The effect of choline on phospholipid metabolism: A literature review and preliminary in vitro experiment

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Content THE EFFECT O F CHOLINE O N PHOSPHOLIPID METABOLISM A L it e r a t u r e Review and P r e lim in a r y i n v i t r o Experim ent A T h e sis P r e se n te d to th e F a c u lty o f th e Departm ent o f B io c h e m istr y and N u t r it io n » The U n iv e r s it y o f S ou th ern C a lif o r n ia In P a r t i a l F u l f il l m e n t o f the R equirem ents f o r th e D egree M aster o f A rts by I r v in g L a s s o ff June 19^1 UMI Number: EP41318 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. U M T Dissertation Publishing UMI EP41318 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest* ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 B io 'SI L.3V7 This thesis, written by W ............... kAM QEF.-..Ir.v.Ins;.............................. under the guidance of h Faculty Committee, and approved by all its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill ment of the requirements for the degree of MASTER GF ARTS Date M a x ..l 2 5 .1 ...................... ... Fapdty Committee / / J Chairmafi ...... THE EFFECT OF CHOLINE O N PHOSPHOLIPID METABOLISM A L it e r a t u r e Review and P r e lim in a r y i n v i t r o E xperim ents TABLE O P CONTENTS PAGE INTRODUCTION AND STATEMENT OP PROBLEM .................................... 1 I LITERATURE REVIEW ....................................................................................... 3; i E vid en ce f o r P h o sp h o lip id S y n th e s is and Turnover | i n L i v e r ................................................................................................ .. . 3 1 i C o n d itio n s I n f lu e n c in g P h o sp h o lip id Turnover . . . . 9j In v i v o ...................................................................................................... 9 1 ! I ll v i t r o .................................................................. 9| i P o s s ib le R outes o f S y n t h e s is or Turnover o f i P h o s p h o l i p i d s ................................... 10 [ 'GENERAL CONSIDERATION OP THE RELATION OP CHOLINE TO PHOSPHOLIPID METABOLISM . . . . . . 13 I G eneral Theory ........................................................ 13 : S u g g e ste d E x p erim en ta l A p p r o a c h e s ............................................. lI|J In v i v o ......................................................................................................... l 4 In v i tr o . . ....................................................................................... V ~ > : PRELIMINARY EXPERIMENTS ON THE IN VITRO EFFECT OP CHOLINE ON THE P320^ TURNOVER OP PHOSPHOLIPIDS IN ! RAT LIVERS . . ........................................................................................ 17 Methods and P rocedure . . . . . . . 17 A n i m a l s ...................................................................................................... 17 I n c u b a t i o n ................................................................................................. 17 V | PAGE F r a c t io n a t io n p ro ced u re ............................................................ '21 In o r g a n ic phosphorus ................................................................... 21j L ip id p h o s p h o r u s ............................................................................. 22j D e te r m in a tio n o f t o t a l and r a d io a c t iv e p h osp h a te . 22; I T o ta l p h o s p h a t e ............................................................................. 22 R a d io a c tiv e p h o sp h ate . . . .................................................... 23; E xp erim en tal R e s u lts "............................................................................. 28 D i s c u s s i o n ...................................................................................................... 29' BIBLIOGRAPHY......................................... ............................................................. 3^ i I LIST OP TABLES TABLE I . I I . I I I . IV. V. C om p ositio n o f C h o li n e - d e f i c i e n t D ie t . . . C om p osition o f M o d ified K r e b s-R in g e r 1s B ica r b o n a te I n c u b a tio n Medium .......................... E f f e c t o f V arying H^SOj^ C o n c e n tr a tio n o f M olybdate Vanadate-PO|^ D e te r m in a tio n . . . C o lo r im e te r R eadings ........................................................ PAGE l 8; 19, 2J 4 - 25: 30 ! LIST OP FIGURES FIGURE PAGE 1 . N o rm a lity o f H ^ S O ]^ ..................................................................... 26 ! 2 . C a lib r a t io n C u r v e .......................................................................... 271 3* E f f e c t o f D i f f e r e n t L e v e ls o f C h olin e on % o f ; P^2 Uptake on Normal and C h o li n e - d e f i c i e n t i L i v e r s ................................................... 31' INTRODUCTION AND STATEMENT OP PROBLEM A v a ila b le e v id e n c e fa v o r s th e h y p o t h e s is t h a t th e 1 I i m eta b o lism and tr a n s p o r t o f f a t t y a c id s i s i n t i m a t e l y ; a s s o c i a t e d w ith p h o s p h o lip id m eta b o lism . There have b e e n a j i number o f s u g g e s t io n s co n c er n in g th e n a tu re o f th e i n t e r - ' r e l a t i o n s h i p betw een p h o s p h o lip id and n e u t r a l f a t m etabolsiiry b u t no one o f th e s e appears t o be c o m p le te ly a d eq u a te. I t ' jhas been w e l l e s t a b l i s h e d t h a t f a t and p h o s p h o lip id m etabo- ; jlism i s m ark ed ly in f lu e n c e d b y th e d ie t a r y l e v e l o f l a b i l e t m eth yl g ro u p s, presum ab ly a c t in g through the in te r m e d ia r y I fo r m a tio n o f c h o l in e , a c o n s t i t u e n t o f l e c i t h i n : ' H2 9 “ 0 - C— — Rx H2^ ' ° ~ P=0H 0-CH2-CH2-N-(CH3 )3 O H L e c it h in c o n t a in s fo u r d i s t i n c t e n t i t i e s : c h o lin e , p h o sp h a te , f a t t y a c id s , and g l y c e r o l . I t i s n o t known w h eth er a m o le c u le o f l e c i t h i n i s formed by th e sim u lta n e o u s co m b in a tio n o f th e f r e e compo- I ;nents t h a t make up th e m o le c u le or w h eth er th e p a r ts may be — - ■ ■■ -2 - •replaced in d e p e n d e n tly . In th e form er c a se th e r a te o f r e placem ent o f the p h osp h ate m o ie ty o f th e p h o s p h o lip id m o le c u le would be th e same as th a t of th e n it r o g e n and f a t t y a c id p o r t i o n s . However, i n th e l a t t e r c a s e , th e r a te o f i tu rn o v er f o r each component m igh t be d i f