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Effect of maternal alcohol consumption on placental blood flow and fetal uptake of zinc, folate, and… Jones, Peter John Harris 1981

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EFFECT OF MATERNAL ALCOHOL CONSUMPTION ON PLACENTAL BLOOD FLOW AND FETAL UPTAKE OF ZINC, FOLATE, AND ANALOGS OF GLUCOSE AND AMINO ACID  PETER JOHN HARRIS JONES B.Sc.  U n i v e r s i t y o f B r i t i s h Columbia, 1978  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Human N u t r i t i o n School of Home Economics We accept t h i s thesis as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1981 (c) P e t e r John H a r r i s Jones, I98I  In p r e s e n t i n g requirements  this thesis  f o r an a d v a n c e d  of  British  it  freely available  agree that for  Columbia,  f u l f i l m e n t of the  degree a t the U n i v e r s i t y  I agree that f o r reference  permission  scholarly  in partial  the Library  shall  and s t u d y .  I  for  that  copying or p u b l i c a t i o n  f i n a n c i a l gain  shall  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 V a n c o u v e r , Canada V6T 1W5  of this  It i s thesis  n o t be a l l o w e d w i t h o u t my  Department o f  (2/79)  thesis  p u r p o s e s may be g r a n t e d by t h e h e a d o f my  permission.  DF-fi  further  f o r extensive copying of t h i s  d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . understood  make  Columbia  written  i i  ABSTRACT The present study was undertaken  t o determine  r e t a r d e d f e t a l growth caused by maternal a l c o h o l p r i o r t o and d u r i n g g e s t a t i o n the t r a n s f e r o f n u t r i e n t s the  whether  consumption  i s due t o an i n t e r f e r e n c e  from the maternal  with  c i r c u l a t i o n into  fetus. Female Sprague-Dawley r a t s were e i t h e r g i v e n 20 p e r c e n t  alcohol  i n d r i n k i n g water and s o l i d d i e t ad l i b i t u m  group),  o r were p a i r - f e d t o the a l c o h o l  group  or were g i v e n water and s o l i d d i e t ad l i b i t u m c o n t r o l group) f o r f o u r weeks. alcohol  folic  of g e s t a t i o n zinc,  folic  acid,  and f e t a l weights,  and non-metabolizable  and the p l a c e n t a l  studied  On day 20  the f e t a l uptake glucose and amino  b l o o d flow were  f e t a l uptake o f the n u t r i e n t s  i n water.  maternal plasma l e v e l s of  a c i d , and z i n c were determined.  placental  a c i d analogs, The  (ad l i b i t u m  They were then mated and the  During the t h i r d week o f g e s t a t i o n  of  ( p a i r - f e d group},  group was changed t o 30 percent a l c o h o l  glucose,  (alcohol  determined.  was i n v e s t i g a t e d by  measuring f e t a l r a d i o a c t i v i t y 90 minutes a f t e r i n j e c t i n g the 3 dams i n t o the femoral v e i n with e i t h e r H labeled f o l i c acid 14 and methyl  ( c x - D - U-  c gluco) pyranoside,  o r zinc-65, or  14 2-amino(l-  C) i s o b u t y r i c a c i d .  The r a d i o a c t i v i t y i n the  maternal plasma and l i v e r 90 minutes a f t e r i n j e c t i o n was a l s o determined.  Blood flow t o the p l a c e n t a s as w e l l  as maternal  c a r d i a c output and r e n a l b l o o d flow were measured-fusing micron diameter microspheres l a b e l e d w i t h 57 Co. The osmolality  15  o f the maternal plasma and maternal muscle water  iii  content were determined i n order t o assess the degree o f dehydration, as a l c o h o l  i n water was o f f e r e d as the s o l e  source o f f l u i d s . No d i f f e r e n c e s and  i n l i t t e r s i z e e x i s t e d between  p a i r - f e d o r alcohol, and ad l i b i t u m c o n t r o l  alcohol  groups,  however l i t t e r s i z e s o f the ad l i b i t u m group were l a r g e r than those o f the p a i r - f e d group.  The 20 day o l d fetuses  were  s i g n i f i c a n t l y s m a l l e r w h i l e the p l a c e n t a s were s i g n i f i c a n t l y l a r g e r i n the a l c o h o l group than i n e i t h e r the p a i r - f e d o r ad l i b i t u m  controls.  No d i f f e r e n c e s  plasma l e v e l s o f glucose, f o l i c treatment groups.  were seen i n maternal  a c i d , o r z i n c among the t h r e e  C a r d i a c output and b l o o d flow t o the  kidneys d i d n o t d i f f e r among t h e t h r e e treatment groups. However b l o o d flow t o the p l a c e n t a ,  whether expressed as  mL/min/g p l a c e n t a o r as mL/min/ e n t i r e p l a c e n t a o r as % cardiac  output was s i g n i f i c a n t l y reduced i n the a l c o h o l  group compared with the p a i r - f e d and ad l i b i t u m c o n t r o l groups. When expressed as mL/min/placenta d i f f e r e n c e s  i n blood flow  were a l s o seen between p a i r - f e d and a_d l i b i t u m c o n t r o l No s i g n i f i c a n t d i f f e r e n c e s  groups.  were ,observed i n the f e t a l uptake  of z i n c o r analogs o f glucose and amino a c i d between the t h r e e treatment groups. alcohol  treated  The f o l a t e taken up by the f e t u s e s  o f the  dams was s i g n i f i c a n t l y h i g h e r than f o r p a i r -  f e d but not ad l i b i t u m c o n t r o l s .  No d i f f e r e n c e s  were observed  i n maternal plasma o r l i v e r c o n c e n t r a t i o n s o f any o f these compounds among the t h r e e groups 90 minutes a f t e r i n j e c t i o n . Plasma o s m o l a l i t y  was i n c r e a s e d  by approximately 7 percent i n  iv  the  alcohol  control  group compared w i t h  groups.  This  indicates  pair-fed  and  ad  libitum  a moderate degree  of  dehydration. The of the  present study demonstrates that  fetuses  exposed to a l c o h o l  impaired transfer  of  c i r c u l a t i o n t o the  the  fetus;  on  b l o o d f l o w i n the  f e t a l g r o w t h and  tested  i s i t due  plasma l e v e l s of these n u t r i e n t s . reduced p l a c e n t a l  The  to  be  due  to a d i r e c t action  lower  of a l c o h o l  of  Thus  ingestion on  the  to  an  maternal  exposed  clear.  growth  maternal  significance  alcohol  development i s not  due  from the  growth-retarding e f f e c t of maternal alcohol still  retarded  i n u t e r o i s not  nutrients nor  the  the  rats the may  fetus.  V  TABLE OF CONTENTS PAGE Abstract  i i  Table of Contents List  v  of Tables  v i i  Acknowledgements  ix  CHAPTER I II  INTRODUCTION  1  LITERATURE REVIEW  4  HUMAN STUDIES: a) R e t r o s p e c t i v e b) P r o s p e c t i v e c) A l c o h o l  Studies  4  Studies  7  Induced A l t e r a t i o n i n M a t e r n a l Metabolism  as C o n t r i b u t i n g t o F e t a l A l c o h o l Syndrome. ANIMAL  10  STUDIES:  a) I n t r o d u c t i o n  15  b) P r e n a t a l E f f e c t s o f A l c o h o l A d m i n i s t r a t i o n i n the Rat  15  c) P r e n a t a l E f f e c t s o f A l c o h o l A d m i n i s t r a t i o n i n t h e Mouse.  19  d) P r e n a t a l E f f e c t s o f A l c o h o l A d m i n i s t r a t i o n i n Other Animals e) Comparison  o f P r e n a t a l A l c o h o l Exposure  22 and  P r e n a t a l M a l n u t r i t i o n on Body C o m p o s i t i o n i n Offspring f ) Comparison  25 o f Growth and Development i n  O f f s p r i n g o f A l c o h o l Fed Versus Malnourished Animals  26  vi  PAGE III  MATERIALS AND METHODS a) P r e l i m i n a r y T r e a t m e n t o f A n i m a l s b) D e t e r m i n a t i o n Folic  o f Plasma A l c o h o l , Glucose,  A c i d , and Z i n c  30  c) P l a c e n t a l B l o o d F l o w D e t e r m i n a t i o n d) F e t a l U p t a k e o f F o l i c of  IV  29  30  A c i d , Z i n c , and Analogs  G l u c o s e and Amino A c i d  32  e) S t a t i s t i c a l A n a l y s i s o f R e s u l t s  33  RESULTS a) F o o d I n t a k e a n d M a t e r n a l Body W e i g h t s b) L i t t e r  35  S i z e , F e t a l Body W e i g h t s , a n d P l a c e n t a l  Weights  36  c) A l c o h o l and N u t r i e n t C o n c e n t r a t i o n s i n M a t e r n a l Plasma  43  d) P l a c e n t a l B l o o d F l o w  43  e) F e t a l U p t a k e o f N u t r i e n t s  44  f)  Plasma and L i v e r C o n c e n t r a t i o n s o f I n j e c t e d Compounds a n d M a t e r n a l P l a s m a O s m o l a l i t y a n d Muscle Dry Weight  V  DISCUSSION  REFERENCES CITED  44 51 62  vii  L I S T OF  TABLES PAGE  Table 1  D a i l y F o o d and  A l c o h o l C o n s u m p t i o n and  Body W e i g h t s o f R a t s G i v e n A l c o h o l , o r Ad L i b i t u m F e d Table 2  Table 3  Pair-Fed,  P r i o r to Pregnancy  D a i l y C a l o r i c Intakes Pair-Fed,  o r Ad L i b i t u m F e d  P r i o r to Pregnancy..  D a i l y F o o d and A l c o h o l C o n s u m p t i o n and o r Ad L i b i t u m F e d  Litter  During Pregnancy....  20  of Gestation  Given  o r Ad L i b i t u m F e d  i n Maternal  Three of G e s t a t i o n Pair-Fed, Blood  42 Zinc  P l a s m a D u r i n g Week  i n Rats Given A l c o h o l ,  o r Ad L i b i t u m F e d  Flow to Placentas  Kidneys,  and  Cardiac  and  45 to the  O u t p u t on Day  of Rats Given A l c o h o l , P a i r - F e d ,  Maternal 20  of  o r Ad  Gestation  Libitum  Fed Table 8  46  F e t a l U p t a k e o f 2-Amino I s o b u t y r a t e , Gluco-pyranoside, Maternal  Table 9  F o l i c A c i d , and  C i r c u l a t i o n a t Day  Concentration -D  40  Placental  E t h y l A l c o h o l , G l u c o s e , F o l i c A c i d , and Concentrations  Table 7  39  S i z e , F e t a l Body W e i g h t s , and  W e i g h t s o f R a t s a t Day  Table 6  Pair-Fed,  of Rats Given A l c o h o l ,  o r Ad L i b i t u m Fed  Alcohol, Pair-Fed,  38  Weekly  During Pregnancy  D a i l y C a l o r i c Intakes Pair-Fed,  Table 5  37  of Rats Given A l c o h o l ,  Body W e i g h t s o f R a t s G i v e n A l c o h o l ,  Table 4  Weekly  Zinc  P l a s m a and  from  the  of Gestation  o f 2-amino I s o b u t y r a t e ,  Gluco-pyranoside,  Maternal  20  Methyl-^D  F o l i c A c i d , and  48  Methyl-©t Zinc  in  L i v e r 90 M i n u t e s a f t e r  Injection of Radioactive  Compounds  49  viii  PAGE T a b l e 10  M a t e r n a l Plasma O s m o l a l i t y and M u s c l e Dry Weight Fed,  ( 7 o f Wet W e i g h t ) i n A l c o h o l , 0  and Ad L i b i t u m Fed R a t s  Pair50  ix  ACKNOWLEDGEMENTS  I Dr.  would  Joseph Leichter  throughout extended as  like  Keeler  of this  i n computer  of the results.  collaboration  her  technical  excellent Financial  Ma  are also  support  advisor  a d v i c e as w e l l and  statistical  t o Dr. Ralph  a s s i s t a n c e and  of the experimental  f o rh i s u s e f u l expressed  assistance  comments a n d  t o Helen  Smith f o r  i n the laboratory.  i s acknowledged  the British  t o my  appreciation i s  grateful  f o rt h e i r  and E n g i n e e r i n g Research  and from  Program.  My  programming  I am v e r y  a n d t o D r . Dan R u r a k Thanks  #A6249  thanks  and encouragement  project.  i n t h e development  suggestions.  Sciences  sincere  t o Dr. M e l v i n Lee f o rh i s v a l u a b l e  and Dr. Stephanie  methods,  my  f o r h i sdirection  the course  f o rthe support  analysis  t o express  from  Council  Columbia  Youth  the National  o f Canada  Grant  Employment  1  INTRODUCTION Concern about the adverse e f f e c t s of maternal a l c o h o l consumption d u r i n g pregnancy on o f f s p r i n g has been s i n c e a n c i e n t times  (1,2).  recorded  However i t has o n l y been i n the  l a s t decade t h a t a r e l a t i o n s h i p has been e s t a b l i s h e d between c h r o n i c maternal a l c o h o l i s m and alies  a s p e c i f i c p a t t e r n of anom-  i n o f f s p r i n g of such women.  T h i s p a t t e r n of anomalies,  termed f e t a l a l c o h o l syndrome, i s c h a r a c t e r i z e d by p r e n a t a l and p o s t n a t a l growth r e t a r d a t i o n , mental d e f i c i e n c y , microcephaly,  c r a n i o f a c i a l anomalies, and o t h e r changes  C h i l d r e n with f e t a l third  a l c o h o l syndrome are u s u a l l y below the  p e r c e n t i l e i n h e i g h t , weight, and  head  circumference,  and do not appear t o demonstrate catch-up growth. average r e p o r t e d I.Q. 106,  (3-5).  The  i s around 65 with a range from 16  to  i n d i c a t i n g t h a t most of the c h i l d r e n were b o r d e r l i n e to  m i l d l y r e t a r d e d i n comparison with S t a n f o r d B i n e t I n t e l l i g e n c e Test 1972  norms (5).  I t has  been r e p o r t e d  i n the medical  literature  (5) t h a t  about 40% of babies born t o heavy d r i n k e r s d i s p l a y e d c h a r a c t e r i s t i c s of the f e t a l a l c o h o l syndrome. i s meant an i n t a k e of 90 mL  (3 oz.)  By heavy d r i n k e r s  o f a b s o l u t e a l c o h o l per  day d u r i n g pregnancy. The  extent to which these i n f a n t s show f e t a l a l c o h o l  syndrome c h a r a c t e r i s t i c s i s probably  determined by the amount  and d u r a t i o n of exposure to a l c o h o l i n u t e r o . of a l c o h o l consumption d u r i n g pregnancy may form of f e t a l damage (5).  Little  Lower l e v e l s  induce  (6) demonstrated  a milder  2  t h a t reduced b i r t h weight due  t o a l c o h o l comsumption i s  independent o f maternal smoking h a b i t s ,  a recognized  birth  weight depressent. S i n c e the r e c o g n i t i o n of the i n c h i l d r e n , numerous s t u d i e s  f e t a l a l c o h o l syndrome  on the e f f e c t s of maternal  a l c o h o l consumption on the progeny have been conducted i n animals.  I t has  been c o n f i r m e d u s i n g  consumption of a l c o h o l d u r i n g f e t a l and the  postnatal  dams d i d not  growth was natally.  not  growth  r a t s t h a t maternal  gestation  (7,8).  results in  D e s p i t e the  fact  observed d u r i n g  t h a t the r e t a r d e d  the  first  w i t h animal models i n d i c a t e  growth i s not  due  t o e i t h e r c a l o r i c under-  p a i r - f e d c o n t r o l animals.  i n g w i t h the m a t e r n a l - f e t a l placenta.  Maternal a l c o h o l growth of the  treated  consumption  f e t u s by  t r a n s f e r of n u t r i e n t s  interfer-  across  Decreased p l a c e n t a l n u t r i e n t a v a i l a b i l i t y ,  r e s u l t of e i t h e r reduced m a t e r n a l plasma n u t r i e n t t i o n or a r e d u c t i o n contribute  the  c a l o r i c i n t a k e s were s i m i l a r i n a l c o h o l  might however cause r e t a r d e d  i n p l a c e n t a l b l o o d flow, may  t o growth r e t a r d a t i o n i n the  To p r o v i d e an e x p l a n a t i o n r e t a r d a t i o n i n the  up  f o u r weeks p o s t -  n u t r i t i o n or t o a s p e c i f i c n u t r i e n t d e f i c i e n c y as  and  that  r e c e i v e a l c o h o l a f t e r d e l i v e r y , catch  These o b s e r v a t i o n s  n u t r i e n t and  retarded  as  further  fetus.  