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Substrate utilization in the ovine fetus in utero Kitts, David D. 1981

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SUBSTRATE UTILIZATION IN THE OVINE FETUS IN UTERO by David Dale K i t t s  B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1974 M.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1976  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE DEPARTMENT OF ANIMAL SCIENCE  We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA March, 1981 ©  David Dale K i t t s , 1981  In p r e s e n t i n g requirements  this thesis  British  it  freely available  for  that  f u l f i l m e n t of the  f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y  of  agree  in partial  Columbia,  I agree  f o r reference  permission  scholarly  that  the L i b r a r y  shall  and s t u d y .  I  f o r extensive  p u r p o s e s may  for  that  shall  Science  Department o f The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  7Q>  W a r c h 1981  of this  It is thesis  n o t be a l l o w e d w i t h o u t my  permission.  Animal  thesis  be g r a n t e d by t h e h e a d o f my  copying or p u b l i c a t i o n  f i n a n c i a l gain  further  copying of t h i s  d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . understood  make  Columbia  written  i i.  Abstract The turnover, interconversion and oxidation of substrates i n the ovine conceptus i n utero were studied making use of isotope d i l u t i o n techniques.  In Experiment 1, s u r g i c a l techniques were standardized f o r the  introduction of vascular catheters i n t o the fetuses at approximately 120130 days of gestation.  Based on maternal and f e t a l blood acid base para-  meters and metabolite and hormone l e v e l s i t was possible to obtain chronic f e t a l preparations which were p h y s i o l o g i c a l l y s t a b l e . In Experiment I I , r a d i o a c t i v e l a b e l l e d substrates were i n j e c t e d introvenausly i n t o the fetus and the disappearance of the l a b e l from the f e t a l c i r c u l a t i o n was monitored against time.  K i n e t i c parameters of subs-  t r a t e metabolism were c a l c u l a t e d by graphic analysis of the s p e c i f i c radioa c t i v i t y - t i m e curves.  The pool s i z e , i r r e v e r s i b l e rate of disposal and  volume of d i s t r i b u t i o n of glucose, l a c t a t e , and amino acids were estimated. The s i n g l e i n j e c t i o n technique employed i n t h i s study f a c i l i t a t e d the calcul a t i o n of 2 a d d i t i o n a l k i n e t i c parameters not reported h i t h e r t o i n the l i t e r a ture.  These include the mean t o t a l residence time and number of cycles the  l a b e l l e d substrates made before being i r r e v e r s i b l y l o s t from the f e t a l circulation.  The f i n d i n g that l a c t a t e and amino acids make more number of  cycles i n t o and out of the f e t a l c i r c u l a t i o n than glucose provides  support  to the concept that the placenta on the f e t a l side i s r e l a t i v e l y impermeable to the former two substrates. administered  Though the rapid disappearance of isotopes  i n t o the fetus was recognized  by e a r l i e r workers, the r e s u l t s  of t h i s study have brought to l i g h t the s i g n i f i c a n c e of r e c y l c i n g of substrates. I t i s suggested that t h i s unique dynamic feature serves as a p h y s i o l o g i c a l control mechanism t o modulate fuel consumption according to n u t r i e n t and  oxygen a v a i l a b i l i t y .  On the other hand, there was very l i t t l e d i f f e r e n c e i n  the i r r e v e r s i b l e rate of disposal when [2- H] or [U-^C] glucose was injected 3  i n d i c a t i n g that there i s only approximately 12.5% of r e c i r c u l a t i o n of glucose w i t h i n the f e t a l t i s s u e s . The appearance i n maternal  c i r c u l a t i o n of only l a b e l l e d glucose  injected into the f e t u s , but not l a c t a t e i n d i c a t e s the i n a b i l i t y of l a c t a t e to cross the placenta from the f e t a l side.  Though 36% of the administered  glucose label appeared i n l a c t a t e , the methodology used i n t h i s study does not d i f f e r e n t i a t e whether the conversion of glucose into l a c t a t e occurred in the fetus i t s e l f or i n the placenta.  The recovery of 8.8% of alanine C  into glucose, though suggestive of gluconeogenic  p o t e n t i a l , may have occurred  by i s o t o p i c cross over rather than true metabolic conversion. In experiment I I I , the CO^ production rates were estimated from 14  the plateau s p e c i f i c a c t i v i t y of blood of [^C] NaHCOg.  C O 2 a f t e r the continuous  infusion  The most important f i n d i n g s i n t h i s study pertains to  the c o n t r i b u t i o n of substrates to o x i d a t i v e metabolism i n the f e t u s . Contrary to the conclusions based on the Fick p r i n c i p l e , the recovery of 14 administered label into  CO^ i n d i c a t e s that glucose, l a c t a t e , a l a n i n e ,  acetate and amino acids contribute 15.2, 14.0, 6.8, 1.7 and 8.2% r e s p e c t i v e l y to f e t a l o x i d a t i v e metabolism.  Though the metabolism of placental t i s s u e s  may have influenced the above values, the r e s u l t s suggest that the metabolic fuel requirements  of the fetus warrant reassessment.  The r e s u l t s of these experiments are discussed with reference to the metabolism of the fetus during a period of gestation where the greatest increment  i n f e t a l growth occurs.  i v. TABLE OF CONTENTS Page T i t l e page Abstract  i i i  Table of Contents L i s t of Tables  iv vii  L i s t of Figures  ix  Acknowledgements  .xi  Introduction  1  Review of L i t e r a t u r e  2  1.  Maternal n u t r i t i o n during pregnancy  2  2.  Placental functions  5  3.  Fetal growth and development  7  4.  Fetal growth and endocrinology  8  A. Growth hormones (OGH and OCS)  8  B. Thyroid hormones  11  C. Adrenal c o r t i c o s t e r o i d s  12  D. I n s u l i n and glucagon  12  E. Hormonal enzyme induction  16  Fetal metabolism  17  A. Techniques f o r metabolic studies i n the fetus  17  B. Fetal c a l o r i c requirements  19  5.  i ) Oxygen consumption and carbon dioxide production i i ) Glucose i i i ) Fetal gluconeogenesis  20 23 26  i v ) Fructose  32  v) Lactate  34  v i ) Amino Acids  36  v i i ) Carbon-nitrogen  balance  Experiment I ; Surgical technique f o r the cannulation of f e t a l saphenous vein and p o s t - s u r g i c a l changes i n blood parameters of the ovine fetus i n utero. Introduction Materials and methods Results Discussion Conclusions Experiment I I : Substrate turnover and i n t e r r e l a t i o n s h i p in the ovine fetus i n utero. Introduction A.  Metabolism of Glucose and Lactate Materials and methods Results Discussion Conclusion  B. Metabolism of Amino Acids Materials and methods Results Discussion Conclusion Experiment I I I : Measurement of carbon dioxide production and substrate o x i d a t i o n by the ovine conceptus i n utero using 14c-labelled compounds. Materials and methods Results Discussion Conclusions  page  General Conclusions Bibliography Appendix B i b l i o g r a p h i c a l Notes  131 137 158  vi.  vn L i s t of Tables Table.  Page  1. Oxygen consumption rates of adults and fetuses of d i f f e r e n t s i z e . 2. Gestational age and body weights of ewes and fetuses.  22 50  3. Maternal and f e t a l p h y s i o l o g i c a l parameters during the experimental period.  72  4. K i n e t i c parameters of glucose metabolism i n the ovine f e t u s , estimated using s i n g l e i n j e c t i o n of a mixture of [U-1 C] and [2-3H] glucose or[U-14c] glucose alone.  80  5. K i n e t i c parameters of glucose and l a c t a t e metabolism i n the ovine f e t u s , estimated by using s i n g l e i n j e c t i o n of [U-14c] glucose or [1-14C] l a c t a t e .  82  6. Glucose-lactate conversions i n the ovine fetus i n utero.  83  7. Estimation of r e c y c l i n g and r e c i r c u l a t i o n of glucose and l a c t a t e i n the ovine fetus i n utero.  84  4  8. Maternal and f e t a l p h y s i o l o g i c a l parameters during the experimental period.  100  9. K i n e t i c parameters of substrate metabolism i n the ovine f e t u s .  103  10. Recycling of amino acids and alanine i n the ovine fetus i n utero.  105  11. Conversion of alanine to l a c t a t e and glucose i n the ovine fetus iii utero.  106  12. Substrate o x i d a t i o n rates i n the ovine fetus iii utero.  lig  viii.  Appendix Table. 1.  Page  Per cent recovery of 14fj-labelled compounds a f t e r treatment with glucose oxidase and anion exchange chromatography.  160  2.  Per cent recovery of [1-14c] l a c t a t e following anion exchange chromatography and t h i n layer chromatography.  161  3.  Per cent recovery of [U-14c] amino acid mixture and alanine following cation exchange chromatography and enzymatic conversion of alanine to l a c t a t e .  162  4.  5 .  6.  Per cent recovery of O^C] whole blood.  NaHC03 i n s a l i n e and 163  Mean and pH i n maternal and f e t a l blood of conscious ewes during and f o l l o w i n g surgery.  1g  Mean hematocrit, blood glucose, l a c t a t e , -hydroxybutyrate and alpha amino nitrogen i n f e t a l blood, following surgery.  153  P O 2 ,  P C O 2  7  IX.  L i s t of Figures Figure. 1. 2. 3.  Page  Post s u r g i c a l changes i n blood gas parameters and hematocrit i n the ovine fetus i n utero.  51  Post s u r g i c a l changes i n plasma metabolite l e v e l s i n the ovine fetus i n utero.  52  Post surgical changes i n plasma Cortisol in the ovine fetus i n utero.  54  levels  4.  Model of glucose and l a c t a t e metabolism i n the ovine f e t u s .  5.  Semi logarithmic p l o t of glucose s p e c i f i c a c t i v i t y versus time f o l l o w i n g i n j e c t i o n of [U-14c] glucose.  6.  7. 8. 9.  Semi logarithmic p l o t of glucose s p e c i f i c a c t i v i t y and l a c t a t e formation versus time f o l l o w i n g the i n j e c t i o n of [U-14C] and [ 2 - 3 H ] glucose.  56  7  '  4  7 5  Linear regression of f e t a l i r r e v e r s i b l e diposal rate of glucose versus blood glucose concentration.  77  Linear regression of f e t a l i r r e v e r s i b l e disposal rate of glucose versus f e t a l body weight.  78  Semi logarithmic p l o t of l a c t a t e s p e c i f i c a c t i v i t y versus time f o l l o w i n g the i n j e c t i o n of [1-14c] lactate.  7  g  10.  Model of amino acid metabolism i n the ovine f e t u s .  11.  Semi logarithmic p l o t of [U-14C] amino acids and [U-14c]alanine s p e c i f i c a c t i v i t y versus time.  102  12.  S p e c i f i c r a d i o a c t i v i t y of f e t a l and maternal blood 14C02 f o l l o w i n g primed dose-infusion of NaHl4C03.  120  13.  Recovery of r a d i o a c t i v i t y of blood 14C02 a f t e r s i n g l e i n j e c t i o n of NaHl4C03-  122  S p e c i f i c r a d i o a c t i v i t y of blood 14C02 a f t e r i n j e c t i o n of 14C-labelled substrates.  123  R e l a t i o n s h i p between rates of oxidation and i r r e v e r s i b l e disposal of glucose and l a c t a t e .  124  14. 15. 16.  Composite p i c t u r e of f e t a l substrate metabolism.  g  7  133  X.  Appendix Figure.  Page  1.  Separation of metabolites by descending paper chromatography (Phenol:water:NH3; 40:40:1 ; w/v/v/)  n64  2.  Quentch curve for 3 H and 14c isotopes.  165  3.  Standard curve for total organic carbon determined by infra-red carbon analyzer.  166  xi Acknowledgements.  I wish to express my gratitude to the many i n d i v i d u a l s that a s s i s t e d me during the course of t h i s study. In p a r t i c u l a r , I would l i k e to acknowledge the Department of Animal Science f o r the use of the animal and laboratory f a c i l i t i e s . . Special thanks i s given to Mr. J . Ciok, animal t e c h n i c i a n , whose expert assistance was c a l l e d upon many times. I wish t o express my sincere gratitude t o Dr. C. R. Krishnamurti, Professor, Animal Science f o r his dedicated p a r t i c i p a t i o n with the animal surgery and assistance with the preparation o f t h i s t h e s i s .  I am  also grateful f o r h i s encouragement and f r i e n d s h i p during my tenure i n the  department. I would l i k e t o record my gratitude t o Mr. G.J. Tompkins  f o r his time spent i n a s s i s t i n g me with animal surgery and experiments, and Ms. Madonna Chan f o r her conscientious help with animal and laboratory work.  experiments  I would l i k e to acknowledge Mr. G. Galzy, f o r h i s  technical assistance and Mr. R. Burton (Pharmaceutical Sciences) f o r help with the computer programs.  A sincere thankyou i s given to Ms. Sannifer  Louie f o r her special a t t e n t i o n i n typing t h i s manuscript. I would l i k e t o thank my family f o r t h e i r support and understanding during the preparation of t h i s manuscript. F i n a l l y I wish to acknowledge my wife E l i z a b e t h , whose patience and understanding, benefaction and devoted assistance enabled me to persue my o b j e c t i v e s . Katharine Heather.  This t h e s i s i s dedicated to Elizabeth and my daughter  1.  Introduction The growth of the ruminant f e t u s , p a r t i c u l a r l y during the l a t e r stages of g e s t a t i o n , has been the subject of extensive i n v e s t i g a t i o n and review.  Since f e t a l metabolism has been  shown to depend on a continuous supply of n u t r i e n t s , the pregnant mother must undergo s p e c i f i c metabolic a l t e r a t i o n s to ensure that the f e t a l metabolic demands are met.  Under normal p r a c t i c a l  feeding conditions the maternal ruminant i s capable of providing a n u t r i t i o n a l environment f o r the fetus which w i l l r e s u l t i n viable offspring.  However i d e n t i f i c a t i o n and u t i l i z a t i o n of  nutrients by the fetus i n regard to i t s c a l o r i c requirements have not been considered  until recently.  The major objectives of t h i s study were to quantitate the metabolism of s p e c i f i c f e t a l substrates, using a chronic f e t a l c a t h e t e r i z a t i o n procedure and i s o t o p i c t r a c e r methodologies.  In  p a r t i c u l a r , the c o n t r i b u t i o n of substrates towards f e t a l o x i d a t i v e metabolism was examined.  2.  Review of the L i t e r a t u r e 1.  Maternal n u t r i t i o n during pregnancy Hammond (1943) o r i g i n a l l y advanced the theory that a v a i l a b l e  n u t r i e n t s are divided between maternal and f e t a l tissues according to t h e i r metabolic needs.  The apparent p a r t i t i o n of nutrients f o r maternal  maintenance and f e t a l growth requirements was postulated t o be e s p e c i a l l y s i g n i f i c a n t when the n u t r i e n t supply was l i m i t e d .  The suggestions brought  f o r t h by Hammond were no doubt instrumental i n the i n i t i a t i o n of numerous studies designed t o examine the importance of adequate n u t r i t i o n during pregnancy on conceptus b i r t h weights and f e t a l v i a b i l i t y (Wallace, 1948; E v e r i t t , 1966; Robinson, 1977; Robinson ejt al_. 1977a). N u t r i t i o n a l stress imposed on the dam during e a r l y pregnancy has been shown to have a more profound d i r e c t e f f e c t on embryo m o r t a l i t y (Edey, 1976) and placental growth (Alexander, 1964) than on absolute f e t a l b i r t h weight ( H u l e t e t a l . , 1969). :  While a small amount of absolute  f e t a l growth (e.g. t o t a l body growth) occurs during e a r l y pregnancy, the s p e c i f i c growth rate (e.g. i n d i v i d u a l organ growth) i s very high (16% per day, Robinson and McDonald, 1979).  The placenta, on the other hand, i s an  a c t i v e l y growing t i s s u e i n e a r l y pregnancy.  Robinson ejt al_. (1979)  reported that a r e s t r i c t i o n i n placental development beyond a c r i t i c a l threshold (e.g. 160 g.) r e s u l t e d i n s i g n i f i c a n t f e t a l growth r e t a r d a t i o n . Alexander (1964;1974) had previously reported high c o r r e l a t i o n s between ovine f e t a l b i r t h weights and placental s i z e . During the 2nd and.3rd months of pregnancy, f e t a l and placental growth c h a r a c t e r i s t i c s change s i g n i f i c a n t l y (Robinson e t a l . , 1977a). Fetuses of ewes fed a d i e t below maintenance a t time o f conception were most vulnerable t o maternal under-nutrition a t t h i s stage of g e s t a t i o n .  3. The fetus makes i t s greatest metabolic demands upon the ewe the l a s t 8 weeks of pregnancy.  during  Robinson et al_.(1977a) reported that at four  and two weeks prepartum, 50 and 75 percent r e s p e c t i v e l y , of f e t a l b i r t h weight i s obtained.  V a r i a b i l i t y i n f e t a l b i r t h weight observed w i t h i n  common breeds has been a t t r i b u t e d to varying l e v e l s of maternal  nutrition  during l a t e pregnancy (Wallace, 1948; Alexander, 1974; M e l l o r and Matheson, 1980).  Reid (1968) and Koong et al_.(1975) have also reported that the  number of fetuses present than maternal  i n utero a f f e c t f e t a l b i r t h weights more so  under-nutrition at 115-120 days gestation.  M e l l o r and  Matheson (1980) reported a 30 to 44 per cent decrease i n f e t a l growth rates three days a f t e r the i n t r o d u c t i o n of maternal u n d e r - n u t r i t i o n . Data obtained from chronic f e t a l preparations have shown s i g n i f i c a n t changes i n f e t a l metabolite and hormone concentrations during maternal  under-  n u t r i t i o n (Tsoulos et_ al_., 1971; Bassett and M a d i l l , 1974a; M e l l o r et a l . , 1977).  P r i o r et a l . (1979) and P r i o r and L i s t e r (1979) demonstrated i n the  bovine, that although maternal metabolism was s i g n i f i c a n t l y a f f e c t e d by the r e s t r i c t i o n of d i e t a r y metabolizable energy to maintenance l e v e l s there was no s i g n i f i c a n t e f f e c t on f e t a l birthweight, f e t a l muscle p r o t e i n or RNA DNA  content.  A s i m i l a r r e s t r i c t i o n on the l e v e l of maternal  intake has been shown-to have l i t t l e e f f e c t on maternal  and  d i e t a r y energy  and f e t a l  glucone-  ogenic enzymatic a c t i v i t y i n v i t r o ( P r i o r and S c o t t , 1977). Several i n v e s t i g a t o r s have documented the s p e c i f i c changes i n maternal  intermediary  metabolism occurring during pregnancy (Bergman, 1963;  Herrera et_ al_., 1969; P r i o r and Christenson, 1978).  E a r l i e r studies have  a l s o shown that the ruminant i s susceptible to severe hypoglycemia and ketosis during the l a s t several weeks of pregnancy and i n f a c t a reduction i n food intake may  reproduce most features of c l i n i c a l ketosis i n the bovine and  4.  pregnancy toxemia i n the ovine species (Kronfeld, 1958; Reid, 1968; Bergman, 1973). The net metabolism of various substrates and the subsequent p a r t i t i o n of energy u t i l i z a t i o n by the pregnant ruminant r e c e i v i n g an adequate plane of n u t r i t i o n time.  do  not appear s u b s t a n t i a l l y a l t e r e d during t h i s  Christenson and P r i o r (1978) reported no s i g n i f i c a n t changes i n  a r t e r i a l plasma glucose concentrations during gestation i n sheep.  Signifi-  cant decreases i n maternal whole blood concentration of several amino acids have, however, been observed i n sheep during t h i s time (Morriss et al_. 1979). These r e s u l t s are of p a r t i c u l a r i n t e r e s t i n view of i n vivo studies that have d i s c l o s e d a maximal rate of maternal  gluconeogenesis  to 4th month of gestation ( P r i o r and S c o t t , 1977).  during the 3rd  Curet et a]_.  (1970)  have a t t r i b u t e d the d e c l i n e i n alpha amino nitrogen l e v e l s observed during gestation to the high c i r c u l a t i n g l e v e l s of estrogen, progesterone  and  Cortisol i n the pregnant animal. Numerous studies have demonstrated that the pregnant uterus during the l a t t e r stages of gestation consumes large q u a n t i t i e s of glucose (Kronfeld, 1958; Bergman, 1963; Reid, 1968; P r i o r and Christenson, 1978). Morriss et al_. (1974) reported logarithmic increases i n both uterine oxygen and glucose uptakes as gestation progresses.  S i g n i f i c a n t increases  in the uptake of glucose and alpha amino nitrogen by the pregnant uterus have been reported i n both ovine (Christensen and P r i o r , 1978) and bovine ( F e r r e l l and Ford, 1980) species.  Bergman (1963) comparing glucose turn-  over rates i n pregnant and nonpregnant sheep, estimated that uterine glucose metabolism accounts for  20 to 40 percent of the t o t a l  turnover r a t e i n ewes with twin pregnancies. reported that 70per cent of maternal by the uterus and i t s contents.  glucose  S e t c h e l l et al_. (1972)  glucose turnover was accounted f o r  P r i o r and Christensen  (1978)  5.  demonstrated f u r t h e r that the number of fetuses i n utero s i g n i f i c a n t l y increased the proportion of maternal  glucose turnover rate to 42 and 62 per cent respect-  i v e l y f o r twins and t r i p l e t s .  They reported also that i n s u l i n markedly reduced  uterine glucose uptake p r i m a r i l y by decreasing the plasma glucose concentrations. These r e s u l t s correspond d i r e c t l y with the increased maternal  glucose entry rates  and turnover time of the glucose pool i n pregnant sheep (Bergman, 1964; Steel and Leng, 1968).  Steel  and Leng (1968) working with pregnant ewes fed ad  1ibitum, a t t r i b u t e d the increase i n glucose entry rates to voluntary increases i n feed intake during pregnancy.  Results concerning the u t i l i z a t i o n of meta-  b o l i z a b l e energy by the pregnant ewe and conceptus confirmed t h i s conclusion (Rattray et al_., 1973).  No s i g n i f i c a n t d i f f e r e n c e s i n the amount of metaboliz-  able energy u t i l i z e d f o r body maintenance and conceptus development were established i n singleton and twin pregnancies.  However, s i g n i f i c a n t d i f f e r e n c e s  were noticed with the t o t a l feed requirements at 140 days gestation.  Further-  more, the e f f i c i e n c y at which the 140 day conceptus u t i l i z e d maternal metab o l i z a b l e energy f o r development was s i g n i f i c a n t l y greater i n ewes with a l e v e l of n u t r i t i o n that was 2x maintenance l e v e l s . 2.  Placental functions P r i o r and L i s t e r  nor uterine  (1979) reported that n e i t h e r f e t a l , placental  weights c o r r e l a t e with cotyledon number, p r i m a r i l y because of the  v a r i a b l e s i z e s of i n d i v i d u a l cotyledons.  Further i t was shown that f e t a l ,  placental and uterine weights strongly c o r r e l a t e d with cotyledon weights  6.  r e f l e c t i n g the p o t e n t i a l f o r utero-placental compensatory growth.  This con-  c l u s i o n i s f u r t h e r exemplified with data from s i n g l e and twin pregnancies and cotyledonary weights (Alexander, 1964).  Although the number of cotyledons i n  placentas o f s i n g l e fetuses was greater than that of twin fetuses there was no s i g n i f i c a n t d i f f e r e n c e i n i n d i v i d u a l placental weights.  Marked h i s t o l o g i c a l  and morphologic changes occur i n the placenta during gestation (Adherne and D u n n i l l , 1966).  Progressive and d i s t i n c t reductions of the trophoblast and  c a p i l l a r y membrane thicknesses as well as a p r o l i f e r a t i o n of the f e t a l  villus  c a p i l l a r y system have been a t t r i b u t e d to the increased a b i l i t y of the placenta t o t r a n s f e r n u t r i e n t s to the fetus (Rosso, 1980).  In a d d i t i o n , an  increased a c t i v i t y of membrane bound ribosomes (Wunderlick et aj_., 1974) and the subsequent synthesis of placental peptide hormones important f o r the regulation of placental substrate metabolism are important f u n c t i o n a l a l t e r a t i o n s occurring during the l a t t e r stages of gestation. Maturation of the placenta i s characterized by a plateauing and eventual reduction i n c e l l u l a r growth of the placenta.  This i s r e f l e c t e d  by a s i g n i f i c a n t d e c l i n e i n the instantaneous growth rate of p l a c e n t a l , uterine and cotyledonary t i s s u e s at approximately  90 days gestation i n  the ewe (Alexander, 1964) and 200 days i n the cow ( P r i o r and L i s t e r , 1979). Functional aspects of the placental t i s s u e s increase however at t h i s time. Kulhanek e t al_. (1974) demonstrated a f i v e - f o l d increase i n the permeability to urea when expressed as a f r a c t i o n o f placental DNA content.  7.  3.  Fetal growth and  development  Fetal growth i s a complex phenomenon, dependent upon a proper balance of maternal, placental and f e t a l f a c t o r s .  The factors c o n t r o l l i n g  f e t a l i n t r a u t e r i n e growth have been studied by many workers.  P r i o r to the  a v a i l a b i l i t y of c h r o n i c a l l y catheterized f e t a l sheep preparations, the majority of the information concerning i n t r a u t e r i n e development was obtained from growth measurements using comparative slaughter techniques. Mathematical equations obtained from these studies have attempted to describe f e t a l growth during the gestational period i n a number of mammalian species, most notable the ovine species (Huggett and Widdas, 1951; Langlands and Sutherland, 1968; Koong et a]_.,1975; Robinson and McDonald, 1979).  The observation that the weight of the avian and mammalian fetus  conforms to a cubic law of growth has been reported since a n t i g u i t y (Roberts, 1906; Huggett and Widdas, 1951; Langlands and Sutherland, 1968).  This  r e l a t i o n s h i p of f e t a l weight and chronological age can be expressed i n the form of a general formula: Wt  1/3  = *  (t-t ) Q  where °< = growth r a t e , derived from slope of the growth curve t = gestation time constant a f t e r conception t = s p e c i f i c time constant reduced a f t e r conception The r e s u l t s of t h i s equation when applied to numerous mammalian species i l l u s t r a t e the remarkable s i m i l a r i t y i n the rate at which i n t r a u t e r i n e growth proceeds i n domestic animals. The changes i n f e t a l crown-rump length (CRL) and weight of i n d i v i d u a l organs have also been studied e x t e n s i v e l y ,  8.  (Wallace, 1948; Joubert, 1956; Richardson and Hebert, 1978; Mellor and Matheson, 1980).  These studies have concluded that the f e t a l growth  gradient occurs a n t e r o - p o s t e r i o r l y along the main axis o f the fetus and c e n t r i p e t a l l y along the limb a x i s .  Meller and Matheson (1980) r e -  ported a l i n e a r r e l a t i o n s h i p between f e t a l CRL and f e t a l weight a f t e r 100 days g e s t a t i o n ; however, a c u r v i - l i n e a r r e l a t i o n s h i p was observed when the t o t a l gestational period was considered. This i s a t t r i b u t e d to the markedly d i f f e r e n t rates a t which i n d i v i d u a l organs grow and the continuous changes i n f e t a l conformation (Wallace, 1948; Rattray e t a l . , 1975). Richardson and Hebert 0 9 7 8 ) , with a l i m i t e d number of fetuses, supported t h i s conclusion with observations made on organs of the nervous system.  Although the cube root of body and t o t a l organ weights gave a  l i n e a r regression with f e t a l age throughout g e s t a t i o n , the cube root of the b r a i n , cerebellum and spinal cord weights resulted i n a sigmoidal trend.  Completion of organogenesis i n v i t a l organs such as those o f the  nervous system has been postulated to be the major f a c t o r l i m i t i n g the gestational length periods (Sacher and S t r a f f e l d t , 1974). Hyperplasia increases as well throughout gestation i n mammalian fetuses (Winnick and Noble, 1965).  P r i o r e t al_. (1979)  have reported  s i g n i f i c a n t increases i n t o t a l f e t a l DNA and DNA/protein and RNA/DNA r a t i o s near the end of gestation i n the bovine fetus.  Hypertrophy as  r e f l e c t e d by DNA/protein and RNA/DNA r a t i o s also increases continuously with f e t a l age. 4.  Fetal growth and endocrinology a)  Growth hormones (OGH and PCS) The source of f e t a l ovine growth hormone (OGH) i s the  9.  f e t a l p i t u i t a r y , as evidenced  from the f e t a l hypophysectomized  studies and the r e s u l t i n g low c i r c u l a t i o n of growth hormone i n f e t a l plasma (Wallace et al_., 1972).  In t h i s study i t was shown  that i s o t o p i c l a b e l l e d growth hormone when administered into the maternal  c i r c u l a t i o n was not detected i n f e t a l plasma.  The pattern  of OGH concentration during gestation i s t r i p h a s i c with high conc e n t r a t i o n a t 100-110 days of gestation (Gluckman e t a l . , 1979). Dramatic increases i n the f e t a l c i r c u l a t i n g hormone concentrations occur i n the l a s t month of pregnancy t o l e v e l s three times the concentrations at 100 days of gestation and ten times the concentrat i o n found p o s t n a t a l l y (Bassett e t a l . , 1970). Bassett and M a d i l l (1974b) attempted to determine the regulatory mechanism of f e t a l OGH secretion by i n f u s i n g glucose continuously f o r a prolonged period of time.  