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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.Sc, University of B r i t i s h Columbia, 1974 M.Sc, University 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 this thesis 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 t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . A n i m a l S c i e n c e Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date Warch 1981 7Q> i i . Abstract The turnover, interconversion and oxidation of substrates in the ovine conceptus in utero were studied making use of isotope d i l u t i o n tech-niques. In Experiment 1, surgical techniques were standardized for the introduction of vascular catheters into the fetuses at approximately 120-130 days of gestation. Based on maternal and f e t a l blood acid base para-meters and metabolite and hormone levels i t was possible to obtain chronic f e t a l preparations which were physiologically stable. In Experiment I I , radioactive labelled substrates were injected introvenausly into the fetus and the disappearance of the label from the fe t a l c i r c u l a t i o n was monitored against time. Kinetic parameters of subs-trate metabolism were calculated by graphic analysis of the s p e c i f i c radio-activity-time 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 single injection technique employed in this study f a c i l i t a t e d the calcu-l a t i o n of 2 additional kinetic parameters not reported hitherto in the l i t e r a -ture. These include the mean total residence time and number of cycles the labelled substrates made before being i r r e v e r s i b l y l o s t from the fe t a l c i r c u l a t i o n . The finding that lactate and amino acids make more number of cycles into 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 fetal side i s r e l a t i v e l y impermeable to the former two substrates. Though the rapid disappearance of isotopes administered into the fetus was recognized by e a r l i e r workers, the results of this study have brought to l i g h t the significance of recylcing of substrates. It i s suggested that this unique dynamic feature serves as a physiological control mechanism to modulate fuel consumption according to nutrient 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 difference in the i r r e v e r s i b l e rate of disposal when [2- 3H] or [U-^C] glucose was injected indicating that there i s only approximately 12.5% of re c i r c u l a t i o n of glucose within the f e t a l tissues. The appearance in maternal c i r c u l a t i o n of only labelled glucose injected into the fetus, but not lactate indicates the i n a b i l i t y of lactate to cross the placenta from the f e t a l side. Though 36% of the administered glucose label appeared in lactate, the methodology used in th i s study does not d i f f e r e n t i a t e whether the conversion of glucose into lactate occurred in the fetus i t s e l f or in 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 isotopic 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 C O 2 after the continuous infusion of [^C] NaHCOg. The most important findings in th i s study pertains to the contribution of substrates to oxidative metabolism in the fetus. 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^ indicates that glucose, lactate, alanine, acetate and amino acids contribute 15.2, 14.0, 6.8, 1.7 and 8.2% respectively to f e t a l oxidative metabolism. Though the metabolism of placental tissues may have influenced the above values, the results suggest that the metabolic fuel requirements of the fetus warrant reassessment. The results of these experiments are discussed with reference to the metabolism of the fetus during a period of gestation where the greatest increment in f e t a l growth occurs. i v. TABLE OF CONTENTS Page T i t l e page i Abstract i i Table of Contents iv L i s t of Tables v i i L i s t of Figures ix Acknowledgements .xi Introduction 1 Review of Literature 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 corticosteroids 12 D. Insulin and glucagon 12 E. Hormonal enzyme induction 16 5. Fetal metabolism 17 A. Techniques for metabolic studies in the fetus 17 B. Fetal c a l o r i c requirements 19 i ) Oxygen consumption and carbon dioxide 20 production i i ) Glucose 23 i i i ) Fetal gluconeogenesis 26 iv) Fructose 32 v) Lactate 34 v i ) Amino Acids 36 v i i ) Carbon-nitrogen balance Experiment I; Surgical technique for the cannulation of f e t a l saphenous vein and post-surgical changes in blood parameters of the ovine fetus in utero. Introduction Materials and methods Results Discussion Conclusions Experiment I I : Substrate turnover and interrelationship in the ovine fetus in 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 oxidation by the ovine conceptus in utero using 14c-labelled compounds. Materials and methods Results Discussion Conclusions General Conclusions Bibliography Appendix Bibliographical Notes v i . page 131 1 3 7 1 5 8 v n L i s t of Tables Table. Page 1. Oxygen consumption rates of adults and fetuses 22 of different size. 2. Gestational age and body weights of ewes and fetuses. 7. Estimation of recycling and rec i r c u l a t i o n of glucose and lactate in the ovine fetus in utero. 8. Maternal and fe t a l physiological parameters during the experimental period. 10. Recycling of amino acids and alanine in the ovine fetus in utero. 11. Conversion of alanine to lactate and glucose in the ovine fetus i i i utero. 50 3. Maternal and fe t a l physiological parameters during the experimental period. 72 4. Kinetic parameters of glucose metabolism in the ovine fetus, estimated using single injection of a mixture of [U-14C] and [2-3H] glucose or[U-14c] glucose alone. 80 5. Kinetic parameters of glucose and lactate metabolism in the ovine fetus, estimated by using single i n j e c t i o n of [U-14c] glucose or [1-14C] lactate. 82 6. Glucose-lactate conversions in the ovine fetus in utero. 83 84 100 9. Kinetic parameters of substrate metabolism in the ovine fetus. 103 105 106 12. Substrate oxidation rates in the ovine fetus l i g i i i utero. v i i i . 161 Appendix Table. Page 1. Per cent recovery of 14fj-labelled compounds after treatment with glucose oxidase and anion exchange chromatography. 160 2. Per cent recovery of [1-14c] lactate following anion exchange chromatography and thin layer chromatography. 3. Per cent recovery of [U-14c] amino acid mixture and alanine following cation exchange chroma- 162 tography and enzymatic conversion of alanine to lactate. 4. Per cent recovery of O^C] NaHC03 in saline and whole blood. 163 5 . Mean P O 2 , P C O 2 and pH in maternal and f e t a l blood of conscious ewes during and following 1g 7 surgery. 6. Mean hematocrit, blood glucose, l a c t a t e , -hydroxybutyrate and alpha amino nitrogen in 153 fe t a l blood, following surgery. I X . L i s t of Figures Figure. Page 1. Post surgical changes in blood gas parameters and hematocrit in the ovine fetus in utero. 51 2. Post surgical changes in plasma metabolite levels in the ovine fetus in utero. 52 3. Post surgical changes in plasma Cortisol levels in the ovine fetus in utero. 54 4. Model of glucose and lactate metabolism in the ovine fetus. 56 5. Semi logarithmic plot of glucose s p e c i f i c a c t i v i t y versus time following injection of 74 [U-14c] glucose. 6. Semi logarithmic plot of glucose s p e c i f i c a c t i v i t y and lactate formation versus time following the injection of [U-14C] and [ 2 - 3 H ] ' 7 5 glucose. 7. 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 8. Linear regression of fe 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 9. Semi logarithmic plot of lactate s p e c i f i c a c t i v i t y versus time following the injection of [1-14c] 7 g lactate. 10. Model of amino acid metabolism in the ovine fetus. g 7 11. Semi logarithmic plot 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. Specific r a d i o a c t i v i t y of f e t a l and maternal blood 14C02 following 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 after single injection of NaHl4C03- 122 14. Specific r a d i o a c t i v i t y of blood 14C02 after injection of 14C-labelled substrates. 123 15. Relationship between rates of oxidation and i r r e v e r s i b l e disposal of glucose and lactate. 124 16. Composite picture of f e t a l substrate metabolism. 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 166 determined by infra-red carbon analyzer. x i Acknowledgements. I wish to express my gratitude to the many individuals that assisted me during the course of th 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 for 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 technician, whose expert assistance was called upon many times. I wish to express my sincere gratitude to Dr. C. R. Krishnamurti, Professor, Animal Science for his dedicated participation with the animal surgery and assistance with the preparation of th i s thesis. I am also grateful for his encouragement and friendship during my tenure in the department. I would l i k e to record my gratitude to Mr. G.J. Tompkins for his time spent in assisting me with animal surgery and experiments, and Ms. Madonna Chan for her conscientious help with animal experiments and laboratory work. I would l i k e to acknowledge Mr. G. Galzy, for his technical assistance and Mr. R. Burton (Pharmaceutical Sciences) for help with the computer programs. A sincere thankyou i s given to Ms. Sannifer Louie for her special attention in typing t h i s manuscript. I would l i k e to thank my family for t h e i r support and understanding during the preparation of th i s manuscript. F i n a l l y I wish to acknowledge my wife Elizabeth, whose patience and understanding, benefaction and devoted assistance enabled me to persue my objectives. This thesis i s dedicated to Elizabeth and my daughter Katharine Heather. Introduction 1. The growth of the ruminant fetus, p a r t i c u l a r l y during the l a t e r stages of gestation, has been the subject of extensive investigation and review. Since f e t a l metabolism has been shown to depend on a continuous supply of nutrients, the pregnant mother must undergo s p e c i f i c metabolic alterations to ensure that the f e t a l metabolic demands are met. Under normal pr a c t i c a l feeding conditions the maternal ruminant is capable of providing a n u t r i t i o n a l environment for the fetus which w i l l r esult in 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 in regard to i t s c a l o r i c requirements have not been considered u n t i l 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 catheterization procedure and isotopic tracer methodologies. In p a r t i c u l a r , the contribution of substrates towards f e t a l oxidative metabolism was examined. 2. Review of the Literature 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 available nutrients are divided between maternal and fe t a l tissues according to th e i r metabolic needs. The apparent p a r t i t i o n of nutrients for maternal maintenance and f e t a l growth requirements was postulated to be especially s i g n i f i c a n t when the nutrient supply was limited. The suggestions brought forth by Hammond were no doubt instrumental in the i n i t i a t i o n of numerous studies designed to examine the importance of adequate nut r i t i o n during pregnancy on conceptus birt h weights and fe 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). Nutritional stress imposed on the dam during early pregnancy has been shown to have a more profound direct effect on embryo mortality (Edey, 1976) and placental growth (Alexander, 1964) than on absolute f e t a l b i r t h weight (Hulet :et a l . , 1969). While a small amount of absolute fe t a l growth (e.g. tota l body growth) occurs during early pregnancy, the sp e c i f i c growth rate (e.g. individual organ growth) i s very high (16% per day, Robinson and McDonald, 1979). The placenta, on the other hand, i s an active l y growing tissue in early pregnancy. Robinson ejt al_. (1979) reported that a r e s t r i c t i o n in placental development beyond a c r i t i c a l threshold (e.g. 160 g.) resulted in s i g n i f i c a n t f e t a l growth retardation. Alexander (1964;1974) had previously reported high correlations 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 characteristics change s i g n i f i c a n t l y (Robinson et a l . , 1977a). Fetuses of ewes fed a diet below maintenance at time of conception were most vulnerable to maternal under-nutrition at this stage of gestation. 3. The fetus makes i t s greatest metabolic demands upon the ewe during the l a s t 8 weeks of pregnancy. Robinson et al_.(1977a) reported that at four and two weeks prepartum, 50 and 75 percent respectively, 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 in f e t a l b i r t h weight observed within common breeds has been attributed to varying levels of maternal n u t r i t i o n during late pregnancy (Wallace, 1948; Alexander, 1974; Mellor and Matheson, 1980). Reid (1968) and Koong et al_.(1975) have also reported that the number of fetuses present in utero affect f e t a l b i r t h weights more so than maternal under-nutrition at 115-120 days gestation. Mellor and Matheson (1980) reported a 30 to 44 per cent decrease i n f e t a l growth rates three days after the introduction of maternal under-nutrition. Data obtained from chronic f e t a l preparations have shown s i g n i f i c a n t changes in f e t a l metabolite and hormone concentrations during maternal under-nu t r i t i o n (Tsoulos et_ al_., 1971; Bassett and M a d i l l , 1974a; Mellor et a l . , 1977). Prior et a l . (1979) and Prior and L i s t e r (1979) demonstrated in the bovine, that although maternal metabolism was s i g n i f i c a n t l y affected by the r e s t r i c t i o n of dietary metabolizable energy to maintenance levels there was no s i g n i f i c a n t effect on f e t a l birthweight, f e t a l muscle protein or RNA and DNA content. A s i m i l a r r e s t r i c t i o n on the level of maternal dietary energy intake has been shown-to have l i t t l e e ffect on maternal and f e t a l glucone-ogenic enzymatic a c t i v i t y in v i t r o (Prior and Scott, 1977). Several investigators have documented the s p e c i f i c changes in maternal intermediary metabolism occurring during pregnancy (Bergman, 1963; Herrera et_ al_., 1969; Prior and Christenson, 1978). E a r l i e r studies have also shown that the ruminant i s susceptible to severe hypoglycemia and ketosis during the l a s t several weeks of pregnancy and in fact a reduction in food intake may reproduce most features of c l i n i c a l ketosis in the bovine and 4. pregnancy toxemia in 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 receiving an ade-quate plane of n u t r i t i o n do not appear substantially altered during t h i s time. Christenson and Prior (1978) reported no s i g n i f i c a n t changes in a r t e r i a l plasma glucose concentrations during gestation in sheep. S i g n i f i -cant decreases in maternal whole blood concentration of several amino acids have, however, been observed in sheep during t h i s time (Morriss et al_. 1979). These results are of particular interest in view of in vivo studies that have disclosed a maximal rate of maternal gluconeogenesis during the 3rd to 4th month of gestation (Prior and Scott, 1977). Curet et a]_. (1970) have attributed the decline i n alpha amino nitrogen levels observed during gestation to the high c i r c u l a t i n g levels of estrogen, progesterone and Cortisol in the pregnant animal. Numerous studies have demonstrated that the pregnant uterus during the l a t t e r stages of gestation consumes large quantities of glucose (Kronfeld, 1958; Bergman, 1963; Reid, 1968; Prior and Christenson, 1978). Morriss et al_. (1974) reported logarithmic increases in both uterine oxygen and glucose uptakes as gestation progresses. Significant increases in the uptake of glucose and alpha amino nitrogen by the pregnant uterus have been reported in 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 in pregnant and nonpregnant sheep, estimated that uterine glucose metabolism accounts for 20 to 40 percent of the t o t a l glucose turnover rate i n ewes with twin pregnancies. Setchell et al_. (1972) reported that 70per cent of maternal glucose turnover was accounted for by the uterus and i t s contents. Prior and Christensen (1978) 5. demonstrated further that the number of fetuses in 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 for 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 primarily by decreasing the plasma glucose concentrations. These results correspond d i r e c t l y with the increased maternal glucose entry rates and turnover time of the glucose pool in pregnant sheep (Bergman, 1964; Steel and Leng, 1968). Steel and Leng (1968) working with pregnant ewes fed ad 1ibitum, attributed the increase in glucose entry rates to voluntary increases in feed intake during pregnancy. Results concerning the u t i l i z a t i o n of meta-bolizable energy by the pregnant ewe and conceptus confirmed this conclusion (Rattray et al_., 1973). No s i g n i f i c a n t differences in the amount of metaboliz-able energy u t i l i z e d for body maintenance and conceptus development were established in singleton and twin pregnancies. However, s i g n i f i c a n t differences were noticed with the tot 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 meta-bolizable energy for development was s i g n i f i c a n t l y greater i n ewes with a level of n u t r i t i o n that was 2x maintenance le v e l s . 