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The effect of litter size on the developmental pattern of cholesterol synthesis in intestinal and white… Kroeger, Steven Hugh 1984

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THE  EFFECT OF  OF  LITTER  CHOLESTEROL  SIZE  ON  THE  SYNTHESIS  ADIPOSE  TISSUE  IN OF  DEVELOPMENTAL  I N T E S T I N A L AND  NEONATAL  PATTERN WHITE  RATS  by STEVEN B.Sc,  A  The  THESIS THE  HUGH  University  of  SUBMITTED  IN  REQUIREMENT MASTER  KROEGER B r i t i s h  Columbia,  PARTIAL FOR  OF  THE  FULFILMENT DEGREE  1976  OF  OF  SCIENCE  in THE  SCHOOL  OF  FAMILY  DIVISION We  accept to  THE  OF  this  AND  HUMAN thesis  the required  UNIVERSITY  NUTRITIONAL  OF  NUTRITION as  conforming  standard  BRITISH  NOVEMBER,  SCIENCES  COLUMBIA  1984  ©Stevem Hugh Kroeger, 1984  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the  requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t  the L i b r a r y s h a l l make  it  and study.  f r e e l y a v a i l a b l e f o r reference  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s .  Iti s  understood t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l n o t be allowed without my  permission.  Department o f H u m a n N u t r i t i o n The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  )E-6  (2/79)  Columbia  written  ABSTRACT This  study  was  performed  to  determine  the  rates  of  3 in  vitro  tion the  cholesterol  into  The  hopefully in  blood  l i t t e r  testine  large the  from  amongst regions  synthesis, the of  litters.  The  a  21.  from  three  l i t t e r  intestine not  l i t t e r ,  and  was  values  seen  day  14.  Previous low  levels  were  day  in  early  7,  thus  testinal  cholesterol  cholesterol of  synthesis  proximal the  the  values. in  intestine  opposite  was  in  postnatal  After  in  found  in  of  the the 7  medium the  the  small  rate  of  different  low with  day  was  higher  and  small  21,  day  14,  synthesis in  the  from  the  that  plasma  l i t t e r s rates low  on  of  i n -  plasma  the  than  rate  that  l i t t e r s ,  l i t t e r .  and  lowest  shown  i n -  decreases on  period  larger  cholesterol  low  large  on  different medium  the  from  weaning,  to  later  was  has  correlate  intestine the  rate  day  rats  in  of  of  days  work  was  (4/dam),  significantly  synthesis  d i s t a l  on  raised  rate  to  and  differences  14  sizes;  the  cholesterol  low  birth  tissue postnatal  the  region  The  larger  on  of  small  to  incorpora-  parameters  rats  distal  H  early  in  and  day  these  by  adipose  the  between  birth  by  white  cause  raised  proximal  again  cholesterol  both  were  (14/dam)  decreased  the  levels  measured  during  studying  elucidate  Rats  as  gluteal  rats  for  cholesterol  increased  varied in  reason  in  in  infant  further  and  synthesis  then  of  sizes.  (8/dam)  in  cholesterol,  intestine  period.  synthesis,  of whereas  i i i  Cholesterol remained medium  at  sized  increased fold  the  21.  This  very  low  rate  l i t t e r s .  continuously rate  of  study  synthesis during  a  synthesis  in  the  different  that has  adipose  up  to  the  21  days  in  contrast,  the  in  the  l i t t e r  in  the  shown  sizes.  large other  that  and  postnatal  l i t t e r  gluteal white  In  intestinal  early  in  two  the  Also,  the  to  of  tissue  by  of  and  synthesis  sizes  threeon  day  cholesterol can  raising  pattern  small  nearly  l i t t e r  rates  adipose period  rate  tissue  be  the  of  altered rats  in  development 3  the  rates  of  corporation results glutaryl  cholesterol synthesis  into  c h o l e s t e r o l are  reported coenzyme  development.  for A  the  in  a c t i v i t y  reductase  for  as  measured  general of this  by  H  agreement  i n with  3-hydroxy-3-methylsame  period  in  in  iv  TABLE  OF  CONTENTS Page  ABSTRACT  i  LIST  OF  TABLES  LIST  OF  FIGURES  i v i  v i i  ACKNOWLEDGEMENTS  v i i i  Chapter I. II.  INTRODUCTION  1  REVIEW  4  OF  THE L I T E R A T U R E  A.  The R o l e  B.  Disorders Associated with Altered Cholesterol Metabolism Developmental Patterns of Cholesterol Synthesis i n Over- and U n d e r - n u t r i t i o n , Cholesterol Synthesis i n the Intestine Cholesterol Synthesis i n Adipose Tissue Methodological Considerations  12 18 21 23  METHODS  30  C. D. E. F. III. A. B. C.  of Cholesterol  AND  i n t h e Body  MATERIALS  4 8  Experimental Design Animals Tissue Preparations and Culture Conditions A n a l y t i c a l Procedures Thin Layer Chromatography Calculations S t a t i s t i c a l Analyses  32 34 36 37 38  RESULTS  40  Preliminary Studies  40  1.  Cell  40  2.  Extraction Efficiency Body Weight D i f f e r e n c e s Cholesterol Synthesis Intestinal Synthesis Adipose Tissue and Cholesterol Synthesis  D. E. F. G. IV. A.  B. C. 1. 2.  V i a b i l i t y  30 31  40 40 44 44 53  V  Page V. A. B. C. D. E. VI.  DISCUSSION  58  O v e r n u t r i t i o n and Undernutrition Methodological Procedure Intestinal Synthesis of Cholesterol Developmental Pattern of Cholesterol Synthes i s  58 59 61 67  Cholesterol  Synthesis  i n Adipose  Tissue  CONCLUSIONS  BIBLIOGRAPHY APPENDIX APPENDIX  APPENDIX  APPENDIX  APPENDIX  APPENDIX  APPENDIX  APPENDIX APPENDIX  A B  - List of Abbreviations Synthesis in and Body - Cholesterol Weights of Rats Raised in Litters of 8 C - Cholesterol Synthesis in and Body Weights of Rats Raised in Litters of 4 D - Cholesterol Synthesis in and Body Weights of Rats Raised in Litters o f 14 E - S t a t i s t i c a l C o m p a r i s o ns o f R a t e s o f Cholesterol S y n t h e s i s Between 2 C o n s e c u t i v e Weeks C o m p a r i s o ns o f R a t e s o f F - S t a t i s t i c a l Cholesterol Synthesis Between P r o x i m a l :and D i s t a l I n te s t i n e C o m p a r i s o ns o f R a t e s o f G - S t a t i s t i c a l Cholesterol S y n t h e s i s Between Rats Raised i n L i t t e r s of 4 and 14 f o r V a r i o u s Tissues C o m p a r i s o ns o f B o d y W e i g h t H - S t a t i s t i c a l Amongst L i t t e r Sizes I - A n a l y s i s o f V a r i a n c e Co n t r a s t t o t h e T i m e F a c t o r a n d I n t e r a c t i o ns w i t h t h e T i m e F a c t o r f o r i C h o l e st e r o l Synthesis i n Proximal I n t e s t i n e and Distal Intestine  68 7/1 101 74  77  78  79  86  87  93 99  v i  LIST  OF  TABLES  Table I  II  I l l  Page Baseline Values f o r the Incorporation of H Water i n t o C h o l e s t e r o l i n Various T i s s u e s C u l t u r e d on I c e .  45  Mean V a l u e s f o r t h e I n c o r p o r a t i o n o f H Water i n t o C h o l e s t e r o l i n I n t e s t i n e and A d i p o s e T i s s u e o f Rats by L i t t e r Size.  54  Mean V a l u e s f o r t h e W e i g h t o f G l u t e a l Adipose Tissue of Rats by L i t t e r Size  56  v i i  LIST  OF  FIGURES  Figure  Page  1  Pathway to B i l e  of Oxidation Acids  of  Cholesterol  2  Pathway of M e t a b o l i t e s f o r the S y n t h e s i s of C h o l e s t e r o l and i t s Products  25  3  Percent C e l l V i a b i l i t y and A d i p o s e Tissue  41  4  I r iv i t r o I n c o r p o r a t i o n o f H Water into Cholesterol i n Various Tissues Time  of  9  Intestinal 3  42 Over  5  Body Weights of Rats R a i s e d Different L i t t e r Sizes  i n  43  6  Incorporation of H Water i n t o C h o l e s t e r o l i n I n t e s t i n e and Adipose Tissue of Rats Raised i n L i t t e r s of Eight  46  7  Cholesterol Synthesis i n Intestine Rats Raised i n Small Litters  of  48  8  Cholesterol Synthesis i n Intestine Rats Raised i n Large Litters  of  50  9  Cholesterol Synthesis i n Distal t e s t i n e i n Rats Raised i n Small Large Litters  3  Inand  51  10  Comparison of the Rates of C h o l e s t e r o l Synthesis i n Proximal Intestine Between Rats R a i s e d i n S m a l l and Large Litters  52  11  C h o l e s t e r o l Synthesis i n Adipose Tissue of Rats Raised i n Small and Large Litters  55  v i i i  ACKNOWLEDGEMENTS  Acknowledgement tion  f o rgenerously  Fellowship  sulting  from  and J i r i  expert  during  the past  I am  a n d Dan R u r a k  I thank  and t h e i r  Smale  help  tragedies  I would  to  f o rh i s patience  advice  on  Barr,  experi-  who  have  and t h e i r worked  especially I also  co-  at the  Mrs.  Freda  express  my  of the Faculty of  a t U.B.C.  f a c i l i t i e s  instruction.  like  i n the preparation of  Hassinali.  Axelson  Sciences  research  and  of setbacks, r e -  t o D r s . Susan  f o rt h e i r  Medicine,  t o D r . James  of their  indebted  a l l the people  and Mr. S a l i m  Pharmaceutical  operation  years Hahn  Assistant  study.  and experimental three  Founda-  Research  For t h e use o f equipment  f o rD e v e l o p m e n t a l  gratitude  use  personal  guidance.  manuscript.  Centre  of this  g r a t i t u d e t o Dr. Peter  procedures  operation  Anne  both  Frohlich  mental this  my  support  c o n s i d e r a t i o n o f t h e number  encountered express  p r o v i d i n g a Graduate  f o rp a r t i a l  In  i s d u e t o t h e B.C. H e a r t  and h i ss t a f f and t h e i r  f o r the  w i l l i n g co-  1  CHAPER  I  INTRODUCTION Within on  studying  and  the last  t h i r t y  the development  humans  during  studies  have  when  certain  biosynthetic  elopment  been  There  metabolic  responses  the  period  early  detailed quences in  and  of l i f e  of early l i f e ,  imbalance  structure  the a b i l i t y  whether  the  pattern  which  under-  dev-  of  during  w i l l  be  lasting  (8-10).  conse-  Malnutrition  or over-nutrition  proportions  of nutrients,  alterations  o r an  has  i n growth,  metabolic  ( 8 , 90.  with  and events  recognized  of development i n this  affected  metabolism  the  t o cope  a r e now  Included  that  (13-18),  period  course  changes  i n permanent  become  by the diet, f e d  demonstrated  time  (11, 12), behavior  the conditions  manently  i t be  suggests  period.  and f u n c t i o n  Hence,  the  have  i n animals  the  pathways  Studies  placed  postnatal  and the adult  altered  nutritional  to result  which  (1-7).  i n the relative  shown  brain  be  processes  out t o determine  processes  may  has been  and e a r l y  and c a t a b o l i c  i n the discussion  early  been  carried  i s evidence  of metabolic  emphasis  of metabolic  the gestational  Many  functional.  years  by  surrounding  as b e i n g through  l i s t  stresses  capable to  of cholesterol.  The  of  perinatal  influencing  adulthood.  of processes  the conditions  the  which  during human  may  early  body  be  l i f e  peri s  synthesizes  2 about  75%  depends  of  on  of  of  entirely  for  of  the  rate  remains  and  low  for  a  in  plasma  the over  (2,22)  to  activity  not  been  3  4  (2).  for  the  clearly  and  levels:  elimination,  (23).  has  way  that  metabolism ascribed  patterns  of  early  post-  variety the  shown from  for  in  the  of  intestine that  the  the  activity  b i r t h a  rats.  the  decrease  both  in  theory,  in  in  pups  a  rise  is animals  factors  contribue  decreased the  of  that  these  three  could  synthesis,  cholesterol  is  hepatic  mechanism(s)  combination  A  and  This  number  causes  cholesterol In  b i r t h  Co.  i n t e s t i n a l mucosa  Reducing  and  blood  after  period  after  increased and  for  on  (19-21).  been  the  not  decreases  also  defined. in  had  and  judged  biochemical  high  by  3-hydroxy-3-methylgluyaryl  levels  The  as  suckling  soon  overall  a  work  pool  demonstrated  under  but  Previous  tissue or  the  amount  contributing  intestine  previously  liver  and  i n d i v i d u a l l y or  elevated  the  body  has  gestation  1.1.1.34) the  in  for  and  this  feedback control  Developmental  synthesis,  cholesterol  responsible  (20).  a  the  studies  that  l i m i t i n g enzyme  during  to  investigated  tissue.  adipose  l i t t e r  acting  for  exert  liver  role  during  been  states  liver  in  has  liver  (HMGR:EC  brown  HMGR  to  cholesterol  reductase  true  function  have  adipose  of  major  synthesis  period  nutritional  rate  a  a  the  animal  although  responsible  cholesterol l i k e l y  from  plays  cholesterol,  natal  of  pool,  which  Tissues  most  produced  cholesterol  or  are  intestine  almost  components  amount  synthesis  the  cholesterol  synthesis.  greatest  Evidence  total  dietary  cholesterol the  the  diet.  to turn-  3. HMGR- i s t h e r a t e pathways: High i l y  ubiquione  l i m i t i n g enzyme  ( 2 4 )a n d d o l i c h o l  levels  of activity  indicate  increased  In  view  of at least  o f these  o f HMGR  facts  (25) synthesis.  do not,  cholesterol  therefore,  synthesis  thepresent  3 to  examine  the incorporation  in  the intestinal  rats  reared  make  i t possible:  a c t i v i t y  i n l i t t e r s  with  the intestine,  ol  synthesis HMGR  and white  ( a ) t o compare rate  activity  adipose  i svery  study  was  H from  H2O i n t o  adipose  tissue  sizes.  27). designed  of synthesis  tissue  cholesterol  of  infant  The d a t a  would  published data  and (b) t o determine  i nwhite  (26,  necessar-  3  of different  the actual  in  where  wall  of  two other  of  cholesterol  therate  during  low and d i f f i c u l t  o n HMGR  of  cholester-  development t o assay.  4  CHAPTER REVIEW  The  following  cholesterol and  tions  dietary  presented. lems  The  A.  The  Role  ity  o f t h e membrane  is  with  component  i n which  structure  (33) , and enzymes  from  yeast  behave  hypothesis (34)  states  are also  the  the absolute  rate  which  when  such  have  probof  by  aggregation  the  fluid-  t o enzymes  (29,  i s changed.  o f human  of their  membrane  oxidase  cholesterol and  hypercholesterolemia  For  platelets cholesterol  (29) and  i n mitochondrial  Roman-Franco  membranes  w i t h i n t h e membrane  fluidity  low or high  cell  Functional properties  as cytochrome  a  affects  of metabolites  membrane  differently  proposed that  of a l l animal  (28).  a f f e c t e d by the concentration  also  concerns  i t i s present  epinephrine-induced  (30)  associated  of cholesterol  ( 3 1 , 32) c o n t a i n e d  are altered  example,  disorders  discussed  and a c c e s s i b i l i t y  and r e c e p t o r s  lattice  morphology func-  determining  i s a  the quantity  30)  i n c e l l  to  m a l n u t r i t i o n . Normal  levels  subject  topics related  o f C h o l e s t e r o l i n t h e Body  and  proteins  reviews  synthesis.  Cholesterol  of  a n d some  associated with  cholesterol  from  and plasma last  LITERATURE  and t o changes  resulting  of cholesterol  altered  THE  presentation  metabolism  metabolism  OF  I I  ATP-ase  membranes content.  An  Santiago-Delphin  w i l l  reduce  .• • '  5 immunocompetence  by a l t e r i n g  membrane  and  consequently  the a b i l i t y  of lymphocytes t o recognize  and  hence  cells.  Heiniger  i s also  reduce'd  target  immunocompetence lesterol  content.  cytolytic of  activity  by T  The  effect  of this  a b i l i t y  Blocking  DNA  on  of the lymphocytes  sterol  synthesis  rather  Using  have  inhibiting  results  that  i n an  endocytosis,  have  been  the  start  increased weeks, in  increased  by  resembled  was  with  the  d i d not alter that  shown  of the diet a n d membrane  f r a g i l i t y , acanthocytes  that cho-  decreased an  inhibitor  f o r 24  reversed  during  suggesting  hours  by the a d d i t i o n  preincubation  the cytotoxic  the effects of effect  than  to inhibition  of the -cells'  same  approach  e t a l . (36)  de  flux  novo o f K+  of c e l l  feeding  and"coconut  the hematocrit  osmotic  acid  i n the morphology  induced  cholesterol  this  and c e s s a t i o n  Changes  there  was  antigens  the cellular  incubated  inhibition  f l u i d i t y  are related to i t s inhibitory  proliferation. shown  lowering  25-hydroxycholesterol,  synthesis  25-hydroxycholesterol  by  experiment  cholesterol or of mevalonic  period.  