Although glucose i s  known to depress OGH i n adult sheep, these workers were unable to achieve the same r e s u l t i n the f e t u s .  L i g g i n s and Kennedy (1968)  reported that t o t a l hypophysectomy was associated with a r e t a r d a t i o n of somatic development, most notably i n bone t i s s u e .  Underdevelopment  of the t h y r o i d and adrenals was also reported i n t h i s study, suggesting that the observed developmental r e t a r d a t i o n may be a r e s u l t of hypothyroidism rather than hypophysectomy.  No growth r e t a r d a t i o n has been  reported i n hypophysectomized porcine fetuses a t 40-50 days of gestation (Stryker and Dzuick, 1975). Wyk et al_. (1974) demonstrated that growth hormone does not d i r e c t l y regulate s k e l e t a l growth, but rather acts i n d i r e c t l y through a generation of intermediate hormones c a l l e d somatomedins.  Somatomedin  10.  i s a peptide hormone reported to influence peripheral a c t i o n of growth hormone and regulate f e t a l growth (Falkner et al.,1979). Hintz e_t al_. (1977) working with infants s u f f e r i n g from a p r o t e i n c a l o r i e d e f i c i e n c y reported low serum l e v e l s of somatomedin i n s p i t e of elevated l e v e l s of growth hormone.  A s i m i l a r observation regarding  these two hormones has been reported by Robinson e_t al_. (1977b) i n f e t a l sheep.  Plasma somatomedin concentration i n fetuses from ewes  that had undergone endometrial  carunculectomy was lower though normal  concentrations of f e t a l growth hormone was observed.  I t would appear  from r e s u l t s reported by Falkner et al_. (1979) i n hypophysectomized and nephrectomized f e t u s e s , that somatomedin a c t i v i t y i n f e t a l sheep i s regulated by s i m i l a r mechanisms i n the adult.  No s i g n i f i c a n t  changes i n somatomedin a c t i v i t y was observed i n control fetuses throughout the gestational period though  reduced f e t a l somatomedin  a c t i v i t y was reported i n hypophysectomized and nephrectomized fetuses. In s p i t e of the exhaustive studies designed to monitor f e t a l OGH  throughout gestation there i s no  d i r e c t r e l a t i o n s h i p between f e t a l  OGH  and i n t r a u t e r i n e weight changes i_n utero.  The i s o l a t i o n and  c h a r a c t e r i z a t i o n of placental e x t r a c t , known as ovine c h o r i o n i c somatomammotrophin, (OCS) of  placental  1975). to OGH  OCS  regulation is  a  stimulated i n t e r e s t and  fetal  polypeptide  growth  in  (Martal  hormone,  the and  area Djiane,  similar  i n f u n c t i o n and possesses both growth and lactogenic p r o p e r t i e s .  This hormone i s secreted p r i m a r i l y i n the maternal predominantly  c i r c u l a t i o n , and i s  a c t i v e as e a r l y as day 16 i n the trophoblast (Martal  and Djiane, 1977), and reaches a maximum at 120 days of g e s t a t i o n .  11.  Martal (1978) reported that the sum of OGH  and OCS  r e l a t e d to f e t a l i n t r a u t e r i n e weight changes.  i s closely  This i s contrary to  the observations of the adult where the sum of growth promoting a c t i v i t i e s (OGH period.  and OCS)  remain constant during the g e s t a t i o n a l  Martal concluded  from these r e s u l t s that OCS c o n t r o l l e d  f e t a l growth during the f i r s t h a l f of gestation and that the combined a c t i v i t i e s of OGH  and OCS regulated f e t a l growth during the  second h a l f of gestation.  Thyroid Hormones The t h y r o i d axis of the f e t a l sheep i s a c t i v e and independent of the maternal  axis.  Experiments i n thyroidectomized  ovine  fetuses have reported undetectable q u a n t i t i e s of t h y r o i d hormones, confirming that a maternal  source does not contribute .to the basal  l e v e l s of f e t a l plasma thyroxine concentrations (Hopkins et a l . , 1971; Erenberg et al_., 1973).  Morphological changes i n the f e t a l  t h y r o i d gland and detectable l e v e l s of thyroxine (T^) have been demonstrated as e a r l y as 50 days of gestation (Thornburn and Hopkins, 1973).  The s i g n i f i c n a c e of f e t a l t h y r o i d hormones on f e t a l  intrau-  t e r i n e growth i s r e l a t e d to the stage of f e t a l maturity at b i r t h (Hopkins et a l . , 1972; Erenberg et a l _ . , 1973).  Thyroidectomized  f e t a l lambs have shown s i g n i f i c a n t growth r e t a r d a t i o n i n o s s i f i c a t i o n centers of hind limbs, reduced muscle development and thymus weights and reduced d i f f e r e n t i a t i o n of wool f o l l i c l e s (Hopkins, 1975).  12.  ADRENAL CORTICOSTEROIDS Studies with adrenalectomized fetuses have shown increased f e t a l growth without hepatic glycogen deposition and high m o r t a l i t y after birth  (Barnes et al_., 1977).  In addition to the induction of  several enzymes c r i t i c a l f o r f e t a l metabolic homeostasis, the glucoc o r t i c o i d s ensure optimal f e t a l maturation at b i r t h  despite gestational  age v a r i a t i o n ( L i g g i n s , 1976). Jost (1961) f i r s t reported the importance of the adrenal cortical  hormones i n inducing f e t a l l i v e r glycogen synthesis i n the  fetal rabbit.  Dramatic increases i n f e t a l plasma c o r t i c o s t e r o i d s ,  most notably C o r t i s o l ,  i n the l a s t few days prepartum have been reported  in ruminant (Bassett and Thorburn, 1969) and porcine (Dvorak, 1972) fetuses.  There i s a close temporal r e l a t i o n s h i p between the observed  increase i n f e t a l plasma C o r t i s o l and hepatic glycogen content (Barnes et al_., 1978), which indicates that the adrenal cortex i s as e s s e n t i a l organ i n the control of hepatic glycogen storage i n the ovine fetus. INSULIN AND GLUCAGON The f e t a l pancreas plays a v i t a l r o l e i n the metabolism of nutrients delivered to the fetus by the maternal organism.  The  precise r o l e that i n s u l i n and glucagon play i n regulating f e t a l  growth  has been studied extensively i n the r a t and sheep (Alexander et a l . , 1971 , 1972, 1973, 1976; Girard et aj_., 1973; Bassett et al_., 1973): I n s u l i n and glucagon influence f e t a l growth i n d i r e c t l y  by i n f l u e n c i n g  the establishment of energy reserves (Milner, 1979) and consequently have been considered t o be the most important growth promoting factors of the fetus.  13.  The ovine placenta i s impermeable to i n s u l i n and glucagon, (Alexander et al_., 1972, 1973 and Sperling et al_., 1973), i n d i c a t i n g that substrate metabolism i s regulated by f e t a l pancreatic a c i t i v i t y . Detectable l e v e l s of f e t a l plasma i n s u l i n and glucagon during most of the gestational period have been reported (Willes et al_., 1969 Alexander et a]_. ,1971).  and  The secretory response of the pancreatic  beta c e l l matures e a r l i e r than that of the alpha secretory c e l l s ( F i s e r et al_. ,1974) which i n part explains the large c i r c u l a t i n g i n s u l i n to glucagon molar r a t i o reported by Girard et al_. (1974). A p o s i t i v e r e l a t i o n s h i p e x i s t s between i n s u l i n secretion and glucose homeostasis (Bassett and M a d i l l 1974a,b; F i s e r et al_. 1974; Simmons et al_., 1978).  W i l l e s et al_. 0969) concluded from studies on  ovine f e t u s e s , with comparatively short postoperative recovery periods, that the f e t a l pancreas does not respond to a glucose or fructose intravenous challenge by secreting i n s u l i n .  This r e s u l t was  in direct  c o n f l i c t to e a r l i e r observations reported by Alexander et al_. (1969), who reported a s i g n i f i c a n t i n s u l i n secretion i n response to a glucose infusion.  F i s e r e t a l _ . (1974)  confirmed Alexander's  original  observa-  t i o n and added that the f e t a l i n s u l i n response increased as pregnancy proceeded, though the magnitude of t h i s response was lower than i n the adult.  significantly  Bassett et al_. (1973) and Bassett and Madill  (1974b) demonstrated that i n s u l i n release by the f e t a l pancreas was stimulated by glucose at concentrations present i n f e t a l plasma. P h i l i p p s et al_. (1978) concluded  from slopes of the i n s u l i n response  curve that the p a n c r e a t i c / - c e l l was s e n s i t i v e to a l t e r a t i o n s i n f e t a l glucose concentrations.  I t was also reported that f e t a l s e n s i t i v i t y to  14.  glucose was equivalent t o maternal responses, though a s i g n i f i c a n t lag time existed before a response was noted.  S i m i l a r i n s u l i n induced  responses were observed i n fetuses from fed and starved ewes (Schreiner et al_., 1980).  Although maternal s t a r v a t i o n was noted to cause a 50%  reduction i n f e t a l plasma glucose concentrations, the k i n e t i c s of f e t a l i n s u l i n secretion were not a f f e c t e d . The apparent biphasic secretory pattern o f i n s u l i n reported by P h i l i p p s et_ al_. (1978), warrants the use of a continuous i n f u s i o n technique rather than an acute i n j e c t i o n of glucose f o r sustaining a p h y s i o l o g i c a l response to i n s u l i n .  S i m i l a r inconsistencies i n the  l i t e r a t u r e regarding fructose and alanine as stimulators o f f e t a l i n s u l i n secretion can be explained on the basis of d i f f e r e n t experimental protocols.  Davis et al_. (1971) and Bassett and M a d i l l (1974b) reported  p o s i t i v e responses i n f e t a l i n s u l i n secretion following fructose i n f u s i o n s ; however, t h i s r e s u l t was not obtainable i n a more recent study by P h i l i p p s e t al_. (1978).  S i m i l a r l y , F i s e r et al_. (1974) reported no  change i n f e t a l glucose, i n s u l i n or glucagon l e v e l s f o l l o w i n g an acute i n j e c t i o n of alanine to the f e t u s .  P h i l i p p s et al_. (1980) employing a  square wave i n f u s i o n technique, reported an induced elevation of i n s u l i n , with maximal response 60 minutes a f t e r the s t a r t of the amino acid infusion. The b i o l o g i c a l a c t i v i t y of f e t a l i n s u l i n has been equally d i f f i c u l t to define.  C o l w i l l et al_. (1970) infused pharmacological  dosages o f i n s u l i n to the fetus and reported minimal decreases i n the concentration of f e t a l plasma glucose, which l e d them to conclude that there was a lack of control i n the rate of glucose u t i l i z a t i o n by the  15.  fetus.  Simmons e t al_. (1978) infused i n s u l i n over a longer period  of time and reported an increase i n f e t a l glucose u t i l i z a t i o n , i n dependent of any changes i n u m b i l i c a l blood flow, f e t a l oxygen consumption and placental clearance. This report disproved previous evidence reported by Alexander e t al_. (1970), describing l i t t l e or no e f f e c t of i n s u l i n on f e t a l glucose u t i l i z a t i o n .  Recently Carson  ejt al_. (1980) demonstrated a dose r e l a t e d increase i n a r t e r i a l venous differences of whole blood glucose and oxygen across the umbilical circulation  during a sustained i n f u s i o n of i n s u l i n to the ovine f e t u s .  Fetal hypoxia was also noticed to occur i n t h i s study and i t was speculated that i n s u l i n , by increasing the u t i l i z a t i o n of glucose by f e t a l t i s s u e s compromised f e t a l oxygenation. The functional secretory mechanisms of glucagon and i t s biological  a c t i v i t y during f e t a l l i f e are not well understood.  The  r e l a t i v e importance of glucagon may be of greatest s i g n i f i c a n c e during the immediate neonatal period. The sudden f a l l i n plasma i n s u l i n at b i r t h and the r i s e i n glucagon are responsible f o r the t r i g g e r i n g of glycogen m o b i l i z a t i o n and i n i t i a t i o n of gluconeogenesis in the neonate (Snell and Walker, 1978).  During t h i s time the develop-  ment of glycogenolys.is and gluconeogenesis i s c r i t i c a l i n order to maintain blood glucose l e v e l s . No c o r r e l a t i o n appears to e x i s t between f e t a l glucose and glucagon concentrations i n the ovine fetus ( F i s e r et al_., 1974).  This  i s supported by r e s u l t s of Alexander e t aj_. (1976) who a l s o observed no apparent change i n plasma glucagon concentrations f o l l o w i n g induced f e t a l hypoglycemia.  insulin  F a i l u r e to stimulate glucagon release by  i n f u s i o n of alanine i n vivo has been reported i n both the rhesus monkey fetus (Chez et a l _ . , 1974) and the ovine fetus (Fisher e t a l . , 1974).  However, independent studies with f e t a l rats (Girard et a l . ,  1971) and f e t a l sheep (Alexander et a l . , 1976) have reported significant  pancreatic release of glucagon with arginine i n f u s i o n s .  Girard et al_. (1973) demonstrated i n the r a t fetus that exogenous noradrenalin stimulated glucagon and i n h i b i t e d release from the pancreas.  insulin  These workers proposed that f e t a l stress  induced at the time of b i r t h was a p o t e n t i a l t r i g g e r i n g mechanism f o r the release of noradrenalin at the pancreatic nerve endings, and would i n turn stimulate the release of glucagon and i n h i b i t the r e l e a s e of i n s u l i n .  I t was f u r t h e r concluded that f e t a l r a t pancreas  does not respond to acute changes i n blood glucose by increasing glucagon release, hence r u l i n g out the p o s s i b i l i t y that postnatal hypoglycemia was a p h y s i o l o g i c a l stimulator of glucagon release.  e)  HORMONAL ENZYME INDUCTION  G l u c o c o r t i c o i d s , catecholamines and glucagon are potential stimulators of enzyme induction whereas i n s u l i n i s an a c t i v e antagonist. Glucocorticoids have been reported to be potent regulators of urea formation  (argininosuccinate synthetase E.C.6.3.4.5.,  Raiha and Suihkonen, 1968), amino a c i d metabolism ( t y r o s i n e aminotransferase E.C.2.6.1.5., Holt and O l i v e r , 1969) and carbohydrate  metabolism (P.E.P. carboxykinase E.C.4.1.1.3.2., Kirby and Hahn, 1973) i n s p e c i f i c mammalian species.  Glucagon and catecholamines  are p o s i t i v e stimulators of tyrosine aminotransferase a c t i v i t y and can reverse the e f f e c t of adrenalectomy i n the r a t fetus (Holt and O l i v e r , 1971).  Catecholamines have a l s o been reported to  stimulate gluconeogenesis by s t i m u l a t i n g phosphoenolpyruvate carboxykinase (Holt and O l i v e r , 1968).  S i g n i f i c a n t enzyme induction  f o l l o w i n g p a r t u r i t i o n i n the neonate (Warnes et_ al_., 1977b) can be a t t r i b u t e d to glucagon release triggered by elevated f e t a l catecholamines (Girard et al_., 1973).  I t i s thus evident that  g l u c o c o r t i c o i d s , glucagon, i n s u l i n and catecholamines p a r t i c i p a t e in a complex i n t e r p l a y which determines the process of enzyme induction i n the f e t a l l i v e r and p o s s i b l y kidney.  Fetal metabolism a)  TECHNIQUES FOR METABOLIC STUDIES IN THE FETUS The pregnant sheep has been a useful animal model f o r studying f e t a l physiology.  The modern era of f e t a l physiology .  research began with the work of Huggett (1927) with e x t e r i o r i z e d animal preparations.  The d e l i v e r y of a pregnant goat by caesarean  section i n t o a bath tub f i l l e d with warm s a l i n e enabled Huggett to measure a v a r i e t y of f e t a l p h y s i o l o g i c a l parameters. of t h i s procedure was performed by Barcroft et al_.  Modification (1939) and  Barcroft and Baron (1946) i n attempts to e l u c i d a t e f u r t h e r the various aspects concerning the metabolism o f the fetus i n utero.  18.  In view of the obvious shortcomings of these  procedures,  p r i m a r i l y the r e l a t i v e l y short time period during which experiments could be performed, f e t a l perfusion procedures were developed. Alexander ejt al_. (1955), employing u m b i l i c a l perfusion of the placenta and Andrews e_t al_. (1961) using perfused l i v e r s attempted to maint a i n a p h y s i o l o g i c a l environment f o r longer durations f o r experimental purposes.  Alexander ejt al_. (1964) f u r t h e r modified these procedures  by i s o l a t i n g the sheep fetus and connecting i t to the u m b i l i c a l c i r c u l a t i o n through an extracorporal c i r c u i t . This development enabled workers to observe the fetus f o r longer periods of time a f t e r separating i t from the placenta.  Numerous aspects of f e t a l  metabolism were i n v e s t i g a t e d by Alexander and coworkers with t h i s procedure. The p o s s i b i l i t y of cannulating ovine f e t a l blood vessels to f a c i l i t a t e p h y s i o l o g i c a l studies i n unrestrained animals and without the influence of anesthetics and surgical stress was f i r s t demonstrated by Blechner et al_.(1960).  However, the vascular catheters  remained f u n c t i o n a l only f o r a short period of time and t h i s necessi t a t e d improvements to be made i n the s u r g i c a l procedures.  Meschia et  a l . (1965a) and Kraner (1965) reported procedures f o r chronic catheteri z a t i o n of f e t a l blood vessels i n unstressed animals f o r the purpose of sampling the fetus f o r longer periods of time.  The question of how  long one must wait f o l l o w i n g surgery to be assured that the n u t r i t i o n and metabolic status of operative fetuses represented normal, unoperated  19.  fetuses was examined by Clapp et al_., 1977; S l a t e r and M e l l o r , 1977; K i t t s et al_., 1979.  Recommendations from these studies indicated that  i t was i n s u f f i c i e n t to simply r e l y on blood gas and pH measurements to determine animal normality since metabolic c o r r e l a t e s of the fetus are of equal concern.  Further i t was suggested that a minumum of 5  postoperative days be given f o r f e t a l and maternal recovery from s u r g i c a l trauma before experiments were i n i t i a t e d . Several sophisticated versions of these techniques are c u r r e n t l y i n use i n studying f e t a l metabolism i n the undisturbed p h y s i o l o g i c a l s t a t e , and consequently have led to the tremendous wealth of information on f e t a l physiology that has been generated from the ovine fetus in_ utero. FETAL CALORIC REQUIREMENTS The t o t a l f e t a l c a l o r i c requirement has been evaluated from c a l c u l a t i o n s based on substrate and oxygen consumption measurements and the determination of c a l o r i c r e q u i s i t e s f o r new t i s s u e a c c r e t i o n , by bomb calorimetry (Rattray e t al_.,1974; B a t t a g l i a and Meschia, 1978). A majority of the information a v a i l a b l e concerning f e t a l substrate u t i l i z a t i o n has o r i g i n a t e d from studies focused on a time period near the end of gestation when the greatest increment of f e t a l substrate requirement occurs.  B a t t a g l i a and Meschia (1978) proposed that the f e t a l  lamb oxidizes a mixture of carbohydrates and amino acids. that the c a l o r i c  yield  of 1  litre  Assuming  of oxygen necessary to combust  t h i s mixture i n toto i s 4.9 k c a l , the d a i l y o x i d a t i v e requirements of the ovine fetus consuming 6 to 9 mis oxygen/min/kg at STP was approximated to be 56 kcal/day/kg.  Rattray et a]_. (1974) d i s c l o s e d that the  130 day ovine fetus contains a c a l o r i c value of 0.9 kcal/g and gains weight at a rate of 36 g/day/kg.  The c a l o r i c requirement . f o r the  20.  formation of new t i s s u e was computed to approximate 32 kcal/g/day.  The  t o t a l c a l o r i c requirements of the ovine fetus has therefore been reported to approximate 88 kcal/day/kg of which 64% i s used f o r o x i d a t i v e purposes and 36% f o r t i s s u e growth ( B a t t a g l i a and Meschia, 1978). i)  OXYGEN CONSUMPTION AND CARBON DIOXIDE PRODUCTION Since the presence of oxygen i s e s s e n t i a l f o r the o x i d a t i v e metabolism of substrates, a tremendous amount of i n t e r e s t has been d i r e c t e d towards accurately d e f i n i n g the mechanisms of oxygen and carbon dioxide exchange between maternal and f e t a l compartments (Meschia et al_., 1965a,b; Motoyama et aj_., 1967; Matalon e t a l . , 1978). Respiratory gases cross the placenta by simple d i f f u s i o n 1977)  (Faber,  and the high a f f i n i t y f o r oxygen i n f e t a l blood (Naughton  et_ al_., 1963)  i s common to a l l mammalian species.  This i s a t t r i b u t e d  to the quantity and unique type of f e t a l hemaglobin (Metcalfe e t a l . , 1972).  These c h a r a c t e r i s t i c s of f e t a l blood account f o r the differences  in the carrying capacity and d i s s o c i a t i o n curves of maternal and f e t a l blood and f a c i l i t a t e the t r a n s f e r of r e s p i r a t o r y gases across the placenta.  Notable d i f f e r e n c e s between maternal and f e t a l  P  cni  ou (oxygen tension at which 50 percent saturation occurs at pH 7.4), have been reported i n a number of mammalian species ( S i l v e r and Comline, 1975) and are l a r g e l y responsible f o r the magnitude of the transplacental gradient f o r PO^.  In ruminant and porcine species,  the transplacental gradient of oxygen i s 10-20 times greater than in equine species.  This i s an i n t e r e s t i n g observation when con-  s i d e r i n g that a l l three species possess a s i m i l a r e p i t h e l i o c h o r i a l placenta ( S i l v e r and Comline, 1975).  S t r u c t u r a l d i f f e r e n c e s i n the  21.  arrangement of the placental vascular c i r c u l a t i o n are responsible f o r the large d i f f e r e n c e s i n maternal-fetal substrate in these animals ( S i l v e r et al_., 1973). blood i s lower than i n the ewe. with the high f e t a l PCO^  gradients  The PG^ i n f e t a l a r t e r i a l  This c h a r a c t e r i s t i c ,  together  and pH, previously led i n v e s t i g a t o r s to  suggest that the fetus e x i s t s i n a hypoxic and a c i d o t i c environment (Vaughn et al_., 1968).  More r e c e n t l y , reports have disputed  this  concept and strongly suggested that anaerobic metabolism does not play an important r o l e i n f e t a l metabolic and Meschia, 1978).  activities (Battaglia  B a t t a g l i a et al_. (1968) demonstrated that f e t a l  oxygenation did not a l t e r s i g n i f i c a n t l y following increased oxygen availability.  Furthermore, i t was  shown that although maternal  was a l t e r e d by increasing the amount of oxygen, the increase  PO^  was  not p r o p o r t i o n a l l y transmitted to the f e t u s . The study of f e t a l uptake and excretion of substances, such as oxygen, has r e l i e d on simultaneous measurements of a r t e r i a l venous d i f f e r e n c e s crossing the u m b i l i c a l and uterine c i r c u l a t i o n s , the blood flow of these c i r c u l a t i o n s , and the a p p l i c a t i o n of the Fick p r i n c i p l e (Meschia et a l . , 1965b, 1967b; James e t a l , 1972) Oxygen  consumption  unanesthetized  in  chronically  catheterized  fetuses varies between 6 and 9 ml/min/kg f e t u s .  This accounts f o r approximately 60% of the t o t a l uterine uptake of oxygen (Meschia _et j f L , 1980).  An inverse r e l a t i o n s h i p e x i s t s  between the arterial-venous d i f f e r e n c e of oxygen and the  blood  flow i n the uterine and u m b i l i c a l c i r c u l a t i o n s , thereby f a c i l i t a t i n g a r e l a t i v e l y constant f e t a l oxygen consumption despite f l u c t u a t i o n s  22.  in blood flow (Meschia et al_., 1967a, Comline and S i l v e r , 1976). The rate of f e t a l oxygen consumption when expressed per u n i t body weight i s greater than the basal l e v e l of the adult (Table 1). B a t t a g l i a and Meschia (1978) have also pointed out that f e t a l oxygen consumption values, when expressed on a basis of f e t a l body weight, are s i m i l a r i n species that d i f f e r i n body s i z e .  This  i s contrary to what i s true i n adult animals ( K l e i b e r , 1947). TABLE 1:  OXYGEN CONSUMPTION RATES OF ADULTS AND FETUSES OF SPECIES OF DIFFERENT SIZE* Oxygen Consumption (ml/min/kg Fetus Adult  Animal Horse Cattle Sheep Rhesus Monkey Guinea Pig  BWT)  7.0 7.4 6.9 7.0 8.5  2.0 2.2 4.0 7.0 9.7  * from B a t t a g l i a and Meschia (1978) S i m i l a r approaches have been taken to assess f e t a l  CO2  production rates across the placenta i n c h r o n i c a l l y catheterized ovine fetuses (James et aj_., 1972).  The carbon dioxide production  reported by these workers u t i l i z i n g the Fick p r i n c i p l e , was ml/min/kg by fetus.  5.65  Single fetuses y i e l d e d a s l i g h t l y higher C O 2  production rate than twins (5.4 vs 5.1 ml/min/kg f e t u s ) and the time f o l l o w i n g surgery before experiments were i n i t i a t e d significant. blood  P C 0 2 >  appeared  No s i g n i f i c a n t changes are observed i n the f e t a l pH or bicarbonate l e v e l s at d i f f e r e n t stages of  gestation i n sheep (Comline and S i l v e r , 1972).  Fetal  in the f e t a l a r t e r y are higher than maternal a r t e r i a l  P C 0  P C 0  ?  2  levels and can  23.  change i n d i r e c t proportion to maternal PCO,,. The pH of f e t a l blood i s always s l i g h t l y lower than that of the mother, although standard bicarbonate concentrations are s i m i l a r (Meschia et a l . , 1970). GLUCOSE Huggett et a[. (1951) f i r s t reported the t r a n s f e r of monosaccharides across the ovine placenta and demonstrated the p r e f e r e n t i a l permeability of the placenta to glucose. C a l c u l a t i o n s made by Widdas (1952) on the data of Huggett et al_. (1951) indicated that the t r a n s f e r of glucose across the placenta of sheep could not be explained by simple d i f f u s i o n mechanisms alone.  The chemical  and s t e r e o - s p e c i f i c c h a r a c t e r i s t i c s regulating placental t r a n s f e r of mono and disaccharides were examined by Walker (1960).  It i s  now believed that the t r a n s f e r of glucose from the mother to the fetus i s mediated by a process of f a c i l i t a t e d d i f f u s i o n , analagous to the mechanisms i n the erythrocyte (Widdas 1961; Boyd et al.,1976). Simmons et al_. (1979) recently concluded from determinations on the rate of placental glucose t r a n s f e r 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 , that the rate of glucose t r a n s f e r was not only l i m i t e d by transport c h a r a c t e r i s t i c s but was a l s o affected by the u t i l i z a t i o n rate of glucose by placental t i s s u e s . I t was not u n t i l the advent of 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 a l preparations that the r e l a t i o n s h i p between f e t a l and maternal glucose concentrations was accurately assessed (James e t al.,1972, Shelley, 1973; Bassett and M a d i l l , 1974a).  The d i f f e r e n c e between  f e t a l and maternal concentration i s common t o a l l animal species  24.  and does not change s i g n i f i c a n t l y with gestation ( S i l v e r , 1977). This observation has been a t t r i b u t e d to a combination tissues ( S i l v e r , 1977; S h e l l e y , 1979).  of placental  Data obtained from the  cow, sheep and horse have indicated that the placental morphology influences the f e t a l to maternal 1973).  glucose gradients ( S i l v e r et a l . ,  Although a l l three species possess s i m i l a r placentas,  the r e l a t i v e i n e f f i c i e n c y of the ruminant placenta, r e s u l t a n t of i t s vascular morphology, i s responsible f o r the low maternal  to  f e t a l substrate gradients reported i n the ruminant ( S i l v e r et a l . , 1973).  The metabolism of glucose by the placenta i s an a d d i t i o n a l  f a c t o r c o n t r o l l i n g f e t a l glucose metabolism.  Alexander et al_. (1955)  demonstrated i n e x t e r i o r i z e d , perfused ovine fetuses a s i g n i f i c a n t proportion of fructose was derived from glucose by placental t i s s u e s . B a t t a g l i a et al_. (1961) f u r t h e r demonstrated that ovine placental cotyledons were p o t e n t i a l consumers of glucose.  S i l v e r (1977)  reported that s i g n i f i c a n t l y greater uptakes of glucose occur i n the ovine uterus than the fetus alone, and S e t c h e l l et al_. (1972) suggested that a considerable amount of glucose removed by the uterus was not oxidized but rather could be used f o r s y n t h e t i c purposes. The r e l a t i o n s h i p between f e t a l glucose uptake and maternal  concentration of glucose has also been studied i n d e t a i l .  Alexander et al_. (1955) f i r s t d i s c l o s e d that an a r t i f i c i a l elevat i o n of maternal  glucose concentration r e s u l t e d i n an increased  t r a n s f e r of glucose to the f e t u s .  Later studies by Alexander et a l .  (1969), i n e x t e r i o r i z e d f e t u s e s , showed that glucose uptake by the fetus was approximately  6 mg/min/kg f e t u s , s i m i l a r to reported  values i n the newborn lamb (5 mg/min/kg lamb, J a r r e t t et al_. 