2. Placental functions Prior and L i s t e r (1979) reported that neither f e t a l , placental nor uterine weights correlate with cotyledon number, primarily because of the variable sizes of individual cotyledons. Further i t was shown that f e t a l , placental and uterine weights strongly correlated with cotyledon weights 6. re f l e c t i n g the potential for utero-placental compensatory growth. This con-clusion i s further exemplified with data from single and twin pregnancies and cotyledonary weights (Alexander, 1964). Although the number of cotyledons in placentas of single fetuses was greater than that of twin fetuses there was no si g n i f i c a n t difference in individual placental weights. Marked hi s t o l o g i c a l and morphologic changes occur in the placenta during gestation (Adherne and Dunnill, 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 fe t a l v i l l u s c a p i l l a r y system have been attributed to the increased a b i l i t y of the placenta to transfer nutrients to the fetus (Rosso, 1980). In addition, 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 for the regulation of placental substrate metabolism are important functional alterations 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 in c e l l u l a r growth of the placenta. This i s reflected by a s i g n i f i c a n t decline i n the instantaneous growth rate of placental, uterine and cotyledonary tissues at approximately 90 days gestation in the ewe (Alexander, 1964) and 200 days in the cow (Prior and L i s t e r , 1979). Functional aspects of the placental tissues increase however at t h i s time. Kulhanek et al_. (1974) demonstrated a f i v e - f o l d increase in the permeability to urea when expressed as a fra c t i o n of 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 factors. The factors controlling f e t a l intrauterine growth have been studied by many workers. Prior to the a v a i l a b i l i t y of chronically catheterized f e t a l sheep preparations, the majority of the information concerning intrauterine development was ob-tained 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 in a number of mam-malian 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 antiguity (Roberts, 1906; Huggett and Widdas, 1951; Langlands and Sutherland, 1968). This relationship of f e t a l weight and chronological age can be expressed in the form of a general formula: Wt 1 / 3 = * ( t - t Q ) where °< = growth rate, derived from slope of the growth curve t = gestation time constant after conception t = s p e c i f i c time constant reduced after conception The results of this 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 in the rate at which intrauterine growth proceeds in domestic animals. The changes in f e t a l crown-rump length (CRL) and weight of individual organs have also been studied extensively, 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 antero-posteriorly along the main axis of the fetus and ce n t r i p e t a l l y along the limb axis. Meller and Matheson (1980) re-ported a linea r relationship between f e t a l CRL and fe t a l weight after 100 days gestation; however, a cur v i - l i n e a r relationship was observed when the total gestational period was considered. This i s attributed to the markedly different rates at which individual organs grow and the continuous changes in f e t a l conformation (Wallace, 1948; Rattray et a l . , 1975). Richardson and Hebert 0978), with a limited number of fetuses, supported this conclusion with observations made on organs of the nervous system. Although the cube root of body and total organ weights gave a linear regression with f e t a l age throughout gestation, the cube root of the brain, cerebellum and spinal cord weights resulted in a sigmoidal trend. Completion of organogenesis in v i t a l organs such as those of the nervous system has been postulated to be the major factor 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 in mammalian fetuses (Winnick and Noble, 1965). Prior et al_. (1979) have reported s i g n i f i c a n t increases in total f e t a l DNA and DNA/protein and RNA/DNA ratios near the end of gestation in the bovine fetus. Hypertrophy as reflected by DNA/protein and RNA/DNA ratios 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. fe 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 resulting low c i r c u l a t i o n of growth hormone in fe t a l plasma (Wallace et al_., 1972). In this study i t was shown that isotopic labelled growth hormone when administered into the maternal c i r c u l a t i o n was not detected in f e t a l plasma. The pattern of OGH concentration during gestation i s triph a s i c with high con-centration at 100-110 days of gestation (Gluckman et a l . , 1979). Dramatic increases in the f e t a l c i r c u l a t i n g hormone concentrations occur in the la s t month of pregnancy to levels three times the concentrations at 100 days of gestation and ten times the concentra-tion found postnatally (Bassett et a l . , 1970). Bassett and Madill (1974b) attempted to determine the regulatory mechanism of f e t a l OGH secretion by infusing glucose continuously for a prolonged period of time. Although glucose i s known to depress OGH in adult sheep, these workers were unable to achieve the same result in the fetus. Liggins and Kennedy (1968) reported that tot a l hypophysectomy was associated with a retardation of somatic development, most notably in bone tissue. Underdevelopment of the thyroid and adrenals was also reported in th i s study, suggesting that the observed developmental retardation may be a result of hypo-thyroidism rather than hypophysectomy. No growth retardation has been reported in hypophysectomized porcine fetuses at 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 skeletal growth, but rather acts i n d i r e c t l y through a generation of intermediate hormones called somatomedins. Somatomedin 10. i s a peptide hormone reported to influence peripheral action of growth hormone and regulate f e t a l growth (Falkner et al.,1979). Hintz e_t al_. (1977) working with infants suffering from a protein-ca l o r i e deficiency reported low serum levels of somatomedin in spite of elevated levels of growth hormone. A similar observation regarding these two hormones has been reported by Robinson e_t al_. (1977b) in f e t a l sheep. Plasma somatomedin concentration in fetuses from ewes that had undergone endometrial carunculectomy was lower though normal concentrations of f e t a l growth hormone was observed. It would appear from results reported by Falkner et al_. (1979) in hypophysectomized and nephrectomized fetuses, that somatomedin a c t i v i t y in f e t a l sheep is regulated by similar mechanisms in the adult. No s i g n i f i c a n t changes in somatomedin a c t i v i t y was observed in control fetuses throughout the gestational period though reduced f e t a l somatomedin a c t i v i t y was reported in hypophysectomized and nephrectomized fetuses. In spite 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 relationship between f e t a l OGH and intrauterine weight changes i_n utero. The i s o l a t i o n and characterization of placental extract, known as ovine chorionic somatomammotrophin, (OCS) stimulated interest i n the area of placental regulation and f e t a l growth (Martal and Djiane, 1975). OCS i s a polypeptide hormone, sim i l a r to OGH in function and possesses both growth and lactogenic properties. This hormone i s secreted primarily in the maternal c i r c u l a t i o n , and i s predominantly active as early as day 16 in the trophoblast (Martal and Djiane, 1977), and reaches a maximum at 120 days of gestation. 1 1 . Martal (1978) reported that the sum of OGH and OCS i s closely related to f e t a l intrauterine weight changes. 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 and OCS) remain constant during the gestational period. Martal concluded from these results that OCS controlled f e t a l growth during the f i r s t half 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 half of gestation. Thyroid Hormones The thyroid axis of the f e t a l sheep i s active and inde-pendent of the maternal axis. Experiments i n thyroidectomized ovine fetuses have reported undetectable quantities of thyroid hormones, confirming that a maternal source does not contribute .to the basal levels of f e t a l plasma thyroxine concentrations (Hopkins et a l . , 1971; Erenberg et al_., 1973). Morphological changes in the f e t a l thyroid gland and detectable levels of thyroxine (T^) have been demonstrated as early as 50 days of gestation (Thornburn and Hopkins, 1973). The significnace of f e t a l thyroid hormones on f e t a l intrau-terine growth i s related to the stage of f e t a l maturity at b i r t h (Hopkins et a l . , 1972; Erenberg et al_., 1973). Thyroidectomized f e t a l lambs have shown s i g n i f i c a n t growth retardation 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 fe t a l growth without hepatic glycogen deposition and high mortality after bir t h (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 gluco-corticoids ensure optimal f e t a l maturation at b i r t h despite gestational age variation (Liggins, 1976). Jost (1961) f i r s t reported the importance of the adrenal c o r t i c a l hormones in inducing f e t a l l i v e r glycogen synthesis in the fe t a l rabbit. Dramatic increases in f e t a l plasma corticosteroids, most notably C o r t i s o l , in 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 relationship between the observed increase in f e t a l plasma Cortisol and hepatic glycogen content (Barnes et al_., 1978), which indicates that the adrenal cortex i s as essential organ in the control of hepatic glycogen storage in the ovine fetus. INSULIN AND GLUCAGON The f e t a l pancreas plays a v i t a l role in the metabolism of nutrients delivered to the fetus by the maternal organism. The precise role that i n s u l i n and glucagon play in regulating f e t a l growth has been studied extensively in the rat and sheep (Alexander et a l . , 1971 , 1972, 1973, 1976; Girard et aj_., 1973; Bassett et al_., 1973): Insulin and glucagon influence f e t a l growth i n d i r e c t l y by influencing the establishment of energy reserves (Milner, 1979) and consequently have been considered to 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), indicating that substrate metabolism i s regulated by f e t a l pancreatic a c i t i v i t y . Detectable levels 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 and Alexander et a]_. ,1971). 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 (Fiser et al_. ,1974) which in part explains the large c i r c u l a t i n g i n -su l i n to glucagon molar r a t i o reported by Girard et al_. (1974). A positive relationship exists between i n s u l i n secretion and glucose homeostasis (Bassett and Madill 1974a,b; Fiser et al_. 1974; Simmons et al_., 1978). Willes et al_. 0969) concluded from studies on ovine fetuses, 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 result 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 in response to a glucose infusion. Fiser e t a l _ . (1974) confirmed Alexander's original observa-tion 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 this response was s i g n i f i c a n t l y lower than in the adult. 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 in f e t a l plasma. Philipps 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 sensitive to alterations in f e t a l glucose concentrations. It 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 to maternal responses, though a s i g n i f i c a n t lag time existed before a response was noted. Similar i n s u l i n induced responses were observed in fetuses from fed and starved ewes (Schreiner et al_., 1980). Although maternal starvation was noted to cause a 50% reduction in f e t a l plasma glucose concentrations, the kinetics of f e t a l i n s u l i n secretion were not affected. The apparent biphasic secretory pattern of i n s u l i n reported by Philipps et_ al_. (1978), warrants the use of a continuous infusion technique rather than an acute injection of glucose for sustaining a physiological response to i n s u l i n . Similar inconsistencies in the l i t e r a t u r e regarding fructose and alanine as stimulators of f e t a l i n s u l i n secretion can be explained on the basis of different experimental protocols. Davis et al_. (1971) and Bassett and Madill (1974b) reported positive responses in f e t a l i n s u l i n secretion following fructose i n -fusions; however, t h i s result was not obtainable in a more recent study by Philipps et al_. (1978). S i m i l a r l y , Fiser et al_. (1974) reported no change in f e t a l glucose, i n s u l i n or glucagon levels following an acute injection of alanine to the fetus. Philipps et al_. (1980) employing a square wave infusion technique, reported an induced elevation of i n s u l i n , with maximal response 60 minutes after the s t a r t of the amino acid infusion. The biological 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 of i n s u l i n to the fetus and reported minimal decreases in the concentration of f e t a l plasma glucose, which led them to conclude that there was a lack of control in the rate of glucose u t i l i z a t i o n by the 15. fetus. Simmons et al_. (1978) infused i n s u l i n over a longer period of time and reported an increase in f e t a l glucose u t i l i z a t i o n , i n -dependent of any changes in umbilical blood flow, f e t a l oxygen con-sumption and placental clearance. This report disproved previous evidence reported by Alexander et al_. (1970), describing l i t t l e or no effect 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 related increase in a r t e r i a l venous differences of whole blood glucose and oxygen across the umbilical c i r c u l a t i o n during a sustained infusion of i n s u l i n to the ovine fetus. Fetal hypoxia was also noticed to occur in th 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 fe t a l tissues 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 re l a t i v e importance of glucagon may be of greatest significance during the immediate neonatal period. The sudden f a l l in plasma in s u l i n at bi r t h and the r i s e in glucagon are responsible for the triggering of glycogen mobilization 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 in order to maintain blood glucose le v e l s . No correlation appears to exi s t between f e t a l glucose and glucagon concentrations in the ovine fetus (Fiser et al_., 1974). This i s supported by results of Alexander et aj_. (1976) who also observed no apparent change in plasma glucagon concentrations following i n s u l i n induced f e t a l hypoglycemia. Failure to stimulate glucagon release by infusion of alanine in vivo has been reported in both the rhesus monkey fetus (Chez et al_., 1974) and the ovine fetus (Fisher et 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 s i g n i f i c a n t pancreatic release of glucagon with arginine infusions. Girard et al_. (1973) demonstrated in the rat fetus that exogenous noradrenalin stimulated glucagon and inhibited i n s u l i n release from the pancreas. These workers proposed that f e t a l stress induced at the time of birth was a potential triggering mechanism for the release of noradrenalin at the pancreatic nerve endings, and would in turn stimulate the release of glucagon and i n h i b i t the release of i n s u l i n . I t was further concluded that f e t a l rat pancreas does not respond to acute changes in blood glucose by increasing glucagon release, hence ruling out the p o s s i b i l i t y that postnatal hypoglycemia was a physiological stimulator of glucagon release. e) HORMONAL ENZYME INDUCTION Glucocorticoids, catecholamines and glucagon are potential stimulators of enzyme induction whereas i n s u l i n i s an active 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 acid metabolism (tyrosine amino-transferase E.C.2.6.1.5., Holt and Oliv e r , 1969) and carbohydrate metabolism (P.E.P. carboxykinase E.C.4.1.1.3.2., Kirby and Hahn, 1973) in s p e c i f i c mammalian species. Glucagon and catecholamines are positive stimulators of tyrosine aminotransferase a c t i v i t y and can reverse the effect of adrenalectomy in the rat fetus (Holt and Oliv e r , 1971). Catecholamines have also been reported to stimulate gluconeogenesis by stimulating phosphoenolpyruvate carbo-xykinase (Holt and Oliv e r , 1968). Significant enzyme induction following par t u r i t i o n in the neonate (Warnes et_ al_., 1977b) can be attributed to glucagon release triggered by elevated f e t a l catecholamines (Girard et al_., 1973). It i s thus evident that glucocorticoids, glucagon, i n s u l i n and catecholamines participate in a complex interplay which determines the process of enzyme induction in the f e t a l l i v e r and possibly kidney. Fetal metabolism a) TECHNIQUES FOR METABOLIC STUDIES IN THE FETUS The pregnant sheep has been a useful animal model for 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 exteriorized animal preparations. The delivery of a pregnant goat by caesarean section into a bath tub f i l l e d with warm saline enabled Huggett to measure a variety of f e t a l physiological parameters. Modification of this procedure was performed by Barcroft et al_. (1939) and Barcroft and Baron (1946) in attempts to elucidate further the various aspects concerning the metabolism of the fetus in utero. 