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Effect of maternal alcohol consumption upon plasma levels of folate, zinc, magnesium, glucose, and amino… Marquis, Sandra Maureen 1982

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EFFECT OF MATERNAL ALCOHOL CONSUMPTION UPON PLASMA LEVELS OF FOLATE, ZINC, MAGNESIUM, GLUCOSE, AND AMINO ACIDS IN RAT DAMS AND THEIR FETUSES by SANDRA MAUREEN MARQUIS B . S c , The U n i v e r s i t y of V i c t o r i a , 1979 A THESIS SUBMITTED IN PARTIAL FULFIEMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Human N u t r i t i o n School of Home Economics We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1982 (c) .Sandra Maureen Marquis, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of HOME ECONOMICS The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e AUGUST 2 T 1982 DE-6 (3/81) ABSTRACT The current study was conducted to determine the e f f e c t of maternal a l c o h o l consumption upon plasma amino a c i d s , glucose, f o l i c a c i d , z i n c and magnesium l e v e l s i n r a t dams and t h e i r f e t u s e s . Female Sprague-Dawley r a t s were d i v i d e d i n t o three d i e t a r y treatment groups. Group 1 (Alcohol) was given a stock d i e t p l u s 10% (v/v) ethanol i n d r i n k i n g water. A f t e r one week, the concentration of ethanol was increased to 20% ( v / v ) . Group 2 ( P a i r - f e d ) was given a stock d i e t p l u s an amount of cornstarch i s o c a l o r i c a l l y equivalent to the ethanol consumed by Group 1. Group 3 (Control) was given the stock d i e t ad l i b i t u m . P a i r - f e d and c o n t r o l r a t s r e c e i v e d water ad l i b i t u m . The three d i e t a r y groups were maintained on t h e i r r e s p e c t i v e regimens f o r 4 weeks, a f t e r which mating of each.animal to male Sprague-Dawley r a t s was begun. The appearance of sperm i n the v a g i n a l v/ashings was taken as day 1 of pregnancy. At th a t time, the a l c o h o l group was changed to 30% (v/v) ethanol. Throughout the experiment d a i l y food consumption by a l c o h o l - t r e a t e d and p a i r - f e d animals was recorded. Weekly body weights of a l l animals were al s o recorded. On day 21 of g e s t a t i o n fetuses and placentae were removed and weighed. Blood samples were c o l l e c t e d by car d i a c puncture from a l l dams and f e t u s e s . A l c o h o l - t r e a t e d fetuses were s i g n i f i c a n t l y l i g h t e r i n body weight than p a i r - f e d or c o n t r o l f e t u s e s . Maternal weight g a i n p r i o r to pregnancy was not c o r r e l a t e d w i t h f e t a l body weight, while maternal weight g a i n throughout pregnancy was p o s i t i v e l y c o r r e l a t e d with, f e t a l weight. Maternal a l c o h o l consumption had no s i g n i f i c a n t e f f e c t upon maternal or f e t a l plasma l e v e l s of f o l i c a c i d , z i n c , and most amino a c i d s . A l c o h o l i i consumption a l s o had no e f f e c t upon maternal plasma glucose and albumin concentrations. F e t a l plasma l a c t i c a c i d was not s i g n i f i c a n t l y a f f e c t e d by maternal a l c o h o l consumption. . However, plasma magnesium l e v e l s were s i g n i f i c a n t l y elevated i n a l c o h o l - t r e a t e d dams. A l s o , plasma p r o l i n e l e v e l s were s i g n i f i c a n t l y reduced and plasma alpha-amino-n-butyric a c i d l e v e l s elevated i n a l c o h o l - t r e a t e d dams. Maternal plasma alpha-amino-n - b u t y r i c a c i d and p r o l i n e l e v e l s c o r r e l a t e d w i t h f e t a l body weight. In a l c o h o l - t r e a t e d f e t u s e s , plasma glucose l e v e l s were s i g n i f i c a n t l y lower than those i n p a i r - f e d and c o n t r o l groups. F e t a l plasma glucose l e v e l s were p o s i t i v e l y c o r r e l a t e d w i t h f e t a l body weight. Plasma a s p a r t i c a c i d l e v e l s were al s o s i g n i f i c a n t l y lower i n a l c o h o l - t r e a t e d fetuses compared to the other two groups. F e t a l plasma l y s i n e and alpha-amino-n - b u t y r i c a c i d l e v e l s c o r r e l a t e d p o s i t i v e l y w i t h f e t a l body weight. No s i g n i f i c a n t d i f f e r e n c e s were found i n i n t e s t i n a l conjugase a c t i v i t y among the three maternal treatment groups, i n d i c a t i n g t h a t a l c o h o l d i d not i n h i b i t the h y d r o l y i s of f o l a t e polyglutamates. Plasma o s m o l a l i t y was s i g n i f i c a n t l y elevated i n a l c o h o l - t r e a t e d dams. However, the e f f e c t of moderate maternal dehydration upon f e t a l growth i s unknown. I t i s concluded t h a t maternal a l c o h o l consumption produces d i s t i n c t changes i n some maternal and f e t a l plasma n u t r i e n t concentrations ( i e . glucose, z i n c , magnesium and some amino a c i d s ) . The s i g n i f i c a n c e of these r e s u l t s i s unknown. However, f e t a l hypoglycemia, produced by chronic maternal a l c o h o l consumption, may c o n t r i b u t e to the growth r e t a r d a t i o n seen i n the F e t a l A l c o h o l Syndrome. i i i TABLE OF CONTENTS Page A b s t r a c t i i Table of Contents i v L i s t of Tables v i i L i s t of Figures v i i i Acknowledgements i x CHAPTER I INTRODUCTION • 1 I I LITERATURE REVIEW A) H i s t o r i c a l P e r s p e c t i v e 3 B) E a r l y Studies on the Teratogenic E f f e c t s of A l c o h o l i n Humans 6 C) E a r l y Studies on the Teratogenic '• E f f e c t s of A l c o h o l i n Animals 7 D) Studies Examining the Mechanisms by which A l c o h o l Causes FAS 10 1) The D i r e c t E f f e c t of Ethanol 11 2) The E f f e c t of Reduced Maternal Food Intake 11 3) The E f f e c t of Ethanol upon Hormonal Function-'. 11 4) The E f f e c t of Ethanol upon P l a c e n t a l Transfer of N u t r i e n t s 12 5) The E f f e c t of Ethanol upon Maternal I n t e s t i n a l N u t r i e n t Absorption 13 6) The E f f e c t of Ethanol upon Maternal Urinary Loss of N u t r i e n t s 14 7) The E f f e c t of Ethanol upon Blood and Tissue Levels of N u t r i e n t s 14 i v Page # a) Zinc 14 b) F o l i c A c i d 14 c) Glucose 15 d) Amino Acids 16 8) Summary.- 18 I I I EXPERIMENTAL DESIGN AND METHODOLOGY A) Treatment of Animals 19 B) C o l l e c t i o n and Pr e p a r a t i o n of Animal Tissues 20 C) Assay Procedures 21 D) S t a t i s t i c a l A n a l y s i s 23 IV RESULTS A) C a l o r i c Intakes and Maternal Body Weights 25 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 28 C) N u t r i e n t Levels i n Maternal Plasma 38 1) F o l i c A c i d 38 2) Zinc 38 3) Magnesium 38 4) Glucose 38 5) Albumin 38 D) . Maternal Plasma Osmolality 39 E) Maternal I n t e s t i n a l Conjugase A c t i v i t y 39 F) Maternal Plasma Amino Acids <• 42 G) N u t r i e n t Levels i n F e t a l Plasma 49 1) F o l i c A c i d 49 v Page # 2) Zinc 49 3) Magnesium 49 4) Glucose 49 5) L a c t i c A c i d 50 H) F e t a l Plasma Amino A c i d s , 52 I) Comparison of Maternal and F e t a l Plasma N u t r i e n t Levels 59 1) F o l i c A c i d 59 2) Zinc 59 3) Magnesium 59 4) Glucose ' 59 J) Comparison of Maternal and F e t a l Plasma Amino A c i d Levels 59 V DISCUSSION 61 VI CONCLUSIONS 75 REFERENCES 77 v i LIST OF TABLES Page # Table 1 D a i l y Food and A l c o h o l Consumption P r i o r t o Pregnancy 30 Table 2 D a i l y C a l o r i c Intake P r i o r to Pregnancy 31 Table 3 D a i l y Food and A l c o h o l Consumption During Pregnancy 32 Table 4 D a i l y C a l o r i c Intake During Pregnancy 33 Table 5 ' Weekly. Maternal Body1 Weights Before and During Pregnancy 34 Table 6 F e t a l Body Weight, L i t t e r S i z e and P l a c e n t a l Weight 37 Table 7 Maternal Plasma F o l a t e , Z i n c , Magnesium, Glucose and Albumin Levels on Day 21 of G e s t a t i o n 40 Table 8 Maternal Plasma Osmolality on Day 21 of Gestation 41 Table 9 Maternal I n t e s t i n a l Conjugase A c t i v i t y . on Day 21 of Gestation 41 Table 10 Maternal Plasma Amino A c i d and Urea Levels on Day 21 of Gestation 44 Table 11 F e t a l Plasma F o l a t e , Z i n c , Magnesium, Glucose and L a c t i c A c i d Levels on Day 21 of Gestation , 51 Table 12 F e t a l Plasma Amino A c i d and Urea Levels on Day 21 of Gestation 54 v i i LIST OF FIGURES Page # Figure 1 Maternal Weight Gain P r i o r to Pregnancy 35 Figure 2 Maternal Weight Gain Throughout Pregnancy 36 Figure 3 Maternal Plasma Amino A c i d Levels . on Day 21 of Ges t a t i o n 46 F i g u r e 4 F e t a l Plasma Amino A c i d Levels on Da}?- 21 of Ges t a t i o n 56 v i i i ACKNOWLEDGEMENTS I wish to express my a p p r e c i a t i o n to my adv i s o r Dr. Joseph L e i c h t e r , f o r h i s d i r e c t i o n and advice throughout t h i s p r o j e c t . I wish a l s o to thank Dr. P a t r i c i a , G a l l o and Dr. Krishnamurti f o r s e r v i n g on my advisory committee, and Dr. Melvin Lee f o r h i s valuable advise and as s i s t a n c e i n c o l l e c t i n g blood samples. Thanks are als o due to Mrs. V i r g i n i a Green of A r t s Computing f o r support i n computer programming and s t a t i s t i c a l a n a l y s i s . Thanks are a l s o expressed to Mrs. Helen Smith f o r her a s s i s t -ance i n the l a b o r a t o r y . L a s t l y , I would l i k e to thank my husband Paul f o r h e l p i n g me w i t h the computer, and f o r a l l h i s patience and support. F i n a n c i a l support i s acknowledged from the B r i t i s h Columbia Youth Employment Program. i x INTRODUCTION A r e l a t i o n s h i p between maternal a l c o h o l consumption and neonatal morbidity and m o r t a l i t y has been observed since ancient times (1,2). However, only i n the past decade has i t been e s t a b l i s h e d that maternal a l c o h o l consumption can produce a d i s t i n c t p a t t e r n of abnormalities i n newborns. This p a t t e r n was f i r s t described i n d e t a i l by Jones and Smith (5,6); they termed i t the F e t a l A l c o h o l Syndrome (FAS). C h a r a c t e r i s t i c s reported i n FAS c h i l d r e n i n c l u d e d pre- and p o s t n a t a l growth r e t a r d a t i o n , c r a n i o f a c i a l anomalies (short p a l p e b r a l f i s s u r e s , m i d f a c i a l h y p o p l a s i a ) , microcephaly, mental d e f i c i e n c y , j o i n t abnormalities and ca r d i a c defects ( 9). Foll o w i n g i t s i n i t i a l d e s c r i p t i o n , growth d e f i c i e n c y i n c h i l d r e n as a r e s u l t of maternal a l c o h o l consumption was confirmed by many researchers (4-6,8-14). However, animal s t u d i e s became necessary to d i f f e r e n t i a t e between the e f f e c t s of a l c o h o l and other r i s k f a c t o r s and to determine the mechanism by which a l c o h o l causes FAS. Studies using r a t s have reported pre- and p o s t n a t a l growth r e t a r d a t i o n i n the o f f s p r i n g of dams given ethanol (15-19,21-28). Increased r a t e of f e t a l r e s o r p t i o n (15,16,19,24,26), and reduced l i t t e r s i z e (16,20,24) have also been reported i n r a t s as a r e s u l t of maternal a l c o h o l consumption. However, i t remains unclear whether the r e t a r d a t i o n i n growth and development found i n FAS i s due to a d i r e c t e f f e c t o f ethanol or one of i t s meta-b o l i t e s , or whether i t i s a consequence of maternal m a l n u t r i t i o n . Animal s t u d i e s employing a p a i r - f e d c o n t r o l group have demonstrated t h a t f e t a l growth r e t a r d a t i o n i s not simply a r e s u l t of underfeeding of the dams (16-18,21-23). However, maternal a l c o h o l consumption might 1 cause retarded f e t a l growth by i n t e r f e r i n g .with maternal or f e t a l n u t r i e n t u t i l i z a t i o n . Ethanol may reduce i n t e s t i n a l n u t r i e n t absorption, p l a c e n t a l t r a n s f e r of n u t r i e n t s , or t i s s u e n u t r i e n t uptake. The present study was designed to determine the e f f e c t of maternal a l c o h o l consumption upon maternal and f e t a l plasma n u t r i e n t l e v e l s i n the r a t model. Rats were given ethanol p r i o r to and throughout g e s t a t i o n and compared to p a i r - f e d and ad l i b i t u m fed c o n t r o l animals. On day 21 of g e s t a t i o n maternal and f e t a l blood samples were c o l l e c t e d and l e v e l s of f o l i c a c i d , z i n c , magnesium, glucose and f r e e amino acids determined i n plasma. The n u t r i e n t s examined were chosen on the b a s i s of both animal and human s t u d i e s demonstrating an a s s o c i a t i o n between a l c o h o l consumption and poor n u t r i e n t s t a t u s . In the nonpregnant animal, a l c o h o l has been shown to decrease i n t e s t i n a l absorption of f o l i c a c i d (39), glucose (40), z i n c (36) and amino acids (37). A l c o h o l increases u r i n a r y l o s s of z i n c (36,38,41,42) and of magnesium (43) i n humans. In animals, a l c o h o l i n h i b i t s h epatic gluconeogenesis (47,48), produces changes i n plasma amino a c i d p a t t e r n s (50-52), and reduces t i s s u e uptake of amino acids (53-58). Thus, i n the present study, c o r r e l a t i o n s between maternal a l c o h o l consumption, plasma n u t r i e n t l e v e l s , and f e t a l growth r e t a r d a t i o n were examined, i n the hopes of p r o v i d i n g an explanation f o r alcohol-induced f e t a l growth r e t a r d a t i o n . 2 LITERATURE REVIEW H i s t o r i c a l P e r s p e c t i v e A r e l a t i o n s h i p between maternal a l c o h o l consumption and neonatal morbidity and m o r t a l i t y has been observed since a n t i q u i t y . Burton's Anatomy of Melancholy, published i n 1621, c i t e d A r i s t o t l e ' s observation t h a t " f o o l i s h and drunken harebrained women most o f t e n b r i n g f o r t h c h i l d r e n l i k e unto themselves, morose and la n g u i d " ( 1). However, through every e ra a t t i t u d e s towards a l c o h o l and i t s e f f e c t s have been debated and r e v i s e d . From 1720 to 1750 England was swept by a "gin epidemic". I t was during t h i s p e r i o d t h a t the f i r s t observations of a f e t a l a l c o h o l syndrome appeared i n the English-language l i t e r a t u r e . In England i n 1751, Mor r i s a t t r i b u t e d a drop i n b i r t h r a t e and increase i n the m o r t a l i t y of c h i l d r e n under 5 years of age to the "enormous use of s p i r i t o u s l i q u o r s " ( 2). In the United States l i t e r a t u r e on a l c o h o l and i t s a c t i o n during pregnancy d i d not develop u n t i l somewhat l a t e r than i t had i n England. However, as e a r l y as 1787 a t r a c t e n t i t l e d "An Enquiry i n t o the E f f e c t s of S p i r i t o u s Liquors upon the Human Body", advised against the consumption of a l c o h o l by pregnant women. ( 2 ) . By 1842 i n the U.S., evidence of damage caused by maternal d r i n k i n g was being used t o support the d r i v e f o r t o t a l abstinence. In 1848, Howe published the f i r s t r e p o r t of e p i d e m i o l o g i c a l research on the t o p i c of p a r e n t a l d r i n k i n g . He examined the f a m i l y h i s t o r i e s of 145 i n s t i t u -t i o n a l i z e d mental d e f e c t i v e s and found that ;;33 had'1 "ihte.roper.ate"' parents (2) . From 1870 to 1920, j o u r n a l s were flooded w i t h a r t i c l e s and l e t t e r s 3 about a l c o h o l . J o u r n a l s , such as The J o u r n a l of I n e b r i e t y and The S c i e n -t i f i c Temperance J o u r n a l , contained r e p o r t s of p a r e n t a l d r i n k i n g r e s u l t i n g i n e p i l e p t i c , insane, i m b e c i l i c and a l c o h o l i c c h i l d r e n . To b u t t r e s s these a s s e r t i o n s , a vast body of survey research accumulated. Long surveyed 200 doctors i n 1876 (2). These doctors a t t r i b u t e d 20 to 30% of i n h e r i t e d mental d e f i c i e n c y to p a r e n t a l a l c o h o l i s m . In 1899, S u l l i v a n (1) studied 21 a l c o h o l i c mothers and compared the m o r t a l i t y r a t e s of t h e i r 125 c h i l d r e n to those of the o f f s p r i n g of t h e i r non-drinking female r e l a t i v e s . He found t h a t the death r a t e of the o f f s p r i n g of the a l c o h o l i c mothers was nearly 2.5 times higher. He d i d emphasize however, that the high m o r t a l i t y r a t e was not s o l e l y due.to a t o x i c e f f e c t of a l c o h o l , but was a l s o r e l a t e d to a high incidence of c h i l d abuse. T h i s , and other s t u d i e s , r a i s e d the question of whether high i n f a n t m o r t a l i t y i n a l c o h o l i c f a m i l i e s was d i r e c t l y r e l a t e d to a l c o h o l , or due to poor maternal care. In 1903, Hodge reported the f i r s t animal experiments on the e f f e c t s of a l c o h o l upon o f f s p r i n g . He t r e a t e d cocker s p a n i e l s w i t h a l c o h o l and reported t h a t the o f f s p r i n g of a l c o h o l - t r e a t e d had l e s s vigour and v i t a l i t y than normal ( 2 ) . Studies using rodents began w i t h Nice i n 1912 and continued u n t i l 1924 ( 2 ) . However, the experimental design of these i n v e s t i g a t i o n s v a r i e d g r e a t l y and r e s u l t s remained i n c o n c l u s i v e . In 1920 P r o h i b i t i o n went i n t o e f f e c t i n the United S t a t e s . In both the U.S. and B r i t a i n , a dramatic drop i n medical w r i t i n g on the e f f e c t s of a l c o h o l upon o f f s p r i n g ensued, and i n t e r e s t s h i f t e d towards the e f f e c t s of environmental f a c t o r s upon development of the newborn (2). Some animal experimentation w i t h a l c o h o l continued, but i t was discounted as crude and u n c o n t r o l l e d . 