Land and Food Systems, Faculty of
DSpace
UBCV
Schaefer, Allan Lee
Doctor of Philosophy - PhD
University of British Columbia
To study the effect of maternal nutrition on fetal protein synthesis, isotopic kinetic studies were undertaken in which indwelling catheters were implanted in the inferior vena cava and saphenous vein of ovine fetuses as well as the jugular vein and femoral artery of ewes at 120-130 days of gestation Following a five day post surgical recovery period an 8h continuous infusion of L-[2,3,5,6-³H] or L-[U-¹⁴ C] tyrosine was made into the fetus and ewe simultaneously. An enzymatic procedure specific for L-tyrosine was used to measure the plateau specific activity in fetal and maternal plasma. Using these values the net placental transfer, endogenous production and net utilization of tyrosine by the fetus were determined employing a two pool kinetic model. For determining the fractional protein synthetic rates in individual tissues the ewes were sacrificed and the fetuses obtained by laparotomy immediately following the continuous infusion of labelled tyrosine. The effect of starvation on tyrosine transfer across the placenta and protein synthesis by the fetus was studied by repeating the above procedures in ewes starved for 48h. Values for the net placental transfer, endogenous production and net utilization of tyrosine were 5.14, 1.29 and 6.42 mmol/d/kg respectively for fetuses of fed ewes, whereas, these values were -0.29, 4.53 and 3.88 mmol/d/kg in fetuses of starved ewes. The plasma concentrations (mg%) of glucose, alpha amino nitrogen and lactate in the fed ewes were 60.18, 10.68 and 5.58 versus 43.66, 7.80 and 7.41 in starved ewes. Glucose, alpha amino nitrogen and lactate values were also seen to change in the fetus from 12.55, 13.38 and 12.98, respectively in the fed versus 8.18, 11.55 and 15.08 in fetuses of starved ewes. The fetal tyrosine net utilization values, corrected for oxidative loss of 5.2%, were used to calculate the whole body protein synthetic values. From an average tyrosine content of carcass protein of 15.71 mmol/kg, the whole body protein synthetic rates were 63 g/d/kg in the fed versus 25 g/d/kg in the starved fetuses. Fetal tissue fractional protein synthetic rates were also affected by maternal starvation. The fractional synthetic rates (%/day) for liver, kidney, lung, heart and skeletal muscle were 78%, 45%, 65%, 14% and 26% for fed versus 12%, 22%, 23%, 11% and 10%/d for the fetuses of starved ewes. The data demonstrate that maternal feed deprivation reduces protein synthesis in the fetus.
PROTEIN SYNTHESIS IN THE OVINE FETUS by ALLAN LEE SCHAEFER B.Sc.(Ag.) r The U n i v e r s i t y of A l b e r t a , 1976 M.Sc.(Ag.), The U n i v e r s i t y of A l b e r t a , 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1983 ©Allan Lee Schaefer 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 The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i ABSTRACT To study the e f f e c t of maternal n u t r i t i o n on f e t a l p r o t e i n s y n t h e s i s , i s o t o p i c k i n e t i c s t u d i e s were undertaken i n which i n d w e l l i n g c a t h e t e r s were implanted i n the i n f e r i o r vena cava and saphenous v e i n of ovine f e t u s e s as w e l l as the j u g u l a r v e i n and femoral a r t e r y of ewes at 120-130 days of g e s t a t i o n F o l l o w i n g a f i v e day post s u r g i c a l recovery p e r i o d an 8h 3 14 continuous i n f u s i o n of L-[2,3,5,6"H] or L-[U- C] t y r o s i n e was made i n t o the f e t u s and ewe s i m u l t a n e o u s l y . An enzymatic procedure s p e c i f i c f o r L - t y r o s i n e was used to measure the p l a t e a u s p e c i f i c a c t i v i t y i n f e t a l and maternal plasma. Using these val u e s the net p l a c e n t a l t r a n s f e r , endogenous p r o d u c t i o n and net u t i l i z a t i o n of t y r o s i n e by the f e t u s were determined employing a two pool k i n e t i c model. For determining the f r a c t i o n a l p r o t e i n s y n t h e t i c r a t e s i n i n d i v i d u a l t i s s u e s the ewes were s a c r i f i c e d and the f e t u s e s o b t a i n e d by laparotomy immediately f o l l o w i n g the continuous i n f u s i o n of l a b e l l e d t y r o s i n e . The e f f e c t of s t a r v a t i o n on t y r o s i n e t r a n s f e r a c r o s s the p l a c e n t a and p r o t e i n s y n t h e s i s by the f e t u s was s t u d i e d by r e p e a t i n g the above procedures i n ewes s t a r v e d f o r 48h. Values f o r the net p l a c e n t a l t r a n s f e r , endogenous p r o d u c t i o n and net u t i l i z a t i o n of t y r o s i n e were 5.14, 1.29 and 6.42 mmol/d/kg r e s p e c t i v e l y f o r f e t u s e s of fed ewes, whereas, these v a l u e s were -0.29, 4.53 and 3.88 mmol/d/kg i n f e t u s e s of s t a r v e d ewes. The plasma c o n c e n t r a t i o n s (mg%) of glucose, alpha amino n i t r o g e n and l a c t a t e i n the fed ewes were 60.18, 10.68 and 5.58 versus 43.66, 7.80 and 7.41 i n s t a r v e d ewes. Glucose, alpha amino n i t r o g e n and l a c t a t e v a l u e s were a l s o seen to change i n the f e t u s from 12.55, 13.38 and 12.98, respectively in the fed versus 8.18, 11.55 and 15.08 in fetuses of starved ewes. The f e t a l tyrosine net u t i l i z a t i o n values, corrected for oxidative loss of 5.2%, were used to calculate the whole body protein synthetic values. From an average tyrosine content of carcass protein of 15.71 mmol/kg, the whole body protein synthetic rates were 63 g/d/kg in the fed versus 25 g/d/kg in the starved fetuses. Fetal tissue f r a c t i o n a l protein synthetic rates were also affected by maternal starvation. The f r a c t i o n a l synthetic rates (%/day) for l i v e r , kidney, lung, heart and skeletal muscle were 78%, 45%, 65%, 14% and 26% for fed versus 12%, 22%, 23%, 11% and 10%/d for the fetuses of starved ewes. The data demonstrate that maternal feed deprivation reduces protein synthesis in the fetus. i v ACKNOWLEDGEMENTS I wish t o thank Drs. B.D. Owen and C R . K r i s h n a m u r t i , past Chairman and A c t i n g Head of the Department of Animal Science, f o r use of the departments f a c i l i t i e s . S i n c e r e a p p r e c i a t i o n i s extended to my s u p e r v i s o r Dr. C R . Kris h n a m u r t i f o r h i s support, encouragement, p a t i e n c e and f r i e n d s h i p throughout t h i s study. I w i l l always have great a d m i r a t i o n f o r h i s pers o n a l example as a teacher and s c i e n t i s t . I would l i k e to extend my g r a t i t u d e to my c o l l e a g u e s Dr. R. Gopinath, Dr. David K i t t s and Ms. Ronna Mathieson f o r s h a r i n g t h e i r knowledge of techniques and procedures. I p a r t i c u l a r l y would l i k e to thank Ronna Mathieson f o r her d e d i c a t e d and s k i l l e d a s s i s t a n c e with animal p r e p a r a t i o n s . A s p e c i a l note of a p p r e c i a t i o n i s extended to Sandy Heindze whose a n a l y t i c a l work on n u c l e i c a c i d s made a s i g n i f i c a n t c o n t r i b u t i o n to t h i s study. I would l i k e to acknowledge the a s s i s t a n c e of E l i z a b e t h Bazley, John Ciok, Ed Mirehouse, C l a r a Shekhtman and Paul W i l l i n g f o r t h e i r care and maintenance of the animals and to Ted Ca t h c a r t and G i l e s Galzy f o r t h e i r advice and a s s i s t a n c e on t e c h n i c a l m atters. I would l i k e to thank the members of my committee f o r t h e i r h e l p f u l comments i n the d r a f t i n g of t h i s t h e s i s and f i n a l l y , to my f a m i l y , f o r t h e i r encouragement and support over the l a s t s e v e r a l years which, i n many ways, gave me the s e l f c o n f i d e n c e to c a r r y out t h i s work. DEDICATION I wish to dedicate t h i s thesis to my wife Jane. Without her support, encouragement, patience and personal s a c r i f i c e t h i s work would not have been done. TABLE OF CONTENTS INTRODUCTION 1 CHAPTER I. REVIEW OF THE LITERATURE 2 (A) Techniques For Assessment Of F e t a l Growth And Metabolism .. 2 (1) N u c l e i c A c i d s 2 (2) Nitrogen R e t e n t i o n 3 (3) Isotope D i l u t i o n Procedures 5 (B) F a c t o r s A f f e c t i n g F e t a l Growth 13 (1) F e t a l Endocrine F a c t o r s 14 (2) P l a c e n t a l F a c t o r s 16 (3) Maternal N u t r i t i o n 21 (C) C o n c l u s i o n s From The L i t e r a t u r e Review 25 CHAPTER I I . TYROSINE UTILIZATION IN THE OVINE FETUS IN UTERO 27 Experiment ( I ) : Development Of A n a l y t i c a l Methodology For The Determination Of T y r o s i n e S p e c i f i c A c t i v i t y . 27 Experiment ( I I ) : I r r e v e r s i b l e Loss And Net U t i l i z a t i o n Of T y r o s i n e 40 Experiment ( I I I ) : T y r o s i n e U t i l i z e d For O x i d a t i o n And Sy n t h e s i s 58 CHAPTER I I I . WHOLE BODY AND TISSUE FRACTIONAL PROTEIN SYNTHESIS IN THE OVINE FETUS IN UTERO 63 Experiment ( I V ) : The C a l c u l a t i o n Of T i s s u e F r a c t i o n a l P r o t e i n S y n t h e s i s 63 CHAPTER IV: EFFECT OF MATERNAL STARVATION ON WHOLE BODY AND v i i TISSUE FRACTIONAL PROTEIN SYNTHESIS IN THE OVINE FETUS IN UTERO 87 Experiment (V): E f f e c t s Of Acute Maternal S t a r v a t i o n ... 87 CHAPTER V: GENERAL SUMMARY AND CONCLUSIONS 106 BIBLIOGRAPHY 110 APPENDIX 142 v i i i LIST OF FIGURES Figure 1: Standard Curve Of L-tyrosine 37 Figure 2: Changes In Spe c i f i c A c t i v i t y Of Fetal Plasma Tyrosine 47 Figure 3: Changes In Spe c i f i c A c t i v i t y Of Ewe Plasma Tyrosine 49 Figure 4: Fetal-maternal Two Compartment Model 52 Figure 5: Kinetic Calculations For a Two.Pool Model 54 Figure 6: The Rate of Production and U t i l i z a t i o n of Tyrosine 62 ix LIST OF TABLES Table 1 : Recovery Of C o l d And L a b e l l e d T y r o s i n e 38 Table 2: Co n c e n t r a t i o n Of L - t y r o s i n e In Plasma 39 Table 3: F e t a l And Maternal Body Weight, G e s t a t i o n a l Age And I n f u s i o n Procedures 42 Table 4: Post S u r g i c a l P h y s i o l o g i c a l Parameters In The Fetus And Ewe 45 Table 5: In v i v o K i n e t i c Parameters Of T y r o s i n e Metabolism . 55 Table 6: Mean P h y s i o l o g i c a l Parameters For The Fetus And Ewe 73 Table 7: R a t i o Of P l a t e a u S p e c i f i c A c t i v i t y And Half L i f e Of Mixed P r o t e i n s 74 Table 8: F r a c t i o n a l And Absolute P r o t e i n S y n t h e t i c Rates .. 75 Tabl e 9: T y r o s i n e And N u c l e i c A c i d C o n c e n t r a t i o n In T i s s u e s 76 Table 10: P r o t e i n S y n t h e t i c C a p a c i t y In F e t a l T i s s u e s 77 Table 11: P h y s i o l o g i c a l Parameters For The Fetus And Ewe .. 90 Table 12: K i n e t i c Parameters Of T y r o s i n e Metabolism 94 Table 13: P l a t e a u S p e c i f i c A c t i v i t y Of T y r o s i n e And P r o t e i n H a l f L i f e 95 Table 14: T i s s u e F r a c t i o n a l P r o t e i n S y n t h e t i c Rates In Fetuses Of Fed And Starved Ewes 96 Table 15: T i s s u e C o n c e n t r a t i o n s Of T y r o s i n e And N u c l e i c A c i d s In Fetuses Of Fed And Starved Ewes 97 Table 16: P r o t e i n S y n t h e t i c C a p a c i t y In F e t a l T i s s u e s 98 Table 17: F r a c t i o n a l (%/d) And Absolute (g/d) Rates Of Protein Synthesis In The Fetuses Of Starved Ewes 1 INTRODUCTION The most rapid rate of growth and protein accretion in the fetus i s during the l a t t e r period of gestation. Maintaining normal growth during t h i s period is important in that intrauterine growth retardation w i l l be re f l e c t e d by poor post-natal growth and development in la t e r l i f e . The a b i l i t y of the fetus to synthesize protein during this period can be affected by many factors, among which the delivery of maternal nutrients to the fetus has been suggested to be of c r u c i a l importance. A considerable e f f o r t has therefore been directed toward investigating f e t a l growth and the maternal n u t r i t i o n a l factors that may influence t h i s process. However, the in vivo study of f e t a l metabolism has been hindered by the b i o l o g i c a l complexity of the fetal-maternal system as well as the technical d i f f i c u l t i e s encountered in measuring f e t a l metabolic parameters. Consequently, many studies of f e t a l growth in domestic ruminants have t r a d i t i o n a l l y been limited to carcass analysis procedures. In recent years, improvements in surgical techniques as well as the increased use of radioactively l a b e l l e d metabolites and isotope d i l u t i o n procedures have greatly f a c i l i t a t e d in vivo investigations of f e t a l metabolism. The purpose of the present study was to u t i l i z e radionuclide k i n e t i c techniques for determining tyrosine u t i l i z a t i o n and the rate of protein synthesis in the ovine fetus in utero. In addition, i t was the intention to use t h i s procedure to investigate the e f f e c t of acute maternal starvation on the a b i l i t y of the fetus to synthesize protein. 2 CHAPTER REVIEW OF THE LITERATURE (A) Techniques for Assessment of Fetal Growth Several approaches to studying f e t a l metabolism in the ovine fetus have been reported. These vary from ex t e r i o r i z e d preparations such as those employed by Barcroft et a l . , (1939) and Barcroft and Baron (1946) to perfusion studies of Alexander et a l . , (1955) and Andrews et a l . , (1961). The chronic catheterization of ovine f e t a l blood vessels has also been reported by several researchers( Meschia et a l . , 1965; Kramer, 1965; Clapp et a l . , 1977; and K i t t s et a l . , 1979). During the course of these studies much information on substrate u t i l i z a t i o n has been gained. Fetal development per se has been described in terms of an increase in body weight with time (Huggett and Widdas, 1951; Langlands and Sutherland, 1968; Fowler, 1968) and an increase in body length, bone size, or crown-rump length with time (Wallace, 1948; Mellor and Matheson, 1979; Spence et a l . , 1982). While these parameters have been useful indicators of f e t a l anatomical development and v i a b i l i t y (Robinson, 1982), increases in body size, weight and length measurements are not t o t a l l y adequate to elucidate the physiological factors influencing f e t a l growth. Subsequently, several attempts have been made to define more objectively the growth process in the fetus. (1) Nucleic Ac ids According to Enesco and Leblond (1962) c e l l u l a r growth occurs by and can be measured as either an increase in c e l l size (hypertrophy) and/or an increase in c e l l number (hyperplasia). Growth occurring by an increase in c e l l u l a r size or hypertrophic 3 growth i s measured by the i n c r e a s e of c y t o p l a s m i c p r o t e i n per u n i t DNA with time whereas h y p e r p l a s i c growth or an i n c r e a s e i n c e l l u l a r number i s measured as an i n c r e a s e d q u a n t i t y of DNA per u n i t weight of t i s s u e , s i n c e the DNA q u a n t i t y per c e l l nucleus has been demonstrated to be e s s e n t i a l l y constant i n mammalian c e l l s (Enesco and Leblond, 1962). However, i n order to use n u c l e i c a c i d s as r e l i a b l e i n d i c a t o r s of c e l l u l a r growth, a t i s s u e must be homogeneous with no e x t r a c e l l u l a r component pr e s e n t . Furthermore, a measured i n c r e a s e i n DNA with time must a c t u a l l y r e f l e c t the f a c t that p o l y p l o i d y r e p r e s e n t s an i n c r e a s e i n cytoplasm. As d i s c u s s e d by Cheek (1975) these two c o n d i t i o n s are not always met, e s p e c i a l l y with s p e c i f i c t i s s u e s such as b r a i n and s k e l e t a l muscle. Notwithstanding these problems, n u c l e i c a c i d a n a l y s i s has been used e f f e c t i v e l y to measure the r a t e of growth i n the f e t a l t i s s u e at v a r i o u s stages of g e s t a t i o n (Cheek, 1975; R a t t r a y et a l . , 1975; Broad and Davies, 1981). (2) Nitrogen Retention As d i s c u s s e d by Munro (1964) and Waterlow et a l . , (1978) growth can e s s e n t i a l l y be d e f i n e d as an i n c r e a s e i n p r o t e i n mass with time. T h i s p r o t e i n a c c r e t i o n i s determined by the balance between the r a t e of p r o t e i n s y n t h e s i s and p r o t e i n degradation (Waterlow et a l . , 1978; Lobley et a l . , 1980). In order to d e f i n e q u a n t i t a t i v e l y the p r o t e i n a c c r e t i o n o c c u r r i n g with f e t a l growth, two procedures have been used. (a) Slaughter Techniques Due to many of the inherent d i f f i c u l t i e s with i n v i v o f e t a l c a t h e t e r i z a t i o n procedures such as c a t h e t e r patency and s u r g i c a l 4 expense as well as the l i m i t a t i o n s on the extent of data co l l e c t e d , slaughter techniques have been used for f e t a l growth and carcass composition studies. This procedure e s s e n t i a l l y involves the use of large treatment groups at various stages of gestation whereby sequential chronological slaughter of the dam and chemical analysis of the conceptus are carr i e d out. Fetal protein content at a p a r t i c u l a r gestational date can then be found by multiplying the quantity of nitrogen in the carcass by 6.25, considering protein i s equal to approximately 16% of body weight (Kleiber, 1975). Subsequent regression analysis on the nitrogen retention data with time can be used to calculate growth rates for the fetus at various ages throughout gestation. This technique has been used for the estimation of ovine f e t a l growth by many researchers (Wallace, 1948; Lodge and Heaney, 1973; Rattray et a l . , 1975; Robinson, 1977; Battaglia and Meschia, 1978; Broad and Davies, 1981; and Meier et a l . , 1981). These procedures indicate a growth rate (nitrogen accretion) in the ovine fetus at 110-140 days of gestation of between 0.65 - 1.6 g nitrogen/day/kg. The disadvantage, of the slaughter technique however, i s that i t i s expensive and laborious and provides no information on the in vivo metabolism of the fetus. For t h i s reason many researchers have perfected umbilical catheterization procedures for the purpose of studying nitrogen metabolism in the fetus. (b) Umbilical Nitrogen Exchange Though the nitrogen balance study has been used extensively in postnatal l i f e to demonstrate whether the body i s in posit i v e 5 or n e g a t i v e n i t r o g e n balance, s e v e r a l m o d i f i c a t i o n s have to be in t r o d u c e d f o r the a p p l i c a t i o n of t h i s technique to f e t a l metabolism. Because of the unique anatomical arrangement of the f e t u s , u m b i l i c a l n i t r o g e n uptake i s measured by the use of u m b i l i c a l v e n o - a r t e r i a l c o n c e n t r a t i o n d i f f e r e n c e i n amino a c i d n i t r o g e n m u l t i p l i e d by u m b i l i c a l blood flow. R e s u l t s from these s t u d i e s have been r e p o r t e d by s e v e r a l r e s e a r c h e r s (Gresham et a l . , 1972; Lemons et a l . , 1976; Meschia and B a t t a g l i a , 1976; Faber and Woods, 1981) and i n d i c a t e a n i t r o g e n r e t e n t i o n i n the ovine f e t u s comparable with r e s u l t s obtained u s i n g s l a u g h t e r techniques. (3) Isotope D i l u t i o n Procedures The use of both s t a b l e and r a d i o a c t i v e i s o t o p e s i n studying s u b s t r a t e metabolism i n man and animals has been e x t e n s i v e and dates back to the experiments of C u r i e (Reid, 1974) and Schoenheimer et a l . , (1939). Reviews by Z i l v e r s m i t (1960), Munro (1964), Waterlow et a l . , (1978), Zak et a l . , (1979) and Waterlow and Stephens (1980) p r o v i d e a c h r o n o l o g i c a l coverage of the use of i s o t o p e s i n metabolic s t u d i e s p a r t i c u l a r l y as they r e l a t e to the i n v e s t i g a t i o n of p r o t e i n metabolism. (a) B a s i c Terms, P r i n c i p l e s and D e f i n i t i o n s As d i s c u s s e d by Jaquez (1972) the use of is o t o p e d i l u t i o n methods i s based on the concept that a b i o l o g i c a l system i s made up of a f i n i t e number of subsystems, each of which i s homogeneous and w e l l mixed. Or, as d e s c r i b e d by S h i p l e y and C l a r k (1972) an animal body i s viewed as an assortment of pools or compartments each made up of i d e n t i c a l molecules which tend 6 to be e n c l o s e d by anatomical boundaries and behave k i n e t i c a l l y l i k e a d i s t i n c t homogeneous w e l l mixed amount of m a t e r i a l . The t o t a l system may be c l o s e d or open and the mathematical theory of behavior of such systems i s r e f e r r e d to as compartmental a n a l y s i s . R a d i o i s o t o p i c t r a c e r s are elements with the same number of protons i n the nucleus but with an a d d i t i o n a l neutron and which undergo nuc l e a r decay demonstrated by the emission of a l p h a , x-ray, gamma and/or beta r a d i a t i o n . Hence, they are o f t e n r e f e r r e d to as r a d i o n u c l i d e s . R a d i o i s o t o p i c t r a c e r s are assumed to behave c h e m i c a l l y and p h y s i o l o g i c a l l y e x a c t l y l i k e t h e i r n a t u r a l c o u n t e r p a r t atoms. The major advantage of u s i n g t r a c e r s to study b i o l o g i c a l compartments l i e s i n the f a c t that due to t h e i r radiodecay p r o p e r t i e s they can be analysed or d e t e c t e d i n q u a n t i t i e s f a r smaller than t h e i r n a t u r a l atoms. Furthermore, the added t r a c e r i s q u a n t i t a t i v e l y so small i t does not d i s t u r b the normal metabolism. The p h y s i o l o g i c a l compartments or pools i n an animal body tend to remain at a constant s i z e under steady s t a t e c o n d i t i o n s , or c o n d i t i o n s where there i s no net a d d i t i o n or l o s s of substance and thus the exchange of an added t r a c e r can be d e s c r i b e d mathematically by e x p o n e n t i a l e q u a t i o n s . (b)Flow and Flux In the s t r i c t sense, the term flow r e f e r s to the t r a n s p o r t of f l u i d and the term f l u x r e f e r s to the t r a n s p o r t of mass between or among compartments. However, as d i s c u s s e d by Lassen and P e r l (1979) the two terms are o f t e n used synonymously in k i n e t i c l i t e r a t u r e . Furthermore, the term f l u x i s o f t e n r e f e r r e d to as "turnover" i n many a r t i c l e s . Z i l v e r s m i t (i960) d e s c r i b e s 7 the term turnover as r e f e r r i n g to the process of renewal of a substance i n a compartment e i t h e r by de novo s y n t h e s i s w i t h i n that compartment or t r a n s f e r to that compartment. T h e r e f o r e , the term turnover should not be used synonymously with or equated with the term s y n t h e s i s . (d) T r a c e r A d m i n i s t r a t i o n In terms of the a d m i n i s t r a t i o n of i s o t o p i c t r a c e r s there are b a s i c a l l y two procedures that are commonly used, the s i n g l e i n j e c t i o n or the continuous i n f u s i o n method. However, c e r t a i n v a r i a t i o n s on these techniques have been employed such as the primed continuous i n f u s i o n , double dosing of isotope which w i l l not be d i s c u s s e d but which are w e l l documented (Munro, 1964; S h i p l e y and C l a r k , 1972; Waterlow et a l . , 1978). (e) S i n g l e I n j e c t i o n The s i n g l e i n j e c t i o n a p p l i c a t i o n of r a d i o a c t i v e i s o t o p e s e n t a i l s the a d m i n i s t r a t i o n ( i n j e c t i o n ) of an i s o t o p e bolus over a r e l a t i v e l y short time (seconds) i n t o the compartment r e f e r r e d to as the p r e c u r s o r pool or, i n the case of a b i o l o g i c a l system, the compartment from which the s y n t h e s i s or t r a n s f e r to a product pool w i l l occur. F o l l o w i n g the i n j e c t i o n , the d e t e r m i n a t i o n of s p e c i f i c a c t i v i t y of t r a c e r over time i n both p r e c u r s o r and product p o o l s w i l l y i e l d a s p e c i f i c a c t i v i t y curve demonstrating a r a p i d r i s e i n the p r e c u r s o r s p e c i f i c a c t i v i t y f o l l o w e d by a r a p i d d e c l i n e due to mixing of the t r a c e r with u n l a b e l l e d molecules. The g e o m e t r i c a l shape of the s p e c i f i c a c t i v i t y curve thus measured w i l l obey f i r s t order k i n e t i c p r o p e r t i e s . To determine the i s o t o p i c f l u x i n the case of a s i n g l e i n j e c t i o n study, the f l u x i s equal to the dose of t r a c e r 8 given d i v i d e d by the area under the p l o t t e d s p e c i f i c a c t i v i t y c urve. T h i s area can be determined by a number of procedures i n c l u d i n g the use of mathematical i n t e g r a t i o n , a planometer, or simply by a c c u r a t e l y