18. In view of the obvious shortcomings of these procedures, primarily 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 umbilical perfusion of the placenta and Andrews e_t al_. (1961) using perfused l i v e r s attempted to main-tain a physiological environment for longer durations for experimental purposes. Alexander ejt al_. (1964) further modified these procedures by i s o l a t i n g the sheep fetus and connecting i t to the umbilical 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 for longer periods of time after separating i t from the placenta. Numerous aspects of f e t a l metabolism were investigated by Alexander and coworkers with this 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 physiological studies in 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 functional only for a short period of time and this necess-itated improvements to be made in the surgical procedures. Meschia et a l . (1965a) and Kraner (1965) reported procedures for chronic catheter-iz a t i o n of f e t a l blood vessels in unstressed animals for the purpose of sampling the fetus for longer periods of time. The question of how long one must wait following 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; Slater and Mellor, 1977; Kit 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 rely on blood gas and pH measurements to determine animal normality since metabolic correlates of the fetus are of equal concern. Further i t was suggested that a minumum of 5 postoperative days be given for f e t a l and maternal recovery from surgical trauma before experiments were i n i t i a t e d . Several sophisticated versions of these techniques are currently in use in studying f e t a l metabolism in the undisturbed physiological state, and consequently have led to the tremendous wealth of information on fe t a l physiology that has been generated from the ovine fetus in_ utero. FETAL CALORIC REQUIREMENTS The total f e t a l c a l o r i c requirement has been evaluated from calculations based on substrate and oxygen consumption measurements and the determination of c a l o r i c requisites for new tissue accretion, by bomb calorimetry (Rattray et al_.,1974; Battaglia and Meschia, 1978). A majority of the information available concerning f e t a l substrate u t i l i z a t i o n has originated 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. Battaglia and Meschia (1978) proposed that the f e t a l lamb oxidizes a mixture of carbohydrates and amino acids. Assuming that the c a l o r i c y i e l d of 1 l i t r e of oxygen necessary to combust this mixture in toto i s 4.9 kcal , the dai l y oxidative requirements of the ovine fetus consuming 6 to 9 mis oxygen/min/kg at STP was approxi-mated to be 56 kcal/day/kg. Rattray et a]_. (1974) disclosed 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 tissue was computed to approximate 32 kcal/g/day. The total 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 for oxidative purposes and 36% for tissue growth (Battaglia and Meschia, 1978). i ) OXYGEN CONSUMPTION AND CARBON DIOXIDE PRODUCTION Since the presence of oxygen i s essential for the oxidative metabolism of substrates, a tremendous amount of interest has been directed towards accurately defining 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 et a l . , 1978). Respiratory gases cross the placenta by simple diffusion (Faber, 1977) and the high a f f i n i t y for oxygen in f e t a l blood (Naughton et_ al_., 1963) i s common to a l l mammalian species. This i s attributed to the quantity and unique type of f e t a l hemaglobin (Metcalfe et a l . , 1972). These characteristics of f e t a l blood account for the differences in the carrying capacity and dissociation curves of maternal and fe t a l blood and f a c i l i t a t e the transfer of respiratory gases across the placenta. Notable differences between maternal and f e t a l Pcni ou (oxygen tension at which 50 percent saturation occurs at pH 7.4), have been reported in a number of mammalian species ( S i l v e r and Comline, 1975) and are largely responsible for the magnitude of the transplacental gradient for 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 interesting observation when con-sidering that a l l three species possess a similar epitheliochorial placenta (Silve r and Comline, 1975). Structural differences in the 21. arrangement of the placental vascular c i r c u l a t i o n are responsible for the large differences in maternal-fetal substrate gradients in these animals (S i l v e r et al_., 1973). The PG^ in f e t a l a r t e r i a l blood i s lower than in the ewe. This c h a r a c t e r i s t i c , together with the high f e t a l PCO^ and pH, previously led investigators to suggest that the fetus exists in a hypoxic and acidotic environment (Vaughn et al_., 1968). More recently, reports have disputed t h i s concept and strongly suggested that anaerobic metabolism does not play an important role in f e t a l metabolic a c t i v i t i e s (Battaglia and Meschia, 1978). Battaglia 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 a v a i l a b i l i t y . Furthermore, i t was shown that although maternal PO^ was altered by increasing the amount of oxygen, the increase was not proportionally transmitted to the fetus. 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 differences crossing the umbilical 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 application of the Fick p r i n c i p l e (Meschia et a l . , 1965b, 1967b; James et a l , 1972) Oxygen consumption i n chronically catheterized unanesthetized fetuses varies between 6 and 9 ml/min/kg fetus. This accounts for approximately 60% of the tot a l uterine uptake of oxygen (Meschia _et j f L , 1980). An inverse relationship exists between the arterial-venous difference of oxygen and the blood flow in the uterine and umbilical 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 fluctuations 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 unit body weight i s greater than the basal level of the adult (Table 1). Battaglia 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 in species that d i f f e r in body siz e . This is contrary to what i s true in adult animals (Kleiber, 1947). TABLE 1: OXYGEN CONSUMPTION RATES OF ADULTS AND FETUSES OF SPECIES OF DIFFERENT SIZE* Animal Oxygen Consumption (ml/min/kg BWT) Adult Fetus Horse 2.0 7.0 Cattle 2.2 7.4 Sheep 4.0 6.9 Rhesus Monkey 7.0 7.0 Guinea Pig 9.7 8.5 * from Battaglia and Meschia (1978) Similar approaches have been taken to assess f e t a l C O 2 production rates across the placenta in chronically 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 5.65 ml/min/kg by fetus. Single fetuses yielded 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 fetus) and the time following surgery before experiments were i n i t i a t e d appeared s i g n i f i c a n t . No s i g n i f i c a n t changes are observed in the f e t a l blood P C 0 2 > pH or bicarbonate levels at d i f f e r e n t stages of gestation in sheep (Comline and S i l v e r , 1972). Fetal P C 0 2 levels in the f e t a l artery are higher than maternal arterial P C 0 ? and can 23. change in direct proportion to maternal PCO,,. The pH of fe 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 similar (Meschia et a l . , 1970). GLUCOSE Huggett et a[. (1951) f i r s t reported the transfer of monosaccharides across the ovine placenta and demonstrated the preferential permeability of the placenta to glucose. Calculations made by Widdas (1952) on the data of Huggett et al_. (1951) indicated that the transfer of glucose across the placenta of sheep could not be explained by simple di f f u s i o n mechanisms alone. The chemical and stereo-specific characteristics regulating placental transfer of mono and disaccharides were examined by Walker (1960). It i s now believed that the transfer 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 in the erythrocyte (Widdas 1961; Boyd et al.,1976). Simmons et al_. (1979) recently concluded from determinations on the rate of placental glucose transfer in chronically catheterized fetuses, that the rate of glucose transfer was not only limited by transport characteristics but was also affected by the u t i l i z a t i o n rate of glucose by placental tissues. I t was not u n t i l the advent of chronically catheterized f e t a l preparations that the relationship between f e t a l and maternal glucose concentrations was accurately assessed (James et al.,1972, Shelley, 1973; Bassett and M a d i l l , 1974a). The difference between f e t a l and maternal concentration i s common to 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 attributed to a combination of placental tissues ( S i l v e r , 1977; Shelley, 1979). Data obtained from the cow, sheep and horse have indicated that the placental morphology influences the f e t a l to maternal glucose gradients (Silver et a l . , 1973). Although a l l three species possess similar 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, resultant of i t s vascular morphology, i s responsible for the low maternal to f e t a l substrate gradients reported in the ruminant (Silver et a l . , 1973). The metabolism of glucose by the placenta i s an additional factor controlling f e t a l glucose metabolism. Alexander et al_. (1955) demonstrated in exteriorized, perfused ovine fetuses a s i g n i f i c a n t proportion of fructose was derived from glucose by placental tissues. Battaglia et al_. (1961) further demonstrated that ovine placental cotyledons were potential 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 in the ovine uterus than the fetus alone, and Setchell et al_. (1972) suggested that a considerable amount of glucose removed by the uterus was not oxidized but rather could be used for synthetic purposes. The relationship between f e t a l glucose uptake and maternal concentration of glucose has also been studied in d e t a i l . Alexander et al_. (1955) f i r s t disclosed that an a r t i f i c i a l eleva-tion of maternal glucose concentration resulted in an increased transfer of glucose to the fetus. Later studies by Alexander et a l . (1969), in exteriorized fetuses, showed that glucose uptake by the fetus was approximately 6 mg/min/kg fetus, similar to reported values in 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 t h a t u m b i l i c a l u p t a k e v a l u e s o f g l u c o s e f rom c h r o n i c a l l y c a t h e t e r i z e d o v i n e f e t u s e s r a n g e d f rom 1 t o 6 m g / m i n / k g f e t u s 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 between u m b i l i c a l g l u c o s e u p t a k e and m a t e r n a l a r t e r i a l g l u c o s e c o n -c e n t r a t i o n s . C o m p a r a t i v e s t u d i e s i n t h e cow, s h e e p , and h o r s e have 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 between m a t e r n a l a r t e r i a l 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 t r a n s f e r r a t e o f g l u c o s e t o t h e f e t u s (Boyd e t a l_. , 1973 ; C o m l i n e and S i l v e r , 1976). T h e r e w o u l d a p p e a r t o be an uppe r l i m i t t o t h e amount t r a n s f e r r e d t o t h e f e t u s f rom t h e m o t h e r , as e v i d e n c e d f rom d a t a p u b l i s h e d on t h e b o v i n e f e t u s ( C o m l i n e and S i l v e r , 1976). I t has a l s o been shown i n e x p e r i m e n t a l l y i n d u c e d m a t e r n a l h y p e r g l y c e m i a , t h a t f e t a l g l u c o s e u p t a k e w i l l p l a t e a u t o 40-60% o f t h e b a s a l l e v e l s ( S h e l l e y 1973; S i l v e r e t a l _ , 1973). No n e t change i n f e t a l g l u c o s e u p t a k e o r m a t e r n a l - f e t a l g l u c o s e g r a d i e n t s i s o b s e r v e d w i t h a d v a n c i n g g e s t a t i o n , a l t h o u g h t h e r e a r e n o t a b l e i n c r e a s e s i n n e t f e t a l oxygen c o n s u m p t i o n and u m b i l i c a l b l o o d f l o w (Boyd e t aJU 1973). The l e v e l o f h y p o x i a , as shown i n s t u d i e s w i t h s p o n t a n e o u s hypoxemic lambs a l s o a f f e c t s f e t a l g l u c o s e and t r a n s p l a c e n t a l g l u c o s e g r a d i e n t s ( C h a r and C r e a s y , 1977). W i t h t h e o n s e t o f m a t e r n a l h y p o g l y c e m i a t h e r e i s a d r a m a t i c f a l l i n b o t h t h e m a t e r n a l and f e t a l g l u c o s e c o n c e n t r a t i o n s , t o 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 (Boyd e t aJU 1973; S c h r e i n e r e t a l * 1978; Anand e t a j_ , 1980). The m a t e r n a l - f e t a l a r t e r i a l g l u c o s e c o n c e n t r a t i o n g r a d i e n t a l s o f a l l s s i g n i f i c a n t l y , and c o n s e q u e n t l y l e a d s t o a r e d u c e d t r a n s f e r r a t e o f g l u c o s e t o t h e 26. fetus (Schreiner et al_., 1978). Schreiner et al_. (1978) proposed that the decline in 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 to restore the glucose con-centration gradient across the placenta and thus increases the umbilical glucose uptake towards normal levels observed in the nonstarved state. Further, the fetus reduces i t s consumption of exogenous glucose as evidenced by the reduction in plasma i n s u l i n levels 2 days after maternal fasting (Bassett and M a d i l l , 1974b). FETAL GLUCONEOGENESIS Whereas hepatic gluconeogenesis i s the major source of blood glucose in the adult ruminant (Bergman, 1973), the s i g n i f i -cance of gluconeogenesis in the fetus, i s not t o t a l l y resolved. 