and  e t a l . (35) have  lymphocytes  cholesterol synthesis,  (35). of  In their  structure  o i l  a  Chen  synthesis a n d Na+,  proliferation.  the r e d c e l l  declined  dogs  with days  Beginning  a p a r a l l e l  and the r e d c e l l s or spur  three  of  after  cholesterol/phospholipid  decreased. with  cells  supplemented  (37). Within  f l u i d i t y  decreased  of red blood diet  of cholesterol  cells.  ats i x  increase,  morphologically Four  weeks  after  the  ratio  6  diet to  was  discontinued,  normal.  l i p i d old  S i m i l a r changes  content  g i r l  a l l red cell  have, more  presenting  parameters  i n red c e l l  r e c e n t l y been  with  had  returned  morphology  reported  and  i n a  4^  year  f a m i l i a l intrahepatic,  cholestasis (38). From stantial  these  and  evidence  other  studies  indicating  in  membranes  i s to maintain  al  integrity  and  Numerous lesterol. phorus  that  cholesterol  activity,  can  also  be  Vitamin  losum  synthesized  irradiation, of the skin  lesterol to  can also  produce  from chain  be  group  c h o l e s t e r o l by to yield  of  the side  of  the various  a  chain  21  of vitamin  D  from  forms  dietary  Provitamin  of  vary-  sources  and  D^,  after  (42).  cho-  phos-  ultragranu-  Dietary  cho-  i n the intestinal  wall  which  are derived  Pregnenolone  the enzymatic  cleavage  compound C-20  from  (28).  o f compounds  between  sites  and  i n the stratum  the intestine metabolized  fluidity.  of calcium  vitamin  i s synthesized  carbon  membrane  exogenous  yields  are the steroids.  subcellular function-  are synthesized  i n t h e body.  which  and  and  i s sub-  of cholesterol  i n i t sseveral  7-dehydrocholesterol  Another cholesterol  D,  i s a v a i l a b l e from  7-dehydrocholesterol, violet  compounds  i s the synthesis  (42).  ing  cellular  to the homeostasis  metabolism  there  the role  cholesterol alters  important  V i t a l  that  (39-41)  and  can, depending  i s produced., of the  followed C-22. on  from  by  oxidation  Further  the type  side  of  oxidation tissue,  yield and  dehydroepiandrosterone,  also  the  corticosteroids  Although which  synthesize  which  necessitate  density  thesis  the  In  delivered  Orwoll  have  (44)  a  gland  2-3  fold  concomitant hormones.  found  cells  In  fold  the  in  steroid  cotropin  i s  3  presence  of  fold  characterized B-containing are  lower  by  the  of  uses  LDL  the  predominantly  of  and  LDL  to results  receptors of  and  a  steroid  magnitude  stimulated  seen  from syn-  corticotropin  synthesis  LDL  that  steroid  presence  synthesis  than  conditions  cholesterol  of  by  maximally  abetalipoproteinemia,  the  complete  to  absence  synthesize  adrenal  increase  steroid  in steroid  stimulation  corticotropin  production  of  number in  the  Illingworth  the  increase  hormone  with  able  corticotropin by  LDL.  in  the  are  cortex  addition  of  analogs,  within  increased  of  lipoproteins (chylomicrons,  unstimulated  tion  in  i t s  the c o r t i -  in  the  LDL.  Patients  i n i t i a l  for  adrenal  that  there  exogenous  i t v i a  absence  increase  plasma,  of  cultured  increase  30-50  the to  produced  steroids  uptake  fact,  cholesterol  in  also  l i p o p r o t e i n s (LDL)  (44).  adrenal  the  and  (43).  cholesterol i s  cells  low  progesterone  which  hormone  (44).  i s probably  rare  a l l  disorder  apoprotein  VLDL,  LDL)  from  cholesterol adequately production  production  results  a  in due  in  and  response  However,  chronic  subnormal  steroid  to  of  a  lack  for for  the  to stimulahormone  lipoprotein  8 and.  hence  Orwoll of  cholesterol  (44)  have  steroid  of  and  lipoprotein  receptors  and  bile  acids,  1).  These they  Nearly back  In  chronic  cholesterol  of  cholic bile  the  initial on  of  cholesterol  in  the  facilitate  the  digestion  liver  by  the  liver  summary,  Cholesterol  is  acids  where  liver  are  they  to and  suppress  cholesterol in  has  membrane  precursor  and  Only topic  vitamin  a  Associated very  because of  cancer  is  absorption and  the  (Figure intestine of  fats.  circulated of  cho-  D,  roles in  adrenal  T  and  the  body:  function,  lymphocytes, sex  and  steroids,  and  acids. Disorders  with  small  structure  as  logy  the  multiple  of  B.  acid  synthesis  p r o l i f e r a t i o n , immunocompetence  bile  LDL  (45).  involved  to  influx  in  produces  reabsorbed  cell a  increases  chenodeoxycholic  transported  bile  the  LDL.  are  the  intra-cellular  requires  from  and  stimulation  stored  resulting  and  Illingworth  stimulation  uptake  acid  (44).  acids  the  lesterol  that  dependent  increased  a l l of  to  is  that  Oxidation  where  proposed  synthesis  cholesterol  availability  diseases and  other  dietary  safe  the  to  brief  such  as  cancers  that  and  Altered  introduction  controversy  intake  say  with  s t i l l  Cholesterol will  be  continues  atherosclerosis, which plasma  dietary  are  given  found  levels  of  cholesterol  over  Metabolism to  this  the-etio-  gallstones, to  be  associated  cholesterol. is  not  colonic  the  only  It cause  Chenodeoxycholic Figure  1.  Pathway Adapted  acid of Oxidation from Sabine,  of J.  Cholesterol to B i l e Acids. R. i n C h o l e s t e r o l (28)-  10 of  heart  provides liver  disease feedback  more  cholesterol  alone, although  cholesterol  death  i n middle  this  do  quantities blood  of  aged  exist  and  of both  herdsmen  camel's  consume  mg%  large  and  and  Attempts  met  20-30%  American  there  the Masai  and  mortality  from  no  t r i a l  populations,  blood  pressure, An  investigated  serum  heart  cholesterol  are not d i r e c t l y  eleven  year  study  the relationship  (53)  high  are the quarts who  greater  raised  predictor of  of  these  than  serum  cho-  morbidity  i n Western  indus-  concentrations,  unlike  630  between  have  five  correlated with of  large  disease.  that  disease  exceptions  Neither  levels  the  coronary  of this  includes  heart  important  coronary  affect  consuming  and m i l k .  cholesterol  an  achieved  cholesterol  i t i s generally accepted provide  of  of Tanganyika  v i r t u a l l y  success  been  the risk  diet  o f meat  dietary  however,  (52, 53).Examples  blood -is.  does,  blood  (51), although  fats  daily  by  has  by  inevitably  varied  not a l l populations  saturated  and  men  w i l l  t o lower  reduction  and  cholesterol  synthesis  subjects with  of the diet  quantities  levels  mortality.  been  (52) whose  milk,  While lesterol  (46).  levels  groups have average 160  the liver  concentration  cholesterol  Somali  a  composition  serum  to  feedback  have  Dietary  of cholesterol  i n hypercholesterolemic  49)  (50).Lipid  this  cholesterol  manipulations 48,  atherosclerosis.  inhibition  and without  produce  (47,  and  Maoris serum  overall  i n New  Zealand  cholesterol  11 concentration was  found  between  mortality of  death  related  due  (HDL)  HDL  An  cholesterol that  risk  hand  persons  high  LDL  several  l o w LDL  factors  have  and that  serum  genetic  influences  (55)  this  lower  risks  from  high  separately  risk  stated  On  that risk  the  other  those  high  than  the  o f HDL  with  improves HDL  cannot  LDL."  cholesterol  i s only  i n the pathogenesis  levels.  coronary  predictor  than  very  i t s e l f ,  of  are high,  Knowledge  populations.  of  have  i s higher.  not  lipoprotein  a better  i f HDL  Other  or the influence  contribute  was  which  density  i t i s not the only  cholesterol  may  was  incidence  i s , by  study  b u t even  dietary  responsible  affects  components,  HDL:  of prediction,  that  makes  the risk  et a l .  o f HDL.  resulting  high  f o r the prediction  are low the r i s k  i s clear  sclerosis  and high  Kannel  at a l l levels  the risk  which  concentration  cholesterol  by  The  and  Western i nd u s t r i a l i z e d  factors  (55).  with  precision  It  LDL  i f HDL  levels  relationship  i n the Maoris  epidemiological  i s modulated  lower,-  remove  as  causes.  f o r predicting  cholesterol  cholesterol  "The  the  factor  LDL  determined  from  inverse  concentrations  disease  cholesterol  disease.  considered  is  blood  a reliable  a r t e r i a l  and other  cholesterol  different  An  cholesterol  cardiovascular  t o serum  Total not  serum  t o cancer  from  population  (54).  and m o r t a l i t y  dietary  of  to the etiology  other of the  of  athero-  factor  factors,  o f some  one  which  including dietary patho-  logical lated  conditions.  to  It  atherosclerosis  creased  turnover  ture  composition  or  LDL-receptors familial  by  The to  for  induce  et  DMH.  of  of  dietary no  Malnutrition  nutritional during  at  that  of  time,  has  only  but  leaves  rei n -  structhat  case  the in  fat  and  and  54,  able the  colon  cancer  which geographical r e l a t i o n -  (58-60)  has  adult  by  not  Under-nutrition been  even  shown  after  Undernutrition a  moderate  development  permanent  was  cancer  61).  evidenced  per-  single  causal  newborn  the  a  examined.  the  a  proposed.  the  a  diets,  with  been  (11,  of  f a c i l i t a t e  colonic  Over-  is  induced  experimental high  in  was  i t s e l f  suggested  fetus  affects  as  experiments  were  with  in  is  by  content,  changes  which  or  the  effects  i t did  of  relating  the  is  in  cholesterol  cholesterol,  have  i s  cancer  the  spread  r e h a b i l i t a t i o n  not  and  although  Patterns  as  (51)  from  Colonic  cancer  permanent  development  restriction  comes  cholesterol  mechanisms  in  cancer  cholesterol  Developmental  result  and  such  alterations  missing  cholesterol  Studies  colonic  and  dietary  cancer,  for  mechanisms,  that  (DMH)  cholesterol  (56).  (57).  that  growth,  adjusted  although  to  a l .  that  lipoproteins  or  component,  colonic  by  incidence  C.  plasma  suggest  showed  development,  ship  of  colonic  variable  induced  other  lipoproteins  defective  to  Cruse  results  were  via  dimethylhydrazine  dietary  possible  hypercholesterolemia  cocarcinogen  with  of  are  Evidence  formed  is  of  distortions  the of  growth brain  gross  and  13 histological mechanisms at  which  structure,.chemical  and  the  behavioral  nervous  rate  of  have  demonstrated  fatty tion  fatty  acid  i s  and  also  a v a i l a b i l i t y  develops  accretion in  the in  of  the  for  both  (62).  i s  infant  s p i n a l cord of  et  the  (62) rate  the  fatty  acids  of  myelina-  activity. (63),  rate  the  a l .  parallels  myelination  c h o l e s t e r o l and  The  by  that  neuro-motor  proper  12).  limited  Clandinin  developing  appearance  needed  metabolic  c h a r a c t e r i s t i c s (11,  system  accretion  process  terol  acid  composition,  Choles-  and  the  during  the  a  rapid  myelination  neuro-motor  period  for  which  described  conducted normal sex,  who  within  months  height, verbal found nerves, ing  (64 ) .  and  were  weight,  of  in  pubertal reflexes,  tests.  the  Their  and  development  Moro  of  weight  follow-up weights  socio-economic  each  at  ages  other.  13  were  development, gait  and  newborns  30%  matched status 19  small have  were  under for  birth  (61).  years  triceps  found. motor  No  r e s u l t s suggest  that  or  date,  Paired  were  seen in  skinfold  and  differences  function,  coordination  also the  Significant deficits  circumference,  I.Q's.  to  two  between  studies  were  fullterm controls and  responses,  development,  normal  (body  studied  head  performance  and  Longterm SGA  ethnicity  subjects  gripping  (SGA)  comparing  race,  the  neurological  age  expected)  3  in  assess  gestational  been  affect  activity.  Differences tests  may  were  cranial  reading  intrauterine  and  spell-  growth  14 retardation  i s  deficits  physical  havior  in  associated  patterns  in  aggressiveness tioned  postnatal et  a l .  stage,  (14)  have  rats,  but  the  rat.  elopment  time  occurs,  species  and  a b i l i t y  to  brain (5,  of  shed in  which  was  when  the  f a l l  in  seen  elopment  has  brain  failed in (1).  pups In  (2),  to  to  from  a  of  in  serum  the  l i t t e r s  contrast,  of  suckling  differing  times  affected  of  been  (3),  ovary  weeks  (4),  of in  and  content  of  vulner-  (3).  weights weight and  A  ovarian rats  the  of de-  c e l l  lipase,  were 3-4  numtry-  diminiday  delay  levels dev(4).  norepinephrine,  during  development  controls  brain  in  intestines  including  and  with  various-  and  body  undernourished  compared  dev-  nutritional  hormone  in  increase  in  brain  content  weight  for  brain  mean  than  DNA  dopamine  the  Byrne  period  the  had  stimulating  of  period.  carboxypeptidases  age  condi-  in  characterized  enzymes,  organ  in  rate  16  extent lower  of  of  of  operant  (11).  by  have  l i t t e r s  total  serotonin  the  (13),  sensitive period  differs  pancreatic  18  the  different species  described  same  gestational  Dobbing,  and  Be-  undernutrition  by  greater  concentrations  by  and  period  systems  in  17),  c r i t i c a l  f o l l i c l e  three  been  the  just  newborn  permanent  development.  the  the  chymotrypsin per  16,  altered  a t t r i b u t e d to  at  show  (15,  pancreas  raised  expressed  normally  Also,  for  probably  t a i l - p i n c h i n g responses  delineated  which  and  Activities  amylase,  as  to  and  cognitive  during  hormone  decrease  be  proposed  fetus  Rats  (3).  psin,  the  7).  crease ber  and  liver  pancreata  at  malnutrition  (1),  6,  not  accounts  Enzyme status  can  effect  as  and  such  further  under-nutrition  longterm  dominance  (18)  the  The  growth  (14),  responses  with  was  (6  pups/litter)  significantly  15 higher in  i n the malnourished  catecholamine  control  animals  er  the  than  dicated both  by  The nutrition  concentration i s probably  a v a i l a b i l i t y similar  groups  than  control between  regulated  of  as  rath-  i si n -  phenylalanine  resulting  described  that  above,  indicate  lower  and  activity,  higher  very  Cholesterol  activity  acids  d i f f e r e n c e s i n metabolism  of  another  amino  and  i n  (1).  i n the production  that  enzyme  t y r o s i n e and  result  but  Differences  the malnourished by  of precursor  levels  animals.  enzyme  different  synthesis  example  of  lesser  patterns  and  the  a  of  of  the case  during  mal-  not  a  simply  product  may  metabolism  metabolism  changes  i t does  amount  as  from  or  be,  emerge.  development i s  i n metabolism  which  result  from m a l n u t r i t i o n . A blood  theory  held  cholesterol  by  several researchers  level  throughout  normal  suckling period  w i l l  levels  at maturity  and  a  plasma  cholesterol  levels  diet in  ( 2 , 8-10,  rats  man.  65,  (9) and  However,  66).  