f e r e n t . Thus the | I i appearance o f l a b e le d p h o sp h a te i n a m o le c u le o f ph osp ho- i j lip id would n o t n e c e s s a r i l y i n d ic a t e th a t th e e n t i r e j 'p h o sp h o lip id m o le c u le had b een s y n t h e s iz e d . The m echanism ; by w hich c h o lin e in f l u e n c e s p h o s p h o lip id s y n t h e s i s i s un- j i q u e s tio n a b ly th rou gh i t s in c o r p o r a t io n i n t o the ph osop ho- i l i p i d m o le c u le . How t h i s in f l u e n c e s th e tu rn o v er o f th e j I p h osp h a te m o ie ty o f p h o s p h o lip id i s a q u e s tio n o f some j I i n t e r e s t . T h e r e fo r e , a stu d y o f th e in f lu e n c e o f c h o lin e Jon p h osp h ate tu rn o v er i n l i v e r p h o s p h o lip id s may add some ^inform ation a s to the m echanism s o f t h e i r s y n t h e s i s or .d e g r a d a tio n . I t i s the purpose o f t h i s paper to r e v ie w the e f f e c t o f c h o lin e on p h o s p h o lip id m eta b o lism and s y n t h e s i s w it h in th e l i v e r , and to p r e s e n t p r e lim in a r y e x p e rim en ts on the i n v l t r o e f f e c t o f c h o lin e on p h o s p h o lip id m eta b o lism as m easured b y p3^0[^ u p ta k e . LITERATURE REVIEW E v id en ce For P h o s p h o lip id S y n t h e s is and Turnover i n L iv e r C o n sid era b le e v id e n c e had accu m u lated i n d i c a t i n g th a t p h o s p h o lip id s y n t h e s i s i s e s p e c i a l l y r a p id i n th e l i v e r . I L in se e d o i l l a b e le d w ith d euteriu m was f e d r a t s by CavanaughJ (1 ) , and th e h ig h e s t p e r c e n ta g e o f deuteriu m was fou n d i n j f a t s o f plasm a. F a ts o f l i v e r c o n ta in e d l e s s e r amounts o f I I th e i s o t o p e . An a n a l y s is o f the r e s u l t s showed th a t th e j l i v e r s e l e c t s f a t s from th e b lo o d so o n a f t e r t h e i r a b so rp - 1 t i o n , and a l s o th a t th e f a t t y a c id s o f p h o s p h o lip id s o f ' • i l l i v e r o r ig i n a t e from th e in g e s t e d d e u t e r o - f a t . S ix hou rs i ia f t e r th e f a t was f e d , d eu teriu m was p r e s e n t i n la r g e amounts i n p lasm a, but i n l a r g e r amounts i n l i v e r phospho l i p i d s . A fte r ten h o u r s, a d e c r e a se was n o te d i n th e deu teriu m c o n te n t o f b lo o d p h o s p h o lip id s w h ile th e r e was l e s s change i n t h a t o f l i v e r l i p i d s . S in c e i t was fou n d th a t ‘plasm a f a t c o n ta in e d te n tim es as much d eu teriu m as plasm a ‘p h o s p h o lip id , i t was s u g g e s te d th a t th e p h o s p h o lip id s o f 'blood were s y n t h e s iz e d i n th e l i v e r and were n ot ta k e n up .from th e i n t e s t i n e . B a r r e tt (2 ) fou n d th a t f a t t y a c id s o f l i v e r p h o s p h o lip id s c o n ta in e d th r ee tim es the amount o f deuteriu m o f body p h o s p h o lip id s a f t e r d e u t e r o - l i n s e e d o i l ,was fe d f o r th r e e d a y s . 1 A fte r i t was d em on strated t h a t l e c i t h i n w ould p r e v e n t I f a t t y l i v e r s i n p a n c r e a te c to m iz e d a n im a ls, B e st (3) showed , th a t c h o lin e was d e f i n i t e l y l i p o t r o p i c . C h o le s t e r o l- p r o - ! 'duced f a t t y l i v e r s were s t u d ie d and i t was found t h a t la r g e ; I ' jdoses o f c h o lin e were n e c e s s a r y t o p r e v e n t su ch f a t t y , l i v e r s (ip)- C h olin e had no marked e f f e c t i n p r e v e n tin g th e ; a c cu m u la tio n o f c h o l e s t e r y l e s t e r s i n th e l i v e r ; how ever, i ! 'it d id p r e v e n t an a c cu m u la tio n o f g l y c e r i d e s , and i n c u r a tiv e exp erim en ts cau sed a r a p id d e c r e a se in g l y c e r i d e s and a s lo w e r d im in u tio n o f c h o l e s t e r y l e s t e r s . In a l l ty p e s o f j , f a t t y l i v e r s , th e p r i n c ip a l e f f e c t o f c h o lin e appeared to 'be on th e g l y c e r id e f r a c t i o n . C h olin e d id n o t i n h i b i t th e j Jd ep osition o f f a t i n th e l i v e r s when r a t s were p o is o n e d w ith p h o sp h o ru s, but d id a c c e le r a t e the rem oval o f f a t from the i t l i v e r du rin g a r e c o v e r y p e r io d . i i j In 1935* Aylward (5 ) d is c o v e r e d t h a t th e p h o s p h o lip id .conten t o f th e l i v e r d e c r e a s e s f o r sev en hours a f t e r a m eal c o n ta in in g f a t and c h o l e s t e r o l . T h is d e c r e a se c o u ld be p r e v e n te d b y the a d m in is t r a t io n o f c h o l i n e . I t was su g g e s t e d th a t e i t h e r c h o lin e p r e v e n te d th e p a r t i a l d is a p p e a r - I ance o f l i v e r p h o s p h o lip id or i t s p r e se n c e i n the d i e t p e r m itte d th e fo r m a tio n of new p h o s p h o lip id to r e p la c e th a t w hich had d is a p p e a r e d . I t was a l s o o b serv ed t h a t c h o l e s t e r o l ■decreased n ew ly -fo rm ed p h o s p h o lip id i n l i v e r ( 6 ) even b e fo r e i th e l i v e r was f a t t y , and . th a t c h o lin e s tim u la te d . _______ . p h o sp h o lip id m eta b o lism under th e s e c o n d i t i o n s . t i In o rd er t o p rove t h a t p h o s p h o lip id s are s y n t h e s iz e d ; { from d i e t s o f c h o lin e ( 7 )> r a t s were f e d th e l i p o t r o p i c j | i su b sta n c e a r s e n o - c h o l in e . A r se n ic was s u b s e q u e n tly found i n | i the l e c i t h i n f r a c t i o n of th e t i s s u e . S t e t t e n ( 8 ) fou n d t h a t 1 ! the c h o lin e o f body p h o s o p h o lip id s was r e p la c e d by i n g e s t e d ! i c h o lin e l a b e le d w ith i s o t o p i c n it r o g e n . The l i v e r was th e ; | ! m ost a c t i v e i n a c c o m p lish in g t h i s tu r n o v e r . A s u f f i c i e n t i jsupply o f c h o lin e p r o t e c t s th e l i v e r (9 ) from c i r r h o s i s r e s u l t i n g from a h ig h a l c o h o l i c d i e t . No e x c e s s i v e d e p o s i t i o n o f l i p i d s nor f i b r o s i s was o b serv ed i n r a t s r e c e i v in g ; c h o l i n e . ! J I t was assumed (1 0 ) th a t any b a se th a t has a c h o lin e - j l i k e a c t i o n on l i v e r f a t sh o u ld be in c o r p o r a te d i n a new p h o s p h o lip id m o le c u le s u b s t i t u t e d f o r c h o l i n e . I n o s i t o l was l c o n s e q u e n tly d is c o v e r e d (1 1 ) i n c e r t a i n t i s s u e p h o sp h o lip id sJ iC ystine and c y s t e i n e , w hich do n o t p o s s e s s l i p o t r o p i c a c t i v i t y (1 2 ) do a c c e le r a t e p h o s p h o lip id tu r n o v e r i n the ;liv e r as m easured by