4 In 1942, Haggard and J e l l i n e k (2) s t a t e d t h a t no evidence had thus f a r been given which showed c o n c l u s i v e l y t h a t maternal a l c o h o l i n t a k e produced any abnormality i n the C h i l d . They a t t r i b u t e d damage to o f f s p r i n g to the poor n u t r i t i o n a l s t a t u s and environment of the a l c o h o l i c mother. In the 1940's and 1950's t o x i c e f f e c t s of a l c o h o l on o f f s p r i n g were g e n e r a l l y discounted. Then i n the 1960's the re d i s c o v e r y of the f e t a l a l c o h o l syndrome began. In 1962, Schaefer (3) reported a case of a Yukon Indian showing signs of a l c o h o l withdrawal s h o r t l y f o l l o w i n g b i r t h . In-1970, U l l e l a n d et a l . (4) r e t r o s p e c t i v e l y i d e n t i f i e d 11 women a l c o h o l i c s and found t h a t 10 of t h e i r 12 c h i l d r e n were small f o r g e s t a t i o n a l age. In 1973, Jones et a l . (5) coined the term f e t a l a l c o h o l syndrome (FAS). In two papers Jones et a l . (5) and Jones and Smith (6) described a p a t t e r n of malformations found i n 16 c h i l d r e n . The c h i l d r e n were of v a r i e d e t h n i c o r i g i n . The mothers were chronic a l c o h o l i c s (see C r i t e r i a f o r the Diagnosis of Alcoholism by the C r i t e r i a Committee, N a t i o n a l C o u n c i l on Alcoholism ( 7 ) ) , who had continued to d r i n k h e a v i l y throughout t h e i r pregnancies. The c h a r a c t e r i s t i c s of FAS described i n these two papers were pre- and p o s t n a t a l growth deficiency,..microcephaly, c r a n i o -f a c i a l a b n o r m a l i t i e s (short p a l p e b r a l f i s s u r e s , m a x i l l a r y h y p o p l a s i a ) , and j o i n t and c a r d i a c a b n o r m a l i t i e s . Jones et a l . (5) noted t h a t b i r t h l e n g t h was more.severely retarded than body weight. Furthermore, the average l i n e a r growth r a t e and weight gain of c h i l d r e n f o l l o w e d f o r one year remained at 65% and 38% of normal r e s p e c t i v e l y . These f i n d i n g s completed an h i s t o r i c a l c y c l e i n which the e f f e c t s of a l c o h o l upon o f f s p r i n g regained s c i e n t i f i c a t t e n t i o n . I f r e p e t i t i o n of such a c y c l e i s to avoided, experiments must be conducted to d e l i n e a t e 5 ethanol's mode of a c t i o n and to d i f f e r e n t i a t e the e f f e c t s of ethanol from environmental and s o c i a l v a r i a b l e s . E a r l y Studies on the Teratogenic E f f e c t s of Al c o h o l i n Humans A neglected 1968 repo r t of Lemoine et a l . (2) was found to independ-e n t l y confirm the f i n d i n g s of Jones et a l . (5). . Lempine et. a l , described c h a r a c t e r i s t i c s of FAS i n 125 French children.. These findings- and. those of Jones et a l . s t i m u l a t e d a s e r i e s of r e t r o s p e c t i v e s t u d i e s confirming the existence of FAS. Jones et a l . (8) reported on an a d d i t i o n a l 23 a l c o h o l i c women. Thirty-two percent of the c h i l d r e n born to these women had fe a t u r e s of FAS. In a review of cases to date, Hanson et a l . (9) described FAS c h a r a c t e r i s t i c s i n a t o t a l of 41 c h i l d r e n . In 1978, Erb and Andresen (10) estimated t h a t 1/3 to 1/2 of the c h i l d r e n born to a l c o h o l i c mothers were a f f e c t e d to some degree w i t h FAS. However, S t r e i s s g u t h et a l . (11) emphasized t h a t there i s a great range i n the number and s e v e r i t y of symptoms seen i n c h i l d r e n w i t h FAS, and that no pa t t e r n of a l c o h o l consumption nor lower l i m i t of amount of a l c o h o l r e q u i r e d to produce FAS had as yet been e s t a b l i s h e d . R e t r o s p e c t i v e s t u d i e s e s t a b l i s h e d the probable r e l a t i o n s h i p between a l c o h o l consumption and FAS c h a r a c t e r i s t i c s , but prosp e c t i v e s t u d i e s were needed to confirm the r e l a t i o n s h i p . Oulette and Rosett (12) surveyed 200 women attending p r e n a t a l c l i n i c s and d i v i d e d them i n t o three groups, based upon t h e i r a l c o h o l consumption ( a b s t i n e n t , moderate, or heavy). The 82 babies born to women of the three groups were subsequently s t u d i e d at b i r t h . A s i g n i f i c a n t l y g r e a t e r incidence of reduced b i r t h weight, le n g t h and head circumference was found i n i n f a n t s of heavy d r i n k e r s . However, Oulette and Rosett concluded t h a t because of the p o s s i b l e 6 i n t e r r e l a t i o n s h i p s among al c o h o l i s m , cigarette-smoking, m a l n u t r i t i o n and socio-economic status i n t h e i r study groups, a conc l u s i v e r e l a t i o n -ship between alcoholism and FAS was not determined. L i t t l e (13) s t u d i e d the r e l a t i o n s h i p between the a l c o h o l intake of 263 women before and during pregnancy and the b i r t h weight of t h e i r o f f s p r i n g . Using m u l t i p l e l i n e a r r e g r e s s i o n , the study c o n t r o l l e d f o r the i n f l u e n c e of maternal age and height, p a r i t y , and cigarette-smoking and the newborns 1 sex and g e s t a t i o n a l age. L i t t l e (13) found t h a t an average maternal intake of one ounce of absolute ethanol/day during the three month p e r i o d p r i o r to pregnancy was as s o c i a t e d w i t h a 90.8 gram decrease i n b i r t h weight. The same amount of ethanol consumed during l a t e pregnancy produced an average b i r t h weight decrease of 160 grams. The f a c t t h a t L i t t l e found a l c o h o l intake during e a r l y pregnancy had no e f f e c t on b i r t h weight, may have been due t o the small sample o f women who drank during t h i s p e r i o d . L i t t l e p o i n t s out that b i r t h weight per se i s not s t r i c t l y e q u ivalent to i n f a n t h e a l t h . However, the " r e d u c t i o n i n b i r t h weight may represent minimal damage w i t h i n a l a r g e r spectrum of growth ret a r d a t i o n . a n d f e t a l i n j u r y . In a r e t r o s p e c t i v e study L i t t l e et a l . (14) found that the mean b i r t h weight of the o f f s p r i n g of a l c o h o l i c s who had abstained throughout pregnancy was 258 grams l e s s than t h a t of c o n t r o l s , while b i r t h weight of c h i l d r e n of non-abstaining a l c o h o l i c s was 493 grams l e s s than t h a t of c o n t r o l s . Again these b i r t h weights were adjusted f o r v a r i a b l e s such as maternal age, weight, smoking h a b i t s , p a r i t y and the i n f a n t ' s sex. E a r l y Studies on the Teratogenic E f f e c t s of A l c o h o l i n Animals Studies t o determine the mechanism by which a l c o h o l causes FAS have now turned p r i m a r i l y t o the use of animals. Animal s t u d i e s allow 7 manipulation not e t h i c a l l y p o s s i b l e i n human s t u d i e s . Animal st u d i e s a l s o have an advantage i n t h a t they allow the study of the e f f e c t s of a s i n g l e f a c t o r (maternal a l c o h o l consumption) upon pregnancy and f e t a l growth. Human s t u d i e s are o f t e n compounded by v a r i a b l e s such as m a l n u t r i t i o n , cigarette-smoking and socio-economic s t a t u s . Animal s t u d i e s e s t a b l i s h e d t h a t a syndrome comparable to the human FAS could be induced i n rodent f e t u s e s . In 1975 Chernoff (15) fed mice a l c o h o l , w i t h l e v e l s ranging from 15-35% of t h e i r t o t a l c a l o r i e s . He found th a t d i f f e r e n t mouse s t r a i n s v a r i e d i n t h e i r s e n s i t i v i t y to a l c o h o l , but th a t a l c o h o l could cause decreased f e t a l weight, increased f e t a l r e s o r p t i o n , d e f i c i e n t o s s i f i c a t i o n and ne u r a l and ca r d i a c a b n o r m a l i t i e s . During t h i s same time, Tze and Lee (16) documented the e f f e c t s o f maternal a l c o h o l consumption upon r a t f e t u s e s . Rats were given 30 grams ethanol/100 ml. water as t h e i r only f l u i d source. The ethanol group was compared to i s o c a l o r i c a l l y p a i r - f e d and ad l i b i t u m f e d c o n t r o l groups. The r a t s were mated 5 weeks a f t e r i n i t i a t i o n of the d i e t a r y regimens and maintained on the regimens throughout pregnancy. Only 50% of mated a l c o h o l - f e d dams d e l i v e r e d , w h i l e 91% of p a i r - f e d and 88% of mated c o n t r o l animals d e l i v e r e d . The e t h a n o l - t r e a t e d dams had s i g n i f i c a n t l y reduced l i t t e r s i z e . Some f e a t u r e s of the f e t a l a l c o h o l syndrome have been reproduced i n animal models, but the r e s u l t s are c o n t r a d i c t o r y because of the wide v a r i e t y of experimental c o n d i t i o n s employed (15-28). In p a r t i c u l a r , experiments have d i f f e r e d i n the mode and time of a l c o h o l a d m i n i s t r a t i o n , and the amount of a l c o h o l consumed. Rider's (24) f i n d i n g s may e x p l a i n some of the v a r i a b i l i t y reported i n the l i t e r a t u r e on the e f f e c t s of maternal a l c o h o l consumption upon rodent f e t u s e s . Rider reported t h a t r a t s can adapt s i g n i f i c a n t l y to 8 a l c o h o l feeding. She compared the reproductive performance of female r a t s s t a r t e d on 11% ethanol (as t h e i r s o l e f l u i d source) 13 weeks before mating, and r a t s given ethanol only from day 1 of g e s t a t i o n . The two groups of pregnant r a t s d i d not d i f f e r i n weight gain throughout g e s t a t i o n . However, the dams s t a r t e d on 11% ethanol on day 1 of g e s t a t i o n had a s i g n i f i c a n t l y reduced number of completed pregnancies as v/ell as s m a l l e r l i t t e r s i z e and weight, and a lower pup s u r v i v a l r a t e , when compared to both ad l i b i t u m fed c o n t r o l s and to dams s t a r t e d on the ethanol t h i r t e e n weeks e a r l i e r . In a d d i t i o n to the t i m i n g of ethanol a d m i n i s t r a t i o n , s t u d i e s have d i f f e r e d i n the mode of a d m i n i s t r a t i o n . Several i n v e s t i g a t o r s have administered ethanol by i n t u b a t i o n . Abel and G r e i z e r s t e i n (21) i n t u b a t e d r a t s w i t h 6 grams ethanol(30%)/kg. body w e i g h t . d a i l y beginning on day 5 of pregnancy. In comparing the a l c o h o l group to a p a i r - f e d group, they found no s i g n i f i c a n t d i f f e r e n c e s i n maternal weight g a i n , p l a c e n t a l weight or l i t t e r s i z e , but they d i d f i n d that the fetuses of intubated dams had s i g n i f i c a n t l y lower body weights and lengths. Ethanol i n t u b a t i o n allows very accurate monitoring of ethanol i n t a k e . However, the a p p l i c a t i o n of such a high concentration of ethanol d i r e c t l y to the g a s t r o i n t e s t i n a l t r a c t may cause some h i s t o l o g i c a l damage which would not otherwise occur i f the ethanol s o l u t i o n were ingested i n s m a l l e r amounts throughout the day or accompanied by food-. A l s o , i n Abel and G r e i z e r s t e i n ' s (21) design, ethanol a d m i n i s t r a t i o n was begun on day 5 of g e s t a t i o n , whereas i n humans, alcoholism i s t y p i c a l l y a chronic c o n d i t i o n . For these reasons, animal experiments which more c l o s e l y simulate the human a l c o h o l i c c o n d i t i o n may be more r e a l i s t i c i n the study of FAS. 9 Numerous workers have administered ethanol i n l i q u i d d i e t s . Henderson and Sehenker (17) f e d female r a t s a l i q u i d d i e t of 6% ethanol f o r 4-21 weeks p r i o r to and throughout g e s t a t i o n . When compared to p a i r - f e d animals, these r a t s showed no d i f f e r e n c e s i n l i t t e r s i z e or g e s t a t i o n a l l e n g t h . However, the ethanol-fed dams had pups which weighed s i g n i f i c a n t l y l e s s than >the-pups of p a i r - f e d dams. Several problems may a r i s e from the use of l i q u i d d i e t s . F i r s t l y , Oisund et a l . (20) found that l i q u i d d i e t s caused an abnormally large intake of water, the e f f e c t s of which are unknown. A l s o , Weiner et a l . (27) suggested that most commercially a v a i l a b l e l i q u i d d i e t s are not n u t r i t i o n a l l y adequate to meet the needs of pregnant r a t s . Animal s t u d i e s i n which ethanol s o l u t i o n s are o f f e r e d as the only f l u i d source are known to decrease food consumption (29) and t o cause moderate dehydration (30). However, these s i t u a t i o n s may a l s o occur i n the human a l c o h o l i c , as a l c o h o l replaces a c e r t a i n percentage of r e q u i r e d c a l o r i e s , and ethanol i s a known d i u r e t i c . Therefore, an experimental design which provides r a t s w i t h ad l i b i t u m food and an ethanol s o l u t i o n ad l i b i t u m may most c l o s e l y resemble the human s i t u a t i o n . . Studies Examining the Mechanisms by which A l c o h o l Causes FAS Since the establishment of FAS c h a r a c t e r i s t i c s i n animal models, an i n c r e a s i n g number of workers have sought to determine the mechanism by which a l c o h o l impairs f e t a l growth. The growth r e t a r d i n g e f f e c t of maternal a l c o h o l i n g e s t i o n may be due to a d i r e c t a c t i o n of ethanol or i t s metabolites upon the f e t u s , reduced maternal food i n t a k e , i n t e r f e r e n c e of a l c o h o l w i t h maternal n u t r i e n t absorption, increased maternal n u t r i e n t l o s s , reduced blood n u t r i e n t l e v e l s during pregnancy, or i n t e r f e r e n c e v/ith 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 10 1) The D i r e c t E f f e c t of Ethanol Brown et a l . (32) kept 9/£>;;day o l d r a t embryos f o r 48 hours (the p e r i o d of organogenesis) i n c u l t u r e s c o n t a i n i n g 150 mg. or 300 mg. ethanol per 100 ml., or c o n t r o l c u l t u r e s c o n t a i n i n g Hanks b a s i c b u f f e r e d s a l t s o l u t i o n . D i f f e r e n t i a t i o n , growth, t o t a l DNA and t o t a l p r o t e i n content were s i g n i f i c a n t l y reduced i n the 300 mg. a l c o h o l group. Growth measures f o r the y o l k sac and p l a c e n t a were not a f f e c t e d , nor were gross a l t e r a t i o n s i n morphogenesis seen. In a d d i t i o n , the r a t i o s o f t o t a l DNA t o t o t a l p r o t e i n were not a f f e c t e d s i g n i f i c a n t l y by ethanol treatment. Thus, c e l l number, but not c e l l s i z e was reduced. Although these f i n d i n g s seem to suggest t h a t f e t a l growth r e t a r d a t i o n i s a d i r e c t r e s u l t of ethanol t o x i c i t y , the mechanism of ethanol's a c t i o n was not i n d i c a t e d . The p o s s i b i l i t y s t i l l e x i s t s t h a t ethanol i n the c u l t u r e medium was i n t e r f e r i n g w i t h the f e t u s e s ' n u t r i e n t supply or u t i l i z a t i o n . A l s o , the f i n d i n g s of L i t t l e et a l . (14) ( i n d i c a t i n g increased incidence of low b i r t h weight babies born t o a l c o h o l i c s who have abstained during pregnancy) suggest t h a t a l c o h o l may have a l a s t i n g e f f e c t upon maternal metabolism or physio-l o g i c a l s t a t e . 