1964).  25.  James e t  a l _ . (1972) r e p o r t e d  g l u c o s e from c h r o n i c a l l y 1 to 6 mg/min/kg fetus umbilical  catheterized  uptake  values  ovine fetuses  ranged  and m a t e r n a l  Comparative studies  arterial  i n the  cow, sheep,  g l u c o s e c o n c e n t r a t i o n and t r a n s p l a c e n t a l fetus  would appear the  fetus  t o be an u p p e r  limit as  to the  and  1973; S i l v e r  et  a l _ , 1973).  or maternal-fetal gestation,  plateau  although there are  o f h y p o x i a , as  lambs a l s o a f f e c t s gradients  dramatic  fetal  fall  onset  is  of maternal  levels  (Shelley  glucose  uptake  advancing  i n net  (Boyd e t  with  the  fetal  fetal  aJU 1973).  spontaneous  hypoxemic  glucose  hypoglycemia there  and f e t a l  50 and 35% o f f e d s t a t e r e s p e c t i v e l y  et  a l * 1978; Anand e t  leads  basal  in fetal  flow  to  1977).  to  consequently  There  a l s o been shown  g l u c o s e and t r a n s p l a c e n t a l  i n both the maternal  glucose concentration  has  increases  blood  glucose  p u b l i s h e d on  observed with  shown i n s t u d i e s  ( C h a r and C r e a s y , With the  change  notable  oxygen c o n s u m p t i o n and u m b i l i c a l The l e v e l  It  t o 40-60% o f t h e  glucose gradients  arterial  1976).  hyperglycemia, that  No n e t  horse  amount t r a n s f e r r e d  1976).  experimentally induced maternal  between  rate of  e v i d e n c e d from d a t a  ( C o m l i n e and S i l v e r ,  glucose uptake w i l l  transfer  a l _ . , 1 9 7 3 ; C o m l i n e and S i l v e r ,  from the mother,  bovine fetus in  (Boyd e t  from  glucose con-  h a v e a l s o shown a p o s i t i v e c o r r e l a t i o n b e t w e e n m a t e r n a l  to the  of  and a l i n e a r r e l a t i o n s h i p e x i s t e d  glucose uptake  centrations.  that umbilical  aj_, 1980). gradient  to a reduced  glucose  (Boyd e t  transfer  a  concentrations,  aJU 1973; S c h r e i n e r  The m a t e r n a l - f e t a l  also falls  is  arterial  significantly,  and  rate of glucose to  the  26.  fetus (Schreiner et al_., 1978).  Schreiner et al_. (1978)  proposed that the decline i n f e t a l glucose concentration to a r e l a t i v e l y constant l e v e l , tends t o restore the glucose concentration gradient across the placenta and thus increases the umbilical glucose uptake towards normal l e v e l s observed i n the nonstarved state.  Further, the fetus reduces i t s consumption  of exogenous glucose as evidenced by the reduction i n plasma i n s u l i n l e v e l s 2 days a f t e r maternal f a s t i n g (Bassett and M a d i l l , 1974b). FETAL GLUCONEOGENESIS Whereas hepatic gluconeogenesis i s the major source of blood glucose i n the adult ruminant (Bergman, 1973), the s i g n i f i cance of gluconeogenesis i n the fetus, i s not t o t a l l y resolved. Incorporation of U-  14  C pyruvate i n t o  14 C glucose was  o r i g i n a l l y reported i n l i v e r s l i c e s of f e t a l sheep, embryonic chicks and postnatal rats and sheep but not i n f e t a l rats and O l i v e r , 1965).  (Ballard  The a c t i v i t y of gluconeogenic enzymes i n the  f e t a l r a t i s low ( B a l l a r d and Hansen, 1967) and although phospoenopyruvate  carboxykinase (PEPCK) a rate l i m i t i n g enzyme,  i s i n d u c i b l e p r i o r t o p a r t u r i t i o n with glucagon, the o v e r a l l pathway i s not detectable ( P h i l l i p p i d e s and B a l l a r d , 1969).  The  key regulatory enzymes of gluconeogenesis are present i n substantial a c t i v i t i e s i n f e t a l sheep l i v e r (Stephenson e_t aj_., 1976; Warnes et_ a h , 1977b) and f e t a l kidney cortex (Stephenson e t a l . , 1976). P r i o r to 100 days of gestation there i s a l i m i t e d i n v i t r o production of endogenous glucose due to the low l e v e l s of f r u c t o s e 1, 6 diphosphatase (F.I.6.D.P.) and glucose 6 phosphatase  (G.6.P.)  a c t i v i t i e s (Stephenson et a l . , 1976).  Warnes et al_. (1977b)  also reported that c y t o s o l i c PEPCK a c t i v i t i e s at mid gestation were only 10 per cent of the a c t i v i t y at b i r t h .  Both l i v e r and  kidney gluconeogenic enzyme a c t i v i t i e s are comparable to adult l e v e l s j u s t p r i o r to p a r t i t i o n  (Stephenson et a l _ . , 1976; Warnes  et al_., 1977b; P r i o r and S c o t t , 1977).  P r i o r and Scott (1977)  f u r t h e r demonstrated the capacity f o r gluconeogenesis i n the bovine fetus as e a r l y as 88 days of gestation.  These workers concluded  that a r e s t r i c t i o n i n maternal d i e t a r y energy during l a t e gestation does not s i g n i f i c a n t l y activity.  a l t e r maternal or f e t a l  gluconeogen  Swiatek (1971) reported that both pyruvate carboxylase  (PC) and PEPCK were l i m i t i n g f a c t o r s i n the absence of gluconeogenes i n the neonatal p i g . Jones and Ashton (1976) demonstrated i n v i t r o that the f e t a l guinea pig has a functional gluconeogenic pathway i n both l i v e r and kidney t i s s u e s , 10 days p r i o r to b i r t h . Two aminotransferases, glutamate-oxaloacetate aminotransferase and glutamate-pyruvate aminotransferase are a c t i v e i n bovine f e t a l l i v e r and kidney t i s s u e s as e a r l y as 45 days o f gestation (Stephenson e t al_., 1976).  Edwards e t al_. (1975)  reported marked increases i n glutamate pyruvate aminotransferase a c t i v i t y i n f e t a l heart muscle with advancing g e s t a t i o n . The incorperation o f gluconeogenic precursors i n t o glucose by l i v e r s l i c e s and organ culture has given support to the occurrence of gluconeogenesis with the enzyme a c t i v i t i e s measured i n v i t r o .  Spontaneous gluconeogenesis from s e r i n e ,  28.  glycerol and to a l e s s e r extent alanine and l a c t a t e was observed in cultured f e t a l r a t l i v e r (Coufulik and Monder, 1976).  Simkins  et al_. (1978) reported a s i m i l a r r e s u l t with galactose added to the medium and f u r t h e r demonstrated that g l u c o c o r t i c o i d s were p o t e n t i a l stimulators of gluconeogenesis from galactose.  Results  obtained from P r i o r and Scott (1977) with f e t a l bovine l i v e r s l i c e s , showed a greater amount of gluconeogenesis from l a c t a t e and pyruvate than from alanine and aspartate.  Furthermore, the incorporation of  these substrates into glucose followed a c u r v i l i n e a r pattern with gestational age, with maximal levels^-occurring a f t e r mid gestation. Although numerous i n v i t r o studies have indicated the presence of a f u n c t i o n a l gluconeogenic pathway i n the ruminant and nonruminant f e t a l species, the extent to which t h i s capacity i s manifested  i n vivo i s uncertain.  Boyd et al_. (1973) reported  that the net f e t a l uptake of glucose and the maternal-fetal  glucose  gradient do not change with g e s t a t i o n , although u m b i l i c a l blood flow and f e t a l oxygen consumption increase during t h i s time. workers, therefore, hypothesized genesis.  Hay  These  the presence of f e t a l gluconeo-  (1979) f u r t h e r speculated that f e t a l gluconeogenesis  could r a i s e f e t a l a r t e r i a l blood glucose concentrations to a l e v e l that would reduce u m b i l i c a l glucose uptake.  Hodgson and Mellor  (1977) estimated from disposal rates of l a b e l l e d glucose, that 60-82% of glucose requirement was p o t e n t i a l l y accounted f o r by gluconeogenesis.  Further studies (Hodgson et al_. 1980)  have  reported that 69% of f e t a l glucose requirements are supplied through f e t a l gluconeogenesis.  Anand et al_. 0979;  1980)  obtained maternal  29.  f e t a l s p e c i f i c a c t i v i t y r a t i o s of glucose following the continuous i n f u s i o n of l a b e l l e d glucose separately into f e t a l and maternal circulations.  The r e s u l t was s i m i l a r to previous work performed  with rats (Girard et aj_., 1977), which demonstrated no s i g n i f i c a n t d i l u t i o n of maternal glucose s p e c i f i c a c t i v i t i e s from f e t a l glucose production. P r i o r and Christenson (1977) reported that alanine accounted f o r 2% of f e t a l glucose entry rate.  This constituted 7%  of alanine turnover and 49% of the net uptake of alanine estimated f o r f e t a l sheep (Lemons et al_.» 1976).  Warnes et al_. (1977a)  u t i l i z i n g s i n g l e i n j e c t i o n of i s o t o p i c tracers f a i l e d to show any gluconeogenesis from l a c t a t e i n the f e t a l lamb.  Recently P r i o r  (1980), employing a continuous i n f u s i o n of r a d i o l a b e l e d l a c t a t e , was successful i n demonstrating s i g n i f i c a n t gluconeogenesis from lactate.  Lactate has been i d e n t i f i e d as an a c t i v e gluconeogenic  precursor i n the newly born lamb (Warnes et al_., 1977b).  Although i t would appear that there i s a p o t e n t i a l f o r f e t a l gluconeogenesis i n most species with the exception of the r a t , suggestions have been made that there are a d d i t i o n a l f a c t o r s regulating gluconeogenesis i n utero.  Warnes et aj_. (1977b)  a t t r i b u t e d the low f e t a l a r t e r i a l PO^, and r e s u l t i n g redox p o t e n t i a l of l i v e r mitochondria (Williamson et al_, 1967), to be a p o t e n t i a l regulator of gluconeogenesis.  Supporting t h i s theory was the  appearance of a c t i v e gluconeogenesis from l a c t a t e 2 minutes f o l l o w i n g natural b i r t h of term lambs.  Jones and Ashton (1976) suggested  30.  that high PO^ of incubation media stimulated gluconeogenesis i n v i t r o i n which guinea pig l i v e r s l i c e s were used.  The apparent  stimulator of gluconeogenesis was considered to be a large increase i n the phosphoenolpyruvate/pyruvate r a t i o .  The r e l a t i o n s h i p  between oxygenation and gluconeogenesis i s also indicated by the i n h i b i t i o n of gluconeogenesis by^-hydroxybutyrate which, by reducing the mitochondrial NAD /NADH r a t i o , may have diminished +  mitochondrial PEP production (Jones and Ashton, 1976). Bohr (1931) o r i g i n a l l y concluded from an i n d i r e c t estimation of the f e t a l r e s p i r a t o r y quotient (R.Q.) that glucose was the sole energy substrate of the fetus.  Experiments conducted  many years l a t e r with f e t a l umbilical perfusion techniques confirmed t h i s conclusion (Alexander et al_., 1966).  However, because the  simultaneous measurement of f e t a l oxygen consumption was not performed, the f e t a l r e s p i r a t o r y quotient was not made i n t h i s study. James e t al_. (1972) determined the oxygen consumption and CO^ production 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 , unanaesthetized fetuses and reported that the f e t a l r e s p i r a t o r y quotient was s i g n i f i c a n t l y less than one. Meschia and coworkers developed another procedure f o r identifying  substrates u t i l i z e d by the ovine f e t u s .  Since f e t a l  oxygen consumption i s an absolute p r e r e q u i s i t e f o r substrate o x i d a t i o n , an expression r e l a t i n g specific  the simultaneous uptake of  substrates and oxygen by the f e t a l u m b i l i c a l  was formulated.  circulation  This expression was referred to as the substrate  oxygen quotient (Tsuolos e t al_.,1971; B a t t a g l i a and Meschia, 1973).  31.  1) Substrate Quotient =  n  x  V  ~  substrate _ n x umbilical uptake of substrate V-A oxygen umbilical uptake of oxygen  A  where n = number of moles of oxygen required f o r complete oxidation of substrate to CC^ and water. This dimensionless quotient represents the f r a c t i o n of oxygen consumption accounted f o r by the complete aerobic oxidation of a substrate crossing the u m b i l i c a l c i r c u l a t i o n . Fetal glucose-oxygen quotient values have ranged from 0.41 to 0.64 in the fed ewe (Tsoulos et. al_., 1971; James et al_., 1972; Boyd et a l . , 1973; Schreiner et al_.» 1978), 0.57 i n the cow (Comline and S i l v e r , 1976) and in the horse ( S i l v e r and Comline (1975).  0.68  This supports the r e s u l t obtained  e a r l i e r i n sheep that glucose uptake, although an important component of f e t a l metabolism, does not account f o r the t o t a l oxygen consumption by the fetus. The wide range observed i n the ewe i s a t t r i b u t e d to the v a r i a b l e l e v e l s of d i e t a r y energy intake of pregnant ewes i n d i f f e r e n t studies and the apparent dependence of the glucose-oxygen quotient on maternal plasma glucose levels.  This i s p a r t i c u l a r l y evident with studies performed on starved ewes.  Glucose-oxygen quotients obtained during s t a r v a t i o n ranged from 0.13 to 0.30 (Tsoulos ejt al_., 1971: Boyd et a l . , 1973; Schreiner et al_., 1978).  Schreiner  et al_. (1978) demonstrated that glucose oxygen quotients decrease r a p i d l y during the f i r s t 2 days of maternal s t a r v a t i o n and remain constant t h e r e a f t e r . The decrease i n placental t r a n s f e r of glucose during maternal f a s t i n g represented a loss of approximately 22% of the normal f e t a l c a l o r i c intake. The glucose-oxygen quotient measured i n i n d i v i d u a l f e t a l organs have demonstrated a s p e c i f i c i t y f o r substrate o x i d a t i o n .  Tsoulos e t al_. (1972)  estimated a cerebral glucose-oxygen quotient of 1.06, i n d i c a t i n g that glucose i s the sole source of energy u t i l i z e d by the f e t a l b r a i n .  Under normal  32.  conditions the cerebral glucose metabolism w i l l account f o r 15% of the f e t a l umbilical glucose uptake (Jones et al_., 1975).  Morriss  et al_. (1973) reported a s i m i l a r glucose oxygen quotient f o r the f e t a l hind limb and concluded that although the hind limb was  less  a c t i v e m e t a b o l i c a l l y than the f e t a l b r a i n , glucose was the predominant substrate o x i d i z e d .  I t was f u r t h e r concluded from these studies  that glucose uptake by i n d i v i d u a l organs i s regulated by f e t a l  arterial  glucose concentrations and i n the case of the f e t a l hind limb, glucose uptake exceeded the requirements of oxidation.  Reviewing the  literature  on the subject, Hay (1979) concluded that glucose u t i l i z a t i o n by i n d i v i d u a l organs was dependent on an assortment of f a c t o r s , including glucose a v a i l a b i l i t y and rates of g l y c o l y s i s , o x i d a t i o n and o v e r a l l fetal  growth.  FRUCTOSE In several mammalian species (sheep, cow and pig) fructose i s the p r i n c i p a l carbohydrate i n f e t a l blood, and i s present i n q u a n t i t i e s that are 3 to 4 times greater than c i r c u l a t i n g glucose concentrations (Randall and L'Ecuver, 1976; Comline and S i l v e r , Bacon and B e l l (1948) f i r s t i d e n t i f i e d  1976).  f r u c t o s e i n blood of f e t a l  sheep and showed that i t e x i s t e d with glucose during the gestation period. Goodwin (1952) demonstrated  f r u c t o s e to be present i n the blood of un-  g u l a t a , but absent i n the blood of carnivora or rodents.  Hitchcock  (1949) observed a gradual disappearance of f r u c t o s e from the lamb circulation,  w i t h i n 36-72 hours of b i r t h .  Huggett et al_. (1951)  proposed from data obtained from e x t e r i o r i z e d f e t a l sheep preparations  33.  with i n t a c t u m b i l i c a l c i r c u l a t i o n s , that fructose enters the f e t a l c i r c u l a t i o n a f t e r i t i s converted from maternal glucose in the placenta and does not return to the maternal c i r c u l a t i o n .  Alexander  et al_. (1955) f u r t h e r demonstrated that the passage of glucose from mother to fetus resulted i n the formation of fructose and at normal blood sugar concentrations.  occurred  Glucose infused d i r e c t l y i n t o  the f e t a l c i r c u l a t i o n i s a c t i v e l y converted to fructose in f e t a l sheep (Warnes et a]_., 1977a) and f e t a l pig (White et al_., 1979).  However,  i t i s unclear whether or not there i s any interconversion of these two sugars.  S e t c h e l l et aJL  (1972) chromatographed blood from f e t a l lambs  that were infused with l a b e l l e d fructose and reported a c t i v i t y i n the glucose molecule i n blood obtained from the u m b i l i c a l v e i n , and heart, but not from the u m b i l i c a l a r t e r y .  fetal  Conversely, Warnes et a l .  (1977a) and White et al_. (1979) f a i l e d to observe t h i s interconversion. Comline and S i l v e r (1970) have disclosed that fructose i s a product of placental metabolism and i s dependent on the plasma glucose concentration in both the mother and f e t u s .  This conclusion i s p a r t i c u l a r l y  evident  during cases of maternal s t a r v a t i o n , when f e t a l fructose l e v e l s f a l l to 43% of the pre-starved 1978).  l e v e l s (Tsoulos et a l . , 1971; Schreiner et al  Meschia and B a t t a g l i a (1978) have speculated from these r e s u l t s  that fructose acts as a glucose reserve, e s p e c i a l l y during periods of maternal s t a r v a t i o n and hypoglycemia. No detectable u m b i l i c a l uptakes of fructose (Tsoulos et a l . , 1971), g l y c e r o l and f a t t y acids (James et al_., 1971) (Morriss e_t al_.j 1974)  have been reported, thus demonstrating that  these substrates are not major metabolic normal conditions.  and ketone bodies  f u e l s of the sheep fetus under  34.  LACTATE Fetal l a c t a t e concentrations are 2-3 times higher than maternal concentrations r e f l e c t i n g the high g l y c o l y t i c capacity of f e t a l and placental  tissues  (Char, and  Creasy,  1976a;  Demigne and Ramesy,1979). S i g n i f i c a n t umbilical venous-arterial differences of l a c t a t e , however, have been reported i n ovine (Burd, et al_., 1975; Char and Creasy, 1976a) and bovine (Comline and S i l v e r , 1976) fetuses, i n d i c a t i n g that l a c t a t e i s taken up by the fetus across the placental c i r c u l a t i o n .  Higher uterine venous-arterial  differences of pyruvate are consistent with higher umbilical a r t e r i a l venous d i f f e r e n c e s , i n d i c a t i n g that pyruvate i s returned to the ewe and i s not u t i l i z e d by the fetus (Char  and Creasy, 1976a).  These  workers also demonstrated s i g n i f i c a n t c o r r e l a t i o n s between f e t a l umbilical and a r t e r i a l l a c t a t e and pyruvate concentrations with maternal a r t e r i a l  levels.  I n i t i a l l y i t was believed that the high f e t a l l a c t a t e l e v e l observed i n f e t a l plasma were the r e s u l t of anaerobic metabolism. However, the appreciable u m b i l i c a l uptake of l a c t a t e suggests the fetus i s a consumer rather than a producer of l a c t a t e (Burd et a l . , 1975; Char and Creasy, 1976a; Comline and S i l v e r , 1976).  The large  lactate-pyruvate r a t i o s observed i n ovine (Burd e t al_., 1975; Char and Creasy, 1976a), fetuses  and  are s i m i l a r  bovine (Demigre and Ramesy, 1979) to maternal lactate-pyruvate r a t i o s i n -  d i c a t i n g that l a c t a t e i s not produced i n excess by the fetus. The source of l a c t a t e a v a i l a b l e f o r f e t a l  utilization  remains questionable. S i g n i f i c a n t c o r r e l a t i o n s reported between  35.  f e t a l a r t e r i a l l a c t a t e and maternal a r t e r i a l l a c t a t e concentrations suggest that maternal t r a n s f e r of l a c t a t e occurs i n utero.  Demigne  Ramesy (1979), however, observed that maternal hyperlactemia influence f e t a l l a c t a t e l e v e l s .  did not  This r e s u l t confirmed a s i m i l a r  observation which showed no t r a n s f e r of l a c t a t e from fetus to under hypoxic conditions ( B r i t t o n et al_., 1967).  ewe  However, Char and  Creasy (1976a) reported p o s i t i v e uterine venous-arterial d i f f e r e n c e s , thereby i n d i c a t i n g that the sheep placenta deposits l a c t a t e i n t o the maternal c i r c u l a t i o n .  Warnes et aj_. (1977a) reported rapid l a b e l l i n g 14  of l a c t a t e molecules f o l l o w i n g the i n j e c t i o n of Ufetal circulation.  C glucose i n t o the  The rate of i r r e v e r s i b l e disposal of l a c t a t e was  s i m i l a r to that of glucose and suggests that placental metabolism as well as f e t a l metabolism of t h i s substrate occurred.  Comline and  S i l v e r (1976) previously reported the net l a c t a t e production  by  uteroplacental t i s s u e i n bovine fetuses was a function of the rate of glucose u t i l i z a t i o n by the uterus.  I t was demonstrated previously  by V i l l e e (1954) that the amount of l a c t a t e produced by f e t a l t i s s u e s l i c e s i n v i t r o did not change appreciably during the l a t e r stages of gestation.  Furthermore, i t was concluded from these studies that  heart t i s s u e produced more l a c t a t e than brain and l i v e r , and  the  amount of l a c t a t e produced was not s t r i c t l y dependent on the type of substrate employed f o r incubation purposes. Burd et al_. (1975) demonstrated from simultaneous measurements of u m b i l i c a l uptake of l a c t a t e and oxygen that the  placenta  produced l a c t a t e i n s u f f i c i e n t q u a n t i t i e s to account f o r approximately 25% (range 0-79%) of f e t a l o x i d a t i v e metabolism.  The consumption  36.  of l a c t a t e by the fetus i n utero was confirmed by Char and Creasy (1976a) (32% i n ovine f e t u s ) and Comline and S i l v e r (1976), (43% i n bovine f e t u s ) . Since the production of placental l a c t a t e i s a f u n c t i o n of the high maternal glucose entry rates reaching the uterus (Setchell et a l . , 1972), discrepancies reported i n the s t o i c h i o m e t r i c measurements of l a c t a t e oxygen quotients can be a t t r i b u t e d to d i s s i m i l a r maternal concentrations.  glucose  Schreiner et al_. (1978) demonstrated t h i s with  s i g n i f i c a n t decreases i n the f e t a l lactate-oxygen quotients obtained from f a s t i n g ewes.  Pyruvate, on the other hand, i s not taken up by  the fetus and, t h e r e f o r e , makes no  c o n t r i b u t i o n as a f e t a l o x i d a t i v e  substrate (Char and Creasy, 1976a). Acetate, an important v o l a t i l e f a t t y acid i n adult ruminant metabolism,  i s taken up by the umbilical c i r c u l a t i o n i n s i g n i f i c a n t  q u a n t i t i e s and accounts f o r 10% of the f e t a l oxygen consumption (Char and Creasy, 1976b).  Comline and S i l v e r (1976) reported a s l i g h t l y  higher r e s p i r a t o r y quotient f o r acetate (16%) i n the bovine fetus. AMINO ACIDS The ovine fetus i s a r a p i d l y growing organism between 100 and 147 days of g e s t a t i o n , increasing i n body'weight at a rate of 1.2- 2 g/hour.  This i s equivalent to 120-210 mg/g/hour of protein  accretion into the f e t a l body (Alexander et_al_., 1970b).  Southgate  (1971) demonstrated from f e t a l r a t carcasses that the amino acid composition of f e t a l p r o t e i n was s i m i l a r i n proportions as weaning and adult carcass protein content.  I t has been well e s t a b l i s h e d  that the primary source of nitrogen f o r growing mammalian fetuses i s  37.  derived from a c i r c u l a t i n g maternal free amino acid  pool (Young  and McFayden, 1973; Smith et al_., 1977; Lemons et al_., 1976).  A  majority of the amino acids transferred across the u m b i l i c a l c i r c u l a t i o n reaches the fetus i n q u a n t i t i e s s u f f i c i e n t f o r adequate growth (Young and McFayden, 1973 and Lemons et al_., 1976).  Com-  parison of amino acid content of lamb carcasses together with placental t r a n s f e r of neutral amino acids led Lemons et al^. (1976) to conclude that some amino acids are transferred i n q u a n t i t i e s i n excess of the growth requirements.  The requirements of l y s i n e and  h i s t i d i n e are, however, s i m i l a r to respective placental t r a n s f e r rates.  Umbilical venous-arterial concentration differences of 22  amino acids i n unstressed ovine fetuses have allowed workers to estimate the f e t a l uptake of amino acid nitrogen.  This uptake (1.5g  N/kg/day), i s a c t u a l l y somewhat greater than the amount of nitrogen required by the fetus f o r growth and urea (Lemons et al_., 1976,  production (1.0 g/kg/day),  Holzman et a]_., 1979).  Numerous studies have attempted to i d e n t i f y and c l a r i f y the mechanisms that regulate amino  acid t r a n s f e r from mother and  placenta.  The placenta has been shown to concentrate a large number of amino acids i n t r a c e l l u l a r ^ , from maternal blood ( H i l l and Young, 1973). Fetal plasma l e v e l s of i n d i v i d u a l amino acids exceed those of the mother by as much as 4 to 5 times (Hopkins e_t al_., 1971; Lemons et a l . , 1976; Smith et al_., 1977).  A s i m i l a r observation has been reported  with f e t a l plasma alpha amino nitrogen concentrations 1970b).  (Alexander et a l . ,  Maternal-fetal plasma concentration gradients are s i m i l a r  for the a c i d i c , branched chain and basic amino acids.  However, the  38.  concentration  gradient between both the maternal and f e t a l plasma and  the placenta i s high f o r the a c i d i c amino acids.  The  fetal-maternal  concentration  gradients are r e l a t i v e l y higher f o r the s t r a i g h t chain  amino acids.  Young and McFayden (1973) have suggested that the high  amino acid l e v e l s observed in f e t a l plasma were a r e f l e c t i o n of the high turnover rate of protein in f e t a l t i s s u e s and the r e l a t i v e differences i n f e t a l organ s i z e , as compared to the mother.  These  workers also concluded that a constant t r a n s f e r of amino acids occurred independently of maternal plasma concentrations.  Holzman  et al_. (1979) recently demonstrated a strong c o r r e l a t i o n between amino acid u m b i l i c a l venous-arterial differences and arterio-venous across the uterine c i r c u l a t i o n .  Furthermore, the  differences  arterio-venous  differences of neutral and basic amino acids were r e l a t e d to the maternal a r t e r i a l  concentration.  Amino a c i d uptake by the placenta i s mediated by d i f f u s i o n and a c t i v e (Na pump and oxygen dependent) transport systems.  Hill  and Young (1973) demonstrated i n the guinea p i g , that t r a n s f e r of amino acids from placental parenchyma to f e t a l plasma was when f e t a l plasma amino a c i d concentrations acid concentrations  blocked  exceeded the free amino  of the placental parenchyma.  Longo et al_. (1973)  reported that placental amino acid transport occurs under anaerobic c o n d i t i o n s , and i s dependent upon glycogenolysis.  Christensen  and  S t r e i c h e r (1948) f i r s t demonstrated that placental t r a n s f e r of i n d i v i d u a l amono acids was concept was  s p e c i f i c to groups of amino acids.  confirmed i n part by Hopkins et al_. (1971) who  This reported  that n e u t r a l , branched chain amino acids belonging to the "L" pref e r r i n g transport system were t r a n s f e r r e d r e a d i l y across the placental  39.  membrane.  Enders e t al_. (1976) attempted to c l a r i f y the mechanisms  that regulate amino acid t r a n s f e r from the placenta, by determining the s p e c i f i c i t y of the p r i n c i p a l placental transport systems.  Three  transport systems f o r neutral amino acids were established i n human placenta, and were reported to represent protein complexes of the syncytiotrophoblast plasma membrane.  With the use of competitive  i n h i b i t i o n techniques, these workers were able to i d e n t i f y separately the transport pathways of s p e c i f i c amino acids.  Recently  Holzman  et al_. (1979) reported that 65 percent of the t o t a l amino acid uptake was represented  by eight neutral amino acids ( a l a n i n e , threonine, 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 ) .  Although g l y c i n e  i s present i n very high concentrations and e x h i b i t s abnormally large venoarterial d i f f e r e n c e s , i t i s not taken up by the uterus i n s i g n i f i c a n t q u a n t i t i e s (Morriss ejt al_., 1979; Holzman e t a l -, 1979). Twenty p e r c e n t o f t o t a l amino a c i d uptake was accounted f o r by basic 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 .  Lemons e t aj_. (1976)  reported no s i g n i f i c a n t u m b i l i c a l uptake of a c i d i c amino a c i d s , glutamate and aspartate, but observed a net f l u x of glutamate out of the fetus i n t o the placenta, i n d i c a t i n g de novo synthesis by f e t a l tissues.  Smith ejt al_. (1977) concluded from u m b i l i c a l venous a r t e r i a l  differences of amino acids that f e t a l t i s s u e s synthesize s u f f i c i e n t glutamate, aspartate, and serine to sustain the synthesis of RNA, DNA and protein but that the remainder of the amino acids needed f o r protein synthesis was supplied from the maternal c i r c u l a t i o n . Due to the r e l a t i v e l y small arterial-venous d i f f e r e n c e of several amino acids and urea, the determination of the u m b i l i c a l uptake of amino acids by the Fick p r i n c i p l e proved d i f f i c u l t .  