r a d io a c t iv e p h o sp h a te . However, th e s e o b s e r v a tio n s were made s e v e r a l hou rs a f t e r a s i n g l e f e e d in g ( w h ile l i p o t r o p i c a c t i v i t y was s t u d ie d s e v e r a l weeks a f t e r r d a i l y f e e d in g . A pproaching th e problem o f p h o s p h o lip id tu r n o v e r ,'d if f e r e n t ly , Artom and a s s o c i a t e s (13> 1^) showed t h a t when r a d io a c t iv e phosphorus and o l i v e o i l.w e r e _fed r a t s , the. i CLiver and i n t e s t i n a l m ucosal l i p i d s .co n ta in ed the g r e a t e s t proportion o f p^^Oi.. Analyzing fu rth er the problem of p-^Oi, ! uptake by l i v e r , i t was shown (1 5 ) t h a t when no f a t was f e d , j th a t th e r e was a sharp in c r e a s e o f p3^Q^ c o n te n t up to te n h ou rs i n l i v e r , a grad u al b u t d e f i n i t e g r e a t e r in c r e a s e i n c o n te n t even a f t e r one-hundred hours i n the c a r c a s s , b u t > sm a ll in c r e a s e i n th e G-.I. t r a c t , w h ich dropped a f t e r , | ! 'fifte e n hours. However, when f a t was a lso fed w ith p^Oj^the; Jturnover in l i v e r was most rap id, but the con ten t decreased j I 'sh arp ly a f t e r t e n h o u rs; th a t o f c a r c a s s c o n tin u e d t o r i s e I 1 ;as b e fo r e ; and th a t o f G .I . t r a c t in c r e a s e d to a p o in t 2.5% ; below t h a t o f l i v e r , b u t f e l l o f f l e s s s h a r p ly . T his ! p r o b a b ly a c c o u n ts fo r Artom’ s f in d in g s (1 3 , llf) c o n c e r n in g ! ’ I jc o n c e n tr a tio n o f p h o s p h o lip id s in w hole r a t r o s e , and a f t e r t w e n t y - f iv e hours ! f e l l o f f g r a d u a lly . These d a ta i n d ic a t e t h a t s y n t h e s i s o f I p h o s p h o lip id s i s d e f i n i t e l y a s s o c i a t e d w ith th e l i v e r . Most s t r i k i n g e v id e n c e o f l i v e r ' s b e in g th e m ain s i t e o f form a t i o n o f p h o s p h o lip id s was shown by F i s h i e r (1 6 ) when p32o^ p as found i n th e l i v e r p h o s p h o lip id s soon a f t e r i n j e c t i o n . I llt s c o n c e n tr a t io n th e r e d im in ish e d , w h ile i t was in c r e a s e d i n th e b lo o d p lasm a. A ft e r h ep atectom y o n ly m inute amounts o f p h o s p h o lip id p32o^ were r e c o v e r e d i n th e plasm a as l a t e as s i x hours a f t e r e x c i s i o n o f th e l i v e r . ! E v id en ce f o r s p e c i f i c a c t i o n o f c h o lin e J_6 ) on the P O h i n i n t e s t i n e . The c o n c e n tr a t io n o f m eta b o lism o f r a t l i v e r p h o s p h o lip id s and t h e ir s y n t h e s i s was shown when w ith an in c r e a s e d i n g e s t i o n o f c h o lin e c h lo r id e (up t o 30 m g .) , th e r e o ccu rred a p r o p o r tio n a te i n c r e a s e i n fo r m a tio n as w e l l as i n rem oval o f p h o s o p h o lip id as m easured by p32Q , . in f lu e n c e o f c h o lin e on phospho- I ^ l i p i d fo r m a tio n d isa p p e a r e d from te n to tw e lv e hours a f t e r jits a d m in is t r a t io n . E ig h t hours a f t e r o r a l fe e d in g o f i m eth io n in e and c h o lin e t o r a t s t h a t had p r e v io u s l y b een p la c e d on a low p r o t e i n d i e t , th e r e was in c r e a s e d r a te o f in c o r p o r a t io n o f p3^0^ i n t o l i v e r p h o s p h o lip id s ( 1 7 ) . A Isin g le la r g e dose o f c h o lin e to r a t s i n s im ila r c o n d it io n s 'stim u la te d l i p i d p h o s p h o r y la tio n w it h in th e l i v e r as I m easured b y r a d io a c t iv e ph osp horus ( 1 8 ) . An in c r e a s e i n th e jrate o f p h o s p h o lip id s y n t h e s i s e v id e n c e d by th e p32o^ u p tak e jwas n o te d (1 9 ) a f t e r la r g e d o se s o f c h o lin e were g iv e n . ■This e f f e c t was n o t d em o n stra ted a f t e r s e v e r a l months o f tr e a tm e n t w ith m e th io n in e ; how ever, i t was t h e o r iz e d th a t th e s t im u l a t i o n o f p h o s p h o lip id tu r n o v e r by the la r g e dose o f th e l i p o t r o p i c ag en t in d ic a t e d a d e f i c i e n c y o f th e l a t t e r m a t e r i a l. The a d d it io n a l amounts o f r a d io - p h o s p h o lip id s formed a f t e r c h o lin e a d m in is t r a t io n ( 2 0 ) do n o t rem ain lo n g i n the l i v e r . A ft e r te n h ou rs th e y are no lo n g e r d e t e c t a b le ; The maximum v a lu e s found f o r th e s p e c i f i c a c t i v i t y o f plasm a p h o s p h o lip id phosphorus a f t e r p32o^ a d m in is t r a t io n were 'h ig h e s t i n dogs fe d c h o lin e th a n i n . dog s.-fe d n o _ .c h o lin e . -. B e st e t a l i n 193^4- (^-) showed th a t c h o lin e a d m in is t r a t io n in c r e a s e s th e amount o f p h o s p h o lip id s i n th e l i v e r b u t i t Loes n ot i n c r e a s e th e p r o p o r tio n i n w hich p h o s p h o lip id s co n t a in in g c h o lin e occu r i n th e p h o s p h o lip id m ix tu r e . Y et, in the l i v e r s o f dogs i n j e c t e d w ith p3^0^, (2 1 ) th e a d m in is tr a t i o n o f c h o lin e i n c r e a s e s the s p e c i f i c a c t i v i t y i n the c h o lin e - c o n t a in in g p h o s p h o lip id s , w hereas i n the n o n -c h o lin e c o n ta in in g f r a c t i o n , the s p e c i f i c a c t i v i t y i s d e f i n i t e l y lo w e r e d . I t seems l i k e l y t h a t th e s t im u la t io n o f l i p i d p h o sp h o r y la tio n i n the l i v e r b y c h o lin e i s e s s e n t i a l l y the e x p r e s s io n o f an in c r e a s e d fo r m a tio n o f c h o lin e - c o n t a in in g p h o s p h o lip id s . I L iv e r seem s t o be a b le t o s y n t h e s iz e p h o s p h o lip id jco n ta in in g p3?o^ b o th i n v iv o and i n v i t r o ( 2 2 ) . The p o s s i b i l i t y th a t in te r c h a n g e may take p la c e may a l t e r the I 'c o n c lu sio n a c c o r d in g to th e s e e x p e r im e n te r s . T his was e v id e n c e d b y th e f a c t t h a t when la b e le d NA^PO^ was shaken w ith a s o l u t i o n o f l e c i t h i n , th e o r g a n ic compound d id n o t p o s s e s s r a d i o a c t i v i t y . T h erefore i t was su rm ised th a t lenzymes are in v o lv e d i n v iv o to in c o r p o r a te phosphorus in t o p h o s p h o lip id s . The e v id e n c e c o n sid e r e d i n the p r e c e d in g s e c t i o n c l e a r l y d em o n stra tes t h a t th e l i v e r i s a m ajor s i t e o f p h o s p h o lip id s y n t h e s i s . jC o n d itio n s I n f lu e n c in g P h o s p h o lip id Turnover In v iv o Cod l i v e r o i l f e e d in g s ( 2 3 , 2I 4 .) p rod u ces changes i n i i c o m p o sitio n b u t n o t i n th e amount o f f a t t y a c id o