pigs only  comparison  to  Hodgson  a l . (68)  diet  et  i n the  cholesterol  l i f e  the duration  result  better when  this not  short-term been  found  that  children  first  three  level  at  age  with  been  have  months  of  7-12  years  the cholesterol  an  low atherogenic  has  been  shown  studies, i n  fed a had  than  high  established i n  performed  l i f e  of  blood  effect  spans,  a  to maintain  challenged  ( 6 7 ) , i t has relatively  i n lower  a b i l i t y  Although  i s that  a  on  humans.  low lower  children  cholesterol blood who  were  16 fed  a high  But  i n the study  three  months  (67)  point  from  less  period the in  cholesterol  out that  only  moderate  their may  a  cholesterol mental time  t o make  the results  of  a proper  ed  l i f e  with  early levels  during  I t would  perinatal  and p o s s i b l y  an a t h e r o g e n i c Green  of Reiser i n l i f e when et a l .  stock  diet  therefore  et a l .  diet  may  only  milk,  fats.  i s low  Also,  observed  a  during  studied  i n  there plasma  postnatal  develop-  unjudicious  since  whereas  which  f o r elevated  be  suckling  suggests  at  regimens  this  based  the important  not present  when  et a l .  u n t i l  the individual  (69) have  with  a  (65) that  related  (69) f e d mother I ) ,  this  increase  effects  middle i s  or  challeng-  diet.  i s inversely  (Group  was  i n i t i a l  Roberts  the  formula  f o r dietary  and Sidelman  challenged  Green  reason  and  of animals  the i n i t i a l  o f Hodgson  Recently, thesis  species  recommendations  on  late  i n nine  Teleologically,  levels  on  polyunsaturated  physiological  stages.  mg%  cow's  milk). the  levels  during  hypercholesterolemia  period  good  mg%  o r f e d on  t o 100-130  during  Carroll  cholesterol  t o 150-180  and contains  laboratory.  be  infants'  o r cow's  levels  not given.  breast-fed  t o marked  suckling  were  rises  cholesterol  ( i . e . breast  cholesterol  1 0 0 mg%  of infants  level  the  blood  of l i f e  than  diet  a  high rats  challenged  t h e hypo-  cholesterol  intake  to adult  blood  fat-high  cholesterol  one o f t h r e e  semisynthetic  diet  cholesterol  diets:  with  15%  diet.  a lard  (Group  I I ) , or a  cholesterol lactation. higher  (Group  of  i n Group  had access  pups  were  most  l i k e l y 60  lard  authors  lower  only  concluded  t h e amount  correlated  with  a  their  subsequent  higher  experiment  cholesterol  indicated  by the lack  milk  cholesterol  food  u n t i l  of  pups  time.  Another  l i f e in  does  adult  study,  demonstrated not protect rats."  These  o r 15  not  that against  i s  Hulbron  idea  inversely induced  of lactation  eating  and therefore  do  the  not exposed  "cholesterol  findings  was  demonstrated  n o t be  diet-induced  10%  dams,  of the maternal  would  by  I  i n l i f e  were  From  containing  e s s e n t i a l l y t h e same performed  diet.  to d i e t a r i l y  was  t h e pups  of any e f f e c t  d a y 14  consuming  this  days  weaning,  d i d not support  response  Pups  a t 30  at  t h e Group  at the beginning  content.  perhaps  were  however,  diet  concentration.  cholesterol  f e d early  However,  because  from  study  were  o f Group I I I  o f t h e dams'  plasma  i n pups  their  levels  fed a diet  Since  of cholesterol  hypercholesterolemia. in  were  weaning  two groups  of consumption  1.5% during  concentrations  u n t i l  i n the other  cholesterol.  and  or t r i g l y c e r i d e  diet  and  no s i g n i f i c a n t  cholesterol  t o day 161 a l l pups  15% l a r d  of gestation  were  cholesterol  than  with  cholesterol  there  and l i v e r  because  a n d 0.5%  that  plasma  diet d a y 18  t o t h e dams'  higher  significantly the  from  I I I dams  i n milk  age. Plasma  day  III)  Although  differences Pups  semisynthetic  to a as  diet  on  solid  a l l three  diet  u n t i l  et a l . given  was  groups that  (70), early  has, i n  hypercholesterolemia  not necessarily  negate  the  18 p o s s i b i l i t y  that  a protective effect  hypercholesterolemia l i f e . tive the  Although feedback  serum  governs  c o n t r o l on  the rate  from  traditional  lesterol  the  total  that  body  found  more  that  remaining  tissues  have  reported  the squirrel  occurs  liver  t o a much  i s the main  influence  factor  that  factor  8%  f o r the rates  which  1 8 % was  14%  have  of  been  et a l . (72) amount  of  produced  only  produced i n the  combined.  Similar  of cholesterol  i n the liver  findings  synthesis  (73), i n which cholesterol degree  cho-  confirm  Turley  bowel  of  intestine  about  of the t o t a l  the small  of the carcass  site  of synthesis  although  and  challenged  the  supplies  Results  site  while  greater  nega-  levels.  have  indicated that  p r o d u c e d 50%  monkey  can  a  or the sole  i n rats  the liver  contributed  fed i n early  exert  and  cholesterol  ( 2 0 , 21)  synthesized,  the skin  been  that  i s a major  the liver  cholesterol  i n the body  cholesterol (19).  recently  does  synthesis  studies  while  the diet  i n the Intestine  (71) and  55-56%,  the intestine  reported  24%;  theory  diet-induced  i t i s not the only  serum  i n vivo  synthesis  contributes  in  i t s own  of synthesis  f o r altering  Results  by  cholesterol i n the diet  Cholesteroll Synthesis  the  invoked  cholesterol levels,  responsible D.  c a n be  against  than  synthesis i n the  intestine. Regulation testine  of cholesterol synthesis  apparently  diversionwas  shown  differs  from that  to result  i n the small i n -  i n the liver.  i n increased  Bile  intestinal  19 cholesterol role sis  for  sis  bile  (74)..  acids  were  which  sis,  o f . HMGR  basal  was  uniform  the  d i s t a l  man  (78),  are  an  i t s e l f  be  portion in  bile  of  the  of  (79),  of  In  bowel to  ileum  the (74).  most  (80),  and  monkey  small  equal  and  the  synthetic  proximalcolon  synthetic  system salt  rates  of  (76)  flux  intestine species  (73).  amounts  are  in  are  studied, active, of  lower  and  A l l other  re-  rates  the  (77).  Hamsters  capable  are  (60%)  cho-  duodenum  i n t e s t i n e (81).  the  reduction  i n t e s t i n e i s most  g a s t r o i n t e s t i n a ltract  small  the  proximal  synthesizing the  by  or  dietary  bile  the  c h o l e s t e r o l ; however,  compared  small  of  synthe-  synthe-  liver  portal  toward  dog  bile  of  marked  by  synthe-  Cholesterol  contrast,  independent  the  a  a  decrease  inhibitor  acids  In  not  levels.  an  i t s length.  activity  rat  gions  and  as  cholesterol synthesis  throughout  regions  colon  circadian  did  hepatic  upper  when  case  the  to  that  obstruction  b i l i a r y  c i r c u l a t e d by shown  found  by  inhibited  exception,  proximal  since  cholesterol  either  with  cholesterol  inhibiting  observed.  can  of  99%  of  not  by  f i n d i n g suggests'/-  be  been  Rates  have  in  in  combination  synthesis  decreasing  (75)  and  This  regulation  enhanced  below  activity  has  rats.  the  a l .  f i r s t  rate  cholesterol  ing  et  feeding,  in  lesterol  and  in  ineffective  but  in  effective only was  synthetic also  acid  Shefer  sitosterol  was  synthesis  of  of  synthesiz-  the  relatively stomach,  esophagus, low d i s t a l  20 Early ized and  studies  almost  entirely  t h e smooth  significant lesterol  HMGR  four  "cell  crypt  axis  through  in  70-84%  i nboth  sterol  v i l l i ;  containing  and  ileum  (83,84). fatty in  acids  both t h e  cholesterol jejunum the  and ileum.  fraction  that  f e da d i e t  (84).  of the v i l l u s / segments  (82).  activity  f e da c o m m e r c i a l  activity  differently  containing  whereas  higher this  pattern  effect could  enzyme  a c t i v i t y  was r e v e r s e d  may b e d u e t o e a s e result  i n increased  i nrats  i n t h e jejunum  a c t i v i t y  when  a diet  containing of the  dietary  diet.  of thepurified  and hence  HMGR i n  changes.  markedly  t h e jejunum,  f e da p u r i f i e d  absorption  suppressed  o f microsomal  exhibited  than  diets  was  t h e enzyme  diet  of digestion  activity.  polyunsaturated  The d i s t r i b u t i o n  i nthe ileum  v i l l i - a n d 16-30%  by various  both  only  non-purified  these found  the least  r a t . , s m a l l i n t e s ' t i r i e 'was__al's'o_modif l e d b y o t h e r  Rats  In  with  i n the intestine i s  a n d c h o l e s t e r o l , HMGR  was s u p p r e s s e d  developed t o  i n t h e lower  possessing  a n d no PUFA was g i v e n ,  ofany  i n t h e mid and upper  t h e HMGR  and jejunum;  V i l l i  o f cho-  The remaining  i s found  i saffected  (PUFA) ileum  place  cholesterol synthesis  of rats  been  levels  synthetic  takes  local-  24,77).  enzyme  of the intestinal  sterol  a c t i v i t y  by the fact  In rats  have  different  was  t o be devoid  limiting  techniques  epithelium  Determining  (23,  c h e l a t i n g agents"  of the total  t h e jejunum  cells  considered  therate  Recently  the latter  complicated  were  incubation  synthetic  with  the crypt  f r a c t i o n s from  the intestinal  crypts of  buffer  wall  activity,  yield  systems  within  muscle  synthesis.  phosphate  indicated cholesterol synthesis  general  while This diet  which  metabolic  activity  i n t h e more  addition  t o this  an  modification  a l t e r a t i o n of thecellular  observed; villous  A significant  cells,  compared  the  animals  E.  Cholesterol The  an  adult  of  that  were  rate rat  kidney,  (85).  by adipose and skin  important  feedback  the  and minor capacity  be g r e a t e r  performed  studies  when  plasma  the  activity  synthesis tissues  tissue  sites  o f minor  of  4%,per  o f HMGR  from  14  was a l s o  of cholesterol such  as  (possibly  synthesis)  there  operating  t o synthesize known,  conmay b e amongst  e t a l .  14  and  increased  three  analogue  lowering  of  increase i n  Cholesterol  C-octanoate fold,  In these  l i p o p r o t e i n s , was  a n d a 5-30 f o l d  C-acetate  (86).  an adenine  of plasma  and kidney.  cholesterol  a s i s shown i n  was a c o n c o m i t a n t  i n lung  tissue,  synthesis.  sites  level  tissue of  gram*of  organs  mechanisms  secretion  cholesterol  (83).  i n adipose  small  by Balasubramaniam  there  when  Tissue  and other  i spresently  hepatic  of  occurred  t h e amount  i srelatively  also  activity  diet.  synthesis  Although  of the gut,  was  cryptic cells,  t o be about  regulatory  t o rats  the  i n HMGR  4-aminopyrazolopyrimidine,  reduces  administered  the length  distribution  a s 2% o f t o t a l  than  studies  which  of the intestine. In  i n Adipose  of cholesterol  as l i t t l e  The  with  Synthesis  tributing  major  along  increase  i sestimated  lung  regions  f e da f a t - c o n t a i n i n g  i n liver  synthesized  may  proximal  i n both  whereas  22 increases  i n the synthesis  observed. activity tion  Plasma i n lung  cholesterol levels and kidney  state  hypothesis  that  synthesis tissues  that  proteins "  blood  cytic  Brown  indented  receptors  with the  i ncertain form  and Goldstein  nonhepatic  o f feedback  i nplasma  ( 8 7 )have  f o r a regulatory  role  lipo-  pits  on t h e c e l l  f o r LDL.  When  themolecules  p i t invaginates  t o form  thec e l l  lysosomal  the apoprotein  t o amino  acids  Free  negative  i n the  surface  which  bind  t o the  a coated  endo-  hydrolysis  and releases the  c h o l e s t e r o l i sproduced  feedback  confirmed  o f LDL  o f LDL c i r c u l a t i n g  Within  f o r the synthesis  which  then  o f b o t h LDL  and cholesterol. (88).  vitro  was  stimulated  the  presence  studies when  have  shown  epididymal  that  f a t pads  o f l i p o p r o t e i n - f r e e serum  o r LDL t o t h e medium  addition  The  and cholesterol  coated  t h e coated  receptors, In  activity  observed  cholesterol. Molecules  fraction.  provides  the administra-  areconsistent  by cholesterol carried  proposal  vescicle.  degrades l i p i d  recently  enter  receptor  a n d HMGR  (86 ) .  original  contain  arenormally  were n o t  was d i s c o n t i n u e d .  l o w l e v e l s o f HMGR  mediated  More  derived  t h e "...data  once  o f t h e r a t a r e due t o an a c t i v e  regulation  their  that  lipids  increased  decreased  of 4-aminopyrazolopyrimidine  authors  VLDL  of saponifiable  suppressed  o f HDL d i d n o t ( 8 5 ) . F e a r s  cholesterol were (85).  synthesis  cultured i n Addition of  synthesis,  whereas  and Umpleby ( 2 1 )  23have,  i n fact,  operation tissues  provided  evidence  of a lipoprotein  i n therat  which  for  receptor  controls  t h e e x i s t e n c e and system  i n nonhepatic  t h e change  i n cholesterol  biosynthesis. Another the  study  potential  capacity  significantly mice  (23).  cholesterol activity  levels  their  lean  litter  i s t h e same  and l i v e r  that  in  white  cholesterol  pathological  bution F.  adipose  levels.  states,  mice  24 h o u r s adipose  study  by a  low-  indicates  produces  proportion-  than  that  adipose  tissue  the activity  tissue positively correlates  tissues,  could  adipose  i nwhite  weight  shows  Therefore,  nonhepatic  The  i n brown for  This  body  also  plasma  was p a r a l l e l e d  levels.  i n obese  i n obese  mates.  t h e mice  which  per unit  t o t h e t o t a l body  Before rates  only,  contribute  i n white  i n HMGR a c t i v i t y  The study  Methodological  higher  i ti s lower  Starving  tissue  i n white  that  while  cholesterol  mice.  o f HMGR  plausible  mice  cholesterol  t h e lean  with  or  obese  adipose  more  level  (23).  i n a decrease  o f plasma  ately  mice  or higher  to  synthesis  have  tissue  ering  cholesterol mice  than  demonstrates  tissues  obese  o f HMGR  from  which  Genetically  i n t h e obese  tissue  reported  of nonhepatic  t o t o t a l body  tissue  resulted  has been  i t  under  make  seems  quite  certain  a significant  cholesterol  conditions contri-  pool.  Considerations  the problems  of cholesterol  associated  synthesis  with  determining  arediscussed,  i t will  absolute be  instructive  at this  cholesterol  synthesis.  are is  incorporated diverse  precursor  and  which  fatty  with  erol  enzymes  produced  membrane  with  which (25).  other  CoA  the 2 The  of  the  which  various endogenous  units  of  catabolism  acid  of  atoms  a l lthe  carbon and  of  of cholesterol  (28).  cholesterol  to the synthesis  f o r the  of  acetyl  of  glucose,  Parts  of  the  starting other  i n i t i a l  steps  of  i n the c y t o s o l ; hence, from  through  the oxidation  does  membranes; carbon  of  not pass  there  unit  the of  two  acyl  mem-  freely  a r e , however,  involves  amino  acetyl  fatty  i s transported  mechanism  from  cholest-  the mitochondrial  CoA  f i r s t  produced  acids,  compounds  through  carnitine, which  i n and  out  of  (25): + Carnitine  mechanism  a c e t y l CoA  two  c i t r i c  Acetyl  the transport  mitochondria Acetyl  The  transported  molecule  f a c i l i t a t e s the  and  the mitochondrial by  Nearly  acetate  are located  to the cytosol.  small  from  required  brane  mechanisms  from  i n mitochrondria  be  the carbon  to the a v a i l a b i l i t y 2).  scheme  2).  