f o r a l c o h o l i n d u c e d growth  f e t u s , the p r e s e n t study was  a v a i l a b i l i t y of n u t r i e n t s t o the T h i s was  a  concentra-  designed t o  determine the e f f e c t of m a t e r n a l a l c o h o l consumption on  model.  the  the  fetus i n a s u i t a b l e rat  a c h i e v e d t h r o u g h i n v e s t i g a t i o n of maternal  3  nutritional  status, p l a c e n t a l blood flow,  t r a n s f e r of n u t r i e n t s  of the  folic  flow t o the  placenta  and  Zinc,  a c i d , and was  folic  of n u t r i e n t s  development  are  concentrations The  radioactive  non-metabolizable  t o measure the  maternal c i r c u l a t i o n to the  known t o h a v e an  (9,10,11).  e f f e c t on  blood micro-  glucose  (methyl-cxl-D g l u c o p y r a n o s i d e  were s e l e c t e d  from the  these nutrients  determined with  and  nutritional  z i n c were measured.  a c i d , and  amino a c i d a n a l o g s  2-amino i s o b u t y r a t e )  and  In o r d e r to determine the  pregnant animals maternal plasma  of glucose,  spheres.  placental  i n r a t s consuming a l c o h o l p r i o r t o  throughout gestation. status  and  and  transfer fetus  fetal  as  growth  4  LITERATURE REVIEW Human S t u d i e s The  a) R e t r o s p e c t i v e  connection  Studies  between maternal a l c o h o l consumption and  f a u l t y development o f the o f f s p r i n g has been s i n c e e a r l y Greek and Roman mythology.  recognized  In Carthage the  b r i d a l couple was f o r b i d d e n t o consume wine on t h e i r wedding n i g h t i n order t h a t d e f e c t i v e c h i l d r e n might not be conceived (1).  S i m i l a r l y i n the nineteenth  century a r e p o r t t o t h e  House of Commons o f England by a s e l e c t committee i n v e s t i g a t i n g drunkenness i n d i c a t e d t h a t i n f a n t s born t o c h r o n i c a l c o h o l i c mothers a c g u i r e d "a s t a r v e d , s h r i v e l l e d , look"  and imperfect  (2). In 1900 S u l l i v a n r e p o r t e d i n c r e a s e d a b o r t i o n and •  s t i l l b i r t h r a t e s among c h r o n i c a l c o h o l i c women and an i n creased (12).  freguency  of epilepsy i n t h e i r surviving o f f s p r i n g  Since t h a t p e r i o d s p o r a d i c c l i n i c a l r e p o r t s have  appeared suggesting  an a s s o c i a t i o n between maternal a l c o h o l i s m  and s e r i o u s a b n o r m a l i t i e s had  i n the o f f s p r i n g (13,14), but none  s u b s t a n t i a l impact u n t i l the r e p o r t s o f Jones and Smith  (3,15).  Since then e x t e n s i v e i n v e s t i g a t i o n , both i n humans  and i n animal models, has been c a r r i e d out i n t h i s In 1973 Jones and Smith  area.  (3,15) d e s c r i b e d a p a t t e r n o f  malformations among o f f s p r i n g o f c h r o n i c a l c o h o l i c mothers and  r e f e r r e d t o t h i s r e c o g n i z a b l e p a t t e r n o f malformations  as the f e t a l a l c o h o l syndrome.  These o f f s p r i n g , of a l c o h o l i c  mothers e x h i b i t e d p r e n a t a l and p o s t n a t a l growth d e f i c i e n c y as w e l l as c r a n i o f a c i a l a b n o r m a l i t i e s short palpebral f i s s u r e s ,  including  microcephaly,  and m a x i l l a r y h y p o p l a s i a .  Joint  5  and  c a r d i a c anomalies were r e p o r t e d i n over 50 percent o f  these c h i l d r e n .  Lack o f p o s t n a t a l growth response t o h i g h  c a l o r i e feeding provided nature  f u r t h e r evidence  o f the immutable  o f the d e l e t e r i o u s p r e n a t a l e f f e c t o f growth r a t e  (15).  Assessment o f maternal n u t r i t i o n a l s t a t u s r e v e a l e d no abnorm a l i t i e s a s i d e from o c c a s i o n a l i r o n d e f i c i e n c y (15).  No  p a t t e r n o f malformation as d e s c r i b e d i n f e t a l a l c o h o l syndrome had been r e p o r t e d i n o f f s p r i n g o f i r o n  deficient  anemic women. I n t e l l e c t u a l development and motor performance was r e p o r t e d by S t r e i s s g u t h i n twelve o f f s p r i n g o f c h r o n i c a l c o h o l i c women (16).  A l l except one o f the c h i l d r e n  exhibited a borderline or retarded i n t e l l i g e n c e l e v e l . was  It  observed t h a t c h i l d r e n with the c l e a r f e a t u r e s of f e t a l  a l c o h o l syndrome were most r e t a r d e d while those with l e s s e r degrees o f dysmorphology appeared l e s s impaired.  The same  workers attempted t o b e t t e r c h a r a c t e r i z e the f e t a l a l c o h o l syndrome by a review o f the c h a r t s o f the N a t i o n a l of N e u r o l o g i c a l Disease  and Strokes  Institute  Perinatal Project  (17).  About 55,000 pregnant women were observed up t o seven years p o s t n a t a l l y and the performance and development o f t h e i r o f f s p r i n g monitored.  A l c o h o l consumption i n the mother's  c l i n i c a l r e c o r d was compared t o c h i l d development. cases  there was an i n d i c a t i o n o f severe  d u r i n g and p r i o r t o pregnancy.  In 23  chronic alcoholism  Each of these  cases was  compared w i t h c o n t r o l n o n - a l c o h o l i c women o f equal s o c i o economic s t a t u s , race, age, education,  parity,  and m a r i t a l  6  status. was  Among the a l c o h o l i c group a 43 percent  observed, as compared with a 2 percent  group.  adverse outcome  r a t e i n the  control  Four of the a l c o h o l exposed o f f s p r i n g d i e d d u r i n g  p e r i n a t a l p e r i o d and  s i x e x h i b i t e d dysmorphology  the  consistent  with the f e t a l a l c o h o l syndrome. These o f f s p r i n g a l s o d i s played  s m a l l e r growth parameters i n the newborn nursery  w e l l as at seven years of age. may  The  as  v a l i d i t y of t h i s work  be g u e s t i o n e d on the grounds t h a t c h r o n i c a l c o h o l i s m  these mothers may  have been complicated  environmental c o n d i t i o n s d e t r i m e n t a l ment o f the o f f s p r i n g .  by  c e r t a i n adverse  to the proper develop-  C h i l d r e n of d i s o r g a n i z e d ,  multiproblem  f a m i l i e s are at g r e a t e r r i s k i n terms of p s y c h o l o g i c a l intellectual  The  I.Q.  f o r c h i l d r e n l i v i n g with r e l a t i v e s  or i n f o s t e r homes averaged 84,  whereas t h a t o f c h i l d r e n  l i v i n g with t h e i r a l c o h o l i c mothers was  73  (17).  Adverse l i f e s t y l e , as w e l l as c o n t r i b u t i n g to  (18).  Elevated  s l e e p i n g h a b i t s may (15,18).  has  the p r e n a t a l e f f e c t s of a l c o h o l  consumption of c a f f e i n e , n i c o t i n e ,  and n a r c o t i c s as w e l l as i r r e g u l a r maternal d i e t a r y  fetus  postnatal  developmental r e t a r d a t i o n of o f f s p r i n g ,  been i m p l i c a t e d as aggravating exposure  and  development than are those from more s t a b l e  family units.  intellectual  of  and  compound the e f f e c t s of a l c o h o l on  the  Jones et a l . (3) found no evidence of drug  use by a l c o h o l i c mothers c o n c e i v i n g o f f s p r i n g c h a r a c t e r i z e d by  f e t a l a l c o h o l syndrome, and w h i l e most women smoked, they  were r e p o r t e d l y not heavy smokers. reported  amounts and  However the accuracy of  types of drugs consumed by  outpatients  7  is  questionable. The  with  initial  documentation of the anomalies a s s o c i a t e d  f e t a l a l c o h o l syndrome prompted a p l e t h o r a of subsequent  c l i n i c a l studies i n various  countries  t i o n i n the p a t t e r n of d e f e c t s was to the degree and nancy.  (16-26).  reported,  Large v a r i a -  possibly linked  t i m i n g of a l c o h o l consumption d u r i n g p r e g -  Small widely  spaced eyes, anteverted  nostrils,  and  a  carp-shaped mouth were common f e a t u r e s c h a r a c t e r i s t i c of f e t a l a l c o h o l syndrome. Hypothalamic and p i t u i t a r y  function i n four s i b l i n g s  e x h i b i t i n g c h a r a c t e r i s t i c s of f e t a l a l c o h o l syndrome i n v e s t i g a t e d by Root et a l . (19) hormone abnormality i n growth.  may  was  to determine whether a  account f o r the observed  reduction  Serum l e v e l s of human growth hormone, i n s u l i n ,  l u t e i n i z i n g hormone, and p a r a t h y r o i d hormone were a l l found w i t h i n normal ranges i n these s i b l i n g s .  I t was  inferred  t h a t p o s t n a t a l growth r e t a r d a t i o n a s s o c i a t e d with a l c o h o l syndrome i s not d i r e c t l y due  fetal  t o hormonal f a c t o r s .  Whether a l c o h o l a f f e c t s f e t a l hormone l e v e l s , o r a l t e r n a t i v e l y somatomedin f u n c t i o n or hormone r e c e p t o r development g e s t a t i o n remains t o be  determined.  b) P r o s p e c t i v e  Studies  Although r e t r o s p e c t i v e s t u d i e s p r o v i d e support  and  good evidence i n  of the adverse e f f e c t s of a l c o h o l on the f e t u s , i t i s  the p r o s p e c t i v e Subjects  during  study which provides  of a p r o s p e c t i v e  a more r i g o r o u s a n a l y s i s .  study are assessed  d u r a t i o n of a l c o h o l i s m p r i o r to d e l i v e r y .  as t o the s e v e r i t y The  occurance  8  of d i f f e r i n g p a t t e r n s o f d r i n k i n g such as heavy e p i s o d i c binges  a t c e r t a i n times d u r i n g g e s t a t i o n may be more r e a d i l y  determined i n t h i s type o f study.  C o r r e l a t i o n s may then be  made between the outcome o f pregnancy and extent o r p a t t e r n of a l c o h o l i s m i n the mother.  P r o s p e c t i v e s t u d i e s have been  used t o i n v e s t i g a t e the e f f e c t o f dosage, d u r a t i o n , type o f beverage consumed and the t h r e s h o l d amount o f a l c o h o l which e l i c i t s adverse e f f e c t s i n u t e r o Mau  and N e t t e r  (20)  (20,  21,  23).  c o l l e c t e d data on a l c o h o l consump-  t i o n from 5200 women a t twenty German h o s p i t a l s d u r i n g t h e f i r s t t r i m e s t e r o f pregnancy and compared a l c o h o l i n t a k e t o the w e l l - b e i n g o f the o f f s p r i n g .  Women consuming a l c o h o l  gave b i r t h t o a l a r g e r number o f o f f s p r i n g o f a g e s t a t i o n a l age  o f l e s s than 260 days, had more a b o r t i o n s , but showed  no h i g h e r i n c i d e n c e o f r e d u c t i o n i n b i r t h weight. use,  however, was e v a l u a t e d  t a b u l a t e d only as 'yes' Thus u n d e r - r e p o r t i n g may  Alcohol  s u b j e c t i v e l y as r e s u l t s were  (4.8 p e r c e n t ) , o r 'no'  (95.2  percent).  o f a c t u a l l e v e l s o f a l c o h o l consumption  have i n f l u e n c e d r e s u l t s . A p r o s p e c t i v e study by L i t t l e  (21)  compared maternal  a l c o h o l consumption o f 263 s u b j e c t s b e f o r e pregnancy, i n e a r l y , and l a t e pregnancy t o b i r t h weights o f o f f s p r i n g . In order t o c o n t r o l f o r smoking, which i s r e l a t e d t o reduced b i r t h weight  (22), s i m i l a r p r o p o r t i o n s o f smokers were  s e l e c t e d f o r each category  o f pregnancy.  height, p a r i t y , n i c o t i n e use, v a r i a b l e s entered  Maternal age,  and a l c o h o l consumption were  i n t o a r e g r e s s i o n eguation which was  computed f o r each o f the three d e f i n e d c a t a g o r i e s o f d r i n k i n g  9  r e l a t i v e t o pregnancy.  S i g n i f i c a n t regression  relationships  were found between two of these d e f i n e d p e r i o d s and weight.  birth-  D a i l y i n g e s t i o n of one ounce o f a b s o l u t e e t h y l  a l c o h o l d u r i n g the t h r e e month p e r i o d p r i o r t o pregnancy a s s o c i a t e d with a 91 g r e d u c t i o n i n b i r t h weight.  The same  amount o f a l c o h o l consumed d u r i n g l a t e pregnancy i a t e d with an average body weight o t h e r parameter  was  assoc-  decrease of 160 g.  of f e t a l h e a l t h except b i r t h weight  determined i n t h i s study and,  was  No was  s i n c e lower b i r t h weights  are  not s t r i c t l y r e l a t e d t o t e r a t o g e n i c outcome, i n t e r p r e t a t i o n of these r e s u l t s must be made w i t h c a u t i o n .  Furthermore,  the  r e l a t i v e l y s m a l l sample s i z e does not permit the robust analyses of more t r a d i t i o n a l s t a t i s t i c a l t e s t s .  However,  t h i s study takes i n t o account some of the c o m p l i c a t i v e f a c t o r s such as smoking, age, height, and p a r i t y which h i n d e r work i n t h i s area, and hence allows a b e t t e r focus on the  specific  e f f e c t s of a l c o h o l i n u t e r o . Kaminski  e t a l . (23) have r e p o r t e d on the r e l a t i o n s h i p  between a l c o h o l consumption of pregnancy  among pregnant women and outcome  from a p r o s p e c t i v e study of more than 9000 b i r t h s .  Mothers consuming more than 1.6  oz a b s o l u t e e t h y l a l c o h o l per  day were compared w i t h those d r i n k i n g l e s s . more than 1.6  Women consuming  oz per day were found t o have more s t i l l b i r t h s  and reduced i n f a n t b i r t h w e i g h t s as compared t o the  lighter  d r i n k i n g group.  in birth-  A c t u a l d i f f e r e n c e s between groups  weight of o f f s p r i n g were r e l a t i v e l y s m a l l even though attained s t a t i s t i c a l significance.  they  P l a c e n t a l weights,  p r e v i o u s l y unreported i n f e t a l a l c o h o l syndrome, were found  10  s i g n i f i c a n t l y reduced i n the h e a v i e r  drinkers.  i c a n t d i f f e r e n c e s i n the number of c o n g e n i t a l were r e p o r t e d  between groups, nor was there  i n mean g e s t a t i o n a l age.  No s i g n i f malformations  any d i f f e r e n c e  When r i s k f a c t o r s i n c l u d i n g  maternal age, p a r i t y , smoking, p r e v i o u s low b i r t h w e i g h t o f f s p r i n g , marital status,  of  and metrorrhagia were taken i n t o  account through a p p r o p r i a t e  s t a t i s t i c a l technigues,  differ-  ences between heavy' and l i g h t d r i n k e r s p e r s i s t e d with to p e r i n a t a l mortality, birthweight,  regard  and p l a c e n t a l weight.  