2) The E f f e c t of Reduced Maternal Food Intake P a i r - f e e d i n g s t u d i e s have shown th a t the f e t a l and neonatal growth r e t a r d a t i o n seen i n r a t s and mice i s not simply the r e s u l t of the reduced food i n t a k e of a l c o h o l - t r e a t e d dams (16-18,21-23). Studies where o f f s p r i n g were s u r r o g a t e - f o s t e r e d a f t e r b i r t h , have shown th a t poor p o s t n a t a l growth i s not a r e s u l t of reduced maternal care (18). 3) The E f f e c t of Ethanol upon Hormonal Function Root et a l . (33) s t u d i e d h y p o t h a l a m i c - p i t u i t a r y f u n c t i o n i n fou r 11 FAS c h i l d r e n . Serum l e v e l s of human growth hormone, i n s u l i n , l u t e i n i z i n g hormone, f o l l i c l e - s t i m u l a t i n g hormone, testosterone and p a r a t h y r o i d hormone were a l l within- normal ranges. The researchers concluded t h a t the post-n a t a l growth r e t a r d a t i o n of FAS was not a r e s u l t of abnormal hypothalamic-p i t u i t a r y f u n c t i o n . 4) The E f f e c t of Ethanol upon P l a c e n t a l N u t r i e n t Transfer Studies on the e f f e c t of ethanol upon 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 u s i n g r a t s have produced c o n f l i c t i n g r e s u l t s . L i n (34) reported a reduced t r a n s f e r of alpha-amino-isobutyric a c i d to f e t u s e s of r a t s administered a l c o h o l from day 6 to day 21 of g e s t a t i o n . Henderson et a l . (35) found a reduced i n v i t r o uptake of v a l i n e by p l a c e n t a l v i l l o u s fragments taken from r a t s which had been intubated w i t h ethanol on days 11-13 or 14-16 of g e s t a t i o n . Henderson et a l . (35) alsaOadministered 6% ethanol i n a l i q u i d d i e t t o r a t s f o r 30 days p r i o r t o mating and throughout g e s t a t i o n . This c h r o n i c ethanol consumption induced a 44% depression i n i n v i t r o p l a c e n t a l v a l i n e uptake t e s t e d on day 20 of g e s t a t i o n . However, i n our l a b o r a t o r y , Jones et a l . (28) found t h a t r a t s given ethanol i n t h e i r d r i n k i n g water f o r 5 weeks p r i o r t o mating and throughout g e s t a t i o n , d i d not d i f f e r from c o n t r o l s i n f e t a l uptake of z i n c , a glucose analog or 2-amino i s o b u t y r a t e . The l a c k of agreement between s t u d i e s may be due to d i f f e r e n c e s i n experimental design. The experiment of L i n (34) i n v o l v e d only short term ethanol a d m i n i s t r a t i o n . Henderson et a l . (35) used an i n v i t r o p l a c e n t a l p r e p a r a t i o n , whereas Jones et a l . (28) fed ethanol to t h e i r r a t s over an extended time p e r i o d and studied i n v i v o f e t a l n u t r i e n t uptake. The f i n d i n g s of Jones et a l . (28,30) may suggest t h a t r a t s c h r o n i c a l l y 12 f e d ethanol are able to maintain normal n u t r i e n t l e v e l s , and t h a t the uptake of n u t r i e n t s by the f e t u s was not a l t e r e d . Jones et a l . (30) reported t h a t a l c o h o l i n g e s t i o n reduced maternal blood flow to placentae, but a l s o t h a t a l c o h o l - t r e a t e d r a t s had s i g n i f i c a n t l y l a r g e r placentae when compared to both p a i r - f e d and c o n t r o l animals. Weiner et a l . (27) a l s o have reported higher p l a c e n t a l weights i n r a t s f e d ethanol i n a l i q u i d d i e t . Jones et a l . (30) suggested that l a r g e r placentae may develop i n a l c o h o l - t r e a t e d dams t o compensate f o r reduced blood flow and to maintain adequate f e t a l n u t r i t u r e . 5) The E f f e c t of Ethanol upon Maternal I n t e s t i n a l N u t r i e n t Absorption Studies have been done w i t h humans and animals on the e f f e c t s of a l c o h o l consumption upon the absorption of n u t r i e n t s from the i n t e s t i n e . In experiments w i t h nonpregnant r a t s , researchers have reported t h a t a l c o h o l consumption increases f e c a l z i n c l o s s (36), and decreases i n t e s t i n a l amino a c i d a c t i v e t r a n s p o r t (37). Increased f e c a l l o s s of z i n c has a l s o been found i n human a l c o h o l i c subjects (38). Lucas et a l . (39) s t u d i e d f o l i c a c i d t r a n s f e r across everted j e j u n a l sacs taken from nonpregnant r a t s . Jejunum which had been taken from r a t s given a 20% ethanol s o l u t i o n f o r 3 weeks showed s i g n i f i c a n t l y elevated r a t e s of f o l i c a c i d t r a n s f e r . However, when jejunum was taken from r a t s which had not been t r e a t e d w i t h a l c o h o l , and put d i r e c t l y i n a 3% ethanol s o l u t i o n , a decrease i n f o l a t e t r a n s f e r r e s u l t e d . The i n t e r a c t i o n of the e f f e c t s of a l c o h o l consumption, n u t r i t i o n a l s t a t u s and impaired absorption of e s s e n t i a l n u t r i e n t s may be very complex. A l c o h o l consumption impairs the absorption of some n u t r i e n t s , which i n t u r n may a f f e c t the absorption of other n u t r i e n t s . Hoyumpa et a l . (40) 13 found t h a t j e j u n a l sacs taken from thiamine d e f i c i e n t r a t s showed an increased t r a n s f e r of (14c)_D-glucose w n e n compared to sacs taken from p a i r - f e d animals. However, Hoyumpa et a l . a l s o found t h a t a s i n g l e dose of ethanol by gavage reduced glucose t r a n s f e r i n sacs taken from both thiamine d e f i c i e n t and p a i r - f e d r a t s . In f a c t , ethanol decreased glucose t r a n s f e r i n the thiamine d e f i c i e n t group to below th a t o f the p a i r - f e d group. 6) The E f f e c t of Ethanol upon Maternal Urinary Loss of N u t r i e n t s A l c o h o l i s a known d i u r e t i c . Increased u r i n a r y l o s s of z i n c by ethanol-fed male r a t s has been observed (36). Several human s t u d i e s have shown th a t a l c o h o l i c s have increased u r i n a r y l o s s of z i n c (38,41,42) and of magnesium (43). 7) The E f f e c t of Ethanol upon Blood and Tissue N u t r i e n t Levels a) . Zinc Decreased t i s s u e z i n c l e v e l s have been found a f t e r 4 weeks of feeding male r a t s a l i q u i d d i e t c o n t a i n i n g 5% ethanol (36). Decreased plasma and l i v e r z i n c l e v e l s have been reported i n male r a t s given a 20% ethanol s o l u t i o n (44). A l s o , depressed serum z i n c l e v e l s were present i n 6 of the 10 human a l c o h o l i c s s t u d i e d by S u l l i v a n and Heaney (42)'. b) F o l i c A c i d H a l s t e d (45) reported t h a t chronic alcoholism i n humans i s ass o c i a t e d w i t h a low d i e t a r y f o l a t e i n t a k e and decreased s a t u r a t i o n of plasma B - g l o b u l i n w i t h f o l i c a c i d . Frank and Baker (46) fed 6 male r a t s P u r i n a Rat Chow and intubated them d a i l y f o r 28 days w i t h 6 grams ethanol/kg. body weight. Blood and 14 l i v e r samples taken from these animals were compared to those of c o n t r o l s maintained on the same chow d i e t and intubated d a i l y w i t h sucrose s o l u t i o n . Bipod f o l a t e l e v e l s of the etha n o l - t r e a t e d group were s i g n i f i c a n t l y lower than those of the c o n t r o l s (871 ng. t o t a l f o l a t e / m l . blood f o r et h a n o l - t r e a t e d animals and 1086 ng./ml. blood f o r c o n t r o l animals). However, l i v e r f o l a t e l e v e l s were increased i n the et h a n o l - t r e a t e d group (117 ug./gram l i v e r p r o t e i n ) when compared to the c o n t r o l s (80 ug./gram l i v e r p r o t e i n ) . c) Glucose Several groups of i n v e s t i g a t o r s have reported t h a t ethanol i n h i b i t s gluconeogenesis (47,48). Krebs et a l . (48) used perfused r a t l i v e r s (from r a t s f a s t e d f o r 48 hours) and reported t h a t 10 mmole ethanol caused a 66% r e d u c t i o n of gluconeogenesis from l a c t a t e , w h i l e 44 mmole ethanol caused a 33% r e d u c t i o n . They proposed t h a t these f i n d i n g s e x p l a i n the hypoglycemia found i n malnourished a l c o h o l i c s . The p o s s i b i l i t y t h a t ethanol has an e f f e c t upon the glucose status of pregnant and neonatal r a t s has been s t u d i e d by Snyder and Singh (49). They gave r a t s a l i q u i d d i e t c o n t a i n i n g ethanol as 36% of t o t a l c a l o r i e s throughout g e s t a t i o n . Maternal blood glucose c o n c e n t r a t i o n i n the ethanol group and blood glucose of t h e i r 2 day o l d pups were compared to blood glucose from p a i r - f e d and ad l i b i t u m f e d c o n t r o l animals. Although maternal blood glucose values f o r ethanol, p a i r - f e d and c o n t r o l groups r e s p e c t i v e l y , were s i m i l a r (45 mg./dl., 43 mg./dl. and 46 mg./dl.), the blood glucose l e v e l s of the 2 day o l d pups were s i g n i f i c a n t l y d i f f e r e n t from each other (51 mg./dl., 67 mg./dl. and 79 mg./dl. blood f o r ethanol-t r e a t e d , p a i r - f e d and c o n t r o l groups r e s p e c t i v e l y ) . Pups of a l c o h o l -t r e a t e d dams weighed 12% l e s s than those of p a i r - f e d dams, and 22% l e s s 15 than those of c o n t r o l dams, d) Amino Acids Recently, i n v e s t i g a t o r s have s t u d i e d the e f f e c t of ethanol i n g e s t i o n upon plasma amino a c i d p a t t e r n s and upon the i n c o r p o r a t i o n of f r e e amino acids i n t o t i s s u e p r o t e i n s . Shaw and Lieber (50) s t u d i e d a l c o h o l i c s who d i d not have l i v e r disease and found that they had increased plasma l e v e l s of alpha-amino-n-butyric a c i d . E r i k s s o n et a l . (51) reported t h a t a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n of 2 grams ethanol/kg. body weight (20% ethanol w/v) to male r a t s decreased the plasma l e v e l s of a l l amino a c i d s , w i t h the exception of glutamine. and glutamate. Furthermore, the r a t i o of some amino acids to the t o t a l plasma amino a c i d pool were changed. The r e l a t i v e amounts of a l a n i n e , asparagine, phenylalanine and threonine were decreased; the r e l a t i v e amounts of glutamate, glutamine, l y s i n e and o r n i t h i n e were increased. Stanko et a l . (52) reported t h a t a l i q u i d d i e t c o n t a i n i n g 34% ethanol administered to r a t s f o r 30 days produced a s i g n i f i c a n t increase i n plasma l e u c i n e , i s o l e u c i n e , and alpha-amino-n-butyric a c i d , and a ••• s i g n i f i c a n t decrease i n plasma a l a n i n e . The e f f e c t of ethanol upon i n c o r p o r a t i o n of amino acids i n t o t i s s u e p r o t e i n s has a l s o been s t u d i e d . Several s t u d i e s (53-55) have shown tha t ethanol i n g e s t i o n by a d u l t rodents reduces i n v i v o hepatic amino a c i d uptake. Morland et a l . (55) reported t h a t hepatic v a l i n e uptake was reduced both i n male r a t s f e d a low p r o t e i n d i e t and i n male r a t s fed a high p r o t e i n d i e t . In a d d i t i o n , maternal a l c o h o l consumption has been shown to decrease i n c o r p o r a t i o n of l e u c i n e i n t o f e t a l b r a i n and hepatic p r o t e i n s (56-58). 16 The s i g n i f i c a n c e of changes i n plasma amino a c i d p a t t e r n and p r o t e i n s y n t h e s i s to f e t a l growth i s unknown. However, s t u d i e s using human subjects have looked at the r e l a t i o n s h i p of maternal and f e t a l plasma amino acids to f e t a l growth and development. Moghissi et a l . (59) s t u d i e d the c o r r e l a t i o n between maternal plasma amino acids (at 32-34 weeks ge s t a t i o n ) and newborn development. They found that maternal g l y c i n e , l y s i n e , and t o t a l amino acids were p o s i t i v e l y c o r r e l a t e d w i t h b i r t h weight, while maternal v a l i n e and threonine showed a negative c o r r e l a t i o n . B a r r e t t et a l . (60) reported that cord blood taken from s m a l l - f o r - g e s t a t i o n a l - a g e newborns had increased l e v e l s of l e u c i n e , v a l i n e , a l a n i n e , c y s t i n e , p r o l i n e , methionine, s e r i n e , g l y c i n e and l y s i n e , when compared to normal b i r t h weight babies. Scott et a l . (61) found t h a t i n s m a l l - f o r - g e s t a t i o n a l - a g e babies plasma branched-chain amino acids (at day 21 postpartum) c o r r e l a t e d i n v e r s e l y w i t h growth and n i t r o g e n r e t e n t i o n . A l o n g - l a s t i n g e f f e c t of p r e n a t a l m a l n u t r i t i o n on amino a c i d p a t t e r n s has been shown i n animal s t u d i e s . Hsu et al.;>(62) s t u d i e d 9 month o l d male o f f s p r i n g of underfed dams. They found that these animals had decreased l e v e l s of a l l plasma amino a c i d s , even though they had been fed ad l i b i t u m since weaning. The most marked decreases were of a l a n i n e , glutamic a c i d , g l y c i n e , p r o l i n e , a r g i n i n e and v a l i n e . The f i n d i n g s of ethanol-induced decreased t o t a l plasma amino a c i d concentrations may be due to a number of f a c t o r s . Ethanol may i n t e r f e r e w i t h the i n t e s t i n a l absorption of some amino acids (37); reduce the uptake and i n c o r p o r a t i o n of some amino acids i n t o l i v e r p r o t e i n s , or e x t r a h e p a t i c t i s s u e s (53-58,63); reduce u t i l i z a t i o n of plasma amino acids f o r gluconeogenesis (64); increase p r o t e o l y s i s i n e x t r a h e p a t i c 17 t i s s u e s , p a r t i c u l a r l y muscle t i s s u e (65,66); or decrease both urea and albumin synt h e s i s (64,67", 68)'? SUMMARY Although i s has been known f o r many years t h a t maternal a l c o h o l consumption has d e l e t e r i o u s e f f e c t s upon f e t a l development, the mechanism of ethanol's a c t i o n i s s t i l l unknown. The p o s s i b i l i t y e x i s t s that ethanol i n t e r f e r e s w i t h maternal and/or f e t a l n u t r i e n t s t a t u s . Studies using nonpregnant s u b j e c t s , have shown th a t a l c o h o l increases u r i n a r y l o s s of minerals, i n h i b i t s hepatic gluconeogenesis, reduces i n t e s t i n a l absorption of n u t r i e n t s and produces changes i n plasma n u t r i e n t concentrations. Thus, the present study was designed to examine the e f f e c t of maternal a l c o h o l consumption upon plasma l e v e l s of s e v e r a l n u t r i e n t s i n r a t dams and t h e i r f e t u s e s . 18 EXPERIMENTAL DESIGN AND METHODOLOGY Treatment of Animals Forty v i r g i n female Sprague-Dawley r a t s were obtained from Biobreeding La b o r a t o r i e s of Canada L t d . (Ottawa, Canada) and housed i n d i v i d u a l l y i n s t a i n l e s s s t e e l screen-bottom cages. On a r r i v a l , the r a t s weighed between 200 and 220 grams. Throughout the experiment they were kept i n a room maintained a t 21°C w i t h a 12-hour on, 12-houE o f f l i g h t i n g c y c l e . A f t e r a one week adjustment p e r i o d , each r a t was randomly assigned to one of three d i e t a r y regimens. Group 1 (Alcohol) r e c e i v e d 10% (v/v) ethanol i n t h e i r d r i n k i n g water and a n u t r i t i o n a l l y adequate s o l i d d i e t ( P u r i n a Rat Chow) ad l i b i t u m . Group 2 (P a i r - f e d ) r e c e i v e d the same amount of P u r i n a Rat Chow as Group 1 had consumed i n the previous 24 hours, mixed w i t h an amount of corn s t a r c h c a l o r i c a i l y e q uivalent to the amount of a l c o h o l consumed by Group 1. The p a i r - f e d group r e c e i v e d water ad l i b i t u m . To enable easy management, the r a t chow was ground f i n e p r i o r to weighing. S p i l l a g e of the ground chow by both the a l c o h o l and p a i r - f e d groups was measured and the food given to the p a i r - f e d animals adjusted a c c o r d i n g l y . Group 3 (Control) r e c e i v e d P u r i n a Rat Chow and water ad  l i b i t u m throughout the study. Animals were fed d a i l y between 0830 and 1030. A f t e r one week, the a l c o h o l given to Group 1 animals was increased to 20% ( v / v ) . The animals i n each group were maintained on t h e i r r e s p e c t i v e feeding regimens f o r 5 weeks, a f t e r which mating of each animal to male Sprague-Dawley r a t s was begun. Males and females were housed together overnight. The presence of sperm i n the v a g i n a l washings the f o l l o w i n g morning was taken as day 1 of pregnancy. 