f i n t e s t i n a l m ucosa. When b e e f k id n e y whose f a t t y a c id s have an a v era g e i io d in e number o f 98 was to c a t s , t h e f a t t y a c id s o f th e . ;p h o sp h o lip id s i s o l a t e d from l i v e r had an io d in e number o f j j 1 labout 124.5• When b e e f m u scle whose f a t t y a c id s p o s s e s s an , io d in e number o f 82 was f e d th e l i v e r p h o s p h o lip id s were I I jmore s a t u r a t e d . T his showed th a t th e c o m p o sitio n o f I I jp h o sp h o lip id s o f d i f f e r e n t t i s s u e s can be in f lu e n c e d b y the . I 1 jtype o f f a t in th e d i e t . Artom (1 3 , llj.) showed t h a t th e 'fe e d in g o f o l i v e o i l seems to prom ote p h o s p h o lip id tu r n - jo v er. An in f lu e n c e o f c a s e i n on p h o s p h o lip id m eta b o lism was observed by Beeston (25)* In creasin g d ie t of ca se in from |5 t o 30% r e s u l t e d i n a p r o g r e s s iv e d e c r e a se i n the amount 'of l i v e r g l y c e r i d e s and an in c r e a s e i n th e amount o f l e c i t h i n and c h o l e s t e r y l o l e a t e i n th e f a t t y l i v e r produced I [by f e e d in g c h o l e s t e r o l . These r e s u l t s s u g g e s te d th a t the [ lip o t r o p ic e f f e c t o f p r o t e i n sh o u ld be d i f f e r e n t i a t e d from th a t o f c h o l i n e . The i n f lu e n c e o f c h o lin e on p h o s p h o lip id tu rn o v er o f l i v e r i n v iv o has a lr e a d y been d i s c u s s e d . j 1- 2. v i t r o D e s t r u c t io n o f c e l l s by th e h om ogenation o f l i v e r (26) r e s u l t e d i n com p lete i n a b i l i t y o f the c e l l s to s y n t h e s iz e I I (p h o sp h o lip id as m easured b y r a d io a c t iv e p h osp h oru s, w hereas j s y n t h e s i s was c a r r ie d out by s u r v iv in g l i v e r s l i c e s . | C a ta b o lic r e a c t io n s upon p h o s p h o lip id s a l s o o ccu rred i n th e | l a t t e r and the f a c t th a t no breakdown was n o t ic e d a f t e r one hour may s u g g e s t t h a t no r a p id a c t io n o f enzymes o ccu rs a t i t h i s tim e . When v a r io u s su g a rs as g lu c o s e , g a l a c t o s e , j mannose and f r u c t o s e (n o t p e n t o s e s ) were added t o R in g e r ’ s i th e r a t e o f in c o r p o r a t io n o f p^^O^ in t o b r a in p h o s p h o lip id s . i s s i g n i f i c a n t l y in c r e a s e d ( 2 7 ) . When t i s s u e s l i c e s were j jin cu b ated i n R in g e r ’ s s o l u t i o n c o n ta in in g g lu c o s e and p3^0[j_,| I th r e e tim es as much p h o s p h o lip id was s y n t h e s iz e d than was ' i produced i n a g l u c o s e - f r e e R in g e r 's . These l a t t e r r e a c t io n s 'do n o t ta k e p la c e a n e r o b i c a l l y or upon th e a d d i t io n o f la z id e , hydrogen s u l f i d e or carbon m onoxide ( 2 8 ) . In the ; : la s t t h r e e c a s e s , the fo r m a tio n o f p h o s p h o lip id s was p r e - i v e n te d by i n h i b i t i n g th e a c t i v i t y o f th e cytochrom e sy stem ( 2 9 ) . A g r e a t e r c o n c e n tr a tio n o f o r g a n ic ph osp horus i s [found when f l u o r id e i s p r e s e n t i n th e medium ( 3 0 ) . t P o s s i b l e R outes o f S y n t h e s is or Turnover o f P h o sp h o lip id s In ord er to d eterm in e what su b s ta n c e s m ust be p r e c u r so r s o f p h o s p h o lip id c h o l in e , a s e r i e s o f e x p e rim en ts ((31) were perform ed u s in g N -^ -la b e le d p r e c u r s o r s . E le v e n and o n e - h a lf p e r c e n t o f a l l p h o sp h o lip id ._ ch o lin e_ was„shown_ — l r to have o r ig i n a t e d from eth an o lam in e o f d i e t . High amounts l o f la b e le d eth a n o la m in e i n p h o s p h o lip id (more than tw ic e j th a t o f g lu ta m ic a c id or o f o th e r amino a c id s ) were a l s o i s o l a t e d when i s o t o p i c g l y c in e was fe d r a t s . Thus g ly c in e I i g iv e s r i s e to e th a n o la m in e , w hich y i e l d s c h o lin e # Amino- e th y l-p h o s p h o r ic a c id e s t e r c o n t r ib u te d i t s P to b oth c e p h a lin s and l e c i t h i n ( 3 2 ) . A lth o u g h no e v id e n c e i s a v a i l- ! ' j |able to show t h a t p h o s p h o lip id s y n t h e s i s i n r a t s may o ccu r ! w ith p h o s p h o r y l- c h o lin e , y e t i t s phosphorus (33) e n t e r s in to ; p h o s p h o lip id f r a c t i o n s and a l s o i n t o b lo o d in o r g a n ic p h o s- 1 1 1 i phorus as in d ic a te d by radio-phosphorus. This may be due to! ! Ip a r tia l h y d r o ly s is o f p h o sp h e ry l c h o l in e . I t was a l s o j i n o t ic e d t h a t t h i s e s t e r i n h i b i t s fo r m a tio n o f th e c e p h a lin f r a c t i o n b u t n o t th e l e c i t h i n f r a c t i o n w h ich may s u g g e s t 'that th e r e a c t i o n , eth a n o la m in e or s e r in e — 4 c h o lin e i s i j i i r r e v e r s i b l e . I ■ G -lycerolphosph ate and p h o sp h o ry l c h o lin e la b e le d w it h p3^0^ were t e s t e d b y l i v e r and k id n e y s l i c e s (29)# I t i s q u e s tio n e d w h eth er th e uptake o f p ^ Q ^ f o llo w e d th e jca ta b o lism o f th e o r g a n ic phosphorus m o le c u le s to in o r g a n ic !pOi.--in w h ich form th e y may have b een in c o r p o r a t e d . However* l s in c e i t i s u n l i k e l y th a t th e re i s o n ly one pathw ay fo r the 1 c o n v e r s io n o f in o r g a n ic p h osp h ate t o p h o s p h o lip id i t i s jassumed t h a t g ly c e r o lp h o s p h a te , p h o s p h o r y lc h o lin e , i p h o sp h o r y la m in o e th a n o l, d i g l y c e r d i e s , and t r i g l y c e r i d e s may_ 1 2 be in t e r m e d ia t e s . Du V igneaud (3^4-) em p loyin g d e u tr o -m e th io n in e showed ; th a t m eth io n in e p r o v id e s m eth yl groups to form c h o l i n e . C h olin e i s o l a t e d from the t i s s u e s c o n ta in e d a s u f f i c i e n t amount o f d eu teriu m to i n d i c a t e t h a t a l l o f th e m eth yl groups o f c h o lin e had come from m e th io n in e . The s u b s t r a t e t h a t i s m e th y la te d was shown (3 5 ) to be e th a n o la m in e , w h ich ; I i s form ed i n th e body b y th e r e d u c tio n o f g l y c i n e or b y th e ' d e c a r b o x y la tio n o f s e r in e (3&). G ly c in e i n turn can a r i s e from th e d e m e th y la tio n o f b e t a i n e . B e ta in e e n a b le s th e r a t I to u t i l i z e h o m o cy stin e i n p la c e o f m e th io n in e , a lth o u g h i t I i s l e s s e f f e c t i v e than e q u iv a le n t amounts o f c h o lin e ( 3 7 )• ; f That eth a n a lo m in e i s e a s i l y c o n v e r te d to c h o lin e i s shown