in te rc h a n g e a b ly to d e s ig n a te th e same p h y s io lo g ic s t a t e . Evidence h as shown t h a t th e r e i s a d e f i n i t e d i s t i n c t i o n t o be made among th e s e te rm s. T here has a ls o been a g e n e ra l f a i l u r e t o r e c o rd q u a n ti ta tiv e ly th e dosage ox v i t a ­ min D i n r e l a t i o n t o body w eight and t o in d i c a t e th e d u r a tio n o f a d m in is tr a tio n . M oreover th e s p e c ie s v a r i a t i o n in s u s c e p ti­ b i l i t y h as v e ry o f te n n o t been ta k e n i n t o a c c o u n t. Then to o , th e p u r i t y o f th e v ita m in D p r e p a ra tio n s used was u n q u estio n ab ly a f a c t o r o f im portance i n a l l th e work on t h i s problem up t o th e p e r io d when c a l c i f e r o l o r v ita m in D2 was made a v a ila b le . Most work t h e r e a f t e r d id n o t d i f f e r e n t i a t e betw een th e e f f e c t s produced by th e e a r ly impure p r e p a ra tio n s and th o se o f p u re c a l­ c ife ro l. F in a lly th e symptans o f in to x ic a ti o n have n o t alw ays been a s c lo s e ly observed as would be d e s ir a b l e , n o r has due acco u n t been ta k e n o f th e in flu e n c e o f d i e t and o f o th e r p h y sio ­ lo g ic a l f a c to rs . Both t o x i s t e r o l and t a c l y s t e r o l a r e c a p a b le o f producing se v e re symptoms o f i n t o x ic a ti o n . F urtherm ore b o th produce h i s t o ­ l o g i c a l and chem ical changes i n th e body t i s s u e s g e n e r a lly t h a t a r e d e f i n i t e l y p a th o lo g ic . —25— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. C a lc if e r o l a ls o i s a b le t o produce to x ic e f f e c t s . H erein l i e s th e j u s t i f i c a t i o n f o r th e assum ption t h a t h y p e rv ita m in o sis D and v ita m in D i n to x ic a tio n a r e one and th e same s t a t e . The com­ mon o ccu rren ce o f h ypercalcem ia i n to x ic c o n d itio n s a t f i r s t le d t o th e assum ption t h a t t h i s phenomenon was r e s p o n s ib le f o r th e to x ic c o n d itio n . Reed and Thacker (70) have c a lle d a t t e n ­ t i o n t o th e fre q u e n t occurrence o f hypercalcem ia o f lo n g du ra­ t i o n w ith o u t sisy symptoms of to x ic ity * From t h e i r s tu d ie s th e y concluded, th e r e f o r e , t h a t v ita m in D in to x ic a tio n and h yper­ calcem ia a r e n o t synonymous, alth o u g h th e two c o n d itio n s may e x i s t sim u lta n e o u sly . The i d e n t i t y o f v ita m in D in to x ic a tio n and h y p erp arattg rro id ism a re n o t th o u g h t to be i d e n t i c a l i n as much as n o t a l l of th e o b je c tiv e and s u b je c tiv e ev id en ces of in to x ic a tio n by one or th e o th e r can be proved by an excess o f th e o th e r . î^ y p erav ita- m in o sis D and th e v ita m in D in to x ic a tio n can n o t be used synony­ m ously inasmuch as th e to x ic th re s h o ld v a r ie s among i n d i v i d u ^ s and th u s h y p e rv ita m in o sis D does n o t in e v ita b ly le a d t o i n t o x i ­ c a tio n . O bviously, a s u b je c t re c e iv in g 30,000 u n its o f v i t a ­ min D p e r kilogram p e r day i s i n a s t a t e o f h y p e rv ita m in o sis D, even though no t o x ic a tio n o c c u rs. Likeiirise th e s u b je c t r e c e iv ­ in g 2,000 u n its p e r kilogram p e r day, may under some circu m stan ces show a to x ic c o n d itio n b u t s c a rc e ly can be s a id to be i n a condi­ t i o n o f h jip e rv ita m in o sis D, s in c e an in c r e a s e o f v ita m in D concen­ t r a t i o n i a th e b lo o d stream may n o t be d em o n strab le. 26— — R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission . I t i s su g g e sted , t h e r e f o r e , t h a t "txypervitam iuosis D" be employed t o d e s ig n a te only th o s e s t a t e s i n w hich th e c o n c e n tra ­ t i o n o f th e v ita m in i n th e b lo o d stream ca n be dem onstrated t o b e d e f i n i t e l y in c r e a s e d . R eg a rd less o f t h e amount in g e s te d , i f i t i s n o t c i r c u l a tin g i t m ust have been m e ta b o liz e d , s to r e d , e x c re te d , o r unabsorbed, and th e r e can be no h y p e rv ita m in o s is. I f under th e s e c o n d itio n s a to x ic s t a t e d e v e lo p s, i t i s ob­ v io u s ly n o t h y p e rv ita m in o sis p e r se a lth o u g h probably a second- cary e f f e c t t h e r e o f . I t i s a p p a re n t t h a t in some r e s p e c ts th e to x ic a c tio n o f v ita m in D i s n o t u n lik e th e d e h y d ra tin g e f f e c t o f p a ra th y ro id e x tr a c t B asset (7 1 )• I t i s a p p a re n t a ls o t h a t th e to x ic a c tio n may a f f e c t any and a l l t i s s u e s b u t th e k id n ey i s m ost commonly in v o lv e d . M oreover, th e e f f e c t s seem to be e x e rte d on t i s s u e c e l l s , and w ith in p h y s io lo g ic a l ra n g e s o f a p p lic a tio n th e r e i s improvement i n c e l l u l a r f u n c tio n . However t h i s p ro c e ss ap p ears t o be r e v e r s i b l e by e i t h e r a d e f ic ie n c y o r an excess a s p o in te d out by F reudenberg ( 7 2 ), W idenbauer (73) claim ed t h a t v ita m in A o r B complex p r o te c te d m ice a g a in s t t o x ic a tio n w ith e i t h e r v ita m in D or d ity d ro ta c h y s te ro l. Numerous r e p o r ts have in d ic a te d t h a t a c tiv a te d e r g o s te r o l i s more to x ic th a n cod l i v e r o i l w ith com parable v ita m in D u n ita g e . I n v e s tig a tio n s w ith mammals have g e n e r a lly in d ic a te d t h a t m assive doses o f v ita m in Dg a r e t o x i c , d em o n stratin g in c re a s e d -27* R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. c a lc iim l e v e l s accompanied by bone d i s s o l u t i o n . , The l e s s f r e ­ q u e n tly s tu d ie d b y -p ro d u c t o f e r g o s te r o l i r r a d i a t i o n d ih y d ro ­ ta c h y s te r o l has been shoim by C o r r e ll and Wise (7U) t o have a low a n t i r a c h i t i c e f f e c t and f o r mammals, i t i s hypercalcém ie and t o x i c . In summing up th e to x ic e f f e c t s o f th e s e compounds, we f in d t h a t sev e re and f a t a l t o x ic a tio n may b e produced b y ex ces­ s iv e amounts o f v ita m in D. The endldence and s e v e r ity may be c o n d itio n e d by: (1 ) th e dose p e r u n i t o f body w eig h t (2) th e d u r a tio n o f a d m in is tr a tio n (3) th e ccsnposition o f th e d i e t , i n b o th m in e ra l and o rg a n ic c o n te n t (it) th e s t a t e o f th e a lim e n ta ry t r a c t (5 ) th e s p e c ie s s u s c e p t i b i l i t y (6) in d iv id u a l s u s c e p t i b i l i t y (7) age (8 ) p r e - e x is tin g p ath o lo g y (9) th e v e h ic le i n w hich th e v ita m in i s a d m in iste re d (10) th e r o u te o f a d m in is tr a tio n (11) th e f u n c tio n a l s t a t e o f th e e n d o crin e system (12) th e p u r i t y o f th e viteanin p r e p a r a tio n (13) th e so u rce o f th e v ita m in (lU ) p ro b ab ly o th e r f a c t o r s n o t y e t re c o g n iz e d There i s a ten d en cy t o d e p o s itio n o f calcium a n d , t o a l e s s e r e x te n t, o f phosphorus i n s o f t t i s s u e s w ith approach t o a to x i c s ta g e . T h is p ro c e ss i s r e v e r s i b l e t o a c o n s id e ra b le e x te n t and a p p a re n tly le a v e s no c h ro n ic i n j u r y . The in c id e n c e o f t o x ic a tio n i n human s u b je c ts i s r e l a t i v e l y low . The th re s h o ld o f to x ic a tio n f o r humans and dogs ap p ears be 20,000 U .S .P . u n i ts to o f v ita m in D p e r kilogram o f body w eig h t p e r day f o r m ost p r e p a ra tio n s used. - 28- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission . Of a l l t i s s u e s s tu d ie d th e kidney ap p e a rs t o m ost v u ln e r a b le , and th e a o r t a n e x t. be th e N e v e rth e le ss h y p e rte n ­ s io n h as n o t b een enco u n tered in human s u b je c ts . The extrem e p a th o lo g ic a l changes d e s c rib e d i n an im als h av e, w ith few e x c e p tio n s , been produced by doses o f v ita m in D g r e a t l y i n ex c ess o f th e h ig h e s t doses recranmended f o r human th e ra p y . I f th e doses a r e c a lc u la te d on th e b a s is o f u n i t w e ig h t. Goormaghtigh and Handosrsky (75) have r e c e n tly shown w ith dogs doses o f v ita m in D (p u re c a l c i f e r o l ) ra n g in g from 1600 t o 2800 u n i t s p e r kilo g ram f o r 37 t o 102 d a y s, th e b lo o d p re s s u re was in c re a s e d from an av erag e o f 134 mm be­ f o r e tre a tm e n t t o l4 ? mm a f t e r tre a tm e n t. They a l s o found t h a t r e n a l damage was produced by doses i n th e ran g e o f 20,000 u n i t s p e r k ilo g ram p e r day o r above. w ere harm less and even b e n e f i c i a l . th e y found t h a t D oses o f 4 ,0 0 0 u n its Even on th e la r g e doses th e a r t e r i o l a r m u scu latu re could re c o v e r w ith no perm anent damage. I n young anim als however r e g r e s s io n o f th e thymus m ig h t o ccur r e s u l t i n g i n c e s s a tio n o f grow th. Changes d e s c rib e d were found only i n th e kidney and t o a l e s s e r e x te n t i n th e s p le e n . L arg er d o ses produced s im ila r changes i n th e n eu rohypophysis, th y r o id , gonads, a d re n a l and p a n c re a s . The a o r t a was n o t a f f e c te d e x c e p t by s t i l l la r g e r do ses. -2 9 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. CHEMISTRY OF THE PROVITAMINB D AND OF THE IRRADIATION PROCESS An im d e rstan d in g o f th e s tr u c tu r e o f th e m o lecu le o f v ita m in D and o f th e chem ical r e a c t io n s le a d in g t o i t s form a­ t i o n as w e ll as o f th e changes b ro u g h t about by v a rio u s chemi­ c a l and p h y s ic a l ag en ts can b e s t be g ain ed by a d is c u s s io n o f th e ch em istry of s te r o ls . T his i s due, o f c o u rse , t o th e f a c t t h a t v ita m in D i s d e riv e d from s t e r o l s , b o th i n n a tu re and i n te c h n ic a l m an u factu re. The c h em istry o f s t e r o l s s in c e th e i s o l a t i o n o f c h o le s te r o l from g a ll- s to n e s by de l a S a lle ab o u t 1769 has been review ed by F ie s e r (76) and B i l l s ( 7 7 ). S te r o ls have been found i n p r a c t i ­ c a l l y a l l anim al and p la n t t i s s u e s —a f a c t w hich h as le d b io l o ­ g i s t s t o th e co n clu sio n t h a t th e y have some im p o rta n t f u n c tio n s i n c e l l u l a r m etabolism . A nother i n d ic a tio n o f t h i s Im portance i t th e f a c t t h a t i t has been amply proved t h a t s y n th e s is may ta k e p la c e i n th e anim al organism . The elem en tary fo rm u las o f m ost o f th e s t e r o l s which have b een i s o l a t e d i n pure form have been d e riv e d , and a few o f th e s e a re in c lu d e d i n th e fo llo w in g t a b l e , w hich l i s t s a ls o th e m e ltin g p o i n t , o p t i c a l r o t a t i o n , and c h ie f so u rce of c e r t a i n o f th e more common s t e r o l s . —30— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. Name C h o le s te ro l .D ih y d ro c h o le ste ro l C o p ro stero l O s tr e a s te r o l L a n o ste ro l A g n o stero l E rg o s te ro l S ito s te ro l S tig m a ste ro l C hincol F u c o s te ro l Tym osterol Formula M.P. C27%60 C27HU8O C27%80 ^29% 8^ C30H40O. C30HU8O C26%itO C29H50 O ^29%8° C29H50 ® C29%8° ^29% ° 190° 1U2 102 1U3 lip. 162 163 IU6 170 lUo 12U no O ccurrence - 38 . 