comparing the weights of the "cut out" s p e c i f i c a c t i v i t y paper p l o t . The d i f f i c u l t y t h a t a r i s e s with t h i s procedure, however, i s i n the i n t e r p r e t a t i o n of the curve and r e l a t i n g the data to p h y s i o l o g i c a l l y meaningful parameters. That i s , the s p e c i f i c a c t i v i t y curve measured w i l l a c t u a l l y r e present the sum or composite of s e v e r a l s p e c i f i c a c t i v i t y curves from s e v e r a l compartments. The d e c i s i o n of how to then f r a c t i o n or " p e e l " the t o t a l s p e c i f i c a c t i v i t y curve i n t o the composite curves i s o f t e n a problem. Furthermore, a s s i g n i n g an a c t u a l b i o l o g i c a l l y meaningful space to a compartment can be d i f f i c u l t . The advent of computer a n a l y s i s has been of some a s s i s t a n c e i n t h i s regard, employing s t a t i s t i c a l a n a l y s i s programs such as the SAAM 27 (Baker and Huebotter, 1972) or other s i m i l a r programs (Aub and Waterlow, 1970; G i b a l d i and P e r r i e r , 1975; Brown, 1980). ( f ) Continuous I n f u s i o n The continuous i n f u s i o n technique f o r r a d i o i s o t o p e a d m i n i s t r a t i o n e s s e n t i a l l y i n v o l v e s the i n f u s i o n of a t r a c e r i n t o the " p r e c u r s o r " pool over an extended p e r i o d of time (hours) to o b t a i n an i s o t o p i c steady s t a t e . The amount of t r a c e r i n f u s e d and the time f o r i n f u s i o n w i l l depend p r i m a r i l y on such f a c t o r s as the pool s i z e of the m e t a b o l i t e concerned and the amount of a c t i v i t y or the s p e c i f i c a c t i v i t y needed f o r a c c u r a t e d e t e c t i o n , as w e l l as f a c t o r s such as c o s t and animal management. The s p e c i f i c a c t i v i t y curve i n such an experiment 9 w i l l r i s e with time u n t i l a p l a t e a u or steady s t a t e has been reached. The r a t e of r i s e of the curve i s mathematically comparable to the r a t e of f a l l or the rate of disappearance of t r a c e r i n a s i n g l e i n j e c t i o n experiment. The f l u x or turnover c a l c u l a t i o n i n a continuous i n f u s i o n experiment i s determined by d i v i d i n g the r a t e of i n f u s i o n by the p l a t e a u s p e c i f i c a c t i v i t y . Again, as with the s i n g l e i n j e c t i o n technique, the i n f u s i o n i s made i n t o a p r e c u r s o r pool and the sampling i s made from the product p o o l . Although r e q u i r i n g more time, which may i n i t s e l f be of importance regarding animal experiments, the continuous i n f u s i o n approach has the advantage i n animal metabolism s t u d i e s when s e v e r a l slower t u r n i n g over p o o l s or compartments e x i s t . In t h i s case, the continuous i n f u s i o n technique f a c i l i t a t e s or accounts f o r the i n f l u e n c e of these p o o l s . In a d d i t i o n , t h e o r e t i c a l l y the continuous i n f u s i o n technique r e q u i r e s fewer samples over a longer p e r i o d than the s i n g l e i n j e c t i o n technique which may a l s o be an advantage when the sample pool s i z e i s l i m i t e d . The a p p l i c a t i o n of i s o t o p e d i l u t i o n procedures i n measuring p r o t e i n s y n t h e s i s i n the mammalian system i n v i v o has been reviewed e x t e n s i v e l y by Waterlow et a l . , (1978); Zak (1979) and Waterlow and Stephens (1980). (g) Whole Body P r o t e i n S y n t h e s i s Rate In measuring whole body p r o t e i n s y n t h e s i s from i s o t o p i c d e t e r m i n a t i o n s of amino a c i d f l u x or turnover, the assumption i s made that amino a c i d turnover i s equal to the intake of amino a c i d s p l u s the amino a c i d s c o n t r i b u t e d from p r o t e i n breakdown p l u s the de novo s y n t h e s i s of amino a c i d s . Conversely, the 10 turnover i s also equal to the quantity of amino acids oxidized and excreted plus the amino acids synthesised to protein plus the amino acids metabolized to other pathways. In an experimental animal, an estimate of the amino acid flux can be determined either from the s p e c i f i c a c t i v i t y curve in plasma or in urine. The flux value obtained in th i s manner i s assumed to be equal to the amino acids used for protein synthesis plus the amino acids oxidized. Or, on rearranging , the whole body protein synthesis i s then equivalent to the flux value minus the oxidative loss. (h) Choice of Amino Ac id In determining both the flux and oxidation values in whole body protein synthesis, the selection of an amino acid for the study i s important. F i r s t of a l l , the determination of a flux value should be made with a representative amino acid or one that i s uniformly d i s t r i b u t e d in major body tissues. Generally, e s s e n t i a l amino acids or near es s e n t i a l amino acids are used for t h i s reason. In conjunction with t h i s decision, the choice of amino acid for a tracer and i t s quantitative d i s t r i b u t i o n in the body or pool size w i l l a f f e c t the amount of tracer required for the study. Secondly, in terms of quanti t a t i v e l y estimating the amount of amino acid oxidized, the choice of tracer i s important as c e r t a i n amino acids w i l l be oxidized p r e f e r e n t i a l l y depending on t h e i r b i o l o g i c a l function. An example of t h i s i s the p r e f e r e n t i a l oxidation of leucine by muscle (Chang and Goldberg, 1978). Furthermore, accurately determining the amount of tracer a c t u a l l y oxidized to labeled C O 2 may be complicated by the p o s i t i o n a l placement of the label on the tracer molecule. For 11 t h i s reason, 1 - l a b e l l e d amino a c i d s are o f t e n u t i l i z e d as the 1-l a b e l appearance in CC^ f o r some amino a c i d s w i l l i n d i c a t e a t r u e i r r e v e r s i b l e l o s s or f l u x unconfounded by r e c y c l i n g of l a b e l v i a TCA i n t e r m e d i a t e s or by r e t e n t i o n of l a b e l i n b i c a r b o n a t e p o o l s ( B u c k l e y and Marquardt, 1981). ( i ) T i s s u e F r a c t i o n a l P r o t e i n S y n t h e s i s As d i s c u s s e d by Zak et a l . , (1979) the degradation and r e s y n t h e s i s of m e t a b o l i t e s are e v i d e n t f o r a l l molecules except DNA. One method to access the turnover of p r o t e i n molecules i s the i s o t o p i c t r a c e r method. T h i s approach r e q u i r e s the d e t e r m i n a t i o n of the s p e c i f i c a c t i v i t y of the amino a c i d both i n the p r o t e i n and i n the p r e c u r s o r p o o l s . However, u n l i k e some m e t a b o l i t e s , the c h o i c e of the p r e c u r s o r p o o l i n p r o t e i n s y n t h e s i s s t u d i e s i s d i f f i c u l t . The most r i g o r o u s approach r e q u i r e s the measurement of s p e c i f i c aminoacyl-tRNA. T h i s approach has been accomplished i n v i t r o (Martin et a l . , 1977). However, the a n a l y s i s of t-RNA i s d i f f i c u l t due f i r s t of a l l to the small c o n c e n t r a t i o n of t-RNA i n c e l l s and secondly to the s h o r t h a l f l i f e of the t-RNA. For these reasons, the i n t r a c e l l u l a r f r e e amino a c i d pool i s commonly used as the i n t r a c e l l u l a r f r e e amino a c i d s are on the d i r e c t pathway f o r p r o t e i n s y n t h e s i s . I t i s noted by Waterlow et a l . , (1978) , however, t h a t t h i s does not h o l d true f o r a l l p r o t e i n s where s p e c i f i c t-RNA sp e c i e s are not "charged" from the i n t r a c e l l u l a r f r e e p o o l . Due to the h e t e r o g e n e i t y of c e l l u l a r p r o t e i n s , there are s e v e r a l a c t u a l f r a c t i o n a l p r o t e i n s y n t h e t i c r a t e s i n a c e l l . The i s o t o p i c d i l u t i o n procedure, whether s i n g l e i n j e c t i o n or 1 2 continuous i n f u s i o n , i s most commonly used to study the mean f r a c t i o n a l s y n t h e t i c r a t e , d e s i g n a t e d k s. The mathematical d e t e r m i n a t i o n of k g i s d e s c r i b e d i n chapter III . The e x p e r i m e n t a l l y determined parameters r e q u i r e d f o r t h i s purpose i n c l u d e a measure of the s p e c i f i c a c t i v i t y i n the p r o t e i n bound and i n t r a c e l l u l a r f r e e amino a c i d p o ols as w e l l as a knowledge of the r a t e of i n c r e a s e i n s p e c i f i c a c t i v i t y i n the p r e c u r s o r p o o l . Assuming that the p r e c u r s o r pool i s represented by the i n t r a c e l l u l a r f r e e amino a c i d p o o l , e x p e r i m e n t a l l y determining the r a t e of i n c r e a s e i n s p e c i f i c a c t i v i t y with time dur i n g an experiment i s l o g i s t i c a l l y d i f f i c u l t and has been approached b a s i c a l l y i n two ways. One method i s to s e q u e n t i a l l y k i l l animals from a p a r t i c u l a r treatment group at s p e c i f i e d times a f t e r an i n j e c t i o n or i n f u s i o n of i s o t o p e . The subsequent a n a l y s i s of the i n t r a c e l l u l a r f r e e s p e c i f i c a c t i v i t y w i l l hence d e f i n e the s p e c i f i c a c t i v i t y time curve (denoted x i ) . The assumption with t h i s method i s that i n d i v i d u a l animal v a r i a t i o n i s n e g l i g i b l e . A l t e r n a t i v e l y , the c h o i c e i s to determine the r a t e of s p e c i f i c a c t i v i t y i n c r e a s e i n the plasma pool (denoted Xp) and assume t h i s v alue w i l l approximate x i . Or, f o r those t i s s u e s deemed to be t u r n i n g over r e l a t i v e l y s l o w l y , and hence where xp would l i k e l y overestimate the r a t e of i n c r e a s e i n p r e c u r s o r s p e c i f i c a c t i v i t y , the use of the r a t i o of p r o t e i n bound to i n t r a c e l l u l a r f r e e s p e c i f i c a c t i v i t y i s used i n p l a c e of x i . ( j ) R a d i o i s o t o p e Use i n The Ovine Fetus In the ovine f e t u s , the use of r a d i o i s o t o p e s has enabled r e s e a r c h e r s to measure the turnover and u t i l i z a t i o n of 13 substrates such as glucose (Prior and Scott, 1977;, Anand et a l . , 1979; Hodgson ' et a l . , 1980; Hay, 1979; K i t t s et a l . , 1982a ), lactate (Warnes et a l . , 1977; K i t t s and Krishnamurti, 1982b), pyruvate (Char and Creasy, 1976), amino acids (Gresham et a l . , 1972; Young et a l . 1979; Meier et a l . , 1981; K i t t s and Krishnamurti, 1982a,b; Schaefer and Krishnamurti, 1982a), and fatty acids and ketones (Char and Creasy, 1976). Much information on the interconversion of substrates has also been obtained using radioactive tracers (Prior and Christenson, 1977; Anand et a l . , 1979; Hodgson et a l . , 1980; Pr i o r , 1980; K i t t s and Krishnamurti. 1982a). However, in terms of nitrogen metabolism, the use of isotopes in the quantitative evaluation of amino acid and nitrogen use, p a r t i c u l a r l y as related to f e t a l protein synthesis and accretion has only recently been attempted (Meier et a l . , 1981; Noakes and Young, 1981). Results from these studies indicate a protein synthetic a b i l i t y in the ovine fetus at 110-130 days of gestation from I5g/day/kg (Meier et a l . , 1981) to 38.6 g/day/kg (Noakes and Young, 1981). (B) Factors Affecting Fetal Growth As discussed by Berg and B u t t e r f i e l d (1976) and Arthur (1981) the absolute f e t a l growth potential i s determined by i t s genotype. However, the f u l l expression of t h i s genetic potential i s u n l i k e l y to be r e a l i z e d due to various environmental constraints largely of f e t a l and maternal o r i g i n (Liggins, 1977; Robinson, 1981). The influence of the f e t a l endocrine system, placental physiology and maternal n u t r i t i o n are l i k e l y to have the greatest impact on f e t a l growth. 1 4 (1) F e t a l Endocrine F a c t o r s The t r a n s p l a c e n t a l t r a n s f e r of maternal pep t i d e hormones to the f e t u s i s e s s e n t i a l l y n i l ( L i g g i n s , 1977). Only very l i m i t e d amounts of t h y r o x i n e have been observed to c r o s s the p l a c e n t a (Erenberg and F i s h e r , 1973; Thorburn and Hopkins, 1973). C o r t i s o l has been rep o r t e d to c r o s s the p l a c e n t a , however, i t i s r a p i d l y converted to the i n a c t i v e form, c o r t i s o n e , by the p l a c e n t a l enzyme II 3 hydroxydehydrogenase (EC 1.1.1) ( O s i n s k i , 1960). The c h r o n o l o g i c a l development and i n t e g r i t y of the f e t a l endocrine system i s e s s e n t i a l f o r normal growth i n u t e r o . Growth Hormone In the ovine f e t u s , plasma growth hormone l e v e l s are r e p o r t e d to f l u c t u a t e throughout g e s t a t i o n (Gluckman et a l . , 1979). In l a t e g e s t a t i o n the l e v e l s are s e v e r a l f o l d g r e a t e r than those i n the a d u l t sheep (Bassett and M a d i l l , 1974). In s p i t e of t h i s , a demonstrated r o l e f o r growth hormone i n the f e t u s remains c o n t r o v e r s i a l ( L i g g i n s , 1977) and i n f a c t , s t u d i e s with f e t a l lambs demonstrate c o n t i n u e d , a l b e i t r e t a r d e d , somatic growth even f o l l o w i n g hypophysectomy ( L i g g i n s and Kennedy, 1968) and the ensuing absence of growth hormone. However, Hurley et a l . , (1977a,b) have suggested that i n the absence of growth hormone, p l a c e n t a l l a ctogen may a c t to s t i m u l a t e somatomedin p r o d u c t i o n and thereby i n d i r e c t l y r e g u l a t e f e t a l growth. Furthermore, the data of Robinson et a l . , (I977a,b) suggest that somatomedin a c t i v i t y i s perhaps more i n f l u e n t i a l than growth hormone per se. The p r e c i s e e f f e c t of growth hormone on f e t a l growth thus remains s p e c u l a t i v e . Thyroxine 15 The growth promoting r o l e of t h y r o x i n e i n the f e t u s has been demonstrated (Hopkins, 1975; L i g g i n s , 1977) however, the extent of involvement appears to be dependent on the s p e c i e s and a l s o on the time of g e s t a t i o n ( L i g g i n s et a l . , 1973). In the sheep f e t u s , the absence of t h y r o x i n e v i a thyroidectomy w i l l r e s u l t i n a s i g n i f i c a n t s t u n t i n g of growth (Hopkins and Thorburn, 1972; Hopkins, 1975). I n s u l i n and Glucagon Research on the r o l e of f e t a l i n s u l i n has been e x t e n s i v e (Bassett et a l . , 1973; G i r a r d et a l . , 1973; Alexander et a l . , 1976; Simmons et a l . , 1978; M i l n e r , 1979). I t i s c u r r e n t l y r e c o g n i z e d that f e t a l i n s u l i n and glucagon are l i k e l y to have the most s i g n i f i c a n t i n f l u e n c e on f e t a l growth, p a r t i c u l a r l y i n l a t e g e s t a t i o n (Bassett and M a d i l l , 1974; L i g g i n s , 1977). The i n c r e a s e d f e t a l i n s u l i n s e c r e t i o n i n response to glucose and i n p a r t i c u l a r amino a c i d l e v e l s i s i n d i c a t i v e of the a n a b o l i c r o l e of f e t a l i n s u l i n ( Milner et a l . , 1972) and a p o s i t i v e c o r r e l a t i o n between b i r t h weight and f e t a l i n s u l i n l e v e l s has been r e p o r t e d ( S h e l l e y , 1973). I n s u l i n p r i m a r i l y a f f e c t s g lucose homeostasis (Bassett and M a d i l l , 1974) as w e l l as energy r e s e r v e s of the f e t u s ( M i l n e r , 1979), and although f e t a l i n s u l i n s e c r e t i o n i s l e s s r e s p o n s i v e to a g l u c o s e l o a d than i n the a d u l t ( F i s e r et a l . , 1974) i t i s nonetheless s e n s i t i v e to a l t e r a t i o n s i n n u t r i e n t d e l i v e r y such as that caused by maternal s t a r v a t i o n ( S c h r e i n e r et a l . , 1980a,b). Glucagon f u n c t i o n appears to be l e s s w e l l understood i n the f e t u s , however, i n view of i t s importance i n promoting 16 glycogenolysis and gluconeogenesis in the neonate (Snell and Walker, 1978), glucagon may be of functional significance during l a t e r periods of gestation . Furthermore, in addition to i t s action in gluconeogenesis and g l y c o l y s i s , glucagon can also stimulate the hepatic production of tyrosine aminotransferase (Holt and O l i v e r , 1971). The observed late maturation of the tyrosine aminotransferase pathway, at least in the sheep fetus, (Linblad, 1977) would further suggest that glucagon i s of functional significance during late gestation. Other f e t a l hormones including glucocorticoids and catecholamines in addition to glucagon appear to function primarily as enzyme inducers, p a r t i c u l a r l y in late gestation (Jost, 1961; Holt and O l i v e r , 1968, 1971; Girard et a l . , 1973). (2) Placental Factors As discussed by Beaconsfield and Ginsburg (1980) the placenta functions b a s i c a l l y to control the a v a i l a b i l i t y of substrates to the fetus. In t h i s role, the placenta functions both as an endocrine organ and as a s t r u c t u r a l l i p i d membrane to regulate maternal-fetal nutrient transfer. (a) Placental Endocrine Function As discussed in recent reviews, the placenta has a proven a b i l i t y to synthesize both steroid and protein hormones and in t h i s way, mimics the role of the p i t u i t a r y , hypothalamus and gonads (Munro, 1980; V i l l e e , 1980). There are presently numerous additional placental proteins which are suspected to have endocrine function (Home and Nisbel, 1979). Although considerable species v a r i a t i o n in the type and quantity of hormones does exist (Davies and Ryan, 1972) the production of 1 7 chorionic gonadotropin and placental lactogen as well as estrogens and progesterone i s suggested to have the most s i g n i f i c a n t impact on f e t a l growth ( V i l l e e , 1980) . Chorionic gonadotropin i s known to be synthesized by the syncytiotrophoblast and i s present throughout gestation (Ikonikoff and C'edard, 1973). Its actions appear to be manifest through an increased production of cyclic-AMP (Le V i l l i e r s et a l . , 1974) and i s suspected to be primarily involved in maintaining the corpus luteum and hence progesterone production throughout pregnancy (Channing, 1970). Iii addition, a role for chorionic gonadotropin in f e t a l steroid synthesis may also be indicated ( V i l l e e , 1969, 1980). The placental production of the protein hormone placental lactogen was f i r s t demonstrated by Grumbach and Kaplan (1964) and Friesen et a l . , (1969). Placental lactogen has since been demonstrated to have a ce r t a i n structural and chemical s i m i l a r i t y to growth hormone (Hurley et a l . , 1977a,b; Martal, 1978). The contention has been made that placental lactogen may f a c i l i t a t e the delivery of adequate glucose and amino acids to the fetus due to i t s l i p o l y t i c , anabolic, and diabetogenic e f f e c t s on the maternal metabolism (Grumbach et a l . , 1968; Handwerger et a l . f 1976; Hurley et a l . , 1977; Chan et a l . , 1978). However, as discussed by Nathanielsz (1976) t h i s role has not as yet been f u l l y proven. The placental production of progesterone from maternal cholesterol has been demonstrated (Schubert and Schade, 1977) and i t has been proposed by V i l l e e (1969) that t h i s progesterone serves as the precursor for the biosynthesis of s t e r o i d hormones 18 by the f e t a l a d r e n a l . Estrogen s y n t h e s i s by the p l a c e n t a has a l s o been observed ( V i l l e e , 1969) and as d i s c u s s e d by V i l l e e (1980) p l a c e n t a l estrogen s y n t h e s i s i s r e p o r t e d l y s t i m u l a t e d by c h o r i o n i c gonadotropin, l u t e i n i z i n g hormone and p l a c e n t a l l a c t o g e n . Estrogen may be i n v o l v e d i n the s t i m u l a t i o n of f e t a l l i v e r p r o t e i n b i o s y n t h e s i s (Page et a l . , 1976) but as d i s c u s s e d by L i g g i n s (1973) may be more important in l a t e g e s t a t i o n as i t a f f e c t s the onset of p a r t u r i t i o n . (b) P l a c e n t a l Transport Mechanisms As emphasized by H i l l and Longo (1980) and 0'shaughnessy (1981) the i n t e g r i t y of p l a c e n t a l n u t r i e n t t r a n s p o r t i s c r u c i a l to the normal development and growth of the embryo and f e t u s . The normal f u n c t i o n of the p l a c e n t a and subsequent f e t a l growth can be a f f e c t e d by s e v e r a l f a c t o r s i n c l u d i n g blood flow (McFadyen, 1979; Clapp et a l . , 1980; F e r r e l and Ford, 1980) as w e l l as numerous p a t h o l o g i c a l mechanisms such as f a u l t y p l a c e n t a t i o n and abnormal v i l l o u s development (Fox, 1981). B a r r i n g e r r o r s i n these mechanisms, the p l a c e n t a l t r a n s p o r t of n u t r i e n t s i s dependent upon s e v e r a l p h y s i c a l and m orphological a s p e c t s of p l a c e n t a l a c t i o n i n c l u d i n g pore s i z e , s u r f a c e area, membrane t h i c k n e s s , molecular s i z e and charge, c o n c e n t r a t i o n g r a d i e n t , temperature and h y d r o s t a t i c pressure ( S h e l l e y , 1979). The n u t r i e n t t r a n s f e r per se seems to f u n c t i o n v i a s e v e r a l mechanisms such as p a s s i v e d i f f u s i o n , f a c i l i t a t e d d i f f u s i o n , a c t i v e t r a n s p o r t , s o l v e n t drag, and p i n o c y t o s i s ( H i l l and Longo, 1980). B e a c o n s f i e l d and Ginsburg (1980) emphasize that many endogenous substances e s s e n t i a l f o r f e t a l development with the e x c e p t i o n of m e t a b o l i t e s such as blood gases (Pearson, 1979) and 19 water (Hytten, 1979) c r o s s the p l a c e n t a a g a i n s t e l e c t r o - c h e m i c a l g r a d i e n t s . A h i g h degree of n u t r i e n t t r a n s p o r t s p e c i f i c i t y can a l s o be seen ( S h e l l e y , 1979; B e a c o n s f i e l d and Ginsburg, 1980). ( i ) T r a n s f e r of Carbohydrates The p l a c e n t a consumes c o n s i d e r a b l e q u a n t i t i e s of glucose ( S h e l l e y , 1979) and i t i s estimated that approximately one t h i r d of the maternal glucose p r o d u c t i o n as w e l l as q u a n t i t i e s produced by the f e t u s are u t i l i z e d by the p l a c e n t a (Anand et a l . , 1979; B e a c o n s f i e l d and Ginsberg, 1980). The Embden-Meyerhof pathway i s operable i n the p l a c e n t a and as a r e s u l t , high l a c t a t e c o n c e n t r a t i o n s can be produced. T h i s l a c t a t e i s r e p o r t e d 0 to be u t i l i z e d by the f e t u s (Burd et a l . , 1975; K i t t s et a l . , 1982a) and may account f o r up to 25% of f e t a l e n e r g e t i c needs. P l a c e n t a l f r u c t o s e c o n c e n t r a t i o n s vary among s p e c i e s and as d i s c u s s e d by S h e l l e y (1979), high c o n c e n t r a t i o n s have been observed i n the p l a c e n t a s of ungulates. However, the p r e c i s e r o l e of f r u c t o s e remains s p e c u l a t i v e . The m a t e r n a l - f e t a l t r a n s f e r of glucose a c r o s s the p l a c e n t a i s a g a i n s t a d e c r e a s i n g c o n c e n t r a t i o n g r a d i e n t ( B a t t a g l i a and Meschia, 1978). This t r a n s f e r i s observed to be f a s t e r than would be p r e d i c t e d by d i f f u s i o n alone ( S h e l l e y , 1979) and a f a c i l i t a t e d t r a n s p o r t mechanism i s suspected. Furthermore, the p l a c e n t a i s impermeable t o d i s a c c h a r i d e s and a l d o s e sugars are a p p a r e n t l y t r a n s p o r t e d more r e a d i l y than ketose sugars. ( i i ) T r a n s f e r of F a t t y A c i d s The l i p i d content of the p l a c e n t a i s c o m p a r a t i v e l y low and although ketone bodies c r o s s the p l a c e n t a l b a r r i e r (Williamson, 1981; Noble et a l . , 1982) l a r g e r f a t molecules, such as 20 l i p o p r o t e i n s , do not appear to c r o s s the p l a c e n t a i n t a c t i n most mammalian s p e c i e s (Van Dyne et a l . , 1962; H u l l , 1977; B e a c o n s f i e l d and Ginsburg, 1980; H u l l and E l p h i c k , 1979, 1981; Leat and H a r r i s o n , 1980; Noble et a l . , 1982). However, l i p o l y s i s i n the p l a c e n t a l i k e l y does occur with the r e s u l t i n g f a t t y a c i d s d e l i v e r e d t o the f e t u s (Noble et a l . , 1979; Shand and Noble, 1979; H u l l and E l p h i c k , 1981; Payne and R a t t r a y , 1982). T h i s would be supported by the presence i n the p l a c e n t a of l i p o p r o t e i n l i p a s e which h y d r o l y s e s maternal serum t r i g l y c e r i d e s ( B e a c o n s f i e l d and Ginsburg, 1980). However, i t must be emphasised that the p l a c e n t a l t r a n s f e r of f a t t y a c i d s to the f e t u s does e x h