j i (3 8 ) even when th e d i e t d id n o t c o n t a in s u f f i c i e n t l a b i l e m eth yl g r o u p s. C h olin e o x id a s e i s red u ced o r i n h i b i t e d b y ■the a c cu m u la tio n o f f a t i n th e l i v e r (39)* T h is i s o f f e r e d ,'as a p o s s i b l e e x p la n a tio n o f th e h ig h c o n te n t o f body ch olin e i 1 jin th e a b sen ce o f a v a i la b l e m eth y l g r o u p s. However, a drop i n th e c h o lin e c o n t e n t o f l i v e r l i p i d s was n o te d (3 8 ) when c h o lin e was o m itte d from th e d i e t . GENERAL CONSIDERATION OP THE RELATION OP CHOLINE TO PHOSPHOLIPID METABOLISM G eneral Theory H ershey (i}.0) showed t h a t the f a t t y l i v e r o f the p a n cr e a te cto m iz ed dog showed a marked d e c r e a se i n f a t w ith the i n g e s t i o n o f l e c i t h i n . C h olin e (I4 I , lf2 ) was th e im p o r ta n t item i n r e d u c in g th e c o n te n t o f f a t i n th e s e f a t t y l i v e r s . A lth ou gh c h o lin e s t im u la te d th e tu r n o v e r or s y n t h e s i s o f p h o s p h o lip id i n r a t l i v e r (Ip ) i t i s n o t i n d i c a te d t h a t t h i s in c r e a s e d s y n t h e s i s b y the l i v e r o c cu rs a t jthe ex p e n se of exogen ou s c h o l in e . S in c e th e form ed phospho l i p i d rem ains o n ly fo r a s h o r t tim e i n the l i v e r , i t i s 1 t h e o r iz e d th a t i t i s r a p id ly t r a n s f e r r e d t o some o th e r t i s s u e . B e st (ip) th o u g h t th a t th e o x id a t io n o f f a t t y a c id s by l i v e r i s a c c e le r a t e d b y c h o l i n e . : M eth ion in e i s l i p o t r o p i c b e c a u se i t was th o u g h t to I p r o v id e th e m eth yl groups f o r th e i n v iv o s y n t h e s is o f 1 c h o lin e (3^)* However, s in c e th e m eth yl grou p s o f a r se n o - c h o lin e or t r i e t h y l c h o l i n e are n o t l a b i l e , i t seems t h a t !th eir l i p o t r o p i c a c t i v i t y m ust depend on the e n t i r e m o le c u le r a th e r than on t h e i r l a b i l e m eth yl groups ( 7 ). E xp erim en ts su p p o rte d th e h y p o th e s is th a t c h o lin e i s p h o sp h o r y la te d and u sed in l e c i t h i n s y n t h e s i s ( ipip). The I p h o sp h o r ic a c id e s t e r o f c h o lin e c h lo r id e i s u n a f f e c t e d by j l i v e r p h o sp h a ta se and i t i s th e r e f o r e p r o t e c t e d from o x id a t i o n i n th e l i v e r . D eu el e t a l (I4 . 5 ) showed t h a t c h o lin e does n o t in c r e a s e th e r a te o f f a t o x id a t io n i f k e to n u r ia d u rin g f a s t i n g i s a c c e p te d as e v id e n c e o f su ch o x id a t io n . \ McLean (I4 .6 ) con firm ed t h i s by show ing t h a t l a r g e r amounts of* jketone b o d ie s were e x c r e t e d d u rin g f a s t i n g by r a t s w ith j f a t t y l i v e r s th an were e x c r e t e d b y normal r a t s ; but th e j e x t e n t o f k e to n u r ia i s n o t p r o p o r tio n a l to th e e x c e s s f a t in th e l i v e r . j From th e fo r e g o in g e x p e r im e n ta l e v id e n c e i t i s assumed t h a t c h o lin e owes i t s l i p o t r o p i c a c t i v i t y to i t s p a r t i c i p a - j 1 t i o n i n th e fo r m a tio n o f p h o s p h o lip id s . P h o sp h o lip id s s e r v e ! to tr a n s p o r t f a t t y a c id s from th e l i v e r and c h o lin e i s r e - iq u ired f o r th e fo r m a tio n o f a t l e a s t some o f t h e s e phospho- i j l i p i d s . I iS u g g e ste d E x p erim en ta l A pproaches ; In v iv o | In ord er t o d em on strate f u l l y what happens to the j 'p h o sp h o lip id m o le c u le under c h o lin e s t i m u l a t i o n i t i s s u g g e s t e d th a t p3.2o^, N -^ - la b e le d c h o lin e and C - ^ - f a t t y a c id s a l l be fe d i n c o n ju n c tio n w it h c h o lin e to normal and c h o lin e d e f i c i e n t r a t s . At c e r t a i n tim e i n t e r v a l s , p h o s p h o lip id s — ' “ “ 1 5 and. f a t t y a c id s c o u ld be i s o l a t e d from l i v e r and the sm a ll gu t so th a t th e r e l a t i v e s p e c i f i c a c t i v i t i e s o f the c h o lin e ,J f a t t y a c id , and phosphorus f r a c t i o n s m ight be d eterm in ed . The same p roced u re may th en be u sed w it h no i n g e s t i o n o f c h o l i n e . These ex p e rim en ts would s e r v e t o e lu c i d a t e th e i m ethods o f s y n t h e s i s or tu rn o v er o f p h o s p h o lip id s , and would| I a l s o se r v e to c l a r i f y th e r o le o f c h o lin e i n th e m eta b o lism | o f phosphorus and f a t t y a c i d s . S in c e we a re n o t y e t su re o f th e mechanism o f fo r m a tio n o f the p h o s p h o lip id m o le c u le i p e r s e , th e r e s u l t s m ig h t i n d ic a t e how and when th e d i f - t f e r e n t components e n t e r i n t o t h i s m o le c u le . | t j In v i t r o i ; I f c h o lin e i s added t o l i v e r s l i c e s i n an in c u b a tio n jmedium c o n ta in in g p32o^ and the r a d i o a c t i v i t y o f th e , ‘ i s o l a t e d p h o s p h o lip id s d eterm in ed i n r e l a t i o n t o th e ■chemical phosphorus i n th e medium, some know ledge would be 'acq u ired as t o th e a c t i o n o f c h o lin e on p h o s p h o lip id form a- I I t i o n . The l i v e r s l i c e s sh o u ld be o b ta in e d from normal and c h o lin e d e f i c i e n t r a t s , so t h a t e f f e c t s o f c h o lin e d e p r iv a t i o n m igh t be e s t a b l i s h e d . A fte r t h i s d a ta i s s e c u r e d , i t m ig h t b e e f f i c a c i o u s t o add N ^ ^ -la b e led c h o lin e and r a d io - c a r b o n - la b e le d f a t t y a c id s to th e medium to determ ine th e m ethods o f phosop ho- i i i l i p i d s y n t h e s i s b y l i v e r , and to d eterm in e w h eth er th e _ _ _ - - io ' tu rn over o f th e d i f f e r e n t c o n s t i t u e n t s o f l e c i t h i n occur in d e p e n d e n tly . t PRELIMINARY EXPERIMENTS O N THE IN VITRO EFFECT OF CHOLINE O N THE P3 2 0^ TURNOVER O F PHOSPHOLIPIDS IN RAT LIVERS ] I J ( Methods and P rocedure ! i ! a . Anim als i The normal r a t s were m ales w eig h in g from 91 1 ° 102 gram s, k e p t on a normal s to c k d i e t , w it h fo o d and w a ter ad lib it u m . 1 . The c h o lin e d e f i c i e n t r a t s w ere m ales w e ig h in g from j 91 to 102 grams and were f e d f o r two weeks on a d i e t l i s t e d I lin Table I . The l o s s in w e ig h t f o r ea ch r a t amounted to about 2 grams i n th e p e r io d o f two w eek s. j j i . £>• In c u b a tio n ! The in c u b a tio n medium was a K r e b s-R in g e r ' s B ica rb o n - 1 a te s o l u t i o n (lj-7), m o d ifie d t o c o n ta in from 0 .2 5 - 0 .5 0 m ic r o c u r ie s o f p-^0j^ p e r $ m l. o f s o l u t i o n as d e s ig n a te d by F i s h i e r e t a l (T able I I ) (I4 . 