Con-  sequently, amino a c i d catabolism i n the fetus was estimated by a  40.  predetermined value of transplacental clearance of urea and the mean plasma urea differences between fetus and mother (Gresham e t al_., 1972a; B a t t a g l i a and Meschia, 1973). 2)  Clearance of urea = Excretion_rate of urea urea urea a  where a  = u r e a  ^urea  =  f e t a l a r t e r i a l urea concentration m a  '  t e r n a  l  a r t e r i a l urea concentration  The rate of ureogenesis i n the ovine and bovine fetus determined by t h i s procedure was 0.54 mg/min/kg and 0.21 mg/min/kg and accounts f o r 25 and 9% of the f e t a l oxygen consumption r e s p e c t i v e l y (Gresham et a l . , 1972a; Comline and S i l v e r , 1976).  These comparative  differences i n the per cent of oxygen consumed f o r amino a c i d catabolism have been a t t r i b u t e d to the lower rate of amino acid breakdown i n the bovine fetus (Comline and S i l v e r , 1976).  Simmons et aj_. (1974) re-  ported that amino acid catabolism i n the ovine fetus was the i n i t i a l adjustment of the fetus to maternal s t a r v a t i o n and at the peak of urea production could account f o r 80% of the f e t a l oxygen consumption. The r e l a t i v e c o n t r i b u t i o n to t o t a l o x i d a t i v e metabolism by a mixture of f e t a l f u e l s has recently been investigated (Shambaugh et al_., 1977a,b).  Evidence r e s u l t i n g from i n v i t r o studies with the  f e t a l r a t indicated a competitive o x i d a t i v e i n t e r a c t i o n of s p e c i f i c f e t a l substrates.  An a l t e r a t i o n i n f e t a l fuel mixture due to maternal  f a s t i n g decreased the r e l a t i v e C0£ production from glucose and l a c t a t e to 45 and 75% r e s p e c t i v e l y of the o r i g i n a l values.  Shambaugh et a l .  (1977 a,b) have f u r t h e r demonstrated that the a d d i t i o n of betahydroxybutyrate to the incubation medium r e s u l t e d i n a s i g n i f i c a n t reduction  41.  i n the C 0 produced from l a c t a t e and glucose. 2  I t was concluded from  these studies that the p r e f e r e n t i a l oxidation of substrates i s determined by the ambient concentration of f u e l s rather than by an i n t r i n s i c adaptation of the t i s s u e . vii)  CARBON-NITROGEN BALANCE The measurement of umbilical substrate uptake coupled with f e t a l carcass analysis enabled i n v e s t i g a t o r s to estimate the carbon and nitrogen balance of the near term fetus and the c o n t r i b u t i o n of i n d i v i d u a l substrates t o t h i s balance. Fetal growth rate j_n utero i s approximately 35 g/kg/day (Gresham e t al_., 1972b, Rattray et al_., 1974).  Based on a f e t a l carcass  carbon content of 9 percent of f e t a l wet weight, the net accumulation of carbon i n the fetus was estimated to be 3.15 g C/kg/day (James e t a l . , 1972).  Carbon excreted as carbon dioxide and urea amounted t o  4.38 gC/kg/day and 0.15 gC/kg/day r e s p e c t i v e l y , representing 60% of the t o t a l umbilical carbon f l u x crossing the placenta to the f e t a l lamb (James ejt al_., 1972 and B a t t a g l i a and Meschia, 1973). The r e l a t i v e d a i l y amount of substrate carbon c o n t r i b u t i n g to the t o t a l carbon f l u x has been estimated from u m b i l i c a l substrate uptake measurements.  Amino a c i d s , based on a C/N r a t i o of an average  protein supply the majority of the carbon (3.16 gC/kg/day), (Gresham et al_., 1972a;  B a t t a g l i a and Meschia, 1973).  The c o n t r i b u t i o n of  carbon by glucose (1.76 gC/kg/day; James e t al_., 1972), l a c t a t e (1.22 gC/kg/day; Burd et al_., 1975; Char and Creasy 1976a) and acetate (0.56 gCAg/day; Char and Creasy, 1976b) i s r e l a t i v e l y small.  42.  The percent c o n t r i b u t i o n of amino a c i d s , glucose, l a c t a t e , and acetate to the t o t a l carbon balance of the fetus i s 41%, 23%, 15%, r e s p e c t i v e l y and t o t a l s approximately 88% of the f e t a l carbon balance. This value  therefore  indicates that other substrates,possibly under  s p e c i f i c environment conditions,cross the sheep placenta i n s i g n i f icant q u a n t i t i e s .  9%  43.  Experiment 1 Surgical technique for the cannulation of f e t a l saphenous vein and post-surgical changes i n blood parameters of the ovine fetus j_n utero Introduction The p o s s i b i l i t y of cannulating ovine blood vessels to f a c i l i t a t e p h y s i o l o g i c a l studies i n unrestrained animals was demonstrated by e a r l y workers i n t h i s f i e l d (Meschia et al_., 1965a).  The techniques and com-  p l e x i t i e s of i n t r a u t e r i n e f e t a l surgery have been reviewed by Kraner (1965), pigs.  and  Mellor  and  Slater  (1973) i n sheep and Randall  (1977) in  Successful cannulation of the f e t a l aorta and vena cava (Comline  and S i l v e r , 1972), c a r o t i d artery and jugular vein (Bassett and M a d i l l , 1974b) and u m b i l i c a l vein (Mellor and Matheson, 1975 and Schreiner et a l . , 1978)  have been reported. For n u t r i t i o n a l and p h y s i o l o g i c a l s t u d i e s , i t i s e s s e n t i a l to  have chronic f e t a l preparations which have recovered from the stress of surgery.  Mellor and S l a t e r (1973) have stated that the r e s u l t s of experi-  ments done on c h r o n i c a l l y catheterized fetuses should be interpreted with caution and emphasized that the d e l i v e r y of f u l l - t e r m lambs f o l l o w i n g surgery i s not absolute proof of i n t r a u t e r i n e normality. time that should elapse before experiments may  The minimum  be undertaken would depend  upon the extent of stress imposed during surgery and the maintenance of the patency of vascular catheters. In t h i s experiment, the technique of ovine f e t a l surgery  was  standardized, and the p o s t - s u r g i c a l status of the fetus was monitored d a i l y to determine the time required f o r the fetus to return to stable levels.  Changes in blood acid-base parameters and metabolite and Cortisol  concentrations normality.  i n plasma were measured as c r i t e r i a of i n t r a u t e r i n e f e t a l  44.  Materials and Methods Animals Twenty-five, 1-2 year o l d pregnant Dorset Horn and Suffolk ewes, weighing 50 to 70 kg were used i n t h i s study.  The estrous cycles  i n these ewes were synchronized by the a p p l i c a t i o n of progestagenimpregnated intravaginal pessaries (Synchromate; G.D. Searle and Co., Chicago).  Intravaginal sponges containing 800 mg progesterone (Sigma,  St. Louis) were also used i n some ewes.  At 90 to 100 days a f t e r mating,  an u l t r a s o n i c detector (Sheepreg, Animark) was used to confirm pregnancy. The ewes were housed i n i n d i v i d u a l pens and fed 650 g of 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 ; 18.58 kJ/g gross energy) twice d a i l y , at 0700 and 1500 hours r e s p e c t i v e l y .  Water and s a l t were made  a v a i l a b l e a t a l l times. Neonatal lambs were rubbed dry of a f t e r b i r t h moisture and weighed as soon as possible a f t e r b i r t h . also taken a t t h i s time.  Crown to rump measurements were  The weights of fetuses at the time of experi-  mentation were estimated from f e t a l b i r t h weights by the regression equation developed by Gresham et al_. (1972b). Animal Preparation The ewes were starved f o r 24 hours and were given i n t r a muscularly 5 ml of an a n t i b i o t i c preparation (Penlong-S P l u s , Rogar/5TB) containing 200,000 i . u . P e n i c i l l i n G and 250 mg dihydrostreptomycin/ml on the day p r i o r to surgery.  Atropine sulphate (BDH Parmaceuticals) was  administered subcutaneously (0.06 mg/kg) 15 minutes p r i o r to surgery to reduce excessive s a l i v a t i o n .  45.  Surgical  Procedure Anaesthesia was induced by the intravenous administration of  thiopental sodium (Abbot) at the rate of 20 mg/kg body weight, and maintained with Halothane (Fluothane, Ayerst) at a concentration of 1.0-1.75% in a closed system.  The anesthetized animal was positioned i n a supine  p o s i t i o n with the head turned l a t e r a l l y and t i l t e d s l i g h t l y downwards to prevent a s p i r a t i o n of s a l i v a or ruminal f l u i d s .  The abdominal area was  d i s i n f e c t e d with s u r g i c a l soap (Surgidine, Ingram and B e l l  Ltd.)  followed by Tincture of Zephiran, and covered with s t e r i l e drapes.  An  intravenous d r i p of Ringer's s o l u t i o n was administered to replace loss of e l e c t r o l y t e s during surgery.  S t r i c t aseptic precautions were maintained  throughout. A midline i n c i s i o n (10-15 cm) s t a r t i n g below the umbilicus and terminating close to the upper margin of the mammary gland was made. i n c i s i n g the peritoneum the gravid uterus was palpated to determine p o s i t i o n of the fe'tus.  After the  The appropriate segment of the uterine horn con-  t a i n i n g the f e t a l hind limbs was brought through the abdominal i n c i s i o n with as l i t t l e handling as p o s s i b l e . An a s s i s t a n t held the hoof of one of the hind limbs against the uterus and an i n c i s i o n (1-2 cm) was made on the l e a s t vascular area of the uterine wall exposing the u t e r i n e mucosa and the f e t a l membranes. The a l l a n t o i c and amniotic membranes were cautiously cut through and the f e t a l hind limb pulled through the uterine opening.  The uterus was held i n such a p o s i t i o n which prevented loss of  amniotic f l u i d .  The f e t a l membranes were secured to the wall of the  uterus with a chromic 3/0 (Ethicon) purse s t r i n g suture.  An i n c i s i o n  (2-3 cm) was made through f e t a l skin on the hock j o i n t and the external  46.  saphenous vein exposed by blunt d i s s e c t i o n and freed of adjacent f a s c i a . S t e r i l e 2% l i d o c a i n e ( S t e r i l a b ) was sprayed on the region t o reduce spasms.  Fetal catheters were made of polyethylene tubing (0.86 mm i . d .  x 1.27 mm o.d.) and enclosed i n a p o l y v i n y l c o l l a r designed t o include s i l k l i g a t u r e s used to anchor the catheter to the l e g and prevent the catheter tubing from kinking.  The catheters were previously s t e r i l i z e d  in Cidex (Arbrook Ltd.) and f i l l e d with s t e r i l e s a l i n e (0.15 M) containing heparin (100 U/ml). S i l k l i g a t u r e s were passed beneath the cleared area of the vein at the proximal and d i s t a l ends of the i n c i s i o n and made into a loose knot around the vein to f a c i l i t a t e handling during cannulation and prevent the flow of blood whenever necessary. of the vein  and a cannula was introduced 18 to 20 cm proximally from the  point of entry into the vein. Ethicon) on  A small opening was made on the wall  The s i l k l i g a t u r e s (2/0 braided s i l k ,  the c o l l a r were anchored to the f a s c i a on e i t h e r side at the  point of i n s e r t i o n of the cannula.  A d d i t i o n a l c o l l a r s on the cannula  served to anchor i t on the f e t a l skin approximately  5 and 8 cm down the  leg. The f e t a l i n c i s i o n was closed using 2/0 braided s i l k and the limb was returned to the uterus taking care t o place the fetus i n i t s original position.  A m p i c i l l i n (Ayert, 500 mg) was i n s t i l l e d  into the  amniotic c a v i t y and the f e t a l membranes and the uterus were closed with continous sutures using chromic catgut 2/0 (Ethicon).  The catheter  was pushed into the uterus f o r a distance of approximately for f e t a l movement.  25 cm t o allow  The sutures were then buried by a continous  to ensure that there was no leak of amniotic fluid'.  suture  The peritoneum,  47.  adjacent muscle layers and the skin were sutured separately using E c h i f l e x 0, P l a i n 0, and P r o l i n e 0 ( E t h i c o n ) , r e s p e c t i v e l y . The catheter was e x t e r i o r i z e d by passing the free end through a hollow p l a s t i c tube placed subcutaneously on the r i g h t flank of the animal.  Catheters were c o i l e d i n s t e r i l e cotton gauze and placed i n s i d e  a s t e r i l e handstitched canvas pouch sutured to the animal. Immediately  f o l l o w i n g surgery, a l l animals received 5.0 ml of  an a n t i b i o t i c preparation (Penlong-S Plus) intramuscularly. They were then returned to t h e i r metabolism cover q u i e t l y .  cages where they were allowed to r e -  The administration of the a n t i b i o t i c was continued d a i l y  f o r 3 days following surgery.  Catheters were wrapped w i t h i n s t e r i l e  gauze d a i l y and f r e e ends were cleansed with an a n t i s e p t i c s o l u t i o n p r i o r to and f o l l o w i n g sampling.  The patency of catheters was maintained d a i l y  by removing heparin from the c a t h e t e r , f l u s h i n g with s t e r i l e 0.15 M s a l i n e and r e f i l l i n g with s t e r i l e heparinized s a l i n e (100 u/ml). A n a l y t i c a l Methods Oxygen and carbon dioxide tensions ( P 0 and PC0 ) and pH of 2  2  maternal and f e t a l venous whole blood were determined a n a e r o b i c a l l y using a Radiometer pH meter (pHM 72) equipped with respective modules and electrodes.  The PC0  t r o l l e d a t 37.5°C. accomplished  2>  P0  2 a n d  p H  e l e c t r o d e s  w e r e  t h e r m o s t a t i c a l l y con-  C a l i b r a t i o n of the PC0 and P 0 electrodes was 2  2  by bubbling a gas mixture with know p a r t i a l pressures of C0  and 0 , corrected f o r d a i l y barometric pressure through the electrodes. 2  Blood gas values were corrected f o r body temperature using appropriate c o r r e c t i o n factors (Severinghaus, 1966).  Fetal hematocrits were  2  48.  determined on a m i c r o c a p i l l a r y centrifuge.  Oxygen saturation (S0£) of  f e t a l blood was estimated from PO^ and pH using the nomogram of Meschia et a i . (1961). Chemical A n a l y s i s Fetal blood was c o l l e c t e d i n i c e - c h i l l e d t e s t tubes containing EDTA c r y s t a l s .  Samples were centrifuged f o r 10 minutes at 1000 xg and  plasma was removed and stored at -20° C. Plasma Cortisol was determined by radioimmunoassay Assays; Travenol Labs., Inc., Cambridge, Mass.).  (Clinical  The antiserum'used i n  the assay k i t had 100% c r o s s r e a c t i v i t y f o r Cortisol.  For the determina-  t i o n of glucose and betahydroxybutyrate, the plasma was deproteinized by the addition of 0.2 N p e r c h l o r i c acid and neutralized with potassium hydrogen carbonate.  A f t e r the removal of the potassium perchlorate  p r e c i p i t a t e , glucose was determined by glucose oxidase (Glucostat, Worthington Biochemical Corp., Freehold), and betahydroxybutyrate by betahydroxybutyrate dehydrogenase (Williamson and Mellanby, 1974).  Fetal  plasma l a c t a t e concentrations were determined on deproteinized f i l t r a t e s using l a c t a t e dehydrogenase (Gutmann and Wahlefeld, 1974).  Total alpha  amino nitrogen was determined c o l o u r i m e t r i c a l l y a f t e r deproteinizing the plasma with tungstic acid  (Mason et  , 1973).  Samples were analyzed i n duplicate and presented as means (+ SEM).  Student's t-test was used to t e s t the s t a t i s t i c a l  significance  of the changes observed during surgery and day 1 a f t e r surgery. To determine the time required f o r the fetus to return to stable l e v e l s , an analysis of variance was done on values between days 1-4 and 5-9 following surgery.  49.  Results The gestational age when surgery was performed, the body weight of the ewes and the morphometric measurements of fetuses are given i n Table 2. During the course of t h i s experiment, 23 fetuses were born a l i v e , 3 were experimentally terminated and 8 were born dead. gestational age i n the operated ewes was 139.3 + 0.8 days.  The average  At the time of  weaning the mean body weight was 22.6 + 0.7 kg as compared to 18-23 kg of those born n a t u r a l l y and reared under i d e n t i c a l conditions. Fetal and maternal pH, PCO^ and PO^ values are presented i n Figure 1 and Appendix Table 5.  During the course of surgery maternal  and f e t a l blood gas parameters were a l t e r e d s u b s t a n t i a l l y .  I t was noticed  that maternal and f e t a l pH values were s i g n i f i c a n t l y ( P < 0.05) lower during surgery, and were associated with a s i g n i f i c a n t ( P < 0.05) increase in f e t a l PO^.  The average increase i n f e t a l PO^ was 2.9 mm Hg/0.1 pH  unit change i n maternal blood. 54 to 68% during surgery.  The f e t a l venous S 0 also increased from 2  In the p o s t - s u r g i c a l period a rapid return of  the blood gas values to stable l e v e l s w i t h i n 24 hours a f t e r surgery was observed  (Figure 1 and Appendix Table 5 ) .  There were no s i g n i f i c a n t  differences i n the f e t a l blood gas parameters between days 1-4 and 5-9 f o l l o w i n g surgery, i n d i c a t i n g that these parameters remained r e l a t i v e l y stable f o r 9 days. The r e l a t i v e changes i n f e t a l hematocrit, plasma metabolites and Cortisol concentrations were used as a d d i t i o n a l c r i t e r i a of i n t r a uterine f e t a l normality (Figures 2, 3 and Appendix Table 6). Fetal hematocrits were elevated during the s u r g i c a l period and d i d not return to s t a b l e l e v e l s u n t i l 5 days a f t e r surgery.  S i m i l a r l y , f e t a l plasma glucose,  l a c t a t e , alpha amino nitrogen and betahydroxybutyrate  increased by 60.2,  Table 2: Gestational age at surgery (days)  Gestational age and body weights of ewes and fetuses (Mean + S.E.M.)  Gestational age at b i r t h (days)  Ewe body weight (Kg)  Recorded b i r t h weight (Kg)  Curved crown to rump length (cm)  122.7  139.3  63.73  3.42  40.2  +1.2  +0.8  +2.18  +0.20  +0.9  Weaning Weight at 60 days 0<9_)  Fetal outcome at b i r t h  22.6  8 born dead  +0.71  23 born a l i v e  51.  Fig.l  Post-surgical changes (± SEM) i n blood gas parameters and hematocrit i n the ovine fetus i n utero. (• i n d i c a t e s day of surgery; F.P0 =fetal P 0 ( f T T M.P0 =maternal P 0 ( o ) ; n=10) o  o  9  7  52.  Q200 tr X  z  UJ  0.175 0.150  20  -4—• 3  4  5  6  DAYS POST-OPERATIVE  7  8  9  53.  16.8, 27.7 and 5.8% r e s p e c t i v e l y , over the l e v e l s i n the p o s t - s u r g i c a l period (Figure 2).  Hematocrit and plasma metabolite concentrations  were s i g n i f i c a n t l y d i f f e r e n t (P-< 0.05)  between post-operative days  1-4  and 5-9, i n d i c a t i n g that metabolite concentrations take longer to reach stable l e v e l s than blood gas  parameters.  Fetal plasma C o r t i s o l c o n c e n t r a t i o n s were r e l a t i v e l y high (4.8 ug/100 ml) during surgery as compared to the p o s t - s u r g i c a l period (Figure 3).  Stable l e v e l s (1.8 ug/100 ml) were reached by the t h i r d  day and s i g n i f i c a n t changes were not observed there a f t e r u n t i l 4-2 days  p r i o r to p a r t u r i t i o n when steep increases were noticed. Discussion Experiment 1 was undertaken to determine the standard s u r g i c a l techniques to cannulate blood vessels i n the ovine fetus i n utero f o r the purpose of studying f e t a l metabolism.  Results i n d i c a t e that f e t a l  development i n utero i s not s i g n i f i c a n t l y a l t e r e d f o l l o w i n g i n t r a u t e r i n e f e t a l surgery.  The f e t a l body weights and crown to rump lengths recorded  at b i r t h correspond to published values at t h i s time of gestation (Joubert, 1956).  Based on physical growth parameters i t appears that both i n t r a -  uterine development and postnatal growth up to weaning are not a f f e c t e d by the s t r e s s of s u r g i c a l procedures used i n t h i s study. Several workers have preferred to employ spinal or epidural anaesthesia to minimize the e f f e c t s of general anaesthetic agents on the fetus (Meschia et al_., 1965a; Towell and L i g g i n s , 1976; and Clapp et a l . , 1977).  Pentobarbital sodium (Nembutol) has been found to be s a t i s f a c t o r y  by Comline and S i l v e r (1970) and Pearson and M e l l o r (1975). was not found s u i t a b l e by Comline and S i l v e r (1970).  Halothane  However, others  (Willes et al_., 1969; Barnes et al_., 1977) have found i t u s e f u l .  In the  F i g . 3 Post-surgical changes (± SEM) in plasma Cortisol levels in the ovine fetus utero. (n=3)  55.  present study, the use of  Halothane at a concentration of 1.0-1.75% with  an oxygen flow rate of 1.0 1/minute f o r maintenance gave adequate depth of anaesthesia and rapid recovery. The increases i n f e t a l PO^ observed during surgery c o r r e l a t e p o s i t i v e l y with changes i n maternal PCO2 and negatively with changes i n maternal and f e t a l pH.  This f i n d i n g i s i n agreement  with the observations  made by Motoyama et al_. (1967), who a t t r i b u t e d t h i s to combined changes in maternal-fetal oxygen t r a n s f e r w i t h i n the placenta and v a r i a t i o n s i n maternal pH.  Increased f e t a l P0^ can be a t t r i b u t e d to maternal a c i d o s i s  (pH 7.258), which decreases the a f f i n i t y of the maternal blood f o r oxygen thus increasing the amount a v a i l a b l e f o r t r a n s p l a c e n t a l exchange. more, the f e t a l P 0  2  Further-  w i l l a l s o increase due to the s h i f t i n the f e t a l  oxygen d i s a s s o c i a t i o n curve because of the e x i s t i n g f e t a l a c i d o s i s (pH 7.277) during surgery.  The increases i n PO^ values during halothane  anaesthesis i n ruminants has a l s o been a t t r i b u t e d to depressed body metabolism  and adequate oxygenation (Gates et al_., 1971).  Acute changes  in maternal blood gas parameters have been reported to r e s u l t i n corresponding v a r i a t i o n s i n f e t a l c i r c u l a t i o n , the magnitude of which would depend on the i n i t i a l maternal l e v e l s (Motoyama et al_.,1967; Matalon et. aj_., 1978). In the post s u r g i c a l p e r i o d , maternal and f e t a l blood gas parameters were w i t h i n the range reported by other i n v e s t i g a t o r s and S i l v e r , 1972; S h e l l e y , 1973).  (Comline  The rapid return of blood gas values  to s t a b l e l e v e l s w i t h i n 24 hours a f t e r surgery i n d i c a t e s that the f e t a l oxygenation has not been a f f e c t e d by the l e v e l of halothane used or surgical trauma. Fetal metabolite and Cortisol i n f e t a l plasma were used as additional c r i t e r i a of i n t r a u t e r i n e normality.  The high P0^ l e v e l s  56.  observed i n t h i s study (Figure 1) precludes hypoxia as the cause o f elevated metabolite l e v e l s .  Increased f e t a l C o r t i s o l output observed  i n t h i s study (Figure 3) and decreased metabolism due to anaesthetic agents (Gates et al_. 1971) may account f o r the elevated metabolites during surgery. The metabolite l e v e l s i n the p o s t - s u r g i c a l period are s i m i l a r to those reported by Shelley (1973) and Jones et al_. (1977).  The rate  at which s p e c i f i c metabolites return to s t a b l e l e v e l s would depend upon homeostatic mechanisms operating i n the mature fetus and metabolic changes.  maternal  The low permeability of the ovine placenta to  l a c t a t e and the high Km f o r l a c t a t e dehydrogenase isoenzyme I i n the ovine f e t a l heart and l i v e r probably account f o r the delayed return of l a c t a t e to basal l e v e l s .  The increase i n f e t a l alpha amino nitrogen  f o l l o w i n g surgery may be ascribed to increased protein breakdown due to the surgical trauma (Clapp ejt a]_. 1977; Young et al_. 1975).  The return  of the concentrations of alpha amino nitrogen to s t a b l e l e v e l s 5-6 days following surgery supports s i m i l a r conclusions from studies on other parameters of nitrogen metabolism such as urea production rates (Gresham et al_. 1972a). days suggested  In the l i g h t of these f i n d i n g s the i n t e r v a l of  10-12  by S l a t e r and Mel l o r (1977) f o r the i n i t i a t i o n of metabolic  studies appears too long.  The f l u c t u a t i o n s i n f e t a l plasma  betahydroxy-  butyrate i n the post s u r g i c a l period are d i f f i c u l t to explain because the e f f e c t s of s u r g i c a l s t r e s s may be masked by increasing g e s t a t i o n a l age occurring simultaneously, p a r t i c u l a r l y i n older fetuses (Jones, 1977). The observed  increase i n f e t a l C o r t i s o l concentrations during  surgery cannot be a t t r i b u t e d to hypoxia since there i s evidence of adequate oxygenation as i n d i c a t e d by high P0£ values.  The f a c t that the  f e t a l adrenal gland i s r e l a t i v e l y unresponsive to ACTH s t i m u l a t i o n ( L i g g i n s ,  57.  1977; Jones e t al_., 1977) and the placenta i s permeable to C o r t i s o l  suggests  that a s i g n i f i c a n t proportion of f e t a l Cortisol may be derived from maternal blood (Jones et al_., 1977).  Fetal a c i d o s i s may also be responsible f o r the  increased Cortisol l e v e l s as shown by Nathanielsz e t al_. (1972). The stable l e v e l s of Cortisol a f t e r 3 days following surgery provide additional support that the fetus and the ewe have recovered from surgical  stress.  The r i s e i n Cortisol l e v e l s 4 days p r i o r to p a r t u r i t i o n i s i n agreement with the observations of Bassett and Thorburn (1969) and may be related to increased adrenal  cortex  s i z e and maternal hormonal changes which  occur s h o r t l y p r i o r to p a r t u r i t i o n ( L i g g i n s , 1977). Conclusions Experiment 1 was undertaken to standardize a surgical  technique  that would allow f o r chronic experimentation of the ovine fetus i n utero. Evaluation of the metabolic status of chronic f e t a l preparations f o r future physiological  studies was also made.  The successful cannulation of f e t a l  blood vessels and maintenance of blood catheters f o r a prolonged period of time, without i n t e r f e r i n g with the normal growth and development of the f e t u s , was accomplished. From the r e s u l t s of maternal and f e t a l blood gas parameters and f e t a l plasma metabolite and Cortisol concentrations i t has been shown that f e t a l metabolite concentrations take s u b s t a n t i a l l y longer to return t o stable l e v e l s than blood gas p a r t i a l pressures and pH.  Therefore, the  normality of c h r o n i c a l l y c a t h e t e r i z e d fetuses cannot be assessed only by blood gas and pH measurements. Using f e t a l blood metabolites i n conjunction with blood gas parameters as c r i t e r i a , i t i s concluded that under the conditions of the  58.  surgical technique used i n t h i s study, i n t r a u t e r i n e f e t a l normality i s reached by the 5th postoperative  day.  59.  Experiment I I Substrate turnover and i n t e r r e l a t i o n s h i p i n the ovine fetus i n utero  Introduction U t i l i z a t i o n of metabolic substrates by the ovine fetus has received considerable a t t e n t i o n i n an attempt to evaluate the c a l o r i c requirements of the ovine fetus i n utero.  Since the i n i t i a l  conclusions  made by Bohr (1931) that glucose was the sole o x i d a t i v e substrate of the ovine f e t u s , p a r t i c u l a r i n t e r e s t has been d i r e c t e d at q u a n t i t a t i n g the metabolism of glucose i n the f e t u s . Current knowledge on substrate metabolism i n the ovine fetus i n utero may be ascribed l a r g e l y to the pioneering work o f B a t t a g l i a , Meschia.and coworkers (Tsoulos e t ^1_., 1971; James et a l _ . , 1972; Boyd et a l . , 1973; Morriss et j i l _ . , 1973).  These workers developed the technique  of u m b i l i c a l c a t h e t e r i z a t i o n and from v e n o a r t e r i a l concentration d i f f e r e n c e and blood flow, estimated the u m b i l i c a l uptake of substrates based on the Fick p r i n c i p l e .  The l i t e r a t u r e on t h i s subject has been reviewed  exhaus-  t i v e l y ( B a t t a g l i a and Meschia, 1978). Recently, the use of t r a c e r methodology f o r studying i n vivo f e t a l substrate metabolism has been reported by many workers.  Warnes e t a l .  (1977a) i n j e c t e d l a b e l l e d substrates into the f e t a l c i r c u l a t i o n and determined k i n e t i c parameters from the disappearance of the label from the c i r culation.  Others infused l a b e l l e d substrates continuously i n t o the f e t a l  c i r c u l a t i o n and estimated substrate u t i l i z a t i o n and interconversion from  60.  the s p e c i f i c a c t i v i t y a f t e r i t reached plateau l e v e l s (Hodgson e t a]_., 1980; P r i o r , 1980; Anand e t a l . , 1979, 1980). The o b j e c t i v e s o f the present experiments were to examine the u t i l i z a t i o n of substrates i n the ovine fetus by t r a c e r techniques.  The  i r r e v e r s i b l e disposal rate provides an estimate of f e t a l substrate u t i l i z a t i o n 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 ovine fetuses.  