8 ° +28.8 +23 .S -ii3 .9 +28.0 +70.6 - 133.0 -4 2 .4 -U 5.0 —2U.0 - 3 8 .Ü +ii7.3 A ll anim al c e l l s Companion o f c h o le s te r o l F eces O y s te rs , g astro p o d s Wool f a t Wool f a t E rg o t, y e a s t F a ts o f h ig h e r p la n ts C alabor b ean s, soy beans Chinchona b a rk A lgae Y east The stu d y o f th e ch em istry o f s t e r o l s has assumed a g r e a t e r im portance i n r e c e n t y e a rs s in c e i t has been found t h a t i n a d d itio n t o v ita m in D c e r t a i n o th e r a ls o s t e r o i d d e r r i v a t i v e s . a r e m o d ified s te r o ls * b i o lo g ic a lly im p o rta n t su b sta n c e s a r e For example c e r t a i n o f th e sex hormones The b i l e a c id s a r e s t e r o i l s , a s a r e c e r t a i n c a rd ia c p o iso n s such a s s tro p h a n th in and th e to a d p o iso n s such a s b u fo to x in . C e rta in o f th e c a rc in o g e n ic hydrocarbons c o n ta in th e phen an th ren e nu cleu s found in a l l s t e r o l s . So f a r as i s known a t p r e s e n t, a l l s t e r o l s a r e m o d ific a ­ t io n s o f th e g e n e ra l s tr u c tu r e shown in fo rm u la I . R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. The r i n g s a r e n o t e r o m e t i c in nature b u t axe n o t n e c e s s a r i l y c o r a v l e t e l y s a t u r a t ec’ . The s i d e e s t e ^ b y R i n F o r nul a I , ' c r i e s be saturated or c o n t a i n typical plant the sterol Indi­ f-nd may one one d o u b l e b o n d . The a c c e p t ­ ed f o r m u l a s of c h o l e s t e r o l Form ulas I I and I I I vitb group, end e rg o ste ro l ere given in as e x a m p l e s and f o r c o m p a r i s o n , sterol, stigm asterol, is â s h o wn i n F o r m u l a IV. 3 “"SSL A H3 -CH=OH-OH-GB /HR 131. -3 i R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. Vr A I t i s n o tic e a b le t h a t th e s e th r e e fo rm u las have a rem ark­ a b le s im i l a r i t y i n s p it e of t h e wide d i v e r s i t y o f th e so u rces o f th e s t e r o l s th e y r e p r e s e n t. I t i s to be n o ted f u r t h e r t h a t th e g r e a t e s t d iff e r e n c e s l i e i n th e s id e c h a in , and in d eed th e v a r i a t io n s in chem ical and p h y s io lo g ic a l b e h a v io r a r e a sso ­ c ia te d more w ith changes in th e s id e c h a in th a n w ith any deep s e a te d rearran g em en ts or s u b s tit u tio n s i n th e r in g system i t ­ s e lf, The n u cleu s i s rem arkably s ta b l e , and only s tro n g methods o f tre a tm e n t w i l l cause r u p tu r e . O ther p r o p e r tie s o f th e s t e r o l s may b e e x p la in e d by th e form ulas g iv en . They a re a l l secondary a lc o h o ls , s in c e th e r in g t o w hich th e hydroxyl group i s a tta c h e d i s s a tu r a te d . Thus th e y a re n e u t r a l r a th e r th a n p h e n o lic . e s te rif ie d . They a re r e a d ily A la r g e p ro p o rtio n o f th e c h o le s te r o l in mammalian t i s s u e s e x i s t s a s e s te r s o f v a r io u s f a t t y a c id s . As would be expected from s t e r i e c o n s id e ra tio n s a s w e ll a s from th e g e n e ra l p r o p e r tie s o f a l l secondary a lc o h o ls , th e e s te r s a r e r e a d i l y s a p o n ifie d . The s i m i l a r i t y o f s tr u c tu r e s I I , I I I and IV su g g e sts t h a t th e p h y s ic a l and chem ical p r o p e r tie s would be expected t o v a ry l i t t l e among th e v a rio u s s te r o ls * T his i s th e c a s e . can be p re p a re d from t h e i r v a r io u s n a tu r a l S te r o ls so u rces w ith o u t g r e a t d i f f i c u l t y , b u t even when such p r e p a ra tio n s y i e l d c ry ­ s t a l l i n e p ro d u c ts th e y a re a lm o st in v a r ia b ly m ix tu re s o f two o r more s t e r o l s . The s e p a ra tio n and p u r i f i c a t i o n o f a s in g le -3 3 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission . s t e r o l i s a p ro c e ss o fte n r e q u ir in g g r e a t in g e n u ity and f r e ­ q u e n tly ta x e s th e re s o u rc e s o f th e ch eaiist to th e u tm ost, s in c e s o l u b i l i t i e s a re alm ost i d e n t i c a l , and d i s t i l l a t i o n ia d iffic u lt. In a d d itio n s t e r o l s form m o lecu lar compounds of c II if 11 II .17 7.4if 10 tt 5 IT II .055 9.52 10.74 11 II 5 .055 9.52 9.76 12 II h II II Tl 3 II It 3 It tl tl 11 f! II II It 13 lU 15 11 It 3 II 11 9 0 ,0 0 0 Stock Young Lo P (b a sa l) R ats II It It Stock Mature .17 Lo P R ats (b a s a l) .17 II 7.44 14.57 12.01 13.91 7.Ü4 8.42 11.10 11.07 .17 12.57 .17 13.42 Lo P = Low Phosphorous —7J4.— Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12.20 13.54 TABLE IV Twenty r a ts (I 6 O-I 7O grams) were placed on a and fe d Jamieson’ s C.L.O. (Vitamin D^) conc. as fo llo w s: No, of mgms Animals stock d ie t 1000 n/ml Ca/lOO ml Serum 10 Days Fed 50,000 u/lO kg 1 day/1 0 days 5 8.78 Fed 100,000 u/lO kg 1 day/10 days 5 10.19 Fed 150,000 u/10 kg 1 day/10 days 5 10.86 Controls 5 10.52 C.L.O. « Cod Liver O il -7 $ - Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE V T h e com p oun ds u s e d i n th e f o llo w in g 1 . P ure c a l c if e r o l in o i l 2 . N a tu r a l f i s h o i l s D3 (D r, e x p e r im e n ts w e r e : ( D r . E w in g ) 2 0 0 , 0 0 0 u /g m E w in g ) 1 $ 0 0 - 1 7 0 0 u /g m 3 . Hydee Yeast Std. Brands 2i4.,000/gm O ne h u n d r ed a n d t w e lv e a n im a ls a b o u t 1 0 0 Two r a t i o n s ( lo w Ca a n d s t o c k r a t i o n ) gm s e a c h mgm s C a /lO O m l s e r u m Supplements Exp. 100 13 days No. of V it. D Source Animals Pure A Stock r a tio n C a lc ife r o l if & U V it. D g B " " U&U ltO,OOG\t/lOkg C Low Ga Ù&4 2,000u/'l0kg U & It It0,000u/l0kg 10.21 10.39 U & it 2,000u/l0kg 10.03 7.69 II it & it itO,OGOu/lOkg 10.01 8.70 G Low Ca II it & it 2,GGGu/lOkg n .it 3 11.06 H It Group Rats II " D " II Yeast E Stock r a tio n Hydee F II II 2,000u/l0kg Exp. 99 1$ days 9.08 10.3 9.31 7.82 8.37 r> O .p j- it & it itG,GGGu/lGkg 9.81 8 .60 Fish J Stock r a tio n O ils it & it 2,GGGu/lGkg 10.18 8 .9 6 K " II it & it itG,GGGu/lGkg 9.lt3 8.31 L Low Ca II it & it 2,GG0u/lGkg 9.96 8.8it M " II it & it itO,GGGu/lOkg lO.ltit 8.79 N Stock r a tio n Control it & it Stock only 9.70 9.78 P Low Ga it & it Low Ca only 9.06 9.13 " " Do " « II — 76 “ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE V I G roup S e x N o . o f S u p p le - D a ily L e v e l R a tio n A n in la ls m e n ts m gm s o r u n its /lO k g _____________________ b ody w t. W t* K g. C o n t r o l s m gm s C a /lO O m l or seru m o D ays d a y s ___ h _______ 7 _______ 1 1 DHT 1 mgm K 10 11.03 1 6 .2 0 19.40 2 1 It 1 mgm K 10 10.10 15.92 18.93 3 1 It .5 mgm It 10 13.02 19.15 19.89 U 1 tt .5 mgm II 10 15.10 19.35 19.53 5 1 tl .5 mgm II 10 9 .5 0 13.78 13.93 6 1 It .5 mgm II 10 13.50 19.05 20.16 7 1 It .5 mgm tl 10 13.30 16.00 17.60 8 1 It .5 mgm II 10 13.24 1 7 .5 0 18.06 9 1 tl .5 mgm It 10 12.00 1 4 .1 0 17.20 10 1 tl .5 mgm It 10 10.61 13.71 15.58 11 1 II .5 mgm It 10 12.33 15.06 17.21 12 1 tl .5 mgm II 10 10.81 12.30 15.30 13 1 tl .2 5 mgm It 10 10.29 11.08 11.70 Hi 1 It .25 mgm It 10 12.01 1 2 .0 1 12.90 15 1 It .25 mgm II 10 13.80 1 4 .2 0 14.24 16 1 tl .25 mgm II 10 14.60 14.89 14.90 17 1 It ,25 mgm II 10 12.01 12.19 13.69 18 1 tl .25 mgm II 10 12.21 12.95 15.08 DHT = D i h y d r o t a c h y s t e r o l -7 7 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. I I D ogs— TABLE V I I G roup Sex N o. of S u p p le - Animals ments Dogs 1 1 2 1 3 1 S td Brands IE D2 in VO II Pure C a lc i­ fe ro l Do in D aily Level É a tio n Remarks Wt. C o n tro ls mgms Ca/lOO ml mgms o r Kg. or serum u n its/lO k g o Days days L 7 115 30 body w t. T issu e Supple­ 10 500,000 m ents 2 3 .L l C a lc i­ 12.31 K f ie d f o r 10 died days 500,000 II II 10 13.62 LOO,000 II II 10 12.81 lL .7 6 19.21 19.65 22.65 l&O "wo h 1 II LOO,000 II 10 12.20 1L.3L 18.70 5 1 II 200,000 II 10 1 2 ,OL 13.20 lL .8 3 6 1 II 200,000 II 10 12.61 13.68 IL .69 7 1 II 200,000 II 10 13.20 1 5 .OL 16. 2L 8 1 II 200,000 II 10 11.08 12.90 lL .2 1 9 1 II 200,000 It 10 13.20 15.30 17.10 1 0 1 II 200,000 II 10 12.80 IL.OO 15.91 11 1 II 100,000 It 10 12.30 d isc o n tin u e d 12 1 II 100,000 II 10 13.00 d isc o n tin u e d 13 1 It 80,000 II 10 10.56 d is a b le d lU 1 II 80,000 II 10 11.50 d ie d 7-2L-LL 20,000 II 10 13.60 IL.OO 10,000 II 10 12.78 13.01 15 1 s td . Brands I E J) inVO 16 1 n IE WO VO = I r r a d i a t e d e r g o s te r o l -= Wesson o i l - V egetable o i l -7B- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE V I I I Group Sex Dogs No. o f S u p p le- D aily L evel R atio n mgms or Animal s ments u n its/lO k g body Trirt. 1 8 .L l II 1 1.70 1 6 .9 0 11 11 13.89 19.81 500,000 (I M 12. 