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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. University of 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 i n THE FACULTY OF GRADUATE STUDIES Divi 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) Peter John Harris Jones, I98I In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 DF-fi (2/79) i i ABSTRACT The present study was undertaken to determine whether retarded f e t a l growth caused by maternal alcohol consumption p r i o r to and during gestation i s due to an interference with the transfer of nutrients from the maternal c i r c u l a t i o n into the fetus. Female Sprague-Dawley rats were either given 20 percent alcohol i n drinking water and s o l i d diet ad libitum (alcohol group), or were pai r - f e d to the alcohol group (pair-fed group}, or were given water and s o l i d diet ad libitum (ad libitum control group) for four weeks. They were then mated and the alcohol group was changed to 30 percent alcohol i n water. During the t h i r d week of gestation maternal plasma levels of glucose, f o l i c acid, and zinc were determined. On day 20 of gestation placental and f e t a l weights, the f e t a l uptake of zinc, f o l i c acid, and non-metabolizable glucose and amino acid analogs, and the placental blood flow were determined. The f e t a l uptake of the nutrients studied was investigated 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 into the femoral vein with either H labeled f o l i c acid 14 and methyl (cx-D- U- c gluco) pyranoside, or zinc-65, or 14 2-amino(l- C) isobutyric acid. 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 also determined. Blood flow to the placentas as well as maternal cardiac output and renal blood flow were measured-fusing 15 57 micron diameter microspheres labeled with Co. The osmolality of the maternal plasma and maternal muscle water i i i content were determined i n order to assess the degree of dehydration, as alcohol i n water was offered as the sole source of f l u i d s . No differences i n l i t t e r size existed between alcohol and pair-fed or alcohol, and ad libitum control groups, however l i t t e r sizes of the ad libitum group were larger than those of the pair-fed group. The 20 day old fetuses were s i g n i f i c a n t l y smaller while the placentas were s i g n i f i c a n t l y larger i n the alcohol group than i n either the pai r - f e d or ad libitum controls. No differences were seen i n maternal plasma leve l s of glucose, f o l i c acid, or zinc among the three treatment groups. Cardiac output and blood flow to the kidneys did not d i f f e r among the three treatment groups. However blood flow to the placenta, whether expressed as mL/min/g placenta or as mL/min/ entire placenta or as % cardiac output was s i g n i f i c a n t l y reduced i n the alcohol group compared with the pai r - f e d and ad libitum control groups. When expressed as mL/min/placenta differences i n blood flow were also seen between pai r - f e d and a_d libitum control groups. No s i g n i f i c a n t differences were ,observed i n the f e t a l uptake of zinc or analogs of glucose and amino acid between the three treatment groups. The fola t e taken up by the fetuses of the alcohol treated dams was s i g n i f i c a n t l y higher than for p a i r -fed but not ad libitum controls. No differences were observed i n maternal plasma or l i v e r concentrations of any of these compounds among the three groups 90 minutes a f t e r i n j e c t i o n . Plasma osmolality was increased by approximately 7 percent in i v t h e a l c o h o l group compared w i t h p a i r - f e d and ad l i b i t u m c o n t r o l groups. T h i s i n d i c a t e s a moderate degree o f d e h y d r a t i o n . The p r e s e n t s t u d y d e m o n s t r a t e s t h a t t h e r e t a r d e d growth o f t h e f e t u s e s exposed t o a l c o h o l i n u t e r o i s n o t due t o an i m p a i r e d t r a n s f e r o f t h e n u t r i e n t s t e s t e d from t h e m a t e r n a l c i r c u l a t i o n t o t h e f e t u s ; n o r i s i t due t o l o w e r m a t e r n a l plasma l e v e l s o f t h e s e n u t r i e n t s . The s i g n i f i c a n c e o f t h e r e d u c e d p l a c e n t a l b l o o d f l o w i n t h e a l c o h o l exposed r a t s on f e t a l growth and development i s n o t c l e a r . Thus t h e g r o w t h - r e t a r d i n g e f f e c t o f m a t e r n a l a l c o h o l i n g e s t i o n may s t i l l be due t o a d i r e c t a c t i o n o f a l c o h o l on t h e f e t u s . V TABLE OF CONTENTS PAGE A b s t r a c t i i T a b l e o f Contents v L i s t o f T a b l e s v i i Acknowledgements i x CHAPTER I INTRODUCTION 1 I I LITERATURE REVIEW 4 HUMAN STUDIES: a) R e t r o s p e c t i v e S t u d i e s 4 b) P r o s p e c t i v e S t u d i e s 7 c) A l c o h o l Induced A l t e r a t i o n i n M a t e r n a l M e t a b o l i s m 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. 10 ANIMAL 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 the 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 Oth e r A n i m a l s 22 e) Comparison o f P r e n a t a l A l c o h o l Exposure 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 O f f s p r i n g 25 f ) Comparison 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 V e r s u s M a l n o u r i s h e d A n i m a l s 26 v i PAGE I I I MATERIALS AND METHODS a) P r e l i m i n a r y Treatment o f Animals 29 b) D e t e r m i n a t i o n o f Plasma 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 30 c) P l a c e n t a l B l o o d Flow D e t e r m i n a t i o n 30 d) F e t a l Uptake o f F o l i c A c i d , Z i n c , and Analogs o f G lucose 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 IV RESULTS a) Food I n t a k e and M a t e r n a l Body Weights 35 b) L i t t e r S i z e , F e t a l Body Weights, and 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 Flow 43 e) F e t a l Uptake 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 and M a t e r n a l Plasma O s m o l a l i t y and Mus c l e Dry Weight 44 V DISCUSSION 51 REFERENCES CITED 62 v i i LIST OF TABLES PAGE Ta b l e 1 D a i l y Food and A l c o h o l Consumption and Weekly Body Weights o f Rats Given 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 P r i o r t o Pregnancy 37 T a b l e 2 D a i l y C a l o r i c I n t a k e s o f Rats 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 P r i o r t o Pregnancy.. 38 T a b l e 3 D a i l y Food and A l c o h o l Consumption and Weekly Body Weights o f Rats 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 Pregnancy 39 T a b l e 4 D a i l y C a l o r i c I n t a k e s o f Rats 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 Pregnancy.... 40 T a b l e 5 L i t t e r S i z e , F e t a l Body Weights, and P l a c e n t a l Weights o f Rats at Day 20 o f G e s t a t i o n 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 42 T a b l e 6 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 Z i n c 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 D u r i n g Week Three o f G e s t a t i o n i n Rats Given 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 45 T a b l e 7 B l o o d Flow t o P l a c e n t a s and to 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 o f G e s t a t i o n o f Rats 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 46 T a b l e 8 F e t a l Uptake o f 2-Amino I s o b u t y r a t e , M e t h y l - ^ 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 , and Z i n c from the M a t e r n a l 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 48 T a b l e 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-© t -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 , and Z i n c i n M a t e r n a l Plasma and L i v e r 90 M i n u t e s a f t e r I n j e c t i o n o f R a d i o a c t i v e Compounds 49 v i i i 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 Muscle Dry Weight ( 7 0 o f Wet Weight) i n A l c o h o l , P a i r -Fed, and Ad L i b i t u m Fed Rats 50 i x ACKNOWLEDGEMENTS I w o u l d l i k e t o e x p r e s s my s i n c e r e t h a n k s t o my a d v i s o r Dr. J o s e p h L e i c h t e r f o r h i s d i r e c t i o n a nd e n c o u r a g e m e n t t h r o u g h o u t t h e c o u r s e o f t h i s p r o j e c t . My a p p r e c i a t i o n i s e x t e n d e d t o Dr. M e l v i n L ee f o r h i s v a l u a b l e a d v i c e as w e l l as f o r t h e s u p p o r t i n computer programming and s t a t i s t i c a l a n a l y s i s o f t h e r e s u l t s . I am v e r y g r a t e f u l t o Dr. R a l p h K e e l e r a n d Dr. S t e p h a n i e Ma f o r t h e i r a s s i s t a n c e and c o l l a b o r a t i o n i n t h e d e v e l o p m e n t o f t h e e x p e r i m e n t a l methods, and t o Dr. Dan Rurak f o r h i s u s e f u l comments and s u g g e s t i o n s . Thanks a r e a l s o e x p r e s s e d t o H e l e n S m i t h f o r h e r e x c e l l e n t t e c h n i c a l a s s i s t a n c e i n t h e l a b o r a t o r y . F i n a n c i a l s u p p o r t i s a c k n o w l e d g e d f r o m t h e N a t i o n a l S c i e n c e s and E n g i n e e r i n g R e s e a r c h C o u n c i l o f Canada G r a n t #A6249 and f r o m t h e B r i t i s h C o l u m b i a Y o u t h Employment Program. 1 INTRODUCTION Concern about the adverse e f f e c t s of maternal alcohol consumption during pregnancy on offspring has been recorded since ancient times (1,2). However i t has only been i n the l a s t decade that a r e l a t i o n s h i p has been established between chronic maternal alcoholism and a s p e c i f i c pattern of anom-a l i e s i n o f f s p r i n g of such women. This pattern of anomalies, termed f e t a l alcohol syndrome, i s characterized by prenatal and postnatal growth retardation, mental deficiency, micro-cephaly, c r a n i o f a c i a l anomalies, and other changes (3-5). Children with f e t a l alcohol syndrome are usually below the t h i r d percentile i n height, weight, and head circumference, and do not appear to demonstrate catch-up growth. The average reported I.Q. i s around 65 with a range from 16 to 106, i n d i c a t i n g that most of the children were borderline to mildly retarded i n comparison with Stanford Binet Intelligence Test 1972 norms (5). It has been reported i n the medical l i t e r a t u r e (5) that about 40% of babies born to heavy drinkers displayed charact-e r i s t i c s of the f e t a l alcohol syndrome. By heavy drinkers i s meant an intake of 90 mL (3 oz.) of absolute alcohol per day during pregnancy. The extent to which these infants show f e t a l alcohol syndrome c h a r a c t e r i s t i c s i s probably determined by the amount and duration of exposure to alcohol i n utero. Lower l e v e l s of alcohol consumption during pregnancy may induce a milder form of f e t a l damage (5). L i t t l e (6) demonstrated 2 that reduced b i r t h weight due to alcohol comsumption i s independent of maternal smoking habits, a recognized b i r t h weight depressent. Since the recognition of the f e t a l alcohol syndrome i n children, numerous studies on the e f f e c t s of maternal alcohol consumption on the progeny have been conducted i n animals. I t has been confirmed using rats that maternal consumption of alcohol during gestation r e s u l t s i n retarded f e t a l and postnatal growth ( 7 , 8 ) . Despite the f a c t that the dams did not receive alcohol a f t e r delivery, catch up growth was not observed during the f i r s t four weeks post-n a t a l l y . These observations with animal models indicate that the retarded growth i s not due to e i t h e r c a l o r i c under-n u t r i t i o n or to a s p e c i f i c n u t rient deficiency as the nutrient and c a l o r i c intakes were s i m i l a r i n alcohol treated and p a i r - f e d control animals. Maternal alcohol consumption might however cause retarded growth of the fetus by i n t e r f e r -ing with the maternal-fetal t r a n s f e r of nutrients across the placenta. Decreased placental nutrient a v a i l a b i l i t y , as a r e s u l t of e i t h e r reduced maternal plasma nutrient concentra-t i o n or a reduction i n p l a c e n t a l blood flow, may further contribute to growth retardation i n the fetus. To provide an explanation f o r alcohol induced growth retardation i n the fetus, the present study was designed to determine the e f f e c t of maternal alcohol consumption on the a v a i l a b i l i t y of nutrients to the fetus i n a s u i t a b l e rat model. This was achieved through i n v e s t i g a t i o n of maternal 3 n u t r i t i o n a l s t a t u s , p l a c e n t a l b l o o d f l o w , and p l a c e n t a l t r a n s f e r o f n u t r i e n t s i n r a t s consuming a l c o h o l p r i o r t o and t h r o u g h o u t g e s t a t i o n . I n o r d e r t o d etermine th e n u t r i t i o n a l s t a t u s o f t h e pregnant a n i m a l s m a t e r n a l plasma c o n c e n t r a t i o n s o f g l u c o s e , f o l i c a c i d , and z i n c were measured. The b l o o d f l o w t o t h e p l a c e n t a was d e t e r m i n e d 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 . Z i n c , f o l i c a c i d , and n o n - m e t a b o l i z a b l e g l u c o s e and amino a c i d a n a l o g s (methyl-c x l-D g l u c o p y r a n o s i d e and 2-amino i s o b u t y r a t e ) were s e l e c t e d t o measure t h e t r a n s f e r o f n u t r i e n t s from th e m a t e r n a l c i r c u l a t i o n t o t h e f e t u s as t h e s e n u t r i e n t s a r e known t o have an e f f e c t on f e t a l growth and development (9,10,11). 