8 ) . G lu cose was added i n a co n c e n t r a t i o n o f 0 .1 $ to in c r e a s e th e uptake o f p32o^ by l i v e r p h o s p h o lip id . V arying amounts o f c h o lin e c h lo r id e r e c r y s t a l l i z e d from a b s o lu te a lc h o h o l and th o r o u g h ly d r ie d were added • to a l l tu b e s e x c e p t th e c o n tr o ls f o r each run. K^PO^ was i Jthe o n ly so u r c e o f phosphorus and e a c h 5 ml* c o n ta in e d T8 TABLE I COMPOSITION OP CHOLINE-DEFICIENT DIET In g e d ie n t Amount A rach in 50 grams C a sein (V itam in f r e e ) 50 grams C otton Seed O il ( F o r t i f i e d w ith V itam in s A, D, E) 5o grams C e r e lo se 35o grams Ca P a n to th e n a te 5 mg Thiamine HC1 2 • 5 mg N i c o t i n i c A cid 10 mg P y r id o x in s 2 • 5 mg R ib o f la v in 5 mg I n o s i t o l 10 mg I 19 TABLE I I COMPOSITION OP MODIFIED KREBS-RING-ER1S BICARBONATE INCUBATION MEDIUM C omp ound I n d iv id u a l c one e n t r a t i on‘ 5 '' I s o t o n i c c o n c e n t r a t i ons S tock j s o l u t i o n NaCl 4 .5 grams/1 0 0 ml 20 ml d i l u t e d to 100 ml - 0 . 9/ 1 100 ml ! 1 KC1 5*75 grams/1 0 0 ml 1 ml d i l u t e d t o 5 ml - 1 .1 5 # k - ml ! 1 Csi Clo 2 6 .1 grams/1 0 0 ml 1 ml d i l u t e d t o 5 ml = 1 . 22/ 3 ml ! . 1 1 KHgPO^ 1 0 .5 5 grams/1 0 0 ml 1 ml d i l u t e d to 5 ml = 2 . 11/ I 1 ml j MgS0^_.7H2 0 1 9 .1 grams 100/m l 1 ml d i l u t e d to 5 ml - 3 . 8 2 / 1 ml N o te: S in c e !•?>% o f Na H COo n e e d e d , th e n 8lj. ml o f sto c k p lu s 16 ml s o l u t i o n c o n ta in in g 0 .2 1 grams Na H C O -s u sed 1 • 3 % x ( 2 _qqc£ - YE = 0*21 g r ) . Composing t h i s 16 ml are a p p r o p r ia te number o f ml o f s o l u t i o n c o n ta in in g enough p32 to y i e l d 0 .2 5 - 0 .5 m ic r o c u r ie s p 3 2 /5 ml medium. 100 mg g lu c o s e was 'added «*^1 mg l / m l . Na HCO^ g a s s e d p r e v io u s l y w ith CO? f o r i h o u r. E n tir e s to c k s o l u t i o n g a s s e d w ith 9 5 / " 5 / C0? m ix tu r e . j w These a r e 5 tim es i s o t o n i c c o n c e n t r a t io n to keep f o r m on th s. 0 .8 1 mg o f IQ^POr or 0 .1 8 mg p h o sp h o ru s. The pH was 7*^4- ~ I j7.3> b e fo r e th e a d d it io n o f l i v e r s l i c e s . Of t h i s medium j 15.0 ml was u sed p e r in c u b a tio n tu b e. A ll tu b es were g a s s e d I w ith 95/^ £ > 2" C02 m ix tu re b e fo r e and a f t e r l i v e r s l i c e s i 1 were p la c e d i n th e tu b e s . ! The an im als were k i l l e d ' w i t h a blow on the h e a d . ! 1 1 The l i v e r was r a p id ly tak en o u t and p la c e d i n a P e t r i d is h 1 Iwhich c o n ta in e d a p ie c e o f f i l t e r paper m o iste n e d w it h a ! few drops o f i s o t o n i c s a l i n e s o l u t i o n . The d is h and th e jcon ten ts were k e p t i n an i c e and w a ter b a th to d e c r e a se r e s p i r a t o r y r eq u ire m en ts o f th e . l i v e r . S l i c e s 0 .3 m m t h ic k j | : jwere made u s in g a s p e c i a l r a z o r and ap p aratu s d e sig n e d to 1 produ ce u n iform s l i c i n g . A fte r rem oving e x c e s s m o istu r e on f i l t e r p a p er, 300 mg s l i c e s c a r e f u l l y w eig h ed , were p la c e d jon e a c h in c u b a t io n tu b e (6 x l" Pyrex t e s t tu b e) c o n t a in in g j '5 ml o f medium, and g a s s in g w it h th e 95$ > O 2 - CO 2 m ix tu re |was im m ed ia te ly i n s t i t u t e d . The tu b es were p la c e d on a .shaking rack f o r fo u r hours i n an o i l b a th m a in ta in e d a t '37° G. The oxygen m ix tu r e was c o n s t a n t ly bu bb led i n f o r the id u ra tio n o f th e in c u b a t io n . A b so r p tio n c o n tr o ls were u sed where th e s l i c e s were p la c e d i n tu b e s c o n t a in in g th e same in c u b a t io n medium c o n ta in in g P^Oji^, g l u c o s e , and % % t r i c h l o r o a c e t i c a c id , and m a in ta in e d i n the o i l b a th as th e r e s p i r i n g l i v e r s w e r e . — 21] I c » F r a c t lo n a t lo n p roced u re ! 1 . In o r g a n ic phosphorus | A fte r in c u b a tio n , th e c o n te n ts o f ea ch tube were j I poured i n t o E rlen m eyer f l a s k s c o n t a in in g s u f f i c i e n t 50$ TCA t o y i e l d a f i n a l c o n c e n tr a tio n o f 5$« A f te r the l a t t e r j and c o n t e n t s were a llo w ed to stan d a t room tem p eratu re fo r 1 t h i r t y m in u tes w ith o c c a s io n a l s h a k in g , the su p e r n a ta n t j l i q u i d was pou red o f f th rou gh f i l t e r p ap er i n t o E rlenm eyer i | t f l a s k s . The resid u e, (sa v e d f o r l i p i d P d e te r m in a tio n ) was ' \ ^washed th r e e tim e s w ith 15 ml p o r t io n s o f 5$' TCA f o r f i f t e e n ' jminutes ea ch , and th e w ash in gs com bined w ith the o r ig i n a l [su p ern a ta n t. T h is c o n ta in e d t o t a l i n s o l u b l e phosphorus 1 :(TAS). T his TAS f i l t r a t e was h a n d led a c c o r d in g t o th e m ethod o f D e lo r y (ip9)• I t was made n e u t r a l t o p h e n o lp h th a - jle in w ith c o n c e n tr a te d NH^OH and an e x c e s s o f 0 .2 ml added. i i 'Two ml o f CaCl2 ( 2 .5 $ ) p e r 10 ml f i l t r a t e w ere added i n jorder, and a llo w e d t o sta n d f o r t h i r t y m in u te s i n the r e f r i g e r a t o r w ith o c c a s io n a l sh a k in g . The s o l u t i o n was j f i l t e r e d th rou gh a s i n t e r e d g l a s s fu n n e l and th e f i l t r a t e jwas d is c a r d e d . The r e s id u e was washed w it h 2 ml p o r t io n s o f 0 .3 N NH^OH, and f i l t r a t e s were a g a in d is c a r d e d . The washed p r e c i p i t a t e was d is o lv e d w ith 2 ml p o r t io n s o f 1 . 2N HNOj and r in s e d through the fu n n e l i n t o a 100 ml v o lu m e tr ic f l a s k , jd ilu te d to 100 ml and sa v e d f o r d e te r m in a tio n o f in o r g a n ic o r th o p h a sp h a te . 