9L 17.72 II LOO,000 H tl 11.13 II 200,000 tt 11 K 1 mgm 1 2 1 II mgm II 3 1 II .5 mgm 1 3 1 6 1 C o n tro ls mgms Ca/lOO ml serum or Days o days 7 10.30 DHT 1 h Wt. Kg. Pure C a lc i­ fe ro l D2 wo 10 10.10 d ied B-12-LL 13.12 DHT = D ih y d ro ta c h y ste ro l WO = Wesson O il TABLE IX Tvto 10 kg dogs p la c e d on Hydes y e a s t e x t r a c t c o n ta in in g 2 ^,0 0 0 u V itam in D/ml . A dm inistered th e o i l i n ground m eat mixed w ith sm a ll amount o f fe e d . 0 Days 5 Days B oots 200,000 u o f V itam in D/ 10 k g /d ay f o r 10 days' IL .O I 1 7 .LO 19.87 19 . 8L Mike Loo,000 u o f V itam in d/ 10 kg/day f o r 10 days IL .82 21.91 25.63 20.96 10 Days 15 Days (Supplem ents sto p p ed h e re ) -7 9 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. I ll C h ic k e n s TABLE X Group Sex No. o f S upple- D aily L evel R atio n Remarks Wt. C o n tro ls mgms Ca/lOO ml or Animals ments mgms or Kg. serum lit u n its/lO k g Davs Chickens body w t. days 7 10 15 Laying 1 Imgm/lOkg 2% Ca Hens 2U.30 DHT 13.91 k 3 0 2 h II Imgm/lOkg " 3 h tl Imgn/lOkg " U It i l Imgm/lOkg " h II Imgia/lOkg " h II Imgra/lOkg " 6 7 3 It Imgm/lOkg " 8 3 II Imgm/lOkg " 9 k II .3mgm/lOkg " 10 h II .3mgm/lOkg " 11 h II .3mgm/lOkg " 12 k II .Smgm/lOkg " 13 k II •3mgm/lOkg " Ih k II .3mgm/lOkg " 13 2 II . 3mgm/lOkg " 16 2 II .3mgm/lOkg " 17 3 II .3mgm/lOkg " 18 3 II .3mgm/lOkg " 19 2 II .25mgm/lOkg " 20 2 II .23mgm/lOkg " II II Non­ la y in g Laying Hens II Laying Hens Non la y in g Non la y in g n 3 8.92 2 19.67 r 17.13 27.92 2 8.30 17.03 2 19,67 28.60 2 8.30 3 13.U7 21.31 3 8.30 15.89 3 13.91 19.56 3 8.92 12.56 2 19.67 2 k .13 2 8.30 l i t . 90 2 19.67 2U.08 2 8.30 lit.9 2 3 10.71 16.11 3 9.68 13.93 3 1 3 .b7 18.73 3 8.30 13.09 3 10.71 11.87 3 9.68 1 1 .ill DHT - D ih y d ro ta c h y ste ro l - o 80- R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout p erm ission . 17.11 TABLE X I Group Sex No, o f Supple- D a ily L evel R a tio n Remarks Wt, C o n tro ls m gm s C a / or 100 ral Animal s ments mgms or Kg. o sérum u n its/lO k g Chickens b o d v w t. Young Irra d . 2% Ca b ir d s 13.20 2 13.10 1 erg Dg boo, 000 3 Ew 2 tl 3 S td . Brands boo, 000 tl tl 2 1 3 .1 0 13.6b 200,000 It It 2 13.10 13.08 Dg 3 II 3 Irra d . erg Dg EW it 5 ii tl 3 S td , Brands 200,000 If It 2 13.10 13.19 3 ^2 Tuna O il Do 200,000 II II 2 1 3 .1 0 lb.90 100,000 II II 2 13.10 13.96 Ew 6 tl 3 Tuna O il Do Ew HLO 7 tr 3 D o Ew b,200 II II 2 13.10 l b . 52 8 11 3 DHT 1 mgm II II 2 13.10 23.23 9 tl 3 DHT .5 mgm It II 2 13.10 18.10 tl 3 DHT ,25 mgm II II 2 13.10 13.50 10 IE “ I r r a d i a t e d E rg o s te ro l O il IW = O b t a i n e d from D r. E w in g HLD = H a liv e r —8 l — R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. IV C ow s TABLE X I I Group Sex No. of Stipple- b a lly Level R ation Remarks Wt. C ontrols 1 mgms Ca/lOO ml serum Animal s ments mgms o r Kg. or Days lit 0 u n its/lO k g or Cows body w t. days 3 5 7 10 15 Hay & Supple­ 500 10.88 1 1 DHT G rain ments .3 mgm 11.2011.681U.2U15.91 11.29 f o r 10 days 2 " 1 DHT .3 mgm 3 " 1 DHT .5 mgm I. " 1 5 " 1 6 " 1 C ontrol Hydee Y east 500,000 S td . Brands 500,000 D g IE 7 " 1 8 " 1 9 " 1 II Hydee Y east 11 II II 12.01 12.0812.U11U.0116.U9 11.81 II II II 11.03 1 1 .83lU .06l6.U 2l6.53 II 10.18 10.2810.2310.1010.19 12.79 1 2 .7 2 1 3 .0U15.9816.03 lU .30 II II II II II tt II 1 0 .Uo 10.U013.361U.U815.98 12.16: 500,000 II II II 13.32 13.3616.0816.8817.76 13.61 250,000 II It II 12.81 12 .9 213.1613.901U.32 13.09 II 12.02 12.0812.1312.0812.10 12.08 Group C ontrol II - 82. R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. B» Phosphorous I E a ts TABLE X I I I Group Sex No. o f S upple- D aily L evel R atio n Remarks Wt. C o n tro ls mgms P/lOO ml Animals ments mgms or Kg. or blood u n lts/lO k g o Days days________ 30________ R ats______________________ body vfb. Lo P Young (b a s a l) R ats 1 mgm 2 .3 9 1 DHT .05 3 .7 9 5 Sp Lo 11 5.81 1 mgm P 2 DHT .05 if.U3 5 M ature Lo P 6 .3 8 1 mgm R ats .165 3.77 DHT k 3 Sp Lo 7.08 1 mgm P .165 5 .6 9 k DHT k Young Lo P 3 .2 8 DHT .5 mgm R ats 2.39 .0 5 5 5 Sp Lo 6 5 .2 8 DHT .S mgm P U.U3 .0 5 5 M ature Lo P 5.08 DHT .5 mgm R ats 7 .1 6 5 3 .7 7 k Sp Lo P 8 DHT .5 mgm .165 5 .6 9 6.63 h II II It II tt II tl tl II It DHT = D ih y d ro ta c h y ste ro l Lo P = Low Phosphorus Sp Lo P = S p e c ia l low phosphorus TABLE XIV T h ir ty r a t s about 00 grams s ta r t e d on sto c k r a t i o n and fe d DHT le v e ls as fo llo w s: N o . o f A n im a ls on each l e v e l and tim e i n t e r v a l L evel B .25 L evel A .5 L evel D .75 L evel D 1 C o n tr o ls m g m /lO k g /d a y /lO m g m /lO k g /d a y /lO 6 days m g m /lO k g /d a y /lO m g m /lO k g /d a y /lO ra ts on s t o c k days days days o n ly - 1 0 D ays 30 Days 3 5 .9 2 6 .1 5 3 6,3h 6.U7 3 7 .2 7 7.51 3 8 .2 8 7 .6 9 3 3 .9 1 It.55 83 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE XV F o u r l a r g e m a tu r e s t o c k a n im a is w e ig h in g e a c h w e r e f e d DHT a s f o l l o w s : gm s N o. o f m gras P / 1 0 0 m l b l o o d 30 D ays A n im a ls R a ts U S to c k 1 m g m /l O k g / d a y / 3 0 200 days U S to c k C o n tr o ls It S . 71 k 3.U9 —8U“ R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. I I D ogs TABLE X V I R atio n Wt. C o n tro ls mgms P/lOO ml b lo o d Group Sex Ko. of Supple­- D a ily Level Days mgms or u n its Animals ments Kg. or /lO kg body vrt 0 Dogs days it 7 111 30 10 2.72 3.21 5 .2 5 11 11 3.08 3 .3 1 3.70 •5 mgm II II Ü .56 3 .5 2 U.53 II .5 mgm II tl U.50 2.80 ii.70 1 II .5 mgm If It ii.o o 5.07 7.55 6 1 II .5 mgm It It 3.70 k.5o 5 .5 2 7 1 II .5 mgm tr 11 2.77 3 .0 3 ii.o e 8 1 II .5 mgm 11 II 3.63 il.22 6.66 9 1 II .5 mgm II II 3 .5 2 U.iU ii.3B 10 1 II .5 mgm II II 6 .3 8 6.U7 6.U7 11 1 II .25 mgm II II 7 .0 5 7.03 6 .9 0 12 1 II .25 mgm II II 3 .Ill 2.69 2 .9 3 . 13 1 II .25 mgm t! II 3.03 3.59 3.10 lit 1 II .25 mgm 11 It 2.55 5.17 6.52 15 1 II .25 mgm tt II 2 .2 k if.lii k.26 16 1 II .25 mgm It It 3 .8 9 ii.o 5 ii.lli 1 1 2 1 II 3 1 II h 1 5 ' DHT 1 mgm K 11 DHT - D ih y d ro ta c h y ste ro l —85— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE X V I I G roup S e x N o . of S u p p le - D a ily A n im a is m e n ts L evel R a t i o n W t. m gm s o r u n i t s /lO kg body w t. K g. C o n t r o l s ” m gm s P /lO O m l b l o o d nr> or Days o 7 - 1 1 2 1 S td . Brands IE Dg i n VO ri 500,000 K 10 ii.oo 6 .7 7 5 0 0 ,0 0 0 II 10 5.L9 6 .7 0 II 10 3 . lit 3.28 3.62 Pure C a lc i- L oo,000 fe ro l Ü2 i n WO 20 3 1 h 1 II LOO,000 It 10 3.01 it.52 )i.9? 5 1 II 200,000 II 10 3.0it 5.07 5.25 6 1 It 200,000 II 10 5.52 5.78 6 .0 0 7 1 II 200,000 II 10 3.U9 it.it9 5.37 8 1 II 200,000 II 10 2.23 3.77 it.63 9 1 II 200,000 II 10 3.1it it.02 5.25 10 1 II 200,000 II 10 1.8L 3.56 it.07 11 1 II 100,000 II 10 1 .1 2 d is c o n tin u e d 12 1 II 100,000 II 10 1 .1 0 d is c o n tin u e d 13 1 II 100,000 II 10 7 .lit d isc o n tin u e d lit 1 II 100,000 II 10 5.it3 d ie d 20,000 II 10 it.35 i t . 25 S td . Brands IE D g i n TO 15 1 16 1 II 20,000 II 10 it.2lt lt.26 17 1 II 10,000 II 10 i t . 91 lt.69 18 1 II 10,000 " 10 5 .0 0 ii.90 IE = I r r a d i a t e d E rg o s te ro l VO = V egetable O il WO = Wesson O il - 86- R ep ro d u ced with p erm ission of th e copyright ow ner. Further reproduction prohibited w ithout p erm ission . TABLE X V I I I R a tio n Wt. C o n tro ls mgms P/lOO mi b lo o d Group Sex Wo. o f S u p p le- D a i l y Level Kg. or Animals m ents mgms o r u n i ts Days o /lO kg body w t. 10 Dogs days 7 10 5.66 S td . 500,000 K 1 3.%3 1 Brands IE Dp i n v6 Pure d ie d 8/b/bb C a lc i- booJ000 2 1 b.07 fe ro l Ü2 i n WO 200,000 It tt It It 3.91 It It 2.95 3.8b b.b5 3 1 " h 1 DHT 5 1 " «5 mgm It tl 3.98 6 1 " .5 mgm tl tl 1 .7 2 1 mgm 5.81 2.12 VO = V egetable O il IE z I r r a d i a t e d E rg o s te ro l DHT = D ih y d ro ta c h y ste ro l WO = Wesson O il TABLE XIX Two dogs fe d sim plem ents o f E x tr a c ts o f %rdee Y east i n ¥ . 0 . c o n ta in in g 2 b ,000 u/m l o f D a s fo llo w s , and b le d a t th e end o f 0 ,§ ,1 0 , and 15 d a y s. mgms P/lOO ml blood Boots 200,000 u /d a y f o r 10 days Supplem ents sto p p ed 10 days 0 Days 5 Days 3.77 b .l6 b .7 b 5.1b b*09 b.56 4 6.03 l5 Days Mike boo,000 u/day f o r 10 days . 8 7 W.O. = Wesson O il -87- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. Ill Chickens TABLE XX Group Sex No, o f Supple- D a ily Level R a tio n Remarks w t . C o n tro ls mgms P / or Animals m ents mgms or Kg. 100 ml u n it s /lO kg o blood Chickens days body w t. 10 Da:^s young S td . 2 6.92 2% Ca 8.87 1 non Brands b o o ,000 k la y in g IE Dp inVO 2 k It II II IE PD Ewing Dp b o o , 000 3 3 4 \ 3 5 3 II 200,000 tt 6 3 II 200,000 tf 200,000 tl 200,000 " 7 h 8 k Std Brands IE Dg in 70 It II II II II 2 6 .0 b 8 .b6 2 6.92 8.37 'd 6 .0 b 7.b3 2 6.92 7.96 2 6 .0 b 6.87 2 6.92 7.19 2 6 .0 b 7.10 IE = I r r a d i a t e d E rg o s te ro l 70 = 7 e g e ta b le O il PD r Parke-D avi s-Ewing —88“ R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE XXI Group Sex No. o f S u p p le- D aily L evel R atio n Remarks Wt. C o n tro ls m pis P/1ÜÜ ml Animals m ents mgms or Kg. or blood u n its/lO k g o Days Chickens body w t. days 7 10 non- "" 3.21 la y in g b 2$ ( 1 mgm 1 DHT 3 II tl tt 2.11 2 b 3 young It tl II b 3 n o n -la y in g 2 6.92 7.8U It tt 6 . 0b 7.60 b 3.21 3.62 li 2.11 3.ii9 b tl 5 3 II .5 mgm tl 6 3 tt •5 mgm It 7 1 tt It II non-laying5.5 3.21 k»92 8 1 It It 11 g.S 2.11 2,79 9 1 tl tt It 1 0 1 11 11 tl 11 1 tl 11 tl 12 1 11 tl tl 13 1 It It tl lb 1 II II tt 15 b rt If II 16 b II 11 Tl 17 1 n 18 1 19 old b 3.26 5 3.26 9.07 b la y in g hen b young n o n -la y in g 2 2.51 7 .1 i2 3.26 2 6.35 la y in g hen old 8.55 ii.99 7.U1 2 6 . 0b young n o n -la y in g 2 6.92 o ld non­ la y in g 5.5 3.21 7.b9 2.21 11 It .25 mgm It 5 .5 2.11 1 It .25 mgm It b 1 tl .25 mgm It 5 21 1 It .25 mgm 11 22 1 It .25 mgm 11 23 1 II .25 mgm It b la y in g hen b young n o n -la y in g 2 2b 1 tl .25 mgm It 25 b It .25 mgm II 26 b It .25 mgm II - 7.1b 6.28 .25 mgm 20 3.62 2 U no n la y in g “ lit or | 1$ ; 7.72 5.81 3,26 2.51 9.39 3.26 6.12 6.77 3.26 9.53 6.28 6.03 2 young n o n -la y in g 2 6.35 6,29 6 . 0b 7.33 2 6.92 7.57 89 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE XXII Group Sex No. o f S u p p le- D aily L evel R atio n Remarks Wt. C o n tro ls mgms P/100 ml Animals m ents mgms or Kg. o r blood u n its/lO k g o Chicken body w t. days Young Tuna 200,000 2 2.