Because of the short  experimental period required and the a v a i l a b i l i t y of computer programs f o r the analysis of data, s i n g l e i n j e c t i o n techniques have p a r t i c u l a r advantages i n f e t a l metabolic studies.  In the present study, t h i s techni-  que was therefore employed to study substrate metabolism and i n t e r r e l a t i o n ship with p a r t i c u l a r emphasis on the re-entry of the t r a c e r i n t o the fetal circulation.  EXPT II A) METABOLISM OF GLUCOSE AND LACTATE Materials and Methods Animals Dorset horn ewes were used i n these experiments.  The main-  tenance of pregnant ewes and i n t r a u t e r i n e surgical procedures were performed as d e t a i l e d i n Experiment 1.  Cannulae were placed i n the j u g u l a r  vein of the ewe the morning p r i o r to the experiment. Metabolic Studies A l l metabolic studies were performed 5-6 hours a f t e r the morning feeding to minimize v a r i a b i l i t y associated with d i f f e r e n c e s i n the post prandial i n t e r v a l .  Adequate recovery from surgery was ensured by monitor-  ing blood gas and pH of both the ewe and fetus.  P r i o r to the experiment  the r a d i o a c t i v e substrate was made t o volume i n 1.0 ml of s t e r i l e s a l i n e  61.  (0.15 M).  The ewe was kept standing during the course of the 3-hour  experiment. Fetal lambs were given a s i n g l e intravenous i n j e c t i o n of 5 0 u C i 14 3 of a mixture o f [U- C] and [2- H] glucose and the cannulae were flushed r 14 immediately with 1.5 ml of s t e r i l e s a l i n e .  In some fetuses,LU- C] glu-  cose or [ 1 - ^ C ] l a c t a t e was i n j e c t e d separately. To monitor rapid changes i n the e a r l y phase of the s p e c i f i c a c t i v i t y - t i m e curve, f e t a l blood sampling was i n i t i a t e d 2 minutes f o l l o w i n g the i n j e c t i o n of l a b e l l e d substrates and was continued u n i n t e r r u p t e d l y f o r 5 minutes. at f i v e minute i n t e r v a l s up to 30 minutes.  This was followed by sampling Thereafter samples were obtained  less frequently up to 3 hours. Maternal blood samples were c o l l e c t e d from 4 minutes f o l l o w i n g the i n j e c t i o n , on a less frequent basis up to 3 hours. Fetal and maternal blood gas and pH values were monitored, as described i n Experiment 1, p e r i o d i c a l l y during the course of the experiment. Whole blood samples were immediately transferred to i c e c h i l l e d t e s t tubes containing EDTA c r y s t a l s . Two hundred m i c r o l i t e r s of whole blood were deproteinized with 3.0 ml of 0.33 N p e r c h l o r i c acid at 0°C.  The  protein p r e c i p i t a t e was removed by c e n t r i f u g a t i o n at 7,000 x g f o r 15 minutes i n a Sorval r e f r i g e r a t e d centrifuge.  The supernatant was pipetted  i n t o t e s t tubes and stored at -40°C. Chemical Methods Frozen deproteinized blood f i l t r a t e s were thawed i n cold tap water and recentrifuged p r i o r to a n a l y s i s .  Supernatants were n e u t r a l i z e d with  KOH and allowed to stand i n i c e f o r 45 minutes.  The i n s o l u b l e KCIO^ p r e c i -  p i t a t e was removed by c e n t r i f u g a t i o n at 0°C and the n e u t r a l i z e d supernatant was transferred to freeze drying f l a s k s .  The KCIO^ p r e c i p i t a t e s were washed  62.  twice with i c e cold water and the combined supernatants and washes were pooled i n respective freeze drying f l a s k s and l y o p h i l i z e d (Lab Conco freeze drier).  Samples were reconstituted i n 1.5 ml of demineralized, d i s t i l l e d  water. I n i t i a l f r a c t i o n a t i o n of blood metabolites into b a s i c , neutral and a c i d i c f r a c t i o n s was accomplished by passing the n e u t r a l i z e d supernatant successively through cation (AG 50X-8 [ H ] 200-400 mesh) and anion (AG 1X-8 +  [formate] 200-400 mesh) (Biorad) exchange resins packed i n p l a s t i c columns, 0.7 x 5.0 cm (Econocolumns,  Biorad). Ion exchange columns were arranged  i n series with a common deionized water r e s e r v o i r supplying each column. Cation and anion  resins were prepared by the a p p l i c a t i o n of 20 volumes  of 2N HC1 and 1.0 N formic acid r e s p e c t i v e l y , followed by a thorough with C0 free demineralized water. 2  washing  Resins were stored i n sealed containers  at 4°C. The bottom of the columns packed with these i o n exchange resins was equipped with a small tygon tubing to which a screw clamp was attached to control the flow rate. 1.  Assay of Glucose S p e c i f i c A c t i v i t y a)  Radiochemical p u r i t y of glucose i n f e t a l whole blood  The neutral f r a c t i o n eluted from the anion exchange column was reconstituted i n 1.5 ml of demineralized d i s t i l l e d water.  For measure-  ment o f r a d i o a c t i v i t y , glucose was q u a n t i t a t i v e l y converted t o gluconic acid byincubating the neutral f r a c t i o n with glucose oxidase (50 units,specific a c t i v i t y 20,000 u n i t s / g , Sigma, St. Louis) and catalase (3,000 u n i t s , s p e c i f i c a c t i v i t y 65,000 units/mg, Bohringer Mannheim Dorval) at 37°C f o r 2 hours. bation  The incu-  mixture was then passed through a reconditioned anion exchange  column and the adsorbed gluconic acid eluted with 8N formic acid.  The  63. recovery of l a b e l l e d glucose under these conditions was 92.09 + 0.71% (Appendix Table 1).  To eliminate possible  contamination of [^C]-glucose  with other neutral compounds, p a r t i c u l a r l y fructose which i s present i n s i g n i f i c a n t amounts i n f e t a l blood, [ C ] - f r u c t o s e and [ C ] - g l y c e r o l were subjected 14  1 4  to ion exchange chromatography separately and i n combination with [^C]-glucose. R a d i o a c t i v i t y i n the gluconic acid f r a c t i o n appeared only when [^C]-glucose was added i n d i c a t i n g the absence o f contamination with fructose or g l y c e r o l . b)  Determination of glucose and fructose concentration i n the neutral f r a c t i o n  Glucose i n the neutral f r a c t i o n was determined by a modification of the glucose oxidase procedure. Fructose i n the neutral f r a c t i o n was determined by the anthrone procedure (Nixon, 1969). 2.  Assay of Lactate S p e c i f i c A c t i v i t y a) Radiochemical p u r i t y of l a c t a t e i n f e t a l whole blood The a c i d i c components adsorbed on the anion exchange r e s i n were-  eluted with 4 N formic a c i d , n e u t r a l i z e d with NaOH and evaporated.  Samples  were reconstituted i n 1.0 ml of demineralized water and an a l i q u o t was used f o r r a d i o a c t i v e measurements.  The v a l i d i t y of t h i s procedure f o r determining [ ^ C ] -  l a c t a t e r a d i o a c t i v i t y was confirmed by t h i n layer chromatography.  Standard  [ ^ C ] - l a c t a t e s o l u t i o n s and samples were applied i n 4 cm streaks on Eastman S i l i c a gel Chromatogram Sheets (Eastman, Kodak) and developed twice i n acetone:n-propanol:water  (6:3:1 v/v) i n the same d i r e c t i o n .  The chromatograms  were scanned on an Actigraph I I I radiochromatograph system (Nuclear Chicago). Radioactive areas corresponding to l a c t a t e standards were cut out, scraped i n t o s c i n t i l l a t i o n v i a l s and suspended i n 10 ml of s c i n t i l l a t i o n f l u i d f o r counting.  No contamination of glucose i n the l a c t a t e f r a c t i o n was detected.  The combined recovery of added [ ^ C ] - l a c t a t e f o r both procedures was 83.0 + 0.63%  (Appendix  Table 2 ) .  64.  b)  Determination of l a c t a t e concentration i n the a c i d i c f r a c t i o n  Lactate i n the a c i d i c f r a c t i o n was determined by the enzymatic procedure described by Gutmann and Wahlefeld (1974). Radioactive Chemicals [ U - C ] glucose, (304 mCi/mmole), [2- H] glucose, (17.9 Ci/mmole), 14  3  [ 1 - C ] l a c t a t e (51 mCi/mmole), and [ U - C ] l a c t a t e (60 mCi/mmole) were 14  14  obtained from Amersham Searle Corp. ( O a k v i l l e , Ont.).  The l i q u i d s c i n t i l l a -  t i o n solution used was composed of 0.5 ml of aqueous sample and 10 ml of s c i n t i l l a t i o n c o c k t a i l (PCS, Amersham Searle Corp.).  The  scintillation  f l u i d used f o r counting r a d i o a c t i v i t y present on s i l i c a gel t h i n layer sheets was prepared by mixing 3.85 l i t r e s of 1,4 dioxane, 3.85 l i t r e s of xylene, 2.30  l i t r e s of absolute ethanol, 800 g of naphthalene, 50 yg of PPO  and 0.5 ug dimethyl-P0P0P  (Amersham Searle Corp.).  Radioactive Counting Procedures Radioactivity  was measured by l i q u i d s c i n t i l l a t i o n spectrometry  (LKB, Rack Beta 1215).  Quenching was determined by external standard r a t i o s .  Automatic quench c a l i b r a t i o n and c a l c u l a t i o n of dpm values were performed f o r both single and doubly l a b e l l e d samples.  Counting e f f i c i e n c i e s were  35-40% and 50-60% f o r [ H ] and [ C ] r e s p e c t i v e l y , with 30% of the [ C] 3  14  14  r a d i o a c t i v i t y appearing i n the [ H] channel. Analysis of Data S p e c i f i c a c t i v i t y values at each sampling time are expressed as a f r a c t i o n of the i n i t i a l dose of tracer and C l a r k , 1972).  injected at zero time (Shipley  I n i t i a l k i n e t i c parameter estimates were obtained using  a Fortran computer program, Autoan, (Sedman and Wagner, 1976) to f i t the data to the equation  65.  n  i=l  where  S.A.  = s p e c i f i c a c t i v i t y at time t ( i n f r a c t i o n dose/mg C);  A = zero time i n t e r c e p t of each exponential component(fraction dose/mgC); n = number of components; i = component i d e n t i f i c a t i o n ; a = rate constant f o r each component (rnin ^); t = time (min). -  The F value and the sum of square deviations were used to evaluate the goodness of f i t of the estimates n  where SA.,SA.  = observed s p e c i f i c a c t i v i t y = estimated s p e c i f i c a c t i v i t y  The set of parameters with the minimal F value was chosen as the i n i t i a l estimate.  The number of exponentials increased u n t i l the per cent improve-  ment i n the goodness of f i t was no longer s i g n i f i c a n t . of Metzler (1969),(NONLIN estimate of the parameters.  The non-linear program  Michigan) was used to give the least squares The squared c o r r e l a t i o n c o e f f i c i e n t (R ) i n d i -  cating the goodness of f i t of the data was c a l c u l a t e d as f o l l o w s :  where  W  =weightedsum of squared observations =V~  W  = sum of weighted squared d e v i a t i o n  • II  w  -jj( -jj Y  ( Y  _  Y  i)  i j 'e.lc j - U T  ) Z  1  j  W  66.  From the result of the f i n a l least squares estimates, the observed data was found to be best described by a biexponential equation. Model Description In this study the term compartment refers to anatomically i d e n t i f i a b l e space  ( e . g . , f e t a l and extrafetal  pool has been used to denote  different  partment (blood; primary pool (a)  compartments, F i g . 4).  The term  locations within the f e t a l com-  and t i s s u e , secondary pool (b))  c a l l y i d e n t i f i a b l e spaces ( e . g . , glucose, l a c t a t e ,  or chemi-  etc.)  Fetal Compartment  Extrafetal compartments Fig. 4  Model of glucose and lactate metabolism in the ovine fetus.  67.  where  k  k  rate constant (pool a to pool b)  b a  rate constant (pool b to pool a)  ab  k  i r r e v e r s i b l e disposal rate constant  oa  r e c i r c u l a t i o n ( t r a c e r re-entering primary pool a f t e r transformation) r e c y c l i n g (tracer re-entering primary  pool without trans-  formati on).  Since the basic o b j e c t i v e of t h i s experiment  i s to study the  disappearance  of the t r a c e r from the f e t a l blood p o o l , the biexponential parameters were formulated i n t o a two pool open exchange system to depict the t r a n s f e r of the label  into  and out of the primary f e t a l blood pool.  No attempts were  made to i d e n t i f y or sample the large number of pools and/or compartments i n which the f e t a l and e x t r a f e t a l compartments may be embedded or are i n communication with each other. Calculations The f o l l o w i n g equations were used f o r estimation of k i n e t i c parameters of substrate metabolism: 1.  Substrate pool  (Q)  =  (White et al_.,  (mgC/kg)  1969)  <V i=l  where A = zero time intercept of s p e c i f i c a c t i v i t y (S.A.); i = exponential component number; n = number of exponential components;  1* = normalized dose.  68.  2.  /*"S.A. (dt)  (mgC/min/kg)  •  3.  1*  I r r e v e r s i b l e rate of disposal (D.R.) =  Volume of d i s t r i b u t i o n (V) =  (Shipley and C l a r k ,  Q blood cone.  1972)  IP.. body weight 0  x  (% body weight) (White et a l . ,  1969)  D R 4.  Metabolic clearance rate (M.C.R.) =  blood cone.  (ml/min)  5.  Half l i f e  I !/2  e*.  (Shipley and C l a r k ,  1972)  (Shipley and C l a r k ,  1972)  (minutes) T  2  =  l/2~  0.693  V  where** a n d $ are rate constants. T-j and T  2  are rapid  and slow components r e s p e c t i v e l y .  The t r a n s f e r of t r a c e r between the f e t a l and maternal compartments was estimated from the r a t i o of the area under the s p e c i f i c - a c t i v i t y - t i m e curves integrated to time i n f i n i t y i n these two compartments.  6.  Per cent of maternal glucose carbon derived from f e t a l glucose carbon ^  S.A. Maternal glucose C (dt) x  S.A.  Fetal glucose C (dt)  100  69.  The re-entry of the label back into the fetal c i r c u l a t i o n may occur by the tracer leaving the f e t a l blood pool and, after a sojourn in some other compartment, returning in i t s original form. referred to as " r e c y c l i n g " .  If  This w i l l be  the tracer returns to the primary f e t a l  blood pool after chemical transformation and reincorporation into newly formed molecules, the process w i l l be referred to as " r e c i r c u l a t i o n " (Hetenyi and Norwich, 1974). Recirculation was estimated according to Dunn et aj_., 1969 by 3  injecting a mixture of glucose labelled with [2- H] and [U-  14  C] and measur-  ing the half l i f e with each isotope.  7.  Recirculation  = 1  ( ^ ) T  (Fraction of i r r e v e r s i b l e disposal rate)  w  ^  1 / 2  h  e  r C]  r  1 4  e  T  =  t  o  t  a  l  h  a  1  f  l  i  f  e  1/2  Recycling is expressed as the fraction of total turnover which re-enters the c i r c u l a t i o n after a sojourn in some other part of the system.  8.  Fraction l o s t i r r e v e r s i b l y  (0) =  M  C  R  K -V  (Gurpide and Mann, 1970)  T  where MCR = metabolic clearance rate (ml/min) Kj = total  turnover rate  constant V = volume of d i s t r i b u t i o n t> = Fraction of total irreversibly  lost.  turnover  70.  9.  The f r a c t i o n 1-0 therefore i n d i c a t e s the f r a c t i o n of t o t a l turnover which returns to the c i r c u l a t i o n through r e c y c l i n g . In the dynamic state of t r a n s f e r of metabolites between the fetus  and the mother, molecules d i f f e r i n t h e i r rates of exchange and mechanisms of transport across the placental b a r r i e r with the r e s u l t that d i f f e r e n t compounds vary i n the time spent w i t h i n and outside the f e t a l vascular space. Therefore the time of residence during one (mean t r a n s i t time, t ) or a l l (mean t o t a l residence time, T) passages of the l a b e l l e d substrate through the f e t a l c i r c u l a t i o n and the number of times a p a r t i c l e returns to the c i r c u l a t i o n a f t e r i t s i n i t i a l passage through i t (number of cycles,2f) were c a l c u l a t e d according to Rescigno and Gurpide  10. Mean t r a n s i t time (T)  = K  (1973).  JT  where Ky = t o t a l turnover rate constant.  11. Mean t o t a l residence time (T) = where Q = pool s i z e D.R. = i r r e v e r s i b l e disposal rate.  12. Number of cycles (Y) = (^-)  - 1  *1  The  conversion  of glucose to l a c t a t e i n the f e t a l compartment  was estimated by the r a t i o of i n t e g r a l s of the s p e c i f i c a c t i v i t y - t i m e curves of these metabolites taken to time i n f i n i t y (Shipley and C l a r k , 1972).  71.  13. Fraction of lactate carbon derived from glucose carbon  /V J o  S.A. lactate C (dt) S.A. glucose C (dt)  o 14. Rate of lactate C derived from glucose  gC.min/kg) =  Equation 13 X lactate i r r e v e r s i b l e disposal rate  15. Per cent of glucose i r r e v e r s i b l e disposal rate going to lactate = Equation 14 Glucose i r r e v e r s i b l e disposal rate  x  100  A l l results were expressed as means (+ SEM).  Statistical  analysis of the data was performed using the paired and independent t-test, where appropriate.  The l i n e a r regression lines and correlation c o e f f i c i e n t s  were calculated by the method of least squares.  Results The mean (+ SEM) gestational age, maternal and f e t a l body weight, blood gas and metabolite measurements recorded at the time of each e x p e r i ment are presented in Table 3.  Very small differences were noted among  maternal and fetal body weights and metabolite concentrations showing uniformity of the preparations used.  Blood gas parameters and metabolite  concentrations were within the range reported previously for well  Table 3:  Maternal  and f e t a l  physiological parameters during the experimental  [U-•  [U- C]  glucose n=7  Parameter M Body weight (Kg)  60.9 ±1.77  Gestational Age (Days)  -  [i- c] 14  C][2- H] glucose n=8  14  14  peri  3  lactate n=8  F  F  M  F  M  r  2.36 ±0.10  65.6 ±2.97  2.91 ±0.98  66.39 ±3.38  2.19 ±0.11  129.14 ±1.99  -  137.5 ±0.98  -  127.1 ±3.113  7.502 ±0.02  7.380 ±0.02  7.466 10.02  7.340 ±0.04  7.488 ±0.02  7.349 ±0.01  pC0 (mm Hg)  30.17 ±2.64  38.09 ±1.36  31.27 ±0.89  41.59 ±1.49  35.21 ±1.52  42.98 ±1.03  p0  33.35 ±1.13  18.61 ±0.47  32.8 ±2.04  20.54 ±0.90  33.16 ±1.29  18.51 ±1.03  -  22.81 ±0.75  -  23.71 ±1.65  pH  2  2  (mm Hg)  TC0  2  (meg/L.)  -  23.60 . ±0.81  Hematocrit {%)  34.50 ±1.02  34.65 ±1.31  31.27 ±0.89  36.12 ±1.09  31.88 ±0.49  38.25 ±1.27  Glucose (mM)  3.390 ±0.12  0.842 ±0.03  3.750 ±0.09  1.006 ±0.02  3.350 ±0.11  0.818 ±0.03  N.D.  3.86 ±0.31  N.D.  4.58 ±0.15  N.D.  3.84 ±0.16  1.18 ±0.08  1.81 ±0.06  1.55 ±0.14  2.022 ±0.04  1.082 ±0.05  1.89 ±0.05  Fructose (mM) Lactate (mM)  Values are means (+ SEM) M = Maternal F = Fetal  73.  oxygenated  near-term fetuses (Experiment 1), i n d i c a t i n g recovery from  s u r g i c a l procedures by the ewe and f e t u s . 1.  D e s c r i p t i o n of s p e c i f i c a c t i v i t y - t i m e curves f o r glucose and l a c t a t e S p e c i f i c a c t i v i t y - t i m e curves, plotted on semi logarithmic  coordinates  f o l l o w i n g the s i n g l e i n j e c t i o n of l a b e l l e d substrates are presented i n Figs. 5, 6 and 9 along with whole blood glucose or l a c t a t e concentrations during the course of the experiments.  The concentrations of glucose and l a c t a t e  were r e l a t i v e l y constant during the experimental period.  In a l l experiments  performed with r a d i o a c t i v e l a b e l l e d glucose or l a c t a t e , the observed data 2 was found t o f i t (R  0.985) a biexponential equation.  A f t e r the i n j e c t i o n  of l a b e l l e d substrates there was an i n i t i a l rapid d e c l i n e i n the s p e c i f i c activity.  A slower l i n e a r d e c l i n e i n the s p e c i f i c a c t i v i t y followed the  i n i t i a l decay f o r the duration of the experiment. t r i t i a t e d glucose was lower than that o f [ U - ^ C ] c u l a r l y marked at the l a t t e r time periods 2.  The s p e c i f i c a c t i v i t y of glucose, which was p a r t i -  (Fig. 6).  K i n e t i c parameters of glucose and l a c t a t e metabolism a) Glucose  metabolism  The k i n e t i c parameters of glucose metabolism c a l c u l a t e d from s p e c i f i c a c t i v i t y - t i m e curves when a mixture of [U-^C] glucose or [ u -  14 C]  and [2-^H]  glucose alone was injected are summarized i n Table  4.  No s i g n i f i c a n t d i f f e r e n c e s i n the glucose pool s i z e and volume of d i s t r i 14 3 bution were noted with [ U - C] and [ 2 - H] l a b e l l e d glucose, when tested by a paired t - t e s t  (P< 0.05).  The mean (+ SEM) i r r e v e r s i b l e disposal  rates and metabolic clearance rates c a l c u l a t e d from  14 [U- CJ  glucose  were 3.541 + 0.25 mg C/min/kg and 140.80 + 4.84 ml/min r e s p e c t i v e l y .  These  74.  100r  ** 4  10  go CiD-  .u 3  E  o  o  < in  w  20  u  co  o o u O u  _2 O  15  5c 10  !  L_  20  60  100  140  Time (min.)  F i g . 5 Semi logarithmic p l o t of glucose s p e c i f i c a c t i v i t y (S.A. versus time f o l l o w i n g i n j e c t i o n of [U-'4C] glucose and whole blood glucose concentration. Values are means (+ SEM; n=7)  180  75.  100 ft  U ^ glucose 2 H]glucose C1 lactate 14  3  = 14  10 O  4 o  U  _u3 E  T  uo aJ  o  r  -5-8  T  20  81 0 8 O  U  15 10  ^  wo  = E  L_  20  Fig.6  60  100  180  140  Time (min)  Semi logarithmic p l o t of glucose s p e c i f i c a c t i v i t y (S.A.) versus time following the i n j e c t i o n of a mixture of [ U - C ] and [2- H] glucose and whole blood glucose concentration. Values are means (± SEM; n=8). 14  J  76.  values were s i g n i f i c a n t l y ( P < 0.05) lower than the values obtained with [ H]-glucose 3  5  (4.07 + 0.16 mgC/min/kg and 155.10 + 5.30 ml/min r e s p e c t i v e l y ) .  The metabolic h a l f l i v e s of the slower decaying components obtained from [ H]-glucose (44.34 + 2.21 min) were s i g n i f i c a n t l y (P < 0.05) lower than 3  from [ C ] - g l u c o s e (50.66 + 2.69 min). 14  In an attempt t o determine i f glucose metabolism was correlated with f e t a l development, [ U - C ] glucose was injected separately i n t o younger 14  fetuses of lower body weight.  The s p e c i f i c a c t i v i t y - t i m e curves obtained  from [U-^ C] glucose i n these fetuses were s i m i l a r to those obtained i n 4  heavier fetuses ( F i g . 5 ) . The mean (+ SEM) i r r e v e r s i b l e disposal rates and metabolic clearance rates estimated i n these fetuses were 2.251 + 0.15 mgC/min/kg and 99.08 + 2.83 ml/min r e s p e c t i v e l y (Table 4). These value were s i g n i f i c a n t l y (P <0.05) lower than those obtained with fetuses of higher body weight.  The mean apparent volume of d i s t r i b u t i o n of glucose  noted i n these experiments was 41.38 + 2.4% and was s i g n i f i c a n t l y greater (P < 0.05) than that observed previously with [ U - C ] glucose i n older 14  fetuses. There was a  p o s i t i v e c o r r e l a t i o n between f e t a l plasma gluocse  concentration and i r r e v e r s i b l e disposal rate ( r = 0.67, P C 0.05, F i g . 7 ) . A s i m i l a r p o s i t i v e c o r r e l a t i o n was observed between f e t a l body weight and glucose i r r e v e r s i b l e disposal rate ( r = 0.61, P < 0.05, F i g . 8 ) . The maternal-fetal r a t i o of the area under the s p e c i f i c a c t i v i t y time curves was 0.092 + 0.002 i n the case of [ u - C ] glucose whereas i t 3 was 0.141 + 0.002 i n the case of [2- H] glucose. 14  77.  Glucose Concentration (mg./iooml.)  F i g . 7 Linear regression of f e t a l i r r e v e r s i b l e disposal rate of glucose (mgC/min/kg) versus blood gluccse concentration (mg/100 ml) a f t e r s i n g l e i n j e c t i o n of [ U - C ] glucose. (Y=0.671x - 3.86; P < 0.05; r=0.66; n=15) 14  7a  Bwt. (kg.)  Fin  P l i n e a r regression of fetal i r r e v e r s i b l e disposal rate of glucose  9  X^B,  W&W!  79  F i g . 9 Semi logarithmic plot of lactate s p e c i f i c a c t i v i t y (S.A.) versus time following the i n j e c t i o n of [ 1 - C ] lactate and whole blood lactate concentrations. Values are means (± SEM; n=8). 14  K i n e t i c parameters of glucose metabolism i n the ovine fetus of "mixture of [ U - ^ C ] , [2-3 ] glucose or [U-^C] glucose  Table 4  H  xperiment  Age (days) and ( Body weight  Irreversible Rate of Disposal (mgC/min/Kg)  Kg) 3.541 ±0.251  140.8 ±4.8  4.070 ±0.162  155.l ±5.3  2.251 ±0.15  99.08 ±2.83  9  [U- C] 14  137.5 ±0.98  C  b  glucose (n=8)  [U- C] glucose (n=7) 14  1  a  129.r ±1.99  Metabolic Clearance Rate (MCR) (ml/min)  C  9  estimated using s i n g l e i n j e c t i o n a W .  Volume of distribution  Pool Size mgC/Kg  (% Bwt.) 30.0 ±2.65  21.72 ±2.16  32.0 ±3.80  25.58 ±3.12  a  a  b  C  (Q)  41.38 ±2.40  b  b  c  (2.36 Kg)  T ,T  Supercripts with d i f f e r e n t alphabets i n the column denote s t a t i s t i c a l - denote h a l f l i f e of f a s t and slow decaying components r e s p e c t i v e l y .  a  24.66 ±1.32  Values = means (± SEM) ' '  a  signi  a  Half l i v e s (Tl/2) (min) T, T 1  1.16 ±0.10  50.66 ±2.69  1.07 ±0.15  44.34 ±2.21  1.47 ±0.13  51.58 ±3.36  a  b  3  81.  b)  Lactate metabolism The metabolic parameters of lactate calculated from [1-^C]  lactate s p e c i f i c activity-time  curves are summarized along with the pooled  [U-^C] glucose data in Table 5.  The lactate pool size was 29.10 +  2.69 mgC/kg which was s i g n i f i c a n t l y (P < 0.05) larger than the glucose pool s i z e .  The i r r e v e r s i b l e rate of disposal of lactate  (2.576 + 0.182  mgC/min/kg) was less than that observed with [U-^C] glucose, which however, was not s i g n i f i c a n t l y d i f f e r e n t .  Lactate metabolic clearance rate (82.03 +  5.62 ml/min) was s i g n i f i c a n t l y (P < 0.05) less than that of glucose. apparent volume of d i s t r i b u t i o n of lactate  (43.93 + 5.11% of f e t a l body  weight) was s i g n i f i c a n t l y larger (P < 0.05) than that of glucose. r a d i o a c t i v i t y was observed in fetal injected into the f e t u s .  Similarly,  The  No  blood glucose when [1-^C] lactate was lactate, label was not detected in the  maternal c i r c u l a t i o n following [1-^C] lactate injection into the f e t u s . The parameters obtained in the single experiment where [U-^C] was injected are very similar to those obtained using [1-^C]  lactate lactate  (Table 5). The extent of glucose conversion to lactate is given in Table 6. The per cent of lactate carbon derived from glucose carbon is 44.16 + 5.40, which results in the formation of 1.223 mgC/min/kg of lactate from glucose.  The per cent of glucose i r r e v e r s i b l e disposal rate which goes  to lactate is 35.92 + 4.30. c.  Re-entry of glucose and lactate carbons The recycling of glucose and lactate which represents the  return of metabolic tracer to the sampled compartment a f t e r a sojurn in some other part of the system was 69.6, 66.1 and 80.2 per cent f o r [U-^C] and [2- H] glucose and [ 1 - C ] lactate respectively 3  14  (Table 7).  In the  able 5 K i n e t i c parameters of glucose and lactate metabolism i n the ovine f e t u s , estimated by using s i n g l e i n j e c t i o n s of [ U - C ] glucose or [ 1 - C ] l a c t a t e . 14  Pool Size (Q) (mgC/kg)  Experiment  [U-  14  2 C] glucose  23.10 ±1.32  a  29.10 ±2.69  L  IH  I r r e v e r s i ble Rate of Disposal (D.R.) (mgC/min/Kg)  Metabolite Clearance Rate (MCR) (ml/min)  Volume of Distribution (v) {% Bwt)  2.935 ±0.210  121.56 ±9.03  35.27 ±1.33  a  2.576° ±0.182  82.03 ±5.62  43.93 ±5.11  L  2.171  75.36  a  a  Half l i f e (min) T]  T  2  1.30 ±.112  50.79 ±2.31  1.36 ±0.17  56.67 ±4.49  1.62  59.33  (n=15)  [1- C] lactate 14  1  (n=8)  [U- C] lactate 14  27.87  45.10  (n=l)  1  2 a  Values = mean (± SEM) 14 Includes a l l [U- C] glucose experiments from table  4  'k Superscripts with d i f f e r e n t alphabets i n the column denote s t a t i s t i c a l s i g n i f i c a n c e ( P < 0.05)  T-j and 1^  =  denotes the h a l f l i f e o f f a s t and slow decaying components respectively.  Table 6  Glucose-lactate conversions in the ovine fetus in utero.  Rate of formation of lactate from glucose (mgC/min/kg)  % glucose going to l a c t a t e  Fetus  % lactate C derived from glucose C'  1  35.5  0.915  26.4  2  29.7  0.765  21.5  3  33.2  0.855  42.0  4  52.2  1.345  32.7  5  44.8  1.154  30.3  6  71.6  1.844  44.5  7  42.11  1.085  54.0  44.16 ±5.40  1.223 ±0.159  35.92 ±4.30  Mean (± SEM)  Equation 13 Equation 14 Equation 15  3  2  Table 7  Estimation of r e c y c l i n g and r e c i r c u l a t i o n of glucose and l a c t a t e i n the ovine fetus. Isotopes injected  Parameter  [ U - C ] glucose (n=8) 14  [2- H] glucose (n=8) 3  [1- C] lactate (n=8) 14  0.199 ±0.011 a  Fraction of t o t a l turnover i r r e v e r s i b l y lost  0.299 ±0.051  0.318 ±0.02  Fraction of t o t a l turnover recycled  0.696 ±0.062  0.661 ±0.02  Mean t r a n s i t time ( t ) (min)  1.71 ±0.35  1.80 ±0.26  2.49 ±0.30  Mean t o t a l residence time (T) (min)  6.21 ±0.90  6.35 ±0.92  12.05 ±1.06  Number o f cycles)  3.30 ±0.46  3.22 ±0.40  4.24 ±0.27  R e c i r c u l a t i o n (% of i r r e v e r s i b l e disposal)  1  a  '  b  a  a  a  a  a  3  a  a  a  12.62±5.44  a  0.802 ±0.010 b  b  b  a  -  values=means (+ SEM) superscripts with d i f f e r e n t alphabets i n rows denote statistical s i g n i f i c a n c e  (P<0.05)  85.  s i n g l e experiment where [U-^ C] l a c t a t e was administered t o the f e t u s , the 4  f r a c t i o n recycled was 79.9 per cent.  No s i g n i f i c a n t d i f f e r e n c e was noted  in the mean t r a n s i t o r t o t a l residence times and the number of cycles to the primary pool between [ U - C ] and [2- H] glucose. l4  3  The mean t o t a l  residence time observed with [1-^ C] l a c t a t e was s i g n i f i c a n t l y greater 4  (P< 0.05) than with [ U - C ] or [2- H] glucose. 