Furthermore, types o f beverages consumed were compared w i t h outcome o f pregnancy i n those mothers d r i n k i n g over 1.6 oz of a l c o h o l p e r day.  I n f a n t s born t o women d r i n k i n g  beer e x h i b i t e d r e d u c t i o n s  i n both mean b i r t h w e i g h t  only and mean  p l a c e n t a l weight when compared with those who drank only wine.  The a c t u a l d i f f e r e n c e s i n b i r t h w e i g h t  were again  r e l a t i v e l y small and w h i l e s t a t i s t i c a l l y s i g n i f i c a n t may not be o f c l i n i c a l importance.  Further  f i n d i n g s showed t h a t the  type o f beverage consumed was r e l a t e d t o maternal s o c i o economic p o s i t i o n , thus s u g g e s t i n g t h a t environmental f a c t o r s may c o n t r i b u t e c) A l c o h o l  t o the r e d u c t i o n  Induced A l t e r a t i o n i n Maternal  Metabolism as C o n t r i b u t i n g Alcohol  i n f e t a l growth.  to Fetal  Syndrome.  Although e x t e n s i v e  documentation of f e t a l  syndrome, both i n r e t r o s p e c t i v e and p r o s p e c t i v e  alcohol s t u d i e s , has  shown a s i g n i f i c a n t a s s o c i a t i o n between maternal a l c o h o l consumption •and adverse outcome o f pregnancy, these s t u d i e s have  11  f a i l e d t o e l l u c i d a t e a mechanism o f a l c o h o l ' s Alcohol  mode o f i n s u l t .  c r o s s e s t h e p l a c e n t a and c i r c u l a t e s through t h e f e t a l  bloodstream  (24).  The o d o r o f a l c o h o l  h a s b e e n n o t e d on t h e  b r e a t h o f a baby a t b i r t h w h i c h d i s p l a y e d fetal alcohol  syndrome.  Ethyl  alcohol  characteristics of  d e t e r m i n a t i o n on c o r d  b l o o d a t b i r t h r e v e a l e d c o n c e n t r a t i o n s o f a s much a s 150 mg per  100 mL  (24).  However, a l c o h o l  h a s n o t y e t b e e n shown t o  d i r e c t l y a f f e c t f e t a l metabolism.  Because f e t a l  development  is  d e p e n d e n t on t h e r e l a t i o n s h i p b e t w e e n t h e m o t h e r a n d f e t u s ,  it  can n o t be r u l e d o u t t h a t  alcohol's  e f f e c t may be a t a s i t e  other than the fetus. A body o f e v i d e n c e i s a c c u m u l a t i n g s u g g e s t i n g alcohol  may c a u s e a p e r s i s t e n t  bolism or physiological development duration studied of  state  that  a l t e r a t i o n i n m a t e r n a l metacontributing  t o adverse  fetal  ( 2 5 , 2 8 ) . T h e s e a l t e r a t i o n s a r e a g g r a v a t e d by  and s e v e r i t y o f a l c o h o l i s m .  Shruygin  the e f f e c t s of maternal alcoholism  19 m o t h e r s .  (25) h a s  i n 42 o f f s p r i n g  Of 2 3 c h i l d r e n b o r n a f t e r t h e i r m o t h e r s h a d  developed advanced a l c o h o l i s m , many d e m o n s t r a t e d s i g n s  14 w e r e m e n t a l l y r e t a r d e d ,  and  of organic impairment o f the c e n t r a l  nervous system e a r l y i n infancy.  In contrast,  19 o f f s p r i n g  born b e f o r e t h e i r mothers had developed advanced s t a g e s o f alcoholism  demonstrated mainly vegetative,  behavioral  disorders.  and  e m o t i o n a l , and  T h e s e • d e v e l o p e d a r o u n d a g e s 9 o r 10 y e a r s  tended t o remit f o l l o w i n g  improvement i n t h e s o c i a l  e n v i r o n m e n t as a r e s u l t o f m a r r i a g e o r employment. Majewski length  (26) h a s s u g g e s t e d a r e l a t i o n s h i p  of alcohol  addiction  between  i n t h e mother and s e v e r i t y o f  12  alcohol i n s u l t i n utero.  He r e p o r t e d t h a t y o u n g e r  a r e t h e most h i g h l y a f f e c t e d by m a t e r n a l the temporal p r o g r e s s i o n that acetaldehyde,  a l c o h o l i s m due t o  of the disease.  I t was s u g g e s t e d  an a l c o h o l m e t a b o l i t e ,  f o r a l c o h o l ' s adverse e f f e c t . may d e p e n d on t h e m a t e r n a l  The e x t e n t  acetaldehyde  i s responsible o f adverse  Mean d a i l y b l o o d  increase with duration of the disease  effect  metabolism capacity,  w h i c h i s t h o u g h t t o be i m p a i r e d w i t h e x t e n d e d a l c o h o l consumption.  siblings  maternal  alcohol levels  may  a s a r e s u l t o f an  i n c r e a s e i n amounts o f a l c o h o l consumed.  Thus t h e h i g h e r  s u s c e p t i b i l i t y o f s i b l i n g s whose m o t h e r s h a d b e e n a l c o h o l i c s f o r a l o n g e r t i m e p e r i o d may be e x p l a i n e d s i m p l y a s due t o the e l e v a t e d a l c o h o l i n s u l t i n utero.  However t h i s  explana-  t i o n i s r e f u t e d by w o r k d o c u m e n t i n g d e c r e a s e d b i r t h i n o f f s p r i n g o f m o t h e r s who h a d a b s t a i n e d consumption d u r i n g pregnancy  (27,28).  weights  from a l c o h o l  Scheiner  r e p o r t e d a case w i t h c o n g e n i t a l malformations  e t al.(27)  as w e l l as  p h y s i c a l and i n t e l l e c t u a l developmental r e t a r d a t i o n i a t e d w i t h f e t a l a l c o h o l syndrome. parents,  although  possibility  I t was r e p o r t e d t h a t t h e  heavy d r i n k e r s f o r s i x t e e n years  s t a i n e d one a n d a h a l f y e a r s  assoc-  p r i o r t o conception.  had abThe  c a n n o t be r u l e d o u t t h a t t h e m o t h e r d i d i n f a c t  d r i n k while pregnant o r that the c h i l d d i s p l a y e d not f e t a l a l c o h o l syndrome b u t a s u g g e s t i v e  p h e n o t y p e w i t h some  other  etiology. Little suggesting  e t a l . (2 8) h a v e p r o v i d e d  f u r t h e r evidence  t h e e x i s t e n c e o f an a l c o h o l s u s c e p t i b l e  component, r e s p o n s i b l e a t l e a s t p a r t i a l l y ,  maternal  f o r the adverse  13  e f f e c t s i n utero.  I n f a n t s born to women who  a l c o h o l i s m p r i o r t o conception,  but had  had  a h i s t o r y of  abstained  during  pregnancy, were compared to i n f a n t s whose mothers drank h e a v i l y throughout the course of n o n - a l c o h o l i c c o n t r o l s . f o r maternal education, o f f s p r i n g , and age  of pregnancy and t o i n f a n t s  The  three groups were matched  smoking d u r i n g pregnancy, race of  of o f f s p r i n g at the time of the  study.  Mean b i r t h weights of c h i l d r e n born t o a b s t i n e n t a l c o h o l i c s was  determined as 258  g l e s s than the mean b i r t h weight of  c h i l d r e n born to c o n t r o l s . the mean b i r t h w e i g h t was p o s s i b i l i t y was  For the d r i n k i n g a l c o h o l i c group  49 3 g below t h a t of c o n t r o l s .  The  examined t h a t the a b s t i n e n t a l c o h o l i c women  may  have r e p o r t e d t h e i r d r i n k i n g i n a c c u r a t e l y , however i t  was  noted t h a t there would be no reason  purposely.  f o r them t o do  In cases where the i n t e r v i e w e r suspected  inadvert-  ent e r r o r , i n t e r n a l v e r i f i c a t i o n s were performed by the events i n the s u b j e c t ' s l i f e d u r i n g the time pregnancy, and  so  listing  surrounding  c r o s s c h e c k i n g her r e c a l l d u r i n g these  events.  In summary, a review of the c l i n i c a l s t u d i e s r e p o r t i n g the e f f e c t s of maternal a l c o h o l consumption on the outcome of pregnancy i n d i c a t e t h a t t h i s phenomenon, although  noted  historically,  has  only r e c e n t l y been e x t e n s i v e l y examined.  Retrospective  and p r o s p e c t i v e i n v e s t i g a t i o n s have as yet  f a i l e d to determine the mode of i n s u l t of a l c o h o l ,  although  the p a t t e r n of malformations c h a r a c t e r i s t i c of f e t a l a l c o h o l syndrome have been w e l l e s t a b l i s h e d .  The  extent of c o n t r i b u -  t i o n of adverse environmental f a c t o r s to f e t a l a l c o h o l  14  s y n d r o m e i s unknown. s e v e r i t y of  Duration of a l c o h o l i s m r e l a t i n g to  f e t a l i n s u l t and  appearance of  fetal  alcohol  syndrome i n o f f s p r i n g whose m o t h e r s s t o p p e d d r i n k i n g p r i o r to conception are both f a c t o r s suggesting t h a t d e l e t e r i o u s e f f e c t o f a l c o h o l may t o the  fetus.  a susceptible  not  be  A l t e r n a t i v e l y , a l c o h o l may  the  restricted directly be  deleterious  component o f m a t e r n a l p h y s i o l o g y o r  to  metabolism.  15  Animal Studies The  a) I n t r o d u c t i o n  advantage o f the animal model i n f e t a l a l c o h o l  syndrome r e s e a r c h i s t o allow i n v e s t i g a t i o n o f the uncompounded e f f e c t o f a l c o h o l on the unborn without  complicative  environmental v a r i a b l e s found i n human s t u d i e s .  E a r l y reports  e v a l u a t i n g the e f f e c t s o f maternal a l c o h o l consumption on f e t a l outcome i n the fowl and the r a t have appeared s i n c e the t u r n o f the century  (29- 34); however dubious method-  ology has o f t e n j e o p a r d i z e d the v a l i d i t y o f these i n v e s t i g a tions.  More recent s t u d i e s employing i s o c a l o r i c a l l y p a i r -  fed c o n t r o l groups, o r a l routes of a l c o h o l a d m i n i s t r a t i o n , and  dosages s i m i l a r t o those encountered i n the human a l c -  o h o l i c have been performed i n r a t s , mice, and other These improvements i n experimental  design have  species.  facilitated  the development of an animal model which c l o s e l y approximates f e t a l a l c o h o l syndrome as manifest  i n the human.  b) P r e n a t a l E f f e c t s o f A l c o h o l A d m i n i s t r a t i o n i n the Rat. Sandor and Amels  (35) made one o f the i n i t i a l  signif-  i c a n t c o n t r i b u t i o n s i n the i n v e s t i g a t i o n o f the e f f e c t o f a l c o h o l on- p r e n a t a l development i n the r a t . administered  through intravenous  A l c o h o l was  i n j e c t i o n on days s i x ,  seven, and e i g h t o f g e s t a t i o n i n one group o f animals and days s i x and seven i n another group. kg body weight were administered  Dosages of 1.5 g per  t o animals o f each group.  Fetuses were removed a t day nineteen  of g e s t a t i o n by  16  ceasarian  s e c t i o n and examined f o r dysmorphology.  doses i n j e c t e d , a l c o h o l i n t h e c o n c e n t r a t i o n body w e i g h t e l i c i t e d t w i c e  Of t h e  of 2 g per kg  t h e number o f m a l f o r m a t i o n s a s  d i d the lower concentration.  Malformations observed  included  s k e l e t a l s t r u c t u r e a n o m a l i e s , most a p p a r e n t i n t h e e x t r e m ities the  and f a c i a l  two h e a v i e r  areas.  Results  dosages a d m i n i s t e r e d  w e r e more d e l e t e r i o u s  than the three  days s i x , seven, and e i g h t were i d e n t i c a l . the  furthermore indicated  that  on days s i x and seven l i g h t e r doses g i v e n  although t o t a l  over  amounts o f a l c o h o l  I n t r a v e n o u s i n j e c t i o n does n o t r e p r e s e n t  n o r m a l mode o f a l c o h o l i n t a k e a n d f a c t o r s s u c h a s  interference increase study.  o f normal n u t r i e n t absorption  and r a t e o f  of blood alcohol l e v e l s are not considered However, r e s u l t s o b t a i n e d  malformation i n t h i s  i n this  show a s i m i l a r p a t t e r n o f  r a t m o d e l a s s e e n i n human f e t a l  alcohol  syndrome. S u b s e q u e n t w o r k i n v o l v e d t h e u s e o f an o r a l r o u t e o f alcohol administration human a l c o h o l i c .  w h i c h i s more a n a l o g o u s t o t h e c h r o n i c  T z e a n d L e e (36) f e d p r e g n a n t r a t s 30 g  a l c o h o l p e r 100 mL w a t e r a s t h e i r o n l y fluid.  A pair-feeding  w e l l as an a l c o h o l  that  d e s i g n was i n c o r p o r a t e d  drinking  w h e r e b y , as  f e d a n d ad. l i b i t u m c o n t r o l g r o u p , a t h i r d  group o f r a t s r e c e i v e d with  available  powdered d i e t and c o r n s t a r c h i s o c a l o r i c  consumed by a l c o h o l  reduced c a l o r i c intake  f e d animals.  i n the alcohol  a c c o u n t e d f o r by t h e e x i s t e n c e  The e f f e c t  of  f e d g r o u p was t h u s  o f t h i s p a i r - f e d group. A l l  a n i m a l s w e r e m a t e d one month a f t e r t h e i n i t i a t i o n  of this  17  d i e t a r y p r o t o c o l and allowed t o d e l i v e r a t term.  Gestation  p e r i o d d i d not vary among the t h r e e treatment groups.  Only  f i f t y p e r c e n t of the a l c o h o l dams known t o have c o p u l a t e d " delivered l i t t e r s .  In comparison,  88 and 91 percent of  c o n t r o l and p a i r - f e d groups,  respectively,  pregnant, produced  The average  litters.  a l c o h o l t r e a t e d mothers was of  assessed as  litter  s i z e of the  s i g n i f i c a n t l y lower than t h a t  p a i r - f e d or c o n t r o l groups.  O f f s p r i n g of the a l c o h o l f e d  group d i s p l a y e d microcephaly, cracked dry s k i n , reddening of areas of the head and body, and a g e n e r a l l y s h r i v e l e d ance.  Malformations r e p o r t e d by e a r l i e r authors  not observed.  appear-  (35) were  A p o s s i b l e c r i t i c i s m of t h i s model may  stem  from the f a c t t h a t the pregnant r a t s were allowed t o d e l i v e r at  term and c a n n i b a l i z a t i o n may  litter  size.  Furthermore  have a f f e c t e d the recorded  r e s o r p t i o n s can not be  determined.  S a c r i f i c e of the animals p r i o r t o d e l i v e r y permits g r e a t e r accuracy of assessment  of l i t t e r  s i z e and number of f e t a l  r e s o r p t i o n s through d e t e r m i n a t i o n of i m p l a n t a t i o n s i t e s a t autopsy. S t u d i e s u s i n g r a t models of f e t a l a l c o h o l syndrome do not c o n s i s t e n t l y r e p o r t a s s o c i a t i o n s between maternal a l c o h o l exposure  and f e t a l i n s u l t i n s p i t e of the  improve-  ments i n design which have emerged over the l a s t decade. Schwetz e t a l , (3 7)  found no p r e n a t a l e f f e c t s i n r a t s given  d r i n k i n g f l u i d c o n t a i n i n g 12 t o 20 percent a l c o h o l . l e v e l of a l c o h o l consumption to  elicit  the anomalies  may  This  have been i n s u f f i c i e n t  observed by other workers.  Peak  18  blood a l c o h o l l e v e l s were r e p o r t e d lower than values r e p o r t e d (38)  to be  40 mg  by other workers.  per  100  mL,  Oisund et a l .  a l s o found no s i g n i f i c a n t d i f f e r e n c e s i n body, b r a i n ,  liver,  kidney, or h e a r t muscle weights between f e t u s e s  c o n t r o l and  a l c o h o l exposed pregnant r a t s .  