must  a  molecule  acids  acids  the  carbon  a r e common  (Figure  synthesis  through  27  to the synthesis  a c e t y l CoA,  The  CoA  amino  leading  compounds  of  (Figure  are derived  general  origin  i s synthesized  acids,  pathway  subject  to outline a  The  i n the  substrates  cholesterol CoA  time  requires  to yield  ^  Acetyl  oxaloacetate  citrate  which  Carnitine f o r the  easily  +  CoA  reaction  permeates the  Some Amino A c i d s  25  Glucose jT A c e t y l CoA <  Citrate  I  Acetoacetyl  t  >Acetate  CoA *C  .Malonyl  Acetone Acetoacetate  HMG  ^"  C o A — > Fatty acids  'HMG  CoA<-  i  Leucine  M e v al ileo n a t e  \  Methylglutaconyl  I  •Isopentenyl tRNA  Dimethylallyl-P-P.  ta c i d s '  DolichoK  I  CoA  f  Prenoic  Ketone b o d i e s  Geranyl-P-P  I  <  • Ubiquinone  Farnesyl-P-P  I  Squalene  Squalene  Lanosterol  2^-dehydrocholesterol  ^ C h o l e s t e r o l esters* 7-hydroxy c h o l e s t e r o l * Bile Figure  acids  2 .  7-dehydrocholesterol  7-aehydrocholesterol  if Cholesterol  Vitamin •Cholesterol s u l f a t e '20-hydroxycholesterol T*  Steroid  Hormones  Pathway o f M e t a b o l i t e s f o r the Synthesis o f C h o l e s t e r o l and i t sP r o d u c t s . Figure adapted from Sabine, J . R. i n C h o l e s t e r o l (28).  26 membranes. be  cleaved  cleavage A  Once by  an enzyme (28).  enzyme  the enzymic  has  entered  exclusive  i t can  to the cytoplasm,  In the presence  reaction  the cytosol  regenerates  o f ATP  acetyl  CoA  citrate  and  coenzyme  and  oxalo-  (25).  acetate In  Mitochondria: Acetyl  In ^•4. a. Citrate  the citrate  CoA  + Oxaloacetate —>  Citrate  +  CoA  Cytosol: Tvm^, ^ * + ATP + CoA  c i t r a t e cleavage. —> enzyme  . , , A c e t y l CoA + +  ADP  +  _ , , , Oxaloacetate  P. l  One  other  mechanism f o r the transport  mitochondria  may  tissues  as  such  vage  enzyme  only  substrate  involve human  activity  acetoacetate  adipose i s low  u t i l i z e d  where  From  scheme  i t c a n be  may  altered  produced the  cholesterol  such The ol  be  as  vitamin  amount ester  governed from  seen  where  Acetyl  enter that  pathway  ubiquinone  by  demands  f o r example,  Investigations  into  leave  acids  the control  w i l l  of  as also  2  pathway. cholesterol away  from  compounds bodies.  cholesterbe  synthesized  and s t e r o i d of  clea-  Figure  and ketone  f o r compounds  bile  the  other  f o r storage  i n lipoproteins  i n  i s not the  are shunted  or f o r packaging the metabolic  CoA  synthesis;  and  out of  the citrate  to produce  available  units  especially  t h e amount  i f substrates  •. d o l i c h o l ,  of cholesterol  cholesterol;  hormones.  (90).  compounds  synthesis D^,  tissue  (89)  f o r cholesterol  indicates this  other  of acetyl  cellular  sex  27 cholesterogenesis  have  the  enzyme  rate  limiting  catalyzes 2).  thereduction  Therefore,  hormonal  the  of this  activity have  been  key  substrate  the  cellular  principally (25). ways  This which  quate  that the of  alter extent  depend  metabolic HMGR ways; levels  been  acetyl  served  for  (89)  a sharp  of other  demand  aged  acetyl for  pathade-  may b e processes CoA.  Thus  the products  cholesterol,  i s met,  of the cell's  other  time.  of at least  do not,  cholesterol  for  metabolic  particular  enzyme  study  point  The p r o v i s i o n o f an  importance  o f HMGR  increase  The  i s derived  branching  ( 2 4 )a n d d o l i c h o l  on mice  o f mammalian  i n the mitochondria  of cytosolic  at that  i sthe limiting  increased  influence  investigation.  CoA w h i c h  including  on t h e r e l a t i v e activities  dietary,  factors  cholesterogenesis  an i n c r e a s e d  exemplified by a  Kandutsch  CoA.  processes,  of a c t i v i t y  indicate  a common  the a v a i l a b i l i t y  ubiquinone  of intense  generated  t o t h e demand  any o f these  may  citrate  t o which  (Figure  numerous  i n several types  acetyl  of substrate  linked  by which  that  s t u d i e s , HMG C o A , i s p r o d u c e d i n  c y t o s o l from  i stherefore  supply  closely  enzyme  HMGR,  synthesis  o f HMG C o A t o m e v a l o n a t e  thesubject  u t i l i z e  t h e enzyme  and time-dependent  i n these  from  around  of cholesterol  t h e mechanisms  lipoprotein,  cells,  centered  two other  ( 2 5 )s y n t h e s i s .  path-  High  therefore, necessarily  synthesis.  performed 3-7 d a y s  This  point has  b y James a n d  i nwhich  i ntheproduction  of  they obdolichol,  28 a  branch  brain  Bensch  using  the  i n the cholesterol synthesis  tissue without  thesis. by  product  a concomitant  et a l .  ( 2 7 )have  a-competitive  confirmed  inhibitor  c o r r e l a t i o n between  enzyme  increase  i n sterol  these  o f HMGR,  activity  pathway, i n  ML  syn-  findings  2 3 6 B, t o  study  and t h e incorpora-  14 tion  of  blasts and  C-acetate (27).  sterol  caution  synthesis  cholesterol  a  enzyme  was d e m o n s t r a t e d  indicates  be e x e r c i s e d  biosynthesis.  rates  substrate limited  which  i nrelating  14  HMGR  C-acetate,  rates  14  or any  of cholesterol  of entry  and Dietschy  of substrate  ( 9 1 )have  pool  into  that  C-labelled  i sconsidered  by an endogenous  activity  a c t i v i t y t o  an u n r e l i a -  synthesis.  o f s t e r o l s may b e u n d e r e s t i m a t e d  dilution  rate  and fibro-  o f c o r r e l a t i o n between  indicator of absolute  Andersen  i nr a t tissue  f o r cholesterol synthesis  Synthetic of  sterol  use of labelled  substrate ble  A lack  should  The  into  because  o r because o f  the c e l l (91).  demonstrated  t r i t i a t e d  3 ( of  H) w a t e r  t o be a s u p e r i o r  t h e absolute  rate  substrate  f o r the  of cholesterol synthesis.  determination The  intra-  3 cellular  compartment  i sr e a d i l y accessible  t o the  H  3 and H water does n o t r e q u i r e e x t e n s i v e metabolism t o be i n c o r p o r a t e d i n t o t h e s t e r o l s . Also, theuse of a  water  high  3 specific of  radioactivity  a dilutional  labelled  water  effect (91).  of  H water  prevents  by themetabolic Studies  comparing  the p o s s i b i l i t y  generation therates  ofuno f apparent  >  29 synthesis ed  that  use  by  t r i t i a t e d  for  this  As this  yielded  purpose  was  various labelled  water  previously  study  the  is  (91,  determine  sis  in  white  and  to  determine  i f raising  has  an  effect  the  the  neonatal  that  the  adipose  intestine  cholesterol  levels  rats  in  14  raised  had  much  tissue  rate  period.  the  (220  l i t t e r s  lower  in  blood  substrate  I,  the  rate  cholesterol  and  intestine  pups  in  of  of  mg%)  the  seen  (2).  Rats  cholesterol  the  to  in  purpose  sized  synthesis is  the  high on  levels  rats l i t t e r s  during  p o s s i b i l i t y plasma  day  raised  of  synthe-  neonatal  different  interest,  source  3  of  cholesterol  +_ 1 3 of  reliable  Chapter  of  Also is  most  suggest-  92).  mentioned  to  on  the  substrates  7 in  (125  in  Wistar  l i t t e r s +_ 9  of  mg%)  (2) . The early were  of  postnatal looked  (82-84) adults more  rates  at  that and  period  in  separately  their  that  actively  cholesterol  may  proximal since  synthetic  the  site  change  synthesis  with  there  rates  which  is a  and  are  throughout distal have  change  intestine  been  different  synthesizing in  diet  the  reports in  the  cholesterol (82).  30 CHAPTER I I I METHODS  A.  Experimental Three  were  different  of  natal  period  the  dam  of  24  so  from  hours.  other  The  a  that  which l i t t e r  possible  gain.  significant raised to  1 on  elaborated  day  21. a  sometimes sizes  of  a  4  metabolic  i n food have  differences 3 to  6  size  than  avoided  14  the  the  of  post-  8 pups, of  having  that  intake  and  already  also,  12-13  i n i t i a l the  rejecting  may  them.  to  result  from  the r e s u l t i n g  demonstrated  raised  on  enough  to touch  established  of  chosen  f o r the  i n t h e dam  those  and  removing  8 pups  were  8 was  small  are produced  and  III-B.  how  during  nor  her with  and  pups/dam)  For the determination  differences  studies  of  t o examine  matter  results  14  i n Section  l i t t e r  more  simply leave  and  fluctuate  1  approach  between  that  rats  i n litters  of  93).  conditions  upon  used  (4, 8  exceeding^large  to  this  Other  ( 2 , 3,  were  t o day  i t was  weight  Living  described  difference  i n litters 18.  manner  delivered  l i t t e r  Using  significant  weight  14  always  the  pups  compare  day  synthesis  nearly  sizes  synthesis  i t i s neither  average, pups  from  l i t t e r  8 pups  cholesterol  rate  because a  i n a  containing  rates  MATERIAL  Design  established  Litters  AND  f o r the animals,  i n the following  section,  which were  w i l l  be  controlled  for  temperature,  and  the size  vided not of  ad  of  t h e number l i t t e r s .  libitum  number  of  hours  influence The tissues  augmented  t o produce of  light  rate was  of  measured  food was  by  to  light,  were  pro-  the pups  o n l y by  no  attempt  was  was  the  f o r controlling  a b i l i t  the  made  to  levels.  synthesis  i n vitro  water  limited  exposure,  activity  and  intake  Except  cholesterol  of exposure  pellet  and  milk.  the animals'  hours  Chow  t o t h e dams;  a r t i f i c i a l l y t h e dam  of  by  i n the various  culturing  an  accurately  3  weighed mining ed  by  portion  of  the specific a  solvent  These  The  day  rats  and  tissues  assayed  l i t t e r s  of  B.  at  and  cholesterol  procedure and  i n Sections the rats  i n l i t t e r s  of  date  and  culture  III-C  were  time  earlier  subsequently  the free  t h e same  an  and  i n the 8 were than  thin  layer  III-D. on  a  light bred  those  isolat-  conditions  and  k i l l e d  deter-  precycle.  and  their  reared i n  14.  Animals Male  exposed and  4  at  raised  of  techniques  a l l experiments  determined  I^O  extraction  described i n detail In  with  activity  l i p i d  chromatography. are  tissue  dark  and  to a  twelve  (0600  Several minimum  female  of  -  rats  three  Wistar hour  1800 were  cycle  hours) mated  l i t t e r s  rats  (Charles River,  of  i n a on  would  light room  (1800  born  0600  maintained  t h e same be  -  Canada)  on  day  so  at  that  t h e same  were  hours) 20°C. a  day.  A l l  l i t t e r s  dam  o n D a y 1.  (small  were  reduced  On  l i t t e r )  Day  to fourteen  following  manner:  the desired  other  of  and added  14 p u p s .  pups  were  another assay, order each  dam,  were  Rats  4 pups  a pool  chosen from  rearranged  u n t i l  the third  day because  shown  that  raised  after  birth  raised C.  Rats tation  midway  hours.  Only  the when  tissues  smaller  k i l l e d through  two r a t s were  the small  2  6 pups,  of  3  i n from  were  o f 14. sized  and M i l l e r  become  pups,  established  different  l i t t e r s  total  l i t t e r s  (93) have  from  s i x hours  heavier than  pups  l i t t e r s .  Preparation were  into  a  f o r each  S i x pups  the l i t t e r  one  t o 2 pups  which  t o assay.  from  four  required  from  t o make  t o make  with  or  one o f t h e  taken  were  four  i nthe  required  l i t t e r  i n each  Wurtman  do n o t n e c e s s a r i l y  i n larger  Tissue  i n  p e r dam  and added  were  o f 8 pups  f o r assay  pups  l i t t e r  s i xpups  with  not  were  to  p e r dam,  obtained from  f o r the l i t t e r s one  pups  o f pups  i n another  randomly  were  were  pups p e r  reduced  pups  t o 7 pups  Since  chosen  l i t t e r )  7 pups  obtained from  t o produce  were  F o r example,  two l i t t e r s  randomly  size  to twelve  l i t t e r )  t h e number  Similarly,  l i t t e r .  (medium  (large  Half  l i t t e r  l i t t e r s .  l i t t e r  3, t h e l i t t e r s  and eight  augmented  up  o r augmented  on  and Culture by c e r v i c a l the dark were  i c e no  and large  Conditions dislocation  cycle,  sacrificed longer than  l i t t e r s  were  starting  and  decapi-  a t 1200  a t one t i m e , ten minutes.  so  that  Also,  a n a l y z e d , one pup  from  each  if  "small  a  k i l l e d , pair.  a  the  "large  may  i c e .  arise  caecum  middle  was  due  three  distal.  cold  Krebs-Hensleit  with  Gluteal  d i s t a l  0 -C0 2  adipose  ice-cold  200  -  300  testine 20-30  mg  exceeded segment were  was  and  mg,  minutes tissue), shaking  (78) and was  on  t h e most  to  f a t and  Intestines  were  proximal,  discarded.  (pH  Prox-  with i c e -  7.40)  equilibrat  longitudinally. and  placed  i n the  the tissue, usually  the quantity sections  adipose  of  available  portion  portion  were  then  Krebs-Hensleit  glucose,  at  37°C  Precision  of  between  available, the i n -  and  proximal  distal  (CGA  duodenum  through  excised  t h e amount  Tissues  incubator  were  buffer.  2mm  When  5 mM  also  weighing  i n oxygenated plus  was  cut  next  the tissues  divisions:  section  be  i n results  wall.  buffer  t h e most  incubated.  length  and,  i n the  proximal  Bicarbonate  cut into  300  time  flushed  depending  pieces.  of  to  f i r s t  differences  were  accurately mg  k i l l e d  t h e most  middle  tissue  k i l l e d ;  i n the pair  the outer  equal  sections  t o be  the intra-abdominal  from  Krebs-Hensleit  After  was  avoided  (95%-5%)  2  f i r s t  pup  and  The  and  the pair  to the length  removed  imal  ed  was  l i t t e r "  excised  into  and  pup  i n t e s t i n e from  were  sectioned  formed  hopefully,  The  pancreas  size  l i t t e r "  This,  which on  l i t t e r  of  tissue  tissue  the  the d i s t a l  incubated buffer i n a  f o r  proximal segment fifteen  (1.0 ml/100  Dubnoff  Scientific)  into  at  mg  metabolic 120  oscillations/minute  prior  to the addition of  10  mCi  i n 50  u l  3  H a  water two  (Amersham,  hour  ice-bath  were  incubation  to stop  performed  incorporation 3  hours  Eight  Oakville,  to determine  whether  proximal  each  linear  300  mg  of  placed  of  over  and  the end  of  i n an  experiments 3  the rate  intestine  containing  were  Preliminary  c h o l e s t e r o l was  f o r liver,  (91, 94). At  the flasks  the reaction.  into  flasks,  period  Ont.)  H  water  a period  adipose  tissue  of  tissue.  were  incubat-  3 ed  with  H  At  0,  2,  and This  1,  water and  3  the reaction procedure  under  the conditions  hours  of  stopped  was  incubation  by  placing  performed  previously 2  flasks  the flasks  f o r each  described.  were  removed  i n an  icebath  of  the three  tissues  during  the three  hour  ment i o n e d . Cell culture method the  v i a b i l i t y  period (79).  adipose  Prior  were  et  suspended  pipette D.  of  was  incubated  and  small  (0.05%)  Corp.,  the method  of  exclusion c e l l  v i a b i l i t y  collagenase (2  mg  previously described  by  Scrapings  i n the trypan  with  Freehold,  from  blue  c e l l  v i a b i l i t y  by  gently  expelling  three  Analytical A  by  blue  to the determination  a l . (95).  ermination  the trypan  Biochemical  collagenase/ml) Krause  using  tissue  (Worthington  was d e t e r m i n e d  the  intestinal  solution  drawing  N.J.)  wall  f o r the  them  into  deta  times.  Procedures  aliquot of  c u l t u r e m e d i a was  taken  to  determine  its  specific  radioactivity  s c i n t i l l a t i o n 111.) were  s c i n t i l l a t i o n transferred  (Beckman The  counter  Model  pellet  by c o u n t i n g i n a Beckman  using  fluid.  to glass  ACS  Contents screw  J B 6 ) a t 3000  was  washed  (Amersham,  with  Arlington  of the culture  cap v i a l s  rpm  and  Heights flasks  centrifuged  f o r 5 minutes  saline,  LS9000  and  centrifuged  decanted.  and  decanted  3  three the  times  tissue  methanol using  was  homogenized  (both from  ( 9 6 ) . The  cleaning Whatman  H water.  