i b i t c o n s i d e r a b l e s p e c i e s s p e c i f i c i t y as w e l l as g e s t a t i o n a l time d i f f e r e n c e s ( H u l l and E l p h i c k , 1979; Zimmerman et a l . , 1979). T h i s i s r e f l e c t e d a l s o by the s p e c i e s v a r i a t i o n i n f e t a l c o n c e n t r a t i o n s of c a r n i t i n e (Hahn et a l . , 1979) needed fo r l i p i d o x i d a t i o n . ( i i i ) T r a n s f e r of Amino Ac i d s The t r a n s f e r of amino a c i d s to the f e t u s i s s e l e c t i v e depending on the amino a c i d group ( H i l l and Young, 1973; Young et a l . , 1979). Y u d i l e v i c h and Eaton (1980) have d e s c r i b e d amino a c i d t r a n s p o r t mechanisms i n the f e t u s as f o l l o w s . B a s i c a l l y , e s s e n t i a l amino a c i d s are t r a n s p o r t e d to the f e t u s r e a d i l y and the n e u t r a l s t r a i g h t c h a i n , g l u c o g e n i c amino a c i d s , formed by tr a n s a m i n a t i o n are t r a n s p o r t e d by the "A" a l a n i n e t r a n s p o r t system which i s sodium and oxygen dependent. The n e u t r a l branched c h a i n and b a s i c amino a c i d s are t r a n s p o r t e d v i a the "L" p r e f e r r i n g system, which i s the most r a p i d t r a n s p o r t system and a l s o d i s p l a y s sodium and oxygen dependency. A c i d i c and n e u t r a l 21 s t r a i g h t c h a i n , n o n - e s s e n t i a l amino a c i d s are l e s s r e a d i l y t r a n s f e r r e d and de novo s y n t h e s i s by the f e t u s i s suspected. Approximately 65% of the amino a c i d uptake by the f e t u s i s r e p r e s e n t e d by e i g h t n e u t r a l amino a c i d s ( a l a n i n e , t h r e o n i n e , s e r i n e , v a l i n e , l e u c i n e , i s o l e u c i n e , glutamine and p r o l i n e ) and approximately 20% by the b a s i c amino a c i d s ( l y s i n e , h i s t i d i n e and a r g i n i n e ) (Enders et a l . , 1976; Holzman et a l . , 1979). Furthermore, no s i g n i f i c a n t uptake of a c i d i c amino a c i d s has been observed (Lemons, 1976) . The f e t u s l i k e l y s y n t h e s i s z e s adequate glutamate and a s p a r t a t e . Thus the f e t u s can be seen to have a high carbohydrate, high p r o t e i n but low f a t n u t r i e n t supply i n most mammalian s p e c i e s . (3) Maternal N u t r i t i o n As d i s c u s s e d by L i g g i n s (1977) the number of c e l l s i n a term f e t u s l i e s w i t h i n f a i r l y narrow l i m i t s and i s the r e s u l t of approximately 42 s u c c e s s i v e d i v i s i o n s of the ovum. However, t h i s f u l l g e n e t i c e x p r e s i o n l i k e l y does not occur as v a r i o u s environmental i n f l u e n c e s l i m i t the growth p r o c e s s . As suggested by S c h r e i n e r et al.,(1980a,b) , f e t a l growth can be s i g n i f i c a n t l y a f f e c t e d by endocrine f a c t o r s as w e l l as s u b s t r a t e a v a i l a b i l i t y . Based on the s t u d i e s of Hammond (1944) one of the most s i g n i f i c a n t f a c t o r s a f f e c t i n g f e t a l growth appears to be the a b i l i t y of the maternal system to supply the f e t u s w i t h n u t r i e n t s . Maternal n u t r i t i o n thus a f f e c t s f e t a l growth p r i m a r i l y by i n f l u e n c i n g the s u b s t r a t e a v a i l a b i l i t y . The i n f l u e n c e of maternal n u t r i t i o n on f e t a l growth has been s t u d i e d e x t e n s i v e l y i n s p e c i e s such as the r a t (Rosso, 1977; Van Marthens e t a l . , 1979; McNurlan e t a l . , 1980; Z a r t a r i a n et a l . , 22 1980), dogs (Kliegman et a l . , 1981) and primates (Cheek, 1975; M e t c o f f , 1980) but a l s o i n domestic s p e c i e s such as sheep ( E v e r i t t , 1 9 6 4 ; Simmons et a l . , 1974; R u s s e l et a l . , 1977; M e l l o r and Matheson, 1979; Hodgson et a l . , 1982; Robinson, 1982) p i g s (Ezekwe, 1982) and bovids ( P r i o r and S c o t t , 1977). D e s p i t e t h i s e f f o r t , there remaines a la c k of consensus both with regard to the magnitude and the mechanism by which maternal n u t r i t i o n can a f f e c t f e t a l growth. One view taken by r e s e a r c h e r s i s that the f e t u s does have demonstrable p r o t e c t i o n a g a i n s t the adverse e f f e c t s of maternal n u t r i e n t d e p r i v a t i o n . S t u d i e s by Z a r t a r i a n et a l . , (1980) would support t h i s concept demonstrating t h a t marginal n u t r i e n t d e p r i v a t i o n of pregnant r a t s w i l l a f f e c t the maternal but not the f e t a l t i s s u e f o rmation. E a r l i e r s t u d i e s by Grumbach et a l . , (1968), Handwerger et a l . , (1976) and Hurley et a l . , (1977a,b) on both r a t s and sheep f u r t h e r support t h i s concept suggesting a r o l e f o r p l a c e n t a l lactogen i n i n f l u e n c i n g maternal metabolism to ensure an adequate d e l i v e r y of n u t r i e n t s to the f e t u s . However, there may be c o n s i d e r a b l e s p e c i e s d i f f e r e n c e s i n t h i s regard as maternal n u t r i e n t d e p r i v a t i o n appears to a f f e c t the f e t u s e s of some s p e c i e s , such as sheep, more a d v e r s e l y (Spence et a l . , 1982) than o t h e r s such as p i g s (Ezekwe, 1982). The concept of t o t a l f e t a l p a r a s i t i s m i n a l l s p e c i e s has been opposed by Metcoff ( 1980), a view which i s a l s o h e l d by Rosso (1977) who suggests that the f e t u s l i k e l y has a l i m i t e d a b i l i t y to i n f l u e n c e the t r a n s p l a c e n t a l d e l i v e r y of n u t r i e n t s . Numerous e f f e c t s of maternal n u t r i e n t d e p r i v a t i o n on f e t a l growth have been r e p o r t e d . I t i s suggested by Morrow et a l . , 23 (1981) that n u t r i e n t d e p r i v a t i o n i s l i k e l y a more s e r i o u s c o n d i t i o n d u r i n g pregnancy, than nonpregnancy, as there i s a more r a p i d onset of maternal k e t o s i s , hypoalaninemia and hypoglycemia . Although the p r e c i s e mechanism i s not w e l l understood, these e f f e c t s are b e l i e v e d to be due to the a c t i o n s of e s t r a d i o l and progesterone (Morrow et a l . , 1981) and/or p l a c e n t a l l a c t o g e n (Grumbach et a l . , 1968). Maternal n u t r i e n t d e p r i v a t i o n has been observed t o cause a r e d u c t i o n i n f e t a l growth r a t e s (Van Marthens et a l . , 1979; M e l l o r and Matheson, 1979; Spence et a l . , 1982), which are u l t i m a t e l y manifest as reduced b i r t h weights as have been observed i n s e v e r a l experimental s t u d i e s with sheep (Russel et a l . , 1967; Espinosa et a l . , 1977) and dogs (Kliegman e t a l . , 1981) as w e l l as r a b b i t s , r a t s , guinea p i g s and hamsters (Cheek, 1975; Hegarty and Kimm 1981). In a d d i t i o n to reduced b i r t h weights, i n v i v o experiments i n d i c a t e that maternal n u t r i e n t d e p r i v a t i o n can l e a d s p e c i f i c a l l y to reduced l i v e r and muscle weights, p r o t e i n s y n t h e s i s and n u c l e i c a c i d c o n c e n t r a t i o n (Sykes and F i e l d , 1972; McNurlan et a l . , 1980; Ezekwe, 1982). A l s o , f e t a l plasma glucose c o n c e n t r a t i o n i s observed to be reduced i n sheep (Simmons et a l . , 1974; M o r r i s et a l . , 1980; S c h r e i n e r et a l . , 1980a,b) and dogs (Kleigman et a l . , 1981). However, the reason f o r the observed r e d u c t i o n i n f e t a l plasma glucose does not appear to be a r e d u c t i o n or change i n gluconeogenic enzymes ( P r i o r and S c o t t , 1977). The experiments of Shambaugh (1977) and Shambaugh et a l . , (1977, 1978) suggest that the f e t u s may d i s p l a y a concomitant i n c r e a s e i n the extent of f a t t y a c i d o x i d a t i o n i n response to maternal f a s t i n g . In a d d i t i o n t o a reduced glucose c o n c e n t r a t i o n 24 i n f e t a l plasma, a reduced f e t a l amino a c i d uptake has a l s o been r e p o r t e d ( M o r r i s et a l . , 1980). Reduced glucose and amino a c i d c o n c e n t r a t i o n s i n f e t a l plasma as a r e s u l t of maternal f a s t i n g appear to be p a r t i c u l a r l y e v ident i n twin pregnancies ( R u s s e l l e t a l . , 1977). Undernourished f e t u s e s are observed to have a comparative lack of subcutaneous f a t (McGowen, 1975). However, the l a c k of subcutaneous f a t , u n l i k e other t i s s u e s , may not be a s e r i o u s long term impediment as p o s t n a t a l r e f e e d i n g can adequately r e h a b i l i t a t e adipose t i s s u e (Tulp et a l . , 1980). Maternal s t a r v a t i o n has a l s o been observed to produce an i n c r e a s e i n urea e x c r e t i o n both i n the mother (Guada et a l . , 1976) and i n the f e t u s (Gresham et a l . , 1972; Espinosa et a l . , 1977; Hodgson et a l . , 1982). The i n c r e a s e i n urea e x c r e t i o n i s l i k e l y a r e f l e c t i o n of p r o t e i n and amino a c i d c a t a b o l i s m , the purpose of which has been suggested to be to spare glucose (Kleigman et a l . , 1981). The q u a n t i t y of feed s u p p l i e d to the maternal system, whether inadequate or i n excess has been shown to be harmful i n both r e s p e c t s ( E v e r i t t , 1964). However, e q u a l l y important as the q u a n t i t y of feed appears to be the q u a l i t y of feed (Metcoff, 1980). Maternal p r o t e i n d e p r i v a t i o n i s known to be harmful to normal f e t a l growth (Wallace, 1948). T h i s appears to be p a r t i c u l a r l y t r u e i f the d e p r i v a t i o n occurs p r i o r to c o n c e p t i o n (Curet, 1973; Lederman and Rosso, 1981) with the r e s u l t being s m a l l e r , lower b i r t h weight o f f s p r i n g . T h i s i s thought to be due to the reduced p l a c e n t a l t r a n s f e r (Rosso, 1980) p a r t i c u l a r l y of n u t r i e n t s such as amino a c i d s (Rosso, 1977) and glucose (Simmons 25 et a l . , 1974). However, a reduced p r o t e i n intake may not be the most s i g n i f i c a n t f a c t o r as d i e t a r y energy , r a t h e r than p r o t e i n d e f i c i e n c y , has been suggested to be of more importance (Lodge and Heaney, 1973; Naismith, 1980). Furthermore, i n a d d i t i o n to the q u a l i t y and q u a n t i t y of feed the mother r e c e i v e s , the frequency of f e e d i n g may be important to the d e l i v e r y of n u t r i e n t s to the f e t u s as demonstrated by S l a t e r and M e l l o r (1981) . A l s o , the time d u r i n g g e s t a t i o n i n which the d e p r i v a t i o n occurs has been r e p o r t e d to be important ( E v e r i t t , 1964; Robinson, 1982). Maternal n u t r i t i o n i s l i k e l y one of the most s i g n i f i c a n t r e g u l a t o r s of f e t a l growth (Metcoff, 1980). The a b i l i t y of the f e t u s to t o l e r a t e maternal n u t r i e n t d e p r i v a t i o n w i l l depend on the age and s p e c i e s of animal as w e l l as the d u r a t i o n and extent of n u t r i t i o n a l s t r e s s . Although the long term permanent e f f e c t s of i n t r a u t e r i n e growth r e t a r d a t i o n have r e c e n t l y been qu e s t i o n e d (Naismith, 1980; Howie, 1982) the f a c t t h at r e f e e d i n g of growth r e t a r d e d f e t u s e s does not r e s u l t i n a demonstrated catchup growth (Widdowson, 1971; Brooke, 1980) i n c o n t r a s t to what i s seen i n the a d u l t animal ( K l e i b e r , 1975; Berg and B u t t e r f i e l d , 1976) would seem to s t r o n g l y support the p o s i t i o n h e l d by many r e s e a r c h e r s that n u t r i t i o n a l r e s t r i c t i o n i n utero causes permanent and i r r e v e r s i b l e damage th a t may w e l l be manifest i n p o s t - n a t a l development (Sykes and F i e l d , 1972; Cheek, 1975; Rosso, 1977; Robinson et a l . , 1977a,b; G a l l e r et a l . , 1980; M e t o c f f , 1980). (C) C o n c l u s i o n s From the L i t e r a t u r e Review From the preceding d i s c u s s i o n , i t i s e v i d e n t t h a t a 26 considerable research e f f o r t has been spent elaborating the metabolic pathways, nutrient exchange and species differences in f e t a l n u t r i t i o n . Despite t h i s considerable e f f o r t , there i s s t i l l a lack of understanding as to the exact biochemical and physiological manner in which maternal n u t r i t i o n a f f e c t s f e t a l growth. In the following chapters, an experimental approach w i l l be described which w i l l f a c i l i t a t e investigations designed to study the ef f e c t of maternal n u t r i t i o n on protein synthesis in the ovine fetus under in utero conditions. 27 CHAPTER I I . TYROSINE UTILIZATION IN THE OVINE FETUS IN UTERO Experiment ( I ) : Development of A n a l y t i c a l Methodology For The Determination of T y r o s i n e S p e c i f i c A c t i v i t y I n t r o d u c t ion From the f o r e g o i n g d i s c u s s i o n , i t i s e v i d e n t that the a p p l i c a t i o n of an i s o t o p i c d i l u t i o n procedure f o r i n v e s t i g a t i n g amino a c i d u t i l i z a t i o n i n v i v o should be done with due c o n s i d e r a t i o n and understanding of s e v e r a l f a c t o r s . These f a c t o r s i n c l u d e the i n f u s i o n method , c h o i c e of i s o t o p e , and a n a l y t i c a l technique to mention a few. Although the k i n e t i c p r i n c i p l e s remain the same, the a p p l i c a t i o n of the i s o t o p i c d i l u t i o n method i n s t u d i e s of f e t a l metabolism i s f u r t h e r complicated by the unique m a t e r n a l - f e t a l u m b i l i c a l exchange system which n e c e s s i t a t e s complex s u r g i c a l procedures f o r i m p l a n t i n g and m a i n t a i n i n g v a s c u l a r c a t h e t e r s . In view of these c o n s i d e r a t i o n s , the d e c i s i o n was made in the present study to make use of the continuous i n f u s i o n technique to study amino a c i d metabolism i n the ovine f e t u s . The advantages of the continuous i n f u s i o n method over the s i n g l e i n j e c t i o n method have been o u t l i n e d p r e v i o u s l y . Furthermore, i t was d e c i d e d to make use of the amino a c i d t y r o s i n e f o r t h i s purpose as t y r o s i n e i s known to be e s s e n t i a l or near e s s e n t i a l f o r a d e v e l o p i n g f e t u s (Young et a l . , 1979) and a l s o i s known to be u n i f o r m l y d i s t r i b u t e d i n major body t i s s u e s (Waterlow et a l . , 1978). In a d d i t i o n , the pool s i z e of t y r o s i n e (2.79 g/16 g c a r c a s s n i t r o g e n , Meier et a l . , 1981b) i n the ovine f e t u s was presumed to be of a magnitude that c o u l d be e q u i l i b r a t e d with so that a steady s t a t e c o u l d be obtained i n a reasonable p e r i o d of 28 time. A major c o n s i d e r a t i o n at the onset of t h i s study was that an a n a l y t i c a l technique was r e q u i r e d to i s o l a t e and recover the L-form of t y r o s i n e from c o l l e c t e d blood and t i s s u e samples f o r determining the s p e c i f i c r a d i o a c t i v i t y . I t appeared i n i t i a l l y that t h i s c o u l d be accomplished by the a p p l i c a t i o n of an enzymatic procedure r e p o r t e d by other workers ( G a r l i c k and M a r s h a l l , 1972). However, on c l o s e r examination, the a v a i l i b l e enzymatic procedure was found to be u n s a t i s f a c t o r y , mainly because of low and v a r i a b l e r e c o v e r i e s and accuracy. The f o l l o w i n g experiment o u t l i n e s the changes and m o d i f i c a t i o n s undertaken i n the present study i n order to improve the enzymatic a n a l y t i c method. Though ion-exchange chromatography has been used e x t e n s i v e l y f o r de t e r m i n a t i o n of amino a c i d s i n plasma and other b i o l o g i c a l m a t e r i a l s , a r a p i d , simple and s p e c i f i c method would be more d e s i r a b l e when the de t e r m i n a t i o n of a s i n g l e amino a c i d in the L form i s of primary i n t e r e s t . In recent years the rate of p r o t e i n s y n t h e s i s i n the whole body and i n i n d i v i d u a l organs has been estimated from the plasma s p e c i f i c r a d i o a c t i v i t y f o l l o w i n g the i n f u s i o n of a l a b e l l e d amino a c i d (Waterlow et a l . , 1978). One of the amino a c i d s used f o r t h i s purpose i s L-t y r o s i n e which can be estimated e n z y m a t i c a l l y u s i n g commercially a v a i l a b l e L - t y r o s i n e decarboxylase (EC.4.1.1.25). The tyramine formed i n the r e a c t i o n i s e x t r a c t e d and measured f l u o r o m e t r i c a l l y f o l l o w i n g the a d d i t i o n of the l - n i t r o s o - 2 -naphthol n i t r i c a c i d reagent (Waalkes and Udenfriend, 1957). The absence of i n t e r f e r e n c e with D - t y r o s i n e has l e d many workers to 29 employ t h i s method of t y r o s i n e e s t i m a t i o n i n s t u d i e s r e l a t e d to p r o t e i n s y n t h e s i s ( G a r l i c ! ; and M a r s h a l l , 1972; James et a l . , 1976). Ambrose et a l . , (1969) d e s c r i b e d a procedure i n which t y r o s i n e was determined f l u o r o m e t r i c a l l y without enzymatic c o n v e r s i o n to tyramine. Subsequently, Ambrose (1974) r e p o r t e d that the s t a b i l i t y of the f l u o r o p h o r e may be improved by the a d d i t i o n of phosphoric a c i d to the r e a c t i o n mixture. However t h i s method w i l l not d i s t i n g u i s h between L and D isomers. During the course of i n v e s t i g a t i o n s on the u t i l i z a t i o n of t y r o s i n e by the ovine f e t u s i n ute r o , we found that the recovery of added t y r o s i n e u s i n g the procedure of G a r l i c k and M a r s h a l l (1972) was c o n s i s t e n t l y low. By combining the enzymatic c o n v e r s i o n of L - t y r o s i n e to tyramine ( G a r l i c k and M a r s h a l l , 1972) with the m o d i f i e d f l u o r o m e t r i c method of Ambrose (1974) we found that the s p e c i f i c i t y , accuracy and p r e c i s i o n of t y r o s i n e d e t e r m i n a t i o n i n plasma was improved. The d e t a i l s of t h i s procedure are d e s c r i b e d below. M a t e r i a l s and Methods Animals and Management Blood samples used i n t h i s study were c o l l e c t e d from 15 Dorset X S u f f o l k c r o s s b r e d ewes aged 1-3 years and weighing 50-75 kg. The ewes were bred n a t u r a l l y and g e s t a t i o n a l records f o r a l l animals were kept. The feeding and maintenance of the ewes used i n t h i s and subsequent experiments was based on N a t i o n a l Research C o u n c i l f e e d i n g recomendations f o r pregnant ewes. A l l ewes were maintained on a d i e t of a l f a l f a cubes and whole b a r l e y u n t i l approximately 70 days of g e s t a t i o n at which time they were 30 moved t o separate metabolic cages and p l a c e d on i n d i v i d u a l f e e d i n g schedules. At t h i s time the ewes were fed a l f a l f a cubes (19.6 % crude p r o t e i n , 27.5% crude f i b e r ) twice d a i l y at 0800 and 1600 h. The average d a i l y consumption was 1.95 kg of cubes/day which p r o v i d e d approximately 18.58 KJ/g g r o s s energy. I o d i z e d block s a l t and water were p r o v i d e d ad l i b i t u m . Approximately four to s i x weeks p r i o r t o surgery the ewes were shorn and given suplementary i n t r a m u s c u l a r i n j e c t i o n s of vit a m i n s (500000 IU Vitamin A; 75000 IU Vitamin D3; and 50 IU Vit a m i n E: Rogas/STB products Inc., London, Ont.). Constant f l u o r e s c e n t l i g h t i n g was maintained f o r a l l animals and the room temperature was kept at 13.9°C (range 11.0 - 15.8) wet bulb and 16°C (range 12.9 - 17.9) dry bulb. S u r g i c a l Procedures F e t a l and maternal c a t h e t e r s were s u r g i c a l y implanted using p o l y e t h y l e n e c a t h e t e r s with s i l a s t i c t i p s (Intramedic "PE-50 and PE-90,"Clay Adams, Parsippany, N,J.) at approximately 110 days g e s t a t i o n . The ewes were s t a r v e d f o r 24 h p r i o r to surgery a f t e r which a n a e s t h e s i a was induced by the intravenous i n f u s i o n of t h i o p e n t a l sodium (Abbot) (20mg/kg body weight) f o r the purpose of i n t u b a t i n g the animal. General a n a e s t h e s i a was subsequently maintained with Halothane (Fluothane, A y e r s t ) and the ewe p l a c e d i n a supine p o s i t i o n . While m a i n t a i n i n g s t e r i l e c o n d i t i o n s a 10 - 15 cm m i d l i n e i n c i s i o n was made which alowed the g r a v i d uterus to be p a l p a t e d and the h i n d limbs of the f e t u s brought through the abdominal i n c i s i o n . C o n v e n t i o n a l v a s c u l a r c a t h e t e r i z a t i o n procedures were then used to i s o l a t e the f e t a l e x t e r n a l saphenous v e i n i n one limb i n t o which a c a t h e t e r was p l a c e d 20 31 cm so t h a t the t i p would l i e i n the i n f e r i o r vena cava. In the o p p o s i t e f e t a l limb both the pedal a r t e r y and e x t e r n a l saphenous v e i n were c a t h e t e r i z e d with the c a t h e t e r s passed approximately 15 cm i n t o each v e s s e l . In a d d i t i o n , from the same m i d l i n e exposure an u m b i l i c a l venous c a t h e t e r was passed through a c o t y l e d e n a r y v e i n . On o c c a t i o n , maternal c a t h e t e r s were p l a c e d i n the femoral a r t e r y v i a a s u p e r f i c i a l a u x i l i a r y a r t e r y on the i n s i d e f l a n k of the ewe and a l s o i n the u t e r i n e v e i n v i a an a u x i l a r y v e i n on the horn of the u t e r u s . S u t u r i n g and anchoring procedures, e x t e r i o i z a t i o n of the c a t h e t e r s and post s u r g i c a l c a r e of the p r e p a r a t i o n s have been d e s c r i b e d i n d e t a i l by K i t t s et a l . , (1979). A f i v e day post s u r g i c a l recovery p e r i o d was given before blood samples were withdrawn. A n a l y t i c a l Procedure Blood was c o l l e c t e d from pregnant ewes and t h e i r f e t u s e s through i n d w e l l i n g c a t h e t e r s i n t o h e p a r i n i z e d p l a s t i c s y r i n g e s and d i s p e n s e d immediately i n t o i c e c h i l l e d pyrex t e s t tubes c o n t a i n i n g EDTA. The tubes were immediately c e n t r i f u g e d a t 500 X g f o r f i v e minutes and the plasma was p i p e t t e d i n t o 10 X 75 mm c u v e t t s , s e a l e d and p l a c e d i n s e a l e d g l a s s c o n t a i n e r s at -20 °C f o r l a t e r a n a l y s i s . A stock s t a n d a r d s o l u t i o n of L - t y r o s i n e (Sigma Chem. Co. S t . L o u i s , Mo.) c o n t a i n i n g 50 vg/ml was made with double d e i o n i z e d d i s t i l l e d water and s t o r e d at 0-4°C. Working standards of 2.5, 5.0, 10.0 and 15.0 vg/ml were prepared f r e s h every week and were s u b j e c t e d to the same treatments as the plasma samples. For e s t i m a t i n g the recovery of 14 r a d i o a c t i v i t y , known q u a n t i t i e s of L-[U- C] t y r o s i n e (450 mCi/mmol; Amersham Chem.Co. O a k v i l l e Ont.) were added to each of 32 the working standards and t r e a t e d s i m i l a r l y . A l l plasma samples were a n a l y s e d i n d u p l i c a t e . The plasma samples were thawed and 400-500 v l were d e p r o t e i n i z e d by adding 1.0 ml of 10% t r i c h l o r o a c e t i c a c i d (TCA, w/v) and c e n t r i f u g e d a t 500 X g f o r 5 minutes. A f t e r p i p e t t i n g o f f the supernatant, the p r e c i p i t a t e was washed twice with 0.5 ml of 10% TCA. The combined supernatants c o n t a i n i n g the f r e e amino a c i d s were e x t r a c t e d with 5 ml of d i e t h y l ether to remove the TCA. The upper ether phase was withdrawn with a Pasteur p i p e t t e and d i s c a r d e d . The remaining ether was evaporated using f o r c e d a i r . To the d e p r o t e i n i z e d , e t h e r - e x t r a c t e d samples, 1.0 ml of c i t r a t e b u f f e r (pH 5.5) and 1.0 ml of L - t y r o s i n e decarboxylase (Sigma) s o l u t i o n (1 U/ml) prepared a c c o r d i n g to G a r l i c k and Marshall(1972) were added to convert the L - t y r o s i n e to tyramine. The mixture was incubated f o r 1 h at 37°C i n a slow moving shaker water bath. The tyramine formed in the r e a c t i o n was e x t r a c t e d i n t o e t h y l a c e t a t e ( c e r t i f i e d ACS, F i s h e r Chem. Co., N.J.). To each tube, 1 g of NaCl and 1 g of Na^O^(anhydrous) were added to r a i s e the pH to above 10. Three ml of e t h y l a cetate were added and the tubes were stoppered, i n v e r t e d and v i g o r o u s l y shaken 100 times. The tubes were c e n t r i f u g e d a t 300 X g f o r 1-2 minutes which produced an upper e t h y l a c e t a t e phase and a lower aqueous phase. I n i t i a l experiments i n which a s i n g l e e t h y l a c e t a t e e x t r a c t i o n s t e p was f o l l o w e d y i e l d e d a low recovery of added t y r o s i n e . A d d i t i o n a l e x t r a c t i o n steps were found to improve the re c o v e r y s u b s t a n t i a l l y with the o p t i m a l number of e x t r a c t i o n s found to be 10. T h e r e f o r e , t h i s step was repeated 10 times u s i n g 33 3.0 ml of e t h y l a c e t a t e every time. The upper e t h y l a c e t a t e phase a f t e r each e x t r a c t i o n was withdrawn with a Pasteur p i p e t t e and p l a c e d i n a 50 ml v o l u m e t r i c f l a s k c o n t a i n i n g 15 ml of chl o r o f o r m ( c e r t i f i e d ACS, F i s h e r ) and 3 ml of 1 :2000 H2SC>4. The 50 ml v o l u m e t r i c f l a s k s were stoppered, i n v e r t e d , v i g o r o u s l y shaken 100 times and l e f t to s e t t l e f o r 1-2 minutes. Under these c o n d i t i o n s , tyramine i s e x t r a c t e d i n t o the upper a c i d i f i e d aqueous phase which was subsequently t r a n s f e r r e d i n t o a 100 ml round bottom evaporation f l a s k . The e x t r a c t i o n s t e p i n v o l v i n g c h l o r o f o r m and a c i d i f i e d water was a l s o repeated 10 times to f a c i l i t a t e the q u a n t i t a t i v e e x t r a c t i o n of tyramine i n t o the upper aqueous phase. The second e x t r a c t i o n procedure r e s u l t s i n a t o t a l volume of 30 ml i n the round bottom f l a s k s . The f l a s k s were p l a c e d on a "rota-vap" f l a s h evaporator (Buchi, Rota-Vapor-R, type KRvr 65/45, Rinco I n s t . Co. Inc. G r e e n v i l l e , 111.) and a f t e r the volume was reduced to approximately 1-2 ml, the contents were t r a n s f e r r e d to a 5 ml vo l u m e t r i c f l a s k . The 100 ml round bottom f l a s k s were r i n s e d with 2 ml of 1:2000 H SO and the 2 4 r i n s e added to the 5 ml v o l u m e t r i c f l a s k . The f i n a l s o l u t i o n was then made up to 5.0 ml and an a l i q u o t (1.0 ml) was an a l y s e d f o r tyramine a c c o r d i n g to the f l u o r o m e t r i c procedure of Ambrose (1974). The reagents used f o r t h i s purpose were ob t a i n e d from the f o l l o w i n g sources: l - n i t r o s o - 2 - n a p h t h o l and sodium pyrophosphate (Na 2HP0 4 : 10 H 20, c e r t i f i e d ACS F i s h e r S c i . Co. N.J.) sodium n i t r i t e (Sigma) n i t r i c a c i d and phosphoric a c i d (ACS) ( A l l i e d Chemical Canada L t d . , Pointe C l a i r e , Quebec). Double d e i o n i z e d water was always used f o r the p r e p a r a t i o n of reagent s o l u t i o n s . 