of  Animals were  fed 12 percent a l c o h o l i n t h e i r d r i n k i n g water to cover 25 percent of t h e i r c a l o r i c i n t a k e .  Alcohol  exposure  extended from three weeks p r i o r to mating, during, a f t e r pregnancy.  Soya o i l was  Abel  (39)  and  low  Abel and  substitute  Again the observed absence  of adverse e f f e c t s of i n utero a t t r i b u t a b l e t o the  and  used as an a l c o h o l  i n the p a i r - f e d group animals.  20-  a l c o h o l exposure may  be  dosage l e v e l i n t h i s model. S i m i l a r l y , Greizerstein  (40)  showed t h a t when  pregnant r a t s were given a l c o h o l by stomach tube throughout pregnancy, no abnormality i n growth of o f f s p r i n g could be a t t r i b u t e d t o doses of 1 or 2 g per kg body weight, f o l l o w i n g the a d m i n i s t r a t i o n  of 4 or 6 g per kg,  b i r t h weight, delayed p o s t n a t a l m o r t a l i t y occurred c o n t r o l progeny.  growth, and  diminished  greater  postnatal  i n a l c o h o l exposed o f f s p r i n g compared t o Generally,  when s u f f i c i e n t dosage l e v e l s  are a d m i n i s t e r e d to pregnant r a t s , s t u d i e s f e t a l outcome.  Henderson et a l . (41)  that alcohol administration  report  adverse  have a l s o demonstrated  i n a l i q u i d d i e t to r a t s  during  pregnancy i s a s s o c i a t e d w i t h adverse e f f e c t s on f e t a l b i l i t y and reported  growth.  but  via-  Maternal b l o o d a l c o h o l l e v e l s were  as 70-200 mg  per  100  mL,  s u b s t a n t i a l l y greater  than those of Schwetz et a l . (37), where no adverse e f f e c t s  19  were noted.  These s t u d i e s suggest t h a t a t h r e s h o l d  of b l o o d a l c o h o l may  be  r e g u i r e d to produce v i s i b l e e f f e c t s .  Although much i n t e r e s t i n maternal a l c o h o l has  level  c e n t e r e d around the f e t u s , the p l a c e n t a  has  consumption recently  a l s o become the focus of a t t e n t i o n i n the r a t model of f e t a l a l c o h o l syndrome. increased  Abel and  Greizerstein  (40)  found  p l a c e n t a l weights i n a l c o h o l exposed pregnant r a t s  when compared with c o n t r o l s , however d i f f e r e n c e s were not s t a t i s t i c a l l y s i g n i f i c a n t . Wiener e t a l . (42), (43) , and heavier (44)  Gordon et al.(44) have r e p o r t e d  placentas  derived  significantly  i n a l c o h o l exposed r a t s .  fed rats a l i g u i d d i e t containing c a l o r i e s f o r two  Skosyreva  Gordon et a l .  26 percent  weeks p r i o r to and  alcohol  during  F e t a l weights d i d not vary between a l c o h o l f e d and groups s u g g e s t i n g t h a t the i n c r e a s e on day  20 of g e s t a t i o n may  gestation. control  i n p l a c e n t a l s i z e found  e x i s t as an adaptive mechanism  compensating f o r the d e l e t e r i o u s e f f e c t of a l c o h o l . c) P r e n a t a l  E f f e c t s of A l c o h o l  Administration  i n the Mouse The subjects  malformations observed i n human f e t a l  alcohol  appear to be more e a s i l y e l i c i t e d i n the mouse  model of maternal a l c o h o l consumption than i n the r a t model. Kronick  (45)  employed a mouse model t o study the e f f e c t s of  acute a l c o h o l exposure on the outcome of pregnancy. treatment c o n s i s t e d of an i n t r a p e r i t o n e a l  Alcohol  i n j e c t i o n of a  25 percent s o l u t i o n of e t h y l a l c o h o l at a does of 3 mL  per  20  100  g body weight.  The s t r e n g t h of t h i s dose induced  hypnosis w i t h animals remaining comatose f o r up t o f o u r hours f o l l o w i n g treatment.  One group of mice r e c e i v e d i n j e c t i o n s  on the e i g h t h and n i n t h day o f g e s t a t i o n , the t e n t h and eleventh  day o f g e s t a t i o n , and a t h i r d group  on only one g e s t a t i o n a l day ranging day  twelve.  another group on  from day seven through  C o n t r o l animals were i n j e c t e d with a s a l i n e  s o l u t i o n and matched with a l c o h o l i n j e c t e d dams. s a c r i f i c e d on g e s t a t i o n a l day 18 and u t e r i n e examined.  Results  Mice were  contents  indicated s u b s t a n t i a l l y elevated  fetal  m o r t a l i t y r a t e s i n day e i g h t and nine as w e l l as day t e n and eleven  a l c o h o l exposed mice as compared t o c o n t r o l s  i n j e c t e d with s a l i n e .  Malformation frequency was  i n a l c o h o l - t r e a t e d groups with the genesis  increased  of s p e c i f i c  malformations dependent on tne time o f i n j e c t i o n d u r i n g gestation.  Coloboma of the i r i s  and e c t r o d a c t y l y o f the  forepaws appeared t o have c r i t i c a l p e r i o d s  at days e i g h t t o  nine and a t day ten, r e s p e c t i v e l y , of g e s t a t i o n . i n t r a p e r i t o n e a l route o f a l c o h o l a d m i n i s t r a t i o n  The used i n  t h i s model i s not c o n s i s t e n t w i t h the o r a l consumption p a t t e r n as seen i n human a l c o h o l i s m , i n methodology.  and may e x i s t as a flaw  A subsequent study by Chernoff  a mouse model employed an o r a l route o f a l c o h o l  (46) u s i n g administra-  t i o n as w e l l as p a i r - f e d c o n t r o l groups. Chernoff two  (46) f e d a l c o h o l through an a l l l i q u i d d i e t t o  s t r a i n s o f mice.  A l c o h o l treatment extended from t h i r t y  days p r i o r t o mating u n t i l day 18 of g e s t a t i o n ,  a t which time  females were s a c r i f i c e d and u t e r i n e contents examined.  One  21  s t r a i n of mice e x h i b i t e d s i g n i f i c a n t l y more f e t a l malformat i o n s than the other, metabolism may  suggesting  t h a t the r a t e of a l c o h o l  d i f f e r between the two  strains.  a l c o h o l dehydrogenase system, r e s p o n s i b l e of a l c o h o l , i s known t o be under g e n e t i c mouse (47).  The  liver  f o r the metabolism c o n t r o l i n the  f e t a l malformations i n c l u d e d i n t r a u t e r i n e  growth d e f i c i e n c y as evidenced by low plete ossification,  n e u r a l and  s k e l e t a l dysmorphogenesis. t i o n together  The  f e t a l weight, incom-  c a r d i a c anomalies,  The  and  p a t t e r n of growth r e t a r d a -  with o c u l a r , n e u r a l ,  c a r d i a c , and s k e l e t a l  anomalies were s i m i l a r t o t h a t of the f e t a l a l c o h o l syndrome observed i n humans. Another animal model employing an o r a l l y a l c o h o l treatment and developed by R a n d a l l administered  a p a i r - f e e d i n g design and  Taylor  d i e t s w i t h 17,  25,  (48).  gestation. was  was  Pregnant mice were of t o t a l  f i v e to day  ten  daily of  I s o c a l o r i c s u b s t i t u t i o n of sucrose f o r a l c o h o l  e f f e c t e d i n the p a i r - f e d group.  dams was  i n mice  or 35 percent  c a l o r i e s s u p p l i e d by a l c o h o l from day  administered  made on day  i n the 25 percent  S a c r i f i c e of the  19 of g e s t a t i o n .  Results  at l e a s t one  malformed f e t u s .  e x t e r n a l examination of the f e t u s e s was  ed i n c l u d e d malformations of the  limbs,  Systematic  Anomalies observ-  cardiovascular  u r o g e n i t a l hydronephrosis, and  As w i t h the study of Chernoff  of  f o l l o w e d by i n t e r n a l  examination under a d i s s e c t i n g microscope.  abnormalities,  showed t h a t  a l c o h o l - c a l o r i e group, f i f t e e n out  s i x t e e n l i t t e r s had  gravid  hydrocephaly.  (46), the anomalies found  i n t h i s mouse model are analagous with those of human o f f s p r i n g  22  d i s p l a y i n g f e t a l a l c o h o l syndrome. Lack o f p r e n a t a l e f f e c t s o f a l c o h o l i n mice has a l s o been r e p o r t e d  ( 3 7 , 4 9 ) , p o s s i b l y due, as seen i n the r a t  model, t o an i n s u f f i c i e n t l e v e l of exposure t o a l c o h o l . Determination of the l e v e l of a l c o h o l exposure i s a c u r r e n t m e t h o d o l o g i c a l problem i n animal models of f e t a l syndrome.  Comparisons o f a l c o h o l i n t a k e between man and  animals a r e d i f f i c u l t ,  plagued by problems r e l a t i n g t o  d i f f e r i n g alcohol metabolizing ingested  alcohol  rates.  Measurement o f  e t h y l a l c o h o l expressed as g per kg o f body weight  per day i s used by some workers, however serum a l c o h o l l e v e l s presently  are most w i d e l y accepted.  The use of serum a l c o h o l  l e v e l s as i n d i c a t o r s o f a l c o h o l i n t a k e obviates c u l t i e s acguired  because o f d i f f e r i n g a l c o h o l pathways and  m e t a b o l i c r a t e s among animal The  the d i f f i -  species.  preceding studies i n d i c a t e that using a s u i t a b l e  animal model o f maternal a l c o h o l exposure, t h e p a t t e r n o f malformation seen i n o f f s p r i n g born t o humans are b e t t e r approximated i n the mouse than i n the r a t .  However, c l e a r  cut adverse e f f e c t s i n c l u d i n g p r e n a t a l growth r e t a r d a t i o n are observed i n the r a t , and the use o f c e r t a i n experimental p r o t o c o l s makes this l a r g e r animal a more a t t r a c t i v e experimental  subject. d) P r e n a t a l  E f f e c t s of Alcohol  Exposure i n  Other Animals Although most o f f e t a l a l c o h o l syndrome r e s e a r c h  using  23  animal models has focused on r a t s and mice, o t h e r have a l s o been the s u b j e c t  of i n v e s t i g a t i o n .  species  Simulating  blood a l c o h o l l e v e l s which would be normal i n the human alcoholic,  Sandor and E l i a s  (50) s t u d i e d the e f f e c t s o f  a l c o h o l i n chick embryos i n e a r l y stages of development.  In  eggs i n j e c t e d w i t h a l c o h o l embryos e x h i b i t e d h i g h e r m o r t a l i t y r a t e s and weight l o s s towards the end of i n c u b a t i o n pared w i t h c o n t r o l s .  However, e x t r a p o l a t i o n  as com-  from the develop-  ment o f the c h i c k embryo t o t h a t o f the embryo i n utero i s tenuous, hence i n most subsequent work mammals were used as experimental  subjects.  Dexter e t a l . (51) f e d miniature S i n c l a i r swine a l c o h o l s o l u t i o n s o r a l l y both p r i o r t o and d u r i n g  gestation.  swine consume l a r g e q u a n t i t i e s o f a l c o h o l d e s p i t e d i e t and c o n s t a n t l y  a v a i l a b l e water, a l l o w i n g  Miniature  adequate  investigation  i n t o the e f f e c t s of prenatal e f f e c t s of a l c o h o l  consumption  i n the presence o f adequate n u t r i t i o n and without d i s t u r b ing  an e s t a b l i s h e d b e h a v i o r .  Results  showed t h a t  by pregnant swine of a l c o h o l a t l e v e l s e q u i v a l e n t  consumption t o one  quart of whiskey p e r day i n the human r e s u l t e d i n a decrease in  l i t t e r s i z e and b i r t h weight, as w e l l as i n c r e a s e d  rates  of occurance of m o r p h o l o g i c a l anomalies and p o s t n a t a l  mort-  ality.  Anomalies observed were s i m i l a r t o those seen i n  human f e t a l a l c o h o l syndrome Ellis  and P i c k  subjects.  (52) used a beagle model t o study the  e f f e c t s o f maternal a l c o h o l consumption on the f e t u s . A l c o h o l was a d m i n i s t e r e d through g a s t r i c i n t u b a t i o n a t  24  s i x d i f f e r e n t dosage l e v e l s i n an attempt to determine a threshold  l e v e l f o r onset of adverse e f f e c t s .  With i n c r e a s -  i n g dosage a h i g h e r occurance of f e t a l m o r t a l i t y and mal- • formation was  observed, with no animal i n the group exposed  t o a l c o h o l at the g r e a t e s t offspring. per 100 1.4  dosage l e v e l d e l i v e r i n g l i v e born  Blood a l c o h o l l e v e l s of t h i s group reached 255  mL.  /Animals consuming the lowest of the  dosages,  g e t h y l a l c o h o l per kg body weight, d i s p l a y e d no  f i c a n t l y increased  r a t e o f malformation or  subject,  signi-  stillbirth,  however p r o g r e s s i v e l y h i g h e r dosages were found to f e t a l w e l f a r e .  detrimental  E x t r a p o l a t i n g these r e s u l t s t o the human  i f a woman weighing 60 kg were to consume 6 oz  100  proof a l c o h o l per day  her  f e t u s may  during  be at r i s k .  The  a l c o h o l syndrome research  gestation,  use  the dog,  of the beagle i n f e t a l  man  however r e s t r i c t i o n s i n group s i z e inherent  certain s t a t i s t i c a l e t a l . (53)  of  i s advantageous i n t h a t a c l o s e  u s i n g an animal of t h i s s i z e may  Ho  of  the w e l l b e i n g  s i m i l a r i t y i n a l c o h o l pharmacokinetics e x i s t s between and  mg  l i m i t the u s e f u l n e s s  in  of  treatments. f e d a l c o h o l t o pregnant monkeys  and  hamsters i n order t o determine i t s d i s t r i b u t i o n i n the maternal and The  f e t a l blood, organs and  c e n t r a l nervous system.  d i s t r i b u t i o n of a l c o h o l i n the p l a c e n t a  and  amniotic  14 f l u i d was  also studied.  ed i n t r a v e n o u s l y  E t h a n o l l a b e l e d with  at doses ranging from 0.5  C was  to 1.5  body weight f o l l o w i n g which animals were k i l l e d by at f i x e d time i n t e r v a l s . and p l a c e n t a l s e c t i o n s was  inject-  g per  bleeding  Radioautography of the b r a i n , performed, and  kg  fetal,  a l c o h o l determinations  25  made on h o m o g e n a t e s o f t h e s e t i s s u e s . that during fluid  Results  l a t e pregnancy both t h e p l a c e n t a  contained  high  concentrations  d i s t r i b u t i o n t o other tissues.  indicated  and  amniotic  o f a l c o h o l compared  with  C o n c e n t r a t i o n s o f unmeta-  b o l i z e d a l c o h o l i n t h e h a m s t e r f e t u s was h i g h e r  i n later  pregnancy than p r i o r t o p l a c e n t a l development,  suggesting  that the placenta  was n o t a b a r r i e r t o a l c o h o l .  Alcohol  m e t a b o l i s m i n t h e monkey was s l o w e r i n t h e f e t u s t h a n i n t h e m o t h e r , a s i n d i c a t e d by h i g h e r fetal  alcohol concentrations  in  t i s s u e s 90 m i n u t e s a f t e r i n j e c t i o n o f t h e d o s e .  