19 S t a r t i n g on day 1 of g e s t a t i o n the a l c o h o l - t r e a t e d r a t s were changed to a regimen of 30% (v/v) ethanol. Pregnant Group 2,animals were p a i r - f e d according to the i n t a k e of pregnant a l c o h o l - t r e a t e d animals at the same stage of g e s t a t i o n . The animals were kept on t h e i r r e s p e c t i v e d i e t s u n t i l day 21 of g e s t a t i o n . Throughout the study weekly body weights of a l l animals were recorded. D a i l y food, a l c o h o l and s t a r c h consumption by a l c o h o l - t r e a t e d and p a i r - f e d animals were recorded. No record of food consumption by the c o n t r o l animals was kept. Food consumption by c o n t r o l animals has been recorded i n s e v e r a l s i m i l a r s t u d i e s i n t h i s l a b o r a t o r y (23,26,29, 31), and i t was not deemed necessary to repeat the measurements. C o l l e c t i o n and P r e p a r a t i o n of Animal Tissues Between 0800 and 1030 on day 21 of g e s t a t i o n each r a t was anesthetized w i t h ether. The chest c a v i t y was opened and approximately 10 ml. of blood was taken by c a r d i a c puncture. The animals were then k i l l e d by c u t t i n g the heart i n two. The duodenum (from the p y l o r i c s p h i n c t e r t o the ligament of T r e i t z ) was removed, r i n s e d w i t h 10 ml. of s a l i n e s o l u t i o n , weighed, and the mucosa removed by s c r a p i n g w i t h a g l a s s s l i d e . Each sample of mucosa was weighed, and immediately homogenized w i t h 19 p a r t s of d i s t i l l e d water. H a l f of each homogenate was used t o determine conjugase a c t i v i t y , u sing the r a d i o a c t i v e method of Krumdieck and Baugh (69). The remaining sample was f r o z e n at -20°C f o r l a t e r determination of t o t a l p r o t e i n . From each r a t a l l f e t u s e s and placentae were removed. Each f e t u s was c a r e f u l l y separated from i t s p l a c e n t a and amniotic sac. U m b i l i c a l cords were t i e d o f f w i t h thread c l o s e to the f e t u s , t o prevent l o s s of f e t a l blood. Fetuses and placentae from each l i t t e r were weighed and an 20 average weight c a l c u l a t e d . Blood was c o l l e c t e d from each f e t u s by-c a r d i a c puncture, using drawn g l a s s acid-washed c a p i l l a r y tubes. From 85 to 225 u l . blood was taken from each f e t u s . A l l blood samples were immediately c e n t r i f u g e d i n r e f r i g e r a t e d c e n t r i f u g e s at 3000 rpm. Plasma was c o l l e c t e d and st o r e d i n a c i d -washed p l a s t i c tubes. Plasma from fetuses of the same l i t t e r was pooled. Plasma t o be used f o r z i n c , magnesium, glucose, f o l i c a c i d , albumin, l a c t i c a c i d and o s m o l a l i t y determinations was immediately f r o z e n and stored at -70°C u n t i l the time of a n a l y s i s . A p o r t i o n of maternal plasma f o r amino a c i d a n a l y s i s was d e p r o t e i n i z e d w i t h 0.1 p a r t s of 50% s u l f o s a l i c y l i c a c i d . F e t a l plasma f o r amino a c i d a n a l y s i s was deprotein-. v i z e d w i t h 3 p a r t s of 2% s u l f o s a l i c y l i c a c i d . These d i l u t i o n s were e s t a b l i s h e d during p r e t r i a l s to o b t a i n the c o r r e c t pH and amino a c i d c o n c e n t r a t i o n , f o r accurate a n a l y s i s . The samples f o r amino a c i d a n a l y s i s were then s t o r e d at -70°C. A l l plasma n u t r i e n t determinations were done w i t h i n 1 to 5 months of the time of sample c o l l e c t i o n . Assay Procedures Determination of plasma glucose, f o l i c a c i d , z i n c , magnesium and f r e e amino acid s was done on both f e t a l and maternal samples. Plasma o s m o l a l i t y and albumin l e v e l s were determined only i n maternal samples. Plasma l a c t i c a c i d concentrations were determined only i n f e t a l samples. Plasma glucose, l a c t i c a c i d , albumin and o s m o l a l i t y , and maternal i n t e s t i n a l p r o t e i n and conjugase a c t i v i t y were determined by the author. Plasma amino a c i d l e v e l s were analyzed by lab t e c h n i c i a n s of the Ch i l d r e n ' s H o s p i t a l , Vancouver B.C. Mrs. S. Horsky of the U.B.C. G e o l o g i c a l Sciences department d i d the z i n c and magnesium analyses. Plasma f o l i c 21 a c i d assays were c a r r i e d out by Dr. P. Cornwell (Department of N u t r i t i o n Sciences, the U n i v e r s i t y of Alabama i n Birmingham). Plasma f r e e amino a c i d s were analyzed by ion-exchange chromatography us i n g a Durrum (Dionex) D-500 amino a c i d analyzer. Each amino a c i d was e l u t e d on an ion-exchange column, then reacted w i t h n i n h y d r i n to produce a purple c o l o u r . O p t i c a l d e n s i t y of the colour was then measured s p e c t r o p h o t o m e t r i c a l l y . Plasma glucose was determined u s i n g the glucose oxidase method (Sigma Chemical Company, Technic a l B u l l e t i n No. 510). Maternal plasma albumin l e v e l s were determined by a c o l o r i m e t r i c assay (Sigma Chemical Company, Technic a l B u l l e t i n No. 630). F e t a l l a c t i c a c i d determination was performed using the l a c t i c a c i d procedure (Sigma Chemical Company, T e c h n i c a l ' B u l l e t i n No. 826-UV). Plasma z i n c and magnesium were determined by atomic absorption spectrophotometry (Perkin-Elmer Atomic Absorption Spectrophotometer, Model 603). Plasma f o l i c a c i d was determined m i c r o b i o l o g i c a l l y w i t h L a c t o b a c i l l u s  c a s e i . T his method i s based on the observation t h a t c e r t a i n micro-organisms r e q u i r e s p e c i f i c vitamins f o r growth. Using a ba s a l medium complete i n a l l respects except f o r the vi t a m i n under t e s t , growth responses of the organism are compared q u a n t i t a t i v e l y i n standard and unknown s o l u t i o n s . O p t i c a l d e n s i t y of the c u l t u r e medium at 650 nm. i s used as a measure of growth and f o l i c a c i d content (70). Maternal i n t e s t i n a l conjugase ( p t e r o y l - p o l y - ^ - g l u t a m y l hydrolase) a c t i v i t y was determined using the method of Krumdieck and Baugh (69). The assay i s based upon the r e l e a s e and q u a n t i t a t i o n of r a d i o a c t i v e glutamic a c i d from l a b e l e d p t e r o y l d i - ^ - g l u t a m y l - t U - C ^ - g l u t a m i c ) a c i d 22 of known s p e c i f i c r a d i o a c t i v i t y . At the end of an i n c u b a t i o n p e r i o d , the unreacted substrate i s removed by adsorption onto c h a r c o a l . The nonadsorbed r a d i o a c t i v e glutamic a c i d i s q u a n t i t a t e d i n a l i q u i d s c i n t i l l a t i o n counter. Conjugase a c t i v i t y i s then expressed as counts per minute per mg. p r o t e i n . T o t a l p r o t e i n of i n t e s t i n a l mucosa samples was assayed by the method of Lowry et a l . (71), using bovine serum albumin as a standard. To 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 g e s t i o n r e s u l t s i n dehydration, plasma o s m o l a l i t y was measured i n maternal samples, using an osmometer ( P r e c i s i o n Systems Inc., Newton, Mass.). S t a t i s t i c a l Analysis.: D i f f e r e n c e s i n plasma n u t r i e n t l e v e l s among the three experimental groups were examined usi n g the Midas Computer Package a n a l y s i s o f v a r i a n c e . One way a n a l y s i s of variance was used. In the case of s i g n i f i c a n t d i f f e r e n c e s , Scheffe Allowances (0.95 and 0.99) w i t h p a i r e d comparisons were used to i d e n t i f y d i f f e r e n c e s among treatment groups. A n a l y s i s of the plasma n u t r i e n t data was a l s o done using Pearson C o r r e l a t i o n C o e f f i c i e n t s ( S t a t i s t i c a l Package f o r the S o c i a l S c i e n c e s ) . Average f e t a l weight ( a l c o h o l - t r e a t e d , p a i r - f e d and c o n t r o l f e t u s e s combined) was examined f o r c o r r e l a t i o n to 62 separate v a r i a b l e s . These v a r i a b l e s i n c l u d e d maternal prepregnancy weight g a i n ; maternal weight g a i n throughout pregnancy; p l a c e n t a l weight; maternal plasma l e v e l s of f o l i c a c i d , z i n c , magnesium, glucose, albumin and 26 amino a c i d s ; and f e t a l plasma l e v e l s of f o l i c a c i d , z i n c , magnesium, glucose and 25 amino a c i d s . Each c o r r e l a t i o n which showed s t a t i s t i c a l s i g n i f i c a n c e (p^.0.05) was examined usi n g a scattergram ( S t a t i s t i c a l Package f o r the S o c i a l 23 S c i e n c e s ) . This was done to e l i m i n a t e the p o s s i b i l i t y t h a t s i g n i f i c a n t c o r r e l a t i o n s were found between data o c c u r r i n g i n clumps. D i f f e r e n c e s i n maternal body weight among the three groups (both p r i o r to and throughout g e s t a t i o n ) were al s o analyzed using the Midas one-way a n a l y s i s of v a r i a n c e . Data on food intake and a l c o h o l consumption were analyzed by repeated measures a n a l y s i s of variance (Midas *ANOVAR) f o r the f a c t o r s of d i e t and time. 24 RESULTS C a l o r i c Intakes and Maternal Body Weights Table 1 shows the average d a i l y food and a l c o h o l consumption of r a t s g iven a l c o h o l or p a i r - f e d p r i o r t o pregnancy. Throughout days 1-7 a l c o h o l - t r e a t e d animals were given a d r i n k i n g s o l u t i o n of 10% a l c o h o l . On day 8 t h i s was increased to 20% ethanol. This increase i n ethanol c o n c e n t r a t i o n almost doubled the mean d a i l y absolute a l c o h o l intake (p<0.05) of a l c o h o l - t r e a t e d animals, and produced a s l i g h t , but not s i g n i f i c a n t , r e d u c t i o n i n t h e i r mean d a i l y food consumption. P r i o r to pregnancy, mean d a i l y food consumption of a l c o h o l - t r e a t e d (and t h e r e f o r e a l s o of p a i r - f e d ) animals only increased s i g n i f i c a n t l y (pKfl-05) between weeks 3 and 4 of the experiment. In Table 2 average food and a l c o h o l consumption p r i o r t o pregnancy are expressed i n c a l o r i e s . During t h i s p e r i o d , the mean d a i l y c a l o r i c i n t a k e of a l c o h o l - t r e a t e d and p a i r - f e d animals was 52.39±5.81 k c a l . average, a l c o h o l or s t a r c h consumption accounted f o r 23.25£.5.27% of t o t a l d a i l y c a l o r i e s . Percent of c a l o r i e s from a l c o h o l or s t r a c h consumption during days 1-7 was 14.20%, which increased to 24.70% during days 8-14 when the a l c o h o l concentration of the d r i n k i n g s o l u t i o n was increased from 10% to 20%. When food consumption was expressed as grams/gram body weight, i t was found t h a t p r i o r to pregnancy, a l c o h o l - t r e a t e d and p a i r - f e d animals consumed from 58 to 65% of the food consumed by c o n t r o l animals i n a s i m i l a r study by Jones et a l . (28). However, expressed as kcal/gram body weight, t o t a l d a i l y c a l o r i c i n t a k e o f a l c o h o l - t r e a t e d and p a i r - f e d animals p r i o r to pregnancy i n the present study was 70 to 92% of the 25 d a i l y c a l o r i c i n t a k e of c o n t r o l r a t s reported by Jones et a l . (28). Table 3 shows the average d a i l y food and a l c o h o l consumption of a l c o h o l - t r e a t e d and p a i r - f e d animals throughout pregnancy. On day 1 of pregnancy the concentration of ethanol i n d r i n k i n g water given to a l c o h o l - t r e a t e d animals was increased t o 30%. This increase d i d not produce an increase i n a l c o h o l consumption of a l c o h o l - t r e a t e d animals during the f i r s t week of pregnancy. Mean d a i l y food consumption of a l c o h o l - t r e a t e d animals during the f i r s t week of pregnancy decreased s l i g h t l y when compared to the week j u s t p r i o r t o pregnancy (however, due t o the la r g e standard d e v i a t i o n s , t h i s decrease was not s i g n i f i c a n t ) . Throughout pregnancy mean d a i l y food consumption of a l c o h o l - t r e a t e d animals increased, although t h i s i n c r e a s e was s i g n i f i c a n t (p<0.05) only between weeks 1 and 2 of g e s t a t i o n . Mean d a i l y a l c o h o l i n t a k e increased s i g n i f i c a n t l y only between weeks 2 and 3. Mean a l c o h o l i n t a k e during the t h i r d week of g e s t a t i o n was 7.5£.3.27 ml. F l u i d i n t a k e by pregnant r a t s has been shown to increase during the f i n a l days of pregnancy. However, other s t u d i e s done i n t h i s l a b o r a t o r y (22,28) have not reported as la r g e an increase i n a l c o h o l consumption as was found i n the present study. The t o t a l c a l o r i c i n t a k e of a l c o h o l - t r e a t e d and p a i r - f e d dams during pregnancy i s presented i n Table 4. The mean d a i l y energy intak e of both groups was 40 k c a l . higher during the l a s t week of g e s t a t i o n , when compared t o the f i r s t week. This was accompanied by an increase of 14% i n the number of d a i l y c a l o r i e s d e r i v e d from a l c o h o l or s t a r c h . During g e s t a t i o n c a l o r i e s d e r i v e d from a l c o h o l or s t a r c h consumption accounted f o r an average of 35.35£7.82% of t o t a l c a l o r i e s . During the f i r s t week of pregnancy, a l c o h o l - t r e a t e d dams consumed 26 52% and p a i r - f e d dams consumed 47% (grams/gram body weight) of the food consumed by c o n t r o l s reported by Jones et a l . (28). This percentage increased throughout g e s t a t i o n . Chow consumed by a l c o h o l - t r e a t e d dams was 74 and 76% of th a t consumed by c o n t r o l dams (Jones et a l . ) f o r the second and t h i r d weeks of g e s t a t i o n , while chow consumed by p a i r - f e d dams was 67 and 63% of t h a t o f c o n t r o l dams (Jones et a l . ) f o r the second and t h i r d weeks of g e s t a t i o n . D i f f e r e n c e s i n percent of chow consumed between a l c o h o l - t r e a t e d and p a i r - f e d dams were'due to the g r e a t e r body weight of p a i r - f e d animals. Mean d a i l y c a l o r i c i n t a k e expressed a kcal./gram body weight, of a l c o h o l - t r e a t e d dams was 69%, 100% and 115% of t h a t of c o n t r o l dams (Jones et a l . , 28) f o r the f i r s t , second and t h i r d weeks of g e s t a t i o n r e s p e c t i v e l y . Mean d a i l y c a l o r i c i n t a k e of p a i r - f e d dams was 63%, 93% and 96% of th a t of c o n t r o l dams (Jones et a l . , 28) f o r the f i r s t , second and t h i r d weeks of g e s t a t i o n . Table 5 and Figures 1 and 2 show the average body weights of a l c o h o l -t r e a t e d , p a i r - f e d and c o n t r o l animals throughout the experiment. Except f o r i n i t i a l weights, the body weights of c o n t r o l animals were s i g n i f i c a n t l y g r e a t e r (p<0.05) than those of the other two experimental groups through-out the e n t i r e experiment. During the 5 weeks p r i o r t o pregnancy there were no s i g n i f i c a n t d i f f e r e n c e s i n body weight between a l c o h o l - t r e a t e d and p a i r - f e d animals. The body weights of both a l c o h o l - t r e a t e d and p a i r - f e d animals dropped s l i g h t l y during the f i r s t week of the experiment. This v/as probably due to adjustment by a l c o h o l - t r e a t e d animals t o the f i r s t i n t r o d u c t i o n of ethanol i n t o t h e i r d r i n k i n g water. A f t e r the f i r s t week, body weight of both a l c o h o l - t r e a t e d and p a i r - f e d animals showed a steady i n c r e a s e . 