14 14 Incorporation of U- C pyruvate into C glucose was o r i g i n a l l y reported in 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 in f e t a l rats (Ballard and Oliver, 1965). The a c t i v i t y of gluconeogenic enzymes in the fe t a l rat i s low (Ballard and Hansen, 1967) and although phospoenopyruvate carboxykinase (PEPCK) a rate l i m i t i n g enzyme, i s inducible prior to par t u r i t i o n with glucagon, the overall pathway i s not detectable (Phi l l i p p i d e s and Ballard, 1969). The key regulatory enzymes of gluconeogenesis are present in substantial a c t i v i t i e s in f e t a l sheep l i v e r (Stephenson e_t aj_., 1976; Warnes et_ ah, 1977b) and f e t a l kidney cortex (Stephenson et a l . , 1976). Prior to 100 days of gestation there i s a limited in v i t r o pro-duction of endogenous glucose due to the low levels of fructose 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 cytosolic 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 levels j u s t p r i o r to p a r t i t i o n (Stephenson et al_., 1976; Warnes et al_., 1977b; Prior and Scott, 1977). Prior and Scott (1977) further demonstrated the capacity for gluconeogenesis i n the bovine fetus as early as 88 days of gestation. These workers concluded that a r e s t r i c t i o n in maternal dietary energy during late gestation does not s i g n i f i c a n t l y a l t e r maternal or f e t a l gluconeogen a c t i v i t y . Swiatek (1971) reported that both pyruvate carboxylase (PC) and PEPCK were l i m i t i n g factors in the absence of gluconeogenes in the neonatal pig. Jones and Ashton (1976) demonstrated in v i t r o that the f e t a l guinea pig has a functional gluconeogenic pathway in both l i v e r and kidney tissues, 10 days prior to b i r t h . Two aminotransferases, glutamate-oxaloacetate aminotransferase and glutamate-pyruvate aminotransferase are active i n bovine feta l l i v e r and kidney tissues as early as 45 days of gestation (Stephenson et al_., 1976). Edwards et al_. (1975) reported marked increases i n glutamate pyruvate aminotransferase a c t i v i t y in f e t a l heart muscle with advancing gestation. The incorperation of gluconeogenic precursors into 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 in v i t r o . Spontaneous gluconeogenesis from serine, 28. glycerol and to a lesser extent alanine and lactate was observed in cultured f e t a l rat l i v e r (Coufulik and Monder, 1976). Simkins et al_. (1978) reported a s i m i l a r result with galactose added to the medium and further demonstrated that glucocorticoids were potential stimulators of gluconeogenesis from galactose. Results obtained from Prior 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 lactate 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 after mid gestation. Although numerous in v i t r o studies have indicated the presence of a functional gluconeogenic pathway in the ruminant and nonruminant f e t a l species, the extent to which this capacity i s manifested in 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 gestation, although umbilical blood flow and f e t a l oxygen consumption increase during t h i s time. These workers, therefore, hypothesized the presence of f e t a l gluconeo-genesis. Hay (1979) further speculated that f e t a l gluconeogenesis could raise f e t a l a r t e r i a l blood glucose concentrations to a level that would reduce umbilical glucose uptake. Hodgson and Mellor (1977) estimated from disposal rates of labelled glucose, that 60-82% of glucose requirement was potentially accounted for 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 ratios of glucose following the continuous infusion of labelled glucose separately into f e t a l and maternal c i r c u l a t i o n s . The result was similar 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. Prior and Christenson (1977) reported that alanine accounted for 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 for f e t a l sheep (Lemons et al_.» 1976). Warnes et al_. (1977a) u t i l i z i n g single injection of isotopic tracers f a i l e d to show any gluconeogenesis from lactate in the f e t a l lamb. Recently Prior (1980), employing a continuous infusion of r a d i o l a b e l e d l a c t a t e , was successful in 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 active gluconeogenic precursor in the newly born lamb (Warnes et al_., 1977b). Although i t would appear that there i s a potential for f e t a l gluconeogenesis in most species with the exception of the ra t , suggestions have been made that there are additional factors regulating gluconeogenesis in utero. Warnes et aj_. (1977b) attributed the low f e t a l a r t e r i a l PO^, and resulting redox potential of l i v e r mitochondria (Williamson et al_, 1967), to be a potential regulator of gluconeogenesis. Supporting t h i s theory was the appearance of active gluconeogenesis from lactate 2 minutes following natural b i r t h of term lambs. Jones and Ashton (1976) suggested 30. that high PO^ of incubation media stimulated gluconeogenesis in v i t r o in 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 in the phosphoenolpyruvate/pyruvate r a t i o . The relationship between oxygenation and gluconeogenesis i s also indicated by the in 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 indirect estimation of the f e t a l respiratory 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 th i s conclusion (Alexander et al_., 1966). However, because the simultaneous measurement of f e t a l oxygen consumption was not per-formed, the f e t a l respiratory quotient was not made in this study. James et al_. (1972) determined the oxygen consumption and CO^ production in chronically catheterized, unanaesthetized fetuses and reported that the f e t a l respiratory quotient was s i g n i f i c a n t l y less than one. Meschia and coworkers developed another procedure for identifying substrates u t i l i z e d by the ovine fetus. Since f e t a l oxygen consumption i s an absolute prerequisite for substrate oxidation, an expression re l a t i n g the simultaneous uptake of spec i f i c substrates and oxygen by the f e t a l umbilical c i r c u l a t i o n was formulated. This expression was referred to as the substrate oxygen quotient (Tsuolos et al_.,1971; Battaglia and Meschia, 1973). 31. 1) Substrate Quotient = n x V ~ A substrate _ n x umbilical uptake of substrate V-A oxygen umbilical uptake of oxygen where n = number of moles of oxygen required for complete oxidation of substrate to CC^ and water. This dimensionless quotient represents the fraction of oxygen consumption accounted for by the complete aerobic oxidation of a substrate crossing the umbilical 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 in the cow (Comline and S i l v e r , 1976) and 0.68 in the horse (S i l v e r and Comline (1975). This supports the result obtained e a r l i e r in sheep that glucose uptake, although an important component of f e t a l metabolism, does not account for the tota l oxygen consumption by the fetus. The wide range observed in the ewe i s attributed to the variable levels of dietary energy intake of pregnant ewes in different 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 starvation 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 rapidly during the f i r s t 2 days of maternal starvation and remain constant thereafter. The decrease in placental transfer of glucose during maternal fasting 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 in individual f e t a l organs have demonstrated a s p e c i f i c i t y for substrate oxidation. Tsoulos et al_. (1972) estimated a cerebral glucose-oxygen quotient of 1.06, indicating that glucose i s the sole source of energy u t i l i z e d by the f e t a l brain. Under normal 32. conditions the cerebral glucose metabolism w i l l account for 15% of the f e t a l umbilical glucose uptake (Jones et al_., 1975). Morriss et al_. (1973) reported a si m i l a r glucose oxygen quotient for the f e t a l hind limb and concluded that although the hind limb was less active metabolically than the f e t a l brain, glucose was the pre-dominant substrate oxidized. I t was further concluded from these studies that glucose uptake by individual organs i s regulated by f e t a l a r t e r i a l glucose concentrations and in the case of the f e t a l hind limb, glucose uptake exceeded the requirements of oxidation. Reviewing the l i t e r a t u r e on the subject, Hay (1979) concluded that glucose u t i l i z a t i o n by individual organs was dependent on an assortment of factors, 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 , oxidation and overall f e t a l growth. FRUCTOSE In several mammalian species (sheep, cow and pig) fructose i s the principal carbohydrate in f e t a l blood, and i s present in quantities 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 , 1976). Bacon and Bell (1948) f i r s t i d e n t i f i e d fructose in blood of f e t a l sheep and showed that i t existed with glucose during the gestation period. Goodwin (1952) demonstrated fructose to be present in the blood of un-gulata, but absent in the blood of carnivora or rodents. Hitchcock (1949) observed a gradual disappearance of fructose from the lamb c i r c u l a t i o n , within 36-72 hours of b i r t h . Huggett et al_. (1951) proposed from data obtained from exteriorized f e t a l sheep preparations 33. with intact umbilical 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 after 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) further demonstrated that the passage of glucose from mother to fetus resulted in the formation of fructose and occurred at normal blood sugar concentrations. Glucose infused d i r e c t l y into the f e t a l c i r c u l a t i o n i s actively 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. Setchell et aJL (1972) chromatographed blood from f e t a l lambs that were infused with labelled fructose and reported a c t i v i t y in the glucose molecule in blood obtained from the umbilical vein, and f e t a l heart, but not from the umbilical artery. Conversely, Warnes et a l . (1977a) and White et al_. (1979) f a i l e d to observe this 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 fetus. This conclusion i s p a r t i c u l a r l y evident during cases of maternal starvation, when f e t a l fructose levels f a l l to 43% of the pre-starved levels (Tsoulos et a l . , 1971; Schreiner et al 1978). Meschia and Battaglia (1978) have speculated from these results that fructose acts as a glucose reserve, especially during periods of maternal starvation and hypoglycemia. No detectable umbilical uptakes of fructose (Tsoulos et a l . , 1971), glycerol and f a t t y acids (James et al_., 1971) and ketone bodies (Morriss e_t al_.j 1974) have been reported, thus demonstrating that these substrates are not major metabolic fuels of the sheep fetus under normal conditions. 34. LACTATE Fetal lactate 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 fe t a l and placental tissues (Char, and Creasy, 1976a; Demigne and Ramesy,1979). Significant umbilical venous-arterial differences of lactate, however, have been reported in ovine (Burd, et al_., 1975; Char and Creasy, 1976a) and bovine (Comline and S i l v e r , 1976) fetuses, indicating that lactate 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 differences, indicating 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 correlations between f e t a l umbilical and a r t e r i a l lactate and pyruvate concentrations with maternal a r t e r i a l l e v e l s . I n i t i a l l y i t was believed that the high feta l lactate level observed in f e t a l plasma were the result of anaerobic metabolism. However, the appreciable umbilical uptake of lactate suggests the fetus i s a consumer rather than a producer of lactate (Burd et a l . , 1975; Char and Creasy, 1976a; Comline and S i l v e r , 1976). The large lactate-pyruvate ratios observed in ovine (Burd et al_., 1975; Char and Creasy, 1976a), and bovine (Demigre and Ramesy, 1979) fetuses are similar to maternal lactate-pyruvate ratios i n -dicating that lactate i s not produced in excess by the fetus. The source of lactate available for f e t a l u t i l i z a t i o n remains questionable. Sign i f i c a n t correlations reported between 35. f e t a l a r t e r i a l lactate and maternal a r t e r i a l lactate concentrations suggest that maternal transfer of lactate occurs in utero. Demigne Ramesy (1979), however, observed that maternal hyperlactemia did not influence f e t a l lactate levels. This result confirmed a s i m i l a r observation which showed no transfer of lactate from fetus to ewe under hypoxic conditions (Britton et al_., 1967). However, Char and Creasy (1976a) reported positive uterine venous-arterial differences, thereby indicating that the sheep placenta deposits lactate into 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 lactate molecules following the injection of U- C glucose into the f e t a l c i r c u l a t i o n . The rate of i r r e v e r s i b l e disposal of lactate was si m i l a r to that of glucose and suggests that placental metabolism as well as f e t a l metabolism of this substrate occurred. Comline and S i l v e r (1976) previously reported the net lactate production by uteroplacental tissue in bovine fetuses was a function of the rate of glucose u t i l i z a t i o n by the uterus. It was demonstrated previously by V i l l e e (1954) that the amount of lactate produced by f e t a l tissue s l i c e s in 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 tissue produced more lactate than brain and l i v e r , and the amount of lactate produced was not s t r i c t l y dependent on the type of substrate employed for incubation purposes. Burd et al_. (1975) demonstrated from simultaneous measure-ments of umbilical uptake of lactate and oxygen that the placenta produced lactate in s u f f i c i e n t quantities to account for approximately 25% (range 0-79%) of f e t a l oxidative metabolism. The consumption 36. of lactate by the fetus in utero was confirmed by Char and Creasy (1976a) (32% in ovine fetus) and Comline and S i l v e r (1976), (43% in bovine fetus). Since the production of placental lactate i s a function of the high ma-ternal glucose entry rates reaching the uterus (Setchell et a l . , 1972), discrepancies reported in the stoichiometric measurements of lactate-oxygen quotients can be attributed to d i s s i m i l a r maternal glucose concentrations. Schreiner et al_. (1978) demonstrated this with s i g n i f i c a n t decreases in the f e t a l lactate-oxygen quotients obtained from fasting ewes. Pyruvate, on the other hand, i s not taken up by the fetus and, therefore, makes no contribution as a f e t a l oxidative substrate (Char and Creasy, 1976a). Acetate, an important v o l a t i l e f a t t y acid in adult ruminant metabolism, i s taken up by the umbilical c i r c u l a t i o n in s i g n i f i c a n t quantities and accounts for 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 respiratory quotient for acetate (16%) in the bovine fetus. AMINO ACIDS The ovine fetus i s a rapidly growing organism between 100 and 147 days of gestation, increasing in 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 rat carcasses that the amino acid composition of f e t a l protein was si m i l a r in proportions as weaning and adult carcass protein content. It has been well established that the primary source of nitrogen for 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 umbilical c i r -culation reaches the fetus in quantities s u f f i c i e n t for adequate growth (Young and McFayden, 1973 and Lemons et al_., 1976). Com-parison of amino acid content of lamb carcasses together with placental transfer of neutral amino acids led Lemons et al^. (1976) to conclude that some amino acids are transferred in quantities in excess of the growth requirements. The requirements of lysine and h i s t i d i n e are, however, sim i l a r to respective placental transfer rates. Umbilical venous-arterial concentration differences of 22 amino acids in 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 actually somewhat greater than the amount of nitrogen required by the fetus for growth and urea production (1.0 g/kg/day), (Lemons et al_., 1976, Holzman et a]_., 1979). Numerous studies have attempted to identify and c l a r i f y the mechanisms that regulate amino acid transfer 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 levels of individual 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 similar observation has been reported with f e t a l plasma alpha amino nitrogen concentrations (Alexander et a l . , 1970b). 