% Ca B ird s 6 .9 2 L iv e r 1 7.67 h O il D3 2 k tl 200,000 3 h tl 100,000 k k $ 3 6 0 V Î1 100,000 H a liv e r O il Do 9,000 b,500 tt tl 2 6 . 0k 6.77 2 6 .9 2 7.U8 It tl 2 6 .0 k 6 .3 s tl tt 2 6 .0 k 6 .3 8 II It 2 6 .9 2 7 .2 0 -9 0 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. IV Covrs TABLE X X I I I G r- Sex No* o f S upple- D aily L evel R atio n Remarks Wt. C o n tro ls mgms P / l ô Ô ml blood oup Animals ments mgms or ICg. o r D ays u n its/lO k g 0 __________ Cows body w t. ____________ ;________ days 3 5 7 10 l5 Hay & $00 4 .8 1 $.07 $ .8 6 $ .9 4 7.14 g r a in 1 1 . $ mgms DHT 2 " 1 3 " 1 ii " 1 $ " 1 C o n tro l (I DHT . $ mgms r! DHT .$ It II mgras 11 C o n tr o l DHT - C o n tro l S u p p le ­ m e n ts 10 d a y s C o n tro l $00 4.47 4.$7 4.77 4 .0 3 4.47 $00 $ .0 9 $.81 6.18 6.$8 7.14 $.63 $00 3.18 3.70 4.22 $.04 $.91 4.07 $00 $.17 $.01 $.28 $ .0 1 4.9$ $.17 D ih y d r o ta c h y s te r o l TABLE X IV Two c o w s f e d H y d e e y e a s t ( S t d . B r a n d s ) 2 ^ ,0 0 0 u n its /g m . B o th w e r e f e d $ 0 0 ,0 0 0 an d 2 $ 0 ,0 0 0 u n its /d a y f o r 10 d ays r e s p e c t iv e ly . S u p p le m e n t sto p p ed 0 D ays 3 D ays $ D ays 7 D ays 10 D ays 1$ D ays 3.67 U.06 U.$$ 6.20 6.79 3.98 Cow A - $ 0 2 $ 0 , 0 0 0 u / d a y / l O k g % rd ee y e a s t as above 3.31 3.83 a . 27 U.69 5.3k h .l2 #U26 $.17 $.01 $.28 $.01 U.9$ $.17 Cow A-U7 $ 0 0 , 0 0 0 u / d a y / l O k g o r l.O ij . k g H y d ee y e a s t m ix e d w i t h g r a i n (g ro u n d c o r n ) C o n tro l H ay & g r a i n -9 1 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE XXV Two cows fe d supplem ents o f S td . B rands I r r a d i a t e d E rg o s te ro l (V itam in D2) V io s te r o l a s fo llo w s : Fed by #10 V et Capsule Potency = 1|00,000 u /g mgms P /1 0 0 ml blood Supplanents Stopped A-53 500,000 u / i o k g /d a y / 25, 000, 000/ f o r 10 days ( 250, 000,000 5.71 5.82 6 . 3U 7.02 7.U6 6.18 A-55 Same as above 5.17 5.25 5.82 6.29 7.02 5.53 A 26 C o n tro l Hay & g r a in 5.17 5.01 5.28 5.01 U.95 5.17 - 92- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. c . P hosphatase I R ats TABLE X X V I T h irty -tw o la r g e anim als w ere p la c e d on sto c k r a t i o n s t e r o l ) as fo llo w s : Group U n its/lO k g No. of R ats C ontrol body w t. Anim als 1 S td . Brands 1000 1 u Source U n its/1 0 0 ml serum Aik Acid 62.1 8.2 7 .6 67.8 Irra d . 2 2000 k 11.2 91.1 E rg o s te ro l 3 Ù000 Ù 9.2 60.9 V io s te r o l- V i t. D 2 ^ 6000 h 9 .2 70,h 3 8000 h 7.2 66.1 6 10,000 h 3 .2 L6.2 7 20,000 h 3 .8 M .o TABLE XXVII S ix ty -f o u r young anim als (60-70 grams) were p la c e d on V itam in D2 on th e fo llo w in g le v e ls s Source S td . Brands V it. Dg (V io s te ro l) U nits/lO kg body w t. No. of Animals U nits A cid Aik 16 3 .3 70.1» 100 16 7 .9 75.7 10 16 10.6 77.8 16 1 0 .1 78.7 1000 u n its D C o n tro ls -9 3 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. TABLE X X V III T w e n ty a n im a is c a lc iu m r a t io n ( y o u n g 6 0 - 7 0 g ra s) w e r e p l a c e d o n a l o w s u p p le m e n te d b y v a r i o u s p e r c e n t a g e s o f R a t i o n 6 S u p p le m e n t L o w Ca ^ % H y d e e Y e a s t 2% (ii8,000 $% (120,000 10% (2U0,000 M o. o f A n im a ls R a ts U n its /lO O m i seru m ' A ik A c id 5 12.9 59.2 fee d u /lO O ) 5 9 .5 Ü8.5 u /lO O 5 6 .0 U1+.6 S 13.5 57.5 u /lO O g ra s) gra s) C o n tr o ls TABLE XXIX O ne h u n d r e d a n d t w e l v e y o u n g a n i m a l s ( 6 0 - 7 0 g r a m s ) w e r e p la c e d on a lo w Ga a n d s t o c k r a t i o n a n d f e d s u p p le m e n ts o f c a l c i f e r o l (V ita m in D g) a n d H a liv e r O i l (V ita m in D ^) a s fo llo w s : S u p p le m e n t R a t io n G roup U n it s N o . o f A n im a ls each t r i a l C a lc ife r o l V i t a m i n Ü2 11 II If u Hydee Y & ast II II II H a liv e r » ft sto c k R a tio n O il A c id m l seru m A ik A 10 h i t .O 72.1 it.7 71.5 B 200 h 2 .5 97.lt 2 .2 55.35 10 k —— l l 5 .i t 9.3 90.0 L ow C a R a tio n C 11 D 200 h 1 .0 8 5 .L 6 .3 82.5 S to c k E 10 k 7 .0 72.1 7.8 78.0 II F 200 k 7 .0 58.6 9.3 90.0 L ow C a R a tio n G 10 h it .O 111.6 6 .3 60.0 11 H 200 k 2 .5 8U.6 22.8 33.0 J 10 h 2 .5 115.7 13.8 13.5 K 200 k 2 .5 8U.6 it.7 82.5 10 k 200 k S to c k II L ow Ca L II II C o n tro l S to c k N L ow Ga P II T r ia l I I U n its /lO O T r ia l I U n its A c id A ik M Controls II h —“ lll.it —— 105.7 15.3 8h.8 7 .0 102.7 6 .3 60.0 7 .0 101.0 7.8 8it.0 ■ _ it -9 h - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. — 111.0 I I Cows TABLE XXX Two c o w s p l a c e d o n e x p e r i m e n t , o n e b e i n g f e d DHT _________________________ a n d t h e o t h e r u s e d a s c o n t r o l ( 1 3 0 0 # e a c h - 6 0 0 K g ) » ____________________ Cow D a ily L e v e l 0 D ays 3 D ays ^ ^ D ays 7 D ays 1 0 D ays # m g m s /lO k g body w t. A-15 . 2 m g /lO k g A-33 C o n tro l g r a in & hay U n its U n its /lO O . m l seru m U n i t s A c id A ik . A c id A ik . A c id A ik . 3 .U2 16.61 •6 16.88 — —— A c id U n its A ik . U n its A c id A ik , it.26 1.26 6.ii2 1.02 3.80 it.7l 7.23 20 .70 9.18 19.02 .92 9.20 it.02 21.10 -9 2 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. DISCUSSION A. C a lc i u m 1» R a t s —— The b lo o d in T a b le s I - c a lc iu m v a lu e s in c o n ta in s th e r e s u lts (c o m p le te r a t i o n ) o f ir r a d ia t e d e r g o ste r o l in a n im a ls a b o u t ^ 0 gram s i n m in is te r e d In on b a s is c o m p a r in g t h e r is e g r a d u a lly [(.0 0 , 0 0 0 n o r m a lly d ecreases le v e l. e x p e c t w here ca u se any fu r th e r T a b le I I m a tu r e r a t s v e g e ta b le and sesa m e w e ig h t w e r e u s e d . r is e fr o m is on sto ck c o n ta in in g in th e on an d lo w .1 6 ^ sam e a s w a s e x c lu d e d . b lo o d in k g .) . th e c o n tr o ls le v e ls . a . 7 5 mgm r i s e a T h is in on t h e h ig h a s o n e w o u ld d o s e s w e r e a d m in is te r e d . c a lc iu m le v e l. se v e r a l grou p s o f young and p h osp h orou s d ie t s a n d a d m in ­ o f d ih y d r o ta c h y s te r o l. o f a r e g u la r b a s a l r a c h it o - p h osp h orou s. p h osp h orou s r a t io n w as th e The r i s e Y oung T h e d o s e s w e i- e a d ­ sh o w n o n a l l c o n s is te d about le v e ls s u p p le m e n t a p p a r e n t ly d i d n o t sh ow s t h e r e s u l t s p la c e d a a n a v e r a g e 1 . 2 ^ mgm i n c r e a s e o f th e in c r e a s e s fed o ils . (u n its p er 10 s u p p le m e n ts o f v a r i o u s l e v e l s d ie t g iv e n s u p p le m e n te d w i t h v a r i o u s su c h m a s s iv e T h e lo w p h o s p h o r o u s d i e t g e n ic are on r a t s The in c r e a s e s w ere n o t a s C o n tin u e d a d m i n i s t r a t i o n is te r e d s tu d ie s o f t h e 8 , 0 0 0 ,0 0 0 u n it l e v e l t o u n it on r a t s e x p e r im e n ta l a n im a ls w it h s l i g h t b lo o d c a lc iu m case of o f b o d y w e ig h t very th e s tu d ie s V. T a b le I sto c k r a tio n th e T h e s p e c i a l lo w t h e b a s a l e x c e p t t h e CaCO^ c a lc iu m on e a c h l e v e l -9 6 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. is d ir e c tly p r o p o r tio n a l t o th e d o se a d m in is te r e d . o n t h e lo w p h o sp h o r o u s (b a s a l) r is e s t h e 1 mgm a n d r e s p e c tiv e ly m a tu r e a n im a ls fo r on t h e t h e 1 mgm a n d s p e c i a l lo w sam e r a t i o n 1 mgm a n d e x h ib ite d t h e 1 mgm a n d . 5 mgm l e v e l s m a tu r e a n im a ls p h osp h oru s, le v e ls in a n im a ls . on a l l th e The le n g th cant a fte r a sto ck mgm l e v e l s , w hereas th e on th e CaCO^) g a v e r i s e s o f ij .li. T h e m a tu r e r a t s on th e o f 2 . 1 m g n s a n d l . l 6 m gm s o n r e s p e c tiv e ly . r a tio n s g re a ter u ses T he lo w b o th in th e in c a lc iu m p h osp h orou s, e x h ib ite d o f tim e o f are th e th e th a n s p e c i a l lo w g r e a test r is e s on a l l e x p e r im e n ta l an d c o n t r o l s u p p le m e n t f e e d i n g r e s u lts a n d lovr p h o s p h o r u s d ia te d b rew e rs y e a s t . sh ow n o s i g n i f i c a n t is in s ig n ifi­ o f b o t h y o u n g an d m a tu r e a n im a ls (b a s a l) The r e s u lts c a lc iu m ch an ges on e i t h e r on t h e r a t ’ s r e s i s t a n c e seem t o agreem en t w ith in r a t i o n s m ix e d w i t h on th e s e l e v e l s T h ese s t u d ie s be a n d 1|.. mgm 1$ d ays. I n T a b le I I I fed r is e s sh o w ed t h e order lis t e d 6 ,0 Y oung a n im a ls p la c e d (le s s r a tio n s . and s to c k .5 . 3 mgm l e v e l s . c a lc iu m T he y o u n g a n im a ls e x h ib ite d g a v e 2 , l | a n d 1 . 1 1 mgm r i s e s . S mgm l e v e l s . p h osp h orou s r a t io n a n d 3 . Oh o n t h e sam e r a t i o n fo r d ie t Y oung a n im a ls to ir r a ­ o f V ita m in D o f th e r a tio n s ch an ges in b lo o d s u c h w o r k e r s a s T a y l o r , W e ld , u sed . c a lc iu m B r a n io n , a n d K ay ( 8 3 ) . The e f f e c t o f C od L i v e r d i e t m ay b e f o u n d i n fro m $ 0 ,0 0 0 to no s ig n ific a n t O il o n m a tu r e r a t s T a b le IV . 1 $0 ,0 0 0 ch an ges On l e v e l s u n its p er 10 in b lo o d fee d on a o f V ita m in sto ck r a n g in g k g o f b o d y w e ig h t t h e r e w e r e c a lc iu m . -9 7 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited without p erm ission. The e f f e c t o f a lo w c a lc iu m m e n ts o f p u r e c a l c i f e r o l , in T a b le V . 