a l c o h o l dehydrogenase l e v e l s a r e r e p o r t e d  Fetal  t o be r e d u c e d when  compared w i t h m a t e r n a l l e v e l s , p o s s i b l y a c c o u n t i n g f o r t h i s higher  f e t a l accumulation of alcohol  ( 5 4 ) . The "^C  labeled  a l c o h o l i n t h e monkey f e t u s was l o c a l i z e d i n t h e l i v e r , pancreas, kidney,  lung,  and h e a r t muscle w i t h  ration i n the cerebellum. geniculate  a high  concent-  The h i p p o c a m p u s , putamen, a n d  body were a l s o s i t e s o f a c c u m u l a t i o n .  malformation i n a l c o h o l exposed fetuses  No o b s e r v e d  was r e p o r t e d  i n this  study. e)  Comparison o f P r e n a t a l Prenatal in  The  Alcohol  Exposure and  M a l n u t r i t i o n on Body C o m p o s i t i o n  Offspring.  mechanism by w h i c h m a t e r n a l a l c o h o l  a f f e c t s f e t a l growth remains unclear.  Studies  consumption reporting  absence o f a s s o c i a t i o n between a l c o h o l consumption p r e g n a n c y and a d v e r s e outcome s u g g e s t t h a t nutrition  i s at least i n part  responsible  during  inadequate f o r the deleterious  26  e f f e c t i n utero  (37,38).  However d i f f e r e n c e s i n body com-  p o s i t i o n of o f f s p r i n g exposed to a l c o h o l i n u t e r o as compared t o food r e s t r i c t e d p a i r - f e d o f f s p r i n g (40,41) suggest t h a t these growth r e t a r d i n g e f f e c t s are not s i m i l a r . i n b r a i n , heart, kidney,  and  Reductions  l i v e r weights were observed i n  f e t u s e s exposed t o a l c o h o l i n utero  (41).  Moreover,  signif-  i c a n t l y e l e v a t e d t o t a l t i s s u e p r o t e i n c o n c e n t r a t i o n s were found i n 20 day  f e t u s e s i n a r a t model of a l c o h o l exposure  i n utero  These r e s u l t s are c o n t r a r y t o  (41).  observations  i n o f f s p r i n g o f malnourished r a t s where l e a n body mass i s g e n e r a l l y reduced or u n a f f e c t e d  (55) .  Abel and G r e i z e r s t e i n (40) have a l s o d e s c r i b e d  metabolic  changes i n growth r e t a r d e d r a t f e t u s e s as a r e s u l t o f a l c o h o l exposure i n u t e r o . was  In the f e t u s e s whole body sodium  content  s i g n i f i c a n t l y e l e v a t e d i n the a l c o h o l t r e a t e d group.  d i f f e r e n c e s were noted i n whole body z i n c , calcium, magnesium content  of f e t u s e s between a l c o h o l and  r e s t r i c t e d p a i r - f e d groups. content  No  or  food  E l e v a t e d whole body sodium  i s not c h a r a c t e r i s t i c of the malnourished r a t  Furthermore, no decrease i n f a t content  (56).  of the a l c o h o l  exposed f e t u s , as i s found i n the f e t u s e s of malnourished r a t s , was  r e p o r t e d i n t h i s study  (40).  These r e s u l t s suggest  t h a t m a l n u t r i t i o n i s not s o l e l y r e s p o n s i b l e f o r the a s s o c i a t e d w i t h i n u t e r o exposure t o a l c o h o l . f) Comparison of Growth and Development i n O f f s p r i n g of A l c o h o l - F e d Malnourished  Animals.  Versus  effects  27  Although d i f f e r e n c e s r e p o r t e d between f e t u s e s rats  (40,41), the  i n body composition have been of a l c o h o l exposed and  s i m i l a r i t y i n the p a t t e r n  developmental r e t a r d a t i o n e f f e c t s may  malnourished  of growth  i n each case suggests t h a t  share a common e t i o l o g y .  Studies  the e f f e c t s of maternal p r o t e i n d e f i c i e n c y  and these  investigating  on n e o n a t a l growth  show s t r i k i n g s i m i l a r i t i e s t o those on the adverse e f f e c t of alcohol. fostered  Young born to p r o t e i n  r e s t r i c t e d mothers but  t o c o n t r o l mothers a t b i r t h e x h i b i t a  retardation  i n growth, which i s not  l i t t e r size  (55).  and  corrected  cross  persistent by  reducing  Delayed appearance of o s s i f i c a t i o n c e n t e r s  r e t a r d e d s k e l e t a l growth were observed w i t h l i t t l e  c a t i o n of c a t c h up growth.  Leichter  and  Leichter  (8), and  D e t e r i n g et a l . (57)  findings  i n a l c o h o l exposed o f f s p r i n g .  Lee  (7), Lee  indiand  have r e p o r t e d s i m i l a r In a d d i t i o n  to retarded  growth, o f f s p r i n g of animals r e c e i v i n g a l c o h o l d u r i n g pregnancy a l s o e x h i b i t a r e t a r d a t i o n maturation, d e s p i t e  the  alcohol after delivery in  i n the  r a t e of s k e l e t a l  f a c t t h a t mothers d i d not (7).  Catch up was  not  growth or s k e l e t a l maturation d u r i n g the  postnatally.  Furthermore, c u l l i n g the  receive  observed e i t h e r  first  f o u r weeks  l i t t e r s i z e from  to three of a l c o h o l exposed o f f s p r i n g t o account f o r p o s s i b i l i t y of p o s t n a t a l s i g n i f i c a n t stimulatory  malnutrition e f f e c t on any  p h y s i c a l growth or maturation measured b r a i n weight, crown rump l e n g t h  and  had  no  the  statistically  of the parameters (8).  eight  of  A reduction  in  t a i l l e n g t h were observed  at t h r e e weeks post partum i n r a t pups exposed t o a l c o h o l u t e r o but weaned t o c o n t r o l dams compared w i t h pups of  in  28  equivalent  age from p a i r - f e d dams (57).  These s t u d i e s i n d i c a t e t h a t the p o s t n a t a l  growth r e t a r d -  a t i o n observed i n f e t a l a l c o h o l syndrome animal models i s s i m i l a r t o t h a t a s c r i b a b l e t o maternal p r o t e i n  malnutrition.  This i s c o n t r a r y  t o i n d i c a t i o n s based on body composition  determinations.  The observed p o s t n a t a l growth r e t a r d a t i o n  appears t o be of d i f f e r e n t o r i g i n because  of r e p o r t s  suggest-  i n g t h a t , w h i l e i n p r o t e i n malnourished r a t s growth hormone synthesis  i s impaired (55), no growth hormone  have been observed i n humans d i s p l a y i n g f e t a l syndrome (58).  Rosso and Kava  alcohol  (59) have suggested t h a t the  mechanism of f e t a l i n s u l t from m a l n u t r i t i o n a decreased m a t e r n a l - f e t a l  abnormalities  r e s u l t s from  t r a n s f e r of n u t r i e n t s .  Although  the use of p a i r - f e e d i n g designs i n animal models of f e t a l a l c o h o l syndrome has i n d i c a t e d t h a t maternal food c i e n c y i s not e n t i r e l y r e s p o n s i b l e growth, a l c o h o l may  adversely  f o r the r e t a r d e d  insuffifetal  a f f e c t the supply of n u t r i e n t s  t o the f e t u s , thus mimicking the e f f e c t of m a l n u t r i t i o n . T h i s may  e x p l a i n the s i m i l a r i t i e s observed i n modes of a c t i o n  of these growth r e t a r d i n g f a c t o r s .  29  MATERIALS AND a) One  METHODS  Preliminary  h u n d r e d and  (Biobreeding  Treatment of  Animals  twenty v i r g i n Sprague-Dawley  Laboratories,  rats  Queb. Canada) w e i g h i n g 200-225 g  were i n d i v i d u a l l y h o u s e d i n s c r e e n b o t t o m cages.  The  living  o e n v i r o n m e n t was dark c y c l e .  m a i n t a i n e d a t 21  A f t e r a one  (v/v)  Group 1  (alcohol)  Chow) ad  Group 2  with  ( p a i r - f e d ) was  group 1 animals d u r i n g  Group 3  w a t e r was  weeks on  this  The  increased  to  amounts  day  t o 30 p e r c e n t .  d e t e r m i n e d w e e k l y and  20 of  Chow) e q u a l t o the  that  previous 2 4 hours, corn  starch.  allowed  the  libitum.  nutritionally After  p r e s e n c e of sperm i n  a l c o h o l content of the  increased  ethyl  four  regimen a l l a n i m a l s were b r e d o v e r n i g h t  v a g i n a l washings e s t a b l i s h e d  was  was  d r i n k i n g w a t e r ad  male Sprague-Dawley r a t s .  time the  alcohol  t o t h e p a i r - f e d g r o u p ad l i b i t u m .  (ad l i b i t u m c o n t r o l )  a d e q u a t e d i e t and  week t h e  substituted with  provided  treatment  adequate d i e t  administered  ( P u r i n a Rat  alcohol isocalorically  Drinking  A f t e r one  light-  animals  10 p e r c e n t e t h y l  d r i n k i n g w a t e r was  n u t r i t i o n a l l y adequate d i e t consumed by t h e  dietary  a nutritionally  libitum.  a l c o h o l c o n t e n t of the percent.  of three  received  i n d r i n k i n g w a t e r and  ( P u r i n a Rat  a twelve hour  week s t a b i l i z a t i o n p e r i o d  w e r e a s s i g n e d a t random t o one groups.  C with  1 of pregnancy.  At  to the that  a l c o h o l group d r i n k i n g water  M a t e r n a l body w e i g h t s w e r e  f o o d and  determined d a i l y throughout the  a l c o h o l consumption experiment.  was  30  b) D e t e r m i n a t i o n o f P l a s m a A l c o h o l , G l u c o s e , F o l i c A c i d , and Z i n c . B l o o d samples maternal taken  (300-400  plasma a l c o h o l ,  uL) f o r t h e d e t e r m i n a t i o n o f  glucose, f o l i c acid,  from t h e t i p o f t h e t a i l  t h e t h i r d week o f g e s t a t i o n .  e a r l y i n the morning d u r i n g The b l o o d s a m p l e s w e r e  ed i n t o h e p a r i n i z e d h e m a t o c r i t t u b e s , rpm  and z i n c were  collect-  c e n t r i f u g e d a t 1500  f o r 5 m i n . , a n d t h e p l a s m a was p l a c e d i n s m a l l  plastic  o  tubes. of  The p l a s m a was k e p t  f r o z e n a t - 5 " C. u n t i l  the time  assay. Plasma a l c o h o l c o n c e n t r a t i o n s were d e t e r m i n e d  a l c o h o l dehydrogenase method  (Sigma C h e m i c a l  Saint Louis, Missouri, Technical B u l l e t i n Glucose  d e t e r m i n a t i o n s were p e r f o r m e d  o x i d a s e method No. 5 1 0 ) .  (Sigma C h e m i c a l  Folic  by the  Company,  No.  331-UV).  using the glucose  Company, T e c h n i c a l  Bulletin  a c i d was m e a s u r e d b y t h e r a d i o a s s a y  Bio-Rad  L a b o r a t o r i e s , Richmond, C a l i f o r n i a ,  Folate,  Bulletin  No. 4 2 0 1 ) ,  absorption spectrophotometry Spectrophotometer,  (Quanta-Count  a n d z i n c was d e t e r m i n e d  of  Determination  and b l o o d f l o w t o t h e p l a c e n t a and o t h e r  and N i e s  (60) and Bruce  g e s t a t i o n t h e r a t s were a n e s t h e t i z e d by  injection  atomic  Model 603).  organs were measured w i t h r a d i o a c t i v e m i c r o s p h e r e s c r i b e d by McDevitt  by  (Perkin-Elmer Atomic Absorption  c) P l a c e n t a l B l o o d F l o w Cardiac output  from  o f 10 mg p e r 100 g b o d y w e i g h t  (61).  as d e s On day 20  intraperitoneal  of Inactin  31 (Promonta, Hamburg, Germany).  A c a n n u l a (PE 10,  Intramedic,  C l a y Adams Co., P a r s i p p a n y , N.J.) was then i n s e r t e d i n t o t h e left  f e m o r a l a r t e r y and a t t a c h e d t o a w i t h d r a w a l pump  (Harvard A p p a r a t u s ,  South N a t i c k , Mass.) t o draw a r e f e r e n c e  sample f o r c a l c u l a t i o n o f t h e b l o o d f l o w r a t e . cannula was  A second  (PE 50, I n t r a m e d i c , C l a y Adams Co., P a r s i p p a n y , N.J.)  i n t r o d u c e d i n t o the l e f t v e n t r i c l e v i a t h e r i g h t  artery  f o r i n j e c t i o n o f the m i c r o s p h e r e s .  carotid  The v e n t r i c u l a r  l o c a t i o n was determined by o b s e r v i n g the c h a r a c t e r i s t i c p r e s s u r e waves u s i n g a p r e s s u r e t r a n s d u c e r and c h a r t r e c o r d e r (Harvard A p p a r a t u s , M i l l i s , Mass.) connected t o t h e cannula of the r i g h t c a r o t i d a r t e r y .  Both cannulas were  filled  with heparinized saline. Microspheres  (New England N u c l e a r , Boston, Mass.)  l a b e l e d w i t h ^ C o and w i t h a mean diameter o f 15 microns were suspended i n 6 p e r c e n t d e x t r a n and mixed t h o r o u g h l y p r i o r t o i n j e c t i o n i n t o the l e f t v e n t r i c l e .  About 40,000 m i c r o s p h e r e s  i n a t o t a l volume o f 0.2 mL were i n j e c t e d over a p e r i o d o f 20 seconds.  S i m u l t a n e o u s l y the r e f e r e n c e sample o f b l o o d  from t h e f e m o r a l a r t e r y was withdrawn a t a r a t e o f 0.445 ml/minute f o r 90 seconds. F o l l o w i n g t h e c o l l e c t i o n o f the r e f e r e n c e sample the r a t was  k i l l e d by i n c i s i o n o f the h e a r t .  Fetuses, placentas,  h e a r t , k i d n e y s , and a muscle sample were i m m e d i a t e l y removed from the c a r c a s s , l i g h t l y  blotted,  and weighed.  Only  o f f o u r f e t u s e s o r more were i n c l u d e d i n t h e study.  litters The  p o s i t i o n o f t h e c a r o t i d cannula was c o n f i r m e d a t t h i s time by d i s s e c t i o n o f the h e a r t .  A l l tissues  i n c l u d i n g the  32  r e f e r e n c e b l o o d sample were a s s a y e d a gamma c o u n t e r 600-120).  for radioactivity i n  (Picker Nuclear, North  Cardiac output  organs were c a l c u l a t e d  H a v e n , Conn. M o d e l  and b l o o d f l o w t o p l a c e n t a s and  using the following  C a r d i a c Output = CPM i n j e c t e d (mL/min)  formulae:  X r e f e r e n c e sample withdrawal r a t e r e f e r e n c e sample CPM  (mL/min)  Organ Blood = r e f e r e n c e sample withdrawal r a t e (mL/min) X organ CPM Flow (mL/min) r e f e r e n c e sample CPM  To  investigate  the p o s s i b i l i t y  that  the level of alcohol  e x p o s u r e i n t h i s s t u d y may r e s u l t i n d e h y d r a t i o n , t h e w a t e r c o n t e n t o f t h e m u s c l e s a m p l e was d e t e r m i n e d constant weight  i n a vacuum o v e n a t 70°C.  by h e a t i n g t o U s i n g a sample o f  a r t e r i a l blood c o l l e c t e d p r i o r to s a c r i f i c e of the animal, another  parameter o f dehydration, plasma o s m o l a l i t y ,  determined  u s i n g an o s m o m e t e r ( P r e c i s i o n  Mass.) .  ...  S y s t e m I n c . , Newton,  ..  d) F e t a l U p t a k e o f F o l i c A c i d , Analogs o f Glucose To analogs  was  determine  Z i n c , and  and Amino  Acid  the f e t a l uptake o f f o l i c  o f glucose  a n d amino a c i d ,  acid,  z i n c , and  r a t s o n d a y 20 o f  gestation  were a n e s t h e t i z e d w i t h e t h e r and i n j e c t e d w i t h e i t h e r : methyl 7,  ( o<- D {U  9 - H folic  ±H  -  acid,  cjgluco) pyranoside  1 uCi  a n d 10 u C i 3', 5',  o r 1 j i C i 2 - a m i n o ( 1 - C) i s o b u t y r i c  65 acid, All  or 8 pCi  Zn i n HC1 s o l u t i o n , p e r 100 g b o d y  compounds w e r e i n j e c t e d  into the l e f t  femoral  weight.  vein.  N i n e t y minutes a f t e r t h e i n j e c t i o n t h e f e t u s e s and maternal l i v e r w e r e r e m o v e d , w e i g h e d , a n d b l o o d was o b t a i n e d f r o m t h e  33  dam b y h e a r t time p o i n t  puncture.  