14  3  The number of cycles  made by the l a c t a t e t r a c e r to the primary pool was higher than that f o r glucose; however, t h i s was not s i g n i f i c a n t . 3  The extent of glucose  14  r e c i r c u l a t i o n using [ H] and [  C] glucose was 12.62 + 5.44% of the  i r r e v e r s i b l e disposal rate (Table 7 ) . Discussion The mathematical  v a l i d i t y of compartmental a n a l y s i s i n v o l v i n g  i s o t o p i c t r a c e r s used i n metabolism  has been discussed elsewhere  (White et al_., 1969; Judson and Leng, 1972; Hetenyi and Norwich, 1974; A t k i n s , 1980).  A fundamental assumption inherent i n the a n a l y s i s o f  t r a c e r k i n e t i c data i s t h a t steady s t a t e conditions p r e v a i l during the experimental period.  The constant l e v e l of blood glucose and l a c t a t e during  the course of these experiments  ( F i g . 5, 6 and 9) i n d i c a t e that steady state  conditions p r e v a i l e d . The volume of d i s t r i b u t i o n of glucose noted i n t h i s study (35.27%) exceeds the e x t r a c e l l u l a r space i n the fetus.  The present value i s lower  than the estimate of 57.4% reported by Warnes e t al_. (1977a) using s i m i l a r i n j e c t i o n techniques.  The l a c t a t e pool s i z e and apparent volume of d i s t r i b u -  t i o n are a l s o lower than those reported by Warnes e t al_. (1977a).  These  differences i n glucose and l a c t a t e space may be due to the lower blood glucose  86.  and higher l a c t a t e i n the fetuses used by Warnes e t a]_. (1977a) than those used i n the present study.  Although the f e t a l blood glucose concentra-  tions are s u b s t a n t i a l l y lower than maternal l e v e l s (Table 3 ) , the volume of d i s t r i b u t i o n i n the fetus exceeds the e x t r a c e l l u l a r f l u i d space of 18% reported i n adult ruminants 1969; 1980).  (Kronfeld and Simenson, 1961; White et a l . ,  This d i f f e r e n c e may be explained by the high glucose content  and metabolic a c t i v i t y of f e t a l erythrocytes ( J a r r e t t et al_., 1964). The i r r e v e r s i b l e disposal rates of glucose and l a c t a t e obtained i n t h i s experiment  (Table 5) are s i m i l a r to the values reported by Warnes  et al_. (1977a), using s i n g l e i n j e c t i o n techniques and t o recent estimates 14 obtained from the continuous i n f u s i o n of  C l a b e l l e d glucose (Anand  et a l . , 1979, Hodgson et aj_., 1980) and l a c t a t e ( P r i o r , 1980).  However,  these values are considerably higher than the estimates obtained by u m b i l i c a l venous-arterial d i f f e r e n c e s and blood flow measurements (Tsoulos et a l . , 1971; James et a l - , 1972; Boyd et aj_., 1973; Comline and S i l v e r , 1976).  The major l i m i t a t i o n of the latter procedure i s that i t does  not take into account endogenous substrate production and thus underestimates 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 placental metabolism  On the other hand, the c o n t r i b u t i o n  and improper mixing and exchange of ^ C with  other substrate pools may have resulted i n an overestimation of i r r e v e r s i ble loss i n the s i n g l e i n j e c t i o n technique employed. Results from t h i s experiment a l s o i n d i c a t e that the metabolism of glucose and l a c t a t e w i t h i n the ovine post-natal l i f e .  fetus  i s more rapid than i n the  For example, White et aj_. (1980) have reported a glucose  pool turnover time i n postabsorptive lambs of 40-50 minutes.  The mean  t o t a l residence time o f glucose from i t s entry i n t o the f e t a l blood pool to i t s f i n a l e x i t was only 6.21 +0.90 minutes (Table 7).  Similarly,  87.  i n the case of l a c t a t e the mean t o t a l residence time i n the fetus was 12.04  only  minutes as compared to the value of 20-30 minutes reported i n the  adult monogastric (Searle and C a v a l e r i , 1972). The data also demonstrate that the i r r e v e r s i b l e disposal rate of glucose i s proportional to the f e t a l blood glucose concentration  ( F i g . 7 ).  I t has been shown by James et al_. (1972) that the f e t a l blood glucose concentration  i s d i r e c t l y c o r r e l a t e d with maternal a r t e r i a l glucose con-  c e n t r a t i o n and u m b i l i c a l uptake.  This r e l a t i o n s h i p may  be p a r t i c u l a r l y  s i g n i f i c a n t i n fetuses of starved ewes, where i t has been documented that maternal s t a r v a t i o n r e s u l t s i n a 35% reduction i n f e t a l plasma glucose l e v e l s (Tsoulos et al_., 1971, Schreiner et al_., 1978).  Such an e f f e c t  would p o t e n t i a l l y reduce f e t a l glucose u t i l i z a t i o n and impair f e t a l growth under conditions of reduced feed intake. Comline and S i l v e r (1976) reported that the a v a i l a b i l i t y of metab o l i c substrates to the fetus would depend not only on the rate of supply but also on the t i s s u e mass involved.  Fetal body mass as a p o t e n t i a l regu-  l a t o r of f e t a l metabolism was observed i n t h i s study. c o r r e l a t i o n (P<  A significant  0.05.) was observed between the i r r e v e r s i b l e disposal rate  of glucose and f e t a l body weight ( F i g . 7).  James et al_. (1972) f a i l e d to  demonstrate a c o r r e l a t i o n between u m b i l i c a l glucose uptake and body weight,which may  fetal  be a t t r i b u t e d to the wide v a r i a t i o n i n the measured  u m b i l i c a l glucose uptakes i n t h e i r study.  White et al_. (1980) reported a  s i g n i f i c a n t c o r r e l a t i o n between the i r r e v e r s i b l e rate of glucose disposal and preweaning body weight.  S i m i l a r l y , F l e c k n e l l et al_. (1980) have  r e c e n t l y reported that the disposal rate of glucose i n the neonatal  pig  was prpportional to both the t o t a l body weight and i n d i v i d u a l organ weights, The p o s i t i v e r e l a t i o n s h i p between f e t a l oxygen consumption and body weight  88.  reported by James et al_. (1972) a l s o supports the increased u t i l i z a t i o n of glucose observed i n t h i s study i n fetuses of higher body weight.  It is  l i k e l y that i f maternal glucose t r a n s f e r to the fetus i s r e s t r i c t e d under  nutrition,  the  fetus  during  may have to r e l y on endogenous sources  of glucose production to compensate f o r the increased metabolic demands during advanced stages of g e s t a t i o n . Glucose r e c i r c u l a t i o n The r e i n c o r p o r a t i o n of i s o t o p i c label into endogenously produced compounds has been referred to as r e c i r c u l a t i o n ( Z i l v e r s m i t 1943; Hetenyi and Norwich, 1974).  et a l . ,  R e c i r c u l a t i o n of a l a b e l l e d substrate  therefore underestimates i t s true rates of i r r e v e r s i b l e d i s p o s a l . 3 14 The simultaneous i n j e c t i o n of [ H] and [ C]  glucose was employed 14  i n t h i s study- to quantitate the extent of r e c i r c u l a t i o n of glucose returning to the blood pool.  C labelled  The metabolic f a t e of t r i t i u m  atoms  has been discussed by many workers (Katz and Dunn, 1967; Judson and Leng, 1972; Katz and Rognstad, 1976).  B r i e f l y , when  [2- H] glucose i s used as  the t r a c e r , t r i t i u m from carbon 2 i s l i b e r a t e d i n the glucose phosphate isomerase reaction between glucose-6-phosphate  and f r u c t o s e 6-phosphate and  i s r a p i d l y l o s t through the exchange with body water (Rose and O'Connell, 3 14 1961). The use of [2- H] glucose i n combination with [U- C] glucose thus y i e l d s a turnover r a t e which gives an estimate of r e c i r c u l a t i o n through both the Cori c y c l e and glycogenolysis (Katz and Dunn, 1967; Judson and Leng, 1972). The small proportion of glucose r e c i r c u l a t i o n (12.64 + 5.1%) i s s i m i l a r to the observations of Anand et a]_. (1979) who reported very l i t t l e  89.  *3  l/i  d i f f e r e n c e i n turnover rate between [2- H] and [Ucontinuous i n f u s i o n study.  C] glucose i n t h e i r  The present values c l o s e l y resemble  those  reported i n adult ruminants, (10%, Annison et al_., 1963; 13%, Judson and Leng, 1972) but are s u b s t a n t i a l l y lower than the value of 26% reported i n neonatal lambs (Makamatsu et al_., 1974).  In rats (Hetenyi and  Mak,  1970) and dogs (Issekutz et al_., 1972) the extent of glucose r e c i r c u l a t i o n has been reported to be 30% and 40% r e s p e c t i v e l y . Glucose-lactate conversions Results of t h i s experiment show that 36% of the glucose pool i s metabolized to l a c t a t e , which accounts f o r 44% of l a c t a t e i r r e v e r s i b l e disposal rate (Table 6).  Warnes et al_. (1977a) also demonstrated 14  rapid l a b e l l i n g of l a c t a t e f o l l o w i n g the i n j e c t i o n of [Uthe f e t a l c i r c u l a t i o n .  a  CJ glucose into  This proportion of l a c t a t e produced from glucose  c l o s e l y resembles the value of 40% reported i n adult sheep.  However,  i t i s possible that part of the glucose conversion to l a c t a t e may have occurred i n the placenta as shown by Burd et al_. (1975) and Char and Creasy (1976a).  The present experimental set up i s not adequate to  d i f f e r e n t i a t e between f e t a l and placental metabolism and i n the absence of umbilical c a t h e t e r i z a t i o n no d e f i n i t e q u a n t i t a t i v e conclusions can be drawn on the  extent of glucose conversion to l a c t a t e by the fetus alone.  When [ l - C ] l a c t a t e was i n j e c t e d no r a d i o a c t i v i t y could be l 4  recovered i n f e t a l blood glucose which agrees with the r e s u l t s of Warnes  90.  e t al_. (1977a).  On the other hand, using continuous  i n f u s i o n of  [U-^C]  l a c t a t e , P r i o r (1980) has recently reported that 22% of the l a c t a t e returns to the f e t a l glucose pool. technique of [1- ^ C] 4  I t i s possible that i n the s i n g l e i n j e c t i o n  l a c t a t e used i n t h i s study, t h e [ ^ C ] carboxyl carbon 4  was e i t h e r l o s t through decarboxylation to acetyl COA and oxidized or d i l u t e d beyond detectable l e v e l s by i t s passage through the oxaloacetate pool.  Further the metabolism of l a c t a t e by the fetus may be too rapid to  detect measurable a c t i v i t y i n glucose with s i n g l e i n j e c t i o n  techniques.  The ovine f e t u s , u n l i k e the a d u l t , synthesizes large q u a n t i t i e s of glycogen  i n l i v e r , heart and muscle t i s s u e s , during l a t e gestation  (Shelley, 1960).  However, the apparent low turnover of the f e t a l  pool (Setchell et aj_., 1972)  glycogen  suggests that glycogen may not contribute  s i g n i f i c a n t l y to f e t a l glucose turnover during the r e l a t i v e l y short e x p e r i mental period employed i n t h i s study.  Glycogenolysis therefore may not be a  major source of f e t a l glucose r e c i r c u l a t i o n . Glucose r e c y c l i n g In dynamic studies with t r a c e r s , the term r e c y c l i n g r e f e r s to the return of the t r a c e r to the sampled pool a f t e r a the system.  (Gurpide and Mann, 1970; Rescigno and Gurpide, 1973; Hetenyi  Norwich, 1974). (pool b) may  sojurn i n other parts of and  In the case of the fetus the blood (pool a) and t i s s u e s  be considered to be embedded i n a multicompartmental system made  up of f e t a l f l u i d s , placenta and maternal  t i s s u e s . The large f r a c t i o n of  glucose and l a c t a t e recycled (70-80%) may  be a t t r i b u t e d to the possible  c o n t r i b u t i o n of placental metabolism of substrates and the inadequate mixing of the label with substrate pools i n the fetus i n the s i n g l e i n j e c t i o n techniques  (Warnes et al_., 1977a).  The f a c t that the  extent of r e c y c l i n g  91.  was not d i f f e r e n t when [ C ] or [ H] l a b e l l e d glucose was used suggests l 4  3  that the glucose molecules re-enter the f e t a l c i r c u l a t i o n without undergoing transformation. The s i g n i f i c a n t l y greater f r a c t i o n o f l a c t a t e recycled coupled with the longer mean t o t a l residence time (Table 7) than glucose and the observation that no l a c t a t e r a d i o a c t i v i t y was found i n the maternal compartment suggest that l a c t a t e i s recycled i n the f e t a l compartment u n t i l i t i s metabolized.  This i s i n p a r t i a l agreement  with the recent report of Kastendieck et al_. (1980) that up to 85% of l a c t a t e was eliminated by u t i l i z a t i o n i n f e t a l sheep.  The impermeability  of the placenta to l a c t a t e ( B r i t t o n et al_., 1967) would a l s o be conducive for recycling.  The longer mean t o t a l residence time of l a c t a t e than  glucose may also be ascribed to i t s s i g n i f i c a n t l y l a r g e r volume of distribution.  The f a c t that the r e c y c l i n g of [U-^ C] l a c t a t e d i d not d i f f e r 4  r  from that o f [1-  14  n  CJ l a c t a t e i s a d d i t i o n a l evidence that t h i s phenomena i s  c h a r a c t e r i s t i c of the l a c t a t e molecule as a whole.  On the other hand,  the observation that glucose returned to the maternal c i r c u l a t i o n suggests that f e t a l glucose r e c y c l i n g i s a process active outside and w i t h i n the fetus. Fetal-maternal glucose t r a n s f e r The t r a n s f e r of glucose from the mother to the fetus has been considered to depend on a s t e r e o s p e c i f i c process o f f a c i l i t a t e d d i f f u s i o n which i s affected by the glucose consumption rate of placental t i s s u e s (Widdas, 1961; Boyd e t aj_., 1976; Simmons e t aJL, 1976, 1979).  It is  evident, however from the r e s u l t s reported i n t h i s experiment that a substantial amount o f f e t a l glucose i s a l s o transferred back to the  92.  maternal c i r c u l a t i o n .  From the per cent of maternal glucose carbon derived  from f e t a l glucose carbon (9.2%), the t r a n s f e r rate of f e t a l glucose to the mother can be estimated assuming a maternal i r r e v e r s i b l e disposal rate of glucose of 108 mg glucose/min i n l a t e gestation (Steele and Leng, 1973). This would amount to 9.95 mg glucose/minute, representing 52% and 10% of the f e t a l and maternal glucose i r r e v e r s i b l e disposal rates r e s p e c t i v e l y . Anand et aj_.  (1979) demonstrated a p o s i t i v e c o r r e l a t i o n between the f e t a l -  maternal t r a n s f e r of glucose and f e t a l glucose concentration. Since the rate of umbilical glucose uptake i s dependent upon the maternal-fetal glucose gradient (James et al_., 1972; Schreiner et al_., 1978), the return of glucose from the f e t a l c i r c u l a t i o n to the mother may represent a mechanism that ensures an optimal rate of glucose supply to the fetus. Although no experiments were done to t e s t the e f f e c t of varying l e v e l s of f e t a l oxygen consumption  on the reverse t r a n s f e r of glucose to  the maternal c i r c u l a t i o n , i t may be reasonable to suggest that glucose made a v a i l a b l e to the fetus i n excess of the oxygen required f o r o x i d a t i v e metabolism would be returned to the mother.  This would be a preventative  measure against possible hypoxia i n the fetus consequent to a sustained increase i n umbilical uptake of glucose (Carson et al_., 1980). Charlton jet al_. (1979) reported large volumes of amniotic f l u i d swallowed by the ovine fetus add s i g n i f i c a n t l y to the o v e r a l l f e t a l metabolism.  The q u a n t i t a t i v e c o n t r i b u t i o n of f e t a l f l u i d s to f e t a l glucose  r e c y c l i n g needs to be studied i n order to describe adequately the r e c y c l i n g of glucose w i t h i n the feto-maternal system.  93.  Conclusions Experiment II a was conducted to study the metabolism of glucose and l a c t a t e i n the ovine fetus using radioistope d i l u t i o n techniques.  It  i s evident from the r e s u l t s that these techniques complement conclusions based on venous a r t e r i a l umbilical d i f f e r e n c e s . It can be concluded that glucose and l a c t a t e are u t i l i z e d at a rapid rate as evidenced by the high rates of i r r e v e r s i b l e disposal and metab o l i c clearance of these compounds.  There i s a c o r r e l a t i o n observed between  the f e t a l glucose i r r e v e r s i b l e disposal rate and blood glucose concentration demonstrating that f e t a l metabolism of glucose i s regulated by the f e t a l blood qlucose concentration. Glucose u t i l i z a t i o n was found to be higher i n older fetuses with a higher body weight than younger ones. The r e c i r c u l a t i o n of glucose has been estimated to be 12.62% of the i r r e v e r s i b l e disposal rate of glucose.  This i s less than reported  in other nonruminant species. Approximately, 36% of glucose i s converted to l a c t a t e . Recycling appears to be an important feature of glucose and l a c tate metabolism i n the fetus occurring to an extent of 70-80% of t o t a l turnover of these compounds.  The absence of r a d i o a c t i v i t y i n maternal blood  f o l l o w i n g l a c t a t e i n j e c t i o n i n d i c a t e s that l a c t a t e r e c y c l i n g occurs w i t h i n the f e t a l compartment, due probably to impermeability of the placenta to lactate. The f a c t that approximately 10% of maternal glucose carbon  was  derived from the f e t a l glucose carbon indicates that glucose r e c y c l i n g occurs both w i t h i n and outside the f e t a l compartment.  94.  EXPT II B) METABOLISM OF AMINO ACIDS M a t e r i a l s and Methods Animals The animals used i n t h i s experiment were s u r g i c a l l y prepared and maintained as described i n Experiment 1. Metabolic Studies The return of the ewe's appetite to presurgical l e v e l s and the d a i l y monitoring of f e t a l and maternal blood gas and pH parameters were used as c r i t e r i a f o r determining the p h y s i o l o g i c a l condition of the animal on the day of experimentation.  The housing and maintenance of the ewes were as  described i n Experiment 1.  A l l experiments were i n i t i a t e d at approximately  5-6 hours a f t e r the morning feeding.  The experimental protocol f o r each  study was as f o l l o w s : a)  [U- CJ amino acid mixture Approximately 50 uCi of [ U - ^ C ] amino acid mixture were introduced  into the f e t a l c i r c u l a t i o n as a bolus i n j e c t i o n i n s t e r i l e phosphate buffer (pH 6.9), and the cannuula dead space was flushed immediately t h e r e a f t e r with  2.0 ml  of  s t e r i l e 0.15 N NaCl.  Maternal and f e t a l whole blood  were sampled at frequent i n t e r v a l s up to 3 hours as d e t a i l e d i n Experiment II and transferred to i c e c h i l l e d t e s t tubes containing EDTA c r y s t a l s . was centrifuged and the plasma was removed and stored a t -40?C.  Blood  Plasma  (500 jui) was deproteinized with double the volume of absolute ethanol and the s o l u t i o n was kept on i c e f o r 30 minutes before c e n t r i f u g a t i o n .  The  deproteinized supernatant was evaporated to dryness i n a warm water bath under a stream of nitrogen.  95.  b)  [ U C ] alanine and [2- H] glucose 14  3  In experiments where [U-^ C] alanine and [2- H] glucose were 4  3  administered simultaneously, 50 jjCi o f each isotope were d i s s o l v e d i n 2.5 ml of s t e r i l e phosphate b u f f e r and i n j e c t e d as a bolus.  Whole blood  (400 u l ) were deproteinized with 6.0 ml of 0.33 N p e r c h l o r i c a c i d . The supernatants were n e u t r a l i z e d with KOH and freeze dried as described in Experiment I I a. Analytical  Procedures  Chemical 1)  Determination of amino acid s p e c i f i c a c t i v i t y  I n i t i a l f r a c t i o n a t i o n of blood metabolites into a c i d i c , neutral and basic f r a c t i o n s was  performed by ion exchange chromatography as  previously described i n Experiment I I a. The amino acids adsorbed on the c a t i o n exchange r e s i n were eluted with 2N triethanolamine i n 20% acetone i n water (Harris et al_., 1961) and evaporated  to dryness under vacuum.  A f t e r r e c o n s t i t u t i n g i n 1.5 ml of  0.1 N HC1, a 500 jul a l i q u o t was taken f o r r a d i o a c t i v e measurements. of C-amino acids applied to the columns was 99.1 + 14  Recovery  0.96% (Appendix  Table 3). An a d d i t i o n a l 500 JJI a l i q u o t was d i l u t e d with equal volume of C0 ~ 2  free water and the t o t a l organic carbon content was quantitated on an i n f r a r e d carbon analyzer (Beckman). Potassium biphthalate standards ing  rang-  from 0-1000 ppm were prepared i n CO,,-free water (Appendix F i g . 3 ) .  96.  2)  Determination of alanine s p e c i f i c a c t i v i t y  For determining the r a d i o a c t i v i t y of [ U - C ] alanine, the basic 14  f r a c t i o n was evaporated to dryness and dissolved i n 1.0 ml 0.05 M T r i s b u f f e r , pH 7.8.  Alanine was q u a n t i t a t i v e l y converted t o l a c t a t e i n a  coupled enzymatic reaction i n v o l v i n g transamination and dehydrogenation by the addition of alanine amino transferase (EC. 2.6.1.2, 10 U/ml, Sigma), l a c t a t e dehydrogenase (EC. 1.1.1. 27, 6 U/ml, Sigma), 2-oxoglutarate (6.7 mM) and NADH (0.2 mM, Sigma).  A f t e r incubation a t 30°C f o r 2 hours,  the mixture was passed through a cation exchange column and the radioa c t i v i t y i n the eluate containing l a c t a t e was counted.  The recovery o f  l a b e l l e d alanine i n t h i s procedure was 82.4 + 0.64% (Appendix Table 3). The concentration of alanine was determined by monitoring the e x t i n c t i o n of NADH a t 340 nm between 40 and 60 minutes (Grassl, 1974). 3)  Determination o f glucose and l a c t a t e s p e c i f i c a c t i v i t y  The s p e c i f i c a c t i v i t y o f glucose and l a c t a t e was determined as described previously (Experiment I I a ) . 4)  Plasma protein determinations  The t o t a l plasma protein content i n maternal and f e t a l plasma was determined by the procedure of Lowry e t al_. (1951).  Bovine  albumin  standards ranged from 0-100 mg protein/100 ml. Samples were read against a Folin-phenol reagent blank a t 600 nm. Radiochemicals [ U - C ] amino acid mixture (50 mCi/mmole) and [ U - C ] alanine 14  1  14  3 (156 mCi/mmole), were supplied by ICN Pharmaceuticals (Montreal). [ 2 , H]  Appendix  (p. 159)  97.  glucose (17.9 Ci/mmole) was obtained from Amersham Searle Corp., O a k v i l l e , Ontario. Analysis of Data The 3-hour sampling time was not s u f f i c i e n t to describe the terminal slope of the [U-^ C] amino acid decay curve accurately. 4  The observed  data  were therefore p l o t t e d on semi logarithmic coordinates and extrapolated to zero s p e c i f i c a c t i v i t y manually.  I n i t i a l l e a s t squares estimates o f the  k i n e t i c parameters were c a l c u l a t e d using a Fortran computer program (AUTOAN).  The f i n a l l e a s t squares estimates were obtained using NONLIN  (Michigan) as described i n Experiment I I a. Model Description The data were f i t t e d to a three-pool model (Waterlow et al_., 1978) in which amino acids are i r r e v e r s i b l y l o s t only through the primary blood pool ( F i g . 10).  The other two pools, which were not sampled are represented  Fetal Compartment  k  F i g . 10  oa  Model of amino acid metabolism  98.  by i n t r a c e l l u l a r free amino acid pool and body proteins.  The transfer of  amino acids from the maternal c i r c u l a t i o n across the placenta and from degradation of body protein through the i n t r a c e l l u l a r pool was assumed to be the sources of amino acids entering the blood pool. Calculations In experiments where [U- C] alanine and [2- H] glucose were 14  3  administered simultaneously, the conversion of alanine to lactate and to glucose was estimated by the r a t i o of integrals of the s p e c i f i c a c t i v i t y time curves as described in Experiment II 1.  Fraction of lactate C derived from alanine C _ J  ^  o o  2.  a.  S.A.  lactate C (dt)  S.A. alanine C (dt)  Rate of lactate C derived from alanine C (mgC/min/kg) = Equation 1 X lactate i r r e v e r s i b l e disposal rate  3.  Percent of alanine C going to lactate C Equation 2 alanine i r r e v e r s i b l e disposal rate  4.  x 100  Fraction of glucose C derived from alanine C  —  J  S.A. glucose C(dt) 1  s*. o  1  S.A. alanine C(dt)  X  6  5  99.  where f- = the corection factor to account for the mean number of o  glucose carbons labelled from alanine (Chochinov et a l . , 1978). 5.  Rate of glucose C derived from alanine C = Equation 4  X  (mgC/min/kg)  glucose i r r e v e r s i b l e disposal rate.  where glucose i r r e v e r s i b l e disposal rate was determined simultaneously 3  using [2- H] glucose. 6.  Per cent of alanine C going to glucose C Equation 5 Alanine i r r e v e r s i b l e disposal rate  x  100  Additional calculations pertaining to the i r r e v e r s i b l e disposal rates and substrate recycling were performed as described in Experiment II A l l results are expressed as means (+ SEM).  Statistical  a.  analysis  of the data was performed by students independent t-test. Results The body weight of ewes and t h e i r fetuses as well as blood gas and metabolite parameters recorded at the time of the experiment are presented in Table  8.  The physiological parameters of the animals were  similar in the series of experiments in which [U-^C] amino acid mixture or [U-  14 o C] alanine/[2- H] glucose was injected. J  Table  8  Maternal  and f e t a l  physiological  parameters d u r i n g the experimental period  Isotope(s)  [ U - ^ C ] Amino A c i d (n=4)  Parameter  Maternal Gestational  age  Body w e i g h t  (kg)  P0  PC0  2  (mm Hg)  pH Total  CO2  Hematocrit Whole  ] A l a n i n e and glucose  Maternal  [2- H] (n=l0) 3  Fetal 133.8+6.5  2.99+0.18  61.30+2.40  2.87  33.10+0.90  20.09+1.04  35.07+1.10  19.70+0.78  34.45+2.54  39.10+1.47  28.93+0.90  41.30+1.22  7.502+0.01  7.376+0.01  7.479+0.01  7.312+0.22  -  23.51+0.52  +0.19  23.36+0.74  34.50+1.29  32.89+0.69  36.15+0.76  3.74+0.03  1.00+0.02  0.133+0.01 1.14+0.04 3.37+0.03  0.296+0.01 1.78+0.02 0.92+0.02  7.03+0.13  3.34+0.26  6.02+0.11  3.77+0.13  0.19+0.02  0.42+0.02  -  -  33.10+0.87  (%)  1 4  62.96+3.30  -  (meq/L)  Fetal  [U-  135.6+1.86  (days)  (mm Hg)  2  Mixture  injected  blood  a l a n i n e (mM) l a c t a t e (mM) g l u c o s e (mM) Plasma  (mg/%)  Plasma P r o t e i n Total  carbon  (2)  V a l u e s = mean (+ SEM) moles c a r b o n / l i t e r  .  101  S p e c i f i c a c t i v i t y - t i m e curves f o r the amino acid mixture and alanine are presented i n F i g . 11. The observed data are best described by a three exponential equation with a corresponding mean R2 value of 0.986  and 0.988 f o r [u- C] amino acid mixtureand alanine r e s p e c t i v e l y . 14  The k i n e t i c parameters obtained from the s i n g l e i n j e c t i o n of a mixture of amino acids and alanine are presented i n Table 9.  The  metabolic h a l f l i f e of the slow component of the amino a c i d mixture was s i g n i f i c a n t l y (P<0.05) higher than those observed with a l a n i n e . h a l f l i f e representing the f a s t component  The  of the s p e c i f i c a c t i v i t y  time curve of the amino acid mixture was s i g n i f i c a n t l y l e s s (P< 0.05) than that of a l a n i n e , i n d i c a t i n g a more rapid removal from the primary free amino acid pool. The alanine pool s i z e was 7.67*0.39 mgC/kg and was not s i g n i f i c a n t l y d i f f e r e n t from the plasma pool s i z e of the amino acid mixture. The apparent volume of d i s t r i b u t i o n of alanine was 66.05+4.09% of f e t a l body weight and was s i g n i f i c a n t l y (P<0.05) greater than that of the amino a c i d mixture (1%).  The i r r e v e r s i b l e disposal rates of amino  acid mixture and alanine were 2.30±0.277 mgC/min/kg and 2.021+0.34mgC/ min/kg r e s p e c t i v e l y . The t o t a l entry rate f o r the amino acid mixture was s i g n i f i c a n t l y greater (P<0.05) than that observed with a l a n i n e . • The extent of r e c y c l i n g of the amino a c i d mixture and alanine was 85.3±0.03 and 71.1±0.05% r e s p e c t i v e l y (Table 10). No s i g n i f i c a n t differences were noted between the mean t o t a l residence time f o r the amino acid mixture and a l a n i n e ; however the number of cycles made by the amino acid carbons was s i g n i f i c a n t l y (P<0.05) greater than by alanine. No 14c a c t i v i t y from alanine or amino acids was detected i n the maternal c i c u l a t i o n f o l l o w i n g the adminstration of these l a b e l l e d substrates.  102  O l OL  "D  U CJ Alanine 14  U cl Amino Acid Mixture l4  < O U c "E < ^  1  0>  o  4  9 "  9  * 9  4  t/5  ?  20  60  140  100  T  180  Time (min)  F i g . 11. Semi logarithmic p l o t of C amino acids and C alanine s p e c i f i c a c t i v i t y (S.A.) versus time. Values are means ("± SEM); Amino Acid (n=4); Alanine(n=10). l4  Table 9  K i n e t i c parameters of amino acid metabolism i n the ovine fetus in. utero .  