2 . L ip id ph osp horus T his p ro ced u re was p erform ed a c c o r d in g t o Schm idt and Tannhauser (5 0 )• The a c id i n s o lu b l e r e s id u e was e x t r a c t e d jwith c o n tin u o u s sh a k in g i n 6 x 1 in c h t e s t tu b e s fo r two 'hours w it h 5 ml o f 3*1 a l c o h o l - e t h e r m ix tu r e . A fte r p o u rin g o f f th e e x t r a c t through f i l t e r paper the r e s id u e was f u r t h e r ^extracted b y r e f l u x i n g w ith 5 ml o f 1:1 ch lo ro fo rm -m eth a n o l m ix tu re f o r t h i r t y m in u tes a t 70°G. The L ip id e x t r a c t s were combined i n a 6 x 1 in c h t e s t tu b e and th en ev a p o r a te d t o d r y n e s s . The l i p i d r e s id u e was d ig e s t e d f o r f i v e m in u tes w ith a sm a ll p o r t io n o f c o n c e n tr a te d E^SO^ u n t i l S0^ fumes f i l l e d th e tu b e , th en a few drops o f 6N HCL0^_ were added Jafter w h ich d i g e s t i o n p r o c ee d e d u n t i l th e s o l u t i o n became jd e c o lo r iz e d . A f te r c o o li n g a s h o r t w h ile , 5 ml H2 O were jadded and th e tube was p la c e d i n a b o i l i n g w a te r b a th f o r f i f t e e n m in u tes i n ord er to h y d r o ly z e p yrop h osp h ate to 'o rth o p h o sp h ate, and s u f f i c i e n t c o n c e n tr a te d Na 0 H was added ,to n e u t r a l i z e th e s o l u t i o n . The c o n te n ts were d il u t e d to jl0 ml i n a 10 ml v o lu m e tr ic f l a s k and s e t a s id e fo r l i p i d - ph osp horus d e te r m in a tio n . d. D e te r m in a tio n o f t o t a l and r a d io a c t iv e ph osp hate 1 . T o ta l p h osp h ate A p roced u re s l i g h t l y m o d ifie d from Sim onsen e t a l : ( 5 l) was u se d f o r c h e m ic a l p h o sp h a te d e te r m in a tio n . The m o d if ic a t io n in v o lv e d an a d ju stm en t o f th e H2S0[|_ c o n c e n tr a - I t i o n to g i v e maximum c o lo r d ev elo p m en t. I t was fou n d th a t a' f i n a l n o r m a lity o f 0 t o 0 .5 N H 2 S0J| c o u ld be u se d to c a rr y out th e p h osp h ate d e te r m in a tio n . j The p h osp h ate sam ple (c o n ta in in g 10 to 100 o f < phosphorus and 0 t o If m i l l i q u i v a l e n t s o f E^SO^) was d i l u t e d ' 1 to 8 ml and 1 ml o f th e ammonium vanad ate r e a g a n t added. j 1 i jThis r e a g e n t was made b y d i s s o l v i n g 2 .5 gm o f ammonium | I I vanad ate i n 500 ml o f h o t w a te r , c o o lin g s l i g h t l y , adding 350 ml o f c o n c e n tr a te d HNO^, and d i l u t i n g t o 1 l i t e r . A fter' th orou gh m ix in g 1 ml o f 5$ ammonium m olybd ate was added, thei j i 'tube m ixed , and the c o lo r rea d i n te n m in u te s, u s in g a No. ; 1|2 f i l t e r w ith th e K lett-Sum m erson c o lo r im e t e r . I | Table I I I and F ig u re 1 show th a t the c o lo r d e v e lo p - 1 |ment was in d ep en d en t o f s u l f u r i c a c id c o n c e n t r a t io n from 0 t jto 0 .5 N and T ab le IV and F ig u re 2 i n d ic a t e th a t th e c o lo r ! 'developm ent f o llo w s th e B eer-Lam bert la w . j l 2* R a d io a c tiv e p h osp h ate D u p lic a te 2 and 3 ml sam ples o f th e in o r g a n ic p h o s- 1 Iphate were m easured i n t o s e p a r a te 5 H t e s t tu b e s , a p p r o p r ia te : 1 amounts o f w a te r, th en van ad ate and m olybdate were added as d e s c r ib e d above t o y i e l d 10 ml i n ea ch tu b e . D u p lic a te 2 ml sam ples o f th e l i p i d phosphorus and a b s o r p tio n c o n t r o ls iwere s i m i l a r l y t r e a t e d , and a l l tu b e s were l e f t s ta n d in g for! 2in TABLE I I I EFFECT OF VARYING HgSO^ CONCENTRATION OF MOLYBDATE VANADATE-PO^ DETERMINATION iip N o rm a lity Of r2so^ m .e . o f H 2S0^ ml o f 12N H2S0if Blank R eadings sample D i f f . 100 0 0 0 19 286 267 jioo 0 .2 1 .6 0 .1 3 3 17 282 265 100 0 .if. 3 .2 0 . 26? 17 278 261 100 1 0 .5 if. 0 0 .3 3 3 17 282 265 100 0 .6 i f .8 O.ifOO 19 270 251 •100 i 0 .8 6 . if 0 .5 3 3 18 218 200 ! a oo 1 .0 8 .0 0 .6 6 7 18 137 119 100 1 .5 1 2 .0 1 .0 0 0 17 3lf 17 100 1 2 .0 1 6 .0 1 .3 3 18 23 5 1 00 3 .0 2lf. 0 2 .0 0 21 0 -2 1 I I i i ■ 2^ 1 I TABLE TV I COLORIMETER READINGS | I ~*p D u p lic a t e s 1 2 ■ A verage D iv is io n /^ P 0 0 0 0 0 10 31 31 31 3 .1 20 53 5ii S k 2 .7 k ° 112 110 i l l 2 .8 6o 159 160 160 2 .7 80 208 208 208 2 .6 120 292 298 295 2 .5 2 .7 \ i 26 I I I 3tg*j j P v I o r m a l i t y o f Hx 5 0„ I F 16 l 1 “ * C o l o r i m e t e r R e a d i n g / O r* o % _ -------------------------------------------------------------------------------------------------------------------- 2B - ten minutes. The phosphate was determined with reference to a blank and a standard containing 100'Y P. T w o or three ml aliquots from the inorganic and digested lip id phosphate Ifractions used in the total phosphate analysis were measured: i i n t o - § ■ o z . t i n o in tm en t d is h e s , 1 drop o f p h e n o p h th a le in and, jl drop o f ’’O rvus " d e te r g e n t were added, f o llo w e d b y drops o f j 1 0 N Na O H to make th e s o l u t i o n j u s t a l k a l i n e . The sam ples ^ •were d r ie d a t 110°C and th e r a d i o a c t i v i t y d eterm in ed i n a j 1 G reiger-M uller c o u n te r w hich in c o r p o r a te d a c o u n tin g tube o f i . i th e t h in g l a s s w a ll (30 mg/cmcL) ty p e . E rrors c a u sed b y i • | s e l f - a b s o r p t i o n were c o n s id e r e d i n c o n s e q u e n t ia l. A ll [samples were rea d a t one tim e , th u s e li m in a t i n g c o r r e c t io n s j | f o r d e c a y . E x p erim en ta l R e s u lts i j The f i r s t e x p e rim en ts were con d u cted w ith l i v e r tis s u e from norm al r a t s w h ich l i v e r s were in c u b a te d as d e s c r ib e d ab ove. Each ex p erim en t was c a r r ie d ou t w it h t i s s u e from a s i n g l e r a t in c u b a te d as p r e v io u s ly i n d ic a t e d i n the p r e se n c e or a b sen ce o f c h o l i n e . The secon d s e r i e s o f e x p e rim en ts i were c a r r ie d o u t w it h l i v e r from c h o lin e d e f i c i e n t r a t s . In o rd er to d eterm in e th e e f f e c t o f a b s o r p tio n o f or th e r e s u l t s , c o n t r o l ex p erim en ts were c a r r ie d out by adding t r i c h l o r o a c e t i c a c id im m e d ia te ly f o llo w in g a d d it io n o f r a d io p h o sp h a te to p r e v e n t th e a c t i v e up take, of— p32()r i n t o . jthe c e l l u l a r com ponents. Each d e te r m in a tio n was done in d u p l i c a t e so t h a t th e r e s u l t s would be c o n s id e r e d v a l i d . Each d e te r m in a tio n o f r a d i o a c t i v i t y was done i n d u p l i c a t e , and ea ch d u p lic a t e rea d tw ic e so th a t f o u r r e a d in g s w ere i l made on a l iq u o t p o r t i o n s . There was c l o s e agreem ent among j j th e r e p l i c a t e s . ' The d a ta d e r iv e d from th e e x p e rim en ts i s s e t f o r t h i n Table V and F ig u re 3 and d is c u s s e d b e lo w . The v a lu e s i n , l i n e 5 o f T able V a r e e x p r e s s e d as s p e c i f i c a c t i v i t y o f the : i l l i p i d f r a c t i o n r e l a t i v e to th e in o r g a n ic f r a c t i o n ; and ! ! ■ i jr e p r e se n ts th e e x t e n t to w h ich l i p i d phosphorus had come j i n t o e q u ilib r iu m w it h in o r g a n ic p h o sp h a te o f the medium. j |The p e r c e n ta g e uptake o f p3^0jj_ by l i v e r in c u b a te d w ith o u t ic h o lin e c h lo r id e i s r e p r e s e n te d i n l i n e 6 as 100$ and th e .p ercen ta g e up take by th o se in c u b a te d w it h c h o lin e i s e x - jp ressed i n r e l a t i o n to the c o n t r o ls i n l i n e 6 . The r e s u l t s [su g g est t h a t c h o lin e a t l e v e l s g r e a t e r than 0 .1 mg/ml may be i n h i b i t o r y to the up take o f p^2 0 [j_ i n t o th e p h o s p h o lip id • f r a c t io n . L e v e ls o f 0 .1 mg/ml on th e o th e r hand seem to [cause a s t i m u l a t i o n o f th e phosphorus tu rn o v er o f t h i s I \ ' f r a c t i o n . D is c u s s io n ! S in c e a t h ig h l e v e l s o f c h o lin e ( in e x c e s s o f 0 .1 mg/ i 1 ml o f m ed ia) p32 0]| u p tak e by .p h o s p h o lip id s _ o f l i v e r .Is.. TABLE V Date 7/7 7/13 7/18 7/21*. 