27 During pregnancy, 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 weight were seen among a l l three treatment groups. Body weight of c o n t r o l animals was considerably g r e a t e r than t h a t o f both a l c o h o l - t r e a t e d (-p<0'°01), and p a i r - f e d (p<.0.05) animals. During the l a s t two weeks of pregnancy, the body weights of the p a i r - f e d animals were g r e a t e r than those of the a l c o h o l - t r e a t e d dams (p^0.005). This was probably due to the i n i t i a l weight l o s s by a l c o h o l ^ t r e a t e d animals during the f i r s t week o f pregnancy. This weight l o s s may have been due t o the regimen of d r i n k i n g water c o n t a i n i n g 30% ethanol which was i n i t i a t e d on day 1 of pregnancy. Weight l o s s by a l c o h o l - t r e a t e d animals was not p a r a l l e l e d by a s i m i l a r weight l o s s by p a i r - f e d animals. During the l a s t week of pregnancy both p a i r - f e d and c o n t r o l groups gained an average of 60 grams, w h i l e a l c o h o l — t r e a t e d animals gained only an average of 30 grams. No c o r r e l a t i o n was found between f e t a l body weight ( a l c o h o l - t r e a t e d , p a i r - f e d and c o n t r o l f e t u s e s combined) and maternal prepregnancy weight g a i n ( a l c o h o l - t r e a t e d , p a i r - f e d and c o n t r o l dams combined). However, a Pearson c o r r e l a t i o n c o e f f i c i e n t o f 0.6473 (p<0.001) was found between f e t a l body weight and maternal weight g a i n throughout pregnancy. 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 Table 6 shows mean l i t t e r s i z e , f e t a l body weight and p l a c e n t a l weight determined on day 21 of g e s t a t i o n . Means and standard d e v i a t i o n s were c a l c u l a t e d using l i t t e r means. Mean f e t a l body weight was s i g n i f i c a n t l y lower (p<0.001) i n the a l c o h o l - t r e a t e d group compared t o the p a i r - f e d or c o n t r o l groups. There was not a s i g n i f i c a n t d i f f e r e n c e i n body weight between p a i r - f e d and c o n t r o l f e t u s e s . Mean l i t t e r s i z e (number of fet u s e s per l i t t e r ) d i d not vary 28 s i g n i f i c a n t l y among the three groups. P l a c e n t a l weights were highest i n the a l c o h o l - t r e a t e d group, and lowest i n the p a i r - f e d group (p^O-05). However, there was not a s i g n i f i c a n t d i f f e r e n c e i n p l a c e n t a l weight between the a l c o h o l - t r e a t e d and c o n t r o l groups. No s i g n i f i c a n t c o r r e l a t i o n e x i s t e d between f e t a l body weight and p l a c e n t a l weight. 29 Table 1_ D a i l y Food and A l c o h o l Consumption P r i o r to Pregnancy (Mean± S.D.) Al c o h o l Group food(g) alcohol(ml) P a i r - f e d Group food(g) s t a r c h (g). No. of r a t s 13 13 10 10 Prepregnancy Days 1-7 8-14 15-21 22-28 29-35 9.39(a) 11.61 8.29(a) £0.83 8.89(a) ±0.96 10.09(b) £0.56 10.40(b) ±0.69 1.20(a) ±0.22 2.10(b) ±0.52 2.66(b) ±0.38 2.57(b)' 10.26 2.74(b) ±0.28 9.39(a) ±0.00 8.29(a) ±0.00 8.89(a) 0.00 10.09(b) +0.00 10.40(b) ±0.00 1.65(a) ±0.00 2.89(b) ±.0.00 3.66(b) ±0.00 3.53(b) ±0.00 3.77(b) ±0.00 a,b Figures i n the same column not sharing the same s u b s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at the p<0.05 l e v e l . 30 Table 2 D a i l y C a l o r i c Intake P r i o r to Pregnancy (Meanjt S.D.) No. of r a t s A l c o h o l Group .food a l c o h o l t o t a l %; !kcal ( k c a l ) (kcal) d a i l y from 13 13 k c a l 13 a l c o h o l 13 P a i r - f e d Group food s t a r c h t o t a l ( k c a l ) ( k c a l ) d a i l y k c a l 10 10 10 Prepregnancy Days 1-7 8-14 15-21 22-28 29-35 39.89 ±6.87 35.22 +3.53 37.77 ±4.08 42.87 + 2.39 44.20 ±2.91 6.61 +1.19 11.55 + 2.86 14.63 ±2.07 14.16 ±1.40 15.05 + 1.54 46.50 14.20 46.77 24.70 52.40 27.92 57.03 24.03 59.25 25.40 39.89 iO.OO 35.22 + 0.00 37.77 +0.00 42.87 10. 00 44.20 + 0.00 6.61 t-0.00 11.55 +0.00 14.63 +0.00 14.16 to. 00 15.05 +0.00 46.50 46.77 52.40 57.03 59.25 31 Table 3 D a i l y Food and A l c o h o l Consumption During Pregnancy (Mean ± S.D.) Pregnancy Days A l c o h o l Group P a i r - f e d Group food(g) alcohol(ml) food(g) starch(g) No. of r a t s 13 13 10 10 1-7 9.11(b) ±2.21 2.91(b) +0.60 9.11(b) +0.00 4.00(b) io.oo 8-14 11.60(c) ±1.89 4.34(b) ±1.09 11.60(c) ±0.00 5.97(b) ±0.00 15-21 12.27(c) ±3.37 7.50(c) ±3.27 12.27(c) + 0.00 10.31(c) ±0.00 b,c Figures i n the same column not sharing the same s u b s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at the p<0.05 l e v e l . 32 Table 4 D a i l y C a l o r i c Intake During Pregnancy (Meani. S.D.) A l c o h o l Group P a i r - f e d Group' food a l c o h o l t o t a l % : k c a l food . s t a r c h t o t a l ( k c a l ) ( k c a l ) d a i l y from 'Ckeal) ( k c a l ) d a i l y k c a l a l c o h o l k c a l No. of r a t s 13 13 13 13 10 10 10 Pregnancy Days 1-7 38.74 16.03 54.77 29.27 38.74 16.03 54.77 +9.38 ±3.30 +.0.00 +0.00 8-14 49.30 23.85 73.15 32.60 49.30 23.85 73.15 ±8.01 16.00 +0.00 ±0.00 15-21 52.14 41.25 93.39 44.17 52.14 41.25 93.39 114.31 +18.00 ±0.00 10.00 33 Table 5 Weekly Maternal Body Weights Before and During Pregnancy (Mean + S.D.) Al c o h o l Group P a i r - f e d Group Co n t r o l Group No. of r a t s 13 10 10 Prepregnancy Days Day 1 222.619.23(a) 225.5111.23(a) 226.1110.16(a) Day 7 223.3115.51(a) 219.3410.22(a) 231.4+13.45(b) Day 14 221.6+17.21(a) 221.2£9.72(a) 242.2116.21(b) Day 21 228.6+17.46(a) 229.2*8.48(a) 251.9+16.32(b) Day 28 236.2116.35(a) 236.3+8.50(a) 256.5116.69(b) Day 35 240.4119.89(a) 241.718.72(a) 261.3117.01(b) Pregnancy Days Day- 1 257.4+19.54(a) 260.8+11.46(a) 270.4121.08(b) Day 7 250.2+23.80(a) 272.5112.54(b) 293.2121.79(c) Day 14 270.5128.28(a) 297.6110.97(b) 313.6121.70(c) Day 21 298.2+36.41(a) 358.0117.83(b) 378.2125.49(c) a,b,c Figures i n the same row not sharing the same s u b s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at l e a s t at the p<0.05 l e v e l . 34 Figure 1 . Mean Maternal Body Weight P r i o r to Pregnancy Figure 2 Mean Maternal Body Weight During Pregnancy 380 t 360 340 320 £ 300 0) 280 260 240 220 (7 A A l c o h o l - t r e a t e d • P a i r - f e d 4* C o n t r o l 7 14 Time (days) 21 36 Table 6 F e t a l Body Weight, L i t t e r Size and P l a c e n t a l Weight (Mean t S.D.) Al c o h o l Group P a i r - f e d Group C o n t r o l Group No. of l i t t e r s 13 10 10 F e t a l body weight (g) 2.89+0.32(a) 3.40±0.30(b) 3.49±0.39(b) L i t t e r s i z e 10.0+3.58 11. 6&U711V 10.3±1.95 P l a c e n t a l •"" weight (g)' 0.53±0.09(a) 0.46+0.04(b) 0.50+0.05(a) a,b Figur e s i n the same row not sharing the same s u b s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at l e a s t at the p<0.05 level/; 37 N u t r i e n t Levels i n Maternal Plasma Mean plasma l e v e l s of f o l i c a c i d , z i n c , magnesium, glucose and albumin i n r a t dams at day 21 of g e s t a t i o n are presented i n Table 7. 1) F o l i c A c i d No d i f f e r e n c e s were observed i n mean plasma f o l i c a c i d l e v e l s among the treatment groups. A s i g n i f i c a n t c o r r e l a t i o n was not found between f e t a l body weight and maternal plasma f o l i c a c i d l e v e l s . 2) Zinc There were no s i g n i f i c a n t d i f f e r e n c e s found i n plasma z i n c concen-t r a t i o n among the three groups of dams, nor was there a s i g n i f i c a n t c o r r e l a t i o n between f e t a l body weight and maternal plasma z i n c l e v e l s . 3) Magnesium Mean maternal plasma magnesium l e v e l s were s i g n i f i c a n t l y higher (p<0.01) i n a l c o h o l - t r e a t e d dams when compared t o both p:air-fed and c o n t r o l animals, but there was not a ; s i g n i f i c a n t d i f f e r e n c e between p a i r -fed and c o n t r o l samples. No c o r r e l a t i o n was found between maternal plasma magnesium con c e n t r a t i o n and f e t a l body weight. 4) Glucose There were no s i g n i f i c a n t d i f f e r e n c e s among the three maternal treatment groups i n plasma glucose concentration. Pearson c o r r e l a t i o n showed th a t no s i g n i f i c a n t c o r r e l a t i o n e x i s t e d between maternal plasma glucose values and f e t a l body weight. 5) Albumin The three groups of dams had s i m i l a r plasma l e v e l s of albumin on day 21 of g e s t a t i o n . No c o r r e l a t i o n was found between maternal plasma 38 albumin l e v e l s and f e t a l body weight. Maternal Plasma Osmolality Mean plasma o s m o l a l i t y i n r a t dams i s shown i n Table 8. Osmolality was highest i n a l c o h o l - t r e a t e d dams and lowest i n p a i r - f e d dams. D i f f e r e n c e s i n degree o f dehydration between a l c o h o l - t r e a t e d and p a i r - f e d dams and between alcohols-treated and c o n t r o l dams were s i g n i f i c a n t at the p<0.01 l e v e l . The d i f f e r e n c e i n plasma o s m o l a l i t y between p a i r - f e d and c o n t r o l animals was not s i g n i f i c a n t . Maternal I n t e s t i n a l Conjugase A c t i v i t y I n t e s t i n a l conjugase a c t i v i t y i s presented i n Table 9. P t e r o y l -poly-^-glutamyl hydrolase a c t i v i t y i n maternal duodenal mucosa was expressed as counts per minute per mg. mucosal p r o t e i n . No s i g n i f i c a n t d i f f e r e n c e s were found among the three groups of animals. 39 Table 7 Maternal Plasma F o l a t e , Zinc, Magnesium, Glucose and Albumin Levels on Day 21 of Gestation (Meanj£ S.D.) A l c o h o l Group v P a i r - f e d Group C o n t r o l Group F o l i c A c i d (ng/ml) 4.99+6.85(4)a 6.69+2.00(4) 12.50+6.61(4) Zinc (mg/ml) 0.96+0.27(7) 1.06+0.36(7) 1.1810.29(8) Magnesium (mg/dl) 3.40+0.86(7,),b 2.17+0.39(7) 2.2010.43(8) Glucose (mg/dl)S 129.11+26.23(13) 117.14+20.29(7) 122.08121.77(13) Albumin (ug/ml) 3.71+0.51(7) 3.28+0.31(7) 3.3-0+0.33(8) a Numbers i n brackets represent the number of animals. b S i g n i f i c a n t l y d i f f e r e n t (p<0.01) from both p a i r - f e d and c o n t r o l values. 40 Table 8 Maternal Plasma Osmolality on Day 21 of Gestation (Mean X. S.D.) A l c o h o l Group P a i r - f e d Group C o n t r o l Group No. of r a t s 7 7 8 Osmolality 296.57+12.25a'; 267.57+16.79 273.38+8.23 (mosmoles/1) a S i g n i f i c a n t l y d i f f e r e n t at p<0.01 from both p a i r - f e d and c o n t r o l . Table 9__Maternal I n t e s t i n a l Conjugase A c t i v i t y on Day 21 of Gestation ""'(Mean ± S.D.) A l c o h o l Group No. of r a t s 9 Conjugase A c t i v i t y 48967±15549a (CPM/mg p r o t e i n ) P a i r - f e d Group C o n t r o l Group 5 8 40276±7376 61355+16673 a There were no s i g n i f i c a n t d i f f e r e n c e s among the three groups. 41 Maternal Plasma Amino Acids The mean l e v e l s of maternal plasma amino acids and urea at day 21 of g e s t a t i o n are shown i n Table 10. Levels of maternal plasma amino acids are a l s o i l l u s t r a t e d i n . F i g u r e 3. In c o n t r o l animals mean plasma amino a c i d l e v e l s v a r i e d from the lowest value of 14.17 u m o l e s / l i t e r f o r alpha-amino-n-butyric a c i d , to the highest value of 976.00 u m o l e s / l i t e r f o r l y s i n e . Generally, mean amino a c i d l e v e l s i n a l c o h o l - t r e a t e d and p a i r -fed animals were s i m i l a r to those i n c o n t r o l animals. However, w i t h i n each of the three groups, there was wide v a r i a t i o n i n amino a c i d l e v e l s . Only seven out of the twenty-five amino acids, examined showed 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 among the three treatment groups. These seven amino acid s were threonine, s e r i n e , p r o l i n e , alpha-amino-n-butyric a c i d , c y s t i n e , a l a n i n e and h i s t i d i n e . Of these seven amino acid s only p r o l i n e and alpha-amino-n-butyric a c i d were s i g n i f i c a n t l y d i f f e r e n t i n a l c o h o l - t r e a t e d dams compared to both p a i r - f e d and c o n t r o l dams. Mean p r o l i n e l e v e l s were s i g n i f i c a n t l y lower (p<0.05), w h i l e mean plasma alpha-amino-n-butyric a c i d l e v e l s were s i g n i f i c a n t l y higher (p<0.01) i n a l c o h o l - t r e a t e d dams compared to the other two groups. Mean plasma threonine l e v e l s were s i g n i f i c a n t l y lower i n p a i r - f e d (310.43 umoles/1.) when compared to c o n t r o l dams (419.00 umoles/i.), while the l e v e l s i n a l c o h o l - t r e a t e d and c o n t r o l dams were s i m i l a r . Mean plasma s e r i n e and alanine l e v e l s i n p a i r - f e d animals were s i g n i f i c a n t l y higher (p<0.01) than those i n both a l c o h o l - t r e a t e d and c o n t r o l groups. Plasma l e v e l s of c y s t i n e and h i s t i d i n e were s i m i l a r i n a l c o h o l - t r e a t e d and p a i r - f e d animals. -Cystine l e v e l s were s i g n i f i c a n t l y lower i n the a l c o h o l - t r e a t e d compared to the c o n t r o l group (p<0.01), and i n the p a i r -42 fed compared to the c o n t r o l group (p<(0.05). C o n t r o l females e x h i b i t e d s i g n i f i c a n t l y higher h i s t i d i n e l e v e l s than a l c o h o l - t r e a t e d (p^0.05), and p a i r - f e d females (p<0.01). Mean plasma l e v e l s of urea v a r i e d g r e a t l y among a l l three treatment groups. P a i r - f e d animals e x h i b i t e d s i g n i f i c a n t l y lower urea l e v e l s than both a l c o h o l - t r e a t e d and c o n t r o l animals (p<0.01). Urea l e v e l s i n a l c o h o l - t r e a t e d animals were s i g n i f i c a n t l y lower than those of c o n t r o l animals (p<0.05). Two of the maternal plasma amino acids showed s i g n i f i c a n t c o r r e l a t i o n s w i t h f e t a l body weight. A p o s i t i v e c o r r e l a t i o n (0.5694, p<0.05) was found between maternal plasma p r o l i n e l e v e l s and f e t a l body weight. A negative c o r r e l a t i o n of -0.6409 (p(0.01) was found between maternal plasma alpha-amino-n-butyric a c i d l e v e l s and f e t a l body weight. 43 Table 10 Maternal Plasma Amino A c i d and Urea Levels on Day 21 of Ge s t a t i o n (Meani: S.D.) No. of r a t s Amino Acids (umol/1)• Alanine ANBA Argi n i n e Asparagine A s p a r t i c A c i d C i t r u l l i n e C y s tine Glutamic A c i d Glutamine Glycin e H i s t i d i n e Hydroxyproline I s o l e u c i n e Leucine Lysine Methionine O r n i t h i n e Phenylalanine P r o l i n e Serine Taurine A l c o h o l Group 6 P a i r - f e d Group 7 Con t r o l Group 6 621.83175.72a 46.50+7.50a 122.00134.69 110.00+10.24 18.83+2.32 50.8318.84 34.17+7.86a 135.33+38.22 972.001111.14 143.83160.41 31.33+3.20a 21.67+5.89 86.83119.39 103.33131.41 818.501161.89 46.67110.63 56.83+18.85 67.33110.29 142.83162.38a 253.17±26.14a 192.83162.77 949.571126.02b 9.0011.00b 124.14+14.88 100.86118.31 22.29+2.98 71.00111.48 38.8616.72a 163.71+26.19 702.00136.77 102.14+38.62 26.2917.18a 29.0017.87 86.57411.33 130.71118.21 790.571205.86 49.2916.65 47.8619.48 76.0018.64 222.57+43.12b 327.57147.60b 177.57+45.28 612.331112.74a 14.1713.87b 147.33*53.28 104.83122.34 18.8314.58 66.50119.61 54.33+10.29b 150.00146.99 583.0010.00 88.17+20.49 42.3317.60b 21.3317.45 97.83114.33 151.17+26.45 976.001207.58 51.00+9.34 56.17114.22 69.33115.49 245.50154.15b 227.83129.90a 166.50153.47 44 Table 10 continued A l c o h o l Group P a i r - f e d Group Co n t r o l Group Threonine Tryptophan Tyrosine V a l i n e 348.17+34.68ayb 56.67±18.02 47.83*11.02 159.00+43.62 310.41+50.96a 53.43*19.41 47.14+9.51 151.29±22.97 419.00i63.45b 76.00+5.66 50.00+16.83 182.50*24.87 Urea 5316.80+280.83a 2524. 00±1081.20b 6850'. 004945. 37c a,b,c Figures i n the same row not sharing the same s u b s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at l e a s t at the p<0.05 l e v e l . 