4 LITERATURE REVIEW Human Studies a) Retrospective Studies The connection between maternal alcohol consumption and faulty development of the offspring has been recognized since early Greek and Roman mythology. In Carthage the b r i d a l couple was forbidden to consume wine on t h e i r wedding night i n order that defective children might not be conceived (1). S i m i l a r l y i n the nineteenth century a report to the House of Commons of England by a select committee investigat-ing drunkenness indicated that infants born to chronic alco-h o l i c mothers acguired "a starved, s h r i v e l l e d , and imperfect look" (2). In 1900 Sulli v a n reported increased abortion and • s t i l l b i r t h rates among chronic a l c o h o l i c women and an i n -creased freguency of epilepsy i n t h e i r surviving offspring (12). Since that period sporadic c l i n i c a l reports have appeared suggesting an association between maternal alcoholism and serious abnormalities i n the o f f s p r i n g (13,14), but none had substantial impact u n t i l the reports of Jones and Smith (3,15). Since then extensive investigation, both i n humans and i n animal models, has been ca r r i e d out i n thi s area. In 1973 Jones and Smith (3,15) described a pattern of malformations among offspring of chronic a l c o h o l i c mothers and referred to t h i s recognizable pattern of malformations as the f e t a l alcohol syndrome. These offspring, of alco h o l i c mothers exhibited prenatal and postnatal growth deficiency as well as c r a n i o f a c i a l abnormalities including microcephaly, short palpebral fissures, and maxillary hypoplasia. Joint 5 and cardiac anomalies were reported i n over 50 percent of these children. Lack of postnatal growth response to high c a l o r i e feeding provided further evidence of the immutable nature of the deleterious prenatal e f f e c t of growth rate (15). Assessment of maternal n u t r i t i o n a l status revealed no abnor-malities aside from occasional iron deficiency (15). No pattern of malformation as described i n f e t a l alcohol syndrome had been reported i n off s p r i n g of iron d e f i c i e n t anemic women. In t e l l e c t u a l development and motor performance was reported by Streissguth i n twelve o f f s p r i n g of chronic a l c o h o l i c women (16). A l l except one of the children exhibited a borderline or retarded i n t e l l i g e n c e l e v e l . I t was observed that children with the clear features of f e t a l alcohol syndrome were most retarded while those with lesser degrees of dysmorphology appeared less impaired. The same workers attempted to better characterize the f e t a l alcohol syndrome by a review of the charts of the National In s t i t u t e of Neurological Disease and Strokes Perinatal Project (17). About 55,000 pregnant women were observed up to seven years postnatally and the performance and development of t h e i r o f f s p r i n g monitored. Alcohol consumption i n the mother's c l i n i c a l record was compared to c h i l d development. In 23 cases there was an indicati o n of severe chronic alcoholism during and p r i o r to pregnancy. Each of these cases was compared with control non-alcoholic women of equal socio-economic status, race, age, education, parity, and marital 6 status. Among the a l c o h o l i c group a 43 percent adverse outcome was observed, as compared with a 2 percent rate i n the control group. Four of the alcohol exposed offspring died during the per i n a t a l period and s i x exhibited dysmorphology consistent with the f e t a l alcohol syndrome. These of f s p r i n g also d i s -played smaller growth parameters i n the newborn nursery as well as at seven years of age. The v a l i d i t y of t h i s work may be guestioned on the grounds that chronic alcoholism of these mothers may have been complicated by certain adverse environmental conditions detrimental to the proper develop-ment of the o f f s p r i n g . Children of disorganized, multiproblem families are at greater r i s k i n terms of psychological and i n t e l l e c t u a l development than are those from more stable family units. The I.Q. for children l i v i n g with r e l a t i v e s or i n foster homes averaged 84, whereas that of children 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 well as contributing to postnatal i n t e l l e c t u a l developmental retardation of offspring, has been implicated as aggravating the prenatal effects of alcohol exposure (18). Elevated consumption of caffeine, nicotine, and narcotics as well as i r r e g u l a r maternal dietary and sleeping habits may compound the e f f e c t s of alcohol on the fetus (15,18). Jones et a l . (3) found no evidence of drug use by a l c o h o l i c mothers conceiving o f f s p r i n g characterized by f e t a l alcohol syndrome, and while most women smoked, they were reportedly not heavy smokers. However the accuracy of reported amounts and types of drugs consumed by outpatients 7 i s questionable. The i n i t i a l documentation of the anomalies associated with f e t a l alcohol syndrome prompted a plethora of subsequent c l i n i c a l studies i n various countries (16-26). Large v a r i a -t i o n i n the pattern of defects was reported, possibly linked to the degree and timing of alcohol consumption during preg-nancy. Small widely spaced eyes, anteverted n o s t r i l s , and a carp-shaped mouth were common features c h a r a c t e r i s t i c of f e t a l alcohol syndrome. Hypothalamic and p i t u i t a r y function i n four s i b l i n g s exhibiting c h a r a c t e r i s t i c s of f e t a l alcohol syndrome was investigated by Root et a l . (19) to determine whether a hormone abnormality may account for the observed reduction i n growth. Serum levels of human growth hormone, i n s u l i n , l u t e i n i z i n g hormone, and parathyroid hormone were a l l found within normal ranges i n these s i b l i n g s . It was i n f e r r e d that postnatal growth retardation associated with f e t a l alcohol syndrome i s not d i r e c t l y due to hormonal factors. Whether alcohol affects f e t a l hormone leve l s , or a l t e r n a t i v e l y somatomedin function or hormone receptor development during gestation remains to be determined. b) Prospective Studies Although retrospective studies provide good evidence i n support of the adverse effects of alcohol on the fetus, i t i s the prospective study which provides a more rigorous analysis. Subjects of a prospective study are assessed as to the severity and duration of alcoholism p r i o r to delivery. The occurance 8 of d i f f e r i n g patterns of drinking such as heavy episodic binges at certain times during gestation may be more readi l y determined i n t h i s type of study. Correlations may then be made between the outcome of pregnancy and extent or pattern of alcoholism i n the mother. Prospective studies have been used to investigate the e f f e c t of dosage, duration, type of beverage consumed and the threshold amount of alcohol which e l i c i t s adverse e f f e c t s i n utero (20, 21, 23). Mau and Netter (20) c o l l e c t e d data on alcohol consump-ti o n from 5200 women at twenty German hospitals during the f i r s t trimester of pregnancy and compared alcohol intake to the well-being of the offspring. Women consuming alcohol gave b i r t h to a larger number of off s p r i n g of a gestational age of less than 260 days, had more abortions, but showed no higher incidence of reduction i n b i r t h weight. Alcohol use, however, was evaluated subjectively as results were tabulated only as 'yes' (4.8 percent), or 'no' (95.2 percent). Thus under-reporting of actual levels of alcohol consumption may have influenced r e s u l t s . A prospective study by L i t t l e (21) compared maternal alcohol consumption of 263 subjects before pregnancy, i n early, and late pregnancy to b i r t h weights of offspring. In order to control for smoking, which i s related to reduced b i r t h weight (22), s i m i l a r proportions of smokers were selected for each category of pregnancy. Maternal age, height, parity, nicotine use, and alcohol consumption were variables entered into a regression eguation which was computed for each of the three defined catagories of drinking 9 r e l a t i v e to pregnancy. S i g n i f i c a n t regression relationships were found between two of these defined periods and b i r t h -weight. Daily ingestion of one ounce of absolute ethyl alcohol during the three month period p r i o r to pregnancy was associated with a 91 g reduction i n b i r t h weight. The same amount of alcohol consumed during late pregnancy was assoc-ia t e d with an average body weight decrease of 160 g. No other parameter of f e t a l health except b i r t h weight was determined i n t h i s study and, since lower b i r t h weights are not s t r i c t l y related to teratogenic outcome, interpretation of these results must be made with caution. Furthermore, the r e l a t i v e l y small sample size 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 ts. However, th i s study takes into account some of the complicative factors such as smoking, age, height, and p a r i t y which hinder work i n t h i s area, and hence allows a better focus on the s p e c i f i c e f f e c t s of alcohol i n utero. Kaminski et a l . (23) have reported on the relationship between alcohol consumption among pregnant women and outcome of pregnancy from a prospective study of more than 9000 b i r t h s . Mothers consuming more than 1.6 oz absolute ethyl alcohol per day were compared with those drinking l e s s . Women consuming more than 1.6 oz per day were found to have more s t i l l b i r t h s and reduced infant birthweights as compared to the l i g h t e r drinking group. Actual differences between groups i n b i r t h -weight of of f s p r i n g were r e l a t i v e l y small even though they attained s t a t i s t i c a l s i g n i f i c a n c e . Placental weights, previously unreported i n f e t a l alcohol syndrome, were found 10 s i g n i f i c a n t l y reduced i n the heavier drinkers. No s i g n i f -icant differences i n the number of congenital malformations were reported between groups, nor was there any difference i n mean gestational age. When r i s k factors including maternal age, parity, smoking, previous low birthweight of offspring, marital status, and metrorrhagia were taken into account through appropriate s t a t i s t i c a l technigues, d i f f e r -ences between heavy' and l i g h t drinkers persisted with regard to p e r i n a t a l mortality, birthweight, and placental weight. Furthermore, types of beverages consumed were compared with outcome of pregnancy i n those mothers drinking over 1.6 oz of alcohol per day. Infants born to women drinking only beer exhibited reductions i n both mean birthweight and mean placental weight when compared with those who drank only wine. The actual differences i n birthweight were again r e l a t i v e l y small and while 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 of c l i n i c a l importance. Further findings showed that the type of beverage consumed was related to maternal socio-economic position, thus suggesting that environmental factors may contribute to the reduction i n f e t a l growth. c) Alcohol Induced A l t e r a t i o n i n Maternal Metabolism as Contributing to Fetal Alcohol Syndrome. Although extensive documentation of f e t a l alcohol syndrome, both i n retrospective and prospective studies, has shown a s i g n i f i c a n t association between maternal alcohol con-sumption •and adverse outcome of pregnancy, these studies 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 mode o f i n s u l t . A l c o h o l 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 t h r o u g h t h e f e t a l b l o o d s t r e a m (2 4 ) . The odor o f a l c o h o l has been 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 which d i s p l a y e d c h a r a c t e r i s t i c s o f f e t a l a l c o h o l syndrome. E t h y l a l c o h o l 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 as much as 150 mg per 100 mL (24). However, a l c o h o l has not y e t been shown t o d i r e c t l y a f f e c t f e t a l m e t a b o l i s m . Because f e t a l development i s dependent on t h e r e l a t i o n s h i p between t h e mother and f e t u s , i t can not be r u l e d out t h a t a l c o h o l ' s e f f e c t may be a t a s i t e o t h e r t h a n t h e f e t u s . 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 t h a t a l c o h o l may cause a p e r s i s t e n t a l t e r a t i o n i n m a t e r n a l meta-b o l i s m o r p h y s i o l o g i c a l s t a t e c o n t r i b u t i n g t o adver s e f e t a l development (25, 28). These a l t e r a t i o n s a re a g g r a v a t e d by d u r a t i o n and s e v e r i t y o f a l c o h o l i s m . S h r u y g i n (25) has s t u d i e d t h e e f f e c t s o f m a t e r n a l a l c o h o l i s m i n 42 o f f s p r i n g o f 19 mothers. 