i n 8.0  Fisher  No.  rinsed  Filtered  Organomation  by adding which  (Evapo-Mix,  shaking  The  ether  free  i n a  flush  cholesterol  mixing  heated  extracted  Ma.)  was  perand  b a r was  a t 70°C  with  8.0  shaken  and shaker  Lee, N.J.)( 9 1 ) .  S c i . Co., N.J.) t h r e e  horizontally  were  ( M / e r N-  i n a metabolic Fort  flute  40°C  to the residue,  glass  was  Instruments,  (Fisher  placed  a  solution  Inc., Northborough,  small  solution  Filters  a nitrogen  o f 5 N NaOH  m i x t u r e s were  the tubes  were  f o r three hours  Buchler  Saponified  t o produce  contained a  (96).  under  Association  2 . 0 ml  vigorously  petroleum  solutions  and evaporated  Saponification  cap v i a l s .  through  ml of the chloroform-methanol  2.0  Evap,  shaken  f i l t e r e d  from  with  bath  vigorously  solution,  into  water  tube  per  1 f i l t e r  times.  formed  was  screw  washing,  (2:1) solution  (50 s t r o k e s  and r i n s e  shaft,  After  ml of a chloroform-  Sc. Co., N.J.)  homogenate  the homogenizer  three  the  the excess  a P o t t e r - E l v e j h e m homogenizer  sample)  then  t o remove  ml of  times  a t 100 o s c i l l a t i o n s  by per  minute  f o r 30  fuging  (Beckman,  organic test E.  minutes.  phase  tubes Thin The  under  were  20  also  run  along  were  developed i n a  with  acetic  to visualize  containing  zole  (PPO)  toluene  bands  x  100  mm  ml  before  volume  N.J.)  of  and  plate.  A  Inc.,  cholesterol  S c i . Co.,  N.J.)  Chromatography  and,  diethyl  after  Co.,  was  plates  petroleum ether:  Instr.  (Amersham internal determine  counter.  Oakville, standard the  (26,  Ont.)  when  drying,  Ann  efficiency  of  were  Arbor.,  (97).  cut of  out a  Vials  under and  0.8%  a  short  placed  2,  were  counting  27)-  (40,000  the  with-  Westbury,  s c i n t i l l a  vigorously  Beckman  LS9000  tissue l i p i d  C-cholesterol DPM) was  was  V.  5,-diphenyloxa-  shaken  i n a  i n  U.  14 s c i n t i l l a t i o n  The  ( P o l y g r a m S i l G,  visualized  were  small  Instrument,  Fisher  of  solution.  hour  plate  (Gelman  5.5  i n a  (84:15:1)  were  bands  vials  f o r one  B  the  patterns The  G  samples.  acid  Rhodamine  tion  left  f o r 5 minutes.  S c i . Co.,  per  system  Mich.)  and  s i l i c a  the  with  lamp.  (Fisher  samples  sprayed  source  rpm  centri-  flush.  148.5-149.5°C,  glacial  2000  by  e v a p o r a t e d i n 13  Brinkmann  cm,., 5  (mp.  Banding  a  mm,  standard  ether:  separated  redissolved  ether  onto  0.25  20- x  at  were  Chromatography  chromatographed  N.Y.)  nitrogen  diethyl  gypsum,  JB6)  d e c a n t e d and  residues  anhydrous  out  Model  was  Layer  Phases  added  as  homogenized  extraction  (79).  to  an  37  F.  Calculations The  calculation * f o r the  incorporation  3  of  H  water  3  into is  cholesterol  p e r f o r m e d as A.  (expressed follows  as  nmoles  H/g  of  tissue/h)  (98):  Determination  of  the s p e c i f i c  a c t i v i t y  of  3  ^ 0  i n the  aliquot to  of  culture the media  the media.  expressed  media  From  was  after  these  done  by  counting  approximately  counts  10  a  mCi  the s p e c i f i c  small was  added  activity  was  as: DPM/nmole  H 0 2  3  B.  The  the  newly  ing  the  synthesized  DPM  specific  calculation  value,  activity  Example  n m o l e s 'of  cholesterol  f o r t h e de 3  of  DPM  of  was  novo  H  incorporated  performed by  cholesterol,  into  d i v i d -  by  the  H^O.  , -rchol. n  DPM/nmole  =  3  nmoles  H  -9 Molecular  weight  Volume  culture  of  Nanomoles  of  H 0  of  H^O  medium : 2.0  2  =  g  18.0 =  g/nmole  2.0 of  ml  H 0 2  = =  =  18.0  2.0  g  2.0  g/18.0  9 in  2 ml  of  culture  =  AS rp be ic it fr ia cr y A tc ot ti av li t DyP Mo f c t ou o ir u 2m. 0 hn e t M fe d =  2.22  x  10  1  0  DPM  0.11 ml  of  x  10  medium  x  10  g/nmole  x  10 g/ nmole _  9  nmoles  38 Specific  a c t i v i t y  =  Total  =  2.22  =  200  DPM-j- n m o l e s x  10  10  H^O  . D P M -r- 0 . 1 1  DPM/nmole  x  10  9  nmoles  H^O 3  Determination For  a  sample  Tissue  of t h e Rate  Incorporation  of  H  where:  Weight  Culture  of  time  =300 =  Arbitrary  DPM  Nanomoles  of  2.0 of  mg  ( 0 . 3 g)  h  isolated  cholesterol  =  3000  DPM  3 DPM e.g.  H  incorporated  . — ~ sample D P M -H  3000  Specific 200  into  isolated  a c t i v i t y  DPM/nmole  =  „ H^O n  15.0  cholesterol: 3 = nmoles H  nmoles  3  H  3 Rate  of  Incorporation  tissue/h)  e.g. Using  (expressed t h e above  as  nmoles  tissue  H/g  weight,  of culture,  3 period into 15.0  and c a l c u l a t e d  values  f o r nmoles  H  incorporated  of significance  f o r differences  i n the rates  synthesis,  weight  cholesterol. nmoles Rate  3  H-^-0.30  of  g 4 - 2.0  h  =  Incorporation 3  =  25.0  G.  nmoles  S t a t i s t i c a l Tests  cholesterol amongst  within  pooled  proximal each  body  l i t t e r  of variance  single  between  Analyses  the various  analysis for  H/g/h  sizes  method  samples  was  and d i s t a l  l i t t e r  size  due  and adipose  were  (100).  performed  to Scheffe  performed  intestine These  tissue  on  using  ( 9 9 ) .A t  t o compare  are  weight an test  differences  individual  analyses  of  days  presented  39  in  Appendices Testing  of  variance  factor tests  E-H. f o r trends  orthogonal  or cubic",  the  rates  of cholesterol  for  these  analyses  (101).  In points present  These  factor  of the r e l a t i o n , between  synthesis. determined  analyses  presented  t h e mean  the standard  by u s i n g  contrast to the  the time  existing  were  t h e graphs  represent  with  the form  quadratic  squares  accomplished  polynomial  and i n t e r a c t i o n s determine  was  error  the time  using  of  i n the results  values  linear,  factor  and  sums o f  i n Appendix section  and t h e e r r o r  o f t h e mean.  These  significance  unique  are presented  time  (101).  i.e.,  Tests  analysis  the  bars r e -  I.  40  CHAPTER  IV  RESULTS  A.  Preliminary 1.  Cell  cells' 3  the  V i a b i l i t y  a b i l i t y  for  gluteal  end  of  a for  with  of  Similarly, the  (Fig.  4)  the  adipose 2.  of  tissue  hour  the  nearly  i s  shown  decreased  at  from  occurring during cells  end  of  linear  intestine  three  over and  Extraction Efficiency  the  At c e l l  to  91%  f i n a l  trypan  three  Sterol hour  the  Figure  hour. blue  v i a b i l i t y  hours.  the  37°C, 95%  excluded and  in  intestine.  period  culture period,  the  i n d i c a t e d by  proximal  incubation  tissue  as  blue,  and  intestinal  at  tissue,  v i a b i l i t y , trypan  decrease  of  89%  was  exclude  adipose  98%  to  Cell  adipose  beginning  creased  to  three  v i a b i l i t y most  Studies  at  desynthesis  period  for  liver. The  a d d i t i o n of  (26,  27)-  14 C-cholesterol indicated the  that  method  to  the  homogenate  as  an  internal  c h o l e s t e r o l e x t r a c t i o n from  described  above  was,  on  the  average,  standard  tissue  85+^  by  3%  ef f i c i e n t . B.  Body  Weight  Raising sulted (Fig.  in 5)  l i t t e r s  a by  and  Differences  the  pups  in  small  but  day  between  7  those  the  different  significant  reared  the in  pups  the  l i t t e r  sizes  difference in reared  medium  and  in  body  reweight  small  large  l i t t e r s  41  100  90 w H H  C D  O C D r H  t  80  o  u O  Gluteal  *  Proximal  Adipose  Tissue  Intestine  70  6o  0 Figure  1.0 3.  •Tissues  Percent Gluteal were  from  2.0  3.0  Time (h) Cell V i a b i l i t y f o r Proximal Adipose Tissue. 21  day  o l d rats  raised  Intestine  i n l i t t e r s  of  and eight  42  1-0  ' Time  Figure * **  4.  In V i t r o I n c o r p o r a t i o n Cholesterol Over Three  Liver  tissue  was  from  adult  3-0  2.0 (h)  of T r i t i a t e d Water Hours. rats  aged  43  and  days.  P r o x i m a l i n t e s t i n e and gluteal adipose tissue were f r o m 21 d a y old r a t s . Each point represents the mean ± S.E.M. o f 6 a s s a y s .  60  •  k Pups/Litter  ^  8 Pups/Litter  43  • l ^ Pups/Litter 50  fclD  30 xi •H CD  >5  O  PQ  20  10  0  T  I  0  1^ Age  Figure  a,b,c,  5.  Body Weights Sizes.  of  Rats  21  (d)  Raised  i n Different  L i t t e r  Means a t t h e same a g e n o t s h a r i n g a common superscript are s i g n i f i c a n t l y different (p < 0.05).  (Appendix  H).  difference those  between  large  differences  number  of  those per  Cholesterol  corresponded the ed  at  free was were  fatty  acid  necessary 1.  to  of  pattern  of  intestine proximal  of  small  much  large  and  larger  differ-  sizes  and  the  s i g n i f i c a n t  sizes  and  the  greater  the body  dif-  the weight.  into free  the  by  TLC  measuring  band  cholesterol  band.  A  by  free  small  amount  of  when  i c e (Table  I ) .  This  background  i t i n any  of  that the  i t was  cultures level,  not  calculations.  Synthesis  Litter  i s shown  magnitude  the  label  even  low  remain-  cholesterol,  band  such  Using  the phospholipids  i n order  the  that  cholesterol  on  of  determined  described,  cholesterol  third  s i g n i f i c a n t  l i t t e r  lower  glycerol.  include  Medium  a  were  was  followed  I n t e s t i n a l a.  as two  the  standard  and  i n the  was  pups  i . e . ,t h e  tritium  system  origin,  however,  of  to the  incubated  small  l i t t e r  expected,  synthesis  the  found  three  l i t t e r ,  rate  developing  there  a  Synthesis  Cholesterol incorporation  well  former  21  the  was  between as  the  day  amongst  pups  there  weight  of  By  were  14  litters,  pups  l i t t e r s .  ferences  day  i n body  o f medium  ence  C.  On  (8 p u p s / l i t t e r ) . synthesis  i n Figure the  6.  i n both Sterol  i n t e s t i n e was  high  The  developmental  sections synthesis at birth,  of  the  i n the and  on  45  TABLE  Baseline into  Values  I  f o r the Incorporation  Cholesterol  i n Various  Tissues  of  3  H  Water  Cultured  on I c e .  H  Incorporation into Cholesterol ( n m o l e s H/g/3.0 h)  Tissue*  0 ..40  + 0 .0 1 * *  Intestine  0 ..40  + 0. 0 1  Tissue  0 ..20  + 0 .02  Proximal Distal Adipose  Intestine  1. . 1 0 + 0 . 1 3  Liver  * **  Tissues Values  are from are  r a t s aged  e x p r e s s e d as  21  days.  t h e mean  ± SEM  of  6 assays.  6 0 Distal Intestine  60  • Proximal I n t e s t i n e  o  O Adipose Tissue  CD -P W CD  /  50  O  o o -p C  •H ^ c  O •P  03  ^  i-l-0  —  30  -  a  /  \  J-l O ft  to 0  o o  S  o  k \ //'  CD -P  20  10  0  J  14  7 Age Figure  6.  21  (d) 3  (8/Litter) Cholesterol  Incorporation i n Various  of  H Water  Tissues  from  into Neonatal  Rats . a,  b  Means script  a t t h e same letter  (p < 0.05 ) .  age  not sharing  are s i g n i f i c a n t l y  a  common  different  super-  46  day  7  was  the  synthetic  further 21  the  below  to  50%  of  rate  less  in  than  synthetic  the  value  proximal 20%  rate  of  had  on  day  1.  On  i n t e s t i n e had  the  value  risen  to  of  80%  14,  declined  day  of  day  1.  the  By  day  day 1  values. Synthetic vated  and  dropped  not  to  rates  levels  week,  increased  those  found  the  rate  significant gions  of  to in  once  distal  day  values from  nearly  during  two  fold  the  i n t e s t i n e on  medium  in  on  days  Groups  (4  and  l i t t e r , was  elevated  were 14  in  on  day  increase,  three  of the  not  was  day  Pups  small  1  ele-  and  7,  third  higher  than  E),  reduced  7,  both  reared  significantly  21.  for  in  in  low  these  proximal  in  pups  on  day  large  l i t t e r s  they  synthetic  rate,  fold  and  five  on  day  had  intestine  in  proximal  The  14  and  d i f f e r e n t from  both  re-  proximal  that  7.  fold  No  14.  i.e., synthesis in  twice  pups/litter)  day  in  and  at  synthesis  l i t t e r s  d i s t a l  and  and  14  on  21  7  rates  sterol  high  7).  (Appendix  l i t t e r s  intestine  on  (Fig.  those  synthetic  seen  l i t t e r s  rates  day  1  the  days  from  large  and,  proximal  again  on  14  days  cholesterol  differences  7 which  d i f f e r e n t on  intestine synthesized  small  synthetic  i n t e s t i n e were  birth.  intestine  differed  distal  day  level  Experimental  Similar  and  a  by  i n t e s t i n e were  b.  raised  to  at  of  the  significantly  low  Distal  in  21  for  the  low  the  rats  and  however,  on  distal did  distal  48  b  0  Distal  %  Proximal  Intestine Intestine  1*  i 0  I 7  I  I 21  lk Age  (d)  3 (4/Litter) Incorporation of H Water into Cholesterol i n D i s t a l and Proximal Intestine of N e o n a t a l Rats Means a t t h e same a g e n o t s h a r i n g a common script l e t t e r are s i g n i f i c a n t l y different ( p < 0.05) . Points  at day  1 are from  t h e medium  l i t t e r .  super-  49»  and  proximal i n t e s t i n e , r e s p e c t i v e l y The  in  differences  l i t t e r s  of  intestine distal  4  or  between  14  pups  and i n . F i g u r e  synthetic  a r e shown  10  of  higher  i n animals  than  raised  i n large  time  the synthetic  Whereas intestine rats the  raised  large  day  l i t t e r  proximal  No  on  l i t t e r s was  those  on  day  on  day  from  14.  the  at nearly  7 only,  i n  large  distal  day  cholesterol  were  large  l i t t e r  were  rats  reared  equal  occurred  the rate  7,  i n the  .of r a t s  rates  between  l i t t e r s ,  change  fold  higher.  different  A  three  at  fold  i n t e s t i n e . On  Synthetic  Irt " t h e  l i t t e r s  three  a t 1 . 5.:f o l d ' t h e r a t e  d i s t a l  were  synthesis  from  synthesized  f o r  i n small  nearly  of proximal  intestine synthesized  i n t e s t i n e , on day  other  synthesis within  raised  raised  so  i n  f o r  that  on  synthesizing  of  rats  raised  l i t t e r s .  Distal  and,  sizes  9  intestine.  significantly  and  ( F i g .10).  the rats  small  not  l i t t e r s  intestine  l i t t e r s  21,  true  i n Figure  cholesterol  l i t t e r s  not  i n small  of  21 was  small  cholesterol -in  day  was  proximal  both  on  from same  the rate  rate  i n rats  s t e r o l synthesis  significantly  which  rates  f o r proximal  intestine, the rates  i n those  (Fig.8).  21,  i n both  day  7,  i n rats  t h e medium  s i g n i f i c a n t differences were  found  the three  between  l i t t e r  more  sizes  cholesterol from  and  t h e medium  the  small  i n the rates  proximal except  and on  than  of  distal  day  21  l i t t e r  l i t t e r s . cholesterol intestine  i n rats  from  0  Distal  #  Proximal  Intestine Intestine  Age  (d) 3  (14/Litter) Cholesterol of Neonatal  Incorporation of H Water into i n D i s t a l and Proximal Intestine R a t s R a i s e d i n L i t t e r s o f 14  Means a t t h e same a g e n o t s h a r i n g a common script letter are s i g n i f i c a n t l y different (p < 0.05) Points  at day  1  are from  t h e medium  l i t t e r .  super-  51  k lk  Pups/Litter Pups/Litter  T  Age  (d) 3  (Distal Intestine) Comparison of the Rates of H Water Incorporation into Cholesterol i n Proximal Intestine Between Rats Raised i n L i t t e r s of 4 and 14 Means a t t h e same a g e n o t s h a r i n g a common script letter are s i g n i f i c a n t l y different (P < 0.05) Point  at day  1  i s from  t h e medium  l i t t e r .  super-  k lk  Pups/Litter Pups/Litter  i  l  I  7  0  14 Age  (d)  3 (Proximal Intestine) Comparison of the Rates of H Water Incorporation into Cholesterol i n Proximal Intestine Between Rats Raised i n L i t t e r s of 4 and 14 Means a t t h e same a g e n o t s h a r i n g a common script l e t t e r are s i g n i f i c a n t l y different (p < 0.05) . Point  at day  1  i s from  t h e medium  l i t t e r .  