2 ,0 0 0 th e sto ck f e d w ith s u p p le ­ O il m ay b e w e r e u s e d n a m e ly U 0 ,0 0 0 Mo s i g n i f i c a n t o r lo w o f v ita m in D in ta k e r e g a r d le s s seen and ' Tw o g r o u p s w e r e c o v e r e d d a y e x p e r im e n ta l p e r i o d . on e i t h e r d ie t and H a liv e r p e r 1 0 k g o f b o (fy w e i g h t . th ro u g h a l 5 le v e l H ydee Y e a s t , Tw o v i t a m i n D l e v e l s u n its w e r e fo u n d and sto c k c a lc iu m of r a tio n d iffe r e n c e s on e it h e r sou rce. 2 . D o g s —— T a b le VI c o n t a in s s u p p le m e n ts th e r e s u lt s w ith o f d ih y d r o ta c h y s te r o l. fo u n d w ere 8 .6 on th e and 1 .1 , 2 5 mgm l e v e l . on th e th e se le v e ls U .S .P . e ffe c ts 1 mgm l e v e l , o b serv ed in th e se r a is in g The d o g s r e c e iv in g in c a lc iu m e ffe c ts su ch a s lo s s tis s u e s su ch r is e mgm l e v e l , o f a n tir a c h itic 50, a c tiv ity and 25 u n its D ih y d r o ta c h y s te r o l i s le v e l. o b v io u s ly C om p ared w i t h v i t a m i n w o u l d r e p r e s e n t U0 , 0 0 0 , r e s p e c tiv e ly . s u p p le m e n ts The le v e ls per 10 k g . c a lc iu m and T here w ere no ap p aren t t o x ic s tu d ie s . o f dogs w ere fe d and pure c a l c if e r o l. 1 1 mgm r i s e m gm s o n t h e b a s is (w e ig h t b a s i s ) an d 1 0 ,0 0 0 u n i t s u n its $ .1 b lo o d c a lc iu m D g a n d D3 t h e s e l e v e l s U .S .P . T he a v e r a g e b lo o d On t h e o f v ita m in D r e s p e c t i v e l y . A grou p a c o m p le te d i e t o f d itg r d r c ta c h y s te r o l r e p r e s e n t 1 0 0 , v e r y p o te n t in 20, 000, dogs fed fe d The r e s u l t s 5 0 0 ,0 0 0 u n its o f ir r a d ia te d v a r ie d are fro m 1 0 ,0 0 0 t o sh ow n i n per 10 kg e r g o ster o l 5 0 0 ,0 0 0 T a b le V I I . sh ow ed a n a v e r a g e ( s e v e r e h y p e r c a lc e m ia ) w it h m ark ed t o x i c o f w e ig h t, as th e liv e r , e m a c ia tio n , s p le e n and d ea th , an d k id n e y s f in a u t o p s y sh ow ed e x tr e m e —98— R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. c a lc ific a tio n » fer o l th e T h e g o d s on U 0 0 ,0 0 0 u n i t s s h o w e d a 6 mgm r i s e 2 0 0 ,0 0 0 l e v e l lo w e r l e v e l s 5 0 0 ,0 0 0 , 1 2 .5 , and lo s s an average r i s e no s ig n if ic a n t p roduce th e sam e b l o o d c a lc iu m o f w h ic h a r e sh o w n i n a n d L o o ,0 0 0 u n i t c o n ta in o b ta in e d Thus on a w e ig h t b a s is in s u p p le m e n ts up i n fu r th e r w ork, o il. o f an e x tr a c t I t w as fe d p er m l). th e ex tr a ct seem ed t o u n it b a s is th a n p u re c a l c i f e r o l th a t c e r ta in to evap­ o f 2 0 0 ,0 0 0 , o f 1 1 a n d 6 m gm s r e s p e c tiv e ly . m ore p a t e n t o r ir r a d ia t e d e r g o s te r o l. p r o d u c ts th e r is e in on t h e sa m e T h is o th e r th a n v i t a ­ b lo o d c a lc iu m . C h ic k e n s — In r a tio n and be a l i t t l e ir r a d ia tio n m in Dg m i g h t h a v e c o n t r i b u t e d e th e r , o i l w as a s s a y e d and fo u n d t o C a lc iu m r i s e s T h is y e a s t 3. to th e r e s u lts a t le v e ls w e r e fo u n d a t t h e L 0 0 ,0 0 0 a n d 2 0 0 ,0 0 0 u n i t l e v e l s m ay i n d i c a t e 20 o f H yd ee Y e a s t . s e v e r a l tim e s w it h ( v i t a m i n D2 i n 2 i+ ,0 0 0 u n i t s on a w e ig h t b a s is r is e . T h is w a s p r e p a r e d b y e x t r a c t in g and ta k in g The T a b le V I I I . Tw o d o g s w e r e f e d o r a tin g ch an ges w ere n o tic e d . rep resen t At At d ih y d r o ta c h y s te r o l a r e r e q u ir e d c a lc iu m S im ila r r e s u lt s w ere c a lc i­ o f 3 m gm s w a s o b s e r v e d * a n d 5 m gm s o f v i t a m i n D2 « t i m e s m o r e v i t a m i n D2 t h a n o f pure o f w e ig h t b u t r e c o v e r e d . 1 + 0 0 ,0 0 0 , a n d 2 0 0 , 0 0 0 l e v e l s 10, le v e l th e se s u p p le m e n te d w i t h .2 5 m gm s. ta in e d s t u d ie s you n g and o ld in a c o m p le te d ih y d r o ta c h y s te r o l a t l e v e l s The r e s u l t s T a b le X . b ir d s w e r e f e d o f t h e b lo o d c a lc iu m F rom t i i i s it can be se en of 1, ch an ges a re t h a t c h ic k e n s -9 9 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. .5 , con ­ resp o n d l i k e dogs to B lo o d Ca r i s e s above th r e e th e v a r io u s of 8.6, le v e ls 5 .1 , le v e ls and 1 .3 o f d ih y d r o ta c h y s te r o l. mg w e r e o f d ih y d r o ta c h y s te r o l. o b serv ed a t th e L a y in g h e n s a sc a n e w h a t s m a l l e r u s e w h i c h m i g h t b e e x p e c t e d had a h ig h e r a s lig h tly i n i t i a l b lo o d c a lc iu m g r e a ter r e sp o n se . le v e l. sa m e r e s p o n s e a s d i d t h e y o u n g b i r d s e r g o ster o l ( v ita m in D g ), liv e r o il kg t o ^ 0 0 ,0 0 0 u n i t s g iv e n in H a liv e r (v ita m in D ^) a t l e v e l s th e y sh o w ed sh ow ed a b o u t t h e on t h e v a r io u s l e v e l s . O th e r g r o u p s o f b ir d s w e r e g iv e n te d in a s m u c h a s T h e m a le s The o ld b ir d s sh o w ed s u p p le m e n ts o il o f ir r a d ia ­ (v ita r a jji D ^ ), kSOO u n i t s p e r r a n g in g fr o m p e r 10 kg b ody w e ig h t. an d Tuna The r e s u lt s are T a b le X I , The b ir d s sh ow ed n o s i g n i f i c a n t o r 2 0 0 ,0 0 0 u n its o il o f 1 .8 a r is e o f v it a m in D2 « 36 r a t io 36 t i m e s u tto r e e f f e c t i v e v ita m in fro m H a liv e r o n 1 + 0 0 ,0 0 0 frc m tu n a l i v e r u n it l e v e l r e p r e s e n te d b etw e en t h e v ita m in D2 and on t h e r is e s W ith v i t a m i n m gm s o n a 2 0 0 , 0 0 0 to c a lc iu m o r t h e v ita m in sa m e u n i t l e v e l . o i l in d ic a te d th e sa m e R e s u lts a 1 w as of e ffe c tiv e n e s s as t h a t o f Tuna o i l . I4 . C o w s— A group o f cow s w e r e f e d and ir r a d ia te d e r g o s te r o l. s u p p le m e n ts The r e s u l t s o f D .H .T ., are W ith d i h y d r o t a c h y s t e r o l a n a v e r a g e r i s e fo u n d on t h e in g ly c lo s e b a s is o f th e . 5 mgm l e v e l f e d . agreem en t w ith t h a t g iv e n in H yd ee Y e a s t , T a b le X I I i o f 5 . 0 0 m gm s w a s T h is is in o f d o g s and c h ic k e n s f o r le v e l, - 100- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. s u r p r is ­ th is T he cow s f e d H y d ee Y e a s t s u p p le m e n t sh o w e d a r i s e 1 . 5 m gm s r e s p e c t i v e l y a t t h e 5 0 0 ,0 0 0 T h ese r e s u l t s w e r e fo u n d t o fed d o g s i n w h ic h a r i s e s and 2 5 0 ,0 0 0 u n it l e v e l s . b e lo w e r th a n t h e o f 11 o f 5*2 and H ÿd ee Y e a s t ex tr a ct an d 6 r e s p e c t iv e ly w ere fo u n d a t t h e iiOOjOOO a n d 2 0 0 , 0 0 0 u n i t l e v e l s . In 5 0 0 ,0 0 0 cow s g iv e n u n it s /lO w a s lo w e r s u p p le m e n ts of ir r a d ia te d kg le v e l a r i s e th a n th e r e s p o n s e in e r g o ste r o l at a o f 5 m gm s w a s o b t a i n e d w h i c h b o th d o g s an d c h ic k e n s . B - P h osp h oru s 1. R a ts— R e s u lts o f s t u d i e s w it h y o u n g a n d m a tu r e r a t s p la c e d p h o sp h o r u s a n d s p e c i a l lo w p h o sp h o r u s d i e t s h y d r o ta c h y ste r o l a re and . 5 mgm l e v e l s d e fin ite g iv e n in T a b le X I I I . m a tu r e a n im a ls . o b ser v ed w ith abundance R e fe r r in g t o T h e so m ew h a t l a r g e r th e d ie t o f c a lc iu m c o n ta in in g in th is s u p p le m e n ts r a n g in g fr o m t h e 1 mgm in c r e a s e s w ith y o u n g and in b lo o d p h o sp h o r u s CaCO, a r e u n d o u b t e d l y d u e t o to 1 .6 0 fo r th e n o ta b le d if f e r e n c e 1 mgm t o c o n te n t) th e phorus d ie t s fo u r le v e ls here is th a t on s t o c k .2 5 m gm s. r a tio n a n d D .H .T . The r e s u lt s o f D .H .T . on th e a d m in is te r e d . sto ck d ie t fo u n d fr o m The ( .6 2 - .6 i|^ e l e v a t i o n s w e r e m uch g r e a t e r t h a n w it h t h e on e i t h e r you n g th e d ie t. T a b le X IV , i n d i c a t e b lo o d p h o s p h o r u s e l e v a t i o n s r a n g in g 3 .1 Ü d i­ one can o b ser v e a t b o th l e v e l s G roups o f y o u n g r a t s w ere p la c e d in s u p p le m e n te d w i t h on t h e lo w p h o sp h o r u s d i e t , b lo o d p h o sp h o r u s r i s e s on lo w P lo w p h o s ­ o r m a tu r e a n im a l s , t h i s m ay i n d i c a t e - 101- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. th a t th e fo o d s u p p lie s im p o r ta n t i n f l u e n c e T h is i s fu r th e r o f p h o sp h o ru s a s w e ll on b lo o d ch a n g es in v o lv in g su p p o rted b y t h e w h ic h h ig h e r p h o s p h o r u s r i s e s m a tu r e r a t s th e sto ck 2. D o g s— r e c e iv in g D .H .T . r e s u lts o f th e c a lc iu m th e se sh o w n i n b lo o d w e r e s u p p le m e n ts i n a c o m p le te r a t i o n on 1 , e le m e n ts . T a b l e XV i n o b serv ed in c o n ju n c tio n w it h .5 , th e b lo o d p h o sp h o ru s v a lu e s s u p p le m e n te d uÿ t h e d a lljy ,2 $ mgm l e v e l s . and a n d 1 . 0 5 m gm s w e r e o b served on t h e th e y a r e n e v e r th e le s s in o f d ogs fe d a d m in is tr a tio n In crea ses above th r e e o f 2 .$ 3 , le v e ls A lth o u g h t h e p h o sp h o r u s c h a n g e s a r e n o t a s c a lc iu m have an r a tio n . T a b le XVI c o n t a i n s D .H .T . as h arm ony w i t h of 1 .7 5 » a d m in is te r e d . c o n s is ta n t a s th e o th er r e s u lt s o f th e in v e s tig a tio n . The r e s u lt s and p u re o f 2 .6 o f fe e d in g c a lc ife r o l are s u p p le m e n ts sh o w n i n m gm s w a s o b t a i n e d a t t h e er g o ster o l and on e o f 1 .6 o f ir r a d ia te d T a b le X V II, 500»000 le v e l m gm s a t 2 0 0 ,0 0 0 e r g o ster o l A p h o sp h o ru s r i s e o f ir r a d ia te d u n its o f pure c a lc i­ o f fu r th e r s tu d ie s on h ig h fe r o l. T a b le X V III c o n t a in s t h e r e s u l t s le v e ls of ir r a d ia te d er g o ster o l th e r e s u lts sh ow a b o u t t h e a s p r e v io u s e x p e r im e n ts a t t h e The r e s u lt s w ith T a b le X IX . and p u re sam e o r d e r o f b lo o d H ere a g a in p h osp h oru s r i s e sam e l e v e l s . an e x tr a c t o f % d ee Y e a st a r e g iv e n B lo o d p h o sp h o r u s r i s e s le v e l and ab ou t 2 .0 c a lc ife r o l. o f l.ij mg o n t h e 2 0 0 ,0 0 0 m g o n t h e 1^0 0 , 0 0 0 l e v e l w e r e - in ob served . 