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 for the acidic amino acids. The fetal-maternal concentration gradients are r e l a t i v e l y higher for the straight chain amino acids. Young and McFayden (1973) have suggested that the high amino acid levels 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 tissues and the r e l a t i v e differences in f e t a l organ s i z e , as compared to the mother. These workers also concluded that a constant transfer of amino acids occurred independently of maternal plasma concentrations. Holzman et al_. (1979) recently demonstrated a strong correlation between amino acid umbilical venous-arterial differences and arterio-venous differences across the uterine c i r c u l a t i o n . Furthermore, the arterio-venous differences of neutral and basic amino acids were related to the maternal a r t e r i a l concentration. Amino acid uptake by the placenta i s mediated by d i f f u s i o n and active (Na pump and oxygen dependent) transport systems. H i l l and Young (1973) demonstrated in the guinea pig, that transfer of amino acids from placental parenchyma to f e t a l plasma was blocked when f e t a l plasma amino acid concentrations exceeded the free amino acid concentrations of the placental parenchyma. Longo et al_. (1973) reported that placental amino acid transport occurs under anaerobic conditions, and i s dependent upon glycogenolysis. Christensen and Streicher (1948) f i r s t demonstrated that placental transfer of i n -dividual amono acids was s p e c i f i c to groups of amino acids. This concept was confirmed in part by Hopkins et al_. (1971) who reported that neutral, branched chain amino acids belonging to the "L" pre-fer r i n g transport system were transferred readily across the placental 39. membrane. Enders et al_. (1976) attempted to c l a r i f y the mechanisms that regulate amino acid transfer from the placenta, by determining the s p e c i f i c i t y of the principal placental transport systems. Three transport systems for neutral amino acids were established in 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 identify 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 total amino acid uptake was represented by eight neutral amino acids (alanine, threonine, serine, valine, leucine, isoleucine, glutamine, and proline). Although glycine i s present in very high concentrations and exhibits abnormally large venoarterial differences, i t i s not taken up by the uterus in s i g -n i f i c a n t quantities (Morriss ejt al_., 1979; Holzman et al -, 1979). Twenty percent of to t a l amino acid uptake was accounted for by basic amino acids, l y s i n e , h i s t i d i n e , and arginine. Lemons et aj_. (1976) reported no s i g n i f i c a n t umbilical uptake of acidic amino acids, glutamate and aspartate, but observed a net flux of glutamate out of the fetus into the placenta, indicating de novo synthesis by fe t a l tissues. Smith ejt al_. (1977) concluded from umbilical venous a r t e r i a l differences of amino acids that f e t a l tissues 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 for 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 difference of several amino acids and urea, the determination of the umbilical 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 acid catabolism in 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 et al_., 1972a; Battaglia and Meschia, 1973). 2) Clearance of urea = Excretion_rate of urea aurea urea where a u r e a = f e t a l a r t e r i a l urea concentration ^urea = m a ' t e r n a l a r t e r i a l urea concentration The rate of ureogenesis in the ovine and bovine fetus deter-mined by t h i s procedure was 0.54 mg/min/kg and 0.21 mg/min/kg and accounts for 25 and 9% of the f e t a l oxygen consumption respectively (Gresham et al . , 1972a; Comline and S i l v e r , 1976). These comparative differences in the per cent of oxygen consumed for amino acid catabolism have been attributed to the lower rate of amino acid breakdown in the bovine fetus (Comline and S i l v e r , 1976). Simmons et aj_. (1974) re-ported that amino acid catabolism in the ovine fetus was the i n i t i a l adjustment of the fetus to maternal starvation and at the peak of urea production could account for 80% of the f e t a l oxygen consumption. The r e l a t i v e contribution to total oxidative metabolism by a mixture of f e t a l fuels has recently been investigated (Shambaugh et al_., 1977a,b). Evidence resulting from in v i t r o studies with the f e t a l rat indicated a competitive oxidative interaction of s p e c i f i c f e t a l substrates. An alte r a t i o n in f e t a l fuel mixture due to maternal fasting decreased the r e l a t i v e C0£ production from glucose and lactate to 45 and 75% respectively of the o r i g i n a l values. Shambaugh et a l . (1977 a,b) have further demonstrated that the addition of betahydroxy-butyrate to the incubation medium resulted in a s i g n i f i c a n t reduction 41. in the C0 2 produced from lactate and glucose. I t was concluded from these studies that the preferential oxidation of substrates i s determined by the ambient concentration of fuels rather than by an i n t r i n s i c adaptation of the tissue. v i i ) CARBON-NITROGEN BALANCE The measurement of umbilical substrate uptake coupled with f e t a l carcass analysis enabled investigators to estimate the carbon and nitrogen balance of the near term fetus and the contribution of individual substrates to t h i s balance. Fetal growth rate j_n utero i s approximately 35 g/kg/day (Gresham et 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 in the fetus was estimated to be 3.15 g C/kg/day (James et al . , 1972). Carbon excreted as carbon dioxide and urea amounted to 4.38 gC/kg/day and 0.15 gC/kg/day respectively, representing 60% of the total umbilical carbon flux crossing the placenta to the f e t a l lamb (James ejt al_., 1972 and Battaglia and Meschia, 1973). The r e l a t i v e d a i l y amount of substrate carbon contributing to the total carbon fl u x has been estimated from umbilical substrate uptake measurements. Amino acids, 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; Battaglia and Meschia, 1973). The contribution of carbon by glucose (1.76 gC/kg/day; James et al_., 1972), lactate (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 contribution of amino acids, glucose, lactate, and acetate to the tot a l carbon balance of the fetus i s 41%, 23%, 15%, 9% respectively and totals approximately 88% of the f e t a l carbon balance. This value therefore indicates that other substrates,possibly under sp e c i f i c environment conditions,cross the sheep placenta in s i g n i f -icant quantities. 43. Experiment 1 Surgical technique for the cannulation of f e t a l saphenous vein and post-surgical changes in 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 physiological studies in unrestrained animals was demonstrated by early workers in t h i s f i e l d (Meschia et al_., 1965a). The techniques and com-pl e x i t i e s of intrauterine f e t a l surgery have been reviewed by Kraner (1965), and Mellor and Slater (1973) in sheep and Randall (1977) in pigs. Successful cannulation of the f e t a l aorta and vena cava (Comline and S i l v e r , 1972), carotid artery and jugular vein (Bassett and M a d i l l , 1974b) and umbilical 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 physiological studies, i t i s essential to have chronic f e t a l preparations which have recovered from the stress of surgery. Mellor and Slater (1973) have stated that the results of experi-ments done on chronically catheterized fetuses should be interpreted with caution and emphasized that the delivery of full-term lambs following surgery i s not absolute proof of intrauterine normality. The minimum time that should elapse before experiments may be undertaken would depend upon the extent of stress imposed during surgery and the maintenance of the patency of vascular catheters. In this experiment, the technique of ovine f e t a l surgery was standardized, and the post-surgical status of the fetus was monitored da i l y to determine the time required for the fetus to return to stable levels. Changes in blood acid-base parameters and metabolite and Cortisol concentrations in plasma were measured as c r i t e r i a of intrauterine f e t a l normality. 44. Materials and Methods Animals Twenty-five, 1-2 year old pregnant Dorset Horn and Suffolk ewes, weighing 50 to 70 kg were used i n th i s study. The estrous cycles in these ewes were synchronized by the application of progestagen-impregnated intravaginal pessaries (Synchromate; G.D. Searle and Co., Chicago). Intravaginal sponges containing 800 mg progesterone (Sigma, St. Louis) were also used in some ewes. At 90 to 100 days after mating, an ultrasonic detector (Sheepreg, Animark) was used to confirm pregnancy. The ewes were housed in individual pens and fed 650 g of a l f a l f a cubes (19.6% crude protein; 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 respectively. Water and s a l t were made available at a l l times. Neonatal lambs were rubbed dry of after b i r t h moisture and weighed as soon as possible after b i r t h . Crown to rump measurements were also taken at this time. 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 for 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 Plus, 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 prior to surgery. Atropine sulphate (BDH Parmaceuticals) was administered subcutaneously (0.06 mg/kg) 15 minutes prior 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 main-tained with Halothane (Fluothane, Ayerst) at a concentration of 1.0-1.75% in a closed system. The anesthetized animal was positioned in a supine position 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 aspiration of sali v a or ruminal f l u i d s . The abdominal area was disinfected with surgical soap (Surgidine, Ingram and Bell Ltd.) followed by Tincture of Zephiran, and covered with s t e r i l e drapes. An intravenous drip of Ringer's solution was administered to replace loss of electrolytes 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) starting below the umbilicus and terminating close to the upper margin of the mammary gland was made. After inci s i n g the peritoneum the gravid uterus was palpated to determine the position of the fe'tus. The appropriate segment of the uterine horn con-taining the f e t a l hind limbs was brought through the abdominal in c i s i o n with as l i t t l e handling as possible. An assistant 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 least vascular area of the uterine wall exposing the uterine 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 in such a position 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 dissection and freed of adjacent f a s c i a . S t e r i l e 2% lidocaine (Sterilab) was sprayed on the region to reduce spasms. Fetal catheters were made of polyethylene tubing (0.86 mm i.d. x 1.27 mm o.d.) and enclosed in a polyvinyl c o l l a r designed to include s i l k ligatures used to anchor the catheter to the leg 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 saline (0.15 M) containing heparin (100 U/ml). S i l k ligatures were passed beneath the cleared area of the vein at the proximal and d i s t a l ends of the inci 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. A small opening was made on the wall of the vein and a cannula was introduced 18 to 20 cm proximally from the point of entry into the vein. The s i l k ligatures (2/0 braided s i l k , Ethicon) on the c o l l a r were anchored to the fascia on either side at the point of insertion of the cannula. Additional 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 to place the fetus in 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 cavity 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 for a distance of approximately 25 cm to allow for f e t a l movement. The sutures were then buried by a continous suture to ensure that there was no leak of amniotic fluid'. The peritoneum, 47. adjacent muscle layers and the skin were sutured separately using Echiflex 0, Plain 0, and Proline 0 (Ethicon), respectively. The catheter was exteriorized by passing the free end through a hollow p l a s t i c tube placed subcutaneously on the right flank of the animal. Catheters were coiled in s t e r i l e cotton gauze and placed inside a s t e r i l e handstitched canvas pouch sutured to the animal. Immediately following 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 cages where they were allowed to re-cover quietly. The administration of the a n t i b i o t i c was continued d a i l y for 3 days following surgery. Catheters were wrapped within s t e r i l e gauze daily and free ends were cleansed with an antiseptic solution prior to and following sampling. The patency of catheters was maintained daily by removing heparin from the catheter, flushing with s t e r i l e 0.15 M saline and r e f i l l i n g with s t e r i l e heparinized saline (100 u/ml). Analytical Methods Oxygen and carbon dioxide tensions (P0 2 and PC02) and pH of maternal and fe t a l venous whole blood were determined anaerobically using a Radiometer pH meter (pHM 72) equipped with respective modules and electrodes. The PC0 2 > P0 2 a n d p H e l e c t r o d e s w e r e thermostatically con-t r o l l e d at 37.5°C. Calibration of the PC0 2 and P0 2 electrodes was accomplished by bubbling a gas mixture with know p a r t i a l pressures of C0 2 and 0 2, corrected for da i l y barometric pressure through the electrodes. Blood gas values were corrected for body temperature using appropriate correction factors (Severinghaus, 1966). Fetal hematocrits were 48. determined on a microcapillary centrifuge. Oxygen saturation (S0£) of feta l blood was estimated from PO^ and pH using the nomogram of Meschia et a i . (1961). Chemical Analysis Fetal blood was collected in i c e - c h i l l e d test tubes containing EDTA crysta l s . Samples were centrifuged for 10 minutes at 1000 xg and plasma was removed and stored at -20° C. Plasma Cortisol was determined by radioimmunoassay ( C l i n i c a l Assays; Travenol Labs., Inc., Cambridge, Mass.). The antiserum'used in the assay k i t had 100% crossreactivity for Cortisol. For the determina-tion of glucose and betahydroxybutyrate, the plasma was deproteinized by the addition of 0.2 N perchloric acid and neutralized with potassium hydrogen carbonate. After the removal of the potassium perchlorate precipitate, glucose was determined by glucose oxidase (Glucostat, Worthington Biochemical Corp., Freehold), and betahydroxybutyrate by betahydroxybutyrate dehydrogenase (Williamson and Mellanby, 1974). Fetal plasma lactate concentrations were determined on deproteinized f i l t r a t e s using lactate dehydrogenase (Gutmann and Wahlefeld, 1974). Total alpha amino nitrogen was determined colourimetrically after deproteinizing the plasma with tungstic acid (Mason et , 1973). Samples were analyzed in duplicate and presented as means (+ SEM). Student's t-test was used to test the s t a t i s t i c a l significance of the changes observed during surgery and day 1 after surgery. To determine the time required for 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 in Table 2. During the course of this experiment, 23 fetuses were born a l i v e , 3 were experimentally terminated and 8 were born dead. The average gestational age in the operated ewes was 139.3 + 0.8 days. At the time of weaning the mean body weight was 22.6 + 0.7 kg as compared to 18-23 kg of those born naturally and reared under identical conditions. Fetal and maternal pH, PCO^ and PO^ values are presented in Figure 1 and Appendix Table 5. During the course of surgery maternal and f e t a l blood gas parameters were altered substantially. It 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 in f e t a l PO^ was 2.9 mm Hg/0.1 pH unit change in maternal blood. The fe t a l venous S0 2 also increased from 54 to 68% during surgery. In the post-surgical period a rapid return of the blood gas values to stable levels within 24 hours after surgery was observed (Figure 1 and Appendix Table 5 ) . There were no s i g n i f i c a n t differences in the f e t a l blood gas parameters between days 1-4 and 5-9 following surgery, indicating that these parameters remained r e l a t i v e l y stable for 9 days. The r e l a t i v e changes in f e t a l hematocrit, plasma metabolites and Cortisol concentrations were used as additional 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 surgical period and did not return to stable levels u n t i l 5 days after surgery. S i m i l a r l y , f e t a l plasma glucose, lactate, alpha amino nitrogen and betahydroxybutyrate increased by 60.2, Table 2: Gestational age and body weights of ewes and fetuses (Mean + S.E.M.) Gestational Gestational Ewe Recorded Curved crown Weaning Fetal outcome age at surgery age at birth body weight birth weight to rump length Weight at birth (days) (days) (Kg) (Kg) (cm) at 60 days 0<9_) 122.7 139.3 63.73 3.42 40.2 22.6 8 born dead +1.2 +0.8 +2.18 +0.20 +0.9 +0.71 23 born alive 51. F i g . l Post-surgical changes (± SEM) in blood gas parameters and hematocrit in the ovine fetus i n utero. (• indicates day of surgery; F.P0 o=fetal P0 o ( f T T M.P09=maternal P0 7 (o); n=10) 52. Q200 tr 0.175 0.150 X z 20 UJ - 4 — • 3 4 5 6 DAYS POST-OPERATIVE 7 8 9 53. 16.8, 27.7 and 5.8% respectively, over the levels in the post-surgical period (Figure 2). Hematocrit and plasma metabolite concentrations were s i g n i f i c a n t l y different (P-< 0.05) between post-operative days 1-4 and 5-9, indicating that metabolite concentrations take longer to reach stable levels than blood gas parameters. Fetal plasma C o r t i s o l concentrations were r e l a t i v e l y high (4.8 ug/100 ml) during surgery as compared to the post-surgical period (Figure 3). Stable levels (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 after u n t i l 4-2 days p r i o r to parturition when steep increases were noticed. Discussion Experiment 1 was undertaken to determine the standard surgical techniques to cannulate blood vessels in the ovine fetus in utero for the purpose of studying f e t a l metabolism. Results indicate that f e t a l development in utero i s not s i g n i f i c a n t l y altered following intrauterine 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 affected by the stress of surgical procedures used in this study. Several workers have preferred to employ spinal or epidural anaesthesia to minimize the effects of general anaesthetic agents on the fetus (Meschia et al_., 1965a; Towell and Liggins, 1976; and Clapp et a l . , 1977). Pentobarbital sodium (Nembutol) has been found to be satisfactory by Comline and S i l v e r (1970) and Pearson and Mellor (1975). Halothane was not found suitable by Comline and S i l v e r (1970). However, others (Willes et al_., 1969; Barnes et al_., 1977) have found i t useful. In the Fig. 