super-  the  large  found  to  (Table  l i t t e r .  synthesize  Trend  cholesterol  a  fold  proximal  the  quadratic  during  described  by  significant In  I)  rate  of  intestine distal  dominant  a  among  same  period.  indicated  was  intestine  the  There  intestine,  the  analysis  curve  quadratic  was  present.  Tissue  and  but  U-shaped were,  contour  of  Cholesterol  has A  the for  con  proxor  best  however,  the  U-shaped  indicated  also  in  in  post  which  variance  synthesis  the  was  of  of  the  means.  confirmed  was  rates  curve  population  and  for  the  during  U-shaped  curve  function.  f o r 22)  Analysis  that  development  (2,  in  function  Adipose  liver  anticipated  differences  S-shaped  rat  reported  indicated  trend  quadratic  d i s t a l  the  was  the  (Appendix  Findings  period  i n t e s t i n e the  2.  1.5  in  relationship  intestine  or  instance,  Analysis  development  similar  imal  at  synthesis  pronounced  trast  this  I I ) . c.  natal  In  that  a  curves.  that  the  cubic  Synthesis  function  Values  for  3 the in  incorporation l i t t e r s  and  of  constant  seen  in  gluteal  4  intestine adipose  t h e s e two  large  l i t t e r s  in  small  large  and  level  tween  tissue,  of  on  8  on  day  only 14  l i t t e r s . did,  and  cholesterol,  per  ( i . e . 6-9  tissue  had  into  pups  l i t t e r  days  l i t t e r s  H  fold  14)  was  51% 21  Adipose  remained less  (Fig.  not  sizes  dam,  6  than  and  40%  however,  a  11).  an  rats  low values  amount  different  weight  of  very  The  of  the  raised  lowest  Rats  from  exhibit  at  I I I ) .  respectively, tissue  rats  the  significantly  (Table and  for  the  rats  raised  increase  be-  raised of  in  in  of  in adipose raised the  the  TABLE  I I  3  In  Vitro  Proximal  Incorporation and  D i s t a l  of  H Water  Intestine  Age (d)  Tissue  1  Prox.  7  2 4 .8  ±  1 .5  b  Prox.  14  1 0 .3  +  0 .9  b  Prox.  21  2 0 .0  +  2.3  b  Dist.  7  Dist.  14  Dist.  Adip.  **  Adip.  **  a, b , c  Synthesis*  Pups/Litter 45. 0  ± 2. 8  22 . 0  ± 5 .3  8. 4  ± 0 .8  14  Pups/Litter  b  16 . 3 +  2 .8b  b  10 . 7 +  0.' 9  a  48 . 6 +  8 .o a  11 . 5 +  1. 9  a  +  1. 6  b  35 . 6  ± 6. l  36. 8  ± 4. 1 ± 6  .ob  b  44 . 0  1 1 .8  ±  1 .6  b  7 .2  ± 0 .7  b  11  21  3 6 .0  +  2 .7  b  65 . 0  ± 9 .6  a  31 . 7 +  7  1 .0 ( 0 . 26  + +  1. 1 ± 0 l 0. l 0 . 0 2 ° )* * * ( 0 . 3 4 + 0 . 2 )  0 .8 ( 0 . 17  + +  0. l 0 .01°)  1 . 2. ± 0 2 ( 0 . 21 + 0.02  0 . l 0 .0 1  1. i . ± 0 . 2 (0. 21 0.02 )  b  b  b  0 .9 + ( 0.1 6 +  b  are expressed  Prox.  =  proximal  Dist.  =  distal  Adip.  =  gluteal  Values  i n each  body  C  )  as  b  nmoles  H/g/h  SEM  ±  0. . i ' 0 .o i a )  1 .8 ( 0 . 28  + +  0 .l 0 .0 2 )  2 .8 ( 0 . 30  +  0 .5 0 .0 3 )  b  SEM  different  superscript  are  ( p < : 0.05) as  a  a  a  a  tissue  t h e same  3 .o b  +  intestine adipose  b  i .5 (0. 21  intestine  i n parentheses are expressed  wt/d ±  )  b  row w i t h  significantly  Values  b  b  Values  not ***  8  Tissue  4 .2  21  *  Adipose  by-  32 . 6 ±  14  Adip.  and G l u t e a l  Pups/Litter  Prox.**  1  Cholesterol  Cholesterol 4  Dist.**  into  nmoles/g  I  0  i 14  I  7 (Age  Figure  11.  1  21  (d)  3 (Adipose Tissue) Comparison o f t h e Rates of H Water I n c o r p o r a t i o n into C h o l e s t e r o l i n White Adipose Tissue Between Rats Raised i n L i t t e r s o f 4 a n d 14 Means a t t h e same a g e n o t s h a r i n g t h e same script letter are s i g n i f i c a n t l y different (p < 0.05).  super-  56  TABLE I I I Weight of G l u t e a l Adipose from Three L i t t e r Sizes  Age (d)  Weight  of Gluteal Number  of  Tissue  Adipose  of  Tissue*  14  7  192.0  ±  7.6  b  187.5  ±  14  267.0  +  8.2  b  296.3  ± 13.6  412.3  ±  8.3  b  402.5  ±  * a,b  Each  value  Values  i s t h e mean  i n each  significantly  row w i t h different  +_ S E M  (mg)  Pups/Litter 8  4  21  Rats  of 6  t h e same  8.0  9.2  86.2  b  b  b  ± 8.4  137.0  ± 12 . 3  a  164.0  ± 16.  a  pups superscript  (p<0.05).  a  are not  0  synthetic the of  rats  rate  synthesis  fold  sterol  increased  progressively 2.3  of  in  between  on age  the  increased  greater;  and  day  each the  small (Day 21,  day  tested  differences and  7, 2.9  1.5  large fold fold  (Fig. in  the  11).  rates  l i t t e r s  also  greater;  day  greater).  As  14,  58  CHAPTER  V  DISCUSSION  Introduction The of and  purpose  cholesterol adipose  different either  raised  A.  a  pattern  state  been  much  a  that  less  probably  raised  with  4  and  t h e amount  a  14  of  pups  the pups  l i t t e r  per  sizes  food  of  i s not  4,  as  a  a  to  the pups  dam,  not  raised  effort  were  procedure  (2, 101,  on  but  s i g n i f i c a n t positive  of  day  7,  4  and  i n body  available i n the  102).  14  so  or  large  obvious  weight and  difference  by  l i t t e r s  the  further, i n favour  This  absorbed  A  i n the  between  pups  weights.  undernutrition.  i s indicated  i n body  t h e medium  14.  were  I n an  studies  raised  state  however, of  whether or  under-eating,  i n l i t t e r  difference  day  when  s i g n i f i c a n t difference  i n l i t t e r s  on  or  pattern  Undernutrition  and  l i t t e r  and  the  development i n i n t e s t i n e  to ascertain  i n several  constitutes  significant  to determine  undernourished.  f o r t h e pups  overnutrition,  as  emerge  over-  t h e pups  produced  was  during  well  would  used  Overnutrition  indicates is  as  of  i n l i t t e r s has  study  synthesis  tissue  Raising dam  this  overnourished or  produce  which  of  state  the  of  of  rats  lack  by  and  of  a  small  the the  small medium  Hahn weight  Walker  difference  However, by  and  day  a  21  state in  for of  the  (2) rats  rats  differences  rats  raised  in  rates 14  could  for  the  growth  rate  small  l i t t e r s  it  l i k e l y  is  for  normal  not  fully  14  were  of  4  raised  access  that  not  they  is  be  as  day  probably to day  the that  for  of  day the  Rats  from  milk  and  amount  volume  growth  after  l i t t e r .  the  required  intake  are  postpartum  raised  in  raised  overnourished  fact  maximal  quantity  pups  8.  between  but  week  rats  The  limiting  for  and  indicates  14,  larger  third  3  in  evident  support  beyond  the  14  trend  l i t t e r s  in  u n t i l  (105). of  l i t t e r s  day  21.  Procedure  conditions  experiments  on  greater  that  while  to  4.  controls  apparent  appear  of  became  the  of  l i t t e r s  4  consumed  before  l i t t e r s be  the  a  same  to  u n t i l  in  to  because  Methodological Culture  the  produced  the  seems  milk  milk  undernourished  did  and  l i t t e r  had  i t  8  size  developed  summary,  B.  enough  rats  in  small  produce  growth  In  were of  of  of  in  l i t t e r s  either  amount  raised  raised  weight  dams  observed  overnutrition  that  the  have  were  indicated  adequate by  the  to  continue  linear  with  incorporation  3 rate (Fig.  of 4),  adipose after  H  water  combined  cells  two  intestinal  into  and  hours c e l l  of  cholesterol  with 92%  of  results  was  which  intestinal  incubation  death  over  (Fig.  probably  the  3  showed  cells 3).  hour 95%  period of  remained Most  experienced  of by  the viable  the the  60  v i l l i  cells  which,  cholesterol,  are  Andersen water  i s the  and  although  less  they  robust  Dietschy  substrate  than  (91)  best  produce the  have  suited  crypt shown  i n most  digitonin  f o r p r e c i p i t a t i n g the s t e r o l s ,  of  having  tissues.  inordinately  fold  that  high  Their  more  cells  (79). tritiated  for studying  synthesis  problem  mammalian  1.5  sterol  method  which  uses  presents  background  a  incorpora-  3 txon on  of  H  water  ice s t i l l  into  sterols.  apparently  Tissues  incorporate  which  are  substantial  cultured  quanti-  3 ties  of  H  pitated is  water  sterol  regenerated  background  when  assayed  method by  counts  (91).  using  no  digitonin-preci-  I f , however,  c l e a v i n g i t from are  the  longer  the  the  free  digitonin  detectable  sterol  these  (91).  3 Apparently, itonin  and  H  water  i s the  source  counts.  The  method  entirely  the  problem  the  of  used of  nonspecifically  the  running  cholesterol  i t in a from  i s tedious  probably  no  precipitated  and  acid of  on  the  of  background  a  silica  G the  plate  free  phospholipids silica  onto  the  time-consuming,  this  method  method.  TLC  and  lipids  the  from  chroma-  separates  than  dig-  avoids  sterol  simply  triglycerides  the  to  study  free  by  which  more- t i m e . - c o n s u m i n g sterol  level  present  sterol  system  fatty  high  regenerating  saponified lipids  Although chromatography plate  the  i n the  digitonin-precipitated  tographing and  binds  is  digitonin-  ,61 C.  I n t e s t i n a l Synthesis Several  d i s t a l  for  82, pups  weaned  a  i n man 84). raised  was  thesis Rates were  of  of not  fold  t o an  on  Prior  within  regions  t h e medium Changes  of  l i t t e r .  sucrose  distal rates  a  sterols  on  of  these day  rats  findings  21, i . e . ,  the  distal  i n the rate  over  there  the three  cholesterol intestine. of  the d i s t a l  i n the d i s t a l  21  proximal  the proximal i n -  d i f f e r e n t between t o day  large  and  no s i g n i f i c a n t  l i t t e r on  intestine.  intestine  the  were  syn-  day  sizes, 7  between  f o r pups  t  Feeding and  cholesterol  been  by  dietary  a p u r i f i e d diet  of  casein,  rate  to rats, of  f e d on  a  than  Sugano  synthesis  i n the proximal  i n the d i s t a l  synthesis  produced  vitamins  higher  i n t e s t i n e . Rats higher  21,  than  synthesized  than  intestine  previously  mineral  demonstrated  21,  increase  day  8  adult,  ( 7 3 ) , and  confirms  l i t t e r  greater  i n the  cholesterol  and  i n d i s t r i b u t i o n of  (82).  precipitable  4 day  large  each  modification  (82)  that  i n t e s t i n e , except  i n t e s t i n e have  mix,  of on  actual  significantly  l i t t e r s .  two  the  the  synthesis,  differences,  in  of  1.5  due  more  study  i n l i t t e r s  i n the proximal  small  the  present  Conversely,  of pups  rate  This  reported  ( 7 8 ) , s q u i r r e l monkeys  The  pups.  testine at  have  Cholesterol  intestine synthesizes  intestine (75,  studies  of  diet  the proximal  et a l .  of d i g i t o n i n -  intestine  r a t chow  along  than had  the synthetic  intestine.  62 Apparently  food  by  types  t h e two  equal,  then  sponsible the  intake  one  of diet. o r more  diet  could  spect  to absorption The  be  the three  between  groups  differences  small.  Compared w i t h  of  synthesis  cause  in  combined .of  age  with  rates  than  l i t t e r might  the synthetic  be  rates  these  expected,  On  on  t o be.  day  7  intes-  differences  same The  two lack  of  i s not  i n body  weight  l i t t e r ,  the  day  rates  but- n o t  7.  A  lack  of  of non-cholesterol i n -  synthesis  appear  appear  i n the d i s t a l  l i t t e r  on  activity.  differences  groups  of the small  intestine  the there  the difference  lower  with r e -  and the  two  of synthesis  does  cells  different.  the effects  cholesterol  proximal  i n proximal  l i t t e r s  these  since  7 and 21.  between  t o be  be r e -  metabolic  of synthesis  those  i s assumed  the  where  days  - i n the large  In the distal  t h e medium  with  namely  are significantly  initestine  magnitude  t h e days  between  at this  of the lower  l i t t e r .  on  altered  could  outlines  are not s i g n i f i c a n t l y  is  hibitors  general  intestine  since  substrate  the d i s t a l  and small  unexpected  proximal  than  follows  not  Alternatively,  digestion  i n the rates  i n d i s t a l  sizes  significant  which  intake  less  and p o s s i b l y  t h e medium  the rates  l i t t e r  active  were  components  observed.  i n the data,  differences  tine in  more  discussion  discrepancies the  dietary  requires  cells  gain  I f caloric  f o r the changes  purified  amongst  and weight  could seen  .be  the  i n the  large  d a y 21 t h e s y n t h e t i c  t o be  o f a much  particularly  i n the small  l i t t e r .  greater  when One  rate  compared  might  presume medium small  The  would  and large sizes,  l i t t e r s .  the diet  somewhere This  conditions  be d e s c r i b e d reason,  unusual  results  Cholesterol testine  l i t t e r  (35.6 ±  distal  follows  i n quantity study  levels with  the only.  cannot,  how-  conditions.  the varied  con-  the seemingly  on d a y 21 i n t h e p r o x i m a l i n -  l i t t e r  than  6.1  i f , i na l l  contained  to relate  of the  on day 21.  synthesis  more  the case  as t h e aforementioned  and maturity  i n the small  s l i g h t l y  i nthe  the values  by t h e pups  and v a r i e d  as simply  seen  be  of the present  an attempt  patterns  of synthesis  between  might  consumed  experimental  sumption  the  range  of nutrients  this  only  f o rthe rates  proportion  ever, For  the values  l i t t e r  l i t t e r same  that  ( 2 0 . 0 ± 2.3  half  nmoles  3  the rate  H/g/h)  3  nmoles  of that  H/g/h) i s  i n t h e medium  and, s i m i l a r l y ,  intestine i n the small  l i t t e r  (36.0 ±  the rate i n 2.7  nmoles  3 H/g/h)  l i t t e r  i s only  a  l i t t l e  (65.0 ±9.6  nmoles  in  the small  l i t t e r  of  a  quantity  greater  more 3  than  half  H/g/h).  The  are probably of milk  which  ( 2 2 ) . Enhanced  in  between  groups  a n d may  commences rats to  amounts  contains  and t h e i r  mother  of inhibitors  amounts  and hence  contained  consume  i n milk.  of  synthesis  that  of Purina  rates  consumption  inhibitors  and 21 i n a l l  attributed to the fact  increasing  synthetic  cholesterol  14  i n t h e medium  to the  o n d a y 15 p o s t n a t a l l y a n d a f t e r  consume  them  be  days  lower  related  cholesterogenesis the gut occurs  of that  three  weaning  that  day  infant  Chow  available  decreasing  64 If  the decreased  responsible 14  f o r the rise  and 21 one w o u l d  higher day  rates  21.  consumption  than  This,  i n synthetic  expect  both  that  i s the only  rates  the large  t h e medium  however,  of milk  between  l i t t e r  and s m a l l  i s not the case  days  would  l i t t e r s  and  factor  have  on  i n fact the  3 upper  value  attained  (48.6 ±  8.0  i n the larger  nmoles l i t t e r  H/h/g, i s less  proximal than  intestine)  the upper  value  3 (65.0  ±  9.6  nmoles  H/g/h,  the  medium  l i t t e r .  Also,  the  medium  (35.6 ±  and  large  are  not s i g n i f i c a n t l y  d i s t a l t h e two  intestine) attained i n lower  values  attained i n  3 6.1  nmoles  H/g/h,  proximal  intestine)  3 31.7  ±  3.0  nmoles  H/g/h,  different.  distal  Compared  intestine) with  the  l i t t e r s  small  3 l i t t e r in  (20.2 ±  2.3  the proximal  nmoles  H/g/h)  the rates  intestine are greater  of  synthesis  i n the large  l i t t e r  3 (48.6 in  ±  8.0  nmoles  the d i s t a l  H/g/h;  however,  the rates  of  i n t e s t i n e are not s i g n i f i c a n t l y  synthesis  different  3 between and  these  31.7 ±  two  3.0  groups  nmoles  3  (36.0 ±  H/g/h,14  rates  of cholesterol synthesis  large  l i t t e r  solely of  on  the basis  sterol  synthesis  Pups and  are lower  this  from  nmoles  l i t t e r ) .  