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 mothers had de v e l o p e d advanced a l c o h o l i s m , 14 were m e n t a l l y r e t a r d e d , and many demonstrated s i g n s o f o r g a n i c impairment o f the c e n t r a l nervous system e a r l y i n i n f a n c y . I n c o n t r a s t , 19 o f f s p r i n g b orn b e f o r e t h e i r mothers had de v e l o p e d advanced s t a g e s o f a l c o h o l i s m demonstrated m a i n l y v e g e t a t i v e , e m o t i o n a l , and b e h a v i o r a l d i s o r d e r s . These•developed around ages 9 o r 10 y e a r s and tended t o r e m i t f o l l o w i n g improvement i n t h e s o c i a l environment as a r e s u l t o f m a r r i a g e o r employment. Majewski (26) has su g g e s t e d a r e l a t i o n s h i p between l e n g t h o f a l c o h o l a d d i c t i o n i n t h e mother and s e v e r i t y o f 12 a l c o h o l i n s u l t i n u t e r o . He r e p o r t e d t h a t younger s i b l i n g s 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 a l c o h o l i s m due t o the t e m p o r a l p r o g r e s s i o n o f t h e d i s e a s e . I t was su g g e s t e d t h a t a c e t a l d e h y d e , an a l c o h o l m e t a b o l i t e , i s r e s p o n s i b l e f o r a l c o h o l ' s a d v e r s e e f f e c t . The e x t e n t o f adverse e f f e c t may depend on t h e m a t e r n a l a c e t a l d e h y d e m e t a b o l i s m c a p a c i t y , which i s thought t o be i m p a i r e d w i t h extended m a t e r n a l a l c o h o l consumption. Mean d a i l y b l o o d a l c o h o l l e v e l s may i n c r e a s e w i t h d u r a t i o n o f t h e d i s e a s e as 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 mothers had been a l c o h o l i c s f o r a l o n g e r time p e r i o d may be e x p l a i n e d s i m p l y as 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 u t e r o . However t h i s e x p l a n a -t i o n i s r e f u t e d by work documenting d e c r e a s e d b i r t h w e i g h t s i n o f f s p r i n g o f mothers who had a b s t a i n e d from a l c o h o l consumption d u r i n g pregnancy (27,28). S c h e i n e r e t a l . ( 2 7 ) r e p o r t e d a case w i t h c o n g e n i t a l m a l f o r m a t i o n s 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 d e v e l o p m e n t a l r e t a r d a t i o n a s s o c -i a t e d w i t h f e t a l a l c o h o l syndrome. I t was r e p o r t e d t h a t t h e p a r e n t s , a l t h o u g h heavy d r i n k e r s f o r s i x t e e n y e a r s had ab-s t a i n e d one and a h a l f y e a r s p r i o r t o c o n c e p t i o n . The p o s s i b i l i t y can not be r u l e d out t h a t t h e mother d i d i n f a c t d r i n k w h i l e pregnant o r t h a t t h e c h i l d d i s p l a y e d n o t f e t a l a l c o h o l syndrome but a s u g g e s t i v e phenotype w i t h some o t h e r e t i o l o g y . L i t t l e e t a l . (2 8) have p r o v i d e d f u r t h e r e v i d e n c e s u g g e s t i n g 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 m a t e r n a l 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 , f o r t h e advers e 13 e f f e c t s i n utero. Infants born to women who had a history of alcoholism p r i o r to conception, but had abstained during pregnancy, were compared to infants whose mothers drank heavily throughout the course of pregnancy and to infants of non-alcoholic controls. The three groups were matched for maternal education, smoking during pregnancy, race of offspring, and age of o f f s p r i n g at the time of the study. Mean b i r t h weights of children born to abstinent alcoholics was determined as 258 g less than the mean b i r t h weight of children born to controls. For the drinking a l c o h o l i c group the mean birthweight was 49 3 g below that of controls. The p o s s i b i l i t y was examined that the abstinent a l c o h o l i c women may have reported t h e i r drinking inaccurately, however i t was noted that there would be no reason for them to do so purposely. In cases where the interviewer suspected inadvert-ent error, i n t e r n a l v e r i f i c a t i o n s were performed by l i s t i n g the events i n the subject's l i f e during the time surrounding pregnancy, and crosschecking her r e c a l l during these events. In summary, a review of the c l i n i c a l studies reporting the effects of maternal alcohol consumption on the outcome of pregnancy indicate that t h i s phenomenon, although noted h i s t o r i c a l l y , has only recently been extensively examined. Retrospective and prospective investigations have as yet f a i l e d to determine the mode of i n s u l t of alcohol, although the pattern of malformations c h a r a c t e r i s t i c of f e t a l alcohol syndrome have been well established. The extent of contribu-t i o n of adverse environmental factors to f e t a l alcohol 14 syndrome i s unknown. D u r a t i o n o f a l c o h o l i s m r e l a t i n g t o s e v e r i t y o f f e t a l i n s u l t and appearance o f f e t a l a l c o h o l syndrome i n o f f s p r i n g whose mothers stopped d r i n k i n g p r i o r t o c o n c e p t i o n a r e b o t h f a c t o r s s u g g e s t i n g t h a t t h e 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 n o t be r e s t r i c t e d d i r e c t l y t o the f e t u s . A l t e r n a t i v e l y , a l c o h o l may be d e l e t e r i o u s t o a s u s c e p t i b l e component o f m a t e r n a l p h y s i o l o g y o r m e t a b o l i s m . 15 Animal Studies a) Introduction The advantage of the animal model i n f e t a l alcohol syndrome research i s to allow investigation of the uncom-pounded e f f e c t of alcohol on the unborn without complicative environmental variables found i n human studies. Early reports evaluating the effects of maternal alcohol consumption on f e t a l outcome i n the fowl and the rat have appeared since the turn of the century (29- 34); however dubious method-ology has often jeopardized the v a l i d i t y of these investiga-tions. More recent studies employing i s o c a l o r i c a l l y p a i r -fed control groups, oral routes of alcohol administration, and dosages s i m i l a r to those encountered i n the human a l c -oholic have been performed i n rats, mice, and other species. These improvements i n experimental design have f a c i l i t a t e d the development of an animal model which closely approximates f e t a l alcohol syndrome as manifest i n the human. b) Prenatal Effects of Alcohol Administration i n the Rat. Sandor and Amels (35) made one of the i n i t i a l s i g n i f -icant contributions i n the investigation of the e f f e c t of alcohol on- prenatal development i n the rat. Alcohol was administered through intravenous i n j e c t i o n on days six, seven, and eight of gestation i n one group of animals and days s i x and seven i n another group. Dosages of 1.5 g per kg body weight were administered to animals of each group. Fetuses were removed at day nineteen of gestation by 16 c e a s a r i a n s e c t i o n and examined f o r dysmorphology. Of t h e 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 o f 2 g p e r kg body w e i g h t e l i c i t e d t w i c e t h e number o f m a l f o r m a t i o n s as d i d t h e lo w e r c o n c e n t r a t i o n . M a l f o r m a t i o n s o b s e r v e d i n c l u d e d 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 apparent i n t h e extrem-i t i e s and f a c i a l a r e a s . R e s u l t s f u r t h e r m o r e i n d i c a t e d t h a t t h e two h e a v i e r dosages a d m i n i s t e r e d on days s i x and seven were more d e l e t e r i o u s t h a n t h e t h r e e l i g h t e r doses g i v e n o v er days s i x , seven, and e i g h t a l t h o u g h t o t a l amounts o f a l c o h o l were i d e n t i c a l . I n t r a v e n o u s i n j e c t i o n does not r e p r e s e n t t h e normal mode o f a l c o h o l i n t a k e and f a c t o r s such as i n t e r f e r e n c e o f normal n u t r i e n t a b s o r p t i o n and r a t e o f i n c r e a s e o f b l o o d a l c o h o l l e v e l s a r e not c o n s i d e r e d i n t h i s s t u d y . However, r e s u l t s o b t a i n e d show a s i m i l a r p a t t e r n o f m a l f o r m a t i o n i n t h i s r a t model as seen i n human f e t a l a l c o h o l syndrome. Subsequent work i n v o l v e d t h e use o f an o r a l r o u t e o f a l c o h o l a d m i n i s t r a t i o n w h i c h i s more analogous t o t h e c h r o n i c human a l c o h o l i c . Tze and Lee (36) f e d pregnant r a t s 30 g a l c o h o l p e r 100 mL w a t e r as t h e i r o n l y a v a i l a b l e d r i n k i n g f l u i d . A p a i r - f e e d i n g d e s i g n was i n c o r p o r a t e d whereby, as w e l l as an a l c o h o l f e d and ad. l i b i t u m c o n t r o l group, a t h i r d group o f r a t s r e c e i v e d 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 w i t h t h a t consumed by a l c o h o l f e d a n i m a l s . The e f f e c t o f red u c e d c a l o r i c i n t a k e i n t h e a l c o h o l f e d group was thus 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 o f t h i s p a i r - f e d group. A l l an i m a l s were mated one month a f t e r t h e i n i t i a t i o n o f t h i s 17 dietary protocol and allowed to d e l i v e r at term. Gestation period did not vary among the three treatment groups. Only f i f t y percent of the alcohol dams known to have copulated" delivered l i t t e r s . In comparison, 88 and 91 percent of control and p a i r - f e d groups, respectively, assessed as pregnant, produced l i t t e r s . The average l i t t e r size of the alcohol treated mothers was s i g n i f i c a n t l y lower than that of p a i r - f e d or control groups. Offspring of the alcohol fed group displayed microcephaly, cracked dry skin, reddening of areas of the head and body, and a generally shriveled appear-ance. Malformations reported by e a r l i e r authors (35) were not observed. A possible c r i t i c i s m of t h i s model may stem from the fact that the pregnant rats were allowed to d e l i v e r at term and cannibalization may have affected the recorded l i t t e r s i z e . Furthermore resorptions can not be determined. S a c r i f i c e of the animals p r i o r to delivery permits greater accuracy of assessment of l i t t e r size and number of f e t a l resorptions through determination of implantation s i t e s at autopsy. Studies using rat models of f e t a l alcohol syndrome do not consistently report associations between maternal alcohol exposure and f e t a l i n s u l t i n spite of the improve-ments i n design which have emerged over the l a s t decade. Schwetz et a l , (3 7) found no prenatal effects i n rats given drinking f l u i d containing 12 to 20 percent alcohol. This l e v e l of alcohol consumption may have been i n s u f f i c i e n t to e l i c i t the anomalies observed by other workers. Peak 18 blood alcohol levels were reported to be 40 mg per 100 mL, lower than values reported by other workers. Oisund et a l . (38) also found no s i g n i f i c a n t differences i n body, brain, l i v e r , kidney, or heart muscle weights between fetuses of control and alcohol exposed pregnant rats. Animals were fed 12 percent alcohol i n t h e i r drinking water to cover 20-25 percent of t h e i r c a l o r i c intake. Alcohol exposure extended from three weeks p r i o r to mating, during, and a f t e r pregnancy. Soya o i l was used as an alcohol substitute i n the p a i r - f e d group animals. Again the observed absence of adverse effects of i n utero alcohol exposure may be attributable to the low dosage l e v e l i n t h i s model. Sim i l a r l y , Abel (39) and Abel and Greizerstein (40) showed that when pregnant rats were given alcohol by stomach tube through-out pregnancy, no abnormality in growth of o f f s p r i n g could be attributed to doses of 1 or 2 g per kg body weight, but following the administration of 4 or 6 g per kg, diminished b i r t h weight, delayed postnatal growth, and greater postnatal mortality occurred i n alcohol exposed offspring compared to control progeny. Generally, when s u f f i c i e n t dosage levels are administered to pregnant rats, studies report adverse f e t a l outcome. Henderson et a l . (41) have also demonstrated that alcohol administration i n a l i q u i d diet to rats during pregnancy i s associated with adverse effects on f e t a l v i a -b i l i t y and growth. Maternal blood alcohol levels were reported as 70-200 mg per 100 mL, substantially greater than those of Schwetz et a l . (37), where no adverse effects 19 were noted. These studies suggest that a threshold l e v e l of blood alcohol may be reguired to produce v i s i b l e e f f e c t s . Although much intere s t i n maternal alcohol consumption has centered around the fetus, the placenta has recently also become the focus of attention i n the rat model of f e t a l alcohol syndrome. Abel and Greizerstein (40) found increased placental weights i n alcohol exposed pregnant rats when compared with controls, however differences 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 et a l . (42), Skosyreva (43) , and Gordon et al.