Labelled substrate injected  [U- C] 14  amino a c i d mixture (n=4)  [u-14c]  alanine (n=10)  Pool s i z e (Q) (mgC/kg)  I r r e v e r s i b l e rate of disposal (D.R.) (mgC/min/kg)  Total entry rate (TER) (mgC/min/kg)  8.25a +1.15  ?..301a +0.277  15.82a +1.30  7.67a +0.39  2.021a +0.34  8.69b +1.14  Volume of distribution (v) [% Bwt)  l  a  66.05b +4.09  Half l i f e (min) T  l  T  2  '  b  3  0.30a +0.03  1.4283 +0.47  81 8 a +2 77  0.88b +0.14  2.99b +0.42  52 . 8 8 b +1 . 7 8  Values = mean (+ SEM) a  T  Supercripts with d i f f e r e n t alphabets i n the column denote s t a t i s t i c a l s i g n i f i c a n c e (P < 0 . 0 5 )  104  Table  10 Recycling of amino acids and alanine in the ovine fetus in u t e r o ' .  Parameter  [U- C] Amino Acid Mixture (n=4) 14  Fraction of total turnover i r r e v e r s i b l y lost  0.146 + 0.021  Fraction of total turnover recycled  0.853 ± 0.030  Mean total residence time (T) (min)  7.22  Number of cycles  6.46 ± 0.93  (v)  a  a  3  14  (n=10)  0.271 ± 0.041  b  0.711 ± 0.052  6.13 ± 1.72  b  a  3.27 ± 0.74  b  Values = means (± SEM)  1  a  ± 0.89  a  [U- C] Alanine  '  b  Superscripts with d i f f e r e n t alphabets in the rows denote s t a t i s t i c a l significance (P < 0.05)  105.  Table 11  Conversion of alanine t o l a c t a t e and glucose i n the ovine fetus i n utero .  Parameter  2 3 A l a n i n e — > Lactate (precursor) (product)  % product C derived from alanine C^  27.07 ±4.85  4.11 ±1.09  Rate of alanine C g going t o product C (mgC/min/kg)  0.696 ±0.131  0.161 ±0.040  34.95 ±4.40  8.85 ±0.36  % of alanine i r r e v e r s i b l e disposal going to product C?  1  o  2 Alanine (precursor)  4 Glucose (product)  Values = means (± SEM) Alanine i r r e v e r s i b l e disposal rate = 2.021 ± 0.34 (mgC/min/kg) Lactate i r r e v e r s i b l e disposal rate = 2.576 ± 0.18 (mgC/min/kg)  4  Glucose i r r e v e r s i b l e disposal rate = 4.05 S.A. product (dt) S.A. alanine (dt) 5 x Product i r r e v e r s i b l e disposal rate  alanine i r r e v e r s i b l e disposal rate  x  ^  ± 0.16 (mgC/min/kg)  106.  In experiments where [ U - ^ C ] alanine and [2- H] glucose were i n j e c t e d 3  simultaneously sion of alanine  i n t o the f e t u s , attempts were made t o quantitate the convercarbon  into  l a c t a t e and glucose carbons.  The mean  3  (+ SEM) i r r e v e r s i b l e disposal rate of [2- H] glucose obtained i n t h i s e x p e r i ment was 4.05 + 0.16 mgC/min/kg and compared favourably with the r e s u l t obtained i n Experiment I I a. A rapid t r a n s f e r of alanine [^C] was observed i n l a c t a t e and glucose, with peak a c t i v i t y occurring 5-10 minutes a f t e r the  injection  o f alanine.  The proportion of l a c t a t e C derived from  alanine C was 27.07 + 4.85% (Table 11). The rate of conversion of alanine C going t o l a c t a t e C was 0.696 + 0.13 mgC/min/kg and was equivalent to 34.95 + 4.4% of the alanine i r r e v e r s i b l e disposal r a t e .  The f r a c t i o n of  glucose C derived from alanine C was 4.11 + 1.09% (Table 11). Based on the 3  i r r e v e r s i b l e disposal rate o f glucose determined simultaneously using [2- H] glucose, the conversion of alanine C going to glucose C occurred a t a rate of 0.161 + 0.04 mgC/min/kg.  This was equivalent to 8.85 + 0.36% of the alanine  i r r e v e r s i b l e disposal r a t e .  Discussion  In t h i s study an attempt was made to assess the u t i l i z a t i o n of amino acids by the ovine fetus i n utero using i s o t o p i c d i l u t i o n  techniques.  The s i n g l e i n j e c t i o n o f a mixture o f [ U - C ] amino acids was used t o estimate 14  the disposal rates from the plasma free amino acid pool.  The simultaneous  i n j e c t i o n of [ u - C ] alanine and [2- H] glucose was made to quantitate the 14  3  conversion of.alanine carbon to other compounds and i t s p o t e n t i a l contribut i o n to gluconeogenesis.  107.  Amino acid metabolism A major concern with the s i n g l e i n j e c t i o n or continuous i n f u s i o n r  of a mixture of [U-  14 - i C] amino acids i s that there are some amino acids that  are not included i n the mixture and y e t account f o r a s i g n i f i c a n t proportion of ninhydrin p o s i t i v e compounds i n plasma (Wolff and Bergman, 1972). Although glutamine, a r g i n i n e , c i t r u l l i n e , o n i t h i n e , N- and 3-methyl h i s t i d i n e and carnosine are excluded  from the 15 amino acids i n j e c t e d i n t o the f e t a l  c i r c u l a t i o n , i t can be c a l c u l a t e d from t h e i r u m b i l i c a l venous-arterial d i f f e r e n c e and blood flow (Lemons e t al_., 1976), that these amino acids cont r i b u t e to only 8% of the u m b i l i c a l amino a c i d uptake.  Thus, i t was assumed  that the r a d i o a c t i v e mixture used i n t h i s experiment c l o s e l y represents the mixture of amino acids that are transferred to the fetus from the u m b i l i c a l circulation. Multicompartmental a n a l y s i s y i e l d e d three exponents which best described the s p e c i f i c a c t i v i t y - t i m e curves of amino acid mixture and r-14  alanine.  S i m i l a r r e s u l t s have been reported with a s i n g l e [  -i  C] amino acid  i n j e c t i o n by Henriques et al_. (1955) i n the r a b b i t or a mixture of [^ C] 4  amino acids by R e i l l y and Green (1975) i n the r a t . Though the pool s i z e of alanine and amino acid mixture was s i m i l a r , the apparent volume of d i s t r i b u t i o n of the amino acid mixture i s less than 1% of the f e t a l body weight, which i s less than the plasma space. This i s s i m i l a r to the value of 2% that can be c a l c u l a t e d from the data of R e i l l y and Green (1975).  On the other hand, the volume of d i s t r i b u t i o n  of alanine was 66.05%, approximately the f e t u s .  equivalent to the t o t a l body water of  The low volume of d i s t r i b u t i o n observed with the amino acid  mixture can be explained on the basis that amino acids are not present i n equal concentrations throughout the body water pool but rather are  108  concentrated 1970).  i n c e r t a i n organs to a greater extent than i n the blood (Munro,  The use of whole blood f o r alanine s p e c i f i c a c t i v i t y  determinations  and plasma f o r amino acid mixture could have also contributed, to some extent to the observed differences i n the volume of d i s t r i b u t i o n . The i r r e v e r s i b l e disposal rate of the amino acid mixture (2.301 min/kg) was  mgC/  s i m i l a r to the u m b i l i c a l uptake measurement (2.73 mgC/min/kg)  reported by Lemons et al_. (1976).  The i r r e v e r s i b l e disposal rate of alanine  i s approximately 40% greater than that reported by P r i o r and  Christenson  (1977) using continuous i n f u s i o n procedures and 30% greater than Lemons et a l . (1976) using u m b i l i c a l venous a r t e r i a l d i f f e r e n c e s . alanine [ C ]  atoms with [^C]  14  atoms of intermediates formed from alanine  would r e s u l t i n isotope d i l u t i o n and thus overestimate of alanine.  This may  The exchange of  the disposal rate  not n e c e s s a r i l y lead to a v e n o - a r t e r i a l d i f f e r e n c e .  The extent of r e c y c l i n g of amino acids observed i n t h i s experiment (Table 10) i s greater than the estimates of 36% reported i n adult rats ( R e i l l y and Green, 1975).  Wolff and Bergman (1972) have indicated  that amino acid r e c y c l i n g involves the i n t r a c e l l u l a r pool and t i s s u e prot e i n pool.  I t i s u n l i k e l y that the large amount of r e c y c l i n g observed  during the 3-hour experimental  period could be a t t r i b u t e d to the protein  pool, the f a s t e s t of which has been reported to have a h a l f l i f e of approximately 24 hours (Young, 1979).  I t , therefore, appears that r e c y c l i n g  involves the i n t r a c e l l u l a r t i s s u e pool, which has been shown to  turnover  very r a p i d l y in order to sustain the rapid growth rate of the fetus (Young, 1979).  This i s f u r t h e r supported by the r e l a t i v e l y greater number of  cycles made by the mixture of amino acids than by l a c t a t e or glucose (Experiment II a ) , though the t o t a l residence time of these compounds i s s i m i l a r (Table  10).  109  I t has been shown that the placenta d e l i v e r s to the mother a s i g n i f i c a n t amount of nitrogen derived from amino acids i n the form of urea and ammonia (Gresham et al_., 1972a; Holzman et al_., 1979). to detect amino acid carbon r a d i o a c t i v i t y i n the maternal  The f a i l u r e circulation  14 f o l l o w i n g the i n j e c t i o n o f  C amino acids to the fetus r a i s e s the question  whether amino acids of f e t a l o r i g i n are excreted across the placental b a r r i e r i n t o the maternal" c i r c u l a t i o n or are metabolized by the fetus and or placenta.  The permeability of the ovine placenta on the maternal  side  has not been completely defined, however, i t would appear from the r e s u l t s of t h i s experiment that the maternal  side of the placenta i s r e l a t i v e l y  impermeable to amino acids. Alanine conversion to l a c t a t e and glucose The use of i n t e g r a l equations and r a t i o of the area under the precursor-product  s p e c i f i c a c t i v i t y - t i m e curves were used i n t h i s study as  a measure of q u a n t i t a t i n g the conversion of alanine carbon to l a c t a t e and glucose carbon i n the ovine f e t u s . The proportion of alanine C going to l a c t a t e C (27%) compares favourably with the r e s u l t (23%) reported by P r i o r and Christenson using a continuous  i n f u s i o n technique.  (1977),  Foster et al_. (1980) have r e c e n t l y  shown i n dogs that carboxyl carbon of alanine exchanges r a p i d l y with lactate.  The rapid exchange between alanine and l a c t a t e i s a r e f l e c t i o n  of glutamate-pyruvate  transaminase a c t i v i t y i n f e t a l t i s s u e s (Stevenson  et al_., 1976) and c o n t r i b u t i o n of alanine to o x i d a t i v e metabolism i n the fetus. The evidence f o r the existence of gluconeogenesis fetus has been a c o n t r o v e r s i a l t o p i c .  i n the ovine  The presence of gluconeogenic  no.  enzymes i n f e t a l ruminant l i v e r ( B a l l a r d et al_., 1965) and the sharp increases i n phosphoenol  pyruvate carboxykinase (EC. 4.1.1.3.2.) a c t i v i t y  between 130-140 days of gestation i n the ovine fetus (Warnes et a l . , 1977b) prompted many workers to f i n d the functional s i g n i f i c a n c e of these enzymes.  Anand et aj_. (1980.) recently reported that no gluconeogenesis  was observed i n the fetus f o l l o w i n g induced f e t a l hypoglycemia with i n s u l i n infusions.  This may not be s u r p r i s i n g , as i n s u l i n i s an antagonist of  gluconeogenesis.  On the other hand, Hodgson et al_. (1980) have estimated  that 69% of f e t a l glucose requirements are supplied through gluconeogenesis. P r i o r (1980) recently has reported, using a continuous i n f u s i o n of [U-^C] l a c t a t e , that 22% of the glucose turnover was derived from l a c t a t e . In t h i s experiment 4% of the glucose disposal rate was derived from alanine C (Table 10), and i s s i m i l a r to the value of 2.3% obtained by P r i o r and Christenson (1977) i n the fetus and 3.5% by Brockman and Berman (1975) i n the adult ewe.  Anand and Sperling (1978) have proposed  that the apparent gluconeogenic a c t i v i t y o r i g i n a t i n g from [ U - C ] a l a n i n e , 14  in the experiments of P r i o r and Christenson (1977) was due to the the  [ C] l4  alanine returning to the mother and then r e c i r c u l a t i n g back to the fetus as [^ C] 4  glucose.  The f a c t that no [^ C]  maternal c i r c u l a t i o n  4  a c t i v i t y was detected i n the  f o l l o w i n g the i n j e c t i o n of [ U - C ] alanine to the 14  fetus i n t h i s experiment, i s s u f f i c i e n t evidence to c o n t r a d i c t t h i s hypothesis. The proportion of alanine C (8.8%) going to glucose C i n t h i s study when expressed as a % of the alanine i r r e v e r s i b l e disposal rate i s s i m i l a r to the value (7-9%) obtained i n adult monogastrics (Chockinov et a l . , 1978; Foster et aj_., 1980), but i s almost h a l f as much (15-20%) reported in adult ruminants (Brockman and Bergman, 1975).  A s i m i l a r estimate (7.3%)  was reported by P r i o r and Christenson (1977) i n the ovine fetus i n t h e i r  Ill  continuous infusion studies.  It  should be noted however that the transfer  of carbon atoms from alanine to glucose may not be a quantitative measure of the true rate of gluconeogenesis because of the "metabolic exchange" or "cross over" of ^ C atoms with 2  1 4  C atoms in the oxaloacetate pool  (Krebs  et al_., 1966). The lack of incorporation of lactate label into glucose in the single injection studies, (this study and Warnes et al_., 1977a) is contrary to the findings of Prior (1980) in a continuous infusion study in which 22% of glucose turnover was reported to be derived from lactate.  The use  of [1-^ C] rather than [U-^C] lactate in this experiment may partly 4  explain the lack of l a b e l l i n g in glucose. As shown in a subsequent experiment (Expt III, mately 63% of [ll-^C] lactate was found to be oxidized.  p. 128  ),  approxi-  The randomization  of lactate carbons during passage through the Krebs cycle would reduce the extent of l a b e l l i n g in glucose even i f lactate conversion to glucose did take place.  These d i l u t i o n effects are l i k e l y to be more pronounced  in single i n j e c t i o n than continuous infusion studies.  Further work is  needed to c l a r i f y the role of lactate as a gluconeogenic precursor in the fetus. Conclusions Experiment II  b  was undertaken to study metabolism of amino  acids in the ovine fetus in utero. The kinetic parameters of amino acid metabolism were obtained following the single injection of [U-^C] amino acid mixture or [u-^C] r  3 -.  alanine [2- HJ glucose. three exponential curve.  The s p e c i f i c activity-time data were f i t t e d to a Based on the rates of total entry and i r r e v e r s i b l e  112  d i s p o s a l , 85% of the amino acid turnover was recycled i n t h i s study.  The  r e l a t i v e impermeability of the placenta to amino acids may account f o r the amount of r e c y c l i n g observed i n t h i s study.  The frequency at which amino  acids returned to the blood pool was shown to be greater than other metabolic compounds; however the mean t o t a l residence time was not longer.  This may  be due to a rapid turnover of the plasma amino-acid pool. The extent of conversion of alanine C to l a c t a t e and glucose C was determined.  Based on the area under the s p e c i f i c a c t i v i t y - t i m e curves of  the precursor and the product i t was estimated that 8 and 27% of the i r r e v e r s i b l e disposal rate of alanine C were converted to glucose C and l a c t a t e C respectively.  Though t h i s conversion i n d i c a t e s that the ovine fetus was  capable of gluconeogenesis, the t r a n s f e r of C atoms from alanine to glucose may not be a q u a n t i t a t i v e measure of the rate of gluconegenesis due to the metabolic exchange of  12 14 C atoms with C atoms i n the oxaloacetate pool.  113 .  Experiment I I I Measurement of carbon dioxide production and substrate oxidation by the ovine fetus i n utero using [' C]-1abel1ed compounds 4  Introduction The metabolic  f u e l s f o r o x i d a t i v e metabolism of the fetus have  been reviewed extensively  by B a t t a g l i a and Meschia (1978).  Using the  elegant technique of u m b i l i c a l c a t h e t e r i z a t i o n , Tsoulos et _al_. (1971), James et aJL  (1972) and Morriss et ^1_. (1973) developed the concept of  substrate -oxygen quotient procedure according to which glucose, l a c t a t e and amino acids have been reported to contribute approximately 46, 20 and 25% r e s p e c t i v e l y to the t o t a l oxygen consumed by the ovine fetus. To provide a d i r e c t measurement of substrate turnover,  isotope  d i l u t i o n techniques have recently been employed i n f e t a l metabolic  studies  (Warnes et a^., 1977a; P r i o r and Christenson, 1977; Anand et al_., 1979, 1980; Hodgson et j H . , 1980; P r i o r , 1980).  However, q u a n t i t a t i v e measurements  of carbon dioxide output from l a b e l l e d substrates i n c h r o n i c a l l y cathet e r i z e d conceptus have not been reported. excretion of  14  CO2 i n breath  In the post-natal l i f e the  f o l l o w i n g the administration of  14  C-labelled  substrates has been used to determine substrate oxidation rates i n sheep (Lindsay and Ford, 1964; Annison et al_. , 1967).  Carbon dioxide  production  rates have also been measured i n adult sheep by the isotope d i l u t i o n 14 procedure from s p e c i f i c r a d i o a c t i v i t y of a i r (Whitelaw, 1974). of plasma  14  CO2 i n blood, urine or expired  A high c o r r e l a t i o n between the s p e c i f i c a c t i v i t y  CO2 and r e s p i r a t o r y  14 C02>during the oxidation of l a b e l l e d  substrates i n man was reported recently by Clague and K e i r (1979).  This  114  coupled with the f a c t that f e t a l r e s p i r a t i o n occurs only through the placental c i r c u l a t i o n prompted the use of isotope d i l u t i o n procedures based 14 on  CO,, a c t i v i t y i n f e t a l blood.  The measurement of f e t a l C 0  2  production  rates and the q u a n t i t a t i o n of the oxidation of substrates, by the ovine fetoplacental t i s s u e s in^utero were investigated i n t h i s experiment. Materials and Methods Animals The animals, surgery and the maintenance practices have been described previously.  In experiments where  [ U - C ] NaHCO^ was 14  two catheters were introduced i n t o the f e t a l c i r c u l a t i o n . used f o r sampling f e t a l blood was  The  infused, catheter  introduced to a distance of 6 - 8 cm i n t o  the external saphenous vein i n one of the hind limbs.  Another catheter  was passed deeply i n t o the external saphenous vein i n the other leg f o r i n f u s i n g r a d i o a c t i v e compounds.  The t i p  of t h i s catheter was found at  autopsy to l i e i n the i n f e r i o r vena cava approximately 1 0 cm from the heart. A minimum of 5 post-operative days elapsed before t r a c e r experiments were undertaken.  Cannulae  were also placed i n the j u g u l a r vein of the ewes one  day p r i o r to the experiment. Metabolic A)  Studies I r r e v e r s i b l e disposal rate of C O 2 The i r r e v e r s i b l e disposal rate of C 0 £ was determined using a  primed dose-continuous i n f u s i o n technique.  The priming dose consisted  115. of 30 uCi of NaH 14 CO^ followed by a continuous i n f u s i o n at the rate of 14 0.5 uCi/min.  Solutions of NaH  C0  3  were prepared i n s t e r i l e s a l i n e and  made s l i g h t l y a l k a l i n e by a d d i t i o n of 0.1 N NaOH. samples f o r C0  2  Fetal and maternal blood  and s p e c i f i c a c t i v i t y determination were taken at 30 minute  i n t e r v a l s p r i o r to and during the period of isotope e q u i l i b r i u m . The t o t a l content of whole blood was c a l c u l a t e d using the Henderson-Hasselbalch  C0  2  equa-  t i o n from PC0 and pH values which were determined w i t h i n 5 min a f t e r 2  c o l l e c t i o n (Radiometer).  The r a d i o a c t i v i t y of  1 4  C0  2  i n blood was deter-  mined by adding 0.5 ml of 6 M p e r c h l o r i c acid from the side arm of Warburg 14  f l a s k s to 0.5 ml of whole blood i n the main chamber.  The  C0  2  which  was  released was trapped i n hyamine hydroxide (Don M i l l s , Ont.) placed i n the central w e l l .  A f t e r standing f o r 1 hour at 25°C an a l i q u o t of hyamine  hydroxide was added to 10 ml of s c i n t i l l a t i o n f l u i d (PCS, Amersham, O a k v i l l e , Ont.) and the r a d i o a c t i v i t y counted by l i q u i d s c i n t i l l a t i o n spectrometry (LKB, Rack Beta 1215).  This procedure gave a recovery of 92.0 ± 2.0% of  known amount of NaH C0 added to blood (Appendix Table 4). To c o r r e c t f o r the retention of C 0 i n slowly mixing pools such as bone, NaH^CO^ was 14  3  1 4  2  14  i n j e c t e d i n t o 3 fetuses i n separate experiments.  Blood  C0  2  radioactivity  was determined at i n t e r v a l s u n t i l no further a c t i v i t y was detected.  From  14 the  C0  2  r a d i o a c t i v i t y per ml of f e t a l blood at each time of c o l l e c t i o n  the t o t a l a c t i v i t y i n the e n t i r e blood volume of the fetus was obtained. The procedure of Faber et a l ^ (1973) was used to estimate the blood volume at the time of experimentation based on body weight at b i r t h using the regression equation of Gresham et ^1_. (1972b).  From the p l o t of t o t a l blood  14 C0  2  r a d i o a c t i v i t y against time the area under the curve at d i f f e r e n t 14  times was computed and expressed as a per cent of administered NaH appearing i n  14  C0  CO^  116.  Oxidation of substrates The oxidation of substrates was determined i n conjunction with experiments II a and b. Approximately  50 uCi of  14 C l a b e l l e d glucose,  l a c t a t e , a l a n i n e , amino a c i d mixture and acetate were i n j e c t e d as a bolus 14 i n t o the f e t a l c i r c u l a t i o n and the s p e c i f i c r a d i o a c t i v i t y of blood was monitored.  C0  2  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 substrates i n blood  was determined by ion-exchange chromatography as described i n Experiment II.  Normalized  s p e c i f i c r a d i o a c t i v i t y - t i m e curves of l a b e l l e d precursors  14 and  C 0 i n blood were described by biexponential equations. 2  Initial  estimates of k i n e t i c parameters were c a l c u l a t e d using AUTOAN Fortran computer program and the f i n a l l e a s t squares f i t t i n g o f the data was done using NONLIN (Michigan), as described i n Experiment II a. Radiochemicals [ U - C ] acetate (51 mCi/mmole) was obtained from ICN Pharmaceu14  t i c a l s , Montreal.  NaH C0 14  3  (58 mCi/mmole) was supplied by Amersham 14  Corp., O a k v i l l e , Ont. The d e t a i l s of amino acid mixture  C-glucose, l a c t a t e , alanine and  are given i n Experiment II a and b.  Calculations The i r r e v e r s i b l e disposal using a continuous  rate of carbon dioxide was determined  14 i n f u s i o n of NaH CO^.  Rate of NaH C0 14  1. I r r e v e r s i b l e disposal rate of C0 = 9  (mgC/min/kg)  3  i n f u s i o n (nCi/min) — ._ mj  Blood  I H  C0  2  plateau  s  Pecifl^nj^^y  117, The rate of oxidation of substrates was c a l c u l a t e d by 2 procedures following s i n g l e i n j e c t i o n s of l a b e l l e d components: A)  From the i s o t o p i c y i e l d i n the product (Heath and Barton, 1973). (i)  Isotopic y i e l d i n product (CO^) = I r r e v e r s i b l e disposal rate of C0 x 2  (ii)  Fraction of substrate oxidized  ( i i i ) Rate of substrate  Isotopic y i e l d i n C0 (uCi) substrate i n j e c t e d (uCi)  =  2  I r r e v e r s i b l e disposal rate of  oxidation  (mgC/min/kg)  substrate x Fraction of substrate oxidized.  where, Aps = Area under the s p e c i f i c r a d i o a c t i v i t y - t i m e curve i n the product (C0 ) f o l l o w i n g i n j e c t i o n of l a b e l l e d substrates. 2  Dose = y X i of substrate i n j e c t e d . B)  From carbon t r a n s f e r quotient r a t i o ( K l e i b e r et al_., 1956). (i)  Rate of substrate oxidation  Fraction of C0  2  from substrate  oxidation x I r r e v e r s i b l e d i s -  (mgC/min/kg)  posal rate of C 0  1 4  (ii)  Fraction of C0 substrate  2  from  oxidation  Per cent C0 substrate  2  derived from  9  s p e c i f i c a c t i v i t y (dt)  £ 14  o (iii)  C0  2  C-substrate s p e c i f i c a c t i v i t y (dt)  Rate of substrate  oxidation  Substrate I r r e v e r s i b l e disposal rate  -x 1  118.  /  3.  Time (Tmax) f o r s p e c i f i c a c t i v i t y of blood '^CG^ to reach a maximum f o l l o w i n g i n j e c t i o n of l a b e l l e d substrates was estimated as f o l l o w s :  Tmax(  1 4  C0 ) ?  =  • log  2  where  • 14 a and B are formation and e l i m i n a t i o n rate constants of CO^ s p e c i f i c a c t i v i t y - t i m e curves ( R i t s c h e l , 1976)  Data were subjected to an a n a l y s i s of variance and where a s i g n i f i c a n t d i f f e r e n c e was notec means were compared using Newmann Keuls m u l t i p l e range t e s t .  Linear regression l i n e s and c o r r e l a t i o n c o e f f i c i e n t s  were calculated by the method of l e a s t squares.  Results The body weights of ewes and fetuses i n the acetate and bicarbonate experiments were 60.0 ± 0.9; 2.67 ± 0.17 and 63.2 ± 1.9; 3.59 ± 0.20 kg respectively.  In conjunction with the studies i n Experiment I I , several  measurements of t o t a l CO^ concentration i n f e t a l whole blood were made. The t o t a l CO^ content of f e t a l venous whole blood ranged from 22 to 24 meq/liter i n a l l experiments. Changes i n the s p e c i f i c a c t i v i t y of f e t a l and maternal blood 14 C O 2 with time f o l l o w i n g primed dose-continuous  14 i n f u s i o n of NaH CO^ 14  are shown i n Figure 12. The s p e c i f i c a c t i v i t y o f f e t a l blood.  C O 2  reached a plateau approximately 90 minutes a f t e r the i n f u s i o n commenced and remained constant t h e r e a f t e r . The mean i r r e v e r s i b l e disposal rate  Table 12  Substrate o x i d a t i o n rates i n the ovine  fetus i n utero .  % Substrate Oxidized  (min)  from Substrate  2  Procedure  Procedure  Procedure Substrate  % C0  Rate of Oxidation (mgC/min/kg)  I.Y.  T.Q.  I.Y.  T.Q.  I.Y.  T.Q.  Glucose (N = 8)  19.60 ± 0.15  30.50 ± 4.20  30.70 ± 4.40  1.06 ± 0.18  1.09 ± 0.20  14.80 ± 2.6  15.20 ± 2.8  Lactate (N - 8)  2.90 + 0.19°  36.83 ± 3.92  36.13 ± 3.91  0.96 ± 0.14  0.98 ± 0.13  14.30 ± 1.6  14.00 ± 1.4  Alanine (N = 10)  19.90 ± 0.85  28.52 ± 3.99  26.93 ± 3.08  0.549 ± 0.09  0.511 ± 0.09  6.49 ± 1.4  6.82 ± 1.2  9.30 ± 1.00  -  23.55 ± 3.22  -  0.59 ± 0.09  -  8.19 ± 0.9  10.30 ± 1.39  15.82 ± 2.81  -  0.10 ± 0.20  Amino acid mixture (N = 4) 2 Acetate (N = 5) a  1  ' ' b  c  a  a  c  c  2  1.70 ± 0.2  2  J  Supercripts with d i f f e r e n t alphabets i n the column denote s t a t i s t i c a l s i g n i f i c a n c e P< 0.05.  Values ± standard e r r o r of the mean. Calculated values based on umbilical uptake of 1.39 g/kg/day.  I.Y. = Isotopic y i e l d  T.Q.  = Transfer quotient.  -  Fetus  l H-r INJECTION OF No H " C ^ TO FETUS  Ewe  1—*—\ 30  60  90  120  X  150  180  210  J  240  TIME (minutes)  F i g . 12 S p e c i f i c r a d i o a c t i v i t y of f e t a l and maternal blood f o l l o w i n g orimed dose-infusion of NaH'^CG^.  ,4  C02  121.  of carbon dioxide i n 6 fetuses was 13.47 ± 1.07 ml/min/kg.  In the 3 single 14  i n j e c t i o n experiments designed to estimate the r e t e n t i o n of  CO2  in  slowly mixing pools, i t was found that only 82.5 ± 5.4% of the adminis14 tered r a d i o a c t i v i t y i n ,NaH of 3 h ( F i g . 13).  COg was recovered i n f e t a l blood at the end  Correction f a c t o r s were therefore applied to the values 14  of s p e c i f i c r a d i o a c t i v i t y of blood  14 CO2 when  C - l a b e l l e d substrates were 14  CO2  i n j e c t e d intravenously. The s p e c i f i c a c t i v i t y of maternal blood  c l o s e l y followed the increase i n the fetus and reached a plateau a f t e r 90 minutes ( F i g . 12).  At steady state c o n d i t i o n s , the r a t i o of the speci14  f i c a c t i v i t y of maternal and f e t a l blood  CO2 was  the 14% of maternal CO2 production can be accounted t r a n s f e r of  0.14 ± .01 i n d i c a t i n g f o r by the placental  CO2. 14  The changes i n the s p e c i f i c a c t i v i t y of blood  CO2 f o l l o w i n g  14 the i n j e c t i o n of  C - l a b e l l e d substrates are shown i n F i g . 14.  The speci-  14  f i c a c t i v i t y of blood CO2 reached a maximum w i t h i n the f i r s t 3 minutes (Tmax) of l a c t a t e i n j e c t i o n which was s i g n i f i c a n t l y {?<• 0.05) more rapid than i n the case of other substrates (Table 12). The peak s p e c i f i c 14 a c t i v i t y of blood CO2 appeared 19.6, 19.9, 10.3 and 9.3 minutes a f t e r the i n j e c t i o n of l a b e l l e d glucose, a l a n i n e , acetate and amino acid mix14 ture r e s p e c t i v e l y . was also higher (P<  Secondly, the peak s p e c i f i c a c t i v i t y of blood 0.05)  CO2  i n l a c t a t e than i n a l l other substrates studied.  The rates of substrate o x i d a t i o n as well as t h e i r c o n t r i b u t i o n to t o t a l carbon dioxide production c a l c u l a t e d by the two  procedures  employed were very s i m i l a r and not s t a t i s t i c a l l y d i f f e r e n t (Tablel2).  It  i s noteworthy t h a t , on an average only 30.60 and 36.48% of glucose and l a c t a t e carbons r e s p e c t i v e l y appeared i n CO2.  The extent of o x i d a t i o n  of alanine or amino acid carbons was 27.73 and 23.55% r e s p e c t i v e l y .  The  1-22.  