34 Another 1.0 ml aliquot was transferred to 10 ml of s c i n t i l l a t i o n fluor (PCS,Amersham) and the radioactivity counted in a liquid s c i n t i l l a t i o n counter (Packard Ins. Comp. Inc. Model A 300C, Downers Group, 111.). The fluorescence of the samples was measured in a fluorometer maintained at 20°C (G.K. Turner Associates, model 3 , Palo Alto, California) with a 354 nm primary f i l t e r and a 580 nm secondary f i l t e r . Students t test was used to compare differences between the effect of 1-3 versus 10 ethyl acetate and chloroform-acidified water extractions. Results and Discussion The standard calibration curve of fluorescence against varying concentrations of L-tyrosine obtained with the method described is shown in Fig. 1. The major difference between the present procedure and that previously described by Garlick and Marshall (1972) is in the number of ethyl acetate and chloroform:acidified water extractions used. Whereas Garlick and Marshall (1972) have used only one extraction with 10 ml of ethyl acetate, we have employed 10 extractions of 3 ml each. The recovery values of 97.6 ±1.6(SE) % for unlabelled and 94.5 ± 1.2 (SE) % for L-(U- 1 4C) tyrosine using 10 extractions are significantly (P <0.05) higher than those obtained when only 1-3 extractions were used (Table 1). The recovery of 1 4C-tyrosine in spiked plasma samples was not significantly different from the recovery obtained with spiked aqueous standards. Secondly, in the present study, the modified 1-nitroso-2-naphthol sodium n i t r i t e reagent reported by Ambrose (1974) for tyrosine determination was used rather than the original reagent 35 of Waalkes and Udenfriend (1957). Acording to Ambrose (1974) the a d d i t i o n of phosphoric a c i d to the r e a c t i o n mixture improves the s t a b i l i t y of the f l u o r o p h o r e . Using these m o d i f i c a t i o n s , t y r o s i n e c o n c e n t r a t i o n s as low as 2.5 vg/ml may be determined. The improvement in s e n s i t i v i t y i s p a r t i c u l a r l y advantageous in f e t a l s t u d i e s i n which o n l y small q u a n t i t i e s of blood can be withdrawn d u r i n g the course of isotope d i l u t i o n t echniques. Furthermore, the c u r r e n t procedure u s i n g Ambrose's method was found t o be more e f f i c i e n t i n terms of time than the f l u o r o m e t r i c method developed by Waalkes and U d e n f r i e n d (1957) and U d e n f r i e n d and Cooper (1952). Using the m o d i f i c a t i o n s f o r tyramine e x t r a c t i o n and f l u o r o m e t r i c a n a l y s i s , 16 d e t e r m i n a t i o n s can be done d a i l y . In terms of s p e c i f i c i t y , the a d d i t i o n of L - t y r o s i n e decarboxylase renders the method s p e c i f i c to L - t y r o s i n e . The e l i m i n a t i o n of contamination with D - t y r o s i n e o f f e r s an advantage over the method r e p o r t e d by Ambrose (1974). A c c o r d i n g to the manufacturer, L - t y r o s i n e decarboxylase w i l l a l s o r e a c t with p h e n y l a l a n i n e . In t h i s study, p h e n y l a l a n i n e , when added at a c o n c e n t r a t i o n of 25 yg/ml, caused l e s s than 1% i n t e r f e r e n c e . The c o e f f i c i e n t of v a r i a t i o n f o r the recovery v a l u e s from both c o l d t y r o s i n e and 1 4 C t y r o s i n e (Table 1) was 6.84% and 5.01% , r e s p e c t i v e l y . T h i s i s comparable to the p r e c i s i o n o b t a i n e d with other amino a c i d techniques such as ion exchange (Blackburn, 1968). Furthermore, the l i n e a r i t y of the standard curve o b t a i n e d with t h i s technique ( F i g . 1) w i t h an r 2 v a l u e of 0.9937 makes the method r e l i a b l e f o r the range of t y r o s i n e c o n c e n t r a t i o n s normally encountered i n the plasma. 36 The present method has been applied to the determination of tyrosine concentrations in plasma samples c o l l e c t e d from f e t a l and maternal vascular s i t e s of ewes between 120 and 130 days of gestation (Schaefer and Krishnamurti, I982b,c). The mean concentration (-SE) of tyrosine in maternal plasma for jugular vein, femoral artery and uterine vein samples was, respectively + + + 50.7-4.2, 55.2-3.3, and 38.5 -4.6 v m o l s / l i t e r . In the f e t a l plasma, tyrosine values for umbilical vein, umbilical artery, i n f e r i o r vena cava and saphenous vein were 103.7-5.5, 72.1-12.0, 93.2^7.6 and 70.0-5.6, respectively. The values obtained were comparable to those reported by other workers, using methods based on ion exchange chromatography (Table 2) (Hopkins et a l . , 1971; Lemons et a l . , 1976; Smith et a l . , 1977; Slater and Mellor, 1979; Holzman et a l . , 1979). The present method has the added advantage that for c a l c u l a t i n g the s p e c i f i c r a d i o a c t i v i t y , the concentration and the a c t i v i t y can be measured simultaneously. I I 1 5 10 15 Tyrosine Concentration pg/ml Legend f o r Figures F i g . l . Standard curve of L - t y r o s i n e determined by enzymatic i s o l a t i o n and f l u o r o m e t r i c a n a l y s i s . A • observed f l u o r o m e t r i c readings * standard d e v i a t i o n at given c o n c e n t r a t i o n s . - p r e d i c t e d best f i t l i n e where y • 2.2998x + 5.1661. The r2 value f o r the p r e d i c t e d l i n e i s 0.9937. Table 1: Recovery of co ld and labe l l ed L- tyros ine as af fected by the number of ext ract ions COLD TYROSINE L - [U -1 4 C] TYROSINE Number of Ext rac t ions Tyrosine Added Number of Samples Analysed Recovery % Standard Er ror Coe f f i c i en t o f Va r i a t i on 1 - 3 10 2.5-15.0 pg/ml 14 28.0 i 4.8 64.7% 17 97.6 i 1.6 6.8% 1 - 3 352700 dpm 11 14.0 i 2.7 63.8% 10 3500 dpm 16 94.5 i 1.2 5.0% Table 2: Mean concentration of tyrosine i n the plasma (umols/liter) obtained from different vascular sites in the pregnant ewe and fetus at 120-130 days of gestation EWE FETUS Study Method of Analysis JV MA Vascular Site UTV UV UA IVC SV Present Enzymatic Study Hopkins et a l . Ion 1971 Exchange Lemons et a l . Ion 1976 Exchange Smith et a l . Ion 1977 Exchange Slater and Ion Mellor 1979 Exchange Holzman et a l . Ion 1979 Exchange 50.7 - 4.2 38 55.2 - 3.3 33.4 100 50 78 38.5 - 4.6 15.9 75 103.7 72.1 - 5.5 -12.0 94.8 90.8 214 201 120 93.2 - 7.6 70.0 -5.6 (SE) 89 (mixed fetal blood) * JV = jugular vein, MA = maternal artery, UTV = uterine vein, UV = umbilical vein UA = umbilical artery, SV = external saphenous vein, IVC = inferior vena cava 40 Experiment ( I I ) : I r r e v e r s i b l e Loss and Net U t i l i z a t i o n of T y r o s i n e I n t r o d u c t i o n The n i t r o g e n requirement of the growing f e t u s i s met p r i m a r i l y by the uptake of f r e e amino a c i d s o r i g i n a t i n g from the maternal c i r c u l a t i o n (Lemons, 1979). I t has been c o n s i s t e n t l y demontrated t h a t the c o n c e n t r a t i o n of f r e e amino a c i d s i s higher i n the f e t a l than i n the maternal plasma (Hopkins et a l . , 1971; H i l l and Young, 1973; Cockburn and G i l e s , 1977; Smith et a l . , 1977; S l a t e r and M e l l o r , 1979). Young et a l . , (1979) have suggested that the higher plasma amino a c i d c o n c e n t r a t i o n i n the f e t u s i n d i c a t e d a f a s t e r turnover r a t e of the t i s s u e p r o t e i n s f o l l o w i n g t h e i r d e p o s i t i o n i n the f e t u s . The present experiment was undertaken to determine the turnover r a t e of t y r o s i n e f o l l o w i n g the i n f u s i o n of l a b e l l e d t y r o s i n e i n t o the f e t a l and/or maternal c i r c u l a t i o n s and to f u r t h e r d i s t i n g u i s h the t y r o s i n e net u t i l i z a t i o n i n f e t a l and non f e t a l t i s s u e s by a p p l y i n g a two pool k i n e t i c model. M a t e r i a l s and Methods Animals and management as w e l l as s u r g i c a l procedures have been d e s c r i b e d i n experiment I (pp29-30). Experimental Procedure F o l l o w i n g a f i v e day post s u r g i c a l recovery p e r i o d , the p h y s i o l o g i c a l s t a b i l i t y of the p r e p a r a t i o n s was v e r i f i e d by m o n i t o r i n g blood gas, blood pH, hematocrit and hemoglobin v a l u e s i n both the ewe and the f e t u s , as w e l l as body temperature i n the ewe. L-[U- 4C] t y r o s i n e (50 pCi) or L-[2,3,5,6- 3H] t y r o s i n e (300 uCi) New England Nuclear, Quebec) was i n f u s e d i n t o the 41 c e n t r a l venous system of the f e t u s f o r a p e r i o d of e i g h t hours without a priming dose using a constant i n f u s i o n pump (Sage 3 14 model 352, Cambridge, Mass.). Both the H and C t y r o s i n e i n f u s a t e s were con t a i n e d i n 20ml of s t e r i l e p h y s i o l o g i c a l s a l i n e and i n f u s e d at a r a t e of 2.2ml/h (which i s approximately 5.5 yCi 4 3 C and 33 CCi H / h ) . In f i v e of the p r e p a r a t i o n s (98,110,690,444 and 453), simultaneous i n f u s i o n s were given so that the f e t u s was i n f u s e d with"''4 C t y r o s i n e and the ewe with H t y r o s i n e . In two p r e p a r a t i o n s (102 and 97), the f e t u s and ewe were i n f u s e d i n d i v i d u a l l y on separate days with an i n t e r v a l of 48h. P r e l i m i n a r y experiments have i n d i c a t e d that no r e s i d u a l a c t i v i t y i n the plasma c o u l d be d e t e c t e d i n the maternal or f e t a l plasma a f t e r 48h f o l l o w i n g ^ C or ^ H t y r o s i n e i n f u s i o n s . In the remainder of the p r e p a r a t i o n s , l a b e l l e d t y r o s i n e was i n f u s e d e i t h e r i n t o the f e t u s or i n t o the ewe f o r the purpose of c a l c u l a t i n g i r r e v e r s i b l e l o s s o n l y . The d e t a i l s of i s o t o p i c a d m i n i s t r a t i o n are given i n Table 3. 42 Table 3. ratal and aatamal body weight, gestational age, Isotope infusion procedures and irreversible loss values of tyrosine Anlaal • tody weight (kg) Oastatlonal Labelled Method of age (days) tyrosine Infusion Infusion Sampling site site Irreversible loss aaol/day/kg IVC SV 5.003 JVCL) JVCR) 1.008 UA IVC 7.1S0 JV(R) JVCL) 0.603 IVC sv 10.S30 JVCL) JV(R) 1.044 UV IVC 3.540 JV(R> JVCL) 0.655 UV IVC 3.010 JV(R) JVCL) 0.677 IVC UV 4.490 4.090 — — — UV IVC 5.700 IVC UA 5.710 98 retus 102 retus 97 retus Ewe retus 110 Ewe retus 110 Ewe (starved) retus 73 71 retus 103 retus 107 retus 106 112 690 444 453 retus Ewe retus Ewe retus l Ewe retus Ewe retus 2.45 68.4 0.88 •4.4 1.93 74.3 2.11 52.3 2.35 49.4 1.81 S3.1 3.29 62.5 1.62 70.1 2.10 72.7 1.23 67.8 1.56 SS.l 1.13 59.0 1.10 59.4 1.39 81.6 120 115 125 120 122 121 117 116 125 127 122 117 119 120 14, 14, Simultaneous 48 h apart 48 h apart Simultaneous Simultaneous *H 14, C « H retus only SH retus only 'H retus only 'H retus only Ewe only Ewe only Simultaneous Simultaneous Simultaneous H 14C »H M C 14„ UV JV(R) JV(R) IVC JVCL) IVC JV(R) SVC JV(R) IVC SV JVCL) IVC JVCL) UA rA UA rA UA JVCL) 8.940 1.237 1.171 5.112 1.316 0.636 1.100 8.283 0.489 MEAN 2 SEl retus 5.79 *0.78 0.96 -0.09 Mean values do not include starved data from #110 IVC-lnferlor vena cava. UA-ueblllealCpedal) artery, UV.umbllicel vein, SV. saphenous vein, JVajugular vein, rAamaternal femoral artery 43 The ewes were allowed free movement in the metabolism cages and normal feeding routines were continued throughout the experiment. During the continuous infusion of tyrosine, blood samples were withdrawn simultaneously from the various catheters at 1.5, 3, 4.5, 6, 7, 7.5 and 8 hr aft e r beginning the infusion and treated as described in experiment I (pp31-32). The t o t a l blood withdrawal over the 8h did not exceed 20 and 18 ml for the ewe and fetus, respectively. A l l blood samples were placed in c h i l l e d pyrex tubes containing EDTA and centrifuged immediately. o The plasma was kept at -20 C u n t i l analyses were ca r r i e d out. A n a l y t i c a l Methods Hemoglobin was determined according to the cyanomethemoglobin method (Hycel Inc.', Houston). Hematocrit was determined by the microhematocrit procedure. L-tyrosine in plasma was quantitatively converted to tyramine by the addition of tyrosine decarboxylase (EC 4.1.1.25: Sigma Chemical Co., Missouri). The tyramine was extracted and analysed by the procedure described under "Experiment I". The r a d i o a c t i v i t y of plasma tyrosine was, measured by placing 1 ml of the extracted tyramine solution into 10 ml of s c i n t i l l a t i o n fluor (PCS, Amersham) and counting in a Packard s c i n t i l l a t i o n counter (Model A300C) using the channel r a t i o method. The method i s s p e c i f i c to L-tyrosine free from contamination by other metabolites. Whole blood samples for determining blood gas and pH was withdrawn from the respective catheters into 1 ml heparinized p l a s t i c syringes and analysed immediately in t r i p l i c a t e on a Radiometer d i g i t a l acid-base analyser (Model pHM72, Radiometer, Copenhagen). 44 Results and Discussion The mean values of the physiological parameters of the ewe and fetus during the experimental period which commenced 5 days after surgery are presented in Table 4. 45 Tabl e 4. Mean p h y s i o l o g i c a l parameters f o r the f e t u s and ewe Parameter Fetus i (N=ll) Ewe (N=ll) Mean SE Mean SE Blood pH 7.39 0.03 7.49 0.04 Blood pC0 2 (mmHg) 33.7 1.8 28.3 1.3 Blood pC»2 (mmHg) 16.5 1.8 24.9 , 0,6 Hct % 34.4 1.4 32.1 1.1 Hb (g%) 10.1 0.5 11.3 0.4 Body temperature (°C) 38.8 0.1 F e t a l b l o o d samples were o b t a i n e d from the i n f e r i o r vena cava. Ewe b l o o d samples were o b t a i n e d from the j u g u l a r v e i n . 46 These values are within the range reported for apparently normal fetuses by many workers (Clapp et a l . , 1980; Battaglia and Meschia, 1978; K i t t s and Krishnamurti, 1982a). Following the 14 continuous infusion of C tyrosine into the fetus, the plasma s p e c i f i c a c t i v i t y gradually increased reaching a plateau after 4 h (Fig.2). IT >1 •p > 0) •rl C •P •rl O (0 < o n o >1 •H •p M-l •H cn O Q) Q* g W & 0) c •rl CO 0 u En 300 J 200 J 100 J f e t u s Blood * * C O , Figure 2, Changes i n s p e c i f i c a c t i v i t y of plasma tyrosine after i n f u s i o n of L-[U- 1 4C3 tyrosine into the fetus (#73). * dpm/200 y l of whole blood (see experiment III for reference to 1 4 C 0 2 data). F=fetus. S o l i d l i n e s f i t t e d by regression equation defined by data points, 48 However, the infusion was continued for 8 h and the mean s p e c i f i c a c t i v i t y of the 4 data points between 6 and 8 h was used to calculate the kinetic parameters. The s p e c i f i c a c t i v i t y of 1 4 C tyrosine in the maternal plasma aft e r the f e t a l plateau had been reached was only 6-7% of 3 that of the fetus. When H tyrosine was infused into the ewe (Fig. 3) the s p e c i f i c a c t i v i t y in the maternal plasma increased gradually and reached a plateau in 6h. •P •r | > •r l •P O C •r l (0 O U >1 •P u •r l m •rl 0) a co e c •rl 10 o u >1 EH s o o 200 -1 100 J I 3 I I 4 5 T I M E ( h ) 7 8 F i g u r e 3. Changes i n s p e c i f i c a c t i v i t y o f plasma t y r o s i n e a f t e r i n f u s i o n o f L-[2,3,5,6- 3H] t y r o s i n e i n t o the ewe (#106). S o l i d l i n e s f i t t e d by r e g r e s s i o n e quation d e f i n e d by data p o i n t s . 50 However, the p l a t e a u s p e c i f i c a c t i v i t y of ~H i n the f e t a l plasma was 60-90% of that of the ewe, i n d i c a t i n g the r a p i d t r a n s f e r of t y r o s i n e from the maternal to the f e t a l c i r c u l a t i o n . The i r r e v e r s i b l e l o s s of t y r o s i n e , obtained by d i v i d i n g the r a t e of i n f u s i o n of the t r a c e r by the steady s t a t e s p e c i f i c a c t i v i t y i n d i c a t e d mean r a t e s f o r a l l p r e p a r a t i o n s of 0.96 and 5.79 mmol/d/kg i n the ewe and f e t u s r e s p e c t i v e l y (Table 3.). The i n utero growth of the f e t u s i s dependent upon the t r a n s f e r of n u t r i e n t s from the maternal system. Q u a n t i t a t i v e measurement of the uptake of n u t r i e n t s by the u m b i l i c a l c i r c u l a t i o n have been t r a d i t i o n a l l y determined by the a p p l i c a t i o n of the F i c k p r i n c i p l e whereby the u m b i l i c a l venous-a r t e r i a l c o n c e n t r a t i o n d i f f e r e n c e i s m u l t i p l i e d by the blood flow ( B a t t a g l i a and Meschia, 1978). More r e c e n t l y , i s o t o p i c procedures have a l s o been used to determine the q u a n t i t a t i v e t r a n s f e r of n u t r i e n t s to the f e t u s ( P r i o r and C h r i s t e n s o n , 1979; Hodgson et a l . , 1980; K i t t s and K r i s h n a m u r t i , 1982a). As p o i n t e d out by Hodgson et a l . , (1980), methods based on the F i c k p r i n c i p l e c o u l d r e s u l t i n an underestimation of f e t a l n u t r i e n t u t i l i z a t i o n u nless the maternal t r a n s f e r i s the only source of n u t r i e n t s and there i s no f e t a l endogenous or de novo s y n t h e s i s . Conversely, the use of i s o t o p i c procedures i n which the f e t a l n u t r i e n t u t i l i z a t i o n i s c a l c u l a t e d as simply i r r e v e r s i b l e l o s s (or e n t r y r a t e s ) c o u l d r e s u l t i n over-e s t i m a t i o n , u n l e s s the r e t u r n of the n u t r i e n t to the mother from the f e t u s i s n e g l i g i b l e . By the simultaneous i n f u s i o n of 3 H and 1 4 C l a b e l l e d compounds i n t o the ewe and f e t u s , r e s p e c t i v e l y , endogenous 51 production, net u t i l i z a t i o n and placental exchange may be calculated by using a two compartment model (Fig. 4 ) (Shipley and Clark, 1972). 52 ENDOGENOUS PRODUCTION F. ENDOGENOUS PRODUCTION UMBILICAL EXCHANGE NET UTILIZATION NET UTILIZATION Figure 4. Schematic i l l u s t r a t i o n of f e t a l - maternal two compartment model. M=Ewe, F=Fetus,(see figure 5 and appendix 3 f o r d e f i n i t i o n of terms) 53 T h i s approach to f e t a l metabolism and i t s experimental v a l i d i t y have been r e c e n t l y d e s c r i b e d i n d e t a i l with r e f e r e n c e to glucose metabolism (Hodgson et a l . , 1980). The two compartment model i s u s e f u l i n a s s e s s i n g the u n i d i r e c t i o n a l u t i l i z a t i o n of m e t a b o l i t e s by the f e t a l and n o n - f e t a l t i s s u e s . The l a t t e r would c o n s i s t of the maternal t i s s u e s p l u s the p l a c e n t a . The e s t i m a t i o n of the i n v i v o k i n e t i c parameters of t y r o s i n e metabolism i n the ewe and f e t u s by using the two compartment model are given i n Table 5 and are estimated a c c o r d i n g to the c a l c u l a t i o n s o u t l i n e d i n F i g . 5. 54 Calculations (1) The production rates of tyrosine were calculated by (a) sampling the l a b e l l e d pool, (b) l a b e l l i n g one pool and sampling another and (c) dissection of flow rates. (a) (b) (c) m m F F f ( m f ) = F + F ( — ) Eq 1 'Wrf m ° f ° F m f + F o f 9 1 a m ™f"4 - ' F T W - ' " F f o + Fmo<F-%-' E<> 2 f f m om fm om a f m a f Where: superscripts r e f e r to infused pool and subscripts refer to sampled pool PR m and PR^ = production rates i n the ewe and fetus, respectively (see appendix 3) r = rate of infusion of tracer = plasma steady state s p e c i f i c a c t i v i t y of tyrosine F^ and = endogenous production rates i n the ewe and fetus, respectively F and F - = net u t i l i z a t i o n rates i n the ewe and fetus, om of . . , respectively F_ = rate of transfer from ewe to fetus fm F c = rate of transfer from fetus to ewe mr Also from F i g . 