H/g/h  4 / l i t t e r  C l e a r l y then  the  i n the intestine i n the  rates  that  might  of the e f f e c t s produced  be by  predicted the  removal  inhibitors.  the large  condition  incapacitating  than  2.7  may  l i t t e r  have  were  undernourished  had the e f f e c t  the intestine i n i t s a b i l i t y  of to  p a r t i a l l y synthesize  65 cholesterol;  so t h a t  of  i n distal  synthesis  (11.8  ±  1.6  pronounced  t o 31.7 as  that  although  ±  there  intestine 3.0  was  a rise  between 3  nmoles  o f t h e medium  day  H/g/h)  i n the  14  and  t r er i s e  l i t t e r  (7.2 ±  21  was  0.7  rate  not  as  to  65.0  i n cholesterol  syn-  3 ±  9.6  nmoles  thesis seen  H/g/h).  The  i n the proximal  between  days  14  marked  intestine  and  21  rise  of the undernourished  (10.7 ±  0.9  t o 48.6  ±  pups  8.0  nmoles  3 H/g/h) may could  be  Also,  this  due  an  due  size  to a  attempt marked  to a release  thesis  as  be  caused  was  from  by milk  demonstrated A  logical  lesterol l i t t e r  t o meet  rise  and age, would  catchup  components  be  only  intestine.  the  that  fact  by  Babicky  as  The  reason  i s more  the proximal  segment. that can of  l i k e l y  Furthermore,  the rates increase  the diet  by  f o r this  intestine  feedback  region  of  Stange  fold  i s altered.  i s why  i n the  may  diet  cho-  large  be  related to almost  of  milk  synthesis,  by c h o l e s t e r o l ,  than  i n the  and Dietschy  when  this  i t d i d i n the  the rate  inhibition  intestine  of  (82) and hence  of cholesterol synthesis seven  time  intestine  to affect  syn-  of the  of c h o l e s t e r o l occurs  consumption  in  l i t t e r  be  (106).  d r a m a t i c a l l y as  i n the proximal  of negative  i n a  proportion  e t a l .  i n the distal  exclusively  way  small  could  of cholesterol  which,  there  requirements.  intestine  to ask at this  absorption  by  a  i n which  growth  the inhibition  d i d not increase  proximal  cellular  i n the proximal  question  synthesis  phenomenon  distal  (82) have  i n the  the cholesterol  shown  intestine content  66 Once synthesis give  again,  the large  are seen  i n the proximal  the impression  that  are abnormally  the  i n the proximal  high. in  Therefore,  ' due  suppression  large  and  weight  though  on  small  available  the previous  differences  were  already  differences  were  just  the  i n t e s t i n e was  the  high  blood  for  pups  from t o be  study.  During  produced  pup  couple  beginning  cholesterol small  litters  supported the f i r s t  cholesterol  by  of  t h e same  blood  time  day  the r e s u l t s  week,  a t a much  rats  greater  period,  rats  their  the  amount  only  gain  In C h a p t e r I I , contributor  7.  been This  of the  rate from  to  reported idea  present  the small in large  the differences  cholesterol- are perhaps  litters.  metabolic  have  from  very  large  but weight  a possible  (2) on  the  i n body  limited  to appear.  b.ut, not.: p r o x i m a l i n t e s t i n e , t h a n Within  and  so that  levels that  and  were  s i t u a t i o n i s that  evident  as  synthesis  inhibitors.  h a d become  very  distal  are  the differences  of days,  suggested  by  the small  of this  to each  of  sizes  differences  7,  between  explanation  appears  i n the  could  exceptionally  litter  synthesis  day  this  of undernutrition  the metabolic  significant  A possible  within  to the e f f e c t s  remained  milk  the three  of  in a l l probability  i n t e s t i n e are  of c h o l e s t e r o l  Even  rates  the differences i n the rates  t h e i n t e s t i n e amongst  /likely-  of  l o w , when  i n the rates  i n t e s t i n e and  the synthetic  intestine rates  fluctuations  litters  distal litters.  i n the  greatest  levels  between  rats ing  raised  i nthe small  t o a chow  cholesterol on  d a y 40  the  diet,  levels  (2).  and large  significant i nthese  Further  studies  period  (2).  differences  two groups  cholesterol synthesized  time  l i t t e r s  w i l l  After  between  blood  a r e no l o n g e r  have  t o verify  apparent whether  by the intestine during  a c t u a l l y becomes  part  o f theblood  wean-  this  cholesterol  pool. D.  Developmental Figure  synthesis the  a  for  raised  pattern,  a c t i v i t y  enzyme  catalyses (Fig.  with  thereduction  the  postnatal  f i r s t  values  experiment from  ( 2 ,22).  t o birth, week  that  i sdelayed.  sent  study  not actively  day  food  and inspection  21 i n d i c a t e d  they  Although weaned  regula-  which acid  i s highest values  and then  during  drops t o  McNamara's ( 2 2 )  rate  does  during  not i n -  the third  therats they  did  i n the prehave  of the intestinal  had been  pattern  pathway  adult  at  by  i s t h e main  activity  (22).  the synthetic  i f weaning  solid  HMGR  and overshoots  t h e low levels observed  were  intestine  levels of synthesis  approximates  f o l l o w i n g weaning"  shows  In  CoA t o mevalonic  t h e enzyme  week  to  o f 8 pups.  t o the developmental  o f HMG  In the liver, prior  of cholesterol  l o w l e v e l s b y d a y 14 f o l l o w e d  i n liver  "....just  crease  t h ehigh  Synthesis  pattern  i n the cholesterol synthesis  2 ) .  adult  i n l i t t e r s  by day 21 i s s i m i l a r  HMGR  tory  rats  of Cholesterol  thedevelopmental  declining t o very  rise  of  6 shows  observed  birth  Pattern  consuming  access  contents  t h e food  on  pellets.  68 It  i s not  unexpected  synthesis  increased  The not  been  reason  i n milk  This  seem  not  increase  inhibitors and  human  which yet  E.  beyond  As  out of  4  did by  and  8.  reveal day  l i t t e r etic  21  an the  sizes.  rate  and  (105)  an  and  as  do  two (22)  uric  the other  which  acid  has  not  contains  two  blocks  the  forma-  unidentified factor  i n Adipose  tissue  found  that  Tissue  t o day  21  o f pups  i n the rates nearly  3  that  l i t t e r s  does  i n the  synthesis  I t i s notable large  since  acid.  tissue  was  (22, 105).  Also,  also  has  an  i n r a t milk  r e s u l t s demonstrate,  increase  i n the  commences.  acid  of  after birth  demonstrated  orotic  period  level  u n t i l  and  pattern  Adipose  explanation  milk  i n liver  tested  acid  levels of  the postnatal  and  mevalonic Bovine  21.  pattern  identified  Synthesis  low  day  been  i n g l u t e a l adipose  Very  on  cholesterol  has  acid,  developmental  liver.  been  of  the presence  postulated  weaning  one  being  the present  synthesis same  have  mevalonic  Cholesterol  but  decrease  u n t i l  before  one  seen  reasonable  not  (105);  of mevalonic  blocks  been a  identified.  inhibitors, tion  do  HMGR  milk  blocks  been  has  again  of  levels  determined,  t o be  rates  the rate  f o r the developmental  inhibitor  synthetic  that  to the  positively  does  then,  were  not  evident  i n pups from of  exhibit  intestine  were  times on  cholesterol  day  14  double  throughi n  large  synthesis that  and  raised  the  of  the  l i t t e r s  l i t t e r s  so  that  the other  when  the  the rate  two  synthi n the  small from  l i t t e r s , large  l i t t e r s rate  from  3 times tissue  higher  on  the rate  LDL  number  pups,  only  weighed there  they  less.  exists  cholesterol of  that of  both  sizes  rather  between and  occurring  than  when  has  rates  pups of  similar.  present,  the  although  the control  the  adipose  contained  nearly  cells  from and  consequently  amount  imply  that  synthesis  as  a  l i t t e r  determined,  t h e same  a t t h e same  an  the small  f a t c e l l  rate  of  regardless of  i n rats  function  of  from c e l l  weight.  r e s u l t s conform  higher  tissue  each  cholesterol levels  had  were  by  expressed  tissue  amount  equal  y e t t o be  that  to the usual  cholesterol synthesis,  serum  l i t t e r s  would  an  i s that  l i t t e r s  smaller  synthesized  which  Also,  the f a t tissue  this  much  cholesterol levels v i a the  p o s s i b i l i t y  much  synthetic as  tissue,  pups  small  l i t t e r s .  synthesis  that  the  the 40%  i n the  the negative-feedback  plasma  as  only  had  from  21  adipose  by  the large  were  was  These  by  Although  was  of  i s probably  cells  cholesterol  number  synthesis  day  large  l i t t e r s  the less  tissue  t h e pups  was  from  the rates  adipose  on  there  the p o s s i b i l i t y  i t ssize,  l i t t e r  when  and  from  of  with  small  Another  i n pups  equal  great  exerted  receptors.  tissue  And,  less  and  factor  synthesis  compared  as  then,  regulating  50%  i n the pups  tissue,  appears  was  I I I ) .  t h e medium  adipose It  l i t t e r s  (Table  was  adipose  there  of  as  judged  ( 2 7 ) . That  inverse by  HMGR  i s ,r a t s  cholesterol synthesis  relationship activity, from  large  i n  adipose  tissue  and,  as  other  work  serum  cholesterol  levels  small  l i t t e r s  lower  high  serum  had  cholesterol  has  shown  (2), concurrent  (125  mg%),  while  synthetic levels  (220  rates mg%)  pups  and (2).  low  from  concurrent  CHAPTER  VI  CONCLUSION Alterations lesterol were  of  synthesis  raised  active  site  pups  synthesis,  on  adult  the  the  of  inhibition and  a  finding  rat  (79),  l i t t e r s .  of  food  are  ficance  of  this  difference  allowed  to  mature  shown  long-lasting  ( 1 0 7,-  109)  .  The  pattern  in  intestine  of  of  pups  developmental  pattern  (22).  of  In  both  decreases creases  from  again  21.  In  may  is  any  of  early  medium HMGR  tissues low is  14  to  be  reared  in  due  rats  real  speculated  signi-  which  studies  cholesterol  days  synthesis  increasing  n u t r i t i o n a l  of  are  have  changes  synthesis  resembles  reported  rate  to  inhibitors  the  several  activity the  man  probably  in  l i t t e r s  It  and  rats  case,  seen  since  reports  shown  when  from  a  the  cholesterol  of  to  was  most  intestine  was  in  growth  be  which The  the  (73),  development  these  b i r t h by  c e l l  adults,  of  rats  non-cholesterol  consumed.  effects  from  cho-  previous  21  situation  in  amounts  day  of  dam.  in  intestine  freedom  phenomenon  per  monkey  on  and  in  l i t t e r s  confirms  proximal  of  pups  weaning,  (80),  reverse  to  14  medium  which  cholesterol  catchup  and  synthesis  This  pattern  demonstrated  after  dog  the  effects  by  4  and  a  greater  combined  of  small  contrast,  site  large  in  developmental been  of  raised  In  have  l i t t e r s  t h i r d ;  (78).  the  in  d i s t a l the  in  the  for  liver  synthesis  later  and  then  that  the  f i r s t  in 1  72 half  of this  inhibition  trend  by  where  cholesterol  which  are contained  rates  may  of  milk  possibly  and hence  The which was  pattern  These  less  ingestion  values  of rats  suggest,  synthesis  l i t t e r s  be  levels  found  subsequent to a of  decreased  i n these  normally  regions  correlate  not prove,  seen  with  that  to the higher  on  i n day  14  intestine. the low  f o r the  i n large  compared  consumption  l i t t e r  of the  (2) reported  rats  i n  inhibitors.  i n the intestine  related  of  inhibitors increase  i n the large  raised  b u t do  cholesterol may  i n both  of synthesis  period  data  altered  of synthesis  cholesterol  postnatal  The  attributed  was  i s the result  and non-cholesterol  i n milk.  day'7  low rates  plasma  the  by  decrease  be  the low rate  reached  rates  early  l i t t e r s .  Therefore,  the higher  of rats  plasma  from  rate small  cholesterol  to rats  from  large  l i t t e r s (2). White unaltered in  rats  from to  adipose  and from  low rate medium  the large  two  l i t t e r from  l i t t e r  small  twice nmoles  l i t t e r s ,  as much 3  H/g  during  synthesis  threefold  sizes.  had 40% less  Even  adipose they  wt  /d)  cholesterol  at  an  t h e 21 day p o s t n a t a l  period  l i t t e r s .  rats  However,  increased the rate  though tissue  on  p e r gram  (14/litter)  i n  continuously  of that  rats  nevertheless  cholesterol  body  synthesized  and small  l i t t e r  d a y 21 t o n e a r l y  other  tissue  from  i n the the  day 21 t h a n produced  of body  large those  nearly  weight  a n d 0.16  up  (0.30  nmoles  3  H/g  of  body of  wt  /d  adipose  rats  from  crease  ( 4 / l i t t e r ) . tissue large  available.  could  cholesterol  l i t t e r s  t h e number  One  speculate  synthesis  represents  of adipocytes  an  when  that  i n the  attempt  the rate 21-day-old  t oi n -  sufficient  food  i s  74  APPENDIX List  of  A  Abbreviations  DPM  disintegrations  HDL  high  HMGR  3 - h y d r o x y - 3 - m e t h y l g l u t a r y l coenzyme  density  per  minute  lipoprotein  3 H  water  t r i t i a t e d  water  LDL  low  density  mCi  m i l l i c u r i e  lipoprotein (22.2  x  10  9  DPM)  - 9 (10  nmole  nanomole  .  PUFA  polyunsaturated  SEM  standard  SGA  small  TLC  thin  layer  VLDL  very  low  error  moles) fatty of  the  f o r gestational  acid.. mean age  chromatography  density  lipoprotein  A  reductase  75.  APPENDIX Cholesterol of  Rats  Synthesis  Raised  i n  B and Body  Weights  i n Litters,oft8  Age  Proximal  Distal  Adipose  Body  (d)  Intestine*  Intestine*  Tissue*  Weight  Mean  Mean  14  Mean  39 . 2  34 . 6  4  57 . 3  25.8  8  40.0  55.2  7  43 . 1  31.4  0  4 8 .1  40.0  0  42.4  33 . 8  0  4 5.0  + 2  36.8  + 4.1  7.0  +  15.3  52 . 5  1.1  16.4  15.4  27 . 5  1.5  12 . 7 :  42 . 9  61.9  0.9  10 . 7  18. 7  3 7 .6  0 .7  17 . 0  17. 6  40.4  1.2  15.7  22 . 0 + 5.3  44.0  +  6.0  1.1  +  0.1  14.5  +  8.5  9.5  0.2  32.6  8.8  7.8  0.4  35.1  8.6  5.4  1.6  32.7  11.2  7.2  1.3  35.2  5.2  5.1  1.6  35.6  7.7  8.0  1.2  34.7  8.4+0.8  7.2+0.7  1.1+0.2  (g)  0.1  1.2  34.3+0.5  7,6. Appendix  B  continued  Age  Proximal  (d)  Intestine  21  *  Distal  Adipose  Intestine*  Tissue  Weight  93.3  1.2  46.4  17 . 0  29.0  1.1  46.7  0.8  47.3  -  51.5  5 9 .4  1.8  46.8  39.5  69.0  0.5  46.3  42 . 3  74 .4  0.9  47.0  35.6  Values  *  16.8  46.5  Mean  Body  +  are  6.1  65.0  expressed  as  +  9.6  nmoles  1.1  H/g/h  +  0.2  +_  SEM  46.8  +  (g)  0.2  77. APPENDIX  Cholesterol of  Rats  Age  Proximal  (d)  Intestine*  7  Mean  Raised  Mean  Intestine*  Adipose  Body  Tissue*  Weight 17 . 7  18.8  17.0  0.9  17.0  26 . 7  30.2  1.1  17.8  29.4  4 6.9  1.0  16.4  24 . 6  39.8  1.2  17.2  26. 3  33.7  1.1  1 7 .4  +  1.5  32.6  +  4.2  1.0  18.1  +  0. 1  17.3  0.9  32 . 8  7 .3  0.4  32 . 6  11. 9  9.9  0.7  31.1  13 . 7  14 . 4  1.4 •  28 . 5  8.6  1 2 .1  0.8  29 . 8  9.7  10.2  0.7  31.4  +  0.9  11.8  +  1.6  0.8  +  0. 1  31.0  + 0.7  11. 7  44 . 9  1.4  52 . 6  18.2  27 . 6  0.9  56.6  29.4  36.2  0 .7  56.5  21.3  29.4  0.6  51.5  18.6  39.7  1.1  53.4  20.6  37.8  0.9  54 . 7  +  2.3  are expressed  36.0  +  2.7  as nmoles  0.9 H/g/h  +  0. 1  +_ S E M  54.2  (g)  + 0.2  10 . 3  20.0  Values  Distal  of 4  0.7  10.3  21  i n Litters  Weights  27.9  7.7  Mean  *  S y n t h e s i s i n and Body  22 . 9  24.8  14  C  + 0.9  78  APPENDIX Cholesterol of  Rats  Synthesis  Raised  D  i n and Body  i n Litters  of  Weights  14  Age  Proximal  (d)  Intestine*  7  16.5  7.2  1.4  15.9  10 . 5  5.8  1.6  15.1  14 . 9  18.3  1.5  15. 3  29.4  10.0  1.6  14.4  10.8  14 . 3  1.8  15 . 6  15.4  13.1  1.3  14 . 