(44) have reported s i g n i f i c a n t l y heavier placentas i n alcohol exposed rats. Gordon et a l . (44) fed rats a l i g u i d diet containing 26 percent alcohol derived calories for two weeks p r i o r to and during gestation. Fetal weights did not vary between alcohol fed and control groups suggesting that the increase i n placental size found on day 20 of gestation may e x i s t as an adaptive mechanism compensating for the deleterious e f f e c t of alcohol. c) Prenatal E f f e c t s of Alcohol Administration in the Mouse The malformations observed i n human f e t a l alcohol subjects 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 alcohol consumption than i n the rat model. Kronick (45) employed a mouse model to study the e f f e c t s of acute alcohol exposure on the outcome of pregnancy. Alcohol treatment consisted of an intraperitoneal i n j e c t i o n of a 25 percent solution of ethyl alcohol at a does of 3 mL per 20 100 g body weight. The strength of thi s dose induced hypnosis with animals remaining comatose for up to four hours following treatment. One group of mice received injections on the eighth and ninth day of gestation, another group on the tenth and eleventh day of gestation, and a t h i r d group on only one gestational day ranging from day seven through day twelve. Control animals were injected with a saline solution and matched with alcohol injected dams. Mice were s a c r i f i c e d on gestational day 18 and uterine contents examined. Results indicated s u b s t a n t i a l l y elevated f e t a l mortality rates in day eight and nine as well as day ten and eleven alcohol exposed mice as compared to controls injected with s a l i n e . Malformation frequency was increased i n alcohol-treated groups with the genesis of s p e c i f i c malformations dependent on tne time of i n j e c t i o n during gestation. Coloboma of the i r i s and ectrodactyly of the forepaws appeared to have c r i t i c a l periods at days eight to nine and at day ten, respectively, of gestation. The intraperitoneal route of alcohol administration used i n this model i s not consistent with the o r a l consumption pattern as seen in human alcoholism, and may exis t as a flaw in methodology. A subsequent study by Chernoff (46) using a mouse model employed an o r a l route of alcohol administra-t i o n as well as pai r - f e d control groups. Chernoff (46) fed alcohol through an a l l l i q u i d diet to two strains of mice. Alcohol treatment extended from t h i r t y days p r i o r to mating u n t i l day 18 of gestation, at which time females were s a c r i f i c e d and uterine contents examined. One 21 s t r a i n of mice exhibited s i g n i f i c a n t l y more f e t a l malforma-tions than the other, suggesting that the rate of alcohol metabolism may d i f f e r between the two s t r a i n s . The l i v e r alcohol dehydrogenase system, responsible for the metabolism of alcohol, i s known to be under genetic control i n the mouse (47). The f e t a l malformations included intrauterine growth deficiency as evidenced by low f e t a l weight, incom-plete o s s i f i c a t i o n , neural and cardiac anomalies, and s k e l e t a l dysmorphogenesis. The pattern of growth retarda-t i o n together with ocular, neural, cardiac, and s k e l e t a l anomalies were s i m i l a r to that of the f e t a l alcohol syndrome observed i n humans. Another animal model employing an o r a l l y administered alcohol treatment and a pair-feeding design i n mice was developed by Randall and Taylor (48). Pregnant mice were administered diets with 17, 25, or 35 percent of t o t a l d a i l y calories supplied by alcohol from day f i v e to day ten of gestation. I s o c a l o r i c substitution of sucrose for alcohol was effected i n the p a i r - f e d group. S a c r i f i c e of the gravid dams was made on day 19 of gestation. Results showed that i n the 25 percent a l c o h o l - c a l o r i e group, f i f t e e n out of sixteen l i t t e r s had at least one malformed fetus. Systematic external examination of the fetuses was followed by i n t e r n a l examination under a dissecting microscope. Anomalies observ-ed included malformations of the limbs, cardiovascular abnormalities, urogenital hydronephrosis, and hydrocephaly. As with the study of Chernoff (46), the anomalies found i n t h i s mouse model are analagous with those of human offspring 22 displaying f e t a l alcohol syndrome. Lack of prenatal e f f e c t s of alcohol i n mice has also been reported (37, 49), possibly due, as seen i n the rat model, to an i n s u f f i c i e n t l e v e l of exposure to alcohol. Determination of the l e v e l of alcohol exposure i s a current methodological problem i n animal models of f e t a l alcohol syndrome. Comparisons of alcohol intake between man and animals are d i f f i c u l t , plagued by problems r e l a t i n g to d i f f e r i n g alcohol metabolizing rates. Measurement of ingested ethyl alcohol expressed as g per kg of body weight per day i s used by some workers, however serum alcohol l e v e l s presently are most widely accepted. The use of serum alcohol levels as indicators of alcohol intake obviates the d i f f i -c u l t i e s acguired because of d i f f e r i n g alcohol pathways and metabolic rates among animal species. The preceding studies indicate that using a suitable animal model of maternal alcohol exposure, the pattern of malformation seen i n off s p r i n g born to humans are better approximated i n the mouse than i n the rat. However, clear cut adverse e f f e c t s including prenatal growth retardation are observed i n the rat, and the use of certain experimental protocols makes this larger animal a more a t t r a c t i v e exper-imental subject. d) Prenatal Effects of Alcohol Exposure i n Other Animals Although most of f e t a l alcohol syndrome research using 23 animal models has focused on rats and mice, other species have also been the subject of investigation. Simulating blood alcohol levels which would be normal in the human alcoholic, Sandor and E l i a s (50) studied the effects of alcohol i n chick embryos i n early stages of development. In eggs injected with alcohol embryos exhibited higher mortality rates and weight loss towards the end of incubation as com-pared with controls. However, extrapolation from the develop-ment of the chick embryo to that of the embryo i n utero i s tenuous, hence i n most subsequent work mammals were used as experimental subjects. Dexter et a l . (51) fed miniature S i n c l a i r swine alcohol solutions o r a l l y both p r i o r to and during gestation. Miniature swine consume large quantities of alcohol despite adequate diet and constantly available water, allowing investigation into the e f f e c t s of prenatal e f f e c t s of alcohol consumption in the presence of adequate n u t r i t i o n and without disturb-ing an established behavior. Results showed that consumption by pregnant swine of alcohol at levels equivalent to one quart of whiskey per day i n the human resulted i n a decrease in l i t t e r s i z e and b i r t h weight, as well as increased rates of occurance of morphological anomalies and postnatal mort-a l i t y . Anomalies observed were s i m i l a r to those seen i n human f e t a l alcohol syndrome subjects. E l l i s and Pick (52) used a beagle model to study the effects of maternal alcohol consumption on the fetus. Alcohol was administered through g a s t r i c intubation at 24 s i x d i f f e r e n t dosage leve l s i n an attempt to determine a threshold l e v e l for onset of adverse e f f e c t s . With increas-ing dosage a higher occurance of f e t a l mortality and mal- • formation was observed, with no animal i n the group exposed to alcohol at the greatest dosage l e v e l d e l i v e r i n g l i v e born offspring. Blood alcohol levels of t h i s group reached 255 mg per 100 mL. /Animals consuming the lowest of the dosages, 1.4 g ethyl alcohol per kg body weight, displayed no s i g n i -f i c a n t l y increased rate of malformation or s t i l l b i r t h , however progressively higher dosages were found detrimental to f e t a l welfare. Extrapolating these results to the human subject, i f a woman weighing 60 kg were to consume 6 oz of 100 proof alcohol per day during gestation, the wellbeing of her fetus may be at r i s k . The use of the beagle in f e t a l alcohol syndrome research i s advantageous i n that a close s i m i l a r i t y i n alcohol pharmacokinetics exists between man and the dog, however r e s t r i c t i o n s i n group size inherent i n using an animal of t h i s s i z e may l i m i t the usefulness of certain s t a t i s t i c a l treatments. Ho et a l . (53) fed alcohol to pregnant monkeys and hamsters i n order to determine i t s d i s t r i b u t i o n i n the maternal and f e t a l blood, organs and central nervous system. The d i s t r i b u t i o n of alcohol i n the placenta and amniotic 14 f l u i d was also studied. Ethanol labeled with C was i n j e c t -ed intravenously at doses ranging from 0.5 to 1.5 g per kg body weight following which animals were k i l l e d by bleeding at f i x e d time i n t e r v a l s . Radioautography of the brain, f e t a l , and placental sections was performed, and alcohol determinations 25 made on homogenates o f t h e s e t i s s u e s . R e s u l t s i n d i c a t e d t h a t d u r i n g l a t e pregnancy b o t h t h e p l a c e n t a and a m n i o t i c f l u i d c o n t a i n e d h i g h c o n c e n t r a t i o n s o f a l c o h o l compared w i t h d i s t r i b u t i o n t o o t h e r t i s s u e s . 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 hamster f e t u s was h i g h e r i n l a t e r pregnancy t h a n p r i o r t o p l a c e n t a l development, s u g g e s t i n g t h a t t h e p l a c e n t a was not a b a r r i e r t o a l c o h o l . A l c o h o l 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 mother, as i n d i c a t e d by h i g h e r a l c o h o l c o n c e n t r a t i o n s i n f e t a l t i s s u e s 90 minutes a f t e r i n j e c t i o n o f t h e dose. F e t a l 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 t o be reduced 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 h i g h e r f e t a l a c c u m u l a t i o n o f a l c o h o l (54). The "^C l a b e l e d 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 , p a n c r e a s , k i d n e y , l u n g , and h e a r t muscle w i t h a h i g h c o n c e n t -r a t i o n i n the c e r e b e l l u m . The hippocampus, putamen, and g e n i c u l a t e 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 . No o b s e r v e d m a l f o r m a t i o n i n a l c o h o l exposed f e t u s e s was r e p o r t e d i n t h i s s t u d y . e) Comparison o f P r e n a t a l A l c o h o l Exposure 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 O f f s p r i n g . The mechanism by which m a t e r n a l a l c o h o l consumption a f f e c t s f e t a l growth remains u n c l e a r . S t u d i e s r e p o r t i n g absence o f a s s o c i a t i o n between a l c o h o l consumption d u r i n g pregnancy and a d v e r s e outcome suggest t h a t i n a d e q u a t e n u t r i t i o n i s a t l e a s t i n p a r t r e s p o n s i b l e f o r t h e d e l e t e r i o u s 26 e f f e c t i n utero (37,38). However differences i n body com-position of o f f s p r i n g exposed to alcohol i n utero as compared to food r e s t r i c t e d p a i r - f e d offspring (40,41) suggest that these growth retarding e f f e c t s are not s i m i l a r . Reductions i n brain, heart, kidney, and l i v e r weights were observed i n fetuses exposed to alcohol i n utero (41). Moreover, s i g n i f -i c a n t l y elevated t o t a l tissue protein concentrations were found i n 20 day fetuses i n a rat model of alcohol exposure in utero (41). These results are contrary to observations i n o f f s p r i n g of malnourished rats where lean body mass i s generally reduced or unaffected (55) . Abel and Greizerstein (40) have also described metabolic changes i n growth retarded rat fetuses as a r e s u l t of alcohol exposure i n utero. In the fetuses whole body sodium content was s i g n i f i c a n t l y elevated i n the alcohol treated group. No differences were noted i n whole body zinc, calcium, or magnesium content of fetuses between alcohol and food r e s t r i c t e d p a i r - f e d groups. Elevated whole body sodium content i s not c h a r a c t e r i s t i c of the malnourished rat (56). Furthermore, no decrease i n fat content of the alcohol exposed fetus, as i s found i n the fetuses of malnourished rats, was reported i n t h i s study (40). These results suggest that malnutrition i s not s o l e l y responsible for the effects associated with i n utero exposure to alcohol. f) Comparison of Growth and Development i n Offspring of Alcohol-Fed Versus Malnourished Animals. 