4: Fmf Fmf F r- ¥ j- +F r mf of fo fm Ffm Ffm and Eq 3 F j. +F F +F -fm om mo mf Since both the values f o r the f r a c t i o n s on the l e f t side of the equation can be obtained from Eq 1 and Eq 2, the remaining values F- . F , . F Fmn and F- can be solved mr rm om or mo to simultaneously. Figure 5: K i n e t i c calculations for a two pool model Table 5. E s t i m a t i o n of i n v i v o k i n e t i c parameters of t y r o s i n e metabolism a f t e r c o n t i n u o u s i n f u s i o n o f l a b e l l e d t y r o s i n e i n t o the f e t u s and ewe Animal # Iso t o p e I n f u s e d endogenous P r o d u c t i o n ( F . o r F ) f o mo Net U t i l i z a t i o n (F .or F ) of om mmol/day/kg mmol/day/kg P l a c e n t a l Exchange F f m Fmf mmol/day/kg Net P l a c e n t a l T r a n s f e r < Ffm * Fmf > mmol/day/kg Fetus 1 4 C 1.051 4.460 98 Ewe 3H 1.121 0.999 Fetus 3H 2.393 4.280 102 Ewe 3H 0.595 0.576 Fetus 3H -0.898 8.182 97 Ewe 3H 1.087 0.842 Fetus 1 4 C -0.144 3.381 110 Ewe 3H 0.682 0.540 Fetus 1 4 C 2.824 1.691 110 Ewe ( s t a r v e d ) 3H 0.650 0.703 Fetus 1 4 C 0.512 23.991 690 Ewe 3H 1.303 0.852 Fetus 1 4 C 0.013 0.331 444 Ewe 3H 1.099 1.093 Fetus 1 4 c 6.084 0.345 453 Ewe 3 H 0.389 0.487 Mean * S E 1 F e t u s 1.29 • 0.89 6.42 Ewe 0.90 • 0.13 0.77 4.031 0.022 2.869 0.010 12.765 0.096 3.725 0.202 60.447 0.627 0.008 0.064 0.708 0.006 3.050 — 0.150 3.406 1.884 9.081 3.525 -1.134 23.479 0.318 -5.739 5.14 i 3.48 0.09 0.14 i 0.10 1 mean v a l u e s do not i n c l u d e d a t a from s t a r v a t i o n experiment (#110) 56 The f r a c t i o n of t y r o s i n e t r a n s f e r r e d from the f e t u s to the ewe (F m f / F mf+F and from the ewe to the f e t u s (F _ / F , +F ) mr mr om f m f^, o m are 0.369 and 0.118 of the f e t a l and maternal turnover r a t e s , r e s p e c t i v e l y . There i s a net p l a c e n t a l t r a n s f e r of 5.4 mmol/d/kg from the ewe to the f e t u s . The endogenous p r o d u c t i o n r a t e s of t y r o s i n e i n the ewe (F ) and f e t u s (F_ ) are 0.90 and 1.29 mo fo mmol/d/kg, r e s p e c t i v e l y . The f e t a l and maternal net t y r o s i n e u t i l i z a t i o n r a t e s which represent the q u a n t i t y of t y r o s i n e used per u n i t time f o r o x i d a t i v e and s y n t h e t i c purposes are 6.42 and 0.77 mmol/d/kg r e s p e c t i v e l y . These r e s u l t s are summarized i n Table 5. The a p p l i c a t i o n of the two compartment model as d e s c r i b e d i s capable of d i s t i n g u i s h i n g the metabolism of t y r o s i n e as e i t h e r o c c u r r i n g i n f e t a l or i n e x t r a f e t a l t i s s u e s . U n l i k e the values based on i r r e v e r s i b l e l o s s alone, the k i n e t i c parameters obt a i n e d u s i n g the model d e s c r i b e d i n F i g u r e 4 take i n t o account the exchange of s u b s t r a t e s between the mother and the f e t u s , as w e l l as the endogenous p r o d u c t i o n by the f e t u s . The net u t i l i z a t i o n of 6.42 mmol/d/kg of t y r o s i n e by the f e t u s t h e r e f o r e r e p r e s e n t s the t i s s u e requirements of the f e t u s f o r metabolic purposes. The r e s u l t s of the present study i n d i c a t e d that there was an endogenous p r o d u c t i o n of 1.29 mmol/d/kg of t y r o s i n e by the f e t u s and that maternal t y r o s i n e i s not the only source. The presence of enzymes f o r the co n v e r s i o n of p h e n y l a l a n i n e to t y r o s i n e has been demonstrated i n human f e t a l l i v e r ( D e l v a l l e and Greengard, 1977) and p h e n y l a l a n i n e i s l i k e l y the source of endogenous t y r o s i n e . T h i s may e x p l a i n the lower u m b i l i c a l uptake 57 of approximately 3 mmol/d/kg r e p o r t e d by Lemons et a l . (1976) on the b a s i s of F i c k p r i n c i p l e compared to the net u t i l i z a t i o n of 6.42 mmol/d/kg observed i n the present study. The present study a l s o demonstrated a r e t u r n of t y r o s i n e to the maternal c i r c u l a t i o n which, as mentioned e a r l i e r , would suggest that the i r r e v e r s i b l e l o s s v a l u e s c o u l d over-estimate the f e t a l t y r o s i n e use. 58 Experiment ( I I I ) : Tyrosine U t i l i z e d for Oxidation and Synthesis Introduct ion It was observed by Lemons et a l . , (1976) that in the ovine fetus, the in utero placental transfer of amino acids measured from umbilical venous-arterial concentration differences times the blood flow i s in excess of the quantity of amino acids deposited as protein in a given time. Gresham et a l . , (1972) studied the rate of urea excretion by the sheep fetus and concluded that amino acid catabolism may account for approximately 25% cf the oxygen consumption. Based on these observations, Battaglia and Meschia (1978) and Lemons (1979) have concluded that a considerable proportion of amino acids taken up by the fetus i s used for oxidative purposes. However, only meagre information i s available on the direct assessment of the extent to which amino acids are oxidized by the ovine fetus ( K i t t s and Krishnamurti, 1982a,b; Meier et a l . , 1981) and the factors which modify the proportion of amino acids used for oxidation versus synthesis. This information i s p a r t i c u l a r l y relevant to animal production because i t has been shown that n u t r i t i o n a l and other environmental factors affect intra-uterine growth which in turn influences b i r t h weight and post-natal development (Cheek, 1975; Metcoff, 1980). Data on the maternal-f e t a l exchange of tyrosine as well as the extent of i t s oxidation calculated from the plateau s p e c i f i c a c t i v i t y of blood 14 . . . C0 2 are presented in t h i s section. Methods Animal care and infusion procedures were as described under Experiment I (pp29-30) and experiment II (pp 43 ). The C0 o 59 content of whole blood samples was determined in 6 preparations by measuring the PC0 2 and the values converted to quantity of 14 CC>2 using the Henderson-Hasselbach equation. The C0 2 content of the whole blood was determined by placing 200yl of freshly drawn blood into Warburg flasks which contained 300yl of hyamine hydroxide (1M) in the center well (Biochemicals Corp., Cleveland) and 500^1 of 30% perchloric acid in the side arm (Ki t t s and Krishnamurti, 1982c). The flask was sealed and the 14 C0 2 present in the sample was liberated by adding the perchloric acid. After incubation for 1 h the hyamine solution was transferred to a s c i n t i l l a t i o n v i a l containing 10ml of PCS s c i n t i l l a t i o n fluor and counted for r a d i o a c t i v i t y . Calculations (1) Fraction of C0 2 from tyrosine oxidation f 1 4 ^ a f C 0 2 4 1 4 C - t y r (2) Rate of tyrosine oxidation = Fraction of C0 2 from tyrosine oxidation X C0 2 production rate Where: C0 2 production rate refers to a value of 8.9 - 2.7 ml/min/kg calculated from f e t a l venous-arterial pC0 2 and pH values using the Henderson-Hasselbalch equation and blood flow estimated according to Setchell and Hinks (1967) (see appendix 5 and 6) . .,. j / o v Rate of tyrosine oxidation (3) Tyrosine oxidized (%) = J Net u t i l i z a t i o n rate of tyr 60 Results Following the infusion of ^ 4 C tyrosine into the fetus the 14 s p e c i f i c a c t i v i t y of CO,, in blood was found to reach a plateau after about 6 h (Fig. 2). From the ra t i o of the plateau s p e c i f i c a c t i v i t y of "'"^CO 14 and C tyrosine, the per cent of tyrosine used for oxidation was calculated to be 5.2 - 1.5% (n=6). This would leave approximately 95% of the tyrosine p o t e n t i a l l y available for protein synthesis in the fetus (Fig. 6). For examples i l l u s t r a t i n g the cal c u l a t i o n of tyrosine oxidation, C0 2 production rates and blood flow used in t h i s experiment the veader i s referred to appendix tables 4, 5 and 6. Di scussion In the present study we have used the r a t i o of the plateau 14 14 s p e c i f i c a c t i v i t y of blood C0 2 and C tyrosine to quantitate the proportion of tyrosine u t i l i z e d by the fetus for oxidative purposes. While th i s methodology has been used extensively in adult human and animal experiments, i t has only recently been applied to the fetus (Meier et a l . , 1981; K i t t s and Krishnamurti, 1982c). Depending on the methodology employed, C0 2 production rates in the fetus have been reported to range from 5.65 (James et a l . , 1972) to 13.5 ml/min/kg (Ki t t s and Krishnamurti, 1982c). Therefore, the fr a c t i o n of tyrosine oxidized would be in the range of 3-8%. The value of 5.2% obtained in t h i s study i s in agreement with the recent observation of Meier et a l . , (1981) who reported that 9% of lysine was oxidized by the sheep fetus in continuous infusion studies. Conversely, approximately 27% of alanine was found to 61 be oxidized by the fetus in single i n j e c t i o n experiments (Kitts and Krishnamurti, 1982c). The extent to which different amino acids are used for oxidation would depend upon the i r s p e c i f i c metabolic function. The kinetic analysis following continuous infusion of l a b e l l e d tyrosine may also be used for estimating the rate of protein synthesis in the fetus using the calcuations of Waterlow et a l . , (1978) For t h i s purpose, i t i s necessary to know the tyrosine concentration in the entire f e t a l body protein. These procedures w i l l be discussed in the next section. 62 ENDOGENOUS PRODUCTION 0.90 m m o l / d a y / k g i ENDOGENOUS PRODUCTION 1.29 m m o l / d a y / k g M I NET PLACENTAL TRANSFER 5.14 mmo l / day /k NET UTILIZATION 0.77 m m o l / d a y / k g I NET UTILIZATION 6.42 mmo l / day /kg 94.8% PROTEIN SYNTHESIS 5.2% OXIDATION Figure 6 . The rate of production and u t i l i za t ion of tyrosine 1n the ewe and the fetus and the rate of net placental transfer j_n utero . M*Ewe, F«Fetus. 63 CHAPTER I I I . WHOLE BODY AND TISSUE FRACTIONAL PROTEIN SYNTHESIS IN THE OVINE FETUS IN UTERO Experiment ( I V ) : The C a l c u a t i o n of T i s s u e F r a c t i o n a l P r o t e i n S y n t h e s i s I n t r o d u c t ion The importance of maternal n u t r i t i o n and environmental c o n d i t i o n s on the growth of the f e t u s and p o s t n a t a l development (Robinson, 1977, 1981; Richardson, 1978; Art h u r , 1981; McDonald et a l . , 1981; M e l l o r and Murray 1981, 1982) has l e d to the development of techniques f o r the measurement of i n t r a - u t e r i n e growth. Morphometric changes i n the sheep f e t u s have been measured at d i f f e r e n t stages of g e s t a t i o n under c h r o n i c ( M e l l o r and Matheson, 1979) or acute c o n d i t i o n s (Koong et a l . , 1975; Robinson and McDonald, 1979). The r a t e of p r o t e i n d e p o s i t i o n i n the sheep f e t u s has been determined by the comparative s l a u g h t e r technique (Lodge and Heaney 1973; Rat t r a y et a l . , 1974, 1975; Broad and Davies, 1981). In a d d i t i o n , the r e t e n t i o n of alpha amino n i t r o g e n was estimated i n c h r o n i c a l l y c a t h e t e r i z e d f e t u s e s from u m b i l i c a l v e n o - a r t e r i a l c o n c e n t r a t i o n d i f f e r e n c e s (Lemons et a l . , 1976; Faber and Woods, 1981). The r a t e of p r o t e i n s y n t h e s i s has a l s o been estimated i n the f e t u s i n utero by the use of i s o t o p i c d i l u t i o n techniques ( Meier et a l . , 1981; Noakes and Young, 1981;Schaefer and Kri s h n a m u r t i , 1982b). T h i s technique has been e x t e n s i v e l y used f o r s t u d y i n g the r a t e of whole body p r o t e i n s y n t h e s i s under i n v i v o c o n d i t i o n s i n post n a t a l l i f e i n man (James et a l . , 1976) p i g ( G a r l i c k et a l . , 1976; Simmons et a l . , 1978; Reeds et a l . , 1980) sheep (Buttery et a l . , 1977; Davis et a l 1981, Bryant and 6 4 Smith, (1982) and c a t t l e (Lobley et a l . , 1980). B a s i c a l l y , a l a b e l l e d amino acid i s infused continuously into the venous system u n t i l the s p e c i f i c a c t i v i t y of the amino acid in the plasma reaches a plateau. The rate of protein synthesis i s estimated from the amino acid flux ( i r r e v e r s i b l e loss) a f t e r adjusting for oxidative l o s s . The v a l i d i t y of the assumptions made in the calculations and the application of the technique under a variety of n u t r i t i o n a l states have been c r i t i c a l l y reviewed (Waterlow et a l . , 1978; Reeds and Lobley, 1980). Apart from the inherent l i m i t a t i o n of using the plateau s p e c i f i c r a d i o a c t i v i t y for calcuating protein synthesis, the application of t h i s technique to the f e t a l system i s further complicated by the b i d i r e c t i o n a l transfer of nutrients between the mother and the fetus (Anand et a l . , 1979; K i t t s and Krishnamurti, 1982a). Under these conditions the flux, calculated from the plasma s p e c i f i c a c t i v i t y of compounds in the foetal compartment alone, would not t r u l y r e f l e c t the u n i d i r e c t i o n a l u t i l i z a t i o n by the fetus. To overcome t h i s problem, a technique based on a two pool kinetic model was proposed by Hodgson et a l . , (1980) in which d i f f e r e n t l y l a b e l l e d isotopes are introduced simultaneously into the maternal and f e t a l systems. The net u t i l i z a t i o n of substrate by the fetus i s obtained from the sum of the net placental exchange and endogenous production. Recently, we have published data (Schaefer and Krishnamurti, 1982a; also see experiment II and III) on the kinetic parameters of tyrosine u t i l i z a t i o n by the sheep fetus in 6 5 utero using the procedure of Hodgson et a l . , (1980). The objective of this study was to use these parameters in conjunction with the extent of tyrosine oxidation(experiment III) to estimate the whole body and f r a c t i o n a l rates of protein synthesis in the ovine fetus under in utero conditions. Materials and Methods Animals Eighteen Dorset X Suffolk ewes of known gestational age were used in t h i s study. Animal management and surgical procedures have been described in experiment I. Protein Turnover Studies To avoid the influence of post surgical trauma on protein turnover, infusions were c a r r i e d out after a minimum of fiv e days following surgery. During t h i s period the physiological s t a b i l i t y of the preparations was v e r i f i e d by monitoring blood gas, pH, hematocrit, hemoglobin and plasma metabolites (Table 6). The protocol of the isotopic infusions has been described in d e t a i l previously (Schaefer and Krishnamurti, 1982a; also see experiment II, I I I , and appendix 1). Analysis of Tissues For estimating the tissue f r a c t i o n a l protein synthetic rates, four of the preparations (#71, 73, 103, 107) were s a c r i f i c e d at the end of the infusion of tyrosine into the i n f e r i o r vena cava of the fetus. The ewe was anesthetised with sodium pentothal and the fetuses were delivered by laparotomy. The infusion was continued u n t i l the umbilical cord was l i g a t e d . The fetus was towel dried, weighed, and the whole carcass transfered to a l i q u i d nitrogen container for quick freezing. 66 F o l l o w i n g l i q u i d n i t r o g e n f r e e z i n g the f e t a l c a r c a s s was s t o r e d i n double s e a l e d p l a s t i c c o n t a i n e r s at - 2 0 ^ f o r approximately four months p r i o r to t i s s u e a n a l y s i s . For t i s s u e a n a l y s i s , the f r o z e n f e t a l c a r c a s s was p l a c e d i n a c o n t a i n e r of crushed i c e and t i s s u e samples of between 3-5 g were d i s s e c t e d with bone s c i s s o r s from the s t i l l f r o z e n c a r c a s s . These froz e n t i s s u e samples were r i n s e d b r i e f l y i n s a l i n e and b l o t t e d dry to remove any p o s s i b l e e x t e r n a l contamination from blood. For the g a s t r o i n t e s t i n a l samples, care was taken to r i n s e the lumen s i d e of the t i s s u e as w e l l to remove any contamination from the lumen c o n t e n t s . The samples were then s e c t i o n e d with d i s s e c t i n g s c i s s o r s and s c a l p e l to obtaine subsamples of approximately 0.1 g which were immediately p l a c e d i n c h i l l e d g l a s s homogenizing tubes to which 1 ml of r e f r i g e r a t e d 10% TCA were added. The g l a s s homogenizing tubes c o n t a i n i n g the t i s s u e sub samples were kept i n 500 ml g l a s s beakers of crushed i c e d u r i n g t i s s u e homogenization. The homogenates were c e n t r i f u g e d at 3000g f o r f i v e minutes a f t e r which the supernatant, c o n t a i n i n g the i n t r a c e l l u l a r f r e e amino a c i d s was t r a n s f e r r e d to c h i l l e d 15ml pyrex t e s t tubes. The p e l l e t was washed twice with 1 ml a l i q u o t s of 10% TCA, rehomogenized, and c e n t r i f u g e d between each washing. The combined supernatants were then analysed f o r t y r o s i n e c o n c e n t r a t i o n and r a d i o a c t i v i t y . For the d e t e r m i n a t i o n of s p e c i f i c a c t i v i t y of t y r o s i n e i n the p r o t e i n bound amino a c i d s , the t i s s u e p e l l e t was a c i d h y d r o l y s e d i n screw capped t e s t tubes by the a d d i t i o n of 2ml 6N HCl and placed i n a 110°C oven f o r 20 h before t y r o s i n e a n a l y s i s . For determining the f e t a l whole c a r c a s s t y r o s i n e content, four f e t u s e s were s e p a r a t e l y 67 homogenized i n an i n d u s t r i a l blender and weighed a l i q u o t s of c a r c a s s homogenates were an a l y z e d f o r p r o t e i n bound t y r o s i n e . For the four f e t u s e s used i n the dete r m i n a t i o n of whole c a r c a s s t y r o s i n e , two were obtained s t i l l b o r n from unknown causes and two were obtained as a r e s u l t of neonatal death due ap p a r e n t l y to hypothermia. A n a l y t i c a l Methods Hemoglobin was determined by the cyanomethemoglobin method ( H y c e l l Inc. Houston) and hematocrit by the microhematocrit procedure. Blood glucose was analysed by the glucose oxidase procedure (Sigma Chemical Comp. S t . L o u i s M i s s o u r i ) . Plasma l a c t a t e was determined by the l a c t a t e dehydrogenase method (Sigma Chemical Comp. K i t #510 and 826 UV), and alpha amino n i t r o g e n a c c o r d i n g to the d i n i t r o f l u o r o b e n z e n e d e r i v a t i v e method of Goodwin (1968). The t o t a l n i t r o g e n i n t i s s u e samples was determined i n a Technicon a u t o a n a l y s e r (Technicon I n s t . Corp., Model I I , Terrytown, N.Y.) f o l l o w i n g wet a c i d d i g e s t i o n . DNA c o n c e n t r a t i o n i n t i s s u e samples was determined a c c o r d i n g t o the method of Gold and Shochat (1980) and RNA by the mod i f i e d Schmidt-Thannhauser technique (Cheek, 1975). DNA standards were prepared from c a l f thymus DNA (Mann Research Labs N.Y.) and RNA standards were obtained from Sigma Chemical Company. L - t y r o s i n e was q u a n t i t a t i v e l y converted to tyramine by the a d d i t i o n of t y r o s i n e decarboxylase (E.C.4.1.25; Sigma). The tyramine was e x t r a c t e d by the procedure of G a r l i c k and M a r s h a l l (1972) with m o d i f i c a t i o n s as d e s c r i b e d by Schaefer and Kri s h n a m u r t i (1982c) ( a l s o see experiment I, pp 27 ) and determined f l u o r o m e t r i c a l l y a c c o r d i n g to the procedure of 68 Ambrose (1974). R a d i o a c t i v i t y of t y r o s i n e was measured by p l a c i n g 1ml of the e x t r a c t e d tyramine s o l u t i o n i n t o 10ml of s c i n t i l l a t i o n f l u o r (PCS, Amersham A r l i n g t o n H e i g h t s , 111.) and counted i n a Packard s c i n t i l l a t i o n counter (Model A 300 C). The d e t e r m i n a t i o n of s p e c i f i c a c t i v i t y of blood 14<-^2 w a S e s t ^ m a t e c ^ ac c o r d i n g to procedures p r e v i o u s l y d e s c r i b e d ( K i t t s and K r i s h n a m u r t i , 1982c; Schaefer and K r i s h n a m u r t i , 1982a; experiment I I I , pp 58 )• C a l c u l a t i o n s The r a t e of whole body p r o t e i n s y n t h e s i s was c a l c u l a t e d by d i v i d i n g the net r a t e of u t i l i z a t i o n of t y r o s i n e , c o r r e c t e d f o r o x i d a t i o n , by the t y r o s i n e content of the f e t a l c a r c a s s p r o t e i n . Rate of p r o t e i n s y n t h e s i s (g/d/kg) = net u t i l i z a t i o n of tyrosine - tyrosine used for oxidation t o t a l tyrosine content of f e t a l carcass protein The net u t i l i z a t i o n r a t e i s the sum of net p l a c e n t a l exchange and endogenous p r o d u c t i o n ( F i g . 4) and was c a l c u l a t e d from the p l a t e a u s p e c i f i c a c t i v i t y of t y r o s i n e i n the maternal and f e t a l compartments f o l l o w i n g the continuous i n f u s i o n of the 3H-t y r o s i n e i n the ewe and 14C-tyrosine i n the f o e t u s . The d e t a i l s of the k i n e t i c procedure and d e t e r m i n a t i o n of the extent of the t y r o s i n e o x i d a t i o n have been d e s c r i b e d (experiment II and I I I ) . The f r a c t i o n a l r a t e of p r o t e i n s y n t h e s i s i n the t i s s u e s was 69 calculated according to two procedures. The f i r s t procedure was according to the method of Waterlow et al.,(l976) using the formula l i s t e d below. Secondly, the tissue fractional protein synthetic rates were calculated by substituting S B/S p for S^/S^ in the forementioned formula. These methods of calculating tissue fractional protein synthetic rates w i l l provide estimates of maximum and minimum rates respectively as shown in Table 6. / S «s R 1— (for muscle ) 1-e B 1 S B / S i R-l " ^ g - R X s t R-l Xi l - e " k s t k s " -Ait — * f o r ** v e r» brain,kidney xi-ks 1-e Xi-k gastrointestinal and lung) S Where: Sfi « specific avtivity of protein bound tyrosine • specific a c t i v i t y of intracellular free tyrosine Xi « rate constant of turnover of tyrosine i n the intracellular free pool •> >p or rate constant of turnover of the infused labeled amino acid i n the extracellular (plasma) pool. fractional synthetic rate: t * time of infusion (d) 2.7182: R * ra t i o pf protein bound to intracellular free tyrosine: S p • specific a c t i v i t y i n plasma In r a p i d l y turning over t i s s u e s , the r i s e to plateau of tyrosine (Xi) was assumed to follow the same exponential curve as the plasma (Xp) (Waterlow et a l . , 1978). *s e 70 S t a t i s t i c a l A n a l y s i s The d i f f e r e n c e s among t i s s u e parameters were determined using the a n a l y s i s of v a r i a n c e and the d i f f e r e n c e s t e s t e d f o r s i g n i f i c a n c e by the Newman-Keuls m u l t i p l e range t e s t ( S t e e l and T o r r i e , 1960). Using " B a r t l e t t s t e s t of homogeneity of v a r i a n c e s " (Sokal and R o h l f , 1969) i t was found that h e t e r o s c e d a s t i c i t y was evident f o r the t i s s u e t y r o s i n e and RNA v a l u e s r e p o r t e d i n t a b l e 9. T h e r e f o r e , t h i s data was r e a n a l y s e d s t a t i s t i c a l l y using a p p r o p r i a t e ANOVA procedures f o r data with hetrogeneous v a r i a n c e s as per the methods of Sokal and Rohlf (1969 ). R e f e r r i n g to Table 6, a l l r e p o r t e d means are from 11 animals with t r i p l i c a t e o b s e r v a t i o n s taken f o r blood gas and pH, d u p l i c a t e f o r hematocrit and hemoglobin and s i n g l e o b s e r v a t i o n s f o r body weight and temperature. For blood g l u c o s e , alpha amino n i t r o g e n and plasma l a c t a t e , v a l u e s are from 12, 16 and 7 animals, r e s p e c t i v e l y , with d u p l i c a t e o b s e r v a t i o n s per parameter. RESULTS The blood parameters of the f e t u s and ewe at the time of experimentation (Table 6) are w i t h i n the p h y s i o l o g i c a l range r e p o r t e d f o r f e t u s e s which have been c a t h e t e r i z e d (Comline and S i l v e r , 1970; B a t t a g l i a and Meschia, 1978; K i t t s et a l . , 1979). The r a t i o of the p l a t e a u s p e c i f i c a c t i v i t y of i n t r a c e l l u l a r f r e e t y r o s i n e (Sj ) to that i n the plasma (Sp) was low (0.14-0.19) i n l i v e r , small i n t e s t i n e , lung, b r a i n and kidney and high (0.45-0.62) i n s k e l e t a l and c a r d i a c muscles (Table 7). The S /S B p 71 i n a l l t i s s u e s was lower than the c o r r e s p o n d i n g S._/S . r a t i o s . B l The h a l f l i f e of mixed p r o t e i n s i n the l i v e r and small i n t e s t i n e was s h o r t e r than in the other t i s s u e s . The net u t i l i z a t i o n of t y r o s i n e by the f e t a l t i s s u e s was 6.42 mmol/d/kg of which 5.2 i l . 6 ( S E ) % was used f o r o x i d a t i o n (experiment I I I ) l e a v i n g 6.00 mmol/d/kg f o r s y n t h e s i s of p r o t e i n s . From the t y r o s i n e content of the e n t i r e f e t a l c a r c a s s p r o t e i n (15.71 mmol/kg±1.30(SE)) the whole body r a t e of p r o t e i n s y n t h e s i s can be c a l c u l a t e d to be approximately 63g/d/kg(see appendix 7). The f r a c t i o n a l s y n t h e t i c r a t e of p r o t e i n s y n t h e s i s (Table 8) was r e l a t i v e l y low i n c a r d i a c and s k e l e t a l muscles and h i g h in small i n t e s t i n e , l i v e r and lungs with the kidney and b r a i n occupying an intermediate p o s i t i o n . The a b s o l u t e r a t e of p r o t e i n s y n t h e s i s i n i n d i v i d u a l t i s s u e s (g/d) was c a l c u l a t e d by m u l t i p l y i n g the t o t a l p r o t e i n c o n c e n t r a t i o n of the t i s s u e by the f r a c t i o n a l s y n t h e t i c r a t e (Table 8 ) . Though the f r a c t i o n a l s y n t h e t i c r a t e of s k e l e t a l muscles i s only 26%/d the t o t a l amount of p r o t e i n s y n t h e s i s e d i n the muscles (12.9 g/d) was higher than in any other s i n g l e t i s s u e . The a b s o l u t e r a t e s were a l s o high i n l i v e r and g a s t r o i n t e s t i n a l t r a c t which s y n t h e s i z e d 9.1 and 12.9 g/d r e s p e c t i v e l y . Other t i s s u e s which i n c l u d e predominantly s k i n , wool and bones were t r e a t e d as a s i n g l e group. The f r a c t i o n a l s y n t h e t i c r a t e s i n t h i s group were c a l c u l a t e d u s i n g a k value equal to s muscles as suggested by Davis et a l . , (1981) or the mean value of other t i s s u e s . The a b s o l u t e r a t e of s y n t h e s i s i n other t i s s u e s ranged from 41.1 to 77.5 g/d. Whole body p r o t e i n 72 synthesis estimated by adding the absolute rate of synthesis in individual tissues was 84.4 - 120.7 g/d or 47.4 - 67.7 g/d/kg of fetus. The tyrosine and nucleic acid concentration in f e t a l tissues are presented in Table 9. The concentration of tyrosine and RNA was higher in l i v e r than in any other tissue, while the DNA concentration was r e l a t i v e l y low. Skeletal and cardiac muscles also had a high l e v e l of tyrosine. The RNA concentration in the lung and kidney was higher than in the muscles and brain. Kidney and lung had the highest concentration of DNA among the tissues analysed. The RNA/DNA r a t i o which is an indicator . of protein synthesising capacity i s higher in the l i v e r than in any other organ (Table 10). The e f f i c i e n c y of protein synthesis was expressed as the amount of protein synthesised per unit RNA per day. The e f f i c i e n c y was high in l i v e r , lung and brain followed by kidney, s k e l e t a l and cardiac muscle. 