45 Figure 3. Maternal Plasma Amino A c i d Levels on Day 21 of Gestation ( u m o l e s / l i t e r ) Alanine Ale 622 ####################################(,a) P F 9 5o Cont 612 ################################### ANBA Ale 47 ### PF 9 # Cont 14 # Argi n i n e A l e 122 PF 124 Cont 147 ####### ####### ######### Asparagine Ale 110 ###### PF 101 ###### Cont 105 ###### A s p a r t i c A c i d A l e 19 # PF 22 '# Cont 19 # Cystine Ale 34 ## •r PF 39 ## Cont 54 ### C i t r u l l i n e Ale 51 ### PF 71 #### Cont 67 #### Glutamic A c i d Ale 135 ######## PF 164 ########## Cont 150 ######### Glutamine A I C 9 7 2 PF 702 ######################################### Cont 583 ################################## Glycine A l e 144 ######## PF 102 ###### Cont 88 ##### 46 Figure 3 continued H i s t i d i n e Ale 31 ## PF 26 ## Cont 42 ## Hydroxyproline Ale 22 # •• 'PF 29 ## Cont 21 # Is o l e u c i n e Al e 87 ##### PF 87 ##### Cont 98 ###### Leucine Ale 130 ######## PF 131 ######## Cont 151 ######### Lysine A I C 8 1 9 P F 7 9 i cont 976 mmmmmm#mmmmm#mmmmm####### Methionine Ale 47 PF 49 Cont 51 ### ### ### O r n i t h i n e Al e 57 •t PF 48 Cont 56 ### ### ### Phenylalanine Ale 67 #### PF 76 #### Cont 69 #### P r o l i n e A l e 143 ######## PF 223 ############# Cont 246 Serine A l e 253 ############### PF 328 ################### Cont 228 ############# 47 Figure 3 continued Taurine A l e 193 ########### PF 178 ########## Cont 167 ########## Threonine Al e 348 #################### PF 310 ################## Cont 167 ########## Tryptophan Al e 57 ### PF 53 ### Cont 76 #### Tyrosine A l e 48 ### PF 47 ### Gont 50 ### V a l i n e A l e 159 ######### PF 151 ######### Cont 183 ########### (a) # corresponds to 17 umoles of amino a c i d per l i t e r of plasma 48 N u t r i e n t Levels i n F e t a l Plasma Mean plasma l e v e l s of f o l i c a c i d , z i n c , magnesium, glucose-and l a c t i c a c i d i n . r a t f e t u s e s at.day"21 o f . g e s t a t i o n . a r e presented.in Table 11. 1) F o l i c A c i d ..' . No d i f f e r e n c e s J i n plasma'folate were\observed among .the--three f e t a l groups. There was not a s i g n i f i c a n t c o r r e l a t i o n between f e t a l bodjr weight and f e t a l plasma f o l i c a c i d l e v e l s ( a l c o h o l - t r e a t e d , p a i r - f e d and c o n t r o l f e t u s e s combined). 2) Zinc Plasma z i n c l e v e l s were s i m i l a r i n a l c o h o l - t r e a t e d and p a i r - f e d animals. Mean plasma z i n c l e v e l s of c o n t r o l f e t u s e s were s i g n i f i c a n t l y -lower than those of a l c o h o l - t r e a t e d (p<0.01) and p a i r - f e d (p<0.001) fe t u s e s . No s i g n i f i c a n t c o r r e l a t i o n was found between f e t a l body weight and f e t a l plasma z i n c values. 3) Magnesium • P a i r - f e d f e t u s e s e x h i b i t e d lower mean plasma magnesium l e v e l s than c o n t r o l f e t u s e s (p<0.05). However, there was not a s i g n i f i c a n t d i f f e r e n c e i n plasma magnesium between a l c o h o l - t r e a t e d and p a i r - f e d , nor between a l c o h o l - t r e a t e d and c o n t r o l f e t u s e s . No c o r r e l a t i o n was found between f e t a l body weight and f e t a l plasma magnesium l e v e l s . 4) Glucose Mean plasma glucose l e v e l s were higher i n both c o n t r o l (p<0.01) and p a i r - f e d (p<0.05) fetu s e s compared to a l c o h o l - t r e a t e d f e t u s e s . The Pearson c o r r e l a t i o n c o e f f i c i e n t of 0.6072 (p<0.01) generated f o r f e t a l body weight 49 and f e t a l plasma glucose, i n d i c a t e d a moderate degree of r e l a t i o n s h i p between the two v a r i a b l e s . 5) L a c t i c A c i d A l l f e t u s e s e x h i b i t e d s i m i l a r mean plasma l a c t i c a c i d l e v e l s . A l s o , no c o r r e l a t i o n was found between f e t a l plasma l a c t i c a c i d c o n c e n t r a t i o n and f e t a l body weight. 50 Table 11 F e t a l Plasma F o l a t e , Z i n c , Magnesium, Glucose and L a c t i c A c i d Levels on Day 21 of Gestation (Mean J L S.D.) A l c o h o l Group P a i r - f e d Group C o n t r o l Group F o l i c A c i d 93.25+12.58(4)a 78.00+17.34 (4) 89.50+15.29(4) (ng/ml) Zinc 4.92t0.73(6)b 5.76+1.24(8)b 2.91+0.57(6)c (ug/ml) lesium 6.30+1.52(6)b,c 5.61+-1.56(8)b 7.18+0.45(6)c (mg/dl) Glucose 21.34+9.42(14)b 34.09+10.57(7)c 38.93+11.63(8) c (mg/dl) L a c t i c A c i d 194.17+3.82(4) 178.33+7.64(3) 190.00+4.33(3) (mg/dl) a Numbers i n brackets represent numbers of l i t t e r s . b,c Figures i n the same row not sharing the same s u b s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at l e a s t at the p<0.05 l e v e l . 51 F e t a l Plasma Amino Acids 1 ••; Tit'a^;pia^a11''eVeTs'<o£Jamiji6'acids and; .urea at -.-day -•21-.;-p'iP--ge's-t-a%^ »a are shown i n Table 12. In a d d i t i o n , f e t a l plasma amino a c i d l e v e l s are i l l u s t r a t e d i n Figure 4. In c o n t r o l fetuses mean plasma amino a c i d l e v e l s were lowest f o r c y s t i n e (30.17 u m o l e s / l i t e r ) and highest f o r l y s i n e (1476.00 u m o l e s / l i t e r ) . G e n e r a l l y , mean amino a c i d l e v e l s i n a l c o h o l -t r e a t e d and p a i r - f e d f e t u s e s were s i m i l a r to those i n c o n t r o l f e t u s e s . However, w i t h i n group v a r i a t i o n i n amino a c i d l e v e l was wide. Thus, only s i x of the tv/enty-four amino acids examined showed 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 among the three treatment groups. These s i x amino acids were a s p a r t i c a c i d , threonine, s e r i n e , t y r o s i n e , phenylalanine and al a n i n e . Plasma a s p a r t i c a c i d l e v e l s were a l i k e i n c o n t r o l and p a i r - f e d f e t u s e s , and higher than those of a l c o h o l - t r e a t e d fetuses (p<0.05). The d i f f e r e n c e s i n mean plasma t y r o s i n e and threonine concentration were only s i g n i f i c a n t between p a i r — f e d and c o n t r o l f e t u s e s . Mean plasma threonine l e v e l s were higher i n c o n t r o l compared to p a i r - f e d fetuses (p<0.01). Mean plasma t y r o s i n e l e v e l s were lower i n c o n t r o l compared to p a i r - f e d f e t u s e s (p<0.05). F e t a l plasma l e v e l s of a l a n i n e , phenylalanine and s e r i n e were s i m i l a r i n a l c o h o l - t r e a t e d and c o n t r o l animals, but s i g n i f i c a n t l y lower than those found i n p a i r - f e d animals. Levels of s i g n i f i c a n c e f o r alanine and phenyl-al a n i n e were p<0.05 f o r ..alcohol-treated compared to p a i r - f e d f e t u s e s , and p<0.01 f o r c o n t r o l compared to p a i r - f e d f e t u s e s . Plasma s e r i n e comparisons were s i g n i f i c a n t l y d i f f e r e n t at the p<0.05 l e v e l . Mean plasma h i s t i d i n e l e v e l s v a r i e d w/idely.^among the three f e t a l groups. However, the standard d e v i a t i o n values f o r p a i r - f e d and c o n t r o l groups were very l a r g e , thus s i g n i f i c a n t d i f f e r e n c e s were not found. 52 Urea l e v e l s were highest i n c o n t r o l animals and lowest i n p a i r - f e d animals, w i t h a l c o h o l - t r e a t e d animals e x h i b i t i n g an intermediate value (p<0.01). S i g n i f i c a n t c o r r e l a t i o n s were found between f e t a l body weight and two of the 24 amino acids examined. A c o r r e l a t i o n of -0.8279 (p<0.05) occurred between f e t a l body weight and f e t a l plasma l e v e l s of alpha-amino-n-butyric a c i d . A c o r r e l a t i o n of -0.5695 (p<0.05) occurred between f e t a l body weight and f e t a l plasma l y s i n e l e v e l s . 53 Table 12 F e t a l Plasma Amino A c i d and Urea Levels on Day 21 of Gestation (Mean ±- S.D.) No. of l i t t e r s Amino Acids (umol/1) Alanine ANBA Ar g i n i n e Asparagine A s p a r t i c A c i d Cystine Glutamic A c i d Glutamine Gl y c i n e H i s t i d i n e Hydroxyproline I s o l e u c i n e Leucine Lysine Methionine O r n i t h i n e Phenylalanine P r o l i n e Serine Taurine Threonine A l c o h o l Group 6 1304.80+141.30a 82.17+21.46 236.17+27.10 190.50+40.36 60.33+6.62a 26.50+10.64 475.00i51.97 1491.50+131.53 265.00+34.53 41.67+25.45 80.50+21.93 232.67+36.34 413.67+66.97 1677.00+203.48 147.00+25.88 126.83+15.05 338.17±22.10a 340.33+126.45 431.17+38.71a 683.33±105.15 514.00+63.00a,b P a i r - f e d Group 7 1594.30+179.25b 25.00+0.00 240.86+24.03 174.43+31.35 80.86+17.41b 32.86t7.29 558.00+120.15 1352.40+182.09 317.86+58.16 229.14+409.46 78.57+17.09 211.14+16.44 398.29+24.50 1545.30+303.21 171.00+27.42 109.43+26.91 390.43+28.42b 395.86t43.42 528.29+64.04b 862.711238.08 447.86+52.30a Con t r o l Group 6 1193.80*108.97a 232.67+55.61 262.67+249.55 79.33*7.74b 30.17+9.37 552.33+82.83 261.00+49.06 318.33t440.93 67.83+26.82 225.33+14.11 403.17+29.41 1476.00+229.33 156.83+20.34 134.50+26.37 307.50+29.97a 445.834:37. 34 440.33+45.04a 876.33*161.24 563. 50+.54.72b 54 Table 12 continued A l c o h o l Group P a i r - f e d Group Co n t r o l Group Tryptophan 87.60139.30 100.71i85.75 Tyrosine 244.17±27.97a,b 281.71+49.41a V a l i n e 514.33+70.85 491.29+72.69 125.67+10.07 208.17+30.31b 501.83+59.74 Urea 5125.30+281.28a 2905.60+1104.00b 7032.00i908.46c a,b,c Figures i n the same row not sharing the same s u b s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at l e a s t at the p<0.05 l e v e l . 55 Figure 4 F e t a l Plasma Amino A c i d Levels on Day 21 of Gestation ( u m o l e s / l i t e r ) Alanine A l e 1305 #################################(a) P F 1 5 9 4 mmmmu#u##u##m##uu######### Cont 1194 ############################### ANBA Al e 82 ## PF 25 # Cont A r g i n i n e Ale 236 ###### PF 241 ###### Cont 233 ###### Asparagine A l e 191 ##### PF 174 #### Cont 263 ####### A s p a r t i c A c i d Ale 60 ## PF 81 ## Cont 79 ## Cystine Ale 25 # PF 33 # Cont 30 # Glutamic A c i d A l e 475 ############ PF 558 ############## Cont 552 ############## Glutamine Ale 1492 ###################################### PF 1352 ################################### Cont Glycin e A l e 265 PF 318 Cont 261 ####### ######## ####### H i s t i d i n e A l e 42 PF 230 Cont 318 # ###### ######## 56 Figure 4 continued Hydroxyproline Ale 81 ## PF 7 9 # # Cont 68 ## Is o l e u c i n e A l e 233 ###### PF 211 ##### Cont 225 ###### Leucine A l e 414 PF 398 Cont 403 ########### ########## ########## Lysine A l e 1677 ##########################################^  PF .1545 ######################################## Cont 1476 ###################################### Methionine A l e 147 #### PF 171 #### Cont 157 #### Ornit h i n e A I C 1 2 7 ###:;< PF 109 ### Cont 135 ### Phenylalanine A l e 338 ######### PF 390 ########## Cont 308 ######## P r o l i n e A l e 340 ######### PF 396 ########## Cont 446 ########### Serine A l e 431 ########### PF 528 ############## Cont 440 ########### Taurine A l e 683 ################## PF 863 ###################### Cont 564 ############## 57 Figure 4 continued Threonine A l e 514 ############# PF 448 ########### Cont 564 ############## Tryptophan A l e 88 ## PF 101 ### Cont 127 ### Tyrosine A l e 244 ###### PF 282 ####### Cont 208 ##### Va l i n e A l e 514 ############# PF 491 ########### Cont 502 (a) # corresponds to 39 umoles of amino a c i d per l i t e r of plasma 58 Comparison of Maternal and F e t a l Plasma N u t r i e n t Levels 1) F o l i c A c i d Mean f o l i c a c i d l e v e l s found i n f e t a l r a t s at day 21 of g e s t a t i o n were consider a b l y g r e a t e r than l e v e l s found i n the r e s p e c t i v e groups of dams. F o l i c a c i d l e v e l s i n a l c o h o l - t r e a t e d and i n c o n t r o l f e t u s e s were approx-imately 7 times the l e v e l s found i n t h e i r dams. P a i r - f e d f e t u s e s had mean plasma f o l a t e l e v e l s 11 times g r e a t e r than vthe l e v e l s i n t h e i r dams.---2) Zinc Mean plasma z i n c l e v e l s were higher i n fetuses than i n dams at day 21 of g e s t a t i o n . Mean z i n c l e v e l s were twice as high i n c o n t r o l fetuses compared to c o n t r o l dams. Mean plasma z i n c l e v e l s were 5 times higher i n p a i r - f e d and a l c o h o l - t r e a t e d f e t u s e s than i n t h e i r corresponding dams. 3) Magnesium At day 21 of g e s t a t i o n , mean plasma magnesium l e v e l s were approx-imately 2-3 times higher i n fetu s e s than i n the dams. 4) Glucose Mean plasma glucose concentrations i n p a i r - f e d and c o n t r o l fetuses were approximately 33% of t h a t seen i n p a i r - f e d and c o n t r o l dams. • R.lasma glucose i n a l c o h o l - t r e a t e d fetuses was 17% of t h a t found i n maternal plasma. Comparison of Maternal and F e t a l Plasma Amino A c i d Levels The mean, amino a c i d l e v e l s of f e t a l plasma i n a l l three treatment groups were g e n e r a l l y : higher', than those i n the.--corresponding_ dams'. The only exception to t h i s was c y s t i n e , f o r which maternal l e v e l s were s l i g h t l y higher than f e t a l l e v e l s . S i g n i f i c a n t d i f f e r e n c e s found i n f e t a l amino 59 a c i d l e v e l s d i d not n e c e s s a r i l y p a r a l l e l d i f f e r e n c e s i n maternal amino a c i d p a t t e r n s , t h i s was true f o r the amino acids c y s t i n e , h i s t i d i n e , a s p a r t i c a c i d and t y r o s i n e . However, plasma threonine, serine.-and a l a n i n e concentrations showed a p a r a l l e l between maternal and f e t a l groups. Mean maternal and f e t a l plasma threonine l e v e l s were lowest i n the p a i r - f e d group, but d i f f e r e d s i g n i f i c a n t l y only from the c o n t r o l s Mean, maternal and fetal;;.plasmc.. - s e r i n e and a l a n i n e l e v e l s were s i g n i f i c a n t l y higher i n p a i r - f e d animals when compared to e i t h e r the a l c o h o l - t r e a t e d or c o n t r o l groups; Maternal plasma l e v e l s of p r o l i n e were s i g n i f i c a n t l y lower i n a l c o h o l -t r e a t e d compared to p a i r - f e d or c o n t r o l dams.- F e t a l plasma p r o l i n e l e v e l s showed the same trend as m a t e r n a l ^ l e v e l s , but the d i f f e r e n c e s i n f e t a l p r o l i n e l e v e l s among the treatment groups were not s i g n i f i c a n t . Plasma phenylalanine l e v e l s i n p a i r - f e d fetuses were s i g n i f i c a n t l y higher when compared to both a l c o h o l - t r e a t e d and c o n t r o l f e t u s e s . In maternal plasma phenylalanine was a l s o highest i n p a i r - f e d animals, but the d i f f e r e n c e s i n maternal l e v e l s were not s i g n i f i c a n t . L a s t l y , mean plasma alpha-amino-n-butyric a c i d l e v e l s were s i g n i f i -c a n t l y higher i n a l c o h o l - t r e a t e d dams. Levels of ANBA were al s o higher i n a l c o h o l - t r e a t e d f e t u s e s , but due to the small number of f e t a l ANBA samples, a s i g n i f i c a n t l e v e l of d i f f e r e n c e was not found. 60 DISCUSSION The f i n d i n g s of t h i s study are c o n s i s t e n t w i t h past work and i n d i c a t e t h a t 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 . No gross f e t a l malformations were observed, but as i n numerous e a r l i e r r e p o r t s (15,17-19,21-23,25), fetuses of a l c o h o l - t r e a t e d dams weighed l e s s than fetuses of both p a i r - f e d and c o n t r o l dams. This weight r e d u c t i o n occurred despite the p a i r - f e e d i n g design employed, i n d i c a t i n g t h a t the reduced weight of a l c o h o l - t r e a t e d f e t u s e s i s not due s o l e l y to reduced maternal food i n t a k e . The N a t i o n a l Academy of Sciences (72) has e s t a b l i s h e d n u t r i e n t requirements f o r growth and g e s t a t i o n of l a b o r a t o r y r a t s . These r e q u i r e -ments are expressed i n terms of n u t r i e n t concentration per kilogram of d i e t . During g e s t a t i o n , the requirements assume an average d a i l y food intak e of 20 grams and an average body weight of 300 grams i n Sprague-Dawley r a t s . In the present study, a l c o h o l - t r e a t e d and p a i r - f e d animals consumed an average of 11 grams of P u r i n a Rat Chow/day throughout g e s t a t i o n . The average weight of a l c o h o l - t r e a t e d and p a i r - f e d dams during g e s t a t i o n was 270 and 297 grams r e s p e c t i v e l y . Comparison of recommended n u t r i e n t intakes by the N a t i o n a l Academy of Sciences (NAS) w i t h the a c t u a l i n t a k e of n u t r i e n t s by animals i n the present study (determined by using P u r i n a Rat Chow composition t a b l e s ) revealed the f o l l o w i n g : 1. A l c o h o l - t r e a t e d and p a i r - f e d dams re c e i v e d an adequate amount of d a i l y d i e t a r y f o l i c a c i d throughout pregnancy. Furthermore, synthesis of f o l i c a c i d by m i c r o f l o r a can occur i n the r a t i n t e s t i n e . Thus, unless ethanol decreased i n t e s t i n a l absorption of f o l i c a c i d , a l l dams i n the 61 present study would be expected to have rece i v e d an adequate amount of f o l i c a c i d . 