27 Although differences i n body composition have been reported between fetuses of alcohol exposed and malnourished rats (40,41), the s i m i l a r i t y i n the pattern of growth and developmental retardation i n each case suggests that these effects may share a common etiology. Studies investigating the e f f e c t s of maternal protein deficiency on neonatal growth show s t r i k i n g s i m i l a r i t i e s to those on the adverse e f f e c t of alcohol. Young born to protein r e s t r i c t e d mothers but cross fostered to control mothers at b i r t h exhibit a persistent retardation i n growth, which i s not corrected by reducing l i t t e r s i z e (55). Delayed appearance of o s s i f i c a t i o n centers and retarded s k e l e t a l growth were observed with l i t t l e i n d i -cation of catch up growth. Leichter and Lee (7), Lee and Leichter (8), and Detering et a l . (57) have reported s i m i l a r findings i n alcohol exposed offspring. In addition to retarded growth, o f f s p r i n g of animals receiving alcohol during preg-nancy also exhibit a retardation in the rate of s k e l e t a l maturation, despite the f a c t that mothers did not receive alcohol a f t e r delivery (7). Catch up was not observed either i n growth or s k e l e t a l maturation during the f i r s t four weeks postnatally. Furthermore, c u l l i n g the l i t t e r s i z e from eight to three of alcohol exposed o f f s p r i n g to account for the p o s s i b i l i t y of postnatal malnutrition had 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 stimulatory e f f e c t on any of the parameters of physical growth or maturation measured (8). A reduction i n brain weight, crown rump length and t a i l length were observed at three weeks post partum i n rat pups exposed to alcohol i n utero but weaned to control dams compared with pups of 28 equivalent age from p a i r - f e d dams (57). These studies indicate that the postnatal growth retard-ation observed i n f e t a l alcohol syndrome animal models i s s i m i l a r to that ascribable to maternal protein malnutrition. This i s contrary to indications based on body composition determinations. The observed postnatal growth retardation appears to be of d i f f e r e n t o r i g i n because of reports suggest-ing that, while i n protein malnourished rats growth hormone synthesis i s impaired (55), no growth hormone abnormalities have been observed i n humans displaying f e t a l alcohol syndrome (58). Rosso and Kava (59) have suggested that the mechanism of f e t a l i n s u l t from malnutrition results from a decreased maternal-fetal transfer of nutrients. Although the use of pair-feeding designs i n animal models of f e t a l alcohol syndrome has indicated that maternal food i n s u f f i -ciency i s not e n t i r e l y responsible for the retarded f e t a l growth, alcohol may adversely a f f e c t the supply of nutrients to the fetus, thus mimicking the e f f e c t of malnutrition. This may explain the s i m i l a r i t i e s observed i n modes of action of these growth retarding factors. 29 MATERIALS AND METHODS a) P r e l i m i n a r y Treatment o f A n i m a l s One hundred and twenty v i r g i n Sprague-Dawley r a t s ( B i o b r e e d i n g L a b o r a t o r i e s , 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 housed i n s c r e e n bottom cages. The l i v i n g o environment was m a i n t a i n e d a t 21 C w i t h a t w e l v e hour l i g h t -dark c y c l e . A f t e r a one week s t a b i l i z a t i o n p e r i o d a n i m a l s were a s s i g n e d a t random t o one o f t h r e e d i e t a r y t r e a t m e n t groups. Group 1 ( a l c o h o l ) r e c e i v e d 10 p e r c e n t e t h y l a l c o h o l (v/v) i n d r i n k i n g w a t e r and a n u t r i t i o n a l l y adequate d i e t ( P u r i n a Rat Chow) ad l i b i t u m . A f t e r one week the e t h y l a l c o h o l c o n t e n t o f the d r i n k i n g w a t e r was i n c r e a s e d t o 20 p e r c e n t . Group 2 ( p a i r - f e d ) was a d m i n i s t e r e d amounts o f n u t r i t i o n a l l y adequate d i e t ( P u r i n a Rat Chow) e q u a l t o t h a t consumed by t h e group 1 a n i m a l s d u r i n g t h e p r e v i o u s 2 4 hours, w i t h a l c o h o l i s o c a l o r i c a l l y s u b s t i t u t e d w i t h c o r n s t a r c h . D r i n k i n g w ater was p r o v i d e d t o t h e p a i r - f e d group ad l i b i t u m . Group 3 (ad l i b i t u m c o n t r o l ) was a l l o w e d t h e n u t r i t i o n a l l y adequate d i e t and d r i n k i n g w a t e r ad l i b i t u m . A f t e r f o u r weeks on t h i s 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 t o male Sprague-Dawley r a t s . The p r e s e n c e o f sperm i n t h e v a g i n a l washings e s t a b l i s h e d day 1 o f pregnancy. At t h a t t i me t h e a l c o h o l c o n t e n t o f the a l c o h o l group d r i n k i n g w ater was i n c r e a s e d t o 30 p e r c e n t . M a t e r n a l body w e i g h t s were de t e r m i n e d weekly and f o o d and a l c o h o l consumption was d e t e r m i n e d d a i l y t h r o u g h o u t the e x p e r i m e n t . 30 b) D e t e r m i n a t i o n o f Plasma 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 (300-400 uL) f o r the d e t e r m i n a t i o n o f m a t e r n a l plasma 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 were t a k e n from t h e t i p o f the t a i l e a r l y i n t h e morning d u r i n g t h e t h i r d week o f g e s t a t i o n . The b l o o d samples were c o l l e c t -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 , c e n t r i f u g e d a t 1500 rpm f o r 5 min., and t h e plasma was p l a c e d i n s m a l l p l a s t i c o t u b e s . The plasma was k e p t f r o z e n a t -5" C. u n t i l the time o f 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 by the a l c o h o l dehydrogenase method (Sigma Chemical Company, S a i n t L o u i s , M i s s o u r i , T e c h n i c a l B u l l e t i n No. 331-UV). Glucose d e t e r m i n a t i o n s were performed u s i n g the g l u c o s e o x i d a s e method (Sigma C h e m i c a l Company, T e c h n i c a l B u l l e t i n No. 510). F o l i c a c i d was measured by the r a d i o a s s a y from 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 , (Quanta-Count F o l a t e , B u l l e t i n No. 4201), and z i n c was det e r m i n e d by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y ( P e r k i n - E l m e r Atomic A b s o r p t i o n S p e c t r o p h o t o m e t e r , Model 603). c) P l a c e n t a l B l o o d Flow D e t e r m i n a t i o n C a r d i a c o u t p u t and b l o o d f l o w t o the p l a c e n t a and o t h e r 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 as des-c r i b e d by M c D e v i t t and N i e s (60) and Bruce (61). On day 20 o f g e s t a t i o n the r a t s were a n e s t h e t i z e d by i n t r a p e r i t o n e a l i n j e c t i o n o f 10 mg p e r 100 g body w e i g h t o f I n a c t i n 31 (Promonta, Hamburg, Germany). A cannula (PE 10, Intramedic, Clay Adams Co., Parsippany, N.J.) was then i n s e r t e d i n t o the l e f t femoral a r t e r y and attached to a withdrawal pump (Harvard Apparatus, South Natick, Mass.) to draw a reference sample f o r c a l c u l a t i o n of the blood flow r a t e . A second cannula (PE 50, Intramedic, Clay Adams Co., Parsippany, N.J.) was introduced i n t o the l e f t v e n t r i c l e v i a the r i g h t c a r o t i d a r t e r y f o r i n j e c t i o n of the microspheres. The v e n t r i c u l a r l o c a t i o n was determined by observing the c h a r a c t e r i s t i c pressure waves us i n g a pressure transducer and chart recorder (Harvard Apparatus, M i l l i s , Mass.) connected to the cannula of the r i g h t c a r o t i d a r t e r y . Both cannulas were f i l l e d w i t h h e p a r i n i z e d s a l i n e . Microspheres (New England Nuclear, Boston, Mass.) l a b e l e d w i t h ^ C o and w i t h a mean diameter of 15 microns were suspended i n 6 percent dextran and mixed thoroughly p r i o r to 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 microspheres i n a t o t a l volume of 0.2 mL were i n j e c t e d over a p e r i o d of 20 seconds. Simultaneously the reference sample of blood from the femoral a r t e r y was withdrawn at a rat e of 0.445 ml/minute f o r 90 seconds. F o l l o w i n g the c o l l e c t i o n o f the reference sample the r a t was k i l l e d by i n c i s i o n of the heart. Fetuses, placentas, heart, kidneys, and a muscle sample were immediately removed from the carcass, l i g h t l y b l o t t e d , and weighed. Only l i t t e r s of four fetuses or more were i n c l u d e d i n the study. The p o s i t i o n of the c a r o t i d cannula was confirmed at t h i s time by d i s s e c t i o n of the heart. A l l t i s s u e s 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 f o r r a d i o a c t i v i t y i n a gamma c o u n t e r ( P i c k e r N u c l e a r , N o r t h Haven, Conn. Model 600-120). C a r d i a c o u t p u t and b l o o d f l o w t o p l a c e n t a s and organs were c a l c u l a t e d u s i n g the f o l l o w i n g f o r m u l a e : Organ Blood = reference sample withdrawal rate (mL/min) X organ CPM Flow (mL/min) reference sample CPM To i n v e s t i g a t e t he p o s s i b i l i t y t h a t t h e l e v e l o f a l c o h o l e xposure 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 wat e r c o n t e n t o f t h e muscle sample was de t e r m i n e d by h e a t i n g t o c o n s t a n t w e i g h t i n a vacuum oven a t 70°C. U s i n g a sample o f a r t e r i a l b l o o d c o l l e c t e d p r i o r t o s a c r i f i c e o f the a n i m a l , a n o t h e r parameter o f d e h y d r a t i o n , plasma o s m o l a l i t y , was det e r m i n e d u s i n g an osmometer ( P r e c i s i o n System I n c . , Newton, Mass.) . ... .. d) F e t a l Uptake o f F o l i c A c i d , Z i n c , and Analog s o f Gluc o s e and Amino A c i d To determine the f e t a l uptake o f f o l i c a c i d , z i n c , and analogs o f g l u c o s e and amino a c i d , r a t s on day 20 o f g e s t a t i o n 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 : 1 u C i me t h y l ( o<- D {U±H - c j g l u c o ) p y r a n o s i d e and 10 u C i 3', 5', 7, 9 - H f o l i c a c i d , o r 1 j i C i 2 - amino ( 1 - C) i s o b u t y r i c 65 a c i d , o r 8 p C i Zn i n HC1 s o l u t i o n , p e r 100 g body w e i g h t . A l l compounds were i n j e c t e d i n t o the l e f t f e m o r a l v e i n . N i n e t y minutes a f t e r t he i n j e c t i o n t h e f e t u s e s and m a t e r n a l l i v e r were removed, weighed, and b l o o d was o b t a i n e d from the Cardiac Output = CPM injec t e d X reference sample withdrawal rate (mL/min) (mL/min) reference sample CPM 33 dam by h e a r t p u n c t u r e . The d e c i s i o n t o use the 90 minute time p o i n t a f t e r l a b e l e d compound i n j e c t i o n was based on a p r e l i m i n a r y s t u d y w h i c h i n d i c a t e d t h a t the c o n c e n t r a t i o n o f l a b e l i n the f e t u s was h i g h e r at 90 minutes than a t e i t h e r 40 o r 60 m i n u t e s . R a d i o a c t i v i t y was de t e r m i n e d i n plasma and l i v e r o f the dam and i n f o u r f e t u s e s l o c a t e d i n p o s i t i o n s 1 and 3 o f each u t e r i n e h orn c o u n t i n g from the t e r m i n a l end. L i t t e r s w i t h e i g h t f e t u s e s o r more were i n c l u d e d i n the s t u d y . To measure the r a d i o a c t i v i t y o f t r i t i u m and the l i v e r and f e t u s e s were homogenized i n 9 p a r t s o f 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 a t 2,000 rpm f o r 10 min. A l i q u o t s o f the s u p e r n a t a n t f r a c t i o n s (0.5 mL) were t r a n s f e r e d t o g l a s s s c i n t i l l a t i o n v i a l s c o n t a i n i n g 10 mL o f s c i n t i l l a t i o n f l u i d (ASC, Amersham, A r l i n g t o n H e i g h t s , 111.) and the r a d i o - „ a c t i v i t y was counted i n a P i c k e r N u c l e a r L i q u i m a t S c i n t i l l a -t i o n Counter. C o r r e c t i o n s f o r quenching were made by the 6 5 ch a n n e l s r a t i o method. I n t h e case o f Zn t h e f e t u s e s , l i v e r s and plasma were p l a c e d i n t e s t tubes and r a d i o a c t i v i t y was counted u s i n g a P i c k e r N u c l e a r A u t o w e l l I I Gamma Spect r o m e t e r . A l l the . . r a d i o a c t i v e compounds were p u r c h a s e d from Amersham, A r l i n g t o n H e i g h t s , 111. 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 R e s u l t s 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 v a r i a n c e , u s i n g the Midas Computer Package (62). In the 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 , S h e f f e ' s i n t e r v a l s (0.9 and 0.95) were used w i t h p a i r e d comparisons t o i d e n t i f y w h i c h 34 group was r e s p o n s i b l e f o r the d i f f e r e n c e . A l e v e l of s i g n i f i -cance of P<0.05 was used f o r a l l t e s t s . 