73 Tabl e 6. Mean p h y s i o l o g i c a l parameters f o r the f e t u s and ewe Parameter Fetus (N=ll) ** Ewe (N=ll) Mean SE Mean SE Blood pH 7.39 0. 03 7.49 0.04 Blood pC0 2 (mmHg) 33.7 1.8 28.3 1.3 Blood p 0 2 (mmHg) 16.5 1. 8 24.9 0.6 Hct % 34.4 1.4 32. 1 1.1 Hb (g%) 10.1 0.5 11. 3 0.4 Body temperature (°C) 38.8 0.1 Body weight (kg)*** 1.78 0.18 65.72 2.71 Plasma glucose (mg%) 12.55 1.56 60.18 5.21 Plasma alph a amino n i t r o g e n (mg%) 13.38 0.70 10.68 0.42 Plasma l a c t a t e (mg%) 12.98 1.26 5.58 0.73 P e t a l blood samples were o b t a i n e d from the i n f e r i o r vena cava ** Ewe blo o d samples were o b t a i n e d from the j u g u l a r v e i n *** p r e d i c t e d f e t a l body weight a t date o f experiment based on the r e g r e s s i o n formula o f Gresham e t a l . , (1972). Table 7: Rat io of p lateau s p e c i f i c a c t i v i t y of 3 H-ty ros ine in d i f fe ren t tyros ine pools and ha l f l i f e ( T 1 / 2 ) of mixed proteins in f e t a l t issues (N-4) L i ve r Small In tes t ine Lung Bra in Kidney Ske le ta l Muscle Cardiac Muscle 0.139 0.152 0.139 0.189 0.152 0.449 0.619 0.056 0.055 0.059 0.042 0.048 0.209 0.181 0.021 0.031 0.022 0.018 0.021 0.013 0.019 0.009 0.009 0.011 0.007 0.012 0.006 0.005 0.201 0.238 0.174 0.103 0.126 0.029 0.032 0.027 0.062 0.047 0.036 0.032 0.008 0.006 0.888 0.707 1.066 1.690 1.540 2.665 4.950 S. = s p e c i f i c a c t i v i t y of i n t r a c e l l u l a r free ty ros ine , S f i = s p e c i f i c a c t i v i t y of prote in bound ty ros ine , S p = s p e c i f i c a c t i v i t y of plasma tyros ine in Table 8. Fractional (%/d) and absolute (g/d) rate of protein synthesis in fetal tissues (N-4) Tissue Mean Weight (g) Mean SE Protein % Mean SE Total Protein g Fractional (k g) Synthetic Rate (%/day) Minimum SE Maximum SE Absolute Rate of Synthesis g/d % of Whole Body Synthesis Heart 20.1 2.6 14.4 0.4 2.8 * Muscle 374 — 13.3 0.3 49.7 Liver 72.1 7.0 16.0 0.4 11.6 Brain 38.2 2.8 7.1 0.4 2.7 Lung 67.5 7.3 13.5 0.8 9.1 Kidney 20.0 3.5 11.4 0.6 2.3 ** GIT 122 — 14.8 0.4 18.1 Other Tissues *** 1068 — 14.8 0.4 158.1 Total 11.2 3.1 14.0 3.0 0.4 0.3 — 0.5 10.3 3.4 26.0 7.0 12.9 11 - 16 9.5 5.2 78.0 15.0 9.1 8 - 11 14.2 3.1 37.3 18.7 1.0 0.9 - 1.3 12.8 6.9 65.0 16.0 5.9 5 - 7.4 10.5 4.9 44.6 11.8 1.0. 0.9 - 1.3 17.6 4.1 71.2 25.7 12.9 7.6 - 16 — — 26.0 49.0 41.1 77.5 49 - 64 84.4 - 120.7 or 47.4 - 67.7 g/dAg ** *** *Based of fetal carcass dissection, muscles were found to contribute 21% of body weight. GIT weight includes reticulum, rumen, omasum, abomasum, small intestine and large intestine. The k g for GIT refers to the mean value from small intestine and reticulum rumen. * Other tissues include those not reported individually. For this purpose two k values are used: one equal to that of muscle (26%) as suggested by Davies et a l . , (1981) and the other equal to the mean of a l l tissues (49%). * Absolute rate of synthesis (g/d) = total protein X k maximum. 4" + S Mean fetal body weight = 1.782 - 0.19 kg. k minimum calculated by substituting S/S for S /S. (see appendix 8). S O P D 1 76 Table 9. The c o n c e n t r a t i o n of t y r o s i n e and n u c l e i c a c i d s i n f e t a l t i s s u e s T i s s u e T y r o s i n e (mg/g) RNA (mg/g) DNA (mg/g) Mean(N=4)SE Mean(N=8)SE Mean(N=8)SE L i v e r 5 .49 a 0 .47 8.80 a 0 .95 2 .62 C 0 .40 Lung 2 . 5 2 C d 0 .28 5 .94 b 0 .38 4 . 4 5 a b 0 .69 Kidney 2 .19 d 0 .26 4.69 0 .55 5 .15 a 0 .63 B r a i n 2 .24* 0 .07 1.90 d 0 .08 3 . l l b C 0 .43 S k e l e t a l Muscle 3 . 6 6 b C 0 .08 3.36 C d 0 .15 2 .43° 0 .37 Cardiac Muscle 3 . 3 3 b c d 0 .27 3.60° 0 .28 3 . 6 1 3 b c 0 .29 a,b,c,d jjggjjg w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n columns are s t a s t i s t i c a l l y d i f f e r e n t at P <_0.01. Tyr o s i n e c o n c e n t r a t i o n r e f e r s t o the p r o t e i n bound t y r o s i n e and t i s s u e weights f o r t y r o s i n e and n u c l e i c a c i d s r e f e r s t o the t i s s u e wet weight. Table 10. Protein synthetic capacity and activity in fetal tissues Tissue RNA Activity RNA/DNA Protein/DNA Protein/RNA Mean Mean Mean (g protein/g RNA/d) Liver 3.36a 61.11* 18.19 14.3 Lung 1.34*- 30.40b 22.76 14.8 Kidney bed 0.91 22.17b 24.33 10.9 Brain cd 0.61 22.88b 37.47 15.3 Skeletal Muscle 1.38b 54.4l a 39.43 10.3 Cardiac Muscle 0.99 39.81b 39.89 5.6 a ' b ' c ' d means with different superscripts within columns are s t a t i s t i c a l l y different P< 0.01 78 D i s c u s s i o n P r o t e i n s y n t h e s i s i n the f e t u s has r e c e n t l y been s t u d i e d by the continuous i n f u s i o n of l a b e l l e d l y s i n e (Noakes and Young, 1981; Meier et a l . , 1981). However, the use of l a b e l l e d l y s i n e n e c e s s i t a t e s a r a t h e r lengthy i n f u s i o n p e r i o d of between 9-13 h as r e p o r t e d by Meier et a l . , (1981). The c h o i c e of l a b e l l e d t y r o s i n e i n the c u r r e n t study was based on i t s smaller pool s i z e than l y s i n e i n the f e t a l plasma (Lemons et a l . , 1976) so that the p l a t e a u s p e c i f i c a c t i v i t y i s reached more r a p i d l y . As r e p o r t e d e a r l i e r (experiment II) the slope of the s p e c i f i c a c t i v i t y - t i m e curve was approximately zero a f t e r s i x hours of i n f u s i o n . In a d d i t i o n , the m o d i f i c a t i o n of a s p e c i f i c enzymatic procedure f o r the d e t e r m i n a t i o n of L - t y r o s i n e (Schaefer and K r i s h n a m u r t i , 1982c) with a higher recovery value than that obtained using the o r i g i n a l procedure of G a r l i c k and Marshal (1972) g r e a t l y f a c i l i t a t e d the d e t e r m i n a t i o n of the s p e c i f i c a c t i v i t y of L - t y r o s i n e . The l i m i t a t i o n s of u s i n g the t y r o s i n e f l u x based on plasma s p e c i f i c a c t i v i t y f o r the e s t i m a t i o n of whole body p r o t e i n s y n t h e s i s have been reviewed (Waterlow et a l . , 1978). The f e t a l system i s f u r t h e r confounded by the h i g h turnover as w e l l as the p o s s i b i l i t y of b i d i r e c t i o n a l t r a n s f e r of the l a b e l a c r o s s the p l a c e n t a . In order to overcome t h i s problem, the net u t i l i z a t i o n of t y r o s i n e by the f e t u s was e s timated by adding the net p l a c e n t a l t r a n s f e r and endogenous p r o d u c t i o n u s i n g the two pool k i n e t i c model as d e s c r i b e d by Hodgson et a l . , (1980). From the net u t i l i z a t i o n r a t e , the amount of t y r o s i n e used f o r o x i d a t i v e purposes i s deducted and the remainder i s assumed to be used f o r 79 p r o t e i n s y n t h e s i s . The use of t y r o s i n e f o r catecholamine and t h y r o i d hormone pr o d u c t i o n i s c o n v e n t i o n a l l y assumed to be q u a n t i t a t i v e l y n e g l i g i b l e i n a d u l t animals (Waterlow et a l . , 1978). T h i s assumption has a l s o been made i n the present study with the f e t a l system. However, t h i s f a c t o r would l i k e l y be worthy of f u r t h e r study. In order t o a c c u r a t e l y c a l c u l a t e t i s s u e f r a c t i o n a l s y n t h e t i c r a t e s the use of the immediate p r e c u r s o r amino a c i d t-RNA s p e c i f i c a c t i v i t y i s d e s i r e d . However, t e c h n i c a l l i m i t a t i o n s to t h i s approach have encouraged workers to make use of a l t e r n a t i v e procedures ( G a r l i c k and.Marshal, 1972; Waterlow et a l . , 1978) whereby the s p e c i f i c a c t i v i t y of the l a b e l l e d amino a c i d i n the plasma or the i n t r a c e l l u l a r f r e e compartments i s used. When employing continuous i n f u s i o n procedures, as i n the present study, f o r c a l c u l a t i n g both whole body and t i s s u e f r a c t i o n a l p r o t e i n s y n t h e t i c r a t e s , the assumption i s made that the animal i s i n a steady s t a t e with r e s p e c t to growth (Zak et a l . , 1979). C o n s i d e r i n g the apparent r a p i d growth of the sheep f e t u s i n utero (Rattray et a l . , 1974, 1975) i t would seem l o g i c a l to q u e s t i o n whether the f e t u s i s a c t u a l l y i n a steady s t a t e c o n d i t i o n . However, based on the comparative s l a u g h t e r data of Ra t t r a y et a l . , (1974), the net alpha amino n i t r o g e n uptake data of Lemons e t a l . , (1976) and the data presented i n the present study, the net p r o t e i n r e t a i n e d by the sheep f e t u s at approximately 120 days of g e s t a t i o n can be estimated to be between 6-10 g/d/kg. Or, on an h o u r l y b a s i s , t h i s would represent at the most approximately 0.4 g/h/kg. In the present study, the s p e c i f i c a c t i v i t y v a l u e s used i n the c a l c u l a t i o n of 80 p r o t e i n s y n t h e t i c r a t e s were obtained between 6 and 8 hours a f t e r commencing the i s o t o p e i n f u s i o n s . T h e r e f o r e , w i t h i n t h i s two hour time span of the experiment, i t i s perhapse j u s t i f i a b l e i n assuming that although the f e t u s i s a d m i t t e d l y i n a non steady s t a t e with respect to growth, there would, however, be at most a 0.8 g/kg change in the t o t a l p r o t e i n accrued (and l e s s i n the s t a r v e d f e t u s e s ) . Or based on an average f e t a l body weight of 1.78 kg (Table 6) t h i s would represent c o n s i d e r a b l y l e s s than a 1% change i n body weight. The r a t i o of S./S i n d i c a t e s the extent of d i l u t i o n of the i P i n f u s e d l a b e l by u n l a b e l l e d t y r o s i n e a r i s i n g from p r o t e i n degradation . I t i s noteworthy that the extent of d i l u t i o n v a r i e s i n d i f f e r e n t t i s s u e s . In muscles f o r example, the r a t i o of s p e c i f i c a c t i v i t y of i n t r a c e l l u l a r f r e e t y r o s i n e to plasma was 45-62 % which i s s i m i l a r to the value of 46% i n the muscle t i s s u e of f e t u s e s r e p o r t e d by (Noakes and Young, 1981) and 49% i n the mature sheep (Bryant and Smith, 1982). On the other hand, the r a t i o of Si/Sp i n the l i v e r (14%) i s much lower than the l i v e r v a l u e s r e p o r t e d i n the f e t u s (42%; Noakes and Young, 1982) or i n growing lambs (30%; D a v i s . e t a l . , 1981). The short h a l f l i f e of the mixed p r o t e i n s i n the l i v e r and small i n t e s t i n e (Table 7) i n d i c a t e s a r a p i d turnover i n these t i s s u e s r e s u l t i n g i n g r e a t e r d i l u t i o n of the l a b e l i n the i n t r a c e l l u l a r p o o l . The i n t r a c e l l u l a r pool i n the s k e l e t a l and c a r d i a c muscles undergoes l e s s d i l u t i o n because of the slower turnover of p r o t e i n s . In a d d i t i o n to the more r a p i d turnover of t i s s u e p r o t e i n s i n the f o e t u s than i n the a d u l t , the use of Sp i n s t e a d of S^ w i l l i n t r o d u c e l a r g e r d i f f e r e n c e s i n the • e s t i m a t i o n of p r o t e i n 81 s y n t h e s i s i n r a p i d l y t u r n i n g over t i s s u e s such as l i v e r and small i n t e s t i n e than i n muscles. R e l a t i n g to the f o r e g o i n g d i s c u s s i o n , when t i s s u e f r a c t i o n a l p r o t e i n s y n t h e s i s i s asessed without determining the t-RNA s p e c i f i c a c t i v i t y , one i s r e q u i r e d to assume a p r e c u r s o r amino a c i d p o o l . As d i s c u s s e d by Waterlow et a l . , (1978) and Zak et a l . , (1979) the most c o n v e n t i o n a l approach, as was done i n the present study, i s to use the i n t r a c e l l u l a r f r e e s p e c i f i c a c t i v i t y as r e p r e s e n t i n g the p r e c u r s o r s p e c i f i c a c t i v i t y . However, as a l s o p o i n t e d out by these authors, depending on the t i s s u e concerned, the p r e c u r s o r pool may i n f a c t be b e t t e r r e p r e s e n t e d by the e x t r a c e l l u l a r s p e c i f i c a c t i v i t y i n the plasma as c e r t a i n t-RNA s p e c i e s appear t o be charged p r e f e r e n t i a l l y from the plasma e x t r a c e l l u l a r pool r a t h e r than the i n t r a c e l l u l a r p o o l . T h e r e f o r e , i n view of the u n c e r t a i n t y i n gen e r a l of the p r e c i s e p r e c u r s o r s p e c i f i c a c t i v i t y p o o l that i s c o n s i d e r e d most r e p r e s e n t a t i v e , a pragmatic approach has been taken i n the present study, as was a l s o r e s e n t l y adopted by Davis et a l . , (1981) and both the S /S as w e l l as the S /S v a l u e s have been B p B i used t o c a l c u l a t e the t i s s u e f r a c t i o n a l s y n t h e t i c r a t e s . T h i s w i l l p r o v i d e estimates of minimum and maximum r a t e s of t i s s u e p r o t e i n s y n t h e s i s r e s p e c t i v e l y (Table 8) between which the t r u e value w i l l l i k e l y l i e . Unless otherwise i n d i c a t e d , a l l d i s c u s s i o n i n the t e x t r e l a t i n g to t i s s u e f r a c t i o n a l p r o t e i n s y n t h e t i c r a t e s r e f e r t o the k g maximum v a l u e s . There was a wide v a r i a t i o n i n f r a c t i o n a l s y n t h e t i c r a t e s (Table 8 ) among the t i s s u e s , an o b s e r v a t i o n s i m i l a r to those r e p o r t e d by Meier et a l . , (1981) and Noakes and Young (1981) 82 Among the t i s s u e s , the small i n t e s t i n e d i s p l a y e d the hig h e s t f r a c t i o n a l s y n t h e t i c r a t e . The swallowing of amniotic f l u i d by the foetus as w e l l as the a b s o r p t i o n of n u t r i e n t s from the g a s t r o i n t e s t i n a l t r a c t has been r e p o r t e d by P i t k i n and Reynolds (1975) and Char and Rudolph (1979). T h i s would suggest that i n l a t e g e s t a t i o n , even under i n utero c o n d i t i o n s , the d i g e s t i v e organs of the foetus are a l r e a d y f u n c t i o n i n g to a c e r t a i n extent. The h i g h Kg v a l u e s , t o t a l RNA c o n c e n t r a t i o n , and high RNA/DNA r a t i o s i n the l i v e r i n d i c a t e the high p r o t e i n s y n t h e t i c c a p a c i t y and turnover of t h i s t i s s u e . The Protein/RNA r a t i o which r e p r e s e n t s the extent to which the p r o t e i n s y n t h e t i c a c t i v i t y i s expressed (Table 10 ) i s low, i n d i c a t i n g that much of the s y n t h e s i z e d p r o t e i n i s exported. The lungs a l s o resemble the l i v e r i n t h i s regard, suggesting the s y n t h e s i s of e x t r a c e l l u l a r s u r f a c t a n t p r o t e i n s . B r a i n and kidney occupy an intermediate p o s i t i o n (Table 8 ) with respect to f r a c t i o n a l r a t e of p r o t e i n s y n t h e s i s . B r a i n t i s s u e shows the lowest RNA/DNA r a t i o (Table 10) suggestive of lower s y n t h e t i c a b i l i t y but a protein/RNA r a t i o as high as l i v e r and lung i n d i c a t i n g that the p r o t e i n s y n t h e t i c c a p a c i t y i s u t i l i z e d to the maximum i n t h i s t i s s u e . Compared to other t i s s u e s , both s k e l e t a l and c a r d i a c muscles showed r e l a t i v e l y low f r a c t i o n a l r a t e s of p r o t e i n s y n t h e s i s (14-26%). These valu e s are comparable to the f r a c t i o n a l p r o t e i n s y n t h e t i c r a t e s of 23-35%/d r e p o r t e d i n newborn or young lambs ( S o l t e s z et a l . , 1973, A r n a l , 1977). As the lambs grow o l d e r the muscle f r a c t i o n a l p r o t e i n s y n t h e t i c r a t e s drop t o 2-7%/d ( Bu t t e r y et al.,1975; A r n a l , 1977; Davis 83 et a l . , 1981 ). The DNA u n i t s i z e (protein/DNA) i s , however, the h i g h e s t i n the muscles and l i v e r , i n d i c a t i v e of hypertrophy of the c e l l s . The high protein/RNA r a t i o i n the muscles l i k e l y r e p r e s e n t s the high p r o t e i n s t a b i l i t y of these t i s s u e s . The a b s o l u t e r a t e s of p r o t e i n s y n t h e s i s i n i n d i v i d u a l t i s s u e s were c a l c u l a t e d by m u l t i p l y i n g the p r o t e i n content by f r a c t i o n a l r a t e s of p r o t e i n s y n t h e s i s . (Table 8 ). The p r o t e i n s y n t h e s i s of other t i s s u e s i n the remainder of the c a r c a s s was not a n a l y s e d i n d i v i d u a l l y but was estimated using e i t h e r a Ks value equal to that of muscle (26%) or a mean value (49%) of a l l t i s s u e s . I t i s p e r t i n e n t to note that the whole body p r o t e i n s y n t h e s i s estimated by adding p r o t e i n s y n t h e s i s i n i n d i v i d u a l t i s s u e s c a l c u l a t e d from f r a c t i o n a l s y n t h e s i s r a t e s and t o t a l p r o t e i n content (45.1-65.3 g/d/kg) i s i n reasonable agreement with the value of 63 g/d/kg estimated from net t y r o s i n e u t i l i z a t i o n r a t e c o r r e c t e d f o r o x i d a t i o n d i v i d e d by the t y r o s i n e content of the f o e t a l c a r c a s s p r o t e i n . O b v i o u s l y , the accuracy of the former procedure would depend on the magnitude of e r r o r i n v o l v e d i n the c a l c u l a t i o n of f r a c t i o n a l s y n t h e t i c r a t e s of i n d i v i d u a l t i s s u e s . T h i s i s p a r t i c u l a r l y t r u e f o r the Ks val u e s of the "other t i s s u e s " c a t e g o r y which makes the s i n g l e l a r g e s t c o n t r i b u t i o n (51-66%) to whole body p r o t e i n s y n t h e s i s (Table 8 ) . T h i s probably i s a r e f l e c t i o n of the growth of s k i n and wool which have been observed to have f r a c t i o n a l s y n t h e t i c r a t e s of 35%/d i n young lambs (Davis et a l . , 1981). The whole body r a t e of p r o t e i n s y n t h e s i s of 45-65 g/d/kg observed i n t h i s study i s s l i g h t l y higher than the values of 38.6 g/d/kg r e p o r t e d by Noakes and Young (1981) and much higher 84 than the value of 15 g/d/kg r e p o r t e d by Meier et a l . , (1981) for f e t u s e s i n l a t e g e s t a t i o n . These values c o n t r a s t s h a r p l y with the low r a t e of whole body p r o t e i n s y n t h e s i s of 6.9g/d/kg r e p o r t e d i n a d u l t sheep (Reeds and Lobley, 1980 ). Values ranging from 45-78.9 g/d/kg have been reported f o r immature r a t s ( G a r l i c k 1980) and 33 g/d/kg f o r newborn lambs ( S o l t e s z et a l . , 1973). When the present values are expresed on a metabolic body s i z e ( b o d y weight ), the whole body p r o t e i n s y n t h e t i c r a t e 75 amounts to 52g/kg" /d which i s c l o s e to the value of 49g/kg* 7 5/d repo r t e d by S o l t e s z et a l . , (1973) f o r newborn lambs. Apart from the inherent v a r i a b l e s a s s o c i a t e d with the p h y s i o l o g i c a l c o n d i t i o n of the p r e p a r a t i o n s , the major d i f f e r e n c e appears to l i e i n the i s o t o p e s employed and the methodology used i n e s t i m a t i n g the k i n e t i c parameters. The foetuses used by Noakes and Young (1981) were i n f u s e d 24-48 h a f t e r surgery which may not p r o v i d e adequate time f o r recovery from the s u r g i c a l trauma ( K i t t s et a l . , 1979). T h i s i s bound to i n f l u e n c e the dynamics of p r o t e i n t u r n o v e r . 14 I t i s a l s o noteworthy that though the "C-lysine was used f o r i n f u s i o n i n both cases, the l y s i n e f l u x v a l u e s and whole body r a t e s of p r o t e i n s y n t h e s i s r e p o r t e d by Meier et a l . , (1981) are approximately three times lower than those obtained by Noakes and Young (1981) f o r foetuses of s i m i l a r age. M e t h o d o l o g i c a l and a n a l y t i c a l d i f f e r e n c e s and v a r i a b i l i t y i n the p h y s i o l o g i c a l s t a t u s of the p r e p a r a t i o n s may account f o r the d i f f e r e n c e s observed. Energy Cost of P r o t e i n S y n t h e s i s i n the Foetus 8 5 The e f f i c i e n c y of whole body protein synthesis i s the r a t i o of the rate of protein deposition to the t o t a l rate of protein synthesis. Based on the comparative slaughter data of Rattray et a l . (1974) i t can be determined that the sheep fetus at 120 days of gestation gains approximately 6-9 g protein/d/kg/ This i s similar to the rate of 9.5 g/d/kg reported by Lemons et a l . , (1976) based on the net alpha amino acid nitrogen uptake by the fetus. In the present study the net alpha amino acid nitrogen uptake by the fetus was determined in one preparation by multiplying the a r t e r i o venous concentration difference by the umbilical blood flow (for blood flow calculations see appendix 6). It was found that 1.59 g/d/kg of alpha amino nitrogen was retained by the fetus which i s equivalent to a protein nitrogen retention of 9.9 g/d/kg. Thus, the rate of whole body protein synthesis in the fetus (47-68 g/d/kg) i s 5-8 times more than the rate of protein deposition (6-10 g/d/kg). However, the significance of protein turnover in the fetus should be considered in r e l a t i o n to i t s contribution to the energy economy of the fetus. Using the value of 4.5 KJ/g as the cost of protein synthesis (Webster, 1977) and a heat production of 368 KJ/d/kg (Battaglia and Meschia, 1978) i t may be calculated that whole body protein turnover contributes at least 55% to t o t a l heat production. This i s similar to the values reported for lambs (25-42%; Soltesz et a l . , , 1973; Davis et a l . 1981) and much higher than in older sheep (4.5-14%; Buttery et al.,1975; MacRae and Reeds, 1977). Further, as suggested by Davis et a l . , (1981) because of the high rate of protein turnover as compared to the rate of 86 deposition, even small changes in the former are l i k e l y to result in larger changes in protein deposition. The methodology used in th i s investigation can be used to investigate intrauterine growth retardation by monitoring changes in f e t a l rates of protein synthesis as influenced by n u t r i t i o n a l and endocrine status. 