The d e c i s i o n t o u s e t h e 90 m i n u t e  a f t e r l a b e l e d compound i n j e c t i o n was b a s e d on a  preliminary  study which i n d i c a t e d that  l a b e l i n t h e f e t u s was h i g h e r 40 o r 60 m i n u t e s . and  R a d i o a c t i v i t y was  determined i n plasma  l i v e r o f t h e dam a n d i n f o u r f e t u s e s  L i t t e r s with eight study.  fetuses  located i n positions  f r o m t h e t e r m i n a l end.  o r more w e r e i n c l u d e d i n t h e  To m e a s u r e t h e r a d i o a c t i v i t y o f t r i t i u m and  l i v e r and f e t u s e s were homogenized i n 9 p a r t s  d i s t i l l e d w a t e r and c e n t r i f u g e d  of  a t 2,000 rpm f o r 10 m i n .  A l i q u o t s o f t h e s u p e r n a t a n t f r a c t i o n s (0.5 mL) w e r e to glass fluid  of  a t 90 m i n u t e s t h a n a t e i t h e r  1 and 3 o f e a c h u t e r i n e h o r n c o u n t i n g  the  the concentration  scintillation  vials  containing  10 mL o f  (ASC, Amersham, A r l i n g t o n H e i g h t s ,  activity  was  scintillation  111.) a n d t h e r a d i o - „  counted i n a P i c k e r Nuclear Liquimat  t i o n Counter.  Corrections  transfered  Scintilla-  f o r q u e n c h i n g w e r e made b y t h e 65  c h a n n e l s r a t i o method. livers was  In the case o f  and plasma were p l a c e d  counted using  Spectrometer.  i n t e s t tubes and  a Picker Nuclear Autowell  e) S t a t i s t i c a l  variance,  radioactivity  I I Gamma  A l l t h e . . r a d i o a c t i v e compounds w e r e p u r c h a s e d  f r o m Amersham, A r l i n g t o n H e i g h t s ,  Results  Zn t h e f e t u s e s ,  Analysis  111. of  Results  were examined s t a t i s t i c a l l y by a n a l y s i s o f  using  t h e Midas Computer Package ( 6 2 ) .  case o f s i g n i f i c a n t  d i f f e r e n c e s , Sheffe's  In the  intervals  0.95) w e r e u s e d w i t h p a i r e d c o m p a r i s o n s t o i d e n t i f y  (0.9 and which  34  group was cance  responsible  o f P < 0 . 0 5 was  f o r the d i f f e r e n c e . used f o r a l l t e s t s .  A  l e v e l of  signifi-  35  RESULTS a) Food I n t a k e and M a t e r n a l Body Weights T a b l e 1 shows t h e average d a i l y food and a l c o h o l  consump-  t i o n and weekly body w e i g h t s o f r a t s g i v e n a l c o h o l , p a i r - f e d , o r ad l i b i t u m f e d p r i o r t o pregnancy.  A s i d e from the f i r s t  week o f t h i s p e r i o d body w e i g h t s o f ad l i b i t u m c o n t r o l a n i m a l s were s i g n i f i c a n t l y g r e a t e r (p<0.05) than those o f e i t h e r a l c o h o l group o r p a i r - f e d group a n i m a l s .  This i s r e f l e c t e d  i n the reduced food consumption o f these l a t t e r two groups. Average e t h y l a l c o h o l consumption d u r i n g t h i s p e r i o d by a l c o h o l group animals amounted t o a p p r o x i m a t e l y 2.5 mL p e r day. T a b l e 2 shows the d a i l y c a l o r i c i n t a k e s o f the t h r e e t r e a t m e n t groups p r i o r t o pregnancy.  Total caloric  intakes  o f t h e a l c o h o l and p a i r - f e d groups were about 80 p e r c e n t o f t h a t o f the ad_ l i b i t u m c o n t r o l s .  The absence o f v a r i a b i l i t y  i n amounts o f f o o d consumed by the p a i r - f e d group i s r e f l e c t i v e o f the p a i r - f e e d i n g design where a l l animals o f t h i s group consumed a l l o f the f o o d g i v e n .  P r i o r t o pregnancy a l c o h o l  p r o v i d e d 25 t o 30 p e r c e n t o f the c a l o r i e s o f the a l c o h o l group. T a b l e 3 shows the average d a i l y f o o d and a l c o h o l consumption and weekly body w e i g h t s o f r a t s g i v e n a l c o h o l , p a i r f e d , o r ad l i b i t u m f e d d u r i n g pregnancy.  S i g n i f i c a n t l y reduced  body w e i g h t s a r e o b s e r v e d i n a l c o h o l (p<0.0001) and i n p a i r f e d (p<0.001) group a n i m a l s when compared w i t h ad l i b i t u m c o n t r o l s d u r i n g each o f the t h r e e weeks o f g e s t a t i o n .  The  absence o f s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n body  36  weights  among t h e t h r e e  reflect  that the f a c t that s i n c e mating o c c u r r e d over a  week p e r i o d , group  may  groups  a t day one  a large v a r i a b i l i t y  have developed.  n o t e d i n body w e i g h t s and p a i r - f e d group  No  gestation The  during  Daily c a l o r i c intakes  two  consumption  4.68+ 0.05  this  mL  each  were  between a l c o h o l  average  a l c o h o l by t h e a l c o h o l g r o u p was s t a n d a r d e r r o r ) p e r day  may  i n body w e i g h t s w i t h i n  significant differences  during  animals.  of gestation  group  of ethyl  (mean +  period.  o f the t h r e e  treatment  during pregnancy  are d i s p l a y e d  alcohol provided  a b o u t 3t*; p e r c e n t o f t h e c a l o r i e s i n t h e  a l c o h o l group.  Total  i n T a b l e 4.  c a l o r i c intakes  During  groups gestation,  of the a l c o h o l  and  p a i r - f e d g r o u p s w e r e 71 p e r c e n t o f t h a t o f ad l i b i t u m controls  during  the l a s t  two weeks o f g e s t a t i o n .  reduced weight controls no  t h e f i r s t week o f g e s t a t i o n  gain  o f t h e s e two  of  groups  o v e r ad l i b i t u m  as s e e n i n T a b l e 3, e v e n t h o u g h  there  appears  during  the  t o be latter  gestation. b) L i t t e r  S i z e , F e t a l Body W e i g h t s ,  Placental Litter  size,  and  Weights.  f e t a l b o d y w e i g h t s , and p l a c e n t a l  o f r a t s on day 20 o f g e s t a t i o n  a r e shown i n T a b l e 5.  d i f f e r e n c e s were observed i n l i t t e r  exposed  during  i s r e f l e c t e d i n the  s i g n i f i c a n t difference i n caloric intake  part  no  This  and 9 1 %  a n i m a l s when c o m p a r e d w i t h  or ad l i b i t u m c o n t r o l groups,  weights Although  s i z e among a l c o h o l  those of e i t h e r p a i r - f e d  a s i g n i f i c a n t reduction  in  TABLE 1 Daily  food  alcohol,  and a l c o h o l  pair-fed,  Means + standard  consumption and weekly body weights o f r a t s  or ad l i b i t u m  fed p r i o r to pregnancy.  deviation.  A l c o h o l Group Food A l c o h o l Consumed C'med. (g) (mL)  Pair-fed  Body Weight (g)  Food Starch Consumed C'med ( ) ( ) g  Number o f Rats  (13)  (13)  (33)  Prepregnancy days 1-7  12.1 +2.1  2.0 +0.3  236.5 + 13.6  8-14  10.3 +2.2  2.5 +0.5  15-21  10.0 +2.8  22-28  10.0 +2.5  a, b,  given  g  Group  Ad l i b i t u m f e d Group  Body Weight ( ) g  g  (12)  (12)  (27)  a  12.1 +0.0  2.5 +0.0  233.l +8.00  242.0 + 15.9  3  10.3 +0.0  3.4 +0.0  244.l +8. 74  2.8 +0.4  252. 7 +16.9  a  10.0 +0.0  3. 7 +0.0  248.3 + 7.77  2.7 +0.3  260.4 120.2  10.0 +0.0  3.6 +0.0  259.0 +9.04  a  f i g u r e s i n the same row not s h a r i n g s i g n i f i c a n t l y d i f f e r e n t at p^.0.05.  Food Body Weight Consumed ( ) ( ) g  (12)  (27)  a  16.9 +4.1  241.7 +11.6  a  a  16.8 +2.6  256.2 + 16.0  b  a  16.6 +3.1  267.9 +19.4  b  3  18.2 +2.6  275.4 +20,5  b  the same l e t t e r are  38  TABLE 2 Daily  c a l o r i c intakes  of rats given alcohol, p a i r - f e d ,  fed p r i o r to pregnancy.  Means + s t a n d a r d  Alcohol  Food Consumed  Group  Alcohol Consumed  o r ad l i b i t u m  deviation  P a i r - F e d Group  Food Consumed  Starch Consumed  Ad L i b i t u m f e d Group Food Consumed  (Calories)  Number o f Rats:  (13)  (13)  51.4  10.0  +9.0 8-14  (12)  (12)  (12)  51.4  10.0  71.8  +2.1  +0.0  +0.0  +17.5  43.8 +9.3  13.6 +3.5  43.8 +0.0  13.6 +0.0  71.4 +11.1  15-21  42.5 +11.9  14.8 +2.8  42.5 +0.0  14.8 +0.0  70.6 +13.1  22-28  42.5 +10.6  14.4 +2.1  42.5 +0.0  14.4 +0.0  77.4 +11.0  Prepregnancy days 1-7  39  TABLE 3 Daily food and alcohol consumption and weekly body weights of rats given alcohol, pair-fed, or ad libitum fed during pregnancy. Means - standard deviation.  Alcohol Group  Pair--fed Group  Food Alcohol Body Consumed C'med Weight (mL) (g) •(g) Number of Rats:  (11)  (ID  (34)  -  269.2 +20.3  a  Ad Libitum fed Group  Food Starch Body Consumed C'med Weight (g) (g) (g)  Food Body Consumed Weight (g) (g)  (10)  (10)  (32)  -  -  267.3 +17.0  a  a  20.5 +6.7  297.0 +25.5  b  b  (6)  (29)  _  275.0 +24.6  Days of Gestation 1  -  9.66 +2.0  4.05 +0.7  273.8 +22.4  a  2-7  9.6 +0.0  5.27 +0.0  276.3 +23.1  8-14  11.6 +1.8  4.7 +0.7  298.4 +26.8  a  11.6 +0.0  6.09 +0.0  299.l +22.8  a  19.0 +7.2  326.9 +28.9  15-20  12.3 +2.2  5.3 +1.0  331.6 +28.8  a  12.3 +0.0  6.90 +0.0  339.6 +29.4  a  20.5 +12  388.l +38.2  a, b, = figures in the same row not sharing the same letter are significantly different at p<0.05.  a  b  40  TABLE  Daily  c a l o r i c intakes  o f r a t s g i v e n a l c o h o l , p a i r - f e d , o r ad l i b i t u m  fed d u r i n g preganancy, Means + standard  Alcohol Food Consumed  4  Group Alcohol Consumed  deviation.  P a i r - f e d Group Food Consumed  Starch Consumed  Ad L i b i t u m f e d Group Food Consumed  (Calories) Number o f Rats:  (11)  (11)  (10)  41.1 +8.5  21. +5.  41.1 +0.0  21.1 +0.0  87.1 +28.5  8-14  49.3 +7.6  24. +4.  49.3 +0.0  24.4 +0.0  80.8 +30.6  15-20  52.3 +9.3  27.6 +7.0  52.3 +0.0  27.6 +0.0  87.1 +51.0  Day of gestation 2-7  (10)  (6)  41  litter  size  ( p < 0 . 0 1 ) was  ad l i b i t u m g r o u p .  found i n the p a i r - f e d over the  The f e t a l  and p l a c e n t a l w e i g h t s w e r e n o t  a f f e c t e d b y t h e i r l o c a t i o n on t h e u t e r i n e  horns.  No e v i d e n c e o f f e t a l m a l f o r m a t i o n was o b s e r v e d i n any of the t h r e e treatment groups, however f e t u s e s exposed  of alcohol  a n i m a l s e x h i b i t e d r e d d e n i n g o f t h e s k i n and a  a l l y more s h r i v e l l e d a p p e a r a n c e  gener-  than those o f e i t h e r p a i r -  f e d o r ad l i b i t u m c o n t r o l groups.  F e t a l body w e i g h t s  on  day 20 o f g e s t a t i o n w e r e s i g n i f i c a n t l y r e d u c e d i n t h e a l c o h o l g r o u p when c o m p a r e d w i t h o  r  ad l i b i t u m c o n t r o l  body w e i g h t s  e i t h e r the p a i r - f e d  ( p < 0.0001) g r o u p s .  Furthermore,  o f t h e p a i r - f e d group were a l s o  lower than t h a t o f the ad l i b i t u m c o n t r o l s ibly reflective group.  o f the reduced  (p<0.0001)  significantly  (p<0.05),  caloric intake  fetal  poss-  o f the former  By c o n t r a s t , p l a c e n t a s o f t h e a l c o h o l g r o u p  were  s u b s t a n t i a l l y h e a v i e r ( p < 0.001) a n d p l a c e n t a s o f t h e p a i r f e d group  lighter  ( p . < 0.05) r e l a t i v e t o t h o s e o f a d l i b i t u m  controls. A n i m a l s were o c c a s i o n a l l y  i n a d v e r t e n t l y omitted a t weekly  weighing during the p r e g e s t a t i o n a l  or gestational periods  hence were e x c l u d e d from t h e t r e a t m e n t group weight.  This i s reflected i n differences  rats or l i t t e r s  i n t h e number o f  b e t w e e n T a b l e s 1 t h r o u g h 5, w h e r e p r i o r t o  day 20 o f g e s t a t i o n , tabulated.  mean b o d y  a c t u a l s a m p l e s i z e may be l a r g e r  than  42  TABLE 5  Litter  size,  f e t a l body w e i g h t s , and p l a c e n t a l weights o f r a t s a t  day 20 g e s t a t i o n standard  given alcohol,  p a i r - f e d , o r ad l i b i t u m - f e d .  error.  Alcohol  Litter  Means +  size  Number o f l i t t e r s  Group  11.3+ 0.32 ' d  35  P a i r - f e d Group  10.8+0.41  Ad l i b i t u m Group  12.3+ 0.43'  33  26  F e t a l body weight 2.01+ 0.03 Number o f l i t t e r s  Placental  2.51+ 0.08  b  33  c  26  weight  (g) Number o f l i t t e r s  a,b,c,=  35  2.33+0.04  0.614+0.02' 11  0.416+ 0.01  b  12  f i g u r e s i n the same row not s h a r i n g the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t a t p<0.05  0,485+0.02° 10  43  c ) A l c o h o l and N u t r i e n t C o n c e n t r a t i o n s i n Maternal  Plasma  Maternal plasma c o n c e n t r a t i o n s of e t h y l a l c o h o l , folic  acid,  i n Table  6.  and  z i n c d u r i n g week t h r e e o f g e s t a t i o n a r e  Average plasma a l c o h o l l e v e l s  d u r i n g days  o f g e s t a t i o n o f t h e a l c o h o l g r o u p w e r e 76+6.6 mg (mean + s t a n d a r d e r r o r ) . glucose, f o l i c  acid,  and  among t h e t h r e e t r e a t m e n t ranges  i n a l l cases  z i n c d i d not groups,  differ  reduced  compared w i t h e i t h e r p a i r - f e d  Furthermore,  placenta a significantly  as mL/min p e r of cardiac  (p<0.001, p<0.001,  when e x p r e s s e d  decrease  as mL/min p e r  b l o o d f l o w was  observed  w i t h ad_ l i b i t u m c o n t r o l s  was  not  statistically  signi-  as mL/min p e r g p l a c e n t a  to the d i f f e r e n c e s i n p l a c e n t a l weights significant  7.  i n t h e a l c o h o l g r o u p when  decreased  expressed  blood  (p<0.05, p<0.001, p<0.05,  i n comparison  (p<0.05), however t h i s  renal  i s presented i n Table  r e s p e c t i v e l y ) o r ad l i b i t u m c o n t r o l  f i c a n t when f l o w was  expected  Flow  c a r d i a c output  significantly  i n p a i r - f e d animals  mL  (9,63,64).  p l a c e n t a , mL/min p e r g p l a c e n t a , o r as p e r c e n t  p<0.05) g r o u p s .  p e r 100  and w e r e w i t h i n  Blood f l o w to the p l a c e n t a , whether expressed  o u t p u t , was  15-17  significantly  d a t a on p l a c e n t a l b l o o d f l o w , m a t e r n a l  f l o w , and m a t e r n a l  shown  Maternal plasma concentrations of  d) P l a c e n t a l B l o o d The  glucose,  between groups.  d i f f e r e n c e s were seen i n b l o o d f l o w t o  the  due No  44  k i d n e y among t h e t h r e e cardiac  output.  elevated  treatment groups, n o r i n the maternal  I t i s i n t e r e s t i n g to note the s u b s t a n t i a l l y  s t a n d a r d e r r o r measurement o f r e n a l B l o o d f l o w  a l c o h o l group compared w i t h control  that  i n the  o f p a i r - f e d o r ad l i b i t u m  groups. e)  F e t a l Uptake o f N u t r i e n t s  F e t a l u p t a k e o f 2-amino i s o b u t y r a t e ,  methyl-ct-D  g l u c o p y r a n o s i d e , f o l i c a c i d , a n d z i n c a t d a y 20 o f g e s t a t i o n f o r a l c o h o l , p a i r - f e d , a n d a d l i b i t u m f e d g r o u p s i s shown i n T a b l e 8.  I n t h e c a s e o f 2 amino i s o b u t y r a t e ,  glucopyranoside  methyl-et-D  and z i n c n o s i g n i f i c a n t d i f f e r e n c e s  u p t a k e w e r e o b s e r v e d o v e r t h e 90 m i n u t e i n c u b a t i o n among t h e t h r e e  treatment groups.  