35 RESULTS a) Food Intake and Maternal Body Weights Table 1 shows the average d a i l y food and a l c o h o l consump-t i o n and weekly body weights of r a t s given a l c o h o l , p a i r - f e d , or ad l i b i t u m fed p r i o r to pregnancy. Aside from the f i r s t week of t h i s p e r i o d body weights of ad l i b i t u m c o n t r o l animals were s i g n i f i c a n t l y greater (p<0.05) than those of e i t h e r a l c o h o l group or p a i r - f e d group animals. This i s r e f l e c t e d i n the reduced food consumption of these l a t t e r two groups. Average e t h y l a l c o h o l consumption during t h i s p e r i o d by a l c o h o l group animals amounted to approximately 2.5 mL per day. Table 2 shows the d a i l y c a l o r i c intakes of the three treatment groups p r i o r to pregnancy. T o t a l c a l o r i c intakes of the a l c o h o l and p a i r - f e d groups were about 80 percent of that of the ad_ l i b i t u m c o n t r o l s . The absence of v a r i a b i l i t y i n amounts of food consumed by the p a i r - f e d group i s r e f l e c t i v e of the p a i r - f e e d i n g design where a l l animals of t h i s group consumed a l l of the food given. P r i o r to pregnancy a l c o h o l provided 25 to 30 percent of the c a l o r i e s of the a l c o h o l group. Table 3 shows the average d a i l y food and a l c o h o l con-sumption and weekly body weights of r a t s given a l c o h o l , p a i r -fed, or ad l i b i t u m fed during pregnancy. S i g n i f i c a n t l y reduced body weights are observed i n a l c o h o l (p<0.0001) and i n p a i r -fed (p<0.001) group animals when compared w i t h ad l i b i t u m c o n t r o l s during each of the three weeks of g e s t a t i o n . The absence of 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 w e i g h t s among the t h r e e groups a t day one o f g e s t a t i o n may r e f l e c t t h a t the f a c t t h a t s i n c e m a t i n g o c c u r r e d over a two week p e r i o d , a l a r g e v a r i a b i l i t y i n body w e i g h t s w i t h i n each group may have developed. No s i g n i f i c a n t d i f f e r e n c e s were n o t e d i n body w e i g h t s d u r i n g g e s t a t i o n between a l c o h o l group and p a i r - f e d group a n i m a l s . The average consumption o f e t h y l a l c o h o l by the a l c o h o l group was 4.68+ 0.05 mL (mean + s t a n d a r d e r r o r ) p e r day d u r i n g t h i s p e r i o d . 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 d u r i n g pregnancy are d i s p l a y e d i n T a b l e 4. D u r i n g g e s t a t i o n , a l c o h o l p r o v i d e d about 3t*; p e r c e n t o f the c a l o r i e s i n the a l c o h o l group. T o t a l c a l o r i c i n t a k e s o f the a l c o h o l and p a i r - f e d groups were 71 p e r c e n t o f t h a t o f ad l i b i t u m c o n t r o l s d u r i n g the f i r s t week o f g e s t a t i o n and 9 1 % d u r i n g the l a s t two weeks o f g e s t a t i o n . T h i s i s r e f l e c t e d i n the r e d u c e d w e i g h t g a i n o f t h e s e two groups o v e r ad l i b i t u m c o n t r o l s as seen i n T a b l e 3, even though t h e r e appears t o be no s i g n i f i c a n t d i f f e r e n c e i n c a l o r i c i n t a k e d u r i n g t h e l a t t e r p a r t o f g e s t a t i o n . b) L i t t e r S i z e , F e t a l Body Weights, and P l a c e n t a l Weights. L i t t e r s i z e , f e t a l body w e i g h t s , and p l a c e n t a l w e i g h t s o f r a t s on day 20 o f g e s t a t i o n are shown i n T a b l e 5. A l t h o u g h no d i f f e r e n c e s were o b s e r v e d i n l i t t e r s i z e among a l c o h o l exposed a n i m a l s when compared w i t h t h o s e 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, a s i g n i f i c a n t r e d u c t i o n i n TABLE 1 Daily food and alcohol consumption and weekly body weights of rats given alcohol, pair-fed, or ad l i b i t u m fed p r i o r to pregnancy. Means + standard deviation. Alcohol Group P a i r - f e d Group Ad l i b i t u m fed Group Food Alcohol Body Food Starch Body Weight Food Body Weight Consumed C'med. Weight Consumed C'med Consumed (g) (mL) (g) ( g) ( g) ( g) ( g) ( g) Number of Rats (13) (13) (33) (12) (12) (27) (12) (27) Pre-pregnancy days 1-7 12.1 +2.1 2.0 +0.3 236.5 a + 13.6 12.1 +0.0 2.5 +0.0 2 3 3 . l a +8.00 16.9 +4.1 241.7 a +11.6 8-14 10.3 +2.2 2.5 +0.5 242.0 3 + 15.9 10.3 +0.0 3.4 +0.0 2 4 4 . l a +8. 74 16.8 +2.6 256.2 b + 16.0 15-21 10.0 +2.8 2.8 +0.4 252. 7 a +16.9 10.0 +0.0 3. 7 +0.0 248.3 a + 7.77 16.6 +3.1 267.9 b +19.4 22-28 10.0 +2.5 2.7 +0.3 260.4 a 120.2 10.0 +0.0 3.6 +0.0 259.0 3 +9.04 18.2 +2.6 275.4 b +20,5 a, b, figures i n the same row not sharing 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. 38 TABLE 2 Daily c a l o r i c intakes of rats given alcohol, p a i r - f e d , or ad l i b i t u m fed p r i o r to pregnancy. Means + standard deviation Alcohol Group Pair-Fed Group Ad Libitum fed Group Food Alcohol Food Starch Food Consumed Consumed Consumed Consumed Consumed (Calories) Number of Rats: (13) (13) Pre-pregnancy days 1-7 51.4 10.0 +9.0 +2.1 8-14 43.8 13.6 +9.3 +3.5 15-21 42.5 14.8 +11.9 +2.8 22-28 42.5 14.4 +10.6 +2.1 (12) (12) (12) 51.4 10.0 71.8 +0.0 +0.0 +17.5 43.8 13.6 71.4 +0.0 +0.0 +11.1 42.5 14.8 70.6 +0.0 +0.0 +13.1 42.5 14.4 77.4 +0.0 +0.0 +11.0 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 Ad Libitum fed Group Food Alcohol Body Food Starch Body Food Body Consumed C'med Weight Consumed C'med Weight Consumed Weight (g) (mL) •(g) (g) (g) (g) (g) (g) Number of Rats: (11) (ID (34) (10) (10) (32) (6) (29) Days of Gestation 1 - - 269.2a - - 267.3a _ 275.0a +20.3 +17.0 +24.6 2-7 9.66 +2.0 4.05 +0.7 273.8a +22.4 9.6 +0.0 5.27 +0.0 276.3a +23.1 20.5 +6.7 297.0b +25.5 8-14 11.6 4.7 298.4a 11.6 6.09 299.l a 19.0 326.9b +1.8 +0.7 +26.8 +0.0 +0.0 +22.8 +7.2 +28.9 15-20 12.3 +2.2 5.3 +1.0 331.6a +28.8 12.3 +0.0 6.90 +0.0 339.6a +29.4 20.5 +12 388.l b +38.2 a, b, = figures in the same row not sharing the same letter are significantly different at p<0.05. 40 TABLE 4 Daily c a l o r i c intakes of rats given alcohol, p a i r - f e d , or ad li b i t u m fed during preganancy, Means + standard deviation. Alcohol Group Food Consumed Pair- f e d Group Alcohol Consumed Food Consumed (Calories) Starch Consumed Ad Libitum fed Group Food Consumed Number of Rats: (11) (11) (10) (10) (6) Day of gestation 2-7 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 41 l i t t e r s i z e (p<0.01) was found i n the p a i r - f e d o v er the ad l i b i t u m group. The f e t a l and p l a c e n t a l w e i g h t s were n o t a f f e c t e d by t h e i r l o c a t i o n on the u t e r i n e h o r n s . 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 o f the t h r e e t r e a t m e n t groups, however f e t u s e s o f a l c o h o l exposed 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 the s k i n and a gener-a l l y more s h r i v e l l e d appearance 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 were s i g n i f i c a n t l y r e d u c e d i n the a l c o h o l group when compared w i t h e i t h e r the p a i r - f e d (p<0.0001) o r ad l i b i t u m c o n t r o l (p< 0.0001) groups. Furthermore, f e t a l body w e i g h t s o f the p a i r - f e d group were a l s o s i g n i f i c a n t l y l o w e r than t h a t o f the ad l i b i t u m c o n t r o l s ( p < 0 . 0 5 ) , poss-i b l y r e f l e c t i v e o f the reduced c a l o r i c i n t a k e o f the former group. By c o n t r a s t , p l a c e n t a s o f the a l c o h o l group were s u b s t a n t i a l l y h e a v i e r (p< 0.001) and p l a c e n t a s o f the p a i r -f e d group l i g h t e r (p.< 0.05) r e l a t i v e t o those o f ad l i b i t u m c o n t r o l s . 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 o m i t t e d a t weekly w e i g h i n g d u r i n g the p r e g e s t a t i o n a l o r g e s t a t i o n a l p e r i o d s hence were e x c l u d e d from the t r e a t m e n t group mean body w e i g h t . T h i s i s r e f l e c t e d i n d i f f e r e n c e s i n the number o f r a t s o r l i t t e r s between T a b l e s 1 t h r o u g h 5, where p r i o r t o day 20 o f g e s t a t i o n , a c t u a l sample s i z e may be l a r g e r than t a b u l a t e d . 42 TABLE 5 L i t t e r s i z e , f e t a l body weights, and placental weights of rats at day 20 gestation given alcohol, p a i r - f e d , or ad l i b i t u m -fed. Means + standard error. Alcohol Group Pai r - f e d Group Ad l i b i t u m Group L i t t e r size 11.3+ 0.32 d' 10.8+0.41 12.3+ 0.43' Number of l i t t e r s 35 33 26 Fe t a l body weight Number of l i t t e r s 2.01+ 0.03 35 2.33+0.04b 2.51+ 0.08c 33 26 Placental weight (g) Number of l i t t e r s 0.614+0.02' 11 0.416+ 0.01 b 0,485+0.02° 12 10 a,b,c,= figures i n the same row not sharing 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 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 M a t e r n a l Plasma M a t e r n a l plasma c o n c e n t r a t i o n s o f 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 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 are shown i n T a b l e 6. Average plasma a l c o h o l l e v e l s d u r i n g days 15-17 o f g e s t a t i o n o f the a l c o h o l group were 76+6.6 mg p e r 100 mL (mean + s t a n d a r d e r r o r ) . M a t e r n a l plasma c o n c e n t r a t i o n s o f g l u c o s e , f o l i c a c i d , and z i n c d i d n o t d i f f e r s i g n i f i c a n t l y among the t h r e e t r e a t m e n t groups, and were w i t h i n e x p e c t e d ranges i n a l l cases (9,63,64). d) P l a c e n t a l B l o o d Flow The 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 r e n a l b l o o d f l o w , and m a t e r n a l c a r d i a c o u t p u t i s p r e s e n t e d i n T a b l e 7. B l o o d f l o w t o the p l a c e n t a , whether e x p r e s s e d as mL/min p e r 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 o f c a r d i a c o u t p u t , 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 when compared w i t h e i t h e r p a i r - f e d (p<0.05, p<0.001, p<0.05, 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 (p<0.001, p<0.001, p<0.05) groups. F u r t h e r m o r e , when e x p r e s s e d as mL/min p e r p l a c e n t a a s i g n i f i c a n t l y d e c r e a s e d b l o o d f l o w was o b s e r v e d i n p a i r - f e d a n i m a l s i n comparison w i t h ad_ l i b i t u m c o n t r o l s (p<0.05), however t h i s decrease was n o t s t a t i s t i c a l l y s i g n i -f i c a n t when f l o w was e x p r e s s e d as mL/min p e r g p l a c e n t a due t o t h e d i f f e r e n c e s i n p l a c e n t a l w e i g h t s between groups. No s i g n i f i c a n t 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 44 k i d n e y among the t h r e e t r e a t m e n t groups, n o r i n the m a t e r n a l c a r d i a c o u t p u t . I t i s i n t e r e s t i n g t o n o t e the s u b s t a n t i a l l y e l e v a t e d 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 i n the a l c o h o l group compared w i t h t h a t o f 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. e) F e t a l Uptake o f N u t r i e n t s F e t a l u ptake 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 , and z i n c a t day 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 , and ad l i b i t u m f e d groups i s shown i n Ta b l e 8. In the case o f 2 amino i s o b u t y r a t e , methyl-et-D g l u c o p y r a n o s i d e and z i n c 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 f e t a l uptake were o b s e r v e d over the 90 minute i n c u b a t i o n p e r i o d among the t h r e e t r e a t m e n t groups. Trends e x i s t i n the cases o f 2-amino i s o b u t y r a t e and z i n c f a v o u r i n g a reduced f e t a l up-tak e by the 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 n o t a c h i e v e d . The f o l a t e t a k e n up by the f e t u s e s o f the a l c o h o l group was s i g n i f i c a n t l y h i g h e r (p<0.05) than t h a t o f the p a i r - f e d , but n o t ad l i b i t u m f e d c o n t r o l s . The p o s i t i o n o f the f e t u s on the u t e r i n e h o r n appeared t o have no e f f e c t on t h e r a t e o f uptake o f any o f the compounds s t u d i e d . 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 and M a t e r n a l Plasma O s m o l a l i t y and Mus c l e Dry Weight. As i n d i c a t e d i n T a b l e 9, no s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d i n the c o n c e n t r a t i o n s o f the f o u r r a d i o a c t i v e compounds 45 TABLE 6 Ethyl alcohol, glucose, f o l i c acid, and zinc concentrations i n maternal plasma during week three of gestation i n rats given alcohol, p a i r - f e d or ad l i b i t u m fed. Means + standard error. Alcohol Group P a i r - f e d Group Ad Libitum fed Group Ethy l alcohol (mg/100 mL) 76+ 6.6 ( l l ) 1 Glucose (mg/100 mL) 99.8+2.9a 96.5+3.0 104.7+ 2.3 (14) (14) (12) F o l i c acid (ng/mL) 46.5+4.2 37.3+3.6 47.9+7.1 (77 (6) (7) Zinc 349+69.8 445+20.8 402+ 83.4 (ppb) (8) (8) (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 differences were observed between treatment groups. 1= figures i n brackets r e f e r to number of rats. 46 TABLE 7 Blood flow to placentas and to the maternal kidneys, and cardiac output on day 20 gestation of rats given alcohol, p a i r - f e d , or ad l i b i t u m fed. Means + standard error. Alcohol Group Pai r - f e d Group Ad l i b i t u m Group Number of l i t t e r s Placental blood flow: mL/min/placenta mL/min/ g placenta % of cardiac output Renal blood flow: % of cardiac output Maternal cardiac output: mL/min/100 g body weight a,b,c= figures i n the same row not sharing 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 ' a t p<0.05. 11 0.170+0.02a 0.227+0.03a 0.270+0.03a 13.2+1.6a 20.5+1.33 12 0.260+ 0.03 0.623+0.07b 0.399+0.05b 10.9+0.61a 20.8+0.98a 10 0.357+ 0.03 C 0.743+ 0.07 b 0.460+0.04b 11.6+0.36a 20.4+ 1.0 a 47 i n the m a t e r n a l plasma o r l i v e r 90 minutes a f t e r i n j e c 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 , f o l i c a c i d , and z i n c among the t h r e e t r e a t m e n t groups. T a b l e 10 shows two parameters o f d e h y d r a t i o n , plasma o s m o l a l i t y and muscle dry w e i g h t f o r 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 groups. 