9/13 9/17 9/22 9/27 Liver Normal Normal Normal Normal Normal Normal Choline d eficien t Choline d eficien t Incubation .time (hrs.) 1 * 1 * k h k 1 . 5 ' 1 * h Cone, of choline (mg per 5 ml 5 media) k k 1 1 0 .5 0 .5 0 .5 Liver wt. (gm) -CH. +CH. .90 .90 -CH. 4CH. .59 .61 -OI. 4CH. .60 .62 -CH. .60 4CH. .60 -CH. 4CH. .21 .12 -CH. .30 4CH. .29 -CH. .29 4CH. .30 -CH. .28 4CH. .27* Amount lip id P (mg/sample) -CH. 4CH. .61* .76 -CH. 4CH. .36 •3k -CH. 4CH. .37 .38 -CH. .1*2 4CH. .1*3 -CH. 4CH. .12 .71* -CH. .99 4CH. .96 -CH. .88 4CH. .88 -CH. .81* 4CH. .86 R S A {%) -CH. 4CH. .63 .23 -CH. 4CH. .62 .33 -CH. 4CH. .69 .28 -CH. 1 .2 4CH. 1 .0 -CH. 4CH. 2.0 .90 -CH. .68 4CH. .85 -CH. 2 .0 4CH. 2 .9 -CH. .22 4C H . .1*0 Rel. uptake (JO -CH. 4CH. 100 36 -CH. 4CH. 100 53 -CH. 4CH. 100 1 * 0 -CH. 100 4CH. 83 -CH. 4CH. 100 1 * 0 -CH. 100 4CH. 125 -CH. 100 4CH. 11*2 -CH. 100 4CH. 180 | * Liver was so fa tty that d iffic u lty in slic in g and consequent greater injury to hepatic tissu e may ^ I have_caused_low-relativ.e_specific_ac.tiv.ities.--------------------------- .. _ . .... _________________________o a - 31 I d z o v 3 X * “ f t £ < C O u a: ( U 0 . 0 IU * t c . *s ' L l > J - 5 IU IX li f t - 4 S 6 * • \E F F S t T OF UlFFFZeMT LCV'CtJ O f <HOt_iWgr ow % oF P t t 0 f UPTAKB 3 9 I V o R M * L A a/|> f r t o H i u E - P C F C C I l? M T n v e « CHoiiNg. t c F i t i e A i r u v e e s N o«,mwl Au i h a i Lia&Rf I L__. 4 _ O f/_ ^6vc. OF OtOLtNF * / F 14. 3 0 ,2 ._____«•? 0 # ____ g.£ / M L i n CUB A T I o n / M e b t l / M 0 i 6 7 O-’ S 6,9 J4 ... 32 ] i n h i b i t e d , i t seems t h a t t o x i c e f f e c t oh l i v e r c e l l s i s ! p rod u ced w it h a c o n seq u en t d e c r e a se i n ph osp horus m etab olism . This seem s to be shown by th e r e c e n t ex p erim en ts o f Artom j (5 2 ) when a f t e r a h ig h p r o t e i n d i e t a n o t i c e a b le i n h i b i t i o n 1 j o f p h o s p h o lip id s y n t h e s i s i n v i t r o was o b ser v ed when c h o lin e ! was added t o s u r v iv in g l i v e r s l i c e s . However, s y n t h e s i s was s tim u la t e d when c h o lin e was added to l i v e r s l i c e s a f t e r i 1 l r a t s were p la c e d on a low p r o t e in d i e t . E thanolam ine a d d it io n ca u sed s i m i la r r e s u l t s and b e ta in e s t im u la t e d I ! 's y n th e s is in d ep en d en t o f th e typ e o f fo o d i n g e s t e d . W e [su ggest t h a t p r o t e i n c a ta b o lis m y i e l d s m eth yl groups a n d /o r ! . 1 jch o lin e p r e c u r s o r s w h ich y i e l d c h o l in e , or h e lp t o form j ■choline, i n v a r y in g amounts dependent on th e amount o f p r o t e i n in g e s t e d . 1 The d a ta add s t r e n g t h to th e th e o r y t h a t th e l i v e r I a s a s i t e o f c h o l in e - c o n t a i n i n g p h o s p h o lip id p r o d u c tio n . ■The fo r m a tio n of p h o s p h o r y l-c h o lin e and i t s c o n seq u en t I n c o r p o r a t io n i n t o p h o s p h o lip id by the i n t a c t l i v e r c e l l may be th e m echanism w hereby t h i s i s a c c o m p lish e d . T his .agrees i n p a r t w ith R i l e y ' s o b s e r v a t io n (3 3 ) t h a t th e phosphorus component o f p h o s p h o r y l-c h o lin e e n t e r s i n t o th e f r a c t i o n o f p h o s p h o lip id . A ls o , a c co r d in g t o Taurog e t a l (5 3 ) p h o s p h o lip id s a r e form ed from in o r g a n ic p h osp h ate from th e e n e rg y o f a e r o b ic o x id a t io n o f th e c e l l . The cytochrom e- cytochrom e o x id a s e sy stem i s p o s s i b l y in v o lv e d .h e r e . ___ . 1 ----------------------------------- 331 i | The work o f P a t te r s o n (5l|-) may y i e l d the c lu e as to J i th e g r e a t e r r e l a t i v e s p e c i f i c a c t i v i t y when c h o lin e d e f i c i e n t l i v e r s i n our e x p e rim en ts were in c u b a te d w it h 0 .1 mg c h o lin e / 1 ml m edia and p32o{j_* a c h o lin e d e f i c i e n t d i e t p rod u ces a j lo w ered p e r c e n ta g e c o n c e n tr a tio n o f p h o s p h o lip id s i n k id n e y s I i and l i v e r s o f young r a t s . P h o sp h o lip id tu rn o v er and p e r - | I c e n ta g e c o n c e n t r a t io n was in c r e a s e d by th e a d d it io n o f j jch o lin e to th e d i e t . T w e n ty -fiv e to t h i r t y - f o u r p e r c e n t more p32oj^_ was r e c o v e r e d from l i v e r p h o s p h o lip id o f an im als j r e c e i v in g c h o l i n e . D eu el (5 5 ) showed t h a t f a t t y l i v e r s I i Iproduced by th e i n g e s t i o n o f b u t t e r f a t were p r e v e n te d b y . i ithe a d d it io n o f c h o lin e to th e d i e t . T his i n d i c a t e s th a t I ,c h o lin e a id s i n f a t t r a n s p o r t th rou gh th e l i v e r . i U sin g l e c i t h i n as an exam ple o f p h o s p h o lip id , th e ;fo llo w in g i s a p ro p o sed scheme o f p h o s p h o lip id fo r m a tio n w it h in l i v e r : eth an olam in e + H^POi, —\ p h o s p h o r y l-c h o lin e + 2 f a t t y a c id s T * / i ; s e r in e from ATP from endogenous s o u r c e s l e c i t h i n BIBLIOGRAPHY BIBLIOGRAPHY 1 . Cavanaugh, B ., and Raper, A.S., Biochem J . , 33 , 17 C 1937^) 2 . B a r r e t t , H.M., B e s t , C.H., and R id o u t, J.H., J. ! 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Asset Metadata

Creator Lassoff, Irving (author)

Core Title The effect of choline on phospholipid metabolism: A literature review and preliminary in vitro experiment

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Degree Program Biochemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-777185

Unique identifier UC11348314

Identifier EP41318.pdf (filename),usctheses-c17-777185 (legacy record id)

Legacy Identifier EP41318.pdf

Dmrecord 777185

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Rights Lassoff, Irving

Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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