3  Mean  16.3  14  Mean  Mean  11.5  +  1.9  Body  Tissue*  Weight  1.5  +  0. 1  15.1  +  14 . 7  2 .2  24 . 0  10 . 6  11. 5  1.7  21.2  9 .9  10 . 6  2 .0  2 2 .8  11.1  8.5  1.8  22 . 6  19.6  18.4  2 .2  21.7  8.2  8.2  1.5  23.2  +  0.9  11.8  +  1.6  1.8  +  0. 1  22.6  +  77.4  33.6  1.6  38.8  61.1  31.1  4 .7  34 . 7  44 . 0  41.4  3.4  38. 7  20.2  19 . 6  1.7  34 . 3  38. 7  27 . 9  2.2  36.2  50.3  36.5  2.9  38.8  48.6  Values  Intestine*  Adipose  4 .5  10.7  21  +2.8  Distal  +  8.0  31.7  are expressed  +  3.0  as nmoles  2.8  H/g/h  +  0. 5  +_ S E M  36.9  +  (g)  0. 3  0. 4  0. 9  APPENDIX E Statistical  Comparisons  B e t w e e n Two C o n s e c u t i v e  Tissue*  Cholesterol  4 1  Day  8  Pups/Litter Day  7  -  Adip .  7  Prox. Dist . Adip.  24 .8 32.. 6 1.0 Day  Prox. Dist. Adip.  10. 3 11.8 0.8  + + +  1 5 4 2b 0 l  14 10. 3 ± 0. 9 a 11.8 ± 1. 6 a 0.8 ± 0. l  0 9 1 6b 0 l  21 20.1 ± 2. 3 a 36.0 ± 2 . 7 a 0.9 ± 0. l  Day  b  b  14 + + +  b  Day  b  b  Prox.  = proximal  Dist.  = distal  Adip.  = g l u t e a l adipose  b  intestine  intestine tissue  Synthesis  Synthesis**  14  Pups/Litter  1 45.0 ± 2.8 b 36.8 ± 4 . 1 b  Dist.  of Cholesterol  Weeks  Day  Prox.  Day  of Rates  -  7 22.0 ± 5 . 3 b 44 . 0 ± 6.0 b 1.1 ± 0 . 1 b Day  14 8.4 ± 0.8 b 7 . 2 ± 0.7 b 1.1 ± 0.2 b Day  7 22 . 0 ± 5. 3 a 44 . 0 ± 6. o b " 1.1 ± 0. 1 Day  14 8.4 ± 0. 8 b 7.2 + 0. 7 a 1.1 ± 0. 2 b Day  21 35.6 ± 6. l 65.0 ± 9. 6 a 1.1 + 0. 2 b a  ** V a l u e s  Day  1  Day  7  16.3 11. 5 1.5  Day  Day  10. 7 11. 8 1.8  are expressed  Pups/Litter  +  +  Day  Day  + +  9b 11.8 ± 1 6 b 1.8 ± 0 l  0.9 1.6 b 0.1b  21 48.6 ± 8 o a 31.7 + 3 . o a 2.8 ± 0 5 b  a, b P a i r e d  values  superscript  are not s i g n i f i c a n t l y  (p <  0.05 ) .  10.7  ± 0  b  Day  b  as nmoles  i n each  14  2.8 1.9 b 0.1b b  14 +  7  H/g/h  ± SEM  r o w w i t h t h e same different  «  80 Appendix S t a t i s t i c a l  1.  Tests  Used  i n the Preceding  C o m p a r i s o n s Amongst D i f f e r e n t 4  f o r Proximal  Source  Error  groups  (within  Ages  Summary  i n Litters  *df  *MS  F  654.8  2  327.4  244.6  15  16.3  899.4  17  20.1  groups) Totals  Group Mean  Day  Mean  14  Day  21  10.3  20.0  -14.5  4.8  Group Day  7  24.8  Day  14  10.3  3  Table  of  Intestine  *SS  Between  E  -9.7  -6-32  zi  fa  <  fa  +.6.32  cX.=  0-05  3.68  81 Appendix 2.  E  continued  Comparisons 4  Amongst  f o r Distal  Error  Ages  i n Litters  of  Intestine.  Source  Between  Different  SS  groups  (within  df  2059.7  2  805.3  15  2865.0  17  MS  F  1 0 2 9 .9  oC=  19.2  0.05  3.68  53 . 7  groups) Totals  Group Mean  D a y 14 11. 8  Mean Group Day 7  32.6  Day  11.8  14  20.8  Comparisons 4  groups  (within  <t±  Amongst D i f f e r e n t  df  Ages  MS  0.1  2  0 . 05  1.6  15  0 .10  1.7  17  groups) Totals  &  .+ 11...5. in  Litters  of  Tissue•  SS  Between Error  -11.5  f o r Adipose  Source  -3 . 4 -24.2  f 3.  Day 21 36.0  F  0.5  OC=0.05  3.68  Appendix 4.  E  continued  Comparisons for  Proximal  Source  Different  Ages  i n Litters  of 8  Intestine  df  SS  F  MS  groups  4600.. 0  3  1 5 3 3 .. 3  (withinc  1 4 4 9 . .4  19  7 6 ..3  6049.. 4  22  Between Error  Amongst  °C=0.05  3.13  20.0  groups) Totals  Group Mean  Day 7 22.0  23.0  Mean  D a y 14 8.4  Day 21 35.6  36.0  9.4  13.6  -13.6  Group Day  1  4 5.0  Day  7  2 2.0  Day  14  -27.2  8.4  Comparisons Amongst  5.  8  SS  Between  groups (within  df  i n  MS  10289.0  3  3429.7  3480.7  19  183.2  13769.7  22  groups ) Totals  Ages  L i t t e r s of  f o r Distal Intestine.  Source  Error  Different  F  18.7  oC=0. 0 5 3. 1 3  83 Appendix  E  continued Group Mean  Mean Day  1  36.8  Day  7  44.0  Day  14  D a y 14 7.2  7.2  29.6  -29.2  36.8  -21.0  -  Day 21 65.0  7.2  1 6.  Day 7 44.0  -57.8  -23.9  Comparisons Amongst for  <  Different  df  SS  Error  + 2 3-.-9  Ages i n L i t t e r s  of  8  Adipose Tissue.  Source  Between  $ * -fs  groups (within  0.05  2  1.67  15  1.72  17  F  MS  0 .03  cC=o. 0 5  0.3  3 .68  0.1  groups) Totals  7.  Comparisons Amongst for  Proximal  Source  Error  groups  (within  df  MS  5022.1  2  2511.1  2279.6  15  151.9  7301.7  17  groups) Totals  Ages i n L i t t e r s  of  14  Intestine.  SS  Between  Different  F  16.5  oC=0.05  3.68  Appendix  E  continued*;  84 Group  Mean  D a y 14 10 . 7  Mean  Day 21 48.6  Group Day  7  16.3  Day  14  10.7  5.6  -32.3 -37.9  + 19. 3 8.  C o m p a r i s o n s Amongst for  Distal  SS  Error  groups  Ages  i n Litters  df  7658.3  2  482.1  15  with  MS  14  3829.2  o£=0.05  F  119.1  32.1  groups Totals  of  Intestine  Source  Between  Different  2090.4  17  Group Mean  D a y 14 11.8  -  Mean  Day 21 31.7  Group Day  7  11. 5  Day  14  11. 8  0.3  -20.2 -19 . 9  +8 .9  3.68  Appendix 9.  E  continued  Comparisons for  Adipose  Amongst  Different  SS  Error  i n Litters  of  14  Tissue.  Source  Between  Ages  groups  df  5.6  2  9.7  15  15.3  17  (within  MS  F  2 . 8  4 ..6 6  0.6  groups) Totals  Group Mean Mean  D a y 14 1.8  Day 21 2.8  -0.3  -1.3  Group Day  7  1.5  Day  14  1.8  -1.0  + 1.21  *SS  =  Sum  of  df  =  degrees  MS  =  mean  squares of  square  freedom  oC=0.05  3.68  86 APPENDIX S t a t i s t i c a l Synthesis  Comparisons Between  of  F Rates  Proximal  and  of  Distal  Cholesterol Pups/Litter  Age (d)  Proximal Intestine  4 4 4  7 14 21  24 .8 10 .3 20 .0  +  8 8 8 8  1 7 14 21  45 .0 22 .0 8 .4 35 . 6  +  14 14 14  7 14 21  16 . 3 10 . 7 48 . 6  +  * **  Values  a, b  Values  df  not  =  are  expressed  degrees  of  i n each  as  + +  + + ±  + +  Intestine  Synthesis  *  Distal Intestine  1. 5 0. 9 2.3 2.8 5 .3 0 .8 6. l 2.8 0 .9 8. o  nmoles  Cholesterol  b  b  b  b  b  b  b  b  b  b  H/g/h  32, 6 11. 8 36. 0  +  36. 8 44 . 0 7. 2 65 . 0  +  11. 5 11. 8 31. 7  ±  + +  + + +  + + ±  4 .2 1. 6 i •7 4 .l 6. o 0. 7 9 .6 1. 9 1. 6 3. o  SEM  freedom row  s i g n i f i c a n t l y  with  the  same  superscript  different  (p <  0.05).  are  d f **  b  b  a  b  a  b  a  b  b  a  10 10 10 10 8 10 9 10 10 10  87 Appendix S t a t i s t i c a l Amongst  Comparisons  Rats  Raised  of  G  Rates  i n Litters  of Cholesterol  of  4,8  and  14  Synthesis  f o r Various  Tissues.  Tissue*  Age (d)  4  Pups/Litter  Prox.  7  2 4 .8 +  1.5  b  Prox.  14  10. 3 +  0.9  b  Prox.  21  20 . 0 +  2.3  Dist.  7  32 . 6 +  Dist.  14  Dist.  21  Adip.  7  Adip. Adip.  *  Cholesterol S y n t h e s i s '** 8 Pups/Litter 14 Pups/Litte: 22 . 0 +  5.3  b  +  0.8  b  b  35 . 6 +  6.1  b  4.2  b  44 . 0 +  6.0  b  11. 8 +  1.6  b  7 .2 ±  0.7  b  36.0  ±  2.7  b  65 . 0 +  9.6  a  1.0  +  o.ib  1.1  ±  14  0 .8 +  o.ib  1.1  21  0.9  +  o.ib  1.1  Prox.  = proximal  Dist.  =  distal  Adip.  =  gluteal  8.4  16. 3 ±  2.8  b  10 . 7 +  0.9  b  +  8.0  a  11. 5 +  1.9  a  11. 8 +  1.6  b  31.7  +  3.0  b  o.ib  1.5  +  a 0 .1  ±  o.i  1.8  +  o.ia  +  o.ib  2.8  +  0 . 5  48.6  b a  intestine  intestine adipose  tissue 3  **  Values  are expressed  ab  Values  i n each  are  not  row  as  with  significantly  nmoles t h e same  different  H/g/h  ±  SEM  superscript (p  0.05).  a  88 Appendix S t a t i s t i c a l 1.  Tests  Used  C o m p a r i s o n s Amongst and  Distal  Source  *SS  Between Error  groups (within  i n the Preceding  Litters  Intestine  G  on  o f 4,  Day  Summary  Table  8 a n d 14 f o r P r o x i m a l  7.  *df  4113.0  5  2178.3  28  6291.3  33  *MS  822.6  F  10 . 6  OC =  0.05  2 . 56  groups) Totals  Group Mean  II 32 . 6  -T • ' .  24 . 8  -7.8  II  32 . 6  III  22 . 0  IV  44 . 0  V  16. 3  Mean  III 22.0  IV 44.0  V 16. 3  VI 11. 5  2.8  -19.2  8.5  13 . 3  10.6  -11.4  16. 3  21.1  -22 . 0  -5 . 7  10. 5  27 . 7  32 . 5  **Group  Confidence  4 .8  intervals  6  subjects  per  6  subjects  i n one  c e l l ,  5  subjects  i n the  other:  f o r pairwise  comparisons  with:  c e l l :  .0  Appendix 2.  G  Comparisons Proximal  and  Source  Error  Amongst Distal  SS  Between  89  continued  groups  (within  Litters  of  Intestine  4, on  df  104.8  5  380.9  29  485.7  34  8  and  Day  14 f o r  14.  MS  F  21.0  oC=  1.6  0.05  2.55  13.1  groups) Totals  3.  Comparisons Proximal  and  Source  Distal  SS  Between Error  Amongst  groups  (within  Litters  Intestine  df  4, on  8 and Day  MS  7209.7  5  1441.9  5914.8  29  204.0  13124.5  34  groups) Totals  of  14 f o r  21.  F  7.1  oC =  0.05  2.55  Appendix  G  90  continued Group Mean  I I 36.0  Mean  I I I 35.6  IV 65.0  V 48.6  VI 31.7  Group I  20.0  II  36.0  III  35.6  IV  65.0  V  48.6  Confidence  -16.0  -15.6 0.4  intervals  subjects  per  6  subjects  i n one  5  subjects  i n t h e others>  Comparisons Adipose  Error  groups  (within  -12.6  4.3  -29.4  -13.0  3.9  16.4  33:3  f o r pairwise  Amongst L i t t e r s on  Day  comparisons  with:  o f 4,  8 a n d 14 f o r  7.  df  MS  F  6  0.84  2  0.40  1.06  15  0.07  1.90  17  groups) Totals  -29.0  c e l l ,  SS  Between  -11.7  cell:  Tissue  Source  -28.6  16.9  6  4.  -45.0  oC=  0.05  3.68  Appendix  G  91  continued Group  Mean Mean  8/litter 1.1  14/litter 1.5  -0.1  •0 . 5  Group 4/litter  1.0  8/litter  1.1  ;  5.  Comparisons Tissue  on  •0.4  -0-4  AmongstLitters  Day  Error  o f 4,  +  0.4  8 a n d 14  f o r Adipose  14.  Source  SS  Between  t < ft  <  groups  (within  df  5.09  2  5.52  15  10.61  17  MS  2.55  F  o£=0.05  6.87  0 .37  groups) Totals  Group Mean  8/litter 1.1  Mean  14/litter 1.8  Group 4/litter  0.8  8/litter  1.1  fa-  -0.3  -1.0 -0.7  -0.9  — jfc  3 . 68  Appendix 6.  G  continued  Comparisons Adipose  Amongst  Tissue  on  Source  Day  groups  df  13.1  (within  o f 4,  8 a n d 14 f o r  21.  SS  Between Error  Litters  6.4  MS  F  2  6.6  15  0.4  15.2  oC=0.05  3.68  groups) Totals  19.5  17  Group Mean  8 / l i t t e r 1.1  Mean  1 4 / l i t t e r 2.8  Group 4 / l i t t e r  0.9  8/litter  1.1  -0.2  -1.9 -1.7  *SS  =  sum  of  squares  df  =  degrees  MS  =  mean  I  =  Proximal  II  =  Distal  III  =  Proximal  IV  =  Distal  V  =  Proximal  VI  =  Distal  of  freedom  square  **Group intestine,  intestine,  4 / l i t t e r  intestine,  intestine,  8 / l i t t e r  8 / l i t t e r  intestine,  intestine,  4 / l i t t e r  1 4 / l i t t e r 1 4 / l i t t e r  9-3,  APPENDIX S t a t i s t i c a l Litter  Age (d)  Comparisons  H  o f Body  Weight  Amongst  Sizes  Body  4  Weight  (g)  Pups/Litter  8 Pups/Li tier  14  Pups/Litter  7  17.3  ±  0.2x  14.5  ±  1.2x  15.1 ±  0.3x  14  31.0  ±  0.7y  34.3 ±  0 .5y  22.6  0.4  21  54.2  ±  0.9z  46.8  0 .2  a,b,c  Values not  x,y,z  row w i t h  significantly  Values are  i n each  i n each  t h e same  different  column  not s i g n i f i c a n t l y  ±  with  (p <  superscript  subscript  (P ^ 0 . 0 5 ) .  ±  are  0.05).  t h e same  different  36.9  b  ±  0.9%  y  Appendix Statistical of  4,  8 a n d 14  Summary 1.  Comparisons  used  Amongst  i n the  Litters  Preceding  Table. Litters  SS  Source  Error  f o r Body,, W e i g h t s  f o r D i f f e r e n t Ages  Comparisons Amongst  Between  H  df  2 4 .8  groups (within  o f 4,  2  22 . 1  14  4 6 .9  16  8 and  14  on  MS  F  1 2 .4  7.75  Day  7.  c£=0.05  3.74  1 .6  groups) Totals  Group Mean  8/litter 14.5  Mean  14/litter 15.1  Group 4/litter  17.3  8/litter  14.5  Confidence  2.8  2.2 -0.6  intervals  f o r pairwise  6  subjects  per  cell:  6  subjects  i n one  cell,  5  subjects  i n the  other:  comparisons  with:  0  Appendix 2.  H  Comparisons Day  95  continued Amongst  Litters  df  SS  Error  4,  8  and  14  on  14.  Source  Between  of  groups  F  MS  436.. 7  2  218.4  4 2 .. 3  15  2.8  4 7 9 .. 0  17  (within  oC=0. 0 5  78.0  3. 68  groups ) Totals  Group Mean  8 / l i t t e r 34.3  Mean  14/litter 22.6  Group 4 / l i t t e r  31.0  8 / l i t t e r  34.3  -  Comparisons  Source  SS  Between Error  Amongst  groups  (within  l i t t e r s  df  < fa  <  of  4,  8 and  +  14  2.6  on  MS  'F  196.5  904.1  2  452.0  35.4  15  2.3  939.5  17  groups) Totals  8.4 11.7  -2.6  3.  3.3  Day  21.  CC=0.05  3.68  Appendix  H  96  continued Group Mean  8 / l i t t e r 46 . 8  Mean  14/litter 36 . 9  Group 4 / l i t t e r  54.2  8/litter  46.8  f  C o m p a r i s o n s Amongst  Source  Between Error  SS  groups  17.3 9.9  -2-4  3  4.  7.4  <f< Different  df  fj Ages  i n Litters  MS  F  4175.0  2  2087.5  50.0  15  3.3  4225.0  17  (within  .2.4  Group Mean Mean  D a y 14 31.0  Day 21 54 . 2  -13 . 7  -36 . 9  Group Day  7  17. 3  Day  14  31.0  -23 . 2  4.  oC=0.05  626.3  groups) Totals  of  3.68  Appendix 5.  H  continued  Comparisons  Amongst  Source  Between  97 Different  SS  df  5835 .9  groups  16.4  Error(within  Ages  i n Litters  1945.3  19  0.9  oC=0.05  F  MS  3  o f 8.  2 2 5 4 .1  3.13  groups) 5852 .3  Totals  22  Group Mean Mean  Day 7 1 4 .5  D a y 14 34 . 3  -7 . 5  -27 . 3  -39.8  -19 . 8  -32 . 3  Day 21 46.8  Group Day  1  7.0  Day  7  14 . 5  Day  14  34 . 3  -12 . 5  < 6.  Comparisons  Source  Between Error  Amongst  D i f f e r e n t Ages  SS  groups (within  df  + 1.7  in  Litters  MS  1472 . 0  2  736 . 0  3 2 .5:  15  2.2  groups) Totals  f,  1504 . 5 • 1 7 .  F  3 3 9 .7  of  14.  OC=0.05  3 . 68  Appendix  H  98  continued  Group Mean  D a y 14 2 2.6  -7.5  Mean  Day 21 3 6.9  Group Day  7  15.1  Day  14  22.6  f,  -21.8 -14.3  "2-3  * $ ± f,  99-  APPENDIX ANOVA and  Orthogonal  Polynomial  Interactions with  Synthesis  i n Proximal  Contrast  t h e Time  Combined  O b s e r v e d Means For 3 (nmoles H/g/h)  Litter  t o t h e Time  Factor  Factor  f o rCholesterol  I n t e s t i n e and D i s t a l  Proximal  Litter  I  Intestine  Intestine  O b s e r v e d Means f o r 3 (nmoles H/g/h)  Combined Time  Size  Age (g) 1  Weighted Unweighted  45.01 45. 01  8  Weighted Unweighted  2 7 . 98 27 . 73  7  Weighted Unweighted  20 . 94 21.00  4  Weighted Unweighted  2 5 . 02 25 .02  14  Weighted Unweighted  9 . 79 9 . 79  14  Weighted Unweighted  30 . 1 5 30 . 15  21  Weighted Unweighted  34 . 72 34 . 72  Tests Sums  of S i g n i f i c a n c e for of  Proximal Intestine  using Unique  Squares  Source of Variation  Sum o f Square  df  Within  4925.45  59  54087.74  1  54087.89  647 .89  316.53  2  158.26  1. 89  0 .159 0 . 000  Cells  Constant Litter  Mean  Square F  Sig. o f F.  0.001  Time  (1)  1588.10  1  1588.10  19 .02  Time  (2)  10603.77  1  10603.77  127 .01  0.000  Time  (3)  476.04  1  476.04  5 .7 0  0.020  L i t t e r  By Time  (1)  1350.91  2  675.45  8 .0 9  0 .001  Litter  By Time  (2)  1017.85  2  508.92  6. 0 9  0,004  Litter  By Time  (3)  28 . 90  2  14 .45  0 .1 7  0 .841  1 GOAPPENDIX  I  (Continued)  Distal  Combined Litter  O b s e r v e d Means 3 (nmoles H/g/h)  Litter  Size  Intestine  f o r  Combined  O b s e r v e d Means f o r 3 (nmoles H/g/h)  Time  Age (d) 1  Weighted Unweighted  3 6 . 79 3 6 . 79  7  Weighted Unweighted  2 8 . 47 29 . 33  8  Weighted Unweighted  36 .76 38 . 24  4  Weighted Unweighted  29 .32 29 .32  14  Weighted Unweighted  1 0 . 38 1 0 . 38  14  Weighted Unweighted  22 .97 22 .97  21  Weighted Unweighted  42 . 98 44 . 2 1  Test  of  Significance for  Sums  of  Squares  Distal  Intestine  using Unique  Square  Source of Variation  Sum o f Square  df  Within  5784.46  58  99.73  63478.45  1  63478.45  6 3 6 .48  0 . 001  2675.48  2  1337.74  13 .41  0 .000 0 . 760  Cells  Constant Litter Time  (1)  Time  (2)  Time  (3)  Mean  F  Sig. of F  1  9 . 38  0 .09  7424.80  1  7424 . 80  7 4 .44  0.001  3598.26  1  3598.26  36 .07  0.001  9 . 38  Litter  By Time  (1)  848.33  2  424.16  4 .25  0.019  L i t t e r  By Time  (2)  399.88  2  199.94  2 .00  0.144  Litter  By Time  (3)  3019.71  2  1509.85  15 . 13  0 .001  10;]>  BIBLIOGRAPHY  Hisatomi,  K.  and  undernutrition rat  brain.  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