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 b o t h plasma o s m o l a l i t y and muscle dry w e i g h t i n a l c o h o l exposed a n i m a l s when compared w i t h e i t h e r p a i r - f e d (p<0.001, p <0.05, 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 (p < 0.001, p < 0.05, r e s p e c t i v e l y ) groups. T h i s r e p r e s e n t s a moderate degree o f d e h y d r a t i o n (about 7 p e r c e n t ) i n the a l c o h o l group. TABLE 8 Fet a l uptake of 2-amino isobutyrate, methyl-cH) glucopyranoside,folic acid, and zinc from the maternal c i r c u l a t i o n at day 20 of gestation. Mean + standard error. Compound injected Alcohol P a i r - f e d Ad l i b i t u m group group controls 2-Amino (1-^C) isobutyric acid DPM/g f e t a l body weight 23,814 + 1444 26,615 + 3007 27,679 + 656 (7) 1 (5) (5) Methyl ( flC-D-fU-^C] gluco)pyranoside DPM/g f e t a l body weight 10,914 + 453 9,840 + 357 9,414 + 574 (11) (10) (6) [ 3',5',7,9-3H] F o l i c acid, K s a l t DPM/g f e t a l body weight 72,406 + 3634 2 61,509 + 1838 2 62,601 + 4669 (11) ( io) (6) Zinc-65 CPM/g f e t a l body weight 1,796 + 177 1,873 + 309 2,601 +'298 (6) (6) (5) Figures i n parentheses indicate the number of l i t t e r s . S i g n i f i c a n t at p<0.05. 49 TABLE 9 Concentration of 2-amino isobutyrate, methyl-<<-D glucopyranoside, f o l i c acid, and zinc i n maternal plasma and l i v e r 90 minutes a f t e r i n j e c t i o n of the radioactive compounds. Means + standard error. Group Alcohol P a i r - Ad Libitum Alcohol P a i r - Ad Libitum fed fed Li v e r Plasma Compound injected: 2-Amino isobutyr-ate (DPM/g) 62100 + 7310 79100 +2940 62800 +4380 20200 +1250 17400 +600 17800 +1030 Number of l i t t e r s Methyl -<*-D gluco-pyranoside (DPM/g) 34700 36500 31400 49500 50600 51600 +1870 +2100 +7540 +2940 +1340 +3540 Numbers of l i t t e r s 10 F o l i c acid (DPM/g) 493100 396500 527500 151400 153800 147600 +41600 +30800 +45300 +13200 +5910 +5530 Number of l i t t e r s 11 Zinc chloride (CPM/g) 14800 +1350 22200 +4040 14400 +1160 1300 +136 1320 +255 960 +126 Number of l i t t e r s a = there were 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 differences between treatment groups. 50 TABLE 10 Maternal plasma osmolality and muscle dry weight (% of wet weight) i n alcohol, p a i r - f e d , and ad l i b i t u m fed r a t s . Means + standard error. Group Plasma osmolality Muscle dry weight (mosm./L) (% of wet weight) Alcohol 302.4 + 4.59 a 25.68 + .58 a Number of l i t t e r s 10 11 Pair- f e d 280.0 + 2.30 b 23.99 + 13 b Number of l i t t e r s 9 12 b b Ad l i b i t u m 278.8+2.05 24.57+22 Number of l i t t e r s 9 10 a, b = figures i n the same column not sharing 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 51 DISCUSSION The f i n d i n g s o f t h i s study i n d i c a t e that p r e n a t a l a l c o h o l exposure i s d e l e t e r i o u s to f e t a l development i n the r a t . Although-no obvious malformations were, observed i n the 20 day g e s t a t i o n f e t u s e s i n any o f the three groups of r a t s , b i r t h weights o f the o f f s p r i n g o f r a t s consuming a l c o h o l p r i o r to and through-out pregnancy were s i g n i f i c a n t l y decreased over r a t s f e d e i t h e r i s o c a l o r i c a l l y or ad l i b i t u m . These f i n d i n g s are c o n s i s t e n t w i t h past work u s i n g a r a t model i n f e t a l a l c o h o l syndrome r e s e a r c h (7,8,41). The d a i l y a l c o h o l consumption, and subsequent b l o o d a l c o h o l l e v e l s , o f the a l c o h o l group dams was s i m i l a r to that of other s t u d i e s i n animals where decreased birth weights were observed (7,8,41). The b l o o d a l c o h o l l e v e l s of a l c o h o l t r e a t e d dams i n the present study (76 mg/100 mL) are however l e s s than i n b l o o d from mothers of i n f a n t s d i s p l a y i n g f e t a l a l c o h o l syndrome, taken s h o r t l y a f t e r b i r t h of the i n f a n t (130 mg/100 mL) (24). The minimum amount of a l c o h o l exposure r e q u i r e d to a f f e c t f e t a l w e l l b e i n g remains to be determined. The absence o f d i f f e r e n c e s i n maternal body weights between a l c o h o l and p a i r - f e d groups at day 20 o f g e s t a t i o n i s r e f l e c t i v e of the i s o c a l o r i c f e e d i n g design of the e x p e r i -ment. Both a l c o h o l and p a i r - f e d groups a l s o e x h i b i t a s i g n i f -i c a n t r e d u c t i o n i n maternal body weight when compared w i t h ad  l i b i t u m group animals, as might be expected by the lower c a l o r i c i n t a k e of the former groups. However the d i f f e r e n c e s i n f e t a l body weights among the three groups at day 20 of g e s t a t i o n 52 suggest t h a t the c a l o r i c r e s t r i c t i o n , a l t h o u g h a c o n t r i b u t i n g f a c t o r , cannot be e n t i r e l y r e s p o n s i b l e f o r the growth r e t a r d a -t i o n i n a l c o h o l exposed f e t u s e s . Other f a c t o r s must e x i s t t o account f o r t h i s r e d u c t i o n i n f e t a l w e i g h t between the two i s o c a l o r i c a l l y f e d groups. To i n v e s t i g a t e whether a l c o h o l consumption b e f o r e and d u r i n g g e s t a t i o n had an e f f e c t on the n u t r i t i o n a l s t a t u s o f the mother, plasma c o n c e n t r a t i o n s o f c e r t a i n n u t r i e n t s were determined. The absence o f any d i f f e r e n c e i n plasma l e v e l s o f g l u c o s e , f o l i c a c i d , o r z i n c d u r i n g the t h i r d week o f g e s t a t i o n s u g g e s t s t h a t m a t e r n a l m e t a b o l i s m 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. F u r t h e r m o r e , the c o n c e n t r a t i o n 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 analogs o f g l u c o s e and amino a c i d 90 minutes a f t e r i n j e c t i o n 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 the t h r e e groups. These f i n d i n g s , t o g e t h e r w i t h 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 s u p p o r t the n o t i o n t h a t a l c o h o l expo-s u r e a t the p r e s e n t l e v e l and d u r a t i o n does n o t a f f e c t m a t e r n a l n u t r i t i o n a l s t a t u s . T h i s i s c o n t r a r y 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 cannot 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 the p r e s e n t s t u d y were s u f f i c i e n t l y 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 s u p p l y , however i t i s l i k e l y t h a t any d e f i c i e n c y would m a n i f e s t i t s e l f i n an o b s e r v a b l e r e d u c t i o n i n m a t e r n a l body w e i g h t . The 53 d e c r e a s e d b i r t h w e i g h t s o f the o f f s p r i n g o f r a t s exposed t o a l c o h o l i n u t e r o o v e r those f e d i s o c a l o r i c a l l y i n t h i s s t u d y can 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 through an a l c o h o l i n d u c e d a l t e r a t i o n o f m a t e r n a l n u t r i e n t u t i l i z a t i o n o r plasma. • c o n c e n t r a t i o n . 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 t h a t a l c o h o l 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 -o p i n g f e t u s , the hemodynamic s t a t u s o f the mother was s t u d i e d at day 20 o f g e s t a t i o n . The use o f r a d i o a c t i v e m i c r o s p h e r e s f o r 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 organ b l o o d f l o w i s a w i d e l y a c c e p t e d p r o c e d u r e (60,61,66). The f r a c t i o n a l d i s t r i -b u t i o n among body t i s s u e s o f a v e n t r i c u l a r l y i n t r o d u c e d p u l s e o f r a d i o a c t i v e m i c r o s p h e r e s i s p r o p o r t i o n a l t o the p e r c e n t a g e o f the c a r d i a c o u t p u t r e a c h i n g those t i s s u e s . C r e a t i o n o f an a r t i f i c i a l organ o f known b l o o d f l o w by means o f r e f e r e n c e s a m p l i n g o f b l o o d a t a f i x e d r a t e a t the time o f the m i c r o -sphere 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 o u t p u t . Thus the b l o o d f l o w t o any t i s s u e can be determined, as b o t h the c a r d i a c o u t p u t and the p e r c e n t a g e o f the c a r d i a c o u t p u t r e a c h -i n g the t i s s u e are known. I n measuring p l a c e n t a l b l o o d f l o w w i t h m i c r o s p h e r e s t h e r e i s some q u e s t i o n as t o the e f f i c i e n c y w i t h w h i c h s m a l l (15 micron) m i c r o s p h e r e s are t r a p p e d i n the p l a c e n t a l c h a n n e l s . However, B j e l l i n e t a l . ( 6 7 ) have 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 g i v e a b e t t e r e s t i m a t e o f p l a c e n t a l b l o o d f l o w than do 50 m i c r o n m i c r o s p h e r e s , due to the tendency o f the l a t t e r t o a x i a l s t r e a m i n g i n b l o o d v e s s e l s . 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 (21 mL/ min/ 100 g body w e i g h t 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 o u t p u t 54 f o r k i d n e y f l o w ) are i n agreement w i t h those o f Bruce (61) and S a p i r s t e i n e t a l . (68) o f 26 and 22 mL/ min/ 100 g f o r c a r d i a c o u t p u t , r e s p e c t i v e l y , and 8.62% f o r k i d n e y b l o o d f l o w ( 6 1 ) . 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 by Bruce (61) o f 1.21 mL/min/g i s g r e a t e r than t h a t o f the ad l i b i t u m c o n t r o l group i n the p r e s e n t s t u d y (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 by Bruce were performed 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 average f e t a l w e i g h t s o f 4.21 g when compared w i t h average f e t a l w e i g h t s o f 2.51 g i n the ad l i b i t u m c o n t r o l group 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 s t u d y . M a t e r n a l c a r d i a c o u t p u t and r e n a l b l o o d f l o w e x p r e s s e d as % c a r d i a c o u t p u t were n o t a f f e c t e d by a l c o h o l co.nsumption o r by p a i r - f e e d i n g when compared w i t h ad 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 measure 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 o u t p u t between non pregnant a l c o h o l exposed and c o n t r o l r a t s f e d ad l i b i t u m . C a r d i a c o u t p u t has been found reduced i n a l c o h o l t r e a t e d non pregnant r a t s , but o n l y when n a r c o t i z i n g dosages were a d m i n i s t e r e d ( 69). I n 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 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 decreases 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 have been r e p o r t e d i n the 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 the r e n a l b l o o d f l o w o f a l c o h o l exposed an i m a l s i n the p r e s e n t s t u d y , 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 groups, i s c o n s i s t e n t w i t h the v a r i a b i l i t y o f f i n d i n g s r e p o r t e d i n the l i t e r a t u r e . 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 b l o o d f l o w t o the p l a c e n t a s i n the a l c o h o l t r e a t e d r a t s , whether e x p r e s s e d i n mL/ min/ p l a c e n t a , o r as p e r c e n t o f c a r d i a c o u t p u t . As the w e i g h t o f each p l a c e n t a was s i g n i f i -c a n t l y g r e a t e r i n the a l c o h o l t r e a t e d animals than i n c o n t r o l s , the 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 even more markedly reduced by a l c o h o l consumption. The r e l a t i v e p r o -p o r t i o n s o f p l a c e n t a l w e i g h t d e r i v e d from m a t e r n a l o r f e t a l components were n o t determined, so i t can n o t be s t a t e d t h a t b l o o d f l o w p e r u n i t o f m a t e r n a l p l a c e n t a l t i s s u e i s reduced. N e v e r t h e l e s s , the t o t a l b l o o d f l o w t o the p l a c e n t a i s dec r e a s e d , i m p l y i n g a reduced d e l i v e r y o f n u t r i e n t s and e s s e n t i a l m e t a b o l i t e s . The r e a s o n f o r the i n c r e a s e d p l a c e n t a l w e i g h t i n the a l c o h o l group i s n o t r e a d i l y apparent. Other f a c t o r s which r e t a r d f e t a l growth, such as u t e r i n e a r t e r y l i g a t i o n and c a l o r i e m a l n u t r i t i o n , are accompanied by reduced p l a c e n t a l w e i g h t (59, 71). I n accordan ce, i t i s n o t e d t h a t i n the p r e s e n t s t u d y the c a l o r i e r e s t r i c t e d group d i s p l a y e d s i g n i f i c a n t l y s m a l l e r p l a c e n t a s than e i t h e r a l c o h o l t r e a t e d o r ad l i b i t u m c o n t r o l groups. The h e a v i e r p l a c e n t a s o f the a l c o h o l group are n o t due t o i n c r e a s e d f l u i d r e t e n t i o n as the p e r c e n t d r y w e i g h t i s the same as i n the c o n t r o l group (72). Wiener e t a l . (42) and S k o s y r e v a (43) have a l s o r e p o r t e d i n c r e a s e d p l a c e n t a l w e i g h t s i n a l c o h o l - e x p o s e d r a t s s i m i l a r t o the p r e s e n t r e s u l t s . Gordon e t a l . (44) a l s o r e p o r t e d h e a v i e r p l a c e n t a s i n a l c o h o l t r e a t e d r a t s and showed t h a t i t i s due t o h y p e r p l a s i a . H i s t o l o g i c a l e x a m i n a t i o n may 56 c a s t some l i g h t on the n a t u r e o f these f i n d i n g s . I t i s p o s s i b l e t h a t the i n c r e a s e d p l a c e n t a l s i z e may e x i s t as a r e s u l t o f mechanisms wh i c h compensate f o r the reduced b l o o d f l o w t o t h i s organ i n the r a t c h r o n i c a l l y exposed t o a l c o h o l . The 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 o f c a r d i a c o u t p u t i n 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 c o n t r a s t s w i t h r e c e n t work by Rosso and Kava (59). These workers r e p o r t e d a d e c r e a s e d p l a c e n t a l b l o o d f l o w i n f o o d r e s t r i c t e d r a t s , w i t h o u t o b s e r v -i n g a change i n the f r a c t i o n o f c a r d i a c o u t p u t r e a c h i n g t h i s o r gan. 