87 CHAPTER IV: EFFECT OF MATERNAL STARVATION ON WHOLE BODY AND TISSUE FRACTIONAL PROTEIN SYNTHESIS IN THE OVINE FETUS IN UTERO Experiment (V): E f f e c t s of Acute Maternal S t a r v a t i o n I n t r o d u c t ion As d i s c u s s e d by Robinson et a l . , (1977a,b); Robinson and McDonald, 1979) and supported by the o b s e r v a t i o n s of Rattray et a l . , (1974, 1975) the h i g h e s t growth p e r i o d and g r e a t e s t n u t r i t i o n a l demand by the ovine f e t u s occur i n the l a s t s e v e r a l weeks pre-partum. The e f f e c t s of maternal n u t r i t i o n on f e t a l metabolism and growth d u r i n g t h i s p e r i o d have r e c e i v e d c o n s i d e r a b l e a t t e n t i o n p a r t i c u l a r l y i n the r a t (Rosso, 1977; G a l l e r et a l . , 1980) and human (Naismith, 1980; Metcoff, 1980) but a l s o i n domestic s p e c i e s such as c a t t l e ( P r i o r and Scott,1977) and sheep ( E v e r i t t , 1964; Sykes and F i e l d , 1972; Robinson et a l . , 1977a,b; Robinson and McDonald, 1979). The common o b s e r v a t i o n i n these s t u d i e s has been that poor maternal n u t r i t i o n w i l l r e s u l t i n a reduced f e t a l b i r t h weight and reduced p l a c e n t a l weight. However, as d i s c u s s e d by Metcoff (1980), f e t a l growth i s complex and b i r t h weight and p l a c e n t a l weight are not t o t a l l y d e s c r i p t i v e . As emphasised by s e v e r a l r e s e a r c h e r s , there has been i n g e n e r a l a lac k of understanding of the s p e c i f i c mechanisms of f e t a l a d a p t a t i o n to n u t r i t i o n a l s t r e s s ( L i n d b l a d , 1977; Naismith, 1980; Brooke, 1980; S c h r e i n e r , 1980b). However, a t t e n t i o n has been d i r e c t e d toward understanding the e f f e c t s of maternal n u t r i t i o n on c e r t a i n s p e c i f i c a s p e c t s of f e t a l metabolism d u r i n g g e s t a t i o n . For example, the e f f e c t s of maternal n u t r i t i o n on f e t a l s u b s t r a t e use p a r t i c u l a r l y glucose 88 and f r u c t o s e (Tsoulos et a l . , 1971; P r i o r and S c o t t , 1977), l a c t a t e ( K i t t s and K r i s h n a m u r t i , I982a,b) and the endocrine f a c t o r s that may c o n t r o l f e t a l m etabolic response (Basset and M a d i l l , 1974) have been examined. The f e t a l metabolic use of amino a c i d s has a l s o been i n v e s t i g a t e d (Lemons et a l . , 1976; Young et a l . , 1979) and based on urea e x c r e t i o n data (Gresham et a l . , 1972; S c h r e i n e r et a l . , 1980a; Hodgson et a l . , 1982) i t has been suggested that reduced maternal n u t r i t i o n i n c r e a s e s the f e t a l c a t a b o l i c use of amino a c i d s (Simmons et a l . , 1974; S c h r e i n e r et a l . , 1980a,b; Hodgson et a l . , 1982). P a r t i c u l a r l y of i n t e r e s t to the f i e l d of a g r i c u l t u r e , i s the understanding of how maternal n u t r i t i o n i n domestic s p e c i e s might a f f e c t the a b i l i t y of the f e t u s to s y n t h e s i s e p r o t e i n i n utero and whether t h i s may have any l a t e n t p o s t n a t a l i n f l u e n c e on the growth r a t e of the o f f s p r i n g . However, the i n v i v o study of f e t a l p r o t e i n s y n t h e s i s i n g e n e r a l u s i n g i s o t o p i c t r a c e r s has only r e c e n t l y been attempted (Meier et a l . , 1981; Noakes and Young, 1981; Schaefer and K r i s h n a m u r t i , I982ab. There i s o n l y l i m i t e d data to assess the e f f e c t of maternal n u t r i t i o n on the a b i l i t y of the f e t u s to s y n t h e s i z e p r o t e i n . Based on a p i l o t study (Schaefer and K r i s h n a m u r t i , 1982a;see animal #110 experiment II and I I I ) , maternal s t a r v a t i o n appeared to demonstrate a dramatic e f f e c t on the f e t a l use of the amino a c i d t y r o s i n e . The c u r r e n t study was t h e r e f o r e undertaken to f o l l o w up t h i s o b s e r v a t i o n and to i n v e s t i g a t e , by means of i s o t o p i c d i l u t i o n and n u c l e i c a c i d a c i d a n a l y s i s techniques, the e f f e c t of maternal s t a r v a t i o n on the q u a l i t a t i v e and q u a n t i t a t i v e manner i n which whole body and 89 t i s s u e f r a c t i o n a l p r o t e i n s y n t h e s i s i n the ovine f e t u s are a f f e c t e d . M a t e r i a l s and Methods Animals and Management E i g h t Dorset X S u f f o l k c r o s s b r e d ewes were used i n the s t a r v a t i o n experiments (see appendix 1 and 2). The general f e e d i n g , management and s u r g i c a l p r e p a r a t i o n of the animals has been d e s c r i b e d i n experiment I. Experimental Procedure The i n f u s i o n of l a b e l l e d t y r o s i n e and blood sampling procedures f o r the animals under normal fee d i n g c o n d i t i o n s have been d e s c r i b e d i n experiment II and I I I . The s t a r v a t i o n experiments were 'conducted i n a s i m i l a r manner i n e i g h t m a t e r n a l - f e t a l p r e p a r a t i o n s . In three of these preparations(Ewe # 110,690,453) continuous i n f u s i o n experiments were done before and a f t e r s t a r v a t i o n of the ewe f o r 48h. In these p r e p a r a t i o n s the f e t u s was i n f u s e d with C t y r o s i n e and the ewe with H t y r o s i n e s i m u l t a n e o u s l y . In three of the p r e p a r a t i o n s (#634,101,712) the ewe was s t a r v e d f o r 48h f o l l o w i n g a 5 day p o s t s u r g i c a l recovery p e r i o d and , a f t e r the 48h s t a r v a t i o n p e r i o d , an i n f u s i o n of both 3H and 1 4 C t y r o s i n e was made i n t o the f e t u s o n l y . A l l p h y s i o l o g i c a l blood parameters as p r e v i o u s l y d e s c r i b e d were measured i n the s t a r v e d animals both before and a f t e r s t a r v a t i o n ( T a b l e 11). For blood glucose, alpha amino n i t r o g e n and plasma l a c t a t e , v a l u e s f o r s t a r v e d animals are from a l l 8 animals with d u p l i c a t e o b s e r v a t i o n s per parameter. Blood sampling and a n a l y t i c procedures were c a r r i e d out as d e s c r i b e d i n Chapter II and I I I . Table 11. P h y s i o l o g i c a l parameters f o r the fetus and ewe in the fed and s t a r v e d c o n d i t i o n Parameter Fed Starved Fetus Ewe Fetus Ewe Mean SE Mean SE Mean SE Mean SE Blood pH 7 .39 0.03 7.49 0.04 7.38 0.04 7.56 0 .04 Blood pC0 2 (mmHg) 33.7 1.8 28.3 1.3 36.5 2.2 26.3 1.3 Blood p 0 2 (mmHg) 16 .5 1.8 24.9 0.6 11.9 0.9 26 .2 2.4 Hct % 34 .4 1.4 32 . 1 1.1 34.5 2.0 31.3 1.4 Hb (g%) 10 .1 0.5 11.3 0.4 10.6. 0.7 11.3 0.4 Body Temp. (°C) 38 .8 0.1 38.4 0.1 Plasma Glucose (mg%) 12.55 a 1.56 60 .18° 5.21 8.18 b 1.02 43 .66 d 5.41 Plasma ct-amino 13,38 a 0 . 70 10 .68° 0.42 11.55 b 1.08 7 .80 d 0.17 n i t r o g e n (mg%) Plasma.Lactate (mg*) 12 .98 1.26 5 .58° 0.73 15.08 1.83 7.41 d 0 .70 Plasma Tyrosine (ug/ml)16,90 1. 20 9 .18 0.91 19.50 4.00 8.83 3.50 a , b f o r the f e t a l v a l u e s , means with d i f f e r e n t s u p e r s c r i p t s w i t h i n rows are s t a t i s t i c a l l y d i f f e r e n t P <.0 .05 . c ' d f o r the ewe va l u e s , means with d i f f e r e n t s u p e r s c r i p t s w i t h i n rows are s t a t i s t i c a l l y d i f f e r e n t P < 0 .05 . 91 C a l c u l a t ions The method of c a l c u l a t i n g i r r e v e r s i b l e l o s s , net p l a c e n t a l t r a n s f e r , endogenous p r o d u c t i o n and net u t i l i z a t i o n of t y r o s i n e has been d e s c r i b e d i n chapter I I . Whole body p r o t e i n s y n t h e s i s was c a l c u l a t e d from the net t y r o s i n e u t i l i z a t i o n r a t e c o r r e c t e d f o r o x i d a t i o n l o s s as o u t l i n e d by Waterlow et a l . , (1978), using a f e t a l c a r c a s s p r o t e i n t y r o s i n e content of 1.93% (Experiment IV) A l l s t a t i s t i c a l comparisons were made using the a n a l y s i s of v a r i a n c e and the d i f f e r e n c e s t e s t e d by the Student-Newman-Keuls m u l t i p l e range t e s t ( S t e e l and T o r r i e , i960). T i s s u e A n a l y s i s For e s t i m a t i n g f r a c t i o n a l t i s s u e s y n t h e t i c r a t e s i n the s t a r v e d animals, three of the ewes were s a c r i f i c e d immediately f o l l o w i n g the continuous i n f u s i o n s and f e t a l t i s s u e analysed as d e s c r i b e d p r e v i o u s l y (experiment I V ) . DNA c o n c e n t r a t i o n i n t i s s u e s was determined by the method of Gold and Shochat (1980) and RNA a c c o r d i n g to the Schmidt-Thannhauser technique d e s c r i b e d by Cheek (1975). R e s u l t s The mean value s of the p h y s i o l o g i c a l and n u t r i t i o n a l parameters f o r the ewe and f e t u s both i n the fed and s t a r v e d animals are presented i n Table 11. S t a r v a t i o n r e s u l t e d i n a s i g n i f i c a n t r e d u c t i o n (P<0.05) i n f e t a l glucose from I2.55mg% to 8.l8mg% and a 14% r e d u c t i o n i n f e t a l alpha amino n i t r o g e n from 13.38mg% to 11.55mg%. S i m i l a r l y , maternal plasma glucose was s i g n i f i c a n t l y reduced (P<0.01) as a r e s u l t of s t a r v a t i o n from 60.l8mg% i n the fed animals to 43.66 mg% i n the s t a r v e d ewes. Maternal alpha amino n i t r o g e n and plasma t y r o s i n e c o n c e n t r a t i o n 92 was a l s o decreased r e s p e c t i v e l y from 10.68 mg% to 7.80 mg% and from 9.18 yg/ml to 8.83 pg/ml f o l l o w i n g s t a r v a t i o n . Furthermore, f e t a l plasma l a c t a t e was i n c r e a s e d f o l l o w i n g s t a r v a t i o n from 12.98mg% i n the fed c o n d i t i o n to I5.08mg% i n the s t a r v e d s t a t e . However, the d i f f e r e n c e s i n plasma l a c t a t e 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 at the 5% l e v e l . The k i n e t i c parameters of t y r o s i n e metabolism i n both the fed and s t a r v e d c o n d i t i o n s i n the ewe and f e t u s are presented i n Table 12. The i r r e v e r s i b l e l o s s of t y r o s i n e was observed to decrease s i g n i f i c a n t l y i n the ewe as a r e s u l t of s t a r v a t i o n from 0.96 to 0.51 mmol/d/kg. Endogenous p r o d u c t i o n of t y r o s i n e i n the ewes decreased s i g n i f i c a n t l y (P<0.10) from 0.90 mmol/d/kg i n the fed ewes to 0.27 mmol/d/kg a f t e r 48h of feed d e p r i v a t i o n . C o nversely, i n the f e t u s , endogenous p r o d u c t i o n rose (P<0.05) from 1.29 mmol/d/kg to 4.53 mmol/d/kg f o l l o w i n g s t a r v a t i o n . The net u t i l i z a t i o n of t y r o s i n e by the ewe and f e t u s were r e s p e c t i v e l y 0.77 and 6.42 mmol/d/kg i n the fed s t a t e versus 0.51 and 3.88 mmol/d/kg as a r e s u l t of s t a r v a t i o n . The net p l a c e n t a l t r a n s f e r , although a p p a r e n t l y much reduced i n the st a r v e d p r e p a r a t i o n s was h i g h l y v a r i a b l e and the d i f f e r e n c e s from the fed animals was 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 . 14 From the r a t i o of the p l a t e a u s p e c i f i c a c t i v i t y of C0 2 14 and C t y r o s i n e , the percent of t y r o s i n e used f o r o x i d a t i o n by the f e t u s e s o f . f e d ewes was c a l c u l a t e d to be 5.2 -1.5 % of net u t i l i z a t i o n . T h i s would leave approximately 95% of the t y r o s i n e p o t e n t i a l l y a v a i l a b l e f o r s y n t h e t i c purposes i n the f e t u s . S t a r v a t i o n r e s u l t e d i n the percent of t y r o s i n e used f o r o x i d a t i o n by the fe t u s to r i s e to 13.7 ^6.45% of net u t i l i z a t i o n 93 l e a v i n g approximately 86% of a v a i l a b l e t y r o s i n e f o r s y n t h e t i c purposes. Based on net u t i l i z a t i o n r a t e s of t y r o s i n e , c o r r e c t e d f o r o x i d a t i v e l o s s , the whole body p r o t e i n s y n t h e t i c r a t e s were determined f o r the f e t u s e s of both the fed and s t a r v e d ewes. While a whole body p r o t e i n s y n t h e t i c r a t e of approximately 63- 8(SE)g/d/kg was observed f o r the f e t u s e s of the fed ewes, t h i s value was reduced to 25- 15g/d/kg i n the f e t u s e s of the s t a r v e d ewes. T i s s u e f r a c t i o n a l s y n t h e t i c r a t e s , both k g minimum and k. maximum as d e s c r i b e d i n experiment IV, f o r f e t u s e s of s t a r v e d ewes are r e p o r t e d i n Table 14. R e f e r r i n g h e n c e f o r t h to k g maximum v a l u e s , s i g n i f i c a n t r e d u c t i o n s i n t i s s u e f r a c t i o n a l s y n t h e t i c r a t e s were evident i n the l i v e r , lung, and kidney t i s s u e s which demonstrated a 7,3,and 2 f o l d r e d u c t i o n r e s p e c t i v e l y i n k values compared to the f e t u s e s of fed ewes. The f e t a l c a r d i a c muscle d i s p l a y e d the l e a s t change i n f r a c t i o n a l s y n t h e t i c r a t e under the i n f l u e n c e of maternal s t a r v a t i o n . The f e t u s e s of s t a r v e d ewes d i s p l a y e d a reduced n u c l e i c a c i d c o n c e n t r a t i o n (Table 15), both RNA and DNA, f o r a l l t i s s u e s except f o r the c a r d i a c muscle and b r a i n . RNA/DNA r a t i o s tended to f o l l o w t h i s same p a t t e r n ( T a b l e 16). Although the protein/RNA and protein/DNA r a t i o s were not reduced i n t i s s u e s of the s t a r v e d f e t u s e s , the RNA a c t i v i t y (g p r o t e i n s y n t h e s i s e d / g RNA/d) was again lower f o r a l l t i s s u e s except the c a r d i a c muscle(Table 16). 94 Ta b l e 12. F e t a l and maternal k i n e t i c parameters o f t y r o s i n e metabolism i n f e d and s t a r v e d c o n d i t i o n s Parameter FED Starved FETUS I r r e v e r s i b l e Loss (mmol/d/kg) Net P l a c e n t a l T r a n s f e r (mmol/d/kg) Endogenous P r o d u c t i o n (mmol/d/kg) Net U t i l i z a t i o n (mmol/d/kg) % T y r o s i n e O x i d a t i o n Mean 5.79 SE 0.78 5.14 3.48 1.29 e 0.89 6.42 3.10 5.2' 1.5 Mean 5.38 -0.29 4 . 5 3 j 3.88 13. 7 b SE 1.08 0.84 0.86 2.19 6.5 I r r e v e r s i b l e Loss (mmol/d/kg) Endogenous P r o d u c t i o n (mmol/d/kg) Net U t i l i z a t i o n (mmol/d/kg) EWE 0.96 a 0.09 0.90 a 0.13 0.77 0.09 0.51 4 0.27J 0.51 0.08 0.24 0.19 a,b,c,d,e,f m e a n s w i t n d i f f e r e n t s u p e r s c r i p t s w i t h i n rows are s t a t i s t i c a l l y d i f f e r e n t (a,b P< 0.10; c,d P< 0.05; e , f P<.0.01) Table 13 : Ratio of plateau specific activity of 3H tyrosine in different tyrosine pools and half l i f e (T 1 / 2> of mixed proteins in fetal tissues of starved ewes Tissue S./S i p p VS i T l / 2 * d a y s ) mean SE mean SE mean SE Liver 0.478 0.276 0.031 0.010 0.043 0.003 5.653 Lung 0.365 0.006 0.026 0.011 0.052 0.002 3.065 Kidney 0.509 0.237 0.040 0.021 0.066 0.003 3.065 Skeletal 0.660 0.488 0.011 0.004 0.023 0.009 6.741 Muscle Heart 0.757 0.224 0.022 0.010 0.026 0.005 6.393 St - specific a c t i v i t y of intracellular free tyrosine; i - specific activity of protein bound tyrosine; S p » specific activity of plasma tyrosine T a b l e 14. Comparison o f f r a c t i o n a l (%/day) r a t e s o f p r o t e i n s y n t h e s i s i n the f e t u s e s o f f e d and s t a r v e d ewes T i s s u e Fed F r a c t i o n a l SE Syn t h e s i s Rate Starved SE L i v e r 78. 0 C 15.0 12.3 d 0.9 Lung 65.0 a 16.0 22.6 b * 1.7 Kidney 44.6 a 11.8 21.6 b 3.3 B r a i n 37. 3 a 18.7 10.4 b 4.0 S k e l e t a l Muscle 26.0 7.0 10.3 3.8 C a r d i a c Muscle 14.0 3.0 10.8 1.7 a,b,c,d m e a n s w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n rows are s t a t i s t i c a l l y d i f f e r e n t (a,b P <.0.10; c,d P<_0.05). T a b l e 1 5 . The c o n c e n t r a t i o n o f t y r o s i n e and n u c l e i c a c i d s i n f e t a l t i s s u e s o f f e d and s t a r v e d ewes T i s s u e Tyrosine (mg/g) FED STARVED Mean(N=4) SE Mean(N=3) SE RNA (mg/g) FED STARVED Mean(N=8) SE Mean(N=5) SE DNA (mg/g) FED STARVED Mean(N=8) SE Mean(N=5) SE L i v e r 5.49 a 0.47 6 . 2 i a 0.46 8.80 a 0.95 5.56a 0.92 2.62° 0.40 3.83 b 0.34 Lung 2.52 G d 0.28 4.66 a 0.73 5.94 b 0.38 bc 2.39 0.37 4.45^ 0.69 5.82 a 0.38 Kidney B r a i n 2.19 d d 2.24 0.26 0.07 5.09 a 2.13 b 0.91 0.04 bc 4.69°° d . 1.90 0.55 0.08 3.83 b 1.97° 0.23 0.21 5.15 a bc 3.11 0.63 0.43 4.96 a bc 3.43 0.32 0.47 S k e l e t a l Muscle 3.66 b C 0.08 4.34 a 0.30 3.36 c d 0.15 2.08° 0.17 2.43° 3.61*° 0.37 1.90 be 3.43 0.25 Cardiac Muscle 3 . 3 3 b c d 0.27 6.97 a 0.56 3.60° 0.28 3.47b 0.59 0.29 0.17 Tyrosine c o n c e n t r a t i o n r e f e r s t o the p r o t e i n bound t y r o s i n e and t i s s u e weights f o r t y r o s i n e and n u c l e i c a c i d s r e f e r t o the t i s s u e wet weight. Data on fed animals were taken from chapter I I I pp5 8 . 00 cr, Table 16. Protein synthetic capacity and activity in fetal tissues of fed and starved ewes RNA/DNA , Protein/DNA Protein/RNA T i s s u e RNA Activity (g protein/g RNA/d) Fed Starved Fed Starved Fed Starved Fed Starved Mean Mean Mean Mean Mean Mean Mean Mean Liver 3.36a 1.45a 61 . l l a 48.20b 18.19 33 .20b° 14.3 4. 1 Lung bc 1.34 0.41° 30 .40b 23.32° 22.76 56 .78^ 14.8 12. 8 Kidney bed 0.91 0.77b 22 .17b 27.44° 24.33 35 .54b 10.9 7. 7 Brain 0.61 C d „ r_bc 0.57 22 .88b 25.57° 37.47 44 .52b 15.3 4. 6 Skeletal Muscle 1.38b ab 1.09 54 . 4 ia 75.37a 39.43 68 .85a 10.3 7. 1 Cardiac Muscle ~ ™bcd 0.99 i . o i b 39 .81b 52.16 39.89 51 .56 a b 5.6 5. 6 a , b ' ° ' means with different superscripts within columns are s t a t i s t i c a l l y different P^.0.01 . Data for fed animals taken from chapter III pp 58-Table 17. Fractional (%/d) and absolute (g/d) rates of protein synthesis in fetal tissues of starved ewes(N=3) Tissue Mean Weight Protein Total Fractional (k s ) Synthetic Absolute Rate t % of Whole ( g ) % Protein Rate (%/day) of Synthesis Body Synthesis Mean SE Mean SE g . . 4> Minimum SE Maximum SE g / d Heart 18.0 1.2 17.9 1.6 3.2 9.2 3.9 10.8 1.7 0.4 1 * Muscle 577 1.2 14.3 0.5 82.6 3.9 2.3 10.3 3.8 8.5 30 Liver 90.0 10.4 18.5 0.5 16.6 7.6 4.6 12.3 0.9 2.0 7 Brain 38.0 3.0 8.8 0.5 3.3 1.3 0.9 10.4 4.0 0.3 1 Lung 95.0 16.1 13.6 0.9 12.9 12.4 4.8 22.6 1.7 2.9 10 Kidney 20.3 2.6 13.6 0.6 2.8 8.8 7.7 21.6 3.3 0.6 2 ** GIT 120 — 15.0 0.4 18.0 2.6 0.7 35.2 10.0 6.3 22 Other Tissues *** 1789 14.8 0.4 264.8 . . . . . . 10.3 17.6 48.4 - 82.7 or 17.6 - 30.1 g / d A g * Based on fetal carcass dissection, muscles were found to contribute 21% of body weight. ** GIT weight includes reticulum, rumen, omasum, abomasum, small intestine and large intestine. k g for GIT refers to the mean value of reticulo rumen (35.2) . *** Other tissues include those not reported individualy. For this purpose two k values are used: one equal to that of muscle (10.3%/d) as suggested by Davies et a l . , (1981) and the other equal to the mean of a l l tissues (17.6%/d). Absolute rate of synthesis (g/d) = total protein X k g maximum. Mean fetal body weight = 2.748 kg - 0.258. k minimum calculated by substituting S /S for Sn/S (see appendix 8). S a p o x 100 D i s c u s s i o n Ef f e e t of S t a r v a t i o n on Plasma N u t r i e n t s Acute maternal s t a r v a t i o n was seen to evoke changes in plasma n u t r i e n t c o n c e n t r a t i o n s i n both the ewe and the f e t u s . The 27% r e d u c t i o n i n maternal plasma glucose c o n c e n t r a t i o n i s s i m i l a r to the magnitude of change r e p o r t e d by s e v e r a l r e s e a r c h e r s (Simmons et a l . , 1974; M e l l o r and Matheson, 1979; M o r r i s s et a l . , 1980; S c h r e i n e r et a l . , 1980a,b) f o r pregnant ewes. A l s o , the 17% r e d u c t i o n i n plasma alpha amino n i t r o g e n i n the s t a r v e d ewes i s c o n s i s t e n t with the observed r e d u c t i o n i n t o t a l plasma amino a c i d s observed by M o r r i s s et a l . , (1980). The i n c r e a s e i n plasma l a c t a t e i n the s t a r v e d ewes i n the present study l i k e l y r e f l e c t s an i n c r e a s e d g l y c o l y s i s i n maternal t i s s u e s . T h i s would be c o n s i s t e n t with the views of Kaplan and Grumbach (1974) suggesting t h i s would be the r e s u l t of an i n c r e a s e d p l a c e n t a l l a c t o g e n p r o d u c t i o n i n the s t a r v e d ewes. In t o t a l , acute maternal s t a r v a t i o n was seen to a l t e r the n u t r i e n t mixture of glucose, alpha amino n i t r o g e n and l a c t a t e that would be d e l i v e r e d to the conceptus. The lower f e t a l plasma glucose and alpha amino n i t r o g e n c o n c e n t r a t i o n s have been observed p r e v i o u s l y i n f e t u s e s of s t a r v e d ewes (Simmons et a l . , 1974; S c h r e i n e r et a l . , 1980a,b; M o r r i s s et a l . , 1980) and are b e l i e v e d to be due to a reduced p l a c e n t a l t r a n s f e r of these n u t r i e n t s (Simmons et a l . , 1974). T r a n s f e r and Production of T y r o s i n e The plasma c o n c e n t r a t i o n of t y r o s i n e i n those ewes measured before and a f t e r s t a r v a t i o n f e l l from 11.05 yg/ml i n the fed s t a t e t o 8.83 vg/ml f o l l o w i n g acute s t a r v a t i o n . T h i s was 101 accompanied by a 70% r e d u c t i o n i n endogenous p r o d u c t i o n of t y r o s i n e by e x t r a f e t a l t i s s u e s . Although these changes i n t y r o s i n e c o n c e n t r a t i o n s and p r o d u c t i o n appear to be r e f l e c t e d by a reduced p l a c e n t a l t r a n s f e r to the f e t u s , the r e d u c t i o n s i n p l a c e n t a l t r a n s f e r were not s t a t i s t i c a l y s i g n i f i c a n t , a p p a r e n t l y due to c o n s i d e r a b l e animal v a r i a t i o n i n t h i s parameter. I t i s noteworthy that u n l i k e the maternal response, the f e t a l plasma c o n c e n t r a t i o n of t y r o s i n e was not reduced as a r e s u l t of maternal s t a r v a t i o n . I f anything, appears to have r i s e n s l i g h t l y from 16.9 ug/ml i n the fed to 19.5 u g / m l i n the s t a r v e d s t a t e s . The reason f o r t h i s appears to be due at l e a s t i n p a r t to a s i g n i f i c a n t (3.5 f o l d ) i n c r e a s e i n endogenous p r o d u c t i o n of t y r o s i n e by the s t a r v e d f e t u s e s . Although the i r r e v e r s i b l e l o s s value c a l c u l a t e d f o r the s t a r v e d f e t u s e s i s s l i g h t l y reduced(Table 12), i t i s i n t e r e s t i n g that the t o t a l t y r o s i n e turnover which i n c l u d e s r e c y c l i n g i s comparable to that i n the f e t u s e s of fed ewes. The i n c r e a s e i n endogenous p r o d u c t i o n i s thus a r e f l e c t i o n of an i n c r e a s e d c o n t r i b u t i o n from degraded p r o t e i n s and the formation of t y r o s i n e from p h e n y l a l a n i n e a r i s i n g from the degradation of f e t a l p r o t e i n under c o n d i t i o n s of maternal s t a r v a t i o n . The b e n e f i t to the f e t u s i n i n c r e a s i n g the endogenous p r o d u c t i o n of t y r o s i n e and hence m a i n t a i n i n g the turnover d u r i n g maternal s t a r v a t i o n would appear to l i e i n the f a c t t h a t more t y r o s i n e c o u l d be used f o r o x i d a t i o n and energy procurement. Indeed, there i s almost a three f o l d i n c r e a s e i n the o x i d a t i o n of t y r o s i n e observed i n the f e t u s e s of s t a r v e d ewes i n the present study, c o n f i r m i n g p r e v i o u s r e p o r t s of i n c r e a s e d amino 1 02 acid catabolism in fetuses of starved ewes based on increased urea formation (Gresham et a l . , 1972; Simmons et a l . , 1974; Hodgson, et a l . , 1982). Increased catabolism of amino acids during starvation would undoubtedly compensate to some extent for the reduced placental transfer of substrates such as glucose, normally used p r e f e r e n t i a l l y for oxidative metabolism. The cost , however, would appear to be a reduced proportion of tyrosine available for protein synthesis. Ti ssue Protein Synthet ic Rates, Degradation and Nucleic Ac i d Concentrations As discussed previously in chapter IV, the r a t i o of S.^ /S.p indicates the extent of d i l u t i o n of the infused label by unlabelled tyrosine a r i s i n g from protein degradation or hydroxyl-ation of phenylalanine. In the fetuses of starved ewes, the S^/S^ r a t i o was observed to increase i n a l l tissues measured (Table 13) This observed increase in S ^ /S p r a t i o s was due primarily to two factors. F i r s t of a l l , a reduction in the turnover rate of c e l l u l a r proteins l i k e l y occurred r e s u l t i n g i n a reduced rate of d i l u t i o n of the tracer with amino acids entering the i n t r a c e l l u l a r pool from degraded proteins. This would result in a reduced rate of removal of l a b e l and hence higher s p e c i f i c a c t i v i t y in the i n t r a c e l l u l a r free pool. Secondly, a reduction in the i n t r a c e l l u l a r free concentration of tyrosine was observed. This i s depicted for example by the l i v e r tissue where the mean l i v e r i n t r a c e l l u l a r free tyrosine concentration for the starved fetuses was 115 pg/g tissue compared to 272 vg/g tissue in the fed fetuses. The i n t r a c e l l u l a r free tyrosine concentration in the l i v e r tissue of the starved fetuses was 103 thus only 42% of that observed i n the fed animals. T h i s would n e c e s s i t a t e a higher s p e c i f i c a c t i v i t y measured in the i n t r a c e l l u l a r f r e e pool of the t i s s u e . These f a c t o r s were a l s o e v i d e n t i n the other t i s s u e s . The reduced c o n c e n t r a t i o n of t i s s u e t y r o s i n e i n the i n t r a c e l l u l a r f r e e pools of s t a r v e d f e t u s e s may be due to i n c r e a s e d r a t e s of e x i t of t y r o s i n e from c e l l s as would be supported by the i n c r e a s e d plasma t y r o s i n e c o n c e n t r a t i o n s i n these animals. The f r a c t i o n a l r a t e s of p r o t e i n s y n t h e s i s i n a l l t i s s u e s of f e t u s e s from s t a r v e d ewes were seen to be reduced compared to f e t a l t i s s u e s from fed ewes (Table 14). Again, t h i s was most prominent i n the more r a p i d l y t u r n i n g over t i s s u e s such as l i v e r , kidney and lung. The combined e f f e c t of the i n c r e a s e d t i s s u e p r o t e i n degradation and reduced p r o t e i n s y n t h e s i s i n a l l f e t a l t i s s u e s of s t a r v e d ewes i s c o n s i s t e n t with the observed s i g n i f i c a n t r e d u c t i o n i n whole body p r o t e i n s y n t h e s i s i n the s t a r v e d f e t u s e s . The a b s o l u t e r a t e s of p r o t e i n s y n t h e s i s i n i n d i v i d u a l t i s s u e s have been c a l c u l a t e d and are presented i n Table 17. Keeping i n mind the p o s s i b l e sources of e r r o r i n these estimates as d i s c u s s e d i n chapter IV, i t i s again noteworthy that the e s t i m a t i o n of whole body p r o t e i n s y n t h e s i s ( g/d/kg) obtained by summation of the i n d i v i d u a l t i s s u e f r a c t i o n a l r a t e s , i s i n reasonable agreement with the value of 25 g/d/kg obtained from the net t y r o s i n e u t i l i z a t i o n r a t e c o r r e c t e d f o r o x i d a t i v e l o s s . The f e t u s e s of s t a r v e d ewes d i s p l a y e d a reduced RNA c o n c e n t r a t i o n , f o r a l l t i s s u e s except f o r the b r a i n . RNA/DNA r a t i o s tended to f o l l o w t h i s same p a t t e r n . Although the 1 04 protein/RNA and protein/DNA r a t i o s were not reduced i n t i s s u e s of the s t a r v e d f e t u s e s , the RNA a c t i v i t y (g p r o t e i n s y n t h e s i s e d / g DNA/d) was again lower f o r a l l t i s s u e s except the c a r d i a c muscle. With regard to s p e c i f i c t i s s u e s i n the s t a r v e d f e t u s e s , most notable i s the l i v e r which demonstrated an int e r m e d i a t e to low f r a c t i o n a l s y n t h e t i c r a t e . Yet, the comparative RNA c o n c e n t r a t i o n (Table 15) and RNA/DNA r a t i o s (Table 16) i n t h i s t i s s u e are i n d i c a t i v e of a hig h p r o t e i n s y n t h e t i c c a p a c i t y . Furthermore, the RNA a c t i v i t y (Table 16) i n t h i s t i s s u e suggests the l i v e r i s s y n t h e s i z i n g c o n s i d e r a b l e amounts of p r o t e i n . However, as was i l l u s t r a t e d by the l i v e r t i s s u e s of f e t u s e s of fed ewes, these parameters l i k e l y i n d i c a t e that much of the s y n t h e s i s e d p r o t e i n i s exported. S i m i l a r to the values seen i n the fe t u s e s of fed ewes the lung t i s s u e of st a r v e d f e t u s e s , although demonstrating a compar a t i v e l y lower RNA c o n c e n t r a t i o n (Table 15) and RNA/DNA r a t i o (Table 16), showed s u b s t a n t i a l l y higher RNA a c t i v i t y . T h i s again suggests the s y n t h e s i s of e x t r a c e l l u l a r s u r f a c t a n t p r o t e i n s . As was the case with the fed f e t u s e s (Table 8) the muscle t i s s u e f r a c t i o n a l s y n t h e t i c r a t e s i n f e t u s e s of s t a r v e d ewes (Table 14) were low. However, the DNA u n i t s i z e (protein/DNA) i s seen t o be the hig h e s t i n t h i s t i s s u e (Table 16) i n d i c a t i n g the hi g h e s t degree of hypertrophy as w e l l as the hig h e s t p r o t e i n s t a b i l i t y i n t h i s t i s s u e . N e v e r t h e l e s s , the a b s o l u t e r a t e of s k e l e t a l muscle p r o t e i n s y n t h e s i s i n the s t a r v e d f e t u s e s was seen to be only 66% of that i n the fed f e t u s e s . 