102- R ep ro d u ced with p erm ission of th e copyright ow ner. Further reproduction prohibited w ithout p erm ission . u n it 3. C h ic k e n s — S t u d i e s w i t h y o u n g m a le a n d f e m a le b i r d s p le te r a tio n a t U0 0 , 0 0 0 s u p p le m e n te d w i t h an d 2 0 0 ,0 0 0 phorous r i s e s . l . I i l w ere ter o l th e u n it l e v e l s O v e r a ll o b ta in e d so u rces I a t t h e 1^.0 0 , 0 0 0 and I I o f D .H .T . c a lc iu m r is e s At a ll o f ir r a d ia te d and r i s e s are le v e ls sh o w n i n an in c r e a s e and fe m a le s sh ow s m a lle r r i s e s e x c e p t a t t h e lo w e r T a b le XX, in b ir d s fe d b lo o d p h o s­ sh ow h ig h e r b lo o d T he m a le s th r o u g h o u t th e s e le v e ls w here th e r e ergos­ ,9 h a n d , 6 3 a t of th r o u g h o u t th a n do th e y o u n g er b ir d s . b o th y o u n g an d o ld mg a n d o f ir r a d ia te d sh o w n f o r y o u n g a n d o l d T he o ld m a le s er g o ster o l a v e r a g e s o f 2 .1 8 u n it le v e l The r e s u l t s a i ’e o n a co m ­ g a v e s i g n i f i c a n t b lo o d p h o s­ r e s p e c tiv e ly , I n T a b le XXI r e s u l t s p h oru s w as n o te d . sou rces ( m a le a n d f e m a le ) 2 0 0 ,0 0 0 u n it l e v e l . s u p p le m e n ts tw o p la c e d seem s t o s tu d ie s be no s ig n ific a n t d iffe r e n c e s . The r e s u lt s o f fe e d in g o f Tuna an d H a liv e r o il The p h o sp h o ru s r i s e s v a r io u s le v e ls . t h e tw o v it a m in U. on v a r io u s l e v e l s here are are in sh o w n i n t h e fo rm T a b le X X II. s m a lle r b u t m o re u n ifo r m w i t h i n No s i g n i f i c a n t d iffe r e n c e s so u rces u sed in th e se are ob served th e b e tw e e n s tu d ie s . C o w s— The s t u d ie s w ith ■5 mgm p e r 1 0 k g l e v e l 2 .0 )1 . tro l s u p p le m e n ts o f V it a m in A ll c o w s a d m i n i s t e r e d D .H .T . g a y s b lo o d p h o sp h o r u s r i s e s w h ic h o f th e m w e r e f a i r l y cow s u se d serv ed s u p p le m e n ts c o n s is te n t a t a s a d o u b le th is le v e l. c o n tr o l an d e x h ib ite d —1 0 3 — R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. at a averaged The con ­ n o b lo o d p h o sp h o ru s c h a n g e s. T he cow s w e r e b le d m e n ts w e r e a d m in is t e r e d w as fe d . H ere .in we fin d a drop in in itia l v a lu e s . $ 0 0 ,0 0 0 to and 5 d a y s a f t e r c o n tra st to th e are o f H yd ee l e a s t a n d 2 $ 0 ,0 0 0 sam e a s lo w e r l e v e l s la s t e ffec t s u p p le ­ s u p p le m e n t o f th e co m p o u n d ^ u n its o b served T a b le X X I I I . a d m in is te r e d o f V ita m in cow sh o w ed n o r i s e . th o se la s tin g sh o w n i n e x e r t a b lo o d p h o sp h o r u s r i s e The c o n tr o l th e b e fo r e b lo o d p h o sp h o r u s b a ck to w a r d s n orm al o r t h e The r e s u l t s S u p p le m e n ts in itia lly in per 10 o f 3 .1 2 g r a in a t le v e ls kg, w ere an d 2 .0 3 ob served r e s p e c tiv e ly * T h ese v a lu e s w ere p r a c t ic a lly on d o g s f e d t h e H ydee Y e a s t e x t r a c t ( H 0 0 ,0 0 0 a n d 2 0 0 , 0 0 0 ) , of T h ese v a lu e s are th e at sh o w n i n T a b l e X X IV . C ow s a d m i n i s t e r e d p er 10 kg le v e l o f 2 .0 9 , as sam e o r d e r ir r a d ia te d e x h ib ite d sh o w n i n as th o se e r g o s t e r o l a t a $ 0 0 ,0 0 0 u n its a n a v e r a g e b lo o d p h o sp h o r u s in c r e a s e T a b l e X X V, T h ese v a lu e s w ere e x h ib ite d b y d ogs a t t h e o f ab ou t th e sam e i r r a d i a t e d er g o ster o l le v e l. C. P h o sp h a ta se 1. R a ts— The p h o sp h a ta se v a lu e s and a d m in is te r e d 1 ,0 0 0 to ir r a d ia te d 2 0 ,0 0 0 u n i t s on m a tu r e r a t s e r g o ster o l The a c id p h o sp h a ta se v a lu e s in a s c e n d in g p h a ta se v a lu e s order on l e v e l s on s t o c k v a r y in g p e r 10 kg o f body w e ig h t a r e X X V I. th e p la c e d sh o w s i g n i f i c a n t o f v i t a m i n D2 d o s a g e . d ecreased fro m sh ow n i n T a b le d ecreases The a lk a lin e a s t h e v i t a m i n D2 l e v e l r a tio n in c r e a s e d . -1 0 b - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. p h os­ T a b l e X X V II s h o w s t h e b o th th e a c id and a lk a lin e p h o sp h a ta se , a s pron oun ced. T h e a n im a ls w e r e f e d s n p p le m e n ts o f i r r a d i a t e d h ig h in sa m e d e c r e a s e i n on lo w a c id p h o sp h a ta se b o th a c id Y oung r a t s p la c e d lo w e r e d a c id on a lo w d iffe r e n c e s in d ie t an d g iv e n S ig n ific a n t d ecreases w a s o b s e r v e d w hen T a b le X X V III, and sto ck e ffe c t at d ie ts a ll s u p p le ­ O il e x h ib it e d le v e ls . T h ere b e t w e e n v i t a m i n s D2 a n d seru m p h o s p h a t a s e r e s u l t s . 5 m g m /lO k g l e v e l cow r e c e i v i n g o f DHT a n d t h e in th e a lk a lin e th e s u p p le m e n ts . sh ow ed n o s i g n i f i c a n t ch a n g es. v a r ia tio n s w ere o b serv ed in ta se c a lc iu m w as n o t C o w s— A grad u al d ecrease th e a c tiv ity . sh o w n i n of T a b l e X X IX . T a b l e XXX s h o w s t h e one fe d as case sh o v æ d a n i n i t i a l l y and a lk a lin e p h o sp h a ta se v a lu e s sh o w n i n 2. d ie t th e c a lc ife r o l. p u r e c a l c i f e r o l % rd ee Y e a s t a n d H a l i v e r w ere no s ig n ific a n t as a norm al s to c k c a lc iu m in th e la t t e r and a lk a lin e p h o sp h a ta se a c t i v i t y H yd ee Y e a s t w a s a d m in is t e r e d m e n te d w it h a lth o e r g o s t e r o l and p u r e Y oung r a t s p la c e d and a lk a lin e a c tiv ity o th e r on tw o u sed a s cow s, a c o n tr o l. p h o sp h a ta se w as o b ser v ed The a c id In th e e ith e r th e - 10^ - p h o sp h a ta se c o n t r o l cow n o a c id in a c tiv ity s ig n ific a n t or a lk a lin e a c tiv ity . R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. p h osp h a­ CONCLUSIONS 1 . R a ts f e d m a s s iv e d o s e s sh ow ed c h a r a c t e r i s t i c 2, o f v it a m in D fro m v a r i o u s r e s is ta n c e D o g s t o w h ic h m a s s iv e d o s e s to ob serv ed to and, a c e r ta in ed b y d e a th . cases, A u to p sy c a lc iu m o f v a r io u s v ita m in w er e a d m in is te r e d w e r e in b lo o d sou rces ch an ges. D p r o d u c ts sh ow a h y p e r c a l c e m ia , g e n e r a l e m a c ia te d c o n d itio n , sh o w ed a g e n e r a l c a l c i f i c a t i o n fo llo w ­ of v a r io u s t i s s u e s . 3= C h ic k e n s a d m i n i s t e r e d v a r jr ln g sh o w ed lo w e r r i s e s V ita m in tiv e th en U . M a s s iv e w as fo u n d t o d oses 6. th a n w ith v ita m in b e a b o u t 36 t im e s m ore e f f e c ­ o f v a r i o u s V ita m in D p r o d u c t s a d m in is te r e d s tu d ie s , it o f ir r a d ia te d th a n a c o r r e s p o n d in g w o u ld a p p e a r i r r a d i a t e d y e a s t w as so m ew h a t m o re a m ou n t o f p u r e V ita m in D . D ih y d r o ta c h y s t e r o l p r o d u c e d s i g n i f i c a n t b lo o d tio n s in to h y p e r c a lc e m ia . o f lim ite d or an e x tr a c t e ffe c tin g D p r o d n ç ts V ita m in D g . On t h e b a s i s yeast o f v ita m in on v it a m in D2 s o u r c e s cow s c a u s e d a d e f i n i t e 5 . d oses y o u n g an d m a tu r e r a t s fed c a lc iu m e le v a ­ n o rm a l a n d lo w p h o sp h o r u s d ie ts . 7. T he a v e r a g e b lo o d ta c h y ste r o l 10 kg. c a lc iu m s u p p le m e n ts u se e x h ib ite d b y d og s o f 1 m g m . ,. 5 m g m s ., o f b od y w e ig h t w ere 8 .6 and mgras 5 - 1 m g r a s, on d ih y d r o ­ . 2 5 mgras p e r and 1 .1 r e s p e c tiv e ly . - 106 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. m gras 8. C h ic k e n s f e d to s u p p le m e n ts sh ow c a lc iu m fo r le v e ls u ses o f 8 .6 o f 1 m gm , T h ese r e s u lt s r is e and . 2 3 m gm . r e s p e c t i v e l y . cow s. o b ta in e d T h ese e f f e c t s are c o m p a r a b le t o b lo o d p h o sp h o r u s a tta in b lo o d p h o sp h o r u s r i s e s b e m ore e f f e c t i v e d oses H ere a g a in t h e v ita m in o f v a r io u s w as fo u n d t o t h a n Dg s u p p le m e n ts o f v a r io u s v ita m in D p r o d u c ts in m a s s iv e d o s e s p r o d u c e d m ark ed b lo o d p h o sp h o r u s r i s e s r a n g in g fro m 2 3 m g m s. 1 3 * Y oung a n d m a tu r e r a t s sh o w n t o have e le v a te d fed n o rm a l a n d lo w th e case p h osp h oru s d ie t s w ere p h o sp h o r u s l e v e l s w hen v a r io u s d ih y d r o ta c h y s te r o l w er e a d m in is te r e d . lit. o f b e tw e e n l.U sh o w s i g n i f i c a n t e l e v a t i o n s w hen f e d m a s s iv e v ita iE in D p r o d u c ts . in th o se o f v a r io u s v ita m in D p r o d u c ts Y oung an d m a tu r e c h ic k e n s w e r e o b s e r v e d t o to a 3 * 0 m gn b l o o d d o g s and c h ic k e n s . w ere o b serv ed t o 1 2 . C ow s f e d on d o g s. . 5 mgm l e v e l p r o d u c e d 1 0 . D ogs a d m in is te r e d m a s s iv e d o s e s 11. $ . 1 m g m s . , a n d 1 . 3 m g m s ., a g r e e w e l l w ith t h o s e in o b ta in e d w it h m g r a s ., m g m ., 9 . D ih y d r o ta c h y s t e r o l on a c a lc iu m o f d ih y d r o ta c tQ r s te r o l w e r e o b s e r v e d The r i s e s d oses of w ere g r e a te s t o f th e norm al r a t io n , D o g s t o t w h ic h d ih y d r o t a c h y s t e r o l w a s a d ia in is t e r e d phorus e le v a tio n s le v e ls o f 1 .0 o f 2 . 3 3 m g m s, m g m ., * 3 m g m ., and 1 . 7 3 m g m s, . 2 3 m g m ., sh ow ed p h o s ­ a n d 1 . 0 3 m gm s a t per 1 0 kgm b o d y w e ig h t r e s p e c t i v e l y . -1 0 7 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 1$. C h ic k e n s a d m in is t e r e d p h osp h oru s in c r e a s e s som ew h at h ig h e r i n s u p p le m e n ts a t a ll le v e ls t h e m a le s t h a n o f D .H .T . u sed . in sh o w ed b lo o d The r i s e s w ere th e fe m a le s . 1 6 . Cows fe d d ih y d ro ta c h y ste ro l on a b a s is .5 mgm p e r 10 kg of body w eight showed an average phosphorus r i s e of 2.0U mgms. 1 7 » A c id a n d a l k a l i n e 1,000 to 20,000 b od y w e ig h t w ere an d lo w c a lc iu m g a v e h ig h e r seru m p h o s p h a ta s e v a lu e s u n it le v e ls sh o w n t o o f v ita m in D be in itia l ( I.E .) s ig n ific a n tly r a t io n s w ere u se d . T h e lo w and f in a l v a lu e s in on r a t s per lo w e r e d . c a lc iu m th e se fed 10 on kg N orm al r a tio n s tu d ie s . 