90 80  1  60  •o w  s O V  40  tr o 20  30  60  __i  90  —i  120  •—  150  180  Time (minutes)  F i g . 13 Recovery of r a d i o a c t i v i t y of blood s i n g l e i n j e c t i o n of NaH^CC^.  1 4  C0  2  after  •032 r L  TIME  Fig. 14  Imin)  S p e c i f i c r a d i o a c t i v i t y (S.A.) of blood C 0 ? a f t e r i n j e c t i o n of C-labe11ed ^ b s t r a t e s i n t o f e t a l c i r c u l a t i o n . ( L = l - C - l a c t a t e ; A=U-14C-acetate; Al=U-14 -alamne; G=U-^C-glucose; AA=U- C-amino acid mixture. The curves are reproduced from a Calcomp p l o t t e r . 14  14  l4  C  14  1-8 r  Irreversible disposal rate.(mgC/min/Kg)  Pia  is  Relationship between rates of oxidation and i r r e v e r s i b l e disposal rates o f glucose and l a c t a t e (•, y=0.40x-0.347; r=0.641; p<0.10; n=8;d, y=0.283x+0.291 ; r=0.604; p<0.10; n=8).  125.  f r a c t i o n of CC^-carbon derived from the o x i d a t i o n of glucose, l a c t a t e , alanine and amino acids was 15.00, 14.15, 6.66  and 8.19%  respectively.  Only 15.80% of acetate carbon was oxidized c o n t r i b u t i n g to 1.70% C0  2  production.  of t o t a l  The l i n e a r regression of rates of glucose and l a c t a t e  oxidation on the i r r e v e r s i b l e rates of disposal i s shown i n F i g . 15.  Discussion C0  2  Disposal i n the Ovine Fetus The C0  2  disposal rates (13.47 ± 1.07 ml/min/kg) are comparable  to the oxygen consumption of 17.5 ml/min/kg by the uterus and i t s contents reported by S e t c h e l l et  (1972) using i s o t o p i c t r a c e r s and  ml/min/kg by Graham (1964) using c a l o r i m e t r i c techniques.  14.2  In t h i s study  attempts were not made to cannulate the u m b i l i c a l blood vessels and the estimated i r r e v e r s i b l e disposal rates of C0  2  would therefore represent  the metabolic a c t i v i t y of the fetus plus c o n t r i b u t i o n from the placental mass.  The determination of the f e t a l C0  2  i r r e v e r s i b l e disposal rate by  s i n g l e i n j e c t i o n techniques was found to be u n s a t i s f a c t o r y .  I t was  evident that because the label was leaving the f e t a l blood pool with such r a p i d i t y , the i n i t i a l slope and i n t e r c e p t d e s c r i b i n g t h i s pool were subjected to considerable e r r o r . 14 continuous i n f u s i o n of  NaH  The C0 C0  3  are  2  production rates obtained from the s l i g h t l y higher than the value  of 11.19 ml/min/kg f o r the fetus plus the utero-placental t i s s u e s e s t i mated from uterine and u m b i l i c a l arterio-venous d i f f e r e n c e s coupled with blood flowUMeschia  et j i l _ . , 1980).  The l a t t e r procedure does not take i n t o  account the r e t e n t i o n and u t i l i z a t i o n of metabolic C0  ?  by the t i s s u e s  126.  and i s l i k e l y to underestimate true production.  In t h i s study, the C0  2  disposal rates have been corrected f o r these factors which r e s u l t s i n s l i g h t l y 14 higher values.  The r e t e n t i o n of 17.5% of 14  the a d m i n i s t r a t i o n of NaH  C0  3  C0  2  i n f e t a l tissues during  c l o s e l y resembles the values of 17-20% i n  adult sheep (Bergman and Hogue, 1967; Annison et a]_.,  1967).  On the other hand, i n adult sheep, i t has been reported that C0  2  disposal rates estimated by isotope d i l u t i o n procedures tend to over-  estimate actual production determined 1974).  by c a l o r i m e t r i c techniques  This has been ascribed to low s p e c i f i c a c t i v i t i e s r e s u l t i n g from  slowly mixing pools and inadequate length of i n f u s i o n .  Though a plateau  14 of  (Whitelaw,  14 C0  2  s p e c i f i c a c t i v i t y was reached w i t h i n 90 min, the NaH  CO3  infus-  ions were continued f o r periods up to 4 hours with no increase i n s p e c i f i c a c t i v i t y i n d i c a t i n g that the duration of i n f u s i o n i s not a major f a c t o r f o r the higher values observed. Further support of the v a l i d i t y of the i r r e v e r s i b l e disposal rates of C0 may be obtained from the r a t i o (0.14) 14 2  of the s p e c i f i c a c t i v i t y of  C0  2  i n maternal blood to that i n f e t a l blood  a f t e r a plateau has been reached ( F i g . 12).  Assuming a maternal C0  2  pro-  duction rate of 362 ml/min during l a t e pregnancy (Whitelaw et aj_., 1972), the rate of placental t r a n s f e r of C0  2  amounts to 13.06 ml/min/kg which  i s s i m i l a r to the value of 13.47 ml/min/kg c a l c u l a t e d from the f e t a l compartment alone. Substrate Oxidation 14 The shorter T  m a x  and the higher s p e c i f i c a c t i v i t y of  C0  2  from  l a b e l l e d l a c t a t e than from l a b e l l e d glucose are s i m i l a r to the f i n d i n g s of Shambaugh et jil.(1977a) i n the t i s s u e s of f e t a l r a t s under i n v i t r o  127.  conditions.  In one experiment where DJ-  C]-lactate was i n j e c t e d instead of  D-^C]-lactate, the rate of o x i d a t i o n was 1.367 to 18.94% of C0  2  production.  mgC/min/kg c o n t r i b u t i n g  This i n d i c a t e s that the higher rate of  C0  2  production from l a c t a t e than from glucose i s not merely due to preferent i a l oxidation of C-l of l a c t a t e .  The absence of hepatic glucokinase i n  the fetus ( B a l l a r d e_t al_., 1969) and the o x i d a t i o n of glucose through an intermediary pool such as l a c t a t e having more carbon than the glucose pool (Table 5 ; Warnes et j f L , 1977a) may  e x p l a i n the slower C0  release from  2  glucose than from l a c t a t e . Two methods of c a l c u l a t i o n were used to quantitate the t r a n s f e r of substrate carbon to CG^-carbon. and Barton, 1973) i s measured.  In the i s o t o p i c y i e l d procedure (Heath  the t o t a l recovery of l a b e l l e d carbon i n the  product  This would give an estimation of the t o t a l f r a c t i o n of the  substrate oxidized by d i r e c t and i n d i r e c t pathways.  From the i r r e v e r s i b l e  disposal rates of the substrates and the f r a c t i o n o x i d i z e d , the rate of oxidation and c o n t r i b u t i o n to C0  2  production are c a l c u l a t e d . The t r a n s f e r  quotient r a t i o expresses the t r a n s f e r of carbon between the precursor and the product but i s independent on the number and s i z e of intermediary pools and rates of intermediary pathways ( K l e i b e r et al_., 1956; Searle et a l . , 1975).  I t i s of i n t e r e s t that the rate of substrate o x i d a t i o n and c o n t r i -  bution to C0  2  production c a l c u l a t e d by the two procedures (Table 12) were  very close to each other. A particularly significant finding  in  this  study pertains  to the extent of substrate o x i d a t i o n and c o n t r i b u t i o n to C0 Oxidation accounts only f o r 15.80  2  production.  - 36.48% of the i r r e v e r s i b l e rates of  disposal of the carbon from substrates studied.  In the s i n g l e experiment  128.  where LU- C>lactate was injected'62.3% was found to be o x i d i z e d . l4  Even  these values may be considered to be a maximum since oxidation rates based on  14  C 0 2 production are l i k e l y to include exchange reactions without net  oxidation taking place (Chang and Goldberg, 1978).  This would mean that  a major proportion of substrate carbon extracted by the f e t a l t i s s u e s i s u t i l i z e d f o r anabolic purposes than f o r o x i d a t i v e metabolism.  The p o s i t i v e  r e l a t i o n s h i p between oxidation rates and i r r e v e r s i b l e rates of disposal ( F i g . 15) i n d i c a t e s that the magnitude of uptake and u t i l i z a t i o n of glucose by the f e t a l t i s s u e s depends l a r g e l y on the materno-fetal ing previous reports (Boyd e_t _al_. , 1973).  t r a n s f e r confirm-  The f a c t that l a c t a t e may be  formed from glucose i n the placenta (Experiment I I a; Warnes et al_., 1977a) may explain the s i m i l a r i t y i n the o x i d a t i v e metabolism o f these compounds i n r e l a t i o n t o t h e i r i r r e v e r s i b l e disposal r a t e s . The f r a c t i o n of t o t a l CO^ derived from the oxidation of glucose (15.0%) i s s i m i l a r to the value of 22% obtained by S e t c h e l l e t al_. (1972) i n f e t a l sheep using t r a c e r techniques.  Kronfeld and Van Soest (1976)  have summarized a v a i l a b l e data on substrate c o n t r i b u t i o n to C 0 and have concluded that approximately  2  production  8-11% of CO2 was derived from oxida-  t i o n o f glucose i n ruminants and 10-50% i n non-ruminants.  The mean value  of 15% c o n t r i b u t i o n by glucose t o t o t a l C 0 production i s w i t h i n the range 2  reported f o r adult monogastric animals. Though the r e l a t i v e importance of various substrates to o v e r a l l f e t a l o x i d a t i v e metabolism would depend on the metabolic fuel mixture  avail-  able (Shambaugh e t £l_., 1977a,b) c o n t r i b u t i o n of l a c t a t e to CO2 production at a level equal to that of glucose i n d i c a t e s that the fetus r e l i e s on other  129.  substrates, i n addition to glucose, f o r oxidation.  A major proportion of  glucose carbon i s therefore used f o r a l t e r n a t i v e purposes.  A similar  explanation would hold good f o r the small c o n t r i b u t i o n (8.19%) of amino acid carbon to CO^  production.  I t i s also noteworthy t h a t , based on the  d i r e c t oxidation of the substrate used i n t h i s study, approximately 40% of t o t a l CO2 production by the fetoplacental t i s s u e s can be detected. suggests t h a t , i n a d d i t i o n to glucose, l a c t a t e , amino acids and other substrate(s) may  be involved i n f e t a l metabolism.  This  acetate,  Though the present  experiment have been designed to study the o x i d a t i v e metabolism of the fetoplacental u n i t as a whole i t i s possible to obtain q u a n t i t a t i v e informat i o n on the u n i d i r e c t i o n a l metabolism of the fetus and placenta  separately  by the simultaneous use of i s o t o p i c tracers and umbilical venoarterial concentration d i f f e r e n c e s coupled with blood flow.  This approach has been  used i n the metabolism of l i v e r (Brockman and Bergman, 1975).  Conclusion Experiment I I I was  i n i t i a t e d to measure the CO2 production  rate  and the r e l a t i v e oxidation rates of s p e c i f i c substrates i n the ovine fetus . in utero.  The rate of CO2 production noted in t h i s experiment represents  the metabolic  a c t i v i t y of the fetus and placental t i s s e s .  It was observed that 14% of the l a b e l l e d bicarbonate into the f e t a l c i r c u l a t i o n was c o l l e c t e d i n the mother. placental t r a n s f e r of C0  2  resembled the C0  placental compartment alone.  2  infused  I t was noted that  production rate from the f e t a l  130.  From the r e s u l t s of the oxidation of s p e c i f i c substrates, i t has been shown that only 15-36% of substrate i r r e v e r s i b l e disposal rates can be accounted f o r by o x i d a t i o n .  I t can be concluded that a greater  proportion of substrate u t i l i z a t i o n i s made a v a i l a b l e f o r synthetic purposes than was h i t h e r t o reported.  Using both the i s o t o p i c y i e l d and t r a n s f e r  quotient procedures to estimate the per cent of CO2 produced from these substrates, i t was concluded that other substrate(s) contribute to the t o t a l f e t a l o x i d a t i v e metabolism. Although the procedure used i n t h i s study estimates the o x i d a t i v e metabolism of the e n t i r e fetoplacental u n i t rather than the fetus i t s e l f , the f a c t that only a small proportion of the substrates u t i l i z e d by the fetus i s oxidized suggests that the q u a n t i t a t i v e c o n t r i b u t i o n of substrates to t o t a l o x i d a t i v e metabolism should be  reassessed.  131. General Conclusions  Surgical techniques were developed and standardized f o r introducing vascular catheters i n t o the fetus so that the metabolism of substrates may be studied i n vivo without s t r e s s . 14 Using s i n g l e i n j e c t i o n s of [U-  3  C] and [2- H] glucose the i r r e v e r -  s i b l e disposal r a t e s , pool s i z e , volume of d i s t r i b u t i o n and other k i n e t i c parameters were determined.  The u t i l i z a t i o n of glucose was also shown to  be r e l a t e d to blood glucose concentration and body mass.  This indicated that  the rate of u t i l i z a t i o n of glucose i s dependent on the umbilical uptake of t h i s substrate from the maternal c i r c u l a t i o n and that f e t a l glucose requirements increase with increases i n f e t a l body mass.  Glucose l a b e l - r e c i r c u l a t e d  within the fetus to an extent of 12.6 per cent of the i r r e v e r s i b l e disposal rate. A substantial amount of f e t a l glucose was found to be recycled through the maternal c i r c u l a t i o n .  Lactate on the other hand, was not  detected i n the maternal c i r c u l a t i o n following i n j e c t i o n i n t o the f e t u s , i n d i c a t i n g a low permeability of l a c t a t e i n the ovine placenta. Recycling of glucose and l a c t a t e i n the fetus occurred to an extent of 70-80% of the t o t a l turnover of these compounds and i t was concluded that t h i s was an important feature of f e t a l glucose and l a c t a t e metabolism. • Studies on the metabolism of a mixture, of amino acids and alanine were performed to quantitate f u r t h e r the u t i l i z a t i o n of amino acids by the fetoplacental u n i t . The rate of i r r e v e r s i b l e disposal of [ U - C ] amino l4  acid mixture was s i m i l a r to that of [ U - C ] glucose and [ 1 - ^ C ] l a c t a t e ; how14  ever, the per cent of substrate turnover recycled was greater. This coupled  132.  with the observation that the number of cycles made by amino acids was twice as much as glucose and lactate leads to the conclusion that the fetal plasma amino acid pool is being replaced at a rapid rate. From the single injection of [U- C] alanine, i t was calculated 14  that 4 per cent of the glucose disposal rate was derived from alanine carbon, indicating the presence of gluconeogenesis.  However, further  investigation  is required before conclusions can be drawn on the physiological circumstances under which gluconeogenesis is manifested. Carbon dioxide rates were determined by a primed dose, continuous infusion technique.  It was concluded from the r a t i o of fetal  to maternal  14 C0  2  s p e c i f i c a c t i v i t y , measured at plateau s p e c i f i c a c t i v i t y  that 14% of  the maternal C0 production rate could be accounted f o r by f e t a l and placental 2  metabolism.  In studies where substrate oxidation was performed in conjunc-  tion with the determination of the i r r e v e r s i b l e disposal rates, i t was shown that only 30, 6.2, 24, 15 and 26% of [U- C] glucose, l a c t a t e , amino 14  a c i d s , acetate and alanine were oxidized to CO,,.  Substrate oxidation observed  in this study accounts for only .40% of the total C0 production of the f e t o 2  placental  tissues. The total inflow and outflow of carbon in the fetoplacental  unit were estimated from the rates of i r r e v e r s i b l e d i s p o s a l , oxidation and interconversion among metabolites and are presented in F i g . 16. values indicated in Fig.16  Using the  with respect to oxidation and interconversion,  the amount of substrate carbon used for anabolic purposes may be estimated. For the purpose of these c a l c u l a t i o n s , the following substrates were assumed to be the major ones involved in f e t a l metabolism (Battaglia and Meschia, 1978).  133.  F i g . 16  Composite p i c t u r e of f e t a l substrate metabolism. Values i n s i d e brackets denote ml/min/Kg and those outside mgC/min/Kg.  134.  Carbon balance in the fetus  Glucose  Lactate  Amino Acids  Total Carbon mgC/min/kg  Irreversible disposal rate (mgC/min/kg) (A)  3.541  2.171  2.301  8.013  Oxidation rate (mgC/min/kg)  1.087  1.353  0.543  2.983  to urea 0.085  2.432  to f r u c t o s e 0.576  Other disposals (mgC/min/kg) (B)  3  to mother 1.771  Used for synthesis (mgC/min/kg) (C = A-B)  0.110  0.821  2.598 or 3.741 gC/day/kg  1.674  1  [U-  C] lactate  2  1 mole amino acid y i e l d s 0.69mole urea (Schulz, 1978) Warnes et a l .  (1977a)  The net retension of carbon in the f e t a l tissues (3.74 gC/day/kg) compares favourably with the value of 3.20 gC/day/kg obtained on the basis of carbon content of the ovine fetus (James et al_., 1972).  It  is note-  worthy that most of the glucose carbon has been used for oxidation or returned to the mother.  Carbon from other substrates, notably amino acids  has been largely used f o r synthesis of f e t a l  tissues.  135.  I t i s a l s o pertinent to discuss the c o n t r i b u t i o n of substrate oxidation to the o v e r a l l c a l o r i c requirement of the fetus.  Depending on  the technique employed, heat production i n the fetus has been reported to be 42-52 (James et al_., 1972; Meschia et al_. ,1967a, oxygen consumption), 65 (Abrams e t a]_., 1970., d i f f e r e n t i a l spirometry) and 90 kcal/day/kg (Graham, 1964, i n d i r e c t c a l o r i m e t r y ) .  The minimum oxygen consumption  needed f o r the proportions of substrates found to be oxidized i n t h i s study i s 6.41 ml/min/kg.  Assuming that the c a l o r i f i c value of oxygen i s 4.9 c a l / m l ,  t h i s would r e s u l t i n the production of 45 kcal/day/kg from o x i d a t i v e metabolism. In addition energy i s also stored i n the form of new t i s s u e s to the extent of 32 kcal/day/kg (Rattray et al_., 1974).  Thus, the t o t a l c a l o r i c require-  ment of the fetus would amount to 77 kcal/day/kg, a value which f a l l s i n the middle of the range reported i n the l i t e r a t u r e .  This may suggest that  o x i d a t i v e metabolism alone i s not s u f f i c i e n t to account f o r the t o t a l c a l o r i c requirement of the f e t u s .  On the other hand, the oxygen uptake of  the fetoplacental u n i t i s approximately 11-12 ml/min/kg  (Meschia et a l . ,  1980) and would r e s u l t i n the production of 78-85 kcal/day/kg from o x i d a t i v e metabolism s u f f i c i e n t to meet the t o t a l c a l o r i c requirement of the f e t u s . The oxygen consumption of 6.41 ml/min/kg used i n the present c a l c u l a t i o n s represents a minimal value to o x i d i z e only those substrates studied i n t h i s investigation.  Therefore, i t i s suggestive that other substrate(s) may be  involved i n f e t a l o x i d a t i v e metabolism. In s p i t e of the f a i l u r e to i d e n t i f y a l l the metabolic sources of f e t a l heat production, i t i s of i n t e r e s t t h a t , i n t h e i r review of f e t a l metabolism, B a t t a g l i a and Meschia (1978) have concluded that "only a n e g l i g i b l e amount of heat l i b e r a t e d by the f e t a l carcass i n the bomb  136.  calorimeter represents energy formerly derived from oxidative metabolism". It has been suggested that " v i r t u a l l y a l l the energy used to fuel oxygen consumption is ultimately dissipated as heat".  fetal  Their conclusions  are based on the observations of Abrams (1970) that heat is transferred from the fetus to the mother.  Though this may be true to the extent that  the energy expended in d i f f e r e n t i a t i o n and maintenance in the prenatal period may be dissipated as heat (Brody, 1945) i t does not imply that virtually  a l l the chemical energy released during oxidation is dissipated  as heat and not used for anabolic purposes.  The metabolic principles of  energy production and u t i l i z a t i o n discussed thoroughly by Mi 11igan (1971) would argue against such a hypothesis.  It may therefore be appropriate to  conclude that the maintenance energy cost of the growing fetus is  relatively  very high and increases with net increase in tissue mass. Conclusions based on the Fick p r i n c i p l e tend to underestimate the true u t i l i z a t i o n of metabolites by the fetus.  On the other hand, tracer  techniques, p a r t i c u l a r l y the single injection procedure, may overestimate disposal rates of substrates.  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"The protein turnover rate in fetal organs: the influence of i n s u l i n . " In: Nutrition and Metabolism of the Fetus and Infant, V Nutricia Symposium, edited by H.K.A. V i s s e r . Rio de Janeiro, B r a z i l : Martinus Nijhoff Publishers, 1979, p. 19-27. Z i l v e r s m i t , D.B., Entenman, C. and F i s h i e r , M.C. On the c a l c u l a t i o n of turnover time and turnover rate from experiments involving the use of l a b e l l i n a agents. J . Gen. Physiol. 26:325-332, 1943.  158. Appendix V a l i d a t i o n of Methods The following procedures were used f o r the separation and q u a n t i t a t i o n of l a b e l l e d metabolites: 1.  I s o l a t i o n and recovery of glucose. Glucose s p e c i f i c a c t i v i t y measurements have r o u t i n e l y  been performed by the formation of glucose d e r i v a t i v e s .  However,  due to the n o n s p e c i f i c i t y of these procedures and the r e l a t i v e l y high concentration of fructose i n f e t a l blood, anion exchange chromatographic procedure followed by the conversion of glucose to gluconic acid enzymatically was standardized. Glucose, i n the presence of other n e u t r a l l y charged metabolites was converted to gluconic acid by an excess of glucose oxidase and catalase and the r e s u l t i n g mixture separated by anion exchange chromatography. To t e s t the p u r i t y and recovery of glucose i n t h i s procedure, a l i q u o t s of the neutral and a c i d i c f r a c t i o n s were applied to paper chromatograms (descending) and developed i n a solvent c o n s i s t i n g of phenol:H20::NH3; 40:40:1; w/v/v  f o r 18 hours.  The separation  of l a b e l l e d glucose from fructose i s given i n Appendix Fig. l . The recovery of l a b e l l e d compounds added to the column i s given in Appendix Table 1 . The m o d i f i c a t i o n of the glucose oxidase procedure as f o l l o w s :  was  Glucose oxidase reagents were prepared by mixing 25  mg of glucose oxidase ( s p e c i f i c a c t i v i t y , 20,000 u n i t s / g , Sigma, St. Louis) and 10 mg of peroxidase ( s p e c i f i c a c t i v i t y , 120 u n i t s / mg, Sigma, St. Louis) i n 10 ml of demineralized water.  The colour  reagent, o-dianisidine-HCl (Sigma) was prepared by weighing 100 mg  159. of dry crystals and dissolving them in 10 ml of d i s t i l l e d water. To each reaction flask containing 400 ul of the neutral  fraction  was added 100 ul of the glucose oxidase-peroxidase and colour reagent.  Flasks were mixed and placed in a water bath at 37°C.  for 40 minutes.  The absorbance was then determined at 406 nm.  A reagent blank and a set of standards ranging in concentration from 5 to 20 ug D-glucose per flask were run with each series of samples. 2.  Isolation and recovery of l a c t a t e . To avoid contamination of lactate with unknown labelled  metabolites, ion exchange chromatography was employed in conjunction with  thin layer chromatography.  Results are given in Appendix  Table 2 . 3.  Isolation and recovery of alanine. [U-14c] amino acids and alanine in p a r t i c u l a r were  separated by cation exchange chromatography.  The separation of  alanine from other amino acids was performed by converting alanine to lactate enzymatically and passing the l a t t e r through a cation exchange column. 4.  Recovery of  14  Results are given in Appendix Table 3. C02-  The oxidation of labelled substrates was monitored by measuring  14  C02 in whole blood.  Whole blood was a c i d i f i e d with  perchloric acid and the liberated l^COg was collected in Hyamine Hydroxide. 5.  Results are given in Appendix Table 4.  Amino acid mixture (15 L-amino a c i d s , in same proportions as a  typical  algal protein hydrolysate)  - g l y c i n e , alanine, s e r i n e , threonine,  p r o l i n e , v a l i n e , i s o l e u c i n e , leucine, phenylalanine, tyrosine, aspartic a c i d , glutamic acid and l y s i n e .  Table 1 : Per cent recovery of r e l a b e l l e d compounds after treatment with glucose oxidase and anionexchange chromatography! Initial  14  (Dpm)  C-metabolite  l - C ] g l u c o n i c acid 14  (N = 6)  U-  14 2 C]glucose  2- H]glucose 3  (N = 6)  14  19.432 ± 241  2  (N = 6)  U- C]glycerol 14  (N = 6)  Neutral Fraction  Recovery  (Dpm)  -  -  Acidic Fraction  Recovery  (Dpm)  (%)  19,093 ±259  98.11 ±0.91  2,509 ±167  1.55 ±0.34  148,484 ±1196  92.09 ±0.71  42,180 ±11.82  210 ±21  .50 ±0.02  39,793 ±665.06  93.72 ±1.56  78,388 ±618  77,119 ±667  98.38 ±0.46  -  -  56,564 ±1,431  96.99 ±2.47  -  . 161,120 ± 398  (N = 10)  U- C]fructose  Activity  59,702 ±8.84  Values = means (± SEM) Rf values are shown in Appendix Fig 1  »  161.  Table  2:  Per cent recovery of 1chromatography and t h i n  C l a c t a t e following anion exchange layer chromatography'.  Contamination from glucose (%)  Initial Activity Dpm  Recovery (%)  Anion Exchange chromatography  15,669 ±252  93.09 ±0.50  N.D.  Thin Layer Chromatography (n-propanol:acetone:H 0 6:3:1 v/v)  14,489 ±208  89.07 ±0.68  0.8 ±0.01  Anion Exchange plus Thin Layer Chromatography  13,857 ±258  83.01 ±0.63  N.D.  Procedure  2  Value = means (± SEM)  162.  Table  3: Per cent recovery of EJ-C] amino acid mixture and alanine following cation exchange chromatography and enzymatic conversion of alanine to l a c t a t e . 1  Procedure  Recovery in Fraction Neutral  Basic  Acidic  Cation exchange chromatography 1) Amino Acid mixture (N = 12)  N.D.  99.1 ±0.96  N.D.  2) Alanine  N.D.  90.3 ±2.46  N.D.  N.D.  1.2 ±0.09  82.4 ±0.64  (N = 12)  Cation exchange and enzymatic conversion of alanine to lactate (N = 12)  Values  = means (± SEM)  163.  Table  4:  Vehicle  Saline (n = 8)  Whole Blood (n = 8)  Per cent recovery [ C] NaHCCu i n s a l i n e and whole blood.  Initial Activity (Dpm)  in  A c t i v i t y recovered Hyamine Hydroxide (Dpm)  % Recovered  168,000 ± 281  154,768 ± 201  91.4 ± 0.8  171,080 ± 310  159,446 ± 240  92.3 ± 1.4  Values = mean (± SEM)  Rf=0.39 Appendix f i g . 1  Rf  sQ56  Separation of metabolites by descending paper chromatography (phenol:water:NH3; 40/40/1; w/v/v.)  g  165.  166.  200  600  Total Carbon Cone. (mg./L) Appendix f i g . 3  1000  Standard curve for total organic carbon determined by infra-red carbon analyzer.  Appendix table  5 .  Mean and pH i n maternal and f e t a l blood of conscious ewes during and f o i l owing surgery P  0  ,  2  P C O 2  1  Days A f t e r Surgery Parameter  1  0  M  7.25S  7.452 ±0.02  3  ±0.02 (5)  B  (7)  3  4  5  6  7  8  7.439 ±0.02 (11)  7.438  7.451  7.450  +0.01  ±0.01  ±0.01  (11)  (ID  (ID  7.445 ±0.01 (9)  7.415 ±0.01 (10)  7.401 ±0.03 (5)  7.371  7.338  7.358  ±0.02  ±0.02  ±0.02  (11)  (11)  7.310 ±0.02 (9)  7.349 ±0.02 (10)  7.311 ±0.01 (4)  7.337 ±0.02  (11)  7.353 ±0.02 (13)  32.67 +0.54 (13)  34.24 ±1.277 (9)  35.69 ±2.35 (10)  34.95 ±1.21 (4)  33.95 ±1.52 (7)  40.51 +1.10 (13)  42.76 ±0.27 (9)  42.78 ±0.58 (10)  47.754 ±1.40 (8)  45.301 ±1.22 (4)  43.917 ±3.64 (6)  17.480 ±0.623 (8)  19.341 ±1.29 (4)  20.294 ±0.844 (6)  pH F  7.348 ±0.02 (8)  7.277° ±0.02 (5)  M  PCO  39.18* ±2.20 (5)  35.85 ±0.625 (8)  32.76 ±1.40 (11)  32.37  30.10  ±2.91  ±1.29  (11)  (ID  35.409 ± 0.9 (5)  41.75 ±1.05 (7)  39.99 +1.03 (10)  41.57  40.58  ±1.02  ±0.77  (11)  (11)  40.334  42.993 ±1.12 (9)  43.676 +1.95 (10)  41.552 (12)  43.727 11.50 (8)  22.037 ±0.94 (9)  21.808 ±0.76 (10)  21.866 ±1.07 (12)  22.292 ±1.153 (8)  F  2  (mm Hg)  F  M PO  D  104.670 ±8.20 (5)  I  H  42.072 ±1.85 (7)  J  20.183 +0.78 (8)  1  ±2.11 (11)  ±1.33  (mm Hg)  25.800 ±0.76 • (5)  K  Data presented as means  22.061 ±0.624 (11)  ±  S.E.M.  Values i n parenthesis are number of observations  7.467 ±0.021 (7)  (6)  41.61 ±2.17 (6)  2  F  9  2  Table : 6  Mean hematocrit, blood glucose, lactate, 3-hydroxybutyrate and alpha amino nitrogen in fetal blood of conscious ewes. 1  Days After Surgery  Hematocrit (%)  Glucose * (mM)  Lactate (mM)  *  8-Hydroxybutyrate (mM) Alpha amino nitrogen (mg/100 ml)  *  *  *  0  1  2  3  4  5  6  38.2 r 1.02 "(5)  37.83 r 1.3 "(8).  35.10 ^ 1.10 "(11)  33.72  r 1.00  34.80 ^ 1.12 "(11)  34.05 r 1.12 "(13)  34.50 i 1.25 "(9)  0.602 + 0.08 (3)  0.602 + 0.02 (5)  0.710 + 0.02 (4)  0.731 + 0.02 (5)  0.901 0.810 + 0.02 + 0.02 (4) (5) .  0.880 + 0.13 (4)  1.90 + 0.17 (3)  1.80 + 0.02 (4)  1.83 + 0.04 (4)  1.85 + 0.02 (5)  1.78 + 0.03 (5)  1.58 + 0.02 (4)  1.62 + 0.03 (4)  0.198 + 0.001 (3)  0.187 + 0.008 (4)  0.172 + 0.001 (4)  0.167 + 0.002 (5)  0.160 + 0.001 (5)  0.165 + 0.003 (4)  0.176 + 0.002 (4)  15.76 + 3.20 (3)  14.40 + 0.71 (4)  +  12.43 0.27 (4)  "(ID  +  11.02 0.45 (5)  +  12.11 0.16 (5)  +  Data presented as means ± S.E.M. Values in parenthesis are number of observations Differences between days 1-4 and 5-9 are s t a t i s t i c a l l y  11.39 0.94 (4)  +  10.98 0.39 (4)  7 33.50 + 1.25 (10)  8 34.64 + 1.26 "(4)  9 34.00 + 1.50  16)  -  0.901 + 0.02 " (4)  0.870 + 0.02 " (4)  -  1.66 + 0.03 ~ (4)  1.70 + 0.04 " (4)  0.173 + 0.002 " (4)  0.159 + 0.001 ~ (4)  10.48 + 0.10 " (4)  10.57 + 1.06 " (4)  —  —  s i g n i f i c a n t (P < 0.05).  

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