o f 2-amino i s o b u t y r a t e  i n fetal period  Trends e x i s t i n t h e cases  and z i n c f a v o u r i n g  a r e d u c e d f e t a l up-  t a k e by t h e a l c o h o l  group o v e r p a i r - f e d and ad l i b i t u m groups  however s t a t i s t i c a l  s i g n i f i c a n c e i s not achieved.  t a k e n up b y t h e f e t u s e s higher  o f t h e a l c o h o l g r o u p was  The f o l a t e significantly  (p<0.05) t h a n t h a t o f t h e p a i r - f e d , b u t n o t a d l i b i t u m  fed controls.  The p o s i t i o n o f t h e f e t u s  on t h e u t e r i n e  horn  a p p e a r e d t o h a v e n o e f f e c t on t h e r a t e o f u p t a k e o f any o f the  compounds  studied.  f ) Plasma and L i v e r C o n c e n t r a t i o n s o f I n j e c t e d Compounds a n d M a t e r n a l P l a s m a O s m o l a l i t y  and  Muscle Dry Weight. As  i n d i c a t e d i n T a b l e 9, n o s i g n i f i c a n t d i f f e r e n c e s  observed i n the concentrations  of the four r a d i o a c t i v e  were  compounds  45  TABLE 6  Ethyl  alcohol,  g l u c o s e , f o l i c a c i d , and z i n c c o n c e n t r a t i o n s i n maternal  plasma d u r i n g week t h r e e o f g e s t a t i o n ad  libitum fed.  Means + s t a n d a r d  Alcohol  Ethyl alcohol (mg/100 mL)  Group  i n rats given a l c o h o l ,  pair-fed or  error.  P a i r - f e d Group  Ad L i b i t u m f e d Group  76+ 6.6 (ll) 1  Glucose (mg/100 mL)  F o l i c acid (ng/mL)  99.8+2.9  a  96.5+3.0  (14)  (14)  46.5+4.2  37.3+3.6  (77  Zinc  349+69.8  (ppb)  (8)  104.7+ 2.3 (12)  47.9+7.1  (6)  (7)  445+20.8  402+ 83.4  (8)  a, No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were observed between treatment groups.  (8)  1= f i g u r e s i n b r a c k e t s r e f e r t o number o f rats.  46  TABLE 7  B l o o d flow t o p l a c e n t a s and t o the m a t e r n a l k i d n e y s , and c a r d i a c output on day 20 g e s t a t i o n o f r a t s g i v e n a l c o h o l , p a i r - f e d , o r ad l i b i t u m f e d . Means + s t a n d a r d  error.  A l c o h o l Group  P a i r - f e d Group  Ad l i b i t u m Group  Number o f l i t t e r s  11  P l a c e n t a l blood mL/min/placenta  0.170+0.02  a  0.260+ 0.03  0.357+ 0.03  C  mL/min/ g p l a c e n t a  0.227+0.03  a  0.623+0.07  b  0.743+ 0.07  b  % o f c a r d i a c output  0.270+0.03  a  0.399+0.05  b  0.460+0.04  12  10  flow:  Renal b l o o d flow: % o f c a r d i a c output 13.2+1.6  a  10.9+0.61  a  11.6+0.36  a  20.5+1.3  3  20.8+0.98  a  20.4+ 1.0  a  Maternal c a r d i a c output: mL/min/100 g body weight  a,b,c= f i g u r e s i n the same row not s h a r i n g the same l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t ' a t p<0.05.  b  47  in  the m a t e r n a l plasma o r l i v e r  2-amino i s o b u t y r a t e ,  90 m i n u t e s a f t e r i n j e c t i o n o f  methyl-**-D g l u c o p y r a n o s i d e ,  folic  acid,  and z i n c among t h e t h r e e t r e a t m e n t g r o u p s . T a b l e 10 shows two p a r a m e t e r s o f d e h y d r a t i o n , osmolality  plasma  and m u s c l e d r y w e i g h t f o r a l c o h o l , p a i r - f e d ,  ad l i b i t u m f e d g r o u p s .  and  There i s a s t a t i s t i c a l l y s i g n i f i c a n t  i n c r e a s e i n both plasma o s m o l a l i t y  and muscle d r y w e i g h t i n  a l c o h o l e x p o s e d a n i m a l s when c o m p a r e d w i t h e i t h e r p a i r - f e d (p<0.001, p < 0 . 0 5 , r e s p e c t i v e l y ) p<0.05, r e s p e c t i v e l y ) groups. degree o f d e h y d r a t i o n  o r ad l i b i t u m c o n t r o l This represents a  (p < 0.001,  moderate  (about 7 p e r c e n t ) i n the a l c o h o l  group.  TABLE 8 F e t a l uptake o f 2-amino i s o b u t y r a t e , c i r c u l a t i o n a t day 20 o f g e s t a t i o n .  Compound  2-Amino  injected  methyl-cH) g l u c o p y r a n o s i d e , f o l i c Mean + standard e r r o r .  Alcohol group  (1-^C) isobutyric  a c i d , and z i n c from the maternal  Pair-fed group  Ad l i b i t u m controls  acid  DPM/g f e t a l body weight  23,814 + 1444  26,615 + 3007  (7)  (5)  1  27,679 + 656 (5)  Methyl ( flC-D-fU-^C] g l u c o ) p y r a n o s i d e DPM/g f e t a l body weight  10,914 + 453  9,840 + 357 (10)  (11) [ 3',5',7,9- H] F o l i c 3  9,414 + 574 (6)  acid, K salt  DPM/g f e t a l body weight  72,406 + 3634  2  (11)  61,509 + 1838  (io)  2  62,601 + 4669 (6)  Zinc-65 CPM/g f e t a l body weight  1,796 + 177 (6)  F i g u r e s i n parentheses i n d i c a t e Significant  at p<0.05.  the number o f l i t t e r s .  1,873 + 309 (6)  2,601 +'298 (5)  49  TABLE 9  C o n c e n t r a t i o n o f 2-amino i s o b u t y r a t e ,  methyl-<<-D g l u c o p y r a n o s i d e ,  a c i d , and z i n c i n m a t e r n a l plasma and l i v e r the r a d i o a c t i v e compounds.  Group  Alcohol  Compound  Means + standard  Pairfed Liver  Ad L i b i t u m  folic  90 minutes a f t e r i n j e c t i o n o f error.  Alcohol  Pairfed Plasma  Ad  Libitum  injected:  2-Amino i s o b u t y r ate (DPM/g)  62100 + 7310  79100 +2940  62800 +4380  20200 +1250  17400 +600  17800 +1030  34700 +1870  36500 +2100  31400 +7540  49500 +2940  50600 +1340  51600 +3540  153800 +5910  147600 +5530  1320 +255  960 +126  Number o f l i t t e r s  Methyl -<*-D glucopyranoside (DPM/g)  10  Numbers o f l i t t e r s  Folic  acid (DPM/g)  493100 +41600  396500 +30800  527500 +45300  Number o f l i t t e r s  Zinc  chloride (CPM/g)  151400 +13200 11  14800 +1350  22200 +4040  14400 +1160  1300 +136  Number o f l i t t e r s  a = there were no s t a t i s t i c a l l y between treatment groups.  significant differences  50  TABLE 10  M a t e r n a l plasma o s m o l a l i t y  and muscle d r y weight (% o f wet weight) i n  a l c o h o l , p a i r - f e d , and ad l i b i t u m f e d r a t s .  Means + standard  error.  Group  Plasma o s m o l a l i t y (mosm./L)  Muscle dry weight (% o f wet weight)  Alcohol  302.4 + 4.59  25.68 + .58  Number o f l i t t e r s  Pair-fed Number o f l i t t e r s  Ad l i b i t u m Number o f l i t t e r s  a  10  11  280.0 + 2.30  b  9  278.8+2.05  23.99 + 1 3  b  12  b  9  a, b = f i g u r e s i n the same column not s h a r i n g the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at p<0.05  b 24.57+22 10  a  51  DISCUSSION The exposure  findings  o f t h i s study i n d i c a t e  i s deleterious  to f e t a l  development  no o b v i o u s m a l f o r m a t i o n s were, o b s e r v e d fetuses  i n any  o f the t h r e e groups  the  offspring of rats  out  pregnancy  either  consistent  syndrome r e s e a r c h and s u b s e q u e n t dams was  (7,8,41).  blood alcohol  s i m i l a r to that  d e c r e a s e d b i r t h weights alcohol  (76 mg/100 mL) of  infants  t o be  The  absence  reflective  ment. icant  Both  intake  o f the  body w e i g h t s  daily alcohol  treated  fetal  exposure  are alcohol  consumption, group  i n animals  (7,8,41).  The  where blood  dams i n t h e p r e s e n t  alcohol  syndrome,  (130 mg/100 mL) required  of differences  taken  (24).  study  shortly  The  to a f f e c t f e t a l  i n m a t e r n a l body  and p a i r - f e d g r o u p s  alcohol  group  findings  l e v e l s , o f the a l c o h o l  o f the i s o c a l o r i c  reduction  libitum  fed  minimum wellbeing  determined.  between a l c o h o l is  These  of  through-  a r a t model i n f e t a l  were o b s e r v e d  o f the i n f a n t  amount o f a l c o h o l remains  gestation  a r e however l e s s t h a n i n b l o o d f r o m m o t h e r s  displaying  after birth  day  p r i o r t o and  of other studies  levels of alcohol  i n the r a t . Although-  decreased over r a t s  The  alcohol  of r a t s , b i r t h weights  o r ad l i b i t u m .  w i t h p a s t work u s i n g  prenatal  i n t h e 20  consuming a l c o h o l  were s i g n i f i c a n t l y  isocalorically  that  a t day  feeding  and p a i r - f e d g r o u p s  i n m a t e r n a l body w e i g h t  a n i m a l s , as m i g h t former  groups.  be  20  of  weights gestation  d e s i g n o f the also  exhibit  a  signif-  when compared w i t h  e x p e c t e d by  the lower  However t h e d i f f e r e n c e s  among t h e t h r e e g r o u p s  experi-  a t day 20  of  in  ad  caloric fetal  gestation  52  suggest that factor, tion  the c a l o r i c r e s t r i c t i o n ,  c a n n o t be e n t i r e l y  responsible  i n a l c o h o l exposed fetuses.  account f o r t h i s isocalorically  reduction  although a c o n t r i b u t i n g f o r the growth  retarda-  O t h e r f a c t o r s must e x i s t t o  i n f e t a l w e i g h t b e t w e e n t h e two  f e d groups.  To i n v e s t i g a t e w h e t h e r a l c o h o l c o n s u m p t i o n b e f o r e during  g e s t a t i o n h a d an e f f e c t on t h e n u t r i t i o n a l  the mother, plasma c o n c e n t r a t i o n s determined. of glucose, gestation  and  status of  o f c e r t a i n n u t r i e n t s were  The a b s e n c e o f any d i f f e r e n c e i n p l a s m a l e v e l s folic  acid, or zinc during  t h e t h i r d week o f  suggests that maternal metabolism o f these n u t r i e n t s  i s n o t a f f e c t e d by a l c o h o l consumption. concentration  Furthermore, the  i n l i v e r o r plasma o f f o l i c  a c i d , z i n c , and  a n a l o g s o f g l u c o s e a n d amino a c i d 90 m i n u t e s a f t e r was n o t s i g n i f i c a n t l y  d i f f e r e n t among t h e t h r e e  These f i n d i n g s , t o g e t h e r  with  injection  groups.  the absence o f d i f f e r e n c e i n  m a t e r n a l body w e i g h t s o f a l c o h o l t r e a t e d and p a i r - f e d a n i m a l s on  day 20 o f g e s t a t i o n  support the notion  sure a t t h e p r e s e n t l e v e l and d u r a t i o n nutritional  status.  This  i s contrary  that alcohol  expo-  does n o t a f f e c t m a t e r n a l t o p a s t work where i n  the non pregnant r a t , a l c o h o l exposure has r e s u l t e d i n reduced plasma l e v e l s o f f o l i c  a c i d , and t h i a m i n  (65).  The p o s s i b i l i t y  c a n n o t be e x c l u d e d t h a t m a t e r n a l s t a t u s o f c e r t a i n n u t r i e n t s w h i c h were n o t measured i n t h e p r e s e n t s t u d y were  sufficiently  a l t e r e d by a l c o h o l consumption t o a f f e c t adequate f e t a l  supply,  however i t i s l i k e l y  itself  that  i n an o b s e r v a b l e r e d u c t i o n  any d e f i c i e n c y w o u l d m a n i f e s t i n m a t e r n a l body w e i g h t .  The  53  decreased b i r t h weights o f the o f f s p r i n g o f r a t s exposed to a l c o h o l i n utero over those f e d i s o c a l o r i c a l l y  i n this  c a n n o t t h e r e f o r e be r e a d i l y e x p l a i n e d t h r o u g h an  study  alcohol  induced a l t e r a t i o n of maternal n u t r i e n t u t i l i z a t i o n  o r plasma. •  concentration. To f u r t h e r i n v e s t i g a t e  the p o s s i b i l i t y  that  alcohol  i n t e r f e r e s w i t h the adequacy o f n u t r i e n t s u p p l y t o the d e v e l oping fetus, at for  t h e hemodynamic  day 20 o f g e s t a t i o n .  s t a t u s o f t h e m o t h e r was  The u s e o f r a d i o a c t i v e  studied  microspheres  e s t i m a t i o n o f c a r d i a c o u t p u t and o r g a n b l o o d f l o w i s a  widely accepted procedure  (60,61,66).  The  fractional  b u t i o n among b o d y t i s s u e s o f a v e n t r i c u l a r l y  introduced pulse  of  r a d i o a c t i v e microspheres  of  the c a r d i a c output reaching those t i s s u e s .  artificial  organ  distri-  i s p r o p o r t i o n a l to the percentage C r e a t i o n o f an  o f known b l o o d f l o w b y means o f r e f e r e n c e  sampling o f b l o o d at a f i x e d r a t e at the time of the microsphere p u l s e p e r m i t s c a l c u l a t i o n o f c a r d i a c output. b l o o d f l o w t o any t i s s u e  c a n be d e t e r m i n e d ,  c a r d i a c o u t p u t and t h e p e r c e n t a g e ing  t h e t i s s u e a r e known.  with microspheres w i t h which  Thus t h e  as b o t h t h e  of the c a r d i a c output reach-  In measuring  placental blood flow  t h e r e i s some q u e s t i o n as t o t h e e f f i c i e n c y  s m a l l (15 m i c r o n ) m i c r o s p h e r e s  p l a c e n t a l channels.  However,  are trapped i n the  B j e l l i n e t a l . ( 6 7 ) h a v e demon-  s t r a t e d t h a t 15 m i c r o n m i c r o s p h e r e s  give a better estimate  of  p l a c e n t a l b l o o d f l o w t h a n do 50 m i c r o n m i c r o s p h e r e s ,  to  the tendency  vessels.  o f the l a t t e r  to a x i a l  streaming i n blood  The f i g u r e s r e p o r t e d p r e s e n t l y  body w e i g h t  due  (21 mL/  m i n / 100 g  f o r c a r d i a c o u t p u t and 11.6% o f c a r d i a c  output  54  for kidney and  f l o w ) a r e i n agreement w i t h those  o f B r u c e (61)  S a p i r s t e i n e t a l . ( 6 8 ) o f 26 a n d 22 mL/ m i n / 100 g f o r  cardiac output, flow(61).  r e s p e c t i v e l y , a n d 8.62% f o r k i d n e y  blood  The p l a c e n t a l b l o o d f l o w v a l u e r e p o r t e d b y B r u c e  (61) o f 1.21 mL/min/g i s g r e a t e r t h a n c o n t r o l group i n t h e p r e s e n t  study  t h a t o f t h e ad l i b i t u m  (0.743 mL/min/g).  However  b l o o d f l o w d e t e r m i n a t i o n s b y B r u c e w e r e p e r f o r m e d on day 22 o f g e s t a t i o n as i s r e f l e c t e d i n a v e r a g e f e t a l w e i g h t s 4.21 g when c o m p a r e d w i t h a v e r a g e f e t a l w e i g h t s  of  o f 2.51 g  i n t h e a d l i b i t u m c o n t r o l g r o u p a t day 20 o f g e s t a t i o n i n the p r e s e n t  study.  Maternal  c a r d i a c output  and r e n a l b l o o d f l o w  expressed  as % c a r d i a c o u t p u t w e r e n o t a f f e c t e d b y a l c o h o l co.nsumption o r b y p a i r - f e e d i n g when c o m p a r e d w i t h a d l i b i t u m f e d c o n t r o l s . S a p i r s t e i n e t a l . (68) , u s i n g 86 Rb t o m e a s u r e b l o o d f l o w , a l s o found no d i f f e r e n c e s i n c a r d i a c output a l c o h o l e x p o s e d and c o n t r o l r a t s output has been found  reduced  between non pregnant  f e d ad l i b i t u m .  Cardiac  i n a l c o h o l t r e a t e d non pregnant  r a t s , b u t o n l y when n a r c o t i z i n g d o s a g e s w e r e a d m i n i s t e r e d ( 6 9 ) . Increases  i n b l o o d f l o w r a t e s t o b r a i n , h e a r t , k i d n e y s , and  l u n g as w e l l as d e c r e a s e s  i n b l o o d f l o w r a t e s t o spleen, muscle,  and k i d n e y s h a v e b e e n r e p o r t e d i n t h e l i t e r a t u r e  (68,70).  