18. Serum phosphatase v a lu e s on cows fe d d ih y d ro ta c h y s te ro l, sh ow ed a g r a d u a l d e c l i n e w hereas th e in th e a lk a lin e p h o sp h a ta se v a lu e s a c i d p h o s p h a t a s e r e m a in e d u n c h a n g e d . - 108 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. BIBLIOGRAPHY 1 . Drummond, J . C ., The F a t-s o lu b le V itam ins in N u tr itio n . Lane M edical L e c t., ( S tan d fo rd U n iv e rs ity P r e s s , 193U)* 2 . M etz, G. A», and Coppens, A ., Long en V itam in D. N ed erl. ty d s c h e r. v . g e n e e sk ., 76, ?69, (I93U)» 3 . Rosenheim, 0 . , and W ebster, T. A ., The R e la tio n of C h o le s te ro l to V itam in D. Biochem. J . , 337j (1926), it. Windaus, A ., and Bock, F ., Uber das P ro -v ita m in aus dem S te r in d e r Schweinschvj-arte, Z ts c h r, f . P h y s io l. Chem., 2h3, 168, (1 9 3 7 ). 3 . Windaus, A ., L in s e r t, C ., L u ttrin g h a u s , A ., and Vv'eidlich, Uber das K r y s t a l l i s i e n t e V itam in D2 . Ann. Chem., it92, 226, (1932) . C t . , 6 . L e t t r e , H ., Zur K enntnis d e r B e s tra h lu n g s -p ro d u c te des E r g o s te r in s . Ann, Chem., g l l , 260, (193U). 7 . Friedm ann, E ., S te r o ls and R e la te d Compounds p p . kS-hl• (Chemical P u b lish in g Company o f N. Y. I n c ., 1 9 3 7 ). 8 . W indaus, A ., W erder, F. von, and G sch aid er, B ., Uber d ie Zahl der K ohlenstoffatom e im M olekul des S i t o s t e r i n s and l i n i g e r a n d e re r S t e r i n e . , B er, d e u tsc h e Chem. G e s e lls c h ., 1006, (1932) . 9 . H o ltz , F . , G is s e l, H ., and Rossmann, E ., Experim ent e l l e und k lin is c h e S tu d ien z e r Behandlung d e r p o s to p e ra tiv e # T e ta n ie m it A .T. 10 D eutsche Z ts c h e r, f , C h ir. 2U2, 521, (193U). 1 0 . A lb rig h t, F , , Bloomberg, E ., D rake, T ., and S ulkow itch, H. W., A Comparison o f th e E f f e c ts of A .T. 10 (d e h y d ra ta c h y s te r o l) and V itam in D on Calcium and Phosphorous Metabo­ lis m in h y p o p arath y ro id ism . J . Chem. I v e s t g . , 1 7 , 317, (1 9 3 8 ). -1 1 . A lb rig h t, F . , S ulkow itch, H. W., and Bloomberg, E ., A Ccmp a ris o n of th e E f f e c ts of V itam in D, d ih y d ro ta c h y s te ro l (A .T .10) and p a ra th y ro id E x tra c t on th e D iso rd e re d M etabol­ ism o f R ic k e ts , J . Chem. I n v e s t i g ., 1 8 , 165, (1 9 3 9 ). - 109 - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 1 2 , S hohl, A. T,^ Fan, H. T ., and F a rb e r, S ., E f f e c t o f A.T, 10 (d ih y d ro ta c h y s te ro l) on V arious Types of E xperim ental R ic h e ts i n R a ts . P ro c, Soc. Exp. B io l, and M ed., 1+2, ^29, (1939). 13» Harnapp, G. 0 . Dur P athogenese d e r S pasm ophilie Behandlungso ersu ch e m it A .T .10 M onatscher. F . K in d e rh ., 63, 262, (1 9 3 3 ). lU . S hohl, A .T ., and B a rb e r, S ., E f f e c t o f A .T .10 (d ih y d ro ta c h y ­ s t e r o l ) on R ic k e ts in R ats Produced by High Calcium , LcwPhosphorus D ie ts . J . N u tr itio n , 21, lU 7, ( I 9 i |l ) . 13» H ess, A. F . , and S uppléé, G. C ., The A ctio n o f I r r a d i a t e d E rg o s te ro l on R ats and C hickens, P ro . Soc. E xper. B io l, and M ed,, 27, 609, (1930). 1 6 . C o rn e ll, J . T ., and Vfise, E. C ., A n ti r a c h i tic P r o p e r tie s of A.T.IO f o r th e R at and Chicken, J . N u tr itio n , 23, 217, (19U2). 1 7 . M assengale, 0 . N. and B i l l s , C. E ., A Q u a n tita tiv e Method f o r th e Assay o f V itam in D w ith C hickens, J . N u tr itio n , 12, i;29, (1936). 1 8 . Gaward, K. H ., The R e la tiv e A n t i r a c h i ti c P o te n tc ie s of V ita ­ min Ü2 ( C a lc if e r o l from E rg o s te ro l) and o f V itam in (from I r r a d i a t e d 7 - d e h y d ro c h o le s te ro l), B u ll. H ealth Organ, (League o f N a tio n s ), 9 , i+23, (19U0-U1). 1 9 . Renys, D. G ., and M a rs h a ll, I . H ., The A n ti r a c h itic A c tiv ity o f V arious Forms o f V itam in D i n t h e C hick, J . N u tr itio n , 13, 323, (1 9 3 8 ). 2 0. R u s s e ll, Vf* C ., T a y lo r, M. ¥ . , and Vfilcox, D. E ., The F a te o f th e A n t i r a c h itic F a c to r i n th e C hicken. I I The E f f e c t o f th e F a c to r A d m in istered by Mouth and I n t r a p e r i t o n e a l l y , J . B io l. Chem., 99, 109, (19 3 2 ). 21. R u s s e ll, W. C ., T a y lo r, M. ¥ . , and W ilcox, D. E ., The F a te o f th e A n tir a c h itic F a c to r in th e Chicken. I l l The E f f e c tiv e L evels and D is tr ib u tio n o f th e F a c to r from Cod L iv e r O il and from I r r a d i a t e d E rg o s te ro l in C e rta in T is su e s of th e Chicken, J , B io l. Chem., 3£7, 733, (193U). 22. McChesney, E. W,, and Homburger, E ., A M o d ific a tio n of th e L ine T e st A p p lic a b le t o Chicken V itam in D A ssay, J . N u tri­ t i o n , 20, 339, (1930). - 110- R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout p erm ission . 2 3. McChesney, E. W., and K acher, H ., An E v a lu a tio n o f V arious Methods f o r B ioassay o f D ih y d ro ta c h y s te ro l, E ndocrinology, 30, 787, (19h2)\ 2k» McChesney, E. W., S tu d ie s o f Calcium and Phosphorus M etabol­ ism in th e C hick. I The Com parative E f f e c ts o f V itam ins Dp and D_ and D ih y d ro ta c h y ste ro l Given O ra lly and In tram u s­ c u la r ly , J . N u tr itio n , 26, 81, (19U3). 25 . McChesney, E. W,, S tu d ie s o f Calcium and Phosphorus M etabol­ ism in th e Chick. I I R e la tiv e A n tir a c h itic E ffe c tiv e n e s s o f V itam ins Dp and D^ and D eh y d ro tach y stero l A dm inistered P a r e n te r a lly , J . N u tr itio n , U8?, (19U3). 26. C o rn e ll, J . T ,, and W ise, E. G., The Com parative T o x ic ity o f C a lc if e r o l, A.T. 1 0 , and Cod L iv e r O il C o n ce n trate f o r C hicks, J . N u tr itio n , _2o, 6 i|l, (19Ù3). 2 7 . Jo n e s, J . H ., R apaport, M ., and Hodes, H. L ., The E f f e c t of I r r a d i a t e d E rg o s te ro l on T h y ro p arath y ro id -ecto m ized Dogs, J . B io l. Chem,, 86, 267, (1930). 28. S ch n eid er, H«, and S teenbock, H ., D iffe re n c e s in Response to V itam in Dg of R ats on C ereal Low Phosphorus D ie ts and S y n th e tic Low Phosphorus D ie ts , J . B io l. Chem., 123, P ro c , CV, (1 9 3 8 ). 29. C o rn e ll, J . T ,, and W ise, E. C ., S tu d ie s on th e R e la tiv e E ffic ie n c y o f V itam in D from S e v e ra l S o u rces. I I In flu e n c e o f V itam in D o f d i f f e r e n t O rig in s on th e Serum P hosphatase o f th e C hick, J . B io l. Chem,, 1 ^ , $81, (1938). 3 0 . R ap ap o rt, S ., and G u est, G. M,, Changes o f O rganic A cids o lu b le phosphorus, D ip h o sp h o g ly ce rate, A d e n o sin e trip h o sp h a te and In o rg an ic Phosphorus in th e Blood C e lls o f R ats During th e Development and th e H ealing of R ic k e ts , J . B io l. Chan., 126, 749, ( 1 9 3 9 ) . 31. Jo n es, J , H ,, The P ro d u c tio n o f H yperoalcem ia w ith sm all Amounts o f V itam in D, J . N u tr itio n , £ 8 , 7, (1944). 32. W addell, J . , The P ro v itam in D o f C h o le s te ro l. I . The A n ti­ r a c h i t i c E ffic a c y of I r r a d i a t e d C h o le s te ro l, J . B io l, Chem,, 105, 711, (1 9 3 4 ). 33. B ethke, R. M ., Record, R, R ,, Kennard, D, C ., The Compara­ t i v e A n tir a c h itic E f f ic ie n c y o f I r r a d i a t e d E rg o s te ro l I r r a ­ d ia te d y e a s t , and Cod L iv e r O il f o r th e Chicken, J , N u tr itio n 6^ 413, (1 9 3 3 ). -Ill- R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout p erm ission . 3U» McChesney, E. W., Comparison o f H ypercalcem ia E f f e c ts of A c tiv a te d S t e r o ls in th e C hick, P ro x . Soc. Exp. B io l, and M ed., 1U7, (19U3). 35» Coward, K, H ., The B io lo g ic a l S ta n d a rd iz a tio n o f th e V ita ­ m in s, (W illiam Wood and C o., B altim o re , Md. 1 9 3 8 )« 36. R u s s e ll, W. C ., and T a y lo r, M. W., The R e la tio n Betvieen th e V itam in A and D In ta k e by th e Hen and th e O utput in Eggs, J . N u tr itio n , W , 6 l3 , (193$). 37« Morgan, A. G ., and S h im o to ri, N ., The A bsorption and R eten­ t i o n by Dogs o f S in g le M assive Doses o f V arious Forms of V itam in D ., J . B io l. Chem., 189, (19U3). 38. Sherman, H. C ., and Booker, L. E ., The Calcium C ontent of th e Body i n R e la tio n to t h a t of th e Food, J . B io l. Chan., 93, 93, (1 9 3 1 ). 39. L an fo rd , C. S . , and Sherman, H. C ., F u rth e r S tu d ie s of th e Calcium C ontent of th e Body as In flu e n c e d By T hat o f th e Food, J . B io l. Chen., ] ^ , 38I , (1 9 3 8 ). UO. Jo n es, J , H ., R ap ap o rt, M ., and Hodes, H. L ., The Source o f Excess Calcium i n H ypercalcem ia Induced by I r r a d i a t e d E rg o s te ro l, J . B io l. Chem. 267, (1930). 111. Greenwald, J . , The R e la tio n of th e C o n c e n tra tio n of C al­ cium to t h a t o f P r o te in and In o rg a n ic P hosphate Serum, J . B io l. Chem., 93, $$1, (1 9 3 1 ). in th e 112. K le in , D ., and R u s s e ll, W. C ., The F a te o f th e A n tira c h i­ t i c F ac to r i n th e Growing C hick. I The A n tir a c h itic F a c to r Balance i n th e Growing C hick, J . B io l. Chem. 9 3 ,6 9 3 ,(1 9 3 1 ). U3. O uthouse, J . , Sm ith, J . , and Twomey, J . , The R e la tiv e E f f e c ts o f C e rta in S ac h a rid e s and o f V itam in D on m in era l m etabolism of R a ts , J . N u tr itio n , 1 ^, 2$7, (1 9 3 8 ). ilU* Me C lean, F. C ., A c tiv a te d S te r o ls in th e T reatm ent of P a ra th jT o id I n s u f f ic ie n c y , J , Am. Med. A s sn ., 117, 609, (19U l). U$. A lb right> A ., S ulkow itch, H. W., Bloomberg, E ., A Comparison o f th e E f f e c ts o f V itam in D, D ih y d ro ta c h y ste ro l (A.T. 1 0 ), and P a ra th y ro id E x tra c t on th e D iso rd e re d M etabolism of R ic k e ts , J . C lin . I n v e s t . , 18, 16$, (19 3 9 ). - 112- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 1|,6. Cohn, W. E ., and G reenberg, D. M ., S tu d ie s in M in eral M etabolism w ith th e A id o f A r t i f i c i a l R a d io a c tiv e I s o to p e s . I l l The In flu e n c e o f V itam in D on th e Phos­ phorus M etabolism o f R a c h itic R a ts , J . B io l. Chem., 128, S c i. P ro c . XVI, {1939) > U7. McDonald, F , G ., and M assengale, 0 . N ., The A n tir a c h itic P o te n tc y o f Eggs From Hens R eceiving M assive Doses of A c tiv a te d E r g o s te ro l, J . B io l. Chem., 59, 79, (1 9 3 2 ). U8. R u s s e ll, W. C ., T a y lo r, M. W., and W ilcox, D. E ., The F a te of th e A n tir a c h itic F a c to r i n C hicken. I I . The Ef­ f e c tiv e n e s s o f th e F a c to r A dm inistered by Mouth and I n t r a p e r i t o n e a l l y , J . B io l. Chem, 99, 109, (1932-33)• I'P; Reed, G= I ^ , S tru c k , H. G ., and Stcclc, I , E ., V itam in D; Chem istry', P h y sio lo g y , Pharmacology, P ath o lo g y , E x p eri­ m en tal and C lin ic a l I n v e s tig a tio n s , (The U n iv e rs ity of Chicago P r e s s , C hicago, I l l i n o i s 1 9 3 9 ). 