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 the cause o f f e t a l 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 a n i m a l s , a s i m i l a r p a t t e r n o f hemodynamic a l t e r a t i o n s w ould be e x p e c t e d i n f o o d r e s t r i c t e d r a t s . In the p r e s e n t work, no d i f f e r e n c e s e x i s t i n c a r d i a c o u t p u t between a l c o h o l t r e a t e d and ad l i b i t u m c o n t r o l s , however Rosso and Kava (59) n o t e a 50 p e r c e n t r e d u c t i o n i n c a r d i a c o u t p u t i n r a t s f e d a c a l o r i e r e s t r i c t e d d i e t ( 50 p e r c e n t o f c o n t r o l s ) as compared w i t h c o n t r o l group a n i m a l s . Thus the mechanism o f b l o o d f l o w r e d u c t i o n i n the p r e s e n t s t u d y e x i s t s t h r o u g h a r e d i s t r i b u t i o n o f a n o r m al 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 r a t , the d i s t r i b u t i o n o f b l o o d f l o w remains c o n s t a n t but the e x p a n s i o n o f b l o o d volume d u r i n g l a t e g e s t a t i o n f a i l s t o o c c u r , r e s u l t i n g i n a lowered c a r d i a c o u t p u t . T h i s l e n d s f u r t h e r e v i d e n c e i n s u p p o r t t h a t the a c t i o n o f a l c o h o l on the f e t u s i n u t e r o i s d i f f e r e n t from t h a t o f m a t e r n a l m a l n u t r i t i o n . As a m a r g i n a l l y f o o d r e s t r i c t e d group, the p a i r - f e d group o f the p r e s e n t s t u d y may be compared w i t h the m a l n o u r i s h e d r a t . The 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 o f p a i r - f e d o v e r 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 s t u d y i s s i m i l a r t o the r e s u l t s o f Rosso and Kava (.59.X., however, 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 o f c a r d i a c o u t p u t r e a c h i n g t h e p l a c e n t a , n o r the c a r d i a c o u t p u t s d i f f e r between p a i r - f e d and ad l i b i t u m groups. T h i s may be due t o the more s e v e r e n a t u r e o f the c a l o r i e r e s t r i c t i o n (50 p e r c e n t as compared w i t h about 20 p e r c e n t i n p a i r - f e d group) o f m a l n o u r i s h e d a n i m a l s (59) o v e r those p a i r -f e d i n the p r e s e n t s t u d y . In these s t u d i e s the p l a c e n t a l a v a i l a b i l i t y o f n u t r i e n t s may be i m p a i r e d as a r e s u l t o f the s i g n i f i c a n t and s u b s t a n t i a l 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 t o t r a n s f e r a r e a s o f the v i l l o u s membrane. 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 t o o r i g i n a t e t h rough a r e d i s t r i b u t i o n o f c a r d i a c o u t p u t . I t i s 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 a v a i l a b i l i t y i s r e s t r i c t e d , t h a t f e t a l growth r e t a r d a t i o n may o c c u r secondary t o 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. R e d u c t i o n o f p l a c -e n t a l b l o o d f l o w has been a s s o c i a t e d w i t h reduced p l a c e n t a l t r a n s f e r o f n u t r i e n t s . N i t z a n e t a l . (71) found r e d u c t i o n s i n p l a c e n t a l t r a n s f e r o f a m i n o i s o b u t y r i c a c i d and deoxy-g l u c o s e i n i n t r a u t e r i n e growth r e s t r i c t e d (IUGR) r a t s , where l i g a t i o n o f a u t e r i n e a r t e r y was performed. However the acute 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 n o t a l l o w compensatory mechanisms t o r e s t o r e n o r m al n u t r i e n t t r a n s f e r . A l c o h o l i n the p r e s e n t s t u d y was a d m i n i s t e r e d p r i o r t o and throughout g e s t a t i o n , thus s u f f i c i e n t time may have e l a p s e d to a l l o w development o f such mechanisms. Fur t h e r m o r e , u t e r i n e a r t e r y l i t a g a t i o n may cause a l a r g e r p l a c e n t a l b l o o d f l o w r e s t r i c t i o n 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 l i m i t s 58 o f t h e s e mechanisms. I t can t h e r e f o r e be argued t h a t a r e d u c t i o n i n p l a c e n t a l n u t r i e n t t r a n s f e r need n o t n e c e s s a r i l y accompany a 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 as e f f e c t e d by c h r o n i c a l c o h o l consumption. As a measure o f f e t a l uptake o f n u t r i e n t s , the p l a c e n t a l t r a n s f e r o f r a d i o l a b e l e d zinc,, f o l a t e , and analogs o f g l u c o s e and amino a c i d were s t u d i e d i n r a t s exposed t o a l c o h o l d u r i n g g e s t a t i o n . . M e t a b o l i t e a nalogs are used because they are h a n d l e d by the t r a n s p o r t systems o f the compounds they r e p r e -s e n t as would the compounds t h e m s e l v e s , however they are n o t degraded i n v i v o thus p r e v e n t i n g the p r o d u c t i o n o f r a d i o -l a b e l e d b y p r o d u c t s . The 90 minute time p o i n t a f t e r i n j e c t i o n o f the r a d i o l a b e l e d compounds was s e l e c t e d by comparison w i t h uptake r a t e s a t 40 and 60 minutes a f t e r i n j e c t i o n . A p r o -p o r t i o n a l l y i n c r e a s e d f e t a l uptake was o b s e r v e d at 90 minutes o v e r each o f the a l t e r n a t e time p o i n t s , i n d i c a t i n g t h a t even i f b a c k - f l o w o f the i n j e c t e d compounds was o c c u r r i n g the n e t f l u x was from mother t o f e t u s . T h i s p e r i o d was thus chosen t o maximize the 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 i n t o the f e t u s compared w i t h the amount i n j e c t e d i n t o the mother. E x c e p t i n the case o f f o l i c a c i d , no d i f f e r e n c e s i n f e t a l uptake r a t e s o f the compounds s t u d i e d were seen i n a l c o h o l exposed f e t u s e s when compared w i t h 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. I t i s u n c e r t a i n why an i n c r e a s e i n f e t a l uptake o f f o l i c a c i d i n a l c o h o l group a n i m a l s o c c u r r e d when compared w i t h the p a i r - f e d group. P o s s i b l y as a r e s u l t o f a d e f i c i e n c y a t an e a r l i e r s t a g e o f g e s t a t i o n a ' c a t c h up' mechanism i s i n e f f e c t . The absence o f a r e d u c t i o n i n f e t a l uptake o f the compounds s t u d i e d s u g g e s t s t h a t the growth r e t a r d a t i o n seen i n o f f s p r i n g o f a n i m a l s c h r o n i c a l l y exposed t o a l c o h o l i s n o t as a r e s u l t o f i n a d e q u a t e p l a c e n t a l n u t r i e n t t r a n s f e r a t l e a s t a t day 20 o f g e s t a t i o n . T h i s s u p p o r t s the concept t h a t compensatory mechanisms t o r e s t o r e adequate n u t r i e n t t r a n s f e r may a r i s e , as s u g g e s t e d by the i n c r e a s e i n p l a c e n t a l s i z e , i n a n i m a l s c h r o n i c a l l y exposed t o a l c o h o l where a 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 has o c c u r r e d . The r e s u l t s o f the p r e s e n t s t u d y are i n c o n t r a s t t o those o f L i n and Maddatu (73) who found a reduced t r a n s f e r . o f a m i n o i -s o b u t y r a t e t o f e t u s e s o f r a t s a d m i n i s t e r e d a l c o h o l from day 6 t o 21 o f g e s t a t i o n . The i n g e s t i o n o f an a l c o h o l s o l u t i o n i n the p r e s e n t s t u d y f o r at l e a s t one month p r i o r t o m a t i n g may have a l l o w e d s u f f i c i e n t time f o r a compensating e f f e c t t o have o c c u r r e d . F u r t h e r m o r e , no i n c r e a s e i n p l a c e n t a l s i z e was n o t e d by L i n and Maddatu (73 ) . A c c o r d i n g t o R i d e r ( 7 4 ) , a d a p t a t i o n t o a l c o h o l i n t a k e seems t o o c c u r as demonstrated by the p o o r e r r e p r o d u c t i v e performance o f the dams p l a c e d on a l c o h o l on day one o f pregnancy as compared w i t h t h a t o f the p r e v i o u s l y a l c o h o l adapted dams, and i n the d e c r e a s e d s u r v i v a l o f the o f f s p r i n g . Henderson e t a l . (75) has a l s o demonstrated i n r a t s t h a t c h r o n i c and acute a l c o h o l a d m i n i s t r a t i o n reduces p l a c e n t a l ^ C - v a l i n e uptake. However, th e s e a u t h o r s used an i n v i t r o p r e p a r a t i o n w h i c h may account f o r the d i f f e r e n c e s i n r e s u l t s . I f m a t e r n a l a l c o h o l consumption does n o t i n t e r f e r e w i t h the f e t a l uptake o f n u t r i e n t s , t o e x p l a i n the o b s e r v e d growth r e t a r d a t i o n i n u t e r o , a l c o h o l must e x e r t i t s d e l e t e r i o u s 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) demon-s t r a t e d t h a t exposure t o a l c o h o l r e t a r d s growth and d i f f e r -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 a l c o h o l can e x e r t 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 con-f o u n d i n g f a c t o r o f n u t r i t i o n . I t i s p o s s i b l e t h a t the f e t u s may be u n a b le t o u t i l i z e n u t r i e n t s once they have been t r a n s -f e r e d i n t o the f e t a l c i r c u l a t i o n . Rawat (77) r e p o r t e d t h a t a l c o h o l consumption by pregnant r a t s r e s u l t e d i n a s i g n i f i c a n t i n h i b i t i o n i n the r a t e o f ( U - ^ ^ C ) - l e u c i n e i n c o r p o r a t i o n i n t o f e t a l c a r d i a c p r o t e i n s . Decreased c a r d i a c t o t a l RNA and un-changed t o t a l DNA i n a l c o h o l exposed neonates has a l s o been r e p o r t e d by Henderson and Sdhenker (78). Thus e v i d e n c e e x i s t s i n s u p p o r t o f a l c o h o l a f f e c t i n g growth i n u t e r o by some mech-anism o t h e r than t h r o u g h r e d u c i n g p l a c e n t a l t r a n s f e r o f n u t r i e n t s . In t h e s e s t u d i e s a l c o h o l i n w a t e r was o f f e r e d as the s o l e s o u r c e o f f l u i d s . As a l c o h o l has a d i u r e t i c a c t i o n and no o t h e r s o u r c e o f w a t e r was a v a i l a b l e t o the a n i m a l s , plasma o s m o l a l i t y and muscle w a t e r c o n t e n t were measured as i n d i -c a t o r s o f d e h y d r a t i o n . Both parameters i n d i c a t e d t h a t a moderate degree o f d e h y d r a t i o n (7 p e r c e n t ) had o c c u r r e d , b u t the s i g n i f i c a n c e o f t h i s f o r the measurement o f p l a c e n t a l b l o o d f l o w , f e t a l n u t r i e n t uptake, and o v e r a l l f e t a l d e v e l -opment remains u n c l e a r . I n summary, m a t e r n a l n u t r i t i o n a l s t a t u s appears t o be u n a f f e c t e d by a l c o h o l t r e a t m e n t under the c o n d i t i o n s o f t h i s e x p e r i m e n t . The o b s e r v e d 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 on day 20 o f g e s t a t i o n i n r a t s c h r o n i c a l l y exposed t o a l c o h o l 6 1 may reduce p l a c e n t a l t r a n s f e r , h e n c e . f e t a l uptake, o f c e r t a i n n u t r i e n t s r e s u l t i n g i n a r e t a r d a t i o n o f f e t a l growth and d e v e l -opment. However, i f such a r e d u c t i o n i n f e t a l uptake o c c u r s , n u t r i e n t s o t h e r than t h o s e s t u d i e d p r e s e n t l y must be i n v o l v e d as no decrease i n the r a t e o f f e t a l uptake was o b s e r v e d f o r z i n c , 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 . A l t e r -n a t i v e l y , compensatory mechanisms, perhaps r e f l e c t e d i n the i n c r e a s e i n p l a c e n t a l w e i g h t emerging as a r e s u l t o f l o n g t e r m a l c o h o l exposure, 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 n u t r i e n t s t o the f e t u s , r e g a r d l e s s o f the 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 . Thus the growth r e t a r d i n g e f f e c t o f m a t e r n a l a l c o h o l i n g e s t i o n may s t i l l be due t o a d i r e c t a c t i o n o f a l c o h o l on the 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 met-a b o l i s m o r f e t a l n u t r i e n t u t i l i z a t i o n . REFERENCES CITED 1) Haggard, H.W. and J e l l i n e c k , E.M. A l c o h o l E x p l o r e d . Garden C i t y , New York, 1942. 2) Report on drunkennes p r e s e n t e d t o the House o f Commons by the S e l e c t Committee, London, 1834. 3) Jones, K. 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