The  l a r g e s t a n d a r d e r r o r measurement i n t h e r e n a l b l o o d f l o w o f a l c o h o l exposed animals  i n the present  study,  i n comparison  w i t h t h a t o f e i t h e r p a i r - f e d o r ad l i b i t u m c o n t r o l is  consistent with the v a r i a b i l i t y  literature.  groups,  of f i n d i n g s reported i n the  The  p r e s e n t s t u d y demonstrates a marked r e d u c t i o n i n  blood flow  t o the placentas  i n the alcohol treated  w h e t h e r e x p r e s s e d i n mL/ m i n / p l a c e n t a , cardiac  output.  cantly greater  o r as p e r c e n t o f  As t h e w e i g h t o f e a c h p l a c e n t a i n the alcohol  treated  rats,  was  signifi-  animals than i n c o n t r o l s ,  t h e b l o o d f l o w p e r u n i t o f p l a c e n t a l w e i g h t i s e v e n more markedly reduced by a l c o h o l consumption. portions  of p l a c e n t a l weight derived  The r e l a t i v e p r o -  from maternal o r f e t a l  components w e r e n o t d e t e r m i n e d , s o i t c a n n o t be s t a t e d  that  blood flow per u n i t o f maternal p l a c e n t a l t i s s u e i s reduced. Nevertheless, implying  the t o t a l blood flow  t o the placenta  a reduced d e l i v e r y o f n u t r i e n t s  i s decreased,  and e s s e n t i a l  metabolites. The  reason f o r the increased  p l a c e n t a l weight i n the  a l c o h o l group i s n o t r e a d i l y apparent. retard  f e t a l g r o w t h , s u c h as u t e r i n e  calorie malnutrition, weight  (59, 71).  Other f a c t o r s  a r t e r y l i g a t i o n and  are accompanied by reduced  I n a c c o r d a n ce,  i t i s noted that  p r e s e n t study t h e c a l o r i e r e s t r i c t e d group significantly  smaller  placentas  or ad l i b i t u m c o n t r o l groups.  placental i n the  displayed  than e i t h e r a l c o h o l The h e a v i e r  a l c o h o l g r o u p a r e n o t due t o i n c r e a s e d  placentas  fluid  W i e n e r e t a l . (42) a n d S k o s y r e v a  reported  increased  that  heavier  o f the  (43) have  group  also  placental weights i n alcohol-exposed  s i m i l a r to the present r e s u l t s . reported  treated  r e t e n t i o n as  t h e p e r c e n t d r y w e i g h t i s t h e same as i n t h e c o n t r o l (72).  which  placentas  Gordon e t a l . (44) a l s o  i n alcohol treated  i t i s due t o h y p e r p l a s i a .  rats  r a t s a n d showed  H i s t o l o g i c a l examination  may  56  c a s t some l i g h t on t h e n a t u r e  of these  t h a t t h e i n c r e a s e d p l a c e n t a l s i z e may mechanisms w h i c h this  organ The  findings.  It i s possible  e x i s t as a r e s u l t  compensate f o r the r e d u c e d b l o o d f l o w t o  i n the r a t c h r o n i c a l l y exposed to  alcohol.  r e d u c t i o n i n p l a c e n t a l f r a c t i o n of c a r d i a c output  a l c o h o l t r e a t e d r a t s r e p o r t e d here by R o s s o a n d K a v a ( 5 9 ) .  These workers  reported a  i n g a change i n t h e f r a c t i o n o f c a r d i a c o u t p u t  decreased observ-  reaching  I f c a l o r i e r e s t r i c t i o n e x i s t e d as t h e c a u s e o f  growth f a i l u r e  i n a l c o h o l t r e a t e d pregnant  animals, a  p a t t e r n o f hemodynamic a l t e r a t i o n s w o u l d be e x p e c t e d restricted rats.  I n t h e p r e s e n t w o r k , no  c a r d i a c output  i n rats  a 50 p e r c e n t  fed a calorie r e s t r i c t e d  controls,  diet  ( 50 animals.  present  a r e d i s t r i b u t i o n of a normal c a r d i a c  o u t p u t , whereas i n the m a l n o u r i s h e d b l o o d f l o w remains constant but during late gestation f a i l s cardiac output.  i n food  reduction i n  Thus t h e m e c h a n i s m o f b l o o d f l o w r e d u c t i o n i n t h e through  fetal similar  ad l i b i t u m  p e r c e n t o f c o n t r o l s ) as c o m p a r e d w i t h c o n t r o l g r o u p  study e x i s t s  this  differences exist in  c a r d i a c o u t p u t b e t w e e n a l c o h o l t r e a t e d and h o w e v e r R o s s o and K a v a (59) n o t e  r a t , the d i s t r i b u t i o n  the expansion  to occur, r e s u l t i n g i n a  This lends f u r t h e r evidence  As  lowered  i n support  that from  malnutrition.  a m a r g i n a l l y food r e s t r i c t e d group, the p a i r - f e d  o f t h e p r e s e n t s t u d y may The  of  o f b l o o d volume  t h e a c t i o n o f a l c o h o l on t h e f e t u s i n u t e r o i s d i f f e r e n t that of maternal  in  c o n t r a s t s w i t h r e c e n t work  p l a c e n t a l blood flow i n food r e s t r i c t e d r a t s , without  organ.  of  be  group  compared w i t h the m a l n o u r i s h e d r a t .  r e d u c t i o n i n p l a c e n t a l blood flow of p a i r - f e d over  ad  57  l i b i t u m c o n t r o l s i n the p r e s e n t study i s s i m i l a r to the  results  R o s s o a n d K a v a (.59.X., h o w e v e r , i n c o n t r a s t , n e i t h e r  the  f r a c t i o n of c a r d i a c output reaching the p l a c e n t a , nor  the  of  c a r d i a c o u t p u t s d i f f e r b e t w e e n p a i r - f e d and T h i s may  be  restriction  due  t o t h e more s e v e r e n a t u r e o f t h e  (50 p e r c e n t as c o m p a r e d w i t h a b o u t  p a i r - f e d group) o f malnourished fed  i n the present In  may  animals  calorie  20 p e r c e n t i n  (59) o v e r t h o s e  the p l a c e n t a l  availability  i m p a i r e d as a r e s u l t o f t h e s i g n i f i c a n t  membrane.  originate  through  and  substantial  to  a redistribution  of cardiac output.  that f e t a l  growth  e n t a l b l o o d f l o w has  occur  restricted  o f a u t e r i n e a r t e r y was  performed.  compensatory mechanisms t o r e s t o r e n o r m a l A l c o h o l i n t h e p r e s e n t s t u d y was  artery  l i t a g a t i o n may  restriction  cause  nutrient  t i m e may  a l l o w development o f such mechanisms.  where  However the  administered prior  thus s u f f i c i e n t  deoxy-  (IUGR) r a t s ,  n a t u r e o f t h i s r e d u c t i o n i n p l a c e n t a l b l o o d f l o w may  to  plac-  placental  a m i n o i s o b u t y r i c a c i d and  glucose i n i n t r a u t e r i n e growth  gestation,  secondary  N i t z a n e t a l . (71) f o u n d r e d u c t i o n s  in placental transfer of  throughout  It is  Reduction of  been a s s o c i a t e d w i t h reduced  transfer of nutrients.  to  availability is  r e t a r d a t i o n may  a r e d u c t i o n i n f e t a l n u t r i e n t uptake.  ligation  the  T h i s r e d u c t i o n i n b l o o d s u p p l y appears  thus p o s s i b l e t h a t i f p l a c e n t a l n u t r i e n t restricted,  pair-  of nutrients  r e d u c t i o n i n the s u p p l y o f b l o o d to t r a n s f e r areas o f villous  groups.  study.  these s t u d i e s  be  ad l i b i t u m  acute  not  allow  transfer. to  and  have e l a p s e d  Furthermore,  uterine  a larger placental blood flow  than t h a t a s c r i b a b l e t o a l c o h o l , beyond the  limits  58  o f t h e s e mechanisms. reduction  I t can  in placental nutrient  accompany a r e d u c t i o n chronic As  alcohol  h a n d l e d by  the  zinc,, f o l a t e ,  degraded i n v i v o  necessarily effected  a t 40  The  and  E x c e p t i n the o f the  c o n t r o l groups. uptake of f o l i c compared w i t h  f e t a l u p t a k e was  repre-  injection  comparison  the  occurring  p e r i o d was  a c i d , no  It i s uncertain  an  minutes  increase  even net  chosen the  mother.  differences  p a i r - f e d o r ad  why  pro-  into  compounds s t u d i e d w e r e s e e n i n  when c o m p a r e d w i t h  with  the  thus  amount i n j e c t e d i n t o t h e  case of f o l i c  A  i n d i c a t i n g that  i n j e c t e d compounds was This  not  radio-  o b s e r v e d a t 90  time p o i n t s ,  in  fetal  alcohol libitum  in  fetal  a c i d i n a l c o h o l g r o u p a n i m a l s o c c u r r e d when  the p a i r - f e d group.  d e f i c i e n c y a t an  during  are  after  amount o f r a d i o l a b e l i n c o r p o r a t e d  compared w i t h  exposed fetuses  s e l e c t e d by  of  60 m i n u t e s a f t e r i n j e c t i o n .  from mother to f e t u s .  uptake rates  glucose  compounds t h e y  90 m i n u t e t i m e p o i n t  alternate  i f b a c k - f l o w of the  t o maximize the  analogs of  the p r o d u c t i o n  compounds was  increased  o v e r each o f the  and  used because they  systems o f the  thus p r e v e n t i n g  radiolabeled  portionally  by  placental  compounds t h e m s e l v e s , h o w e v e r t h e y a r e  labeled byproducts.  uptake rates  a  i n r a t s exposed to a l c o h o l  analogs are  transport  s e n t as w o u l d t h e  fetus  t r a n s f e r need not  a measure o f f e t a l u p t a k e o f n u t r i e n t s , the  gestation. . Metabolite  f l u x was  argued that  consumption.  amino a c i d w e r e s t u d i e d  of the  be  i n p l a c e n t a l b l o o d f l o w as  t r a n s f e r of radiolabeled and  therefore  P o s s i b l y as  e a r l i e r stage of gestation  mechanism i s i n e f f e c t .  The  a  a r e s u l t of 'catch  absence of a r e d u c t i o n  a  up' in  fetal  uptake o f the retardation  compounds s t u d i e d  as  concept that  supports  compensatory mechanisms t o r e s t o r e  adequate  placental size,  20  of gestation.  a r i s e , as  s u g g e s t e d by  i n p l a c e n t a l b l o o d f l o w has  r e s u l t s o f the p r e s e n t s t u d y are Maddatu  (73)  sobutyrate to fetuses of gestation.  who  The  have a l l o w e d  by  F u r t h e r m o r e , no  n o t e d by L i n and to a l c o h o l  intake  month p r i o r t o  aminoiday  mating  one  performance of the  o f p r e g n a n c y as  previously  a l c o h o l a d a p t e d dams, and  i n the  and  acute a l c o h o l  placental ^C-valine  uptake.  in v i t r o preparation  w h i c h may  size (74),  demonstrated dams p l a c e d  compared w i t h  H e n d e r s o n e t a l . (75)  chronic  in placental  seems t o o c c u r as  day  i n rats that  those  solution  According to Rider  a l c o h o l on  offspring.  alcohol  increase  Maddatu (73).  the p o o r e r r e p r o d u c t i v e  o f the  to  s u f f i c i e n t time f o r a compensating e f f e c t  to have o c c u r r e d .  adaptation  occurred.  a l c o h o l from  i n g e s t i o n o f an  in  alcohol  i n contrast  of rats administered  the  increase  found a reduced t r a n s f e r . o f  i n t h e p r e s e n t s t u d y f o r a t l e a s t one  was  the  i n animals c h r o n i c a l l y exposed to  where a r e d u c t i o n  may  nutrient  This  n u t r i e n t t r a n s f e r may  6 t o 21  growth  a r e s u l t of inadequate p l a c e n t a l  t r a n s f e r a t l e a s t a t day  o f L i n and  the  seen i n o f f s p r i n g o f animals c h r o n i c a l l y exposed  to a l c o h o l i s not  The  suggests that  that  of  on the  decreased s u r v i v a l  has  also  demonstrated  administration  reduces  However, t h e s e a u t h o r s u s e d account f o r the  an  differences  in results. I f maternal alcohol the  c o n s u m p t i o n does n o t  f e t a l uptake of n u t r i e n t s , to e x p l a i n  retardation i n utero,  a l c o h o l must e x e r t  the  interfere with observed growth  i t s deleterious  60  e f f e c t i n some a l t e r n a t e manner.  Brown e t a l . (76)  s t r a t e d that exposure to a l c o h o l retards  demon-  g r o w t h and  differ-  e n t i a t i o n i n c u l t u r e d r a t embryos, s u g g e s t i n g t h a t can  exert  a d i r e c t e f f e c t on  f e t a l growth, w i t h o u t the  founding f a c t o r of n u t r i t i o n . may  be  fetal  circulation.  a l c o h o l c o n s u m p t i o n by inhibition fetal  It i s possible  unable to u t i l i z e n u t r i e n t s  f e r e d i n t o the  i n the  reported  by  that  rate of  trans-  Rawat  that  (77)  (U-^^C)-leucine  reported  (78).  t o t a l RNA  into  and  also  un-  been  Thus e v i d e n c e e x i s t s  i n support of a l c o h o l a f f e c t i n g growth i n utero anism other than through reducing  significant  incorporation  i n a l c o h o l e x p o s e d n e o n a t e s has Sdhenker  fetus  once t h e y have been  Decreased cardiac  H e n d e r s o n and  con-  the  pregnant r a t s r e s u l t e d i n a  cardiac proteins.  c h a n g e d t o t a l DNA  alcohol  by  some mech-  placental transfer  of  nutrients. In these s t u d i e s source of f l u i d s .  As  a l c o h o l i n w a t e r was a l c o h o l has  o t h e r s o u r c e o f w a t e r was osmolality cators  and  (7 p e r c e n t ) h a d  f o r t h e measurement o f  f e t a l n u t r i e n t u p t a k e , and  opment r e m a i n s  by  experiment. on  day  20  plasma india  occurred,  but  placental  overall fetal  devel-  unclear.  I n summary, m a t e r n a l n u t r i t i o n a l unaffected  sole no  Both parameters i n d i c a t e d that  s i g n i f i c a n c e of t h i s  blood flow,  animals,  m u s c l e w a t e r c o n t e n t w e r e m e a s u r e d as  of dehydration.  the  a d i u r e t i c a c t i o n and  a v a i l a b l e to the  moderate degree o f d e h y d r a t i o n the  o f f e r e d as  status  a l c o h o l t r e a t m e n t under the The  observed reduction  of gestation  appears to  conditions  of  i n placental blood  i n r a t s c h r o n i c a l l y exposed to  be this flow  alcohol  61  may  reduce p l a c e n t a l  nutrients opment.  transfer, hence.fetal  resulting i n a retardation  o t h e r than those s t u d i e d  as n o d e c r e a s e i n t h e r a t e zinc,  of fetal  However, i f s u c h a r e d u c t i o n  nutrients  uptake, o f c e r t a i n  i n f e t a l uptake  presently  of f e t a l  growth and  must be  increase  compensatory  mechanisms, perhaps  i n p l a c e n t a l weight emerging  term a l c o h o l nutrients  involved  Alter-  r e f l e c t e d i n the  as a r e s u l t o f  long  e x p o s u r e , may r e s t o r e n o r m a l t r a n s f e r o f a l l  to the fetus, regardless  cental blood flow. maternal alcohol of alcohol  occurs,  u p t a k e was o b s e r v e d f o r  f o l a t e , o r a n a l o g s o f g l u c o s e o r amino a c i d .  natively,  devel-  o f the reduction  Thus t h e g r o w t h r e t a r d i n g  i n g e s t i o n may s t i l l  i n pla-  effect of  b e due t o a d i r e c t  on t h e f e t u s , e i t h e r a f f e c t i n g p r i m a r y f e t a l  abolism or f e t a l nutrient  utilization.  action met-  REFERENCES CITED 1)  H a g g a r d , H.W. a n d J e l l i n e c k , E.M. 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