5 0. Eddy, W, H ., and D a lld o r f, G. The A v itam in o ses. The Chem ical, C l in ic a l and P a th o lo g ic a l A spects of th e V ita ­ min D eficien c y D is e a se s . (W illiam s and W ilk in s, B a l t i ­ m ore, Md., 19 3 7). 5 1 . Kern, R ., Montgomery, M ., and S t i l l , E. U ., The E f f e c t o f Large Doses o f I r r a d i a t e d E r g o s te ro l Upon N itro g e n , Calcium, and Phosphorus M etabolism in R a ts . J . B io l. Chem. 93, 365, (1931). 5 2 . T a y lo r, N. B ., and Weld, C. B ., The M o b iliz a tio n and E x c re tio n o f Calcium Follovdng Overdosage w ith I r r a d i a t e d E r g o s te ro l, B r i t . J . E xper. P a th ,, 13, 109, (1 9 3 2 ). 5 3 . B auer, W. , M arble, A ., and C la f lin , D ., S tu d ie s on th e Mode o f A ction o f I r r a d i a t e d E r g o s te r o l, IV In Hyper­ p a ra th y ro id is m , J . Chem. I n v e s t , , U , U7, (1932), 5ii. Duncan, G, W,, and Huffman, C, F , , The E f f e c t o f D aily M assive Doses o f V io s te r o l Upon Calcium and Phosphorus M etabolism and Blood Calcium and In o rg a n ic Phosphorus i n C alves, J , D airy S c i , , 3 J , 83, (193U). 5 5 . W a llis , G. C ., Palm er, L, S ,, and G u llic k so n , T, W*, The R e la tio n of V itam in D to Calcium and Phosphorus R e te n tio n i n C a ttle as Shown by B alance T r i a l s , J . D airy S c ., 18, 213, (1 9 3 5 ). " -113- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 5 6. Patw hordan, V. N ., and C h itr e , P. G . , The E f f e c t o f Hyperv ita m in o s is D on th e Calcium and Phosphorus M etabolism i n th e A lbino R a ts , P ro c. Soc. Exp. B io l, and M ed., (In d ia ) 3 , 63, (1 9 3 8 ). 5 7. Reed, C. I . , On th e N ature of th e T oxic A ctio n of V itam in D. P ro c. S oc, Exper, B io l, and Med. 791, (1 9 3 8 ). 58. N ico lay sen , R ,, S tu d ie s on th e Mode o f A ction o f V itandn D. I . I n v e s tig a tio n s Upon th e Phosphorus Compounds in M uscles, L iv e r, and Kidneys as In flu e n c e d by D if f e r e n t L ev els of V itam in D and Phosphorus i n th e D ie t, Biochem. J , , 30, 1329, (1936). 5 9. N ico lay sen , R ., I I . The In flu e n c e o f V itam in D on th e F a e c a l O utput of Endogenous Calcium and Phosphorus in th e R a t, B io­ chem. J . 31, 107, ( 1 9 3 7 ) . 60. N ico lay sen , R ., I I I . The In flu e n c e o f V itam in D on th e Ab­ s o rp tio n o f Calcium and Phosphorus in th e R a t, B iochan. J . , 31 , 122 , (1937 ). 61 . N ico lay sen , X ylose, and t e s t i n e and o f th e R a t, R ., IV, The A bsorption o f Calcium C h lo rid e , Sodium S u lf a te From I s o l a t e d Loops o f Small In ­ o f Calcium C h lo rid e from th e Abdominal C avity Biochem. J . , 323, (1 9 3 7 ). 62. N ico lay sen , R ., V The A bsorption o f P hosphates from th e I s o l a t e d Loops of th e Sm all I n t e s t i n e in th e R a t, Biochem. J. 1086, (1937). 6 3 . N ico lay sen , R ,, The Carbon D ioxice O utput in Awake and N arc o tize d R ats in R ic k e ts and IVhen R ic k e ts Has Been Qured, Skandinav. A rch, f . P h y s io l. 79, (1 9 3 9 ). 6U. Schm idt, G .L .A ., and G reenberg, D. M ., O ccurrence, T ra n s p o rt, and R e g u la tio n of Calcium, Magnesium, and Phosphorus in th e Animal Organism s, P h y s io l. R e v ., 1 ^ , 297, (1935). 65. P h i l l i p s , R. A ., R obertson, D. F ., C arson, W, C ., and Irvrin, G. E ., The E f f e c t o f I r r a d i a t e d iîrg o s te ro l on th e Thrombo­ c y te s and th e C oagulation o f Blood, Ann. I n t . M ed., I4., 113k, (1931) . 66. S u r e ,.B ., and K ik, M. C ., H e in a to p o ie tic F u n ctio n i n Hyperv ita m in o s is . V. V itam in D D e fic ie n c y , P rox, Soc. Exp. B io l, and M ed., 28, 196, (1931). -Ilk - R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 67. T aylor, N. B ., Vfe ld , C. B ., Branion, H. D ., and Kay, H. D ., A Study o f th e A ction o f Irra d ia ted E rgosterol and i t s R ela tio n sh ip to Parathj'Toid Function, Canal, M .A .J., 2l|., 763, (1931). 68. Leenhardt, E ., Baucomont, J . , and Gayla, J , , Recherches Sur l e Phosphore e t l e s Glycerophosphatases du Sang des Enfants R a ch itiq u es. V ariation s Observées au Cours du Traitement par l e s Rayons U lt r a - v io le t s , Rev, Franc, de P e d ia t ., 13, lUU, (1937). 69 . Dreyer, I . , and Reed, C. I . , Treatment o f A r th r itis w ith M assive Doses o f Vitamin D. Arch. Phys. Therapy, I 6 , 537, (1 9 3 5 ). 7 0 . Reed, C. I . , and T hacker, E, A ., The E f f e c t o f In tra v e n o u s and I n t r a p e r i t c n e a l I n je c tio n s o f I r r a d i a t e d E r g o s te ro l, Am. J . P h y s io l., 96, 21, (19 3 1 ). 71 . B a s s e tt, S. H ., Mineral Exchanges of Man. V. Balances of E lectroljj-tes in a Case of Hyperthyroidism, J . N u tr itio n , 9 , 323 , (1 9 3 5 ). 72 . Freudenberg, E ., R a ch itis und Spasmophile. , Monatschr. f . K inderh., 303 , (1937). 73 . Widenbauer, F ., K orrelation der Vitamin A and B m it dem C a lcin o se -fa k to r, K lin. Wchnscher., 1U,901, (1935). 7U. C o rn ell, J. T, and Wise, E. G.j A n ti-r a c h itic P rop erties o f A.T. 10 fo r the Rat and Chicken, J . N u tritio n , 23, 217, (19U2). 75 . Goorraaghtigh, N ., and Handovslqy, H ., E ffe c t o f Vitamin Dp (C a lc ife r o l) on th e Dog, Arch. P a th ., 26, III4 J 4 , (1938). 76 . F ie s e r , L. F ., The Chemistry of Natural Products R elated t o Phenanthrene. 2nd ed. (Rheinhold P ub lishing Corp. New York, N. Y. 1937). 77 . B i l l s , C. E ., and McDonald, F. G ., Experiments on th e S yn th esis and I s o la tio n of Vitamin D. Read b efore th e American A sso cia tio n fo r th e Advancement o f S cien ce, New O rleans, December 30, ( 1931 ) , 78» Heinbron, I . M., Kamm, E. D ., and Morton, R. A ., The Ab­ so rp tio n Spectrum of C h olesterol and I t s P o s s ib le B io lo g i­ c a l S ig n ific a n c e w ith R eference to Vitamin D. I . P relim i­ nary O bservations. J. S oc. Chem. In d u st. , l|.5j 932, (1926). -115" R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. 79, B i l l s , C. E ., Physiology of the S t e r o ls , Including Vitamin D, P h y s io l. R ev., I^ , 1 , (1 9 3 5 ). 80, Smith, E. E ,, and McLean, F. C ., E ffe c t o f Hyperthy­ roidism upon Growth and Chemical Composition of Bone, Endocrinology, 23, 5H6, (1 9 3 8 ), 81, Benjamin, H. R ., and H ess, A. F ., The Forms o f th e Cal­ cium and Inorganic Phosphorus in Human and Animal S era. I . Normal, R a c h itic , Hypercalcémie,^ and other C onditions, J. B io l. Chem,, 3 ^ , 27, (1 9 3 3 ). 82, G elfan, S ., E ffe c t o f V io s te r o l Upon Oxygen Consumption o f Frog's M uscle, Am, J , P h y sio l, 113, U6Uj (1935). 83* T a y lo r, N. B ,, Weld, C« B ,, and Sykes, J , F ,, Observa­ tio n s Upon the A usoi'ptlon o f Calcium in Normal A nim als, B r i t . J , Exper, P a th ,, lU , 33S, (1933). - 116- R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. ACKNOY£LEDGEMENTS 1. To Dr. Huffman fo r h is kind support in supplying feed in g and m aintaining the cows fo r th e se s tu d ie s . 2. To Dr. Davidson fo r h is g en ero sity and help in ob tain in g, feed in g and housing th e b ird s used in th e se s tu d ie s . 3« To Standard Brands Company fo r supplying our ir r a d ia te d e r g o ste r o l (1 ,0 0 0 ,0 0 0 ,0 0 0 u n its ) used in th e se s tu d ie s . i|. To E. R. Squibb and Company fo r th e ir kindness in preparing and donating some of th e various vitam in D products used in th e se s tu d ie s. 5* To sen io r problem stu d en ts Robert TiT. S tip e k , James M. I s b e s te r and Charles Leng, fo r th e ir help in th e carryin g out o f some o f th e a n a ly tic a l and animal work. 6. To th e V eterinary and Poultry'' and P hysiology Departments in fu rn ish in g housing quarters fo r th e dogs used in th e se s tu d ie s . 7. To th e assay lab oratory fo r th e assays run on various m a teria ls used. 8. To W illiam H. G ill fo r the many kindnesses and help in ob tain ­ ing equipment fo r maintenance o f the various animals used in th ese s tu d ie s . R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission. ACKNOV^Lï;DGEMENTS 1. To Dr. D. T. Ewing o f th e Chemistry Department fo r h is kind a s sista n c e and g en ero sity in supplying of variou s vitaiuin D so u rces. 2. To Dr. Huffman fo r h is kind support in supplying feed in g and m aintaining the cows fo r th ese s tu d ie s . 3. To Dr. Davidson fo r h is g en ero sity and help in ob­ ta in in g , feed in g and housing th e b ird s used in th ese s tu d ie s . U. To Standard Brands Company for supplying our ir r a ­ d iated er g o ste r o l (1 ,0 0 0 ,0 0 0 ,0 0 0 u n its ) used in th ese s tu d ie s . 5. To E. R. Squibb and Company fo r t h e ir kindness inpreparing and donating some of th e variou s vitam in D products used in th e se s tu d ie s . 6. To sen io r problsn stu d en ts Robert Vii. S tip ek , James W. Isb e ster and Charles Lenz, fo r t h e ir help in th e carrying out of some o f the a n a ly tic a l and animal work. 7. To the V eterinary and Poultry and P hysiology Depart­ ments in fu rn ish in g housing quarters for the dogs used in th ese s tu d ie s . 8. To the assa y laboratory fo r the assa y s run on various m a teria ls used. 9. To W illiam H. G ill fo r th e many kindnesses and help in obtaining equipment fo r maintenance o f th e various animals used in th ese s tu d ie s . R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited w ithout p erm ission.