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 for maintenance gave adequate depth of anaesthesia and rapid recovery. The increases in f e t a l PO^ observed during surgery correlate p o s i t i v e l y with changes in maternal PCO2 and negatively with changes in maternal and f e t a l pH. This finding i s in agreement with the observations made by Motoyama et al_. (1967), who attributed this to combined changes in maternal-fetal oxygen transfer within the placenta and variations in maternal pH. Increased f e t a l P0^ can be attributed to maternal acidosis (pH 7.258), which decreases the a f f i n i t y of the maternal blood for oxygen thus increasing the amount available for transplacental exchange. Further-more, the f e t a l P 0 2 w i l l also increase due to the s h i f t in the f e t a l oxygen disassociation curve because of the existing f e t a l acidosis (pH 7.277) during surgery. The increases in PO^ values during halothane anaesthesis in ruminants has also been attributed to depressed body metabolism and adequate oxygenation (Gates et al_., 1971). Acute changes in maternal blood gas parameters have been reported to result in correspond-ing variations in 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 levels (Motoyama et al_.,1967; Matalon et. aj_., 1978). In the post surgical period, maternal and f e t a l blood gas parameters were within the range reported by other investigators (Comline and S i l v e r , 1972; Shelley, 1973). The rapid return of blood gas values to stable levels within 24 hours a f t e r surgery indicates that the f e t a l oxygenation has not been affected by the level of halothane used or surgical trauma. Fetal metabolite and Cortisol in f e t a l plasma were used as additional c r i t e r i a of intrauterine normality. The high P0^ levels 56. observed in this study (Figure 1) precludes hypoxia as the cause of elevated metabolite levels. Increased f e t a l C o r t i s o l output observed in 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 levels in the post-surgical 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 stable levels would depend upon homeostatic mechanisms operating i n the mature fetus and maternal metabolic changes. The low permeability of the ovine placenta to lactate and the high Km for lactate dehydrogenase isoenzyme I in the ovine f e t a l heart and l i v e r probably account for the delayed return of lactate to basal l e v e l s . The increase i n f e t a l alpha amino nitrogen following 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 stable levels 5-6 days following surgery supports si m i l a r conclusions from studies on other parameters of nitrogen metabolism such as urea production rates (Gresham et al_. 1972a). In the l i g h t of these findings the interval of 10-12 days suggested by Slater and Mel l o r (1977) for the i n i t i a t i o n of metabolic studies appears too long. The fluctuations in f e t a l plasma betahydroxy-butyrate in the post surgical period are d i f f i c u l t to explain because the effects of surgical stress may be masked by increasing gestational age occurring simultaneously, p a r t i c u l a r l y in older fetuses (Jones, 1977). The observed increase in f e t a l Cortisol concentrations during surgery cannot be attributed to hypoxia since there i s evidence of adequate oxygenation as indicated by high P0£ values. The fact that the f e t a l adrenal gland i s r e l a t i v e l y unresponsive to ACTH stimulation (Liggins, 57. 1977; Jones et al_., 1977) and the placenta i s permeable to Cortisol 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 acidosis may also be responsible for the increased Cortisol levels as shown by Nathanielsz et al_. (1972). The stable levels of Cortisol after 3 days following surgery provide additional support that the fetus and the ewe have recovered from surgical stress. The r i s e in Cortisol levels 4 days prior to parturition i s in agreement with the observations of Bassett and Thorburn (1969) and may be related to increased adrenal cortex size and maternal hormonal changes which occur shortly prior to parturition (Liggins, 1977). Conclusions Experiment 1 was undertaken to standardize a surgical technique that would allow for chronic experimentation of the ovine fetus in utero. Evaluation of the metabolic status of chronic f e t a l preparations for future physiological studies was also made. The successful cannulation of f e t a l blood vessels and maintenance of blood catheters for a prolonged period of time, without interfering with the normal growth and development of the fetus, was accomplished. From the results of maternal and f e t a l blood gas parameters and fet a l plasma metabolite and Cortisol concentrations i t has been shown that fe t a l metabolite concentrations take substantially longer to return to stable levels than blood gas p a r t i a l pressures and pH. Therefore, the normality of chronically catheterized fetuses cannot be assessed only by blood gas and pH measurements. Using f e t a l blood metabolites in 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 in this study, intrauterine f e t a l normality i s reached by the 5th postoperative day. 59. Experiment II Substrate turnover and interrelationship in the ovine fetus in utero Introduction U t i l i z a t i o n of metabolic substrates by the ovine fetus has received considerable attention i n an attempt to evaluate the ca 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 oxidative substrate of the ovine fetus, pa r t i c u l a r interest has been directed at quantitating the metabolism of glucose in the fetus. Current knowledge on substrate metabolism i n the ovine fetus in utero may be ascribed largely to the pioneering work of Batt a g l i a , Meschia.and coworkers (Tsoulos et ^ 1_., 1971; James et al_., 1972; Boyd et a l . , 1973; Morriss et j i l _ . , 1973). These workers developed the technique of umbilical catheterization and from venoarterial concentration difference and blood flow, estimated the umbilical 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 th i s subject has been reviewed exhaus-t i v e l y (Battaglia and Meschia, 1978). Recently, the use of tracer methodology for studying in vivo f e t a l substrate metabolism has been reported by many workers. Warnes et a l . (1977a) injected labelled substrates into the f e t a l c i r c u l a t i o n and deter-mined kinetic parameters from the disappearance of the label from the c i r -culation. Others infused labelled substrates continuously into 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 fter i t reached plateau levels (Hodgson et a]_., 1980; P r i o r , 1980; Anand et a l . , 1979, 1980). The objectives of the present experiments were to examine the u t i l i z a t i o n of substrates in the ovine fetus by tracer techniques. The ir r e v e r s i b l e disposal rate provides an estimate of fe t a l substrate u t i l i z a -tion in chronically catheterized 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 for the analysis of data, single injection techniques have particular advantages in f e t a l metabolic studies. In the present study, this 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 particular emphasis on the re-entry of the tracer into the fe t a l c i r c u l a t i o n . EXPT II A) METABOLISM OF GLUCOSE AND LACTATE Materials and Methods Animals Dorset horn ewes were used in these experiments. The main-tenance of pregnant ewes and intrauterine surgical procedures were per-formed as detailed in Experiment 1. Cannulae were placed in the jugular vein of the ewe the morning prior to the experiment. Metabolic Studies A l l metabolic studies were performed 5-6 hours after the morning feeding to minimize v a r i a b i l i t y associated with differences in 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. Pri o r to the experiment the radioactive substrate was made to volume in 1.0 ml of s t e r i l e saline 61. (0.15 M). The ewe was kept standing during the course of the 3-hour experiment. Fetal lambs were given a single intravenous in j e c t i o n of 50uCi 14 3 of a mixture of [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 saline. In some fetuses,LU- C] glu-cose or [1-^C] lactate was injected separately. To monitor rapid changes in the early phase of the s p e c i f i c activity-time curve, f e t a l blood sampling was i n i t i a t e d 2 minutes following the injection of labelled substrates and was continued uninterruptedly for 5 minutes. This was followed by sampling at f i v e minute intervals up to 30 minutes. Thereafter samples were obtained less frequently up to 3 hours. Maternal blood samples were collected from 4 minutes following the injection, 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 ice c h i l l e d test tubes containing EDTA cr y s t a l s . Two hundred mi c r o l i t e r s of whole blood were deproteinized with 3.0 ml of 0.33 N perchloric acid at 0°C. The protein precipitate was removed by centrifugation at 7,000 x g for 15 minutes in a Sorval refrigerated centrifuge. The supernatant was pipetted into test 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 analysis. Supernatants were neutralized with KOH and allowed to stand in ice for 45 minutes. The insoluble KCIO^ preci-pitate was removed by centrifugation at 0°C and the neutralized supernatant was transferred to freeze drying f l a s k s . The KCIO^ precipitates were washed 62. twice with ice cold water and the combined supernatants and washes were pooled in respective freeze drying flasks and lyophilized (Lab Conco freeze d r i e r ) . Samples were reconstituted in 1.5 ml of demineralized, d i s t i l l e d water. I n i t i a l fractionation of blood metabolites into basic, neutral and acidic fractions was accomplished by passing the neutralized supernatant successively through cation (AG 50X-8 [H +] 200-400 mesh) and anion (AG 1X-8 [formate] 200-400 mesh) (Biorad) exchange resins packed in 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 reservoir supplying each column. Cation and anion resins were prepared by the application of 20 volumes of 2N HC1 and 1.0 N formic acid respectively, followed by a thorough washing with C0 2 free demineralized water. Resins were stored in sealed containers at 4°C. The bottom of the columns packed with these ion 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 Specific A c t i v i t y a) Radiochemical purity of glucose in fe t a l whole blood The neutral fraction eluted from the anion exchange column was reconstituted in 1.5 ml of demineralized d i s t i l l e d water. For measure-ment of r a d i o a c t i v i t y , glucose was quantitatively converted to gluconic acid byincubating the neutral fraction with glucose oxidase (50 units,specific a c t i v i t y 20,000 units/g, Sigma, St. Louis) and catalase (3,000 units, 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 for 2 hours. The incu-bation mixture was then passed through a reconditioned anion exchange column and the adsorbed gluconic acid eluted with 8N formic acid. The 63. recovery of labelled 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 in s i g n i -f i c a n t amounts in f e t a l blood, [ 1 4C]-fructose and [ 1 4C]-glycerol were subjected to ion exchange chromatography separately and in combination with [^C]-glucose. Radioactivity in the gluconic acid fraction appeared only when [^C]-glucose was added indicating the absence of contamination with fructose or glycerol. b) Determination of glucose and fructose concentration in the  neutral f r a c t i o n Glucose in the neutral fraction was determined by a modification of the glucose oxidase procedure. Fructose in the neutral fraction was determined by the anthrone procedure (Nixon, 1969). 2. Assay of Lactate Specific A c t i v i t y a) Radiochemical purity of lactate in f e t a l whole blood The acidic components adsorbed on the anion exchange resin were-eluted with 4 N formic acid, neutralized with NaOH and evaporated. Samples were reconstituted in 1.0 ml of demineralized water and an aliquot was used for radioactive measurements. The v a l i d i t y of this procedure for determining [^C]-lactate r a d i o a c t i v i t y was confirmed by thin layer chromatography. Standard [^C]-lactate solutions and samples were applied in 4 cm streaks on Eastman S i l i c a gel Chromatogram Sheets (Eastman, Kodak) and developed twice in acetone:n-propanol:water (6:3:1 v/v) in the same di r e c t i o n . The chromatograms were scanned on an Actigraph III radiochromatograph system (Nuclear Chicago). Radioactive areas corresponding to lactate standards were cut out, scraped into s c i n t i l l a t i o n v i a l s and suspended in 10 ml of s c i n t i l l a t i o n f l u i d for counting. No contamination of glucose in the lactate f r a c t i o n was detected. The combined recovery of added [^C]-lactate for both procedures was 83.0 + 0.63% (Appendix Table 2). 64. b) Determination of lactate concentration in the acidic fraction Lactate in the acidic fraction was determined by the enzymatic procedure described by Gutmann and Wahlefeld (1974). Radioactive Chemicals [U- 1 4C] glucose, (304 mCi/mmole), [2- 3H] glucose, (17.9 Ci/mmole), [1- 1 4C] lactate (51 mCi/mmole), and [U- 1 4C] lactate (60 mCi/mmole) were obtained from Amersham Searle Corp. (Oakville, Ont.). The l i q u i d s c i n t i l l a -tion 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 cocktail (PCS, Amersham Searle Corp.). The s c i n t i l l a t i o n f l u i d used for counting r a d i o a c t i v i t y present on s i l i c a gel thin 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 ra t i o s . Automatic quench c a l i b r a t i o n and calculation of dpm values were performed for both single and doubly labelled samples. Counting e f f i c i e n c i e s were 35-40% and 50-60% for [ 3 H ] and [ 1 4C] respectively, with 30% of the [ 1 4C] rad i o a c t i v i t y appearing in the [ H] channel. Analysis of Data Specific a c t i v i t y values at each sampling time are expressed as a fraction of the i n i t i a l dose of tracer injected at zero time (Shipley and Clark, 1972). I n i t i a l kinetic 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 (in fraction dose/mg C); A = zero time intercept 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 for 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 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 in the goodness of f i t was no longer s i g n i f i c a n t . The non-linear program of Metzler (1969),(NONLIN Michigan) was used to give the least squares estimate of the parameters. The squared correlation 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 calculated as follows: where W =weightedsum of squared observations =V~ w - j j ( Y - j j _ Y i ) n where SA.,- = observed s p e c i f i c a c t i v i t y SA. = estimated s p e c i f i c a c t i v i t y W = sum of weighted squared deviation • II ( Y i j ' T e . l c 1 j ) Z - W U j 66. From the result of the f inal 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 ident i -f iable space (e.g. , fetal and extrafetal compartments, F ig. 4). The term pool has been used to denote different locations within the fetal com-partment (blood; primary pool (a) and t issue, secondary pool (b)) or chemi-ca l ly identif iable spaces (e .g. , glucose, lactate, etc.) Fetal Compartment Extra-fetal compartments F ig. 4 Model of glucose and lactate metabolism in the ovine fetus. 67. rate constant (pool a to pool b) rate constant (pool b to pool a) i r r e v e r s i b l e disposal rate constant rec i r c u l a t i o n (tracer re-entering primary pool after trans-formation) recycling (tracer re-entering primary pool without trans-formati on). Since the basic objective of this experiment i s to study the disappearance of the tracer from the f e t a l blood pool, the biexponential parameters were formulated into a two pool open exchange system to depict the transfer of the label into and out of the primary f e t a l blood pool. No attempts were made to id e n t i f y or sample the large number of pools and/or compartments in which the f e t a l and extrafetal compartments may be embedded or are i n communication with each other. Calculations The following equations were used for estimation of kinetic para-meters of substrate metabolism: 1. Substrate pool (Q) = (White et al_., 1969) (mgC/kg) <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. where k b a kab koa 68. 