2. Throughout g e s t a t i o n a l c o h o l - t r e a t e d and p a i r - f e d dams consumed an average of 0.33 mg. zinc/day and 23.0 mg. magnesium/day. These amounts exceed the e s t a b l i s h e d requirements of 0.24 mg. zinc/day and 8.0 mg. magnesium/day set by the NAS (72). 3. The N a t i o n a l Academy of Sciences (72) has e s t a b l i s h e d a d a i l y requirement f o r 13 amino aci d s t o support optimum growth and g e s t a t i o n of r a t s . These amino acids are a r g i n i n e , asparagine, glutamic a c i d , h i s t i d i n e , i s o l e u c i n e , l y s i n e , l e u c i n e , methionine, p h e n y l a l a n i n e / t y r o s i n e , p r o l i n e , threonine, tryptophan and v a l i n e . The a d d i t i o n of a r g i n i n e , asparagine, glutamic a c i d and p r o l i n e to diets, produces growth responses presumed to be due to the i n a b i l i t y of the r a t to synthesize s u f f i c i e n t q u a n t i t i e s o f these amino a c i d s during r a p i d growth (72). Comparison of the d a i l y requirements e s t a b l i s h e d by the NAS (72) f o r these 13 amino a c i d s , to l e v e l s consumed by r a t s i n the present study, revealed that the average inta k e of a r g i n i n e , h i s t i d i n e , i s o l e u c i n e , l e u c i n e , l y s i n e , threonine, tryptophan and v a l i n e by a l l three groups of r a t s exceeded requirements. However, the d a i l y i n t a k e of methionine/cystine (85.8 mg.) and phenylalanine (111.7 mg.) by both alcohol-=-treate,d and p a i r - f e d dams was below the requirement e s t a b l i s h e d by the NAS (120 mg. and 160 mg. r e s p e c t i v e l y ) . When intake of methionine/cystine and phenylalanine were expressed r e l a t i v e to body weight, the amounts consumed by both a l c o h o l - t r e a t e d ' and p a i r - f e d dams were s t i l l below the requirements. 4. The N a t i o n a l Academy of Sciences (72) estimated t h a t during g e s t a t i o n r a t s r e q u i r e 28 kcal./lOO grams body weight ( k c a l . are expressed as gross energy a v a i l a b l e i n the d i e t ) . In the present study, a l c o h o l -62 t r e a t e d animals consumed an average of 27.3 kcal./lOO grams body weight, and p a i r - f e d animals consumed an average o f 24.8 kcal./lOO grams body weight throughout g e s t a t i o n . Based on the NAS recommendations, the d a i l y i n t a k e of f o l i c a c i d , z i n c , magnesium and most amino acids" by alcohol-='treated and p a i r - f e d dams was adequate t o support g e s t a t i o n . However, the lower food and c a l o r i e i n t a k e of a l c o h o l - t r e a t e d and p a i r - f e d dams r e s u l t e d i n s i g n i -f i c a n t l y l e s s weight g a i n by these two groups compared to c o n t r o l dams. P a i r - f e e d i n g r e s u l t e d i n very s i m i l a r prepregnancy r a t e s of weight g a i n by a l c o h o l - t r e a t e d and p a i r - f e d animals. However, throughout pregnancy a l c o h o l - t r e a t e d dams gained s i g n i f i c a n t l y l e s s weight than p a i r - f e d dams (p<0.005). Several other i n v e s t i g a t o r s have reported s i m i l a r f i n d i n g s (16,21). This may i n d i c a t e t h a t ethanol at a l e v e l of 30% (v/v) i n water reduces maternal n u t r i e n t u t i l i z a t i o n to a grea t e r degree than 10 or 20% ethanol s o l u t i o n s . Maternal dehydration, produced by a l c o h o l consumption, may a l s o be increased by the higher concentration of a l c o h o l . A l t e r n a t i v e l y , c a l o r i e s derived from a l c o h o l may be l e s s a v a i l a b l e than c a l o r i e s d e r i v e d from cornstarch. R e s u l t s of t h i s experiment i n d i c a t e that weight g a i n p r i o r to pregnancy i s not c o r r e l a t e d w i t h f e t a l weight, w h i l e maternal weight g a i n during pregnancy i s p o s i t i v e l y c o r r e l a t e d w i t h f e t a l body weight (r=0.6473, p^O.001). In humans, Metcoff (73) has estimated t h a t t o t a l maternal weight g a i n during pregnancy c o n t r i b u t e s 6% to the variance i n b i r t h weight. In t h i s study no r e d u c t i o n i n l i t t e r s i z e of a l c o h o l - t r e a t e d dams occurred. This f i n d i n g corroborates those of s e v e r a l other workers (17, 18,21-23,34). However, Singh and Snyder (74) d i d r e p o r t a r e d u c t i o n i n 63 l i t t e r s i z e of r a t dams f e d a l i q u i d d i e t s t a r t i n g on day 1 of pregnancy, w i t h ethanol p r o v i d i n g 36% of t o t a l c a l o r i e s . An exp l a n a t i o n f o r t h i s r e d u c t i o n i n l i t t e r s i z e may be provided by the f i n d i n g s of Rider (24). Rider compared the reproductive performance of r a t dams f e d 11% ethanol ( i n d r i n k i n g water) f o r 13 days p r i o r to and throughout g e s t a t i o n t o the reproductive performance of dams s t a r t e d on an 11% ethanol s o l u t i o n on day 1 of pregnancy. The dams s t a r t e d on ethanol on day 1 of pregnancy had a reduced number of completed pregnancies and sm a l l e r l i t t e r s than the dams f e d ethanol f o r the longer p e r i o d of time. A l s o , Abel and G r e i z e r s t e i n (21) noted t h a t r a t dams intubated d a i l y w i t h 6 grams ethanol/kg. body weight, from day 5 of g e s t a t i o n , showed a higher r a t e of f e t a l r e s o r p t i o n than dams int u b a t e d w i t h 6 grams ethanol/kg. body weight commencing on day 1 of g e s t a t i o n . These and the present r e s u l t s suggest t h a t short term a l c o h o l consumption reduces the reproductive p o t e n t i a l of r a t dams, whereas the dams can adapt somewhat to chronic a l c o h o l consumption. However, f e t a l growth i s compromised by both short term and chronic maternal a l c o h o l consumption. In the present study, no increase i n p l a c e n t a l weight was found i n a l c o h o l - t r e a t e d dams compared to c o n t r o l s . Both Jones et a l . (30) and Weiner e t a l . (27) have reported higher p l a c e n t a l weights i n a l c o h o l -t r e a t e d dams compared to p a i r - f e d and c o n t r o l dams. Jones et a l . (30) suggested t h a t increased p l a c e n t a l weight may represent a compensation f o r reduced blood flow t o the pl a c e n t a . However, i n the present study evidence of hypoglycemia was found i n a l c o h o l - t r e a t e d f e t u s e s , i n d i c a t i n g t h a t compensatory p l a c e n t a l hypertrophy i s not adequate to maintain normal f e t a l blood glucose l e v e l s . No c o r r e l a t i o n was found between p l a c e n t a l weight and f e t a l body weight. 64 Our r e s u l t s show th a t maternal duodenal conjugase a c t i v i t y (necessary f o r the h y d r o l y s i s of f o l a t e polyglutamates p r i o r to a b s o r p t i o n ) , i s not impaired by a l c o h o l consumption. A l c o h o l - t r e a t e d dams would t h e r e -f o r have r e c e i v e d an adequate supply of f o l i c a c i d both from d i e t a r y sources and from i n t e s t i n a l m i c r o f l o r a . However, from the present r e s u l t s i t cannot be concluded t h a t the d i e t a r y treatments had no e f f e c t upon maternal plasma f o l a t e . Although s i g n i f i c a n t d i f f e r e n c e s were not found i n maternal plasma f o l a t e l e v e l s , the means f o r a l c o h o l - t r e a t e d and p a i r - f e d groups were one h a l f or l e s s of the mean of the c o n t r o l group. In a d d i t i o n , the samples s i z e s were s m a l l , and the standard d e v i a t i o n of the a l c o h o l - t r e a t e d group was-large. Determination of f o l a t e l e v e l s i n a l a r g e r sample may r e v e a l d i f f e r e n c e s among the three treatment groups. L i n (75) reported t h a t plasma f o l a t e l e v e l s were s i g n i f i c a n t l y lower i n a l c o h o l - t r e a t e d and p a i r - f e d dams compared t o c o n t r o l dams at day 21 of g e s t a t i o n . However, i n agreement w i t h the present r e s u l t s , she found ho d i f f e r e n c e s i n plasma f o l a t e l e v e l s between a l c o h o l - t r e a t e d and p a i r - f e d dams. Also s i m i l a r t o our r e s u l t s , L i n (75) found no s i g n i f i c a n t d i f f e r e n c e s i n plasma f o l a t e among a l c o h o l - t r e a t e d , p a i r - f e d and c o n t r o l fetuses at day 21 of g e s t a t i o n . These and the present r e s u l t s i n d i c a t e t h a t ethanol does not seem to i n h i b i t p l a c e n t a l t r a n s p o r t of f o l a t e . However, we cannot conclude from the present study t h a t ethanol has no e f f e c t upon maternal or f e t a l f o l a t e s t a t u s . Ethanol may a f f e c t e r y t h r o c y t e , bone marrow or l i v e r f o l a t e l e v e l s . Frank and Baker (46) reported t h a t a l c o h o l consumption by male r a t s produced a decrease i n blood t o t a l f o l a t e l e v e l s when compared t o p a i r - f e d animals. Switzer 65 et a l . (76) reported t h a t ethanol s i g n i f i c a n t l y decreased mean erythrocyte f o l a t e l e v e l s i n dogs, but had no e f f e c t upon mean plasma f o l a t e l e v e l s , when compared to p a i r - f e d animals. Carney and S h e f f i e l d (77) reported t h a t mean serum f o l a t e l e v e l s i n a l c o h o l i c s d i d not d i f f e r from those i n no n a l c o h o l i c s u b j e c t s . However, there i s a l s o some evidence t h a t ethanol d i r e c t l y reduces the formation of normoblastic bone marrow, and decreases h e p a t i c r e t e n t i o n of f o l i c a c i d (78). Thus, the e f f e c t s of ethanol on f o l a t e s t a t u s may vary depending upon whether blood, plasma, bone marrow or l i v e r f o l a t e i s measured: No s i g n i f i c a n t d i f f e r e n c e s were found i n plasma z i n c l e v e l s among the --three groups of dams. This i s i n cont r a s t t o s e v e r a l r e p o r t s of abnormally reduced serum z i n c c oncentration (41) or increased r e n a l clearance of z i n c (42) found i n human a l c o h o l i c s . Our r e s u l t s are a l s o at variance w i t h those of Wang and P i e r s o n (44), who reported decreased plasma z i n c l e v e l s i n male r a t s g iven ethanol f o r 9 weeks, compared to c o n t r o l s . However, s i m i l a r to the present study, Suh and F i r e k (79) found n'6 s i g n i f i c a n t d i f f e r e n c e s i n plasma z i n c l e v e l s between pregnant r a t s given a 24% ethanol s o l u t i o n and a p a i r - f e d group. Discrepancies among st u d i e s may be due to d i f f e r e n c e s i n the l e v e l s of d i e t a r y z i n c consumed. In the present study, a l c o h o l - t r e a t e d dams consumed d a i l y amounts of z i n c i n excess of requirements f o r pregnant animals. D a i l y consumption of z i n c was not reported i n other s t u d i e s . In the present study, plasma z i n c l e v e l s d i d not d i f f e r s i g n i f i c a n t l y between a l c o h o l - t r e a t e d and p a i r - f e d f e t u s e s . Hurley and Mutch (80) reported t h a t mean plasma z i n c l e v e l s i n normal r a t fetuses at day 21 of ges t a i o n was approximately 440 ug./lOO ml. This value i s comparable to the mean l e v e l s of plasma z i n c found i n the present study i n a l c o h o l -66 t r e a t e d and p a i r — f e d f e t u s e s (492 and 576 ug./100 ml r e s p e c t i v e l y ) . These r e s u l t s i n d i c a t e that maternal a l c o h o l consumption d i d not reduce the a v a i l a b i l i t y of z i n c to the a l c o h o l - t r e a t e d f e t u s e s . However, Hurley and Mutch (80) fed r a t dams a z i n c d e f i c i e n t d i e t only from days 6 t o 14 of g e s t a t i o n and compared pregnancy outcome of these animals to t h a t of c o n t r o l s f e d a d i e t c o n t a i n i n g adequate z i n c . They found t h a t z i n c d e f i c i e n c y produced a high r a t e of s t i l l b i r t h s and neonatal a b n o r m a l i t i e s , and a r e d u c t i o n i n the weight of day 16 and day 21 f e t u s e s . Despite these f i n d i n g s , 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 concentration of plasma or t i s s u e z i n c occurred at any age between c o n t r o l and z i n c - d e f i c i e n t f e t u s e s . Thus i t seems t h a t f e t a l plasma z i n c concentration i s not a s e n s i t i v e index of z i n c s t a t u s . An e f f e c t of ethanol upon z i n c s t a t u s may only be evident by studying some other body z i n c p o o l , such as zinc-dependent metalloenzymes. The present study i n d i c a t e s t h a t ethanol does not reduce maternal or f e t a l plasma magnesium l e v e l s . Magnesium l e v e l s were found to be s i g n i f i c a n t l y higher i n a l c o h o l - t r e a t e d dams when compared to both p a i r - f e d and c o n t r o l dams. The high plasma magnesium l e v e l s i n the a l c o h o l - t r e a t e d group were unexpected, because a l c o h o l consumption has been reported to cause increased u r i n a r y l o s s of magnesium, and to be a s s o c i a t e d w i t h magnesium d e f i c i e n c y (43). However, Suh and F i r e k (79) reported t h a t when female r a t s were given a 24% ethanol s o l u t i o n f o r 2-8 weeks p r i o r to g e s t a t i o n and compared to p a i r - f e d r a t s , no d i f f e r e n c e s were found i n plasma magnesium l e v e l s between the two groups. The r e s u l t s of the present study are i n agreement w i t h those of Abel and G r e i z e r s t e i n (21). They reported t h a t i n t u b a t i o n of pregnant r a t s w i t h 6 grams ethanol (30% w/v)/kg. body weight, from 67 days 5 to 19 of g e s t a t i o n , produced s i g n i f i c a n t l y higher mean plasma magnesium l e v e l s when compared t o p a i r - f e d dams (approximately 2.3 mg./lOO ml. and 1.97 mg./lOO ml. r e s p e c t i v e l y ) , at day 20 of g e s t a t i o n . - 'These, and the present f i n d i n g s may be due to increased absorption of magnesium or t o increased m o b i l i z a t i o n of magnesium from maternal bone and muscle. In humans, a l c o h o l i n g e s t i o n i s known to cause degeneration of muscle t i s s u e (65,66). Maternal a l c o h o l consumption had no e f f e c t upon f e t a l plasma magnesium concentration. Despite an e l e v a t i o n i n the plasma magnesium l e v e l s of a l c o h o l - t r e a t e d dams, increased plasma magnesium l e v e l s were not found i n a l c o h o l - t r e a t e d f e t u s e s . Abel and G r e i z e r s t e i n (21) a l s o reported no d i f f e r e n c e s i n magnesium concentration of amniotic f l u i d , p lacentae, or whole fetuses between a l c o h o l - t r e a t e d and p a i r - f e d animals, desp i t e s i g n i f i c a n t l y higher plasma magnesium l e v e l s i n a l c o h o l - t r e a t e d dams compared to p a i r - f e d dams. These r e s u l t s may be an i n d i c a t i o n of a s a t u r a t i o n mechanism of p l a c e n t a l t r a n s p o r t of magnesium. A l t e r n a t i v e l y , some of the magnesium i n the plasma of a l c o h o l - t r e a t e d dams may be present i n a form which i s u n a v a i l a b l e f o r p l a c e n t a l t r a n s p o r t ( i e . complexed w i t h p r o t e i n ) . I t was a n t i c i p a t e d t h a t a l c o h o l consumption might cause hypoglycemia due to i n h i b i t i o n of hepatic gluconeogenesis (47,48). A l c o h o l - t r e a t e d dams consumed fewer c a l o r i e s (kcal./gram body weight) than d i d c o n t r o l dams. A low i n t a k e of c a l o r i e s would be expected- to cause m o b i l i z a t i o n of maternal body f a t and r e l e a s e of muscle alanine t o provide substrates f o r gluconeogenesis. However, Scholz et a l . (81) reported t h a t p e r f u s i o n of r a t l i v e r s w i t h a l a n i n e and o l e a t e ( a c o n d i t i o n designed to mimic hypoglycemia) caused s t i m u l a t i o n of gluconeogenesis, while a d d i t i o n of 68 ethanol to the perfusate diminished the s t i m u l a t i o n o f gluconeogenesis. In the present study, 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 maternal plasma glucose among the three treatment groups. Thus, i n h i b i t i o n of gluconeogenesis does not seem t o be a f a c t o r i n pregnant dams consuming a l c o h o l . In agreement w i t h our r e s u l t s , Snyder and Singh (49) found no s i g n i f i c a n t d i f f e r e n c e s i n maternal blood glucose l e v e l s among a l c o h o l -t r e a t e d , p a i r - f e d and c o n t r o l dams, when blood samples were taken at term. A l s o , i n a study reported by Wallgren et a l . (82), no d i f f e r e n c e s i n plasma glucose l e v e l s were detected between a group of male r a t s g i ven a 3-9% ethanol s o l u t i o n and a p a i r - f e d group. I n c o n t r a s t to glucose l e v e l s i n maternal plasma, the mean f e t a l plasma glucose l e v e l s i n the present study were s i g n i f i c a n t l y lower i n a l c o h o l - t r e a t e d compared t o both p a i r - f e d and c o n t r o l groups. Snyder and Singh (49) a l s o found s i g n i f i c a n t l y reduced blood glucose l e v e l s i n the newborn pups of t h e i r a l c o h o l - t r e a t e d dams when compared to e i t h e r p a i r -f e d or c o n t r o l pups. The reason t h a t a l c o h o l - t r e a t e d fetuses should'display,"evidence df hypoglycemia while t h e i r dams d i d not, i s not r e a d i l y apparent. Glucose i s t r ansported across the p l a c e n t a v i a f a c i l i t a t e d d i f f u s i o n (83). Jones et a l . (28) found t h a t a l c o h o l consumption by r a t dams d i d not d i m i n i s h f e t a l uptake of a non-metabolizable glucose analog (methyl-(alpha-D(U- 1 4C)gluco)pyranoside). Thus r e d u c t i o n of glucose t r a n s p o r t across the p l a c e n t a by ethanol seems u n l i k e l y . In f e t a l r a t s a l c o h o l dehydrogenase a c t i v i t y has not been detected u n t i l approximately day 18 of g e s t a t i o n (84). F o l l o w i n g day 18 i t i s not known what e f f e c t ethanol o x i d a t i o n may have on f e t a l gluconeogenesis. 69 However, there i s evidence t h a t metabolic response to ethanol v a r i e s w i t h age. H o l l s t e d t et a l . (85) found t h a t ethanol i n j e c t e d i n t r a -p e r i t o n e a l l y (27.1 mM/kg. body weight), produced hypoglycemia i n r a t s weighing 25 grams, but not i n 50, 100, 200 or 250 gram r a t s . The e f f e c t of hypoglycemia beginning on p r e n a t a l day 18 upon f e t a l growth i s unknown. However, because f e t a l growth i s very r a p i d during the l a s t few days of g e s t a t i o n , hypoglycemia during t h i s time may r e s u l t i n s i g n i f i c a n t growth r e t a r d a t i o n . Further s t u d i e s examining f e t a l growth and plasma glucose l e v e l s p r i o r to day 18 of g e s t a t i o n may c l a r i f y t h i s f i n d i n g . Are a l c o h o l derived c a l o r i e s a v a i l a b l e to the f e t u s ? In the present study, a l c o h o l - t r e a t e d dams consumed approximately 33% of d a i l y c a l o r i e s as ethanol throughout g e s t a t i o n . In humans, i t has been estimated t h a t approximately 75% of ethanol taken up by the l i v e r i s r e l e a s e d i n t o the c i r c u l a t i o n as acetate (31). Acetate can be u t i l i z e d by maternal t i s s u e s as an energy source. However, i t i s unknown i f acetate crosses the p l a c e n t a or can be u t i l i z e d by f e t a l t i s s u e s . Plasma o s m o l a l i t y was increased s i g n i f i c a n t l y i n a l c o h o l - t r e a t e d dams compared t o both p a i r - f e d and c o n t r o l dams. S i m i l a r f i n d i n g s were reported by Jones et a l . (28). Redetzki et a l . (86) found a d i r e c t r e l a t i o n s h i p between blood a l c o h o l concentration and serum o s m o l a l i t y i n 50 a l c o h o l i c p a t i e n t s . They reported t h a t f a c t o r s which c o n t r i b u t e d to the increase i n o s m o l a l i t y i n c l u d e d increased l e v e l s of serum l a c t i c a c i d , acetate and a l c o h o l congeners. In the present study, no d i f f e r e n c e s i n plasma l a c t a t e were found among the three f e t a l groups. However, because l a c t a t e was measured i n plasma which had not been d e p r o t e i n i z e d immediately, the values obtained 70 may not be r e l i a b l e . The e f f e c t of maternal dehydration upon f e t a l growth i s unknown. However, Jones et a l . (30) reported t h a t alcohol-induced maternal •'< dehydration i n r a t s was a s s o c i a t e d w i t h reduced blood flow t o placentae. This r e d u c t i o n i n blood flow may reduce the supply o f n u t r i e n t s a v a i l a b l e t o fetuses of a l c o h o l - t r e a t e d dams. Only seven of the maternal plasma amino acid s and s i x of the f e t a l plasma amino acid s showed s i g n i f i c a n t d i f f e r e n c e s among the three treatment groups. I n the dams, reduced plasma threonine, c y s t i n e and h i s t i d i n e l e v e l s were apparently a r e s u l t of reduced food i n t a k e , as the l e v e l s of these amino acid s were s i m i l a r i n a l c o h o l - t r e a t e d and p a i r - f e d dams, but s i g n i f i c a n t l y lower than those of c o n t r o l dams. Reduction i n these plasma amino acids i n response to p r o t e i n - c a l o r i e m a l n u t r i t i o n has been found i n other s t u d i e s . Worthington et a l . (87) found a 68% r e d u c t i o n i n serum c y s t i n e , and a 32% r e d u c t i o n i n serum threonine i n a d u l t monkeys a f t e r 4 weeks of severe p r o t e i n - c a l o r i e m a l n u t r i t i o n . In a d d i t i o n , p r o t e i n - c a l o r i e m a l n u t r i t i o n r e s u l t s i n an increase i n muscle r e l e a s e of alanine (88). I n the present study, p a i r - f e d dams had s i g n i f i c a n t l y g r e a t e r plasma alanine and s e r i n e l e v e l s than both a l c o h o l - t r e a t e d and c o n t r o l dams. An increase i n serum s e r i n e (6%) and a l a n i n e (44%) were a l s o found i n the study of Worthington et a l . (87) i n response to p r o t e i n - c a l o r i e m a l n u t r i t i o n . . A decrease i n the r a t i o of plasma e s s e n t i a l to n o n e s s e n t i a l amino acids has a l s o been found i n p r o t e i n - c a l o r i e m a l n u t r i t i o n . Comparison of t h i s r a t i o i n the maternal plasma of the three treatment groups, showed t h a t indeed i t was l e s s i n p a i r - f e d (0.75) compared to a l c o h o l -71 t r e a t e d (0.92) and c o n t r o l (1.38) animals. Thus, i t i s p o s s i b l e t h a t p a i r - f e d dams may have been s u f f e r i n g from m i l d p r o t e i n - c a l o r i e mal-n u t r i t i o n . However, t h i s had no s i g n i f i c a n t e f f e c t on the body weight of p a i r - f e d f e t u s e s when compared to c o n t r o l f e t u s e s . S i g n i f i c a n t l y elevated l e v e l s of a l a n i n e , phenylalanine, t y r o s i n e and s e r i n e were found i n p a i r - f e d fetuses compared to c o n t r o l s . The e l e v a t i o n of f e t a l plasma s e r i n e and alanine are a r e f l e c t i o n of s i m i l a r e l e v a t i o n s of these amino acid s i n the plasma of p a i r - f e d dams. A l c o h o l consumption d i d produce changes i n a few maternal and f e t a l plasma amino acids i n a l c o h o l - t r e a t e d animals when compared to p a i r - f e d animals. These amino acids i n c l u d e d maternal alpha-amino-n-butyric a c i d and p r o l i n e , and f e t a l a s p a r t i c a c i d and alpha-amino-n-butyric a c i d . Plasma l e v e l s of p r o l i n e were s i g n i f i c a n t l y depressed i n a l c o h o l -t r e a t e d dams. Mean p r o l i n e l e v e l s were a l s o depressed i n a l c o h o l - t r e a t e d f e t u s e s , although due to the la r g e standard d e v i a t i o n v a lue, d i f f e r e n c e s i n f e t a l plasma p r o l i n e were not s i g n i f i c a n t . A d e f i c i e n c y of p r o l i n e may a f f e c t f e t a l s k e l e t a l development. P r o l i n e i s a precursor i n the formation of c o l l a g e n , and has been shown to be an e s s e n t i a l amino a c i d f o r r a t s during r a p i d growth (72). In a d d i t i o n , s t u d i e s have reported r e t a r d a t i o n of s k e l e t a l development i n the o f f s p r i n g of a l c o h o l - t r e a t e d dams (15,22,25). L e i c h t e r and Lee (22) measured arm le n g t h , l e g le n g t h , p e l v i c width, pelvic:;lehgthj.-;skull width and s k u l l l e n g t h i n the pups of a l c o h o l - t r e a t e d , p a i r - f e d and c o n t r o l dams at 30, 37, 44 and 51 days postconception. FOE every parameter, at every measurement p e r i o d , s k e l e t a l maturity of the a l c o h o l - t r e a t e d pups was s i g n i f i c a n t l y l e s s than t h a t of p a i r - f e d and c o n t r o l pups. Mean plasma a s p a r t i c a c i d l e v e l s were s i g n i f i c a n t l y depressed i n 72 a l c o h o l - t r e a t e d fetuses but not alcohol-^treated dams. This may i n d i c a t e t h a t a l c o h o l reduces p l a c e n t a l t r a n s p o r t o f some amino a c i d s . I n a study of normal p l a c e n t a l f u n c t i o n , Eaton and Y u d i l e v i c h (90) perfused i s o l a t e d guinea p i g placentae w i t h t r i t i a t e d amino acid s and measured p l a c e n t a l uptake. They found t h a t uptake of a s p a r t i c a c i d was minimal (approximately 5% compared t o 55% f o r i s o l e u c i n e ) . Other amino acids which showed small percentage uptake by the placentae were glutamic a c i d (approximately 1%) and t a u r i n e (approximately 13%). In the present study, plasma glutamic a c i d and t a u r i n e l e v e l s were lower i n a l c o h o l -t r e a t e d f e t u s e s compared to p a i r - f e d and c o n t r o l f e t u s e s , although the d i f f e r e n c e s were not s t a t i s t i c a l l y s i g n i f i c a n t . Reduction of the supply of a c i d i c amino acid s t o a l c o h o l - t r e a t e d f e t u s e s may be a r e s u l t of reduced maternal blood flow to placentae. Reduced supply of a c i d i c amino acids may have relevance t o f e t a l develop-ment. Studies have suggested t h a t glutamic a c i d i s an e s s e n t i a l amino a c i d f o r r a p i d l y growing r a t s (72). A s p a r t i c a c i d i s an e s s e n t i a l precursor i n pyrimidine and purine s y n t h e s i s . H o l t e t a l . (89) reported t h a t kwashiokor i n young c h i l d r e n was accompanied by a decrease i n plasma alpha-amino-n-butyric a c i d , and an increase i n plasma p r o l i n e . In c o n t r a s t , a l c o h o l - t r e a t e d dams i n our study had s i g n i f i c a n t l y elevated plasma alpha-amino-n-butyric a c i d and depressed plasma p r o l i n e l e v e l s . A c o r r e l a t i o n a l s o e x i s t e d between f e t a l plasma alpha-amino-n-butyric a c i d (ANBA) l e v e l s and f e t a l body weight. These r e s u l t s i n d i c a t e t h a t a l c o h o l has a unique e f f e c t upon plasma amino acids i r r e s p e c t i v e of reduced food i n t a k e . P r e l i m i n a r y f i n d i n g s by Shaw and coworkers (91,92) i n d i c a t e t h a t the r a t i o of the concentration of plasma alpha-amino-n-butyric a c i d to 73 l e u c i n e (A/L) may be used as a t e s t f o r alco h o l i s m . In humans, they found t h a t the r a t i o of these two amino acids was elevated f o r s e v e r a l weeks a f t e r c e s s a t i o n of chronic d r i n k i n g , but was not a f f e c t e d by acute a l c o h o l i n g e s t i o n . The elevated r a t i o was observed i n both w e l l -nourished and po o r l y nourished a l c o h o l i c s . Baboons fed a l c o h o l along w i t h a p r o t e i n d e f i c i e n t d i e t developed an abnormal A/L . r a t i o when a l c o h o l was g i v e n , but not when a l c o h o l was omitted (91). Furthermore, i t was found t h a t i n p a t i e n t s w i t h acute and chronic h e p a t i t i s , the r a t i o was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s (91). Thus i n the present study, e l e v a t e d plasma l e v e l s of ANBA i n a l c o h o l -t r e a t e d r a t s may r e f l e c t chronic a l c o h o l consumption r a t h e r than the occurrence of l i v e r damage. Evidence t h a t l i v e r damage d i d not occur, in., the dams i s supported by the f a c t t h a t changes i n plasma p r o t e i n and amino acid s g e n e r a l l y a s s o c i a t e d w i t h a l c o h o l i c l i v e r disease d i d not occur. Low l e v e l s of serum albumin have been a s s o c i a t e d w i t h l i v e r c i r r h o s i s (31). In the present study, no s i g n i f i c a n t d i f f e r e n c e s were found i n maternal plasma albumin among the three treatment groups. A l s o , i n p a t i e n t s w i t h chronic a c t i v e l i v e r disease, an abnormal e l e v a t i o n of serum phenylalanine and t y r o s i n e and a decrease i n the r a t i o of serum branched-chain amino acid s to aromatic amino acid s were found (93). These changes were not seen i n the a l c o h o l - t r e a t e d animals of the present study. 74 CONCLUSIONS The present study has shown th a t ethanol consumption by r a t s f o r 5 weeks p r i o r t o and throughout g e s t a t i o n r e s u l t s i n reduced f e t a l weight. This r e d u c t i o n was independent of t o t a l maternal food i n t a k e . F e t a l growth r e t a r d a t i o n was not associated w i t h any abnormal f i n d i n g s i n maternal plasma f o l i c a c i d , z i n c , glucose or albumin; or i n f e t a l plasma f o l i c a c i d , z i n c , or•magnesium. Hov/ever, i n the f u t u r e , o b s ervation of parameters other than plasma n u t r i e n t l e v e l s may show th a t a l c o h o l consumption does a f f e c t the s t a t u s of these n u t r i e n t s i n e i t h e r r a t dams or f e t u s e s . For example, a l c o h o l consumption may reduce i n c o r p o r a t i o n of f o l a t e i n t o e r y t h r o c y t e s , or may reduce hepatic z i n c and f o l a t e r e t e n t i o n . A l c o h o l consumption was found to increase maternal plasma o s m o l a l i t y . The e f f e c t of t h i s upon f e t a l growth i s unknown. Increase i n maternal plasma magnesium was found as a r e s u l t of a l c o h o l consumption. This may be a r e s u l t of muscle t i s s u e m o b i l i z a t i o n . Plasma' l e v e l s of p r o l i n e were decreased i n a l c o h o l - t r e a t e d dams, which may compromise f e t a l c o l l a g e n formation. Levels of a s p a r t i c a c i d were reduced i n a l c o h o l - t r e a t e d f e t u s e s , which may have an e f f e c t on p y r i m i d i n e and purine syn t h e s i s and u l t i m a t e l y the formation of DNA and RNA and c e l l m u l t i p l i c a t i o n . A p o s i t i v e c o r r e l a t i o n was found between maternal plasma p r o l i n e l e v e l s and f e t a l body weight, and a negative c o r r e l a t i o n between maternal plasma alpha-amino-n-butyric a c i d l e v e l s and f e t a l body weight. A negative c o r r e l a t i o n was a l s o observed between f e t a l plasma ANBA and f e t a l body weight. These plasma amino acids may prove to be u s e f u l i n d i c a t o r s of maternal alcoholism during pregnancy and of the r i s k of 75 f e t a l a l c o h o l syndrome o c c u r r i n g i n the developing f e t u s . However, the mechanisms—whereby a l c o h o l a f f e c t s amino a c i d metabolism remain to be e l u c i d a t e d . 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