105 P r o t e i n A c c r e t i o n i n Starved Fetuses The r a t e of p r o t e i n d e p o s i t i o n i n the s t a r v e d f e t u s e s was not determined d i r e c t l y i n the present study. However, based on the crown rump measurements of M e l l o r and Matheson (1979), M e l l o r and Murray (1982) and Spence et a l . , (1982) maternal n u t r i e n t d e p r i v a t i o n appears to have a severe growth r e t a r d a t i o n e f f e c t . In the present study, a s i g n i f i c a n t r e d u c t i o n i n whole body p r o t e i n s y n t h e s i s was observed i n the s t a r v e d f e t u s e s . T h i s was accompanied by an i n c r e a s e i n the t i s s u e degradation r a t e as evidenced by the S./S r a t i o s . Not w i t h s t a n ding these f a c t o r s , based on an energy requirement of 4.5 KJ/g p r o t e i n s y n t h e s i s e d (Webster, 1977) the p r o t e i n s y n t h e s i s i n the s t a r v e d f e t u s e s would s t i l l r e q u i r e a s i g n i f i c a n t p o r t i o n of the energy budget of the f e t u s , and t h i s at a time when t r a n s p l a c e n t a l t r a n s f e r of s u b s t r a t e s f o r o x i d a t i v e metabolism i s reduced. 106 Chapter Vj_ GENERAL SUMMARY AND CONCLUSIONS Although numerous s t u d i e s have examined the e f f e c t of maternal s t a r v a t i o n on f e t a l growth and development, there s t i l l seems to be a c o n s i d e r a b l e amount of c o n t r o v e r s y i n t h i s a r e a. Based on the i n i t i a l o b s e r v a t i o n s of Hammond (1944) the concept was developed that the maternal n u t r i e n t s d u r i n g pregnancy are d i v i d e d between the mother and f e t u s ( e s ) a c c o r d i n g to need. Subsequently, the a b i l i t y of the f e t u s to s u c c e s s f u l l y p a r a s i t i z e the maternal system and thus ensure p r e f e r e n t i a l n u t r i e n t d e l i v e r y , even i n the event of maternal n u t r i e n t d e p r i v a t i o n has been developed ( Z a r t a r i a n et a l . , 1980; Howie, 1982; Ezekwe, 1982). There has, however, i n recent years been the r e c o g n i t i o n that the p a r t i t i o n i n g of n u t r i e n t s between the f e t u s and mother i s complex, and i s dependent upon many f a c t o r s among which are the time of g e s t a t i o n (Wallace, 1948; R u s s e l l et a l . , 1967; Curet, 1972; L i n d b l a d , 1977; Robinson, 1982); the n u t r i e n t concerned (Graham, 1964; T u l p et a l . , 1980; S h i e l d s et a l . , 1980; Naismith, 1980; Z a r t a r i a n et a l . , 1980); the prepregnancy c o n d i t i o n of the mother ( E v e r i t t , 1964; Lederman and Rosso, 1981) and the s p e c i e s and t i s s u e concerned (Waterlow and Stephen, 1968; Sykes and F i e l d , 1972; McNurlan et a l . , 1980; Hegarty and Kim, 1981). Thus there remains a need f o r more a c c u r a t e and p r e c i s e data d e s c r i b i n g the s p e c i f i c manner i n which f e t a l growth occurs and how the f e t u s may adapt to a changing n u t r i e n t environment d u r i n g g e s t a t i o n . C o n s i d e r i n g the s i g n i f i c a n t p r o p o r t i o n of t h e i r pre-marketing time spent i n - u t e r o , t h i s understanding may 107 be p a r t i c u l a r l y relevant for animals of a g r i c u l t u r a l importance as t h i s knowledge may serve as a sound basis for recommending feeding and management practices. With th i s in mind, the present study was undertaken to develop a procedure for more accurately determining an important aspect of f e t a l growth, namely, the whole body and tissue f r a c t i o n a l protein synthesis. The major accomplishments of the present study were as follows: (1) An isotopic d i l u t i o n technique involving the continuous infusion of the l a b e l l e d amino acid tyrosine was successfully employed in determining the whole body and tissue f r a c t i o n a l protein synthetic rates in the ovine fetus. At the onset of t h i s study such an approach to understanding f e t a l protein metabolism had not been completed. (2) The use of the isotopic d i l u t i o n technique necessitated the improvement of an enzymatic analytic procedure for tyrosine. This was accomplished as described and subsequently f a c i l i t a t e d greater accuracy and consistency in the analysis of t h i s amino acid. (3) By employing a two compartment kinetic model to analyse the data, more accurate and extensive information regarding maternal-fetal tyrosine exchange was obtained which would not have been possible employing either the commonly used Fick p r i n c i p l e or turnover c a l c u l a t i o n methods often used by researchers. 108 (4) T h i s two compartment model demonstrated a s i g n i f i c a n t endogenous p r o d u c t i o n of t y r o s i n e by the f e t u s , a f a c t not p r e v i o u s l y demonstrated i n v i v o . (5) By measuring the s p e c i f i c a c t i v i t y of f e t a l whole blood 14 C0 2 at steady s t a t e c o n d i t i o n s i t was p o s s i b l e to c a l c u l a t e the p r o p o r t i o n of t y r o s i n e used by the f e t u s f o r o x i d a t i o n . I t was thus p o s s i b l e to determine more a c c u r a t e l y the q u a n t i t y of t y r o s i n e u t i l i z e d by the f e t u s , c o r r e c t e d f o r o x i d a t i v e l o s s , f o r p r o t e i n s y n t h e s i s . (6) The combined c a l c u l a t i o n of both t i s s u e f r a c t i o n a l s y n t h e t i c r a t e s and n u c l e i c a c i d a n a l y s i s p r o v i d e d a method of examining the q u a n t i t a t i v e and q u a l i t a t i v e aspects of p r o t e i n s y n t h e s i s c a p a b i l i t y i n i n d i v i d u a l f e t a l t i s s u e s . (7) Due to the p e r f e c t i o n of the forementioned techniques, i t was p o s s i b l e to examine s e v e r a l p r e c i s e i n v i v o e f f e c t s of acute maternal s t a r v a t i o n on f e t a l metabolism. Observed s i g n i f i c a n t e f f e c t s were seen i n the f e t a l r a t e s of t y r o s i n e o x i d a t i o n , endogenous t y r o s i n e p r o d u c t i o n and whole body and t i s s u e f r a c t i o n a l p r o t e i n s y n t h e t i c r a t e s . Although the long term damage caused by i n t r a u t e r i n e growth r e t a r d a t i o n has been qu e s t i o n e d (Winick and Noble, 1966; Robinson, 1969; S h i e l d s et a l . , 1980) the r e s u l t s of the present study suggest that maternal n u t r i t i o n i s a major determinant of f e t a l growth and that n u t r i e n t d e p r i v a t i o n d u r i n g g e s t a t i o n w i l l 109 adversely a f f e c t f e t a l in utero growth. 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Details of animal preparations used in kinetic studies and outcome of surgery Ewe and Fetus # Experiment Number IL Details of Kinetic Experiments FED : STARVED 2 Pool Oxid TFS : IL 2 Pool Oxid TFS 37 CF 71 1,11.IV X - — X 73 i . i i , i n , i v X - X X 74 CF 96 A 97 I,II,iv X X "* 98 I,II,III,IV X X X 101 V - - * * 102 1.11,1V X 103 I.II.IV X - - X 104 CF 105 I.IV.V M 106 I.II X 107 I.II.IV X - X 108 CF 109 CF 110 I,II,III,IV, V X X X 111 CF 112 I.H.V M ~ 113 CF 115 A 444 I,II,III,IV X X X 453 I,II,III,IV,V X X X 594 CF 609 CF 634 I.V - — — 658 A 690 I,II,III,IV ,v X X X 712 V - — X M IL •= irreversible loss, 2 Pool - data used in 2 pool calculations for net utilization, net placental transfer, endogenous production and whole body protein synthesis, Oxid « 14C02 data collected for calculating tyrosine oxidation, TFS « data used in tissue fractional protein Bynthesis calculations, CF - catheter failure, no data obtained, A " abortion of fetus prior to experimentation, M " blood metabolites measured (glucose, alpha amino nitrogen and lactate) due to only one catheter being patent in the fetus and therefore no continuous infusion study was possible. X indicates data used in study. 144 Appendix 2. Details of animal preparations used for nuc l e i c a c i d and nitrogen analysis FetUS # FED STARVED Experiment RNA DNA Nitrogen RNA DNA Nitrogen Humber RNA DNA Nitrogen RNA DNA Nitrogen 71 IV X X x - - — 73 IV X X x 73(twin) IV X X x 74 IV ,V whole carcass x 79 IV ,V whole carcass X X X X 101 V X X X 101(twin) V 102 IV X X X 103 IV X X x 103(twin) IV X X X 104 IV ,V whole carcass X 107 IV X X x 107(twin) IV X X x 190 IV,V whole carcass X X X X 634 V X X X 634(twin) V X X X 712 V X indicates data used i n study 145 Appendix 3. Example of 2-Pool k i n e t i c c a l c u l a t i o n s Equations f o r the 2-Pool compartment model as per Hodgson e t al.,(1980) a l s o see Figure 4 and 5 pp ) . Production r a t e s of " f " : PR„ = r f f J f Ffm ] ,. = r m I F f m + Fom«» = F^ + F I F~~ +~F J f — — fo mofc fm om* f m a f a f Where: PR and PR,. = production r a t e s of m and f r e s p e c t i v e l y m f r = the r a t e of i n f u s i o n of i s o t o p i c t r a c e r and the s u p e r s c r i p t t o r i n d i c a t e s which p o o l i s i n f u s e d a = s p e c i f i c a c t i v i t y and the s u p e r s c r i p t denotes which pool was i n f u s e d and the s u b s c r i p t i s the p o o l sampled F r a c t i o n of f going t o m (F ): F F mf mf mf " F + F = F + F — — mf of f o fm F F fm fm F r a c t i o n of m going t o f ( F , ): F, + F = F fm fm om mo mf 146 Seven steps f o r equation s o l u t i o n (I) Experimentally determine a mT, ct fT, a^T, a mT m m t m (t = p l a t e a u or steady s t a t e time a f t e r commencing the i s o t o p e i n f u s i o n ) (II) C a l c u l a t e the production r a t e of m and f PR = D m where D m = isotope dose i n f u s e d i n t o pool m m — — a™? m PR = D where D = isotope dose i n f u s e d i n t o pool f ( I I I ) Determine the f r a c t i o n of f converted t o m by the f o l l o w i n g formula: f — 1 f I F , + F J PR = D- *• mf of^ m r where mf a T m F + F mf of = f r a c t i o n of f going to m (IV) Determine the f r a c t i o n of m converted t o f by the f o l l o w i n g formula: PR, = D m I F , + F J • Tin rm fm om where fm otfT = f r a c t i o n o f m converted t o f F,, + F fm om (V) C a l c u l a t e F and F„ from the f o l l o w i n g simultaneous equations: mo f o -m PR = D m F 1 * , m t j [ ^ 1 fm + FonT = F + F , I F _ + F J mo f o I mf or a T m PR f = D m fm * r F f m s h i + Fom^ = F F + F F . + F fo n o i fin om «* note: the simultaneous equations i n (v) and (VI) can be s o l v e d by u s i n g the 2nd order determinant method o f K r e y s z i g ( 1 9 6 7 ) o r by l i n e a r algebra as per p l e y and Clark (1969) a T f (VI) C a l c u l a t e F _ and F^ from the f o l l o w i n g equations: mf fm mf mf fm fm F + F mf of F + F f o fm F , + F fm om F + F . mo mf 147 (VII) Calculate F and F , from the above results om or Numerical example from preparation #110 A continuous infusion of 50 yCi L-Cu-^-^C] tyrosine was made into the umbilical vein simultaneously with a continuous infusion of 300 yCi L-[2,3,5,6-3H] tyrosine into the maternal jugular vein. Sampling was done from the saphenous vein in the fetus and opposite jugular vein i n the ewe. The infusion was for 8 h. The infusate = 6581345 dpm/ml at an infusion rate of 2.2 ml/h. The 3 H infusate = 30214250 dpm/ml and infused at a rate of 2.2 ml/h. ct^T Time Plasma dpm/ml plasma dpm/yg tyrosine tyrosine (yg/ml) 6 14.38 3313 230 7 13.90 3300 237 7.5 14.55 3888 267 8 14.40 4202 292 X 14.78 257 (I) a^T = 257 dpm ^C/yg tyrosine and by similiar methodology a mT was found 3 f 14 mm 3 to be 247 dpm H/yg tyrosine, a T = 3 dpm C/yg tyr and ct_ T = 257 dpm H / J. • m t / yg tyrosine (II) PR = D f = 241316 dpm/min = 939 yg/min f a fT 257 dpm/yg tyr i body wt. of 2.11 kg = 445.0 yg/min A g i molecular wt. of tyr of 181.19 g/mole = 2.456 ymole/min/kg or 3.537 mmol/day/kg PR = D m = 1107856 dpm/min = 4485 yg/min m 6 a T 247 dpm/yg tyr m T by body wt. of 54.43 kg = 82.4 yg/minAg * by molecular weight of tyr of 181.19 g/mole = 0,4548ymole/min/kg or 0.655 mmol/d/kg _ f mf 1 (III) Fraction of f going to m: I I PR =D f l Fmf + F o f J m r a T oc T or on rearranging: F (PR ) ( m ) S i ! " L - ^ Therefore: F , + F . D mf of Fmf = (4485 yg/min) (3 dpm/yg tyr) = 0.0558 Fmf + F o f 241316 dpm/min m (IV) F r a c t i o n o f m going t o f : PR f =D ?fm I + F J 148 fm om a T f or on r e a r r a n g i n g : F f m = (939 y g / m i n ) (257 dpm/pg t y r ) =0.2178 F + F 1107856 dpm/min fm om (V) F and F, (Endogenous Production) mo f o [ ^ 1 [ ^ 1 PR m = D f L Fmf + F o f J = F M O + F F O [ F ^ + F Q f J a T m P R = 4485 pg min and F n c c o m mf = 0.0558 F _ + F mf of Therefore: 4485 pg/min = F + F _ (0.0558) mo f o and: P R f = F = 4485 pg/min - 0.0558 F £ mo fo r _ ^ J r r » 1 D m I F , + F = F £ + F I F_ + F * t fm oml f o mo fm om a f T F f m : PR, = 939 pg/min and F + F = 0.2178 f rm om : 939 pg/min = F^ + 0.2178 F ^ fo mo : F, = 939 pg/min - 0.2178 F f o mo o r : F = 4485 pg/min - (0.0558(939 pg/min - 0.2178 F )) mo mo : F = 4485 - (52.4 pg/min - 0.0122 F ) mo mo : F = 4432.6 pg/min + 0.0122 F mo mo : 0.9878 F = 4432.6 pg/min o r : F = 4487 pg/min mo mo and: F^ = 939 pg/min - (0.2178) (4487 pg/min) = 939 pg/min - 977 pg/min f o : = -38.3 pg/min fo 149 (VI) F , and F_ ( P l a c e n t a l Exchange) mf fm F F F, F, mf = mf fm = rm F , + F , F^ + F^ F^_ + F F + F _ mf of f o fm fm om mo mf Therefore: 0.0558 = F , and: 4487 yg/min + F _ = F f m mt mf -38.3 yg/min + F^ 0.2178 fm : -2.14 yg/min + 0.0558 F^ = F . and: 977.3 yg/min + 0.2178 F = F„ fm mf mf fm -: F = -2.14 yg/min + (0.0558 (977.3 yg/min + 0.02178 F J ) mf mf .: F _ = -2.14 yg/min + (54.5 yg/min + 0.0122 F ,) mr * mf : F , = 52.4 + 0.0122 F ^ mf mf : 0.9878 F ^ = 52.4 mf : F , = 53.05 yg/min * body wt. of 54.43 kg * 181.19 X60 X 24 mf : F , = 7.746 ymol/d/kg = 0.007746 mmol/dAg mr and: F, = 977.3 yg/min + 0.2178 (53.05) = 977.3 yg/min + 11.56 rm : F^ = 988.9 yg/min i body wt. i 181.19 X 60 X 24 rm : F^ = 3724.6 ymol/d/kg = 3.7246 mmol/d/kg rm (VII) F and F ^ (Net U t i l i z a t i o n ) om of F = F + (F , - F. ) Therefore: F = 4487 yg/min + (53.05 yg/min)-(988.9 yg/min) om mo mf fm om = 4487 + (-935.85 yg/min) = 3551.15 yg/min o r : 0.5185 mmol/d/kg and: F _ = F_ + (F^ - F ) = -38.3 yg/min + ((9889 yg/min)-(53.05 yg/min) of fo fm mf = -38.3 yg/min + 935.85 yg/min Therefore: F . = 897.55 yg/min = 3.3807 mmol/d/kg of and: Net P l a c e n t a l Exchange = F, - F = 988.9 yg/min - 53.05 yg/min fm mf = 935.85 yg/min o r : 3.5249 mmol/dAg 150 Appendix 4. Tyrosine o x i d a t i o n Equations 14 (I) % t y r o s i n e o x i d i z e d = CO from t y r o s i n e . t y r o s i n e net u t i l i z a t i o n r a t e 14 (II) C0 2 from t y r o s i n e = (% C0 2 d e r i v e d from t y r ) ( t o t a l CO^ production) ( I I I ) % C0 2 d e r i v e d from o x i d a t i o n of t y r o s i n e = a^T ^CO^ x a^T plasma t y r o s i n e f 14 14 (IV) a fT C0 2 = C0 2 dpm/ml whole blood pg C0 2 i n whole blood/ml note: C0 2 p r o d u c t i o n r a t e used i s 8.9 ml/min/kg (see appendix 5) Example f e t u s #110 (fed) pC0 2 as determined from the mean of t r i p l i c a t e values during the 8 h i n f u s i o n = 44.3 mmHg Using the "Siggard Andersen" nomogram (Annals. N.Y. Academy of S c i . 133: 41-48. 1966) w i t h a d d i t i o n a l i n f o r m a t i o n on blood pH, Hb, Hct and body temperature, the t o t a l C0 2 i n blood can be c a l c u l a t e d as 27.2640 mm o l / l i t e r 1000ml = 27.26 mmol of C0 2 = 27260 ymoles of C0 2 Therefore: 1 ml = 27.26 pinoles of C0 2 and: 1 pinole of C0 2 = 44 pg Therefore: 27.260 pmoles = 1199 pg of C0 2 a l s o : from the use of Warburg f l a s k technique (see Experiment I I I i n t e x t ) 200 p i of f e t a l whole blood c o n t a i n 140 dpm ( 1 4C0 2) Therefore: 1 ml whole blood = 700 dpm 14 and: the s p e c i f i c a c t i v i t y of C0 2 = dpm/pg C0 2 = 700 dpm _ Q 5 Q 2 Q A p m / V g c o 1199 pg C0 2 2 14 and: s p e c i f i c a c t i v i t y of C0_ , . . ~ j • -, ' 2 = f r a c t i o n of C0 2 d e r i v e d from t y r s p e c i f i c a c t i v i t y o f t y r f _ 1 4 ^ ft T C O o r : % CO d e r i v e d from t y r = f 2 X 100 = 0.5838 dpm/pg CO f 14 • X 100 ctfT c t y r 2 5 ? d p m / v ( g t y r = 0.0022715 or 0.2% of C0 2 i s d e r i v e d from t y r 151 Therefore: knowing the mean C0 2 production r a t e i s 8.9 ml/min/kg o r : 1000 ml CO^ contains or weighs 1977 mg then: 1 ml CO^ contains or weighs 1.977 mg and 8.9 ml CO^ c o n t a i n or weigh 17.59 mg = 17590 ug/min/kg and: a l s o 1 mole or 181.19 g t y r c o n t a i n 108 g carbon o r : 181.19 yg t y r contains 108 yg carbon and: 1 yg t y r contains 0.5961 yg carbon and: F , or net u t i l i z a t i o n of t y r i s 897.55 yg/min or v 2.11 kg body wt. = 425.38 yg/min/kg of t y r = (425.38)(0.5961) yg/min/kg of carbon Therefore: t y r net u t i l i z a t i o n r a t e = 253.55 yg/min/kg of carbon Therefore: CO^ production r a t e = 17590 yg/min/kg or : 12/44 X 17590 = 4797.27 yg C/minAg Therefore: CO^ de r i v e d from o x i d a t i o n of t y r = (4797.3)(0.2) 100 yg = 9.595 yg C/min/kg Therefore: t y r used which i s o x i d i z e d = 9.595 yg C/min/kg X 100 = 3.8% 253.55yg C/minAg 152 Appendix 5. CO p r o d u c t i o n r a t e s The C0 2 p r o d u c t i o n r a t e of 8.9 ml/min/kg used i n the present study i s the mean from CC>2 production r a t e s from 3 f e t a l p r e p a r a t i o n s i n the fed and 4 preparations i n the s t a r v e d f e t u s e s . These r a t e s were c a l c u l a t e d from the u m b i l i c a l veno-a r t e r i a l C0 2 d i f f e r e n c e s u s i n g the " F i c k " p r i n c i p l e Example c a l c u l a t i o n f e t u s 690 (fed) T o t a l C0 2 i n the saphenous v e i n = 21.38 m m o l / l i t e r T o t a l C0 2 i n the pedal a r t e r y = 19.40 m m o l / l i t e r Therefore: 21.38mmol/l - 19.40 mmol/1 = 1.98 mmol/1 or : 0.00198 mmol/ml X u m b i l i c a l blood flow of 206.8 ml/min (see appendix 6) = 0.4094 mmol/min and: 1 mmol CO^ contains 12 mg carbon : 0.4094 mmol C0 2 = 4.913 mg C/min and i f 12 mg C corresponds t o 44 mg C0 2 then: 4.913 mg C corresponds t o 4.913 mg C/min X 44/12 = 18.014 mg C0 2/min and: 1000ml CC>2 = 1.977g = 1977 mg Therefore: 18.014 mg CCL/min = 1000 ml „ ... . . n ,,_ , . . 3 2 X 18.014 mg CO /min = 9.112 ml CO /min 1977 mg or : d i v i d e d by body wt. f o r f e t u s #690 of 1.125 kg = 8.099 ml CO^/min/kg 153 Appendix 6. Blood flow c a l c u l a t i o n s Blood flow c a l c u l a t i o n s used i n determining C0 2 production r a t e s were a l s o determined i n 3 fed and 4 starved fetuses from the " F i c k " p r i n c i p l e using the steady s t a t e t y r o s i n e s p e c i f i c a c t i v i t y v e n o - a r t e r i a l d i f f e r e n c e s as per the method of S e t c h e l l and Hinks (1967). Example c a l c u l a t i o n f e t u s 690 (fed) Blood flow (ml/min) = m/(d - e ) j 3 14 Where: m = r a t e of i n f u s i o n of t r a c e r ( H and C t y r ) i n t o the artery(mCi/min) d = ct^T t y r i n the v e i n e = cx^T t y r i n the a r t e r y j = carbon (g) as t y r / m l of a r t e r i a l blood f o r f e t u s 690: m = 195366 dpm/min (i n f u s e d i n t o the i n f e r i o r vena cava) d = 361 dpm/pg t y r (saphenous vein) e = 270 dpm/yg t y r (pedal a r t e r y ) j = 17.42 pg t y r / m l plasma = 0.09614 pmoles/ml and: 1 pinole t y r = 108 pg C Therefore: 0.09614 pmoles = 10.3831 pg C/ml Therefore: blood flow = 195366 dpm/min (361 - 270 dpm/pg t y r ) (10.3831 pg C/ml) = 195366 dpm/min = 206.8 ml/min 944.86 dpm/pg C/ml and: * body wt. of 1.125 kg = 183.82 ml/min/kg 154 Appendix 7. Whole body protein synthesis calculations The whole body protein synthesis values were calculated from the net ut i l i z a t i o n rates of tyrosine as determined from the two pool model described in appendix 3. The tyrosine oxidation values were also needed for this purpose (appendix 4). Example calculation fetus #110 (fed) net u t i l i z a t i o n = 3.3807 mmol/d/kg oxidation = 3.8% Therefore: synthesis = 3.3807 - (3.3807)(0.038) = 3.2522 mmol/d/kg and: 1kg fetal carcass contains 15.71 mmol tyr (from the whole carcass tyr determinations of fetus # 74,79,104 and 190 mean values, see appendix 2) Therefore: net synthesis fraction of 3.2522 mmol tyr/d/kg 15.71 mmo3 tyr/kg : fraction = 0.20702 of a kg of fetal carcass synthesized/d and: i f protein i s 16% of 1 kg or 160 g/kg then: 160 g X 0.20702 = 33.12 g/d/kg 155 Appendix 8. Tissue f r a c t i o n a l p r o t e i n s y n t h e s i s c a l c u l a t i o n s The t i s s u e f r a c t i o n a l p r o t e i n s y n t h e s i s c a l c u l a t i o n s were conducted according t o the procedures of Waterlow e t a l . , (1978) based on the f o l l o w i n g formula: B i X i X i -k ( 1-e s ) , , - X i t . ( 1-e ) X i -k Where: S = s p e c i f i c a c t i v i t y of p r o t e i n bound amino a c i d ( t y r ) B S. I = s p e c i f i c a c t i v i t y of i n t r a c e l l u l a r f r e e t y r S_ = s p e c i f i c a c t i v i t y of plasma f r e e t y r (note Sg/S^ r a t i o s used i n P X i = k = s t = e t e x t t o c a l c u l a t e minimum r a t e s , see chapter IV) r a t e constant of turnover of the i n f u s e d amino a c i d i n the i n t r a c e l l u l a r pool ( i n the case of l i v e r , X i = Xp or the r a t e constant of turnover of the i n f u s e d amino a c i d i n the e x t r a c e l l u l a r pool (plasma). f r a c t i o n a l s y n t h e s i s r a t e time of i n f u s i o n (8 h or 0.333 days) = 2.7182 Example c a l c u l a t i o n fetus # 107, l i v e r t i s s u e : (I) t o determine X i X (independent v a r i a b l e ) Time (h) 1.5 3 4.5 6 7.5 8 Y (dependent v a r i a b l e ) S p e c i f i c A c t i v i t y i n plasma (dpm/yg t y r 100 189 290 406 461 491 2 Therefore: us i n g a r e g r e s s i o n formula, r=0.9959, slope = 61.06, i n t e r c e p t = 12.46 and: the slope = Xp = X i = 61.06 = r a t e constant 156 Therefore: f o r f e t u s #107: S_ = 20 dpm/yg t y r , S = 127 dpm/yg t y r , t = 0.333days, e = 2.7182, X i = Xp = 61.06 -(k )(0.333), k Therefore: 20 dpm/yg t y r _ 61.06 . (1-2.7182 s ' ) -(61.06)(0.333) s 127 dpm/yg t y r 61.06 -k (1-2.7182 ) 61.06 (II) a s s i g n values t o k t o determine S/S. r a t i o s and p l o t s B i fii nfi M o 7 1 R , - ( 0 . 5 ) ( 0 . 3 3 3 ) (a) i f k = 0.5 then S/S. = ' d-2-7182 - _ 0 ^ _ 1 61.06-0.5 l-2.7182- ( 6 1-° 6 ) (°- 3 3 3 )) 61.06-Therefore S/S. = 0.1321 B i (b) s i m i l a r l y , i f k =0.75, S/S. = 0.1914 s B i (c) s i m i l a r l y , i f k = 1.0, S_/S. = 0.2468 s B i but, our experimental value f o r Sg/S^ = 0.1574 ( I I I ) Therefore, u s i n g a r e g r e s s i o n formula o f the three assigned k g v a l u e s , t o g i ve a r e g r e s s i o n l i n e t o which our e x p e r i m e n t a l l y determined values 2 can be f i t . Regression of the above three p o i n t s f i v e r = 0.9998, slope = 0.2294 and i n t e r c e p t = 0.0180 and t h e r e f o r e our Sg/S^ value of 0.1574 = a k o f 0.6076 or approximately 61 %/day. s
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Animal Science
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Protein synthesis in the ovine fetus
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