1* 2. Irreversible rate of disposal (D.R.) = (mgC/min/kg) /*"S.A. (dt) • (Shipley and Clark, 1972) 3. Volume of d i s t r i b u t i o n (V) = Q x IP.0. blood cone. body weight (% body weight) (White et a l . , 1969) D R 4. Metabolic clearance rate (M.C.R.) = (ml/min) 5. Half l i f e I blood cone. (Shipley and Clark, 1972) (minutes) T !/2 e*. (Shipley and Clark, 1972) 2 = 0.693 l/ 2 ~ V where** and$ are rate constants. T-j and T 2 are rapid and slow components respectively. The transfer of tracer 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) S.A. Fetal glucose C (dt) x 100 69. The re-entry of the label back into the fetal circulation may occur by the tracer leaving the fetal blood pool and, after a sojourn in some other compartment, returning in i ts original form. This wi l l be referred to as "recycl ing". If the tracer returns to the primary fetal blood pool after chemical transformation and reincorporation into newly formed molecules, the process wil l be referred to as "recirculat ion" (Hetenyi and Norwich, 1974). Recirculation was estimated according to Dunn et aj_., 1969 by 3 14 injecting a mixture of glucose labelled with [2- H] and [U- C] and measur-ing the half l i f e with each isotope. 7. Recirculation = 1 ( ^ ) T 1 / 2 r 1 4 C] (Fraction of irreversible ^ disposal rate) w h e r 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 circulation after a sojourn in some other part of the system. 8. Fraction lost irreversibly (0) = M C R (Gurpide and Mann, 1970) KT-V where MCR = metabolic clearance rate (ml/min) Kj = total turnover rate constant V = volume of distr ibution t> = Fraction of total turnover irreversibly lost . 70. 9. The fraction 1-0 therefore indicates the fraction of to t a l turnover which returns to the c i r c u l a t i o n through recycling. In the dynamic state of transfer of metabolites between the fetus and the mother, molecules d i f f e r in t h e i r rates of exchange and mechanisms of transport across the placental barrier with the result that different compounds vary in the time spent within 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 total residence time, T) passages of the labelled 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 after i t s i n i t i a l passage through i t (number of cycles,2f) were calculated according to Rescigno and Gurpide (1973). 10. Mean t r a n s i t time (T) = J-KT where Ky = to t a l turnover rate constant. 11. Mean total residence time (T) = where Q = pool size 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 lactate in the f e t a l compartment was estimated by the r a t i o of integrals of the s p e c i f i c activity-time curves of these metabolites taken to time i n f i n i t y (Shipley and Clark, 1972). 71. 13. Fraction of lactate carbon derived from glucose carbon /V S.A. lactate C (dt) J o S.A. glucose C (dt) o 14. Rate of lactate C derived from glucose gC.min/kg) = Equation 13 X lactate irreversible disposal rate 15. Per cent of glucose irreversible disposal rate going to lactate = Equation 14 x 100 Glucose irreversible disposal rate Al l results were expressed as means (+ SEM). Stat ist ical analysis of the data was performed using the paired and independent t-test, where appropriate. The linear regression lines and correlation coefficients were calculated by the method of least squares. Results The mean (+ SEM) gestational age, maternal and fetal body weight, blood gas and metabolite measurements recorded at the time of each experi-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 fetal physiological parameters during the experimental peri Parameter [U-14C] glucose n=7 [U-• 1 4C][2- 3H] glucose n=8 [i - 1 4 c ] lactate n=8 F M F M F M r Body weight (Kg) 60.9 ±1.77 2.36 ±0.10 65.6 ±2.97 2.91 ±0.98 66.39 ±3.38 2.19 ±0.11 Gestational Age (Days) -129.14 ±1.99 -137.5 ±0.98 -127.1 ±3.113 pH 7.502 ±0.02 7.380 ±0.02 7.466 10.02 7.340 ±0.04 7.488 ±0.02 7.349 ±0.01 pC02(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 2 (mm Hg) 33.35 ±1.13 18.61 ±0.47 32.8 ±2.04 20.54 ±0.90 33.16 ±1.29 18.51 ±1.03 TC02 (meg/L.) -23.60 . ±0.81 -22.81 ±0.75 -23.71 ±1.65 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 Fructose (mM) N.D. 3.86 ±0.31 N.D. 4.58 ±0.15 N.D. 3.84 ±0.16 Lactate (mM) 1.18 ±0.08 1.81 ±0.06 1.55 ±0.14 2.022 ±0.04 1.082 ±0.05 1.89 ±0.05 Values are means (+ SEM) M = Maternal F = Fetal 73. oxygenated near-term fetuses (Experiment 1), indicating recovery from surgical procedures by the ewe and fetus. 1. Description of sp e c i f i c activity-time curves for glucose and lactate Specific activity-time curves, plotted on semi logarithmic coordinates following the single injection of labelled substrates are presented in Figs. 5, 6 and 9 along with whole blood glucose or lactate concentrations during the course of the experiments. The concentrations of glucose and lactate were r e l a t i v e l y constant during the experimental period. In a l l experiments performed with radioactive labelled glucose or lactate, the observed data 2 was found to f i t (R 0.985) a biexponential equation. After the injection of labelled substrates there was an i n i t i a l rapid decline in the spec i f i c a c t i v i t y . A slower lin e a r decline in the spec i f i c a c t i v i t y followed the i n i t i a l decay for the duration of the experiment. The s p e c i f i c a c t i v i t y of t r i t i a t e d glucose was lower than that o f [ U - ^ C ] glucose, which was p a r t i -c u l a r l y marked at the l a t t e r time periods (Fig. 6). 2. Kinetic parameters of glucose and lactate metabolism  a) Glucose metabolism The kinetic parameters of glucose metabolism calculated from sp e c i f i c activity-time curves when a mixture of [U-^C] and [2-^H] 14 glucose or [u- C] glucose alone was injected are summarized in Table 4. No s i g n i f i c a n t differences i n the glucose pool size and volume of d i s t r i -14 3 bution were noted with [U- C] and [2- H] labelled glucose, when tested by a paired t-test (P< 0.05). The mean (+ SEM) i r r e v e r s i b l e disposal 14 rates and metabolic clearance rates calculated from [U- CJ glucose were 3.541 + 0.25 mg C/min/kg and 140.80 + 4.84 ml/min respectively. These 74. 100r 10 go . u CiD-3 E o o < in  w * * 4 u c-o u O u _2 O 20 o o 5c 15 10! L_ 20 60 100 Time (min.) 140 180 Fig. 5 Semi logarithmic plot of glucose s p e c i f i c a c t i v i t y (S.A. versus time following injection of [U-'4C] glucose and whole blood glucose concentration. Values are means (+ SEM; n=7) 75. 100 O U u 3 _ E uo aJ -5-8 ft 10 4 o T U 1 4 ^ glucose 23H]glucose = 14C1 lactate o r T 81 0 8 O ^ U wo = E 20 15 10 L_ 20 6 0 100 140 Time (min) 180 Fig.6 Semi logarithmic plot of glucose time following the inj e c t i o n of glucose and whole blood glucose (± SEM; n=8). s p e c i f i c a c t i v i t y (S.A.) versus a mixture of [U- 1 4C] and [2- JH] concentration. Values are means 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 [ 3H]-glucose 5 (4.07 + 0.16 mgC/min/kg and 155.10 + 5.30 ml/min respectively). The metabolic half l i v e s of the slower decaying components obtained from [ 3H]-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 from [ 1 4C]-glucose (50.66 + 2.69 min). In an attempt to determine i f glucose metabolism was correlated with f e t a l development, [U- 1 4C] glucose was injected separately into younger fetuses of lower body weight. The sp e c i f i c activity-time curves obtained from [U-^ 4C] glucose in these fetuses were similar to those obtained in heavier fetuses (Fig. 5). The mean (+ SEM) i r r e v e r s i b l e disposal rates and metabolic clearance rates estimated in these fetuses were 2.251 + 0.15 mgC/min/kg and 99.08 + 2.83 ml/min respectively (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 in 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- 1 4C] glucose in older fetuses. There was a positive correlation 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, Fig. 7). A simi l a r positive correlation 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, Fig. 8). The maternal-fetal r a t i o of the area under the sp e c i f i c a c t i v i t y -time curves was 0.092 + 0.002 in the case of [u- 1 4C] glucose whereas i t 3 was 0.141 + 0.002 in the case of [2- H] glucose. 77. Glucose Concentration (mg./iooml.) Fig. 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) after single injection of [U- 1 4C] glucose. (Y=0.671x - 3.86; P < 0.05; r=0.66; n=15) 7a Bwt. (kg.) Fin P l inear regression of fetal irreversible disposal rate of glucose 9 W&W! X ^ B , 79 Fig. 9 Semi logarithmic plot of lactate specif ic act iv i ty (S.A.) versus time following the injection of [1- 1 4C] lactate and whole blood lactate concentrations. Values are means (± SEM; n=8). Table 4 Kinetic parameters of glucose metabolism in the ovine fetus estimated using single i n j e c t i o n of "mixture of [U-^C], [2-3 H] glucose or [U-^C] glucose a W . Age xperiment (days) and ( Body weight Kg) Irreversible Rate of Disposal (mgC/min/Kg) Metabolic Volume of Pool Size Half l i v e s (Tl/2) Clearance Rate di s t r i b u t i o n mgC/Kg (min) (MCR) (ml/min) (% Bwt.) (Q) T 1 T, [U- 1 4C] 137.5C ±0.98 glucose (n=8) 3.5419 ±0.251 140.8 9 ±4.8 30.0a ±2.65 21.72a ±2.16 1.16 ±0.10 50.66a ±2.69 4.070b ±0.162 155.l b ±5.3 32.0a ±3.80 25.58a ±3.12 1.07 ±0.15 44.34b ±2.21 2.251C ±0.15 99.08 C ±2.83 41.38b ±2.40 24.66a ±1.32 1.47 ±0.13 51.583 ±3.36 [U- 1 4C] glucose (n=7) 129.r ±1.99 (2.36 Kg) 1 Values = means (± SEM) a' b' c Supercripts with di f f e r e n t alphabets in the column denote s t a t i s t i c a l signi T ,T - denote half l i f e of fast and slow decaying components respectively. 81. b) Lactate metabolism The metabolic parameters of lactate calculated from [1-^C] lactate specif ic 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 igni f icant ly (P < 0.05) larger than the glucose pool s ize. The irreversible 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 ignif icant ly different. Lactate metabolic clearance rate (82.03 + 5.62 ml/min) was significantly (P < 0.05) less than that of glucose. The apparent volume of distr ibution of lactate (43.93 + 5.11% of fetal body weight) was s igni f icant ly larger (P < 0.05) than that of glucose. No radioactivity was observed in fetal blood glucose when [1-^C] lactate was injected into the fetus. S imilar ly, lactate, label was not detected in the maternal circulation following [1-^C] lactate injection into the fetus. The parameters obtained in the single experiment where [U-^C] lactate was injected are very similar to those obtained using [1-^C] 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 irreversible 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 after a sojurn in some other part of the system was 69.6, 66.1 and 80.2 per cent for [U-^C] and [2-3H] glucose and [1- 1 4C] lactate respectively (Table 7). In the able 5 Kinetic parameters of glucose and lactate metabolism in the ovine fetus, estimated by using single injections of [U- 1 4C] glucose or [1- I HC] lactate . Experiment Pool Size (Q) (mgC/kg) Irreversi ble Rate of Disposal (D.R.) (mgC/min/Kg) Metabolite Clearance Rate (MCR) (ml/min) Volume of Distribution (v) {% Bwt) Half l i f e (min) T] T 2 14 2 [U- C] glucose 23.10a ±1.32 2.935a ±0.210 121.56a ±9.03 35.27a ±1.33 1.30 50.79 ±.112 ±2.31 (n=15) 29.10L ±2.69 27.87 2.576° ±0.182 82.031 ±5.62 2.171 75.36 43.93L ±5.11 45.10 1.36 ±0.17 56.67 ±4.49 1.62 59.33 [1- 1 4C] lactate (n=8) [U- 1 4C] lactate (n=l) 1 Values = mean (± SEM) 2 14 Includes a l l [U- C] glucose experiments from table 4 a'k Superscripts with different alphabets in the column denote s t a t i s t i c a l significance (P< 0.05) T-j and 1^ = denotes the half l i f e of fast and slow decaying components respectively. Table 6 Glucose-lactate conversions in the ovine fetus in utero. Fetus % lactate C derived from glucose C' Rate of formation of lactate from glucose (mgC/min/kg)2 % glucose going to lactate 3 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 Mean (± SEM) 44.16 ±5.40 1.223 ±0.159 35.92 ±4.30 Equation 13 Equation 14 Equation 15 Table 7 Estimation of recycling and recirculation of glucose and lactate in the ovine fetus. Isotopes injected Parameter [U- 1 4C] glucose (n=8) [2- 3H] glucose (n=8) [1- 1 4C] lactate (n=8) Fraction of to t a l turnover i r r e v e r s i b l y l o s t 0.299a ±0.051 0.3183 ±0.02 0.199a ±0.011 Fraction of to t a l turnover recycled 0.696a ±0.062 0.661a ±0.02 0.802b ±0.010 Mean t r a n s i t time (t) (min) 1.71a ±0.35 1.80a ±0.26 2.49b ±0.30 Mean total residence time (T) (min) 6.21a ±0.90 6.35a ±0.92 12.05b ±1.06 Number of cycles) 3.30a ±0.46 3.22a ±0.40 4.24 a ±0.27 Recirculation (% of ir r e v e r s i b l e disposal) 12.62±5.44 -1 values=means (+ SEM) a ' b superscripts with different alphabets in rows denote statistical significance (P<0.05) 85. single experiment where [U-^4C] lactate was administered to the fetus, the fraction recycled was 79.9 per cent. No s i g n i f i c a n t difference was noted in the mean t r a n s i t or total residence times and the number of cycles to the primary pool between [U- l 4C] and [2- 3H] glucose. The mean total residence time observed with [1-^ 4C] lactate was s i g n i f i c a n t l y greater (P< 0.05) than with [U- 1 4C] or [2- 3H] glucose. The number of cycles made by the lactate tracer to the primary pool was higher than that for glucose; however, th i s was not s i g n i f i c a n t . The extent of glucose 3 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 ir 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 analysis involving isotopic tracers used in metabolism has been discussed elsewhere (White et al_., 1969; Judson and Leng, 1972; Hetenyi and Norwich, 1974; Atkins, 1980). A fundamental assumption inherent in the analysis of tracer kinetic data i s that steady state conditions prevail during the experimental period. The constant level of blood glucose and lactate during the course of these experiments (Fig. 5, 6 and 9) indicate that steady state conditions prevailed. The volume of d i s t r i b u t i o n of glucose noted in t h i s study (35.27%) exceeds the ex t r a c e l l u l a r space in the fetus. The present value i s lower than the estimate of 57.4% reported by Warnes et al_. (1977a) using si m i l a r i n j e c t i o n techniques. The lactate pool size and apparent volume of d i s t r i b u -tion are also lower than those reported by Warnes et al_. (1977a). These differences in glucose and lactate space may be due to the lower blood glucose 86. and higher lactate in the fetuses used by Warnes et a]_. (1977a) than those used in the present study. Although the f e t a l blood glucose concentra-tions are substantially lower than maternal levels (Table 3 ) , the volume of d i s t r i b u t i o n in the fetus exceeds the ext r a c e l l u l a r f l u i d space of 18% reported in adult ruminants (Kronfeld and Simenson, 1961; White et a l . , 1969; 1980). This difference 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 (Jarrett et al_., 1964). The i r r e v e r s i b l e disposal rates of glucose and lactate obtained in t h i s experiment (Table 5) are simi l a r to the values reported by Warnes et al_. (1977a), using single injection techniques and to recent estimates 14 obtained from the continuous infusion of C labelled glucose (Anand et a l . , 1979, Hodgson et aj_., 1980) and lactate ( P r i o r , 1980). However, these values are considerably higher than the estimates obtained by umbilical venous-arterial differences 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 under-estimates unidirectional u t i l i z a t i o n . On the other hand, the contribution of placental metabolism and improper mixing and exchange of ^ C with other substrate pools may have resulted in an overestimation of i r r e v e r s i -ble loss in the single injection technique employed. Results from t h i s experiment also indicate that the metabolism of glucose and lactate within the ovine fetus i s more rapid than in the post-natal l i f e . For example, White et aj_. (1980) have reported a glucose pool turnover time in postabsorptive lambs of 40-50 minutes. The mean total residence time of glucose from i t s entry into 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). S i m i l a r l y , 87. in the case of lactate the mean total residence time in the fetus was only 12.04 minutes as compared to the value of 20-30 minutes reported in the adult monogastric (Searle and Cavaleri, 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 (Fig. 7 ). It 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 correlated with maternal a r t e r i a l glucose con-centration and umbilical uptake. This relationship 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 in fetuses of starved ewes, where i t has been documented that maternal starvation results in a 35% reduction in f e t a l plasma glucose levels (Tsoulos et al_., 1971, Schreiner et al_., 1978). Such an effect would potentially 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 meta-boli c substrates to the fetus would depend not only on the rate of supply but also on the tissue mass involved. Fetal body mass as a potential regu-la t o r of f e t a l metabolism was observed in t h i s study. A s i g n i f i c a n t correlation (P< 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 (Fig. 7). James et al_. (1972) f a i l e d to demonstrate a correlation between umbilical glucose uptake and f e t a l body weight,which may be attributed to the wide variation in the measured umbilical glucose uptakes in t h e i r study. White et al_. (1980) reported a s i g n i f i c a n t correlation 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 , Flecknell et al_. (1980) have recently reported that the disposal rate of glucose in the neonatal pig was prpportional to both the total body weight and individual organ weights, The positive relationship between f e t a l oxygen consumption and body weight 88. reported by James et al_. (1972) also supports the increased u t i l i z a t i o n of glucose observed in this study in fetuses of higher body weight. It i s l i k e l y that i f maternal glucose transfer to the fetus i s r e s t r i c t e d during under n u t r i t i o n , the fetus may have to rely on endogenous sources of glucose production to compensate for the increased metabolic demands during advanced stages of gestation. Glucose r e c i r c u l a t i o n The reincorporation of isotopic label into endogenously produced compounds has been referred to as r e c i r c u l a t i o n (Zilversmit et a l . , 1943; Hetenyi and Norwich, 1974). Recirculation of a labelled substrate therefore underestimates i t s true rates of i r r e v e r s i b l e disposal. 3 14 The simultaneous in j e c t i o n of [ H] and [ C] glucose was employed 14 in t h i s study- to quantitate the extent of r e c i r c u l a t i o n of C labelled glucose returning to the blood pool. The metabolic fate 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 tracer, t r i t i u m from carbon 2 i s liberated in the glucose phosphate isomerase reaction between glucose-6-phosphate and fructose 6-phosphate and i s rapidly l o s t through the exchange with body water (Rose and O'Connell, 3 14 1961). The use of [2- H] glucose in combination with [U- C] glucose thus y i e l d s a turnover rate which gives an estimate of r e c i r c u l a t i o n through both the Cori cycle 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 similar to the observations of Anand et a]_. (1979) who reported very l i t t l e 89. *3 l/i difference in turnover rate between [2- H] and [U- C] glucose in t h e i r continuous infusion study. The present values closely resemble those reported in adult ruminants, (10%, Annison et al_., 1963; 13%, Judson and Leng, 1972) but are substantially lower than the value of 26% reported in 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% respectively. Glucose-lactate conversions Results of this experiment show that 36% of the glucose pool i s metabolized to lactate, which accounts for 44% of lactate i r r e v e r s i b l e disposal rate (Table 6). Warnes et al_. (1977a) also demonstrated a 14 rapid l a b e l l i n g of lactate following the in j e c t i o n of [U- CJ glucose into the f e t a l c i r c u l a t i o n . This proportion of lactate produced from glucose closely resembles the value of 40% reported in adult sheep. However, i t i s possible that part of the glucose conversion to lactate may have occurred in 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 in the absence of umbilical catheterization no d e f i n i t e quantitative conclusions can be drawn on the extent of glucose conversion to lactate by the fetus alone. When [ l - l 4 C ] lactate was injected no r a d i o a c t i v i t y could be recovered in f e t a l blood glucose which agrees with the results of Warnes 90. et al_. (1977a). On the other hand, using continuous infusion of [U-^C] lactate, Prior (1980) has recently reported that 22% of the lactate returns to the f e t a l glucose pool. I t i s possible that in the single injection technique of [1- ^ 4C] lactate used in t h i s study, the[^ 4C] carboxyl carbon was either lost through decarboxylation to acetyl COA and oxidized or diluted beyond detectable levels by i t s passage through the oxaloacetate pool. Further the metabolism of lactate by the fetus may be too rapid to detect measurable a c t i v i t y in glucose with single injection techniques. The ovine fetus, unlike the adult, synthesizes large quantities of glycogen in l i v e r , heart and muscle tissues, during late gestation (Shelley, 1960). However, the apparent low turnover of the f e t a l glycogen pool (Setchell et aj_., 1972) 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 experi-mental period employed in 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 recycling In dynamic studies with tracers, the term recycling refers to the return of the tracer to the sampled pool after a sojurn in other parts of the system. (Gurpide and Mann, 1970; Rescigno and Gurpide, 1973; Hetenyi and Norwich, 1974). In the case of the fetus the blood (pool a) and tissues (pool b) may 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 tissues. The large f r a c t i o n of glucose and lactate recycled (70-80%) may be attributed to the possible contribution of placental metabolism of substrates and the inadequate mixing of the label with substrate pools i n the fetus in the single in j e c t i o n techniques (Warnes et al_., 1977a). The fact that the extent of recycling 91. was not diff e r e n t when [ l 4C] or [ 3H] labelled glucose was used suggests that the glucose molecules re-enter the fe t a l c i r c u l a t i o n without under-going transformation. The s i g n i f i c a n t l y greater fraction of lactate recycled coupled with the longer mean total residence time (Table 7) than glucose and the observation that no lactate r a d i o a c t i v i t y was found in the maternal compartment suggest that lactate i s recycled in 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 lactate was eliminated by u t i l i z a t i o n in f e t a l sheep. The impermeability of the placenta to lactate (Britton et al_., 1967) would also be conducive for recycling. The longer mean total residence time of lactate than glucose may also be ascribed to i t s s i g n i f i c a n t l y larger volume of d i s t r i b u t i o n . The fact that the recycling of [U-^4C] lactate did not d i f f e r r 14 n from that of [1- CJ lactate i s additional evidence that this phenomena i s characteristic of the lactate 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 recycling i s a process active outside and within the fetus. Fetal-maternal glucose transfer The transfer of glucose from the mother to the fetus has been considered to depend on a stereospecific process of 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 tissues (Widdas, 1961; Boyd et aj_., 1976; Simmons et aJL, 1976, 1979). I t i s evident, however from the results reported in this experiment that a substantial amount of fe t a l glucose i s also 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 transfer 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 in late 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 respectively. Anand et aj_. (1979) demonstrated a positive correlation between the f e t a l -maternal transfer 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 feta 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 test the effect of varying levels of f e t a l oxygen consumption on the reverse transfer 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 available to the fetus in excess of the oxygen required for oxidative metabolism would be returned to the mother. This would be a preventative measure against possible hypoxia in the fetus consequent to a sustained increase in 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 overall f e t a l meta-bolism. The quantitative contribution of f e t a l f l u i d s to f e t a l glucose recycling needs to be studied in order to describe adequately the recycling of glucose within the feto-maternal system. 93. Conclusions Experiment II a was conducted to study the metabolism of glucose and lactate in the ovine fetus using radioistope d i l u t i o n techniques. It i s evident from the results that these techniques complement conclusions based on venous a r t e r i a l umbilical differences. It can be concluded that glucose and lactate 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 meta-bolic clearance of these compounds. There i s a correlation 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 in 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 lactate. Recycling appears to be an important feature of glucose and lac-tate metabolism in the fetus occurring to an extent of 70-80% of t o t a l turn-over of these compounds. The absence of r a d i o a c t i v i t y in maternal blood following lactate i n j e c t i o n indicates that lactate recycling occurs within the f e t a l compartment, due probably to impermeability of the placenta to lactate. The fact that approximately 10% of maternal glucose carbon was derived from the f e t a l glucose carbon indicates that glucose recycling occurs both within and outside the f e t a l compartment. 94. EXPT II B) METABOLISM OF AMINO ACIDS Materials and Methods Animals The animals used in this experiment were surg i c a l l y prepared and maintained as described in Experiment 1. Metabolic Studies The return of the ewe's appetite to presurgical levels and the da 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 for determining the physiological condition of the animal on the day of experimentation. The housing and maintenance of the ewes were as described in Experiment 1. A l l experiments were i n i t i a t e d at approximately 5-6 hours after the morning feeding. The experimental protocol for each study was as follows: 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 injection in s t e r i l e phosphate buffer (pH 6.9), and the cannuula dead space was flushed immediately thereafter 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 intervals up to 3 hours as detailed in Experiment II and transferred to ice c h i l l e d test tubes containing EDTA c r y s t a l s . Blood was centrifuged and the plasma was removed and stored at -40?C. Plasma (500 jui) was deproteinized with double the volume of absolute ethanol and the solution was kept on ice for 30 minutes before centrifugation. The deproteinized supernatant was evaporated to dryness in a warm water bath under a stream of nitrogen. 95. b) [U 1 4C] alanine and [2- 3H] glucose In experiments where [U-^ 4C] alanine and [2- 3H] glucose were administered simultaneously, 50 jjCi of each isotope were dissolved in 2.5 ml of s t e r i l e phosphate buffer and injected as a bolus. Whole blood (400 ul) were deproteinized with 6.0 ml of 0.33 N perchloric acid. The supernatants were neutralized with KOH and freeze dried as described in Experiment II 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 fractionation of blood metabolites into a c i d i c , neutral and basic fractions was performed by ion exchange chromatography as previously described in Experiment II a. The amino acids adsorbed on the cation exchange resin were eluted with 2N triethanolamine in 20% acetone in water (Harris et al_., 1961) and evaporated to dryness under vacuum. After reconstituting i n 1.5 ml of 0.1 N HC1, a 500 jul aliquot was taken for radioactive measurements. Recovery of 1 4C-amino acids applied to the columns was 99.1 + 0.96% (Appendix Table 3). An additional 500 JJI aliquot was diluted with equal volume of C0 2~ free water and the to t a l organic carbon content was quantitated on an infrared carbon analyzer (Beckman). Potassium biphthalate standards rang-ing from 0-1000 ppm were prepared in CO,,-free water (Appendix Fig. 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- 1 4C] alanine, the basic fraction was evaporated to dryness and dissolved in 1.0 ml 0.05 M Tris buffer, pH 7.8. Alanine was quantitatively converted to lactate in a coupled enzymatic reaction involving transamination and dehydrogenation by the addition of alanine amino transferase (EC. 2.6.1.2, 10 U/ml, Sigma), lactate dehydrogenase (EC. 1.1.1. 27, 6 U/ml, Sigma), 2-oxoglutarate (6.7 mM) and NADH (0.2 mM, Sigma). After incubation at 30°C for 2 hours, the mixture was passed through a cation exchange column and the radio-a c t i v i t y in the eluate containing lactate was counted. The recovery of labelled alanine in this procedure was 82.4 + 0.64% (Appendix Table 3). The concentration of alanine was determined by monitoring the extinction of NADH at 340 nm between 40 and 60 minutes (Grassl, 1974). 3) Determination of glucose and lactate 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 of glucose and lactate was determined as described previously (Experiment II a). 4) Plasma protein determinations The total plasma protein content in maternal and fe t a l plasma was determined by the procedure of Lowry et al_. (1951). Bovine albumin standards ranged from 0-100 mg protein/100 ml. Samples were read against a Folin-phenol reagent blank at 600 nm. Radiochemicals [U- 1 4C] amino acid mixture 1 (50 mCi/mmole) and [U- 1 4C] alanine 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., Oakville, 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-^4C] amino acid decay curve accurately. The observed data were therefore plotted 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 least squares estimates of the kinet i c parameters were calculated using a Fortran computer program (AUTOAN). The f i n a l least squares estimates were obtained using NONLIN (Michigan) as described in Experiment II 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 (Fig. 10). The other two pools, which were not sampled are represented Fetal Compartment k oa Fig. 10 Model of amino acid metabolism 98. by intracel lu lar free amino acid pool and body proteins. The transfer of amino acids from the maternal circulation across the placenta and from degra-dation of body protein through the intracel lular pool was assumed to be the sources of amino acids entering the blood pool. Calculations In experiments where [U- 1 4C] alanine and [2-3H] glucose were administered simultaneously, the conversion of alanine to lactate and to glucose was estimated by the ratio of integrals of the specif ic act iv i ty -time curves as described in Experiment II a. 1. Fraction of lactate C derived from alanine C ^ S.A. lactate C (dt) _ J o S.A. alanine C (dt) o 2. Rate of lactate C derived from alanine C (mgC/min/kg) = Equation 1 X lactate irreversible disposal rate 3. Percent of alanine C going to lactate C Equation 2 x 100 alanine irreversible disposal rate 4. Fraction of glucose C derived from alanine C S.A. glucose C(dt) 6 — 1 1 X s*. S.A. alanine C(dt) 5 J o 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 (mgC/min/kg) = Equation 4 X glucose irreversible disposal rate. where glucose irreversible disposal rate was determined simultaneously 3 using [2- H] glucose. 6. Per cent of alanine C going to glucose C Equation 5 Alanine irreversible disposal rate x 100 Additional calculations pertaining to the irreversible disposal rates and substrate recycling were performed as described in Experiment II a. Al l results are expressed as means (+ SEM). Stat ist ica l analysis of the data was performed by students independent t-test. Results The body weight of ewes and their 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 14 o or [U- C] alanine/[2- JH] glucose was injected. T a b l e 8 M a t e r n a l and f e t a l p h y s i o l o g i c a l p a r a m e t e r s d u r i n g t h e e x p e r i m e n t a l p e r i o d . I s o t o p e ( s ) i n j e c t e d P a r a m e t e r [ U - ^ C ] Amino A c i d M i x t u r e (n=4) [ U - 1 4 ] A l a n i n e and [ 2 - 3 H ] g l u c o s e (n=l0) M a t e r n a l F e t a l M a t e r n a l F e t a l G e s t a t i o n a l age ( d a y s ) 135 .6+1 .86 133 .8+6 .5 Body w e i g h t ( k g ) 6 2 . 9 6 + 3 . 3 0 2 .99+0 .18 6 1 . 3 0 + 2 . 4 0 2 .87 +0.19 P 0 2 (mm Hg) 3 3 . 1 0 + 0 . 9 0 2 0 . 0 9 + 1 . 0 4 35 .07+1 .10 19 .70+0 .78 P C 0 2 (mm Hg) 3 4 . 4 5 + 2 . 5 4 39 .10+1 .47 2 8 . 9 3 + 0 . 9 0 41 .30+1 .22 pH 7 .502+0 .01 7 .376+0.01 7 .479+0.01 7 .312+0 .22 T o t a l C O 2 (meq/L) - 2 3 . 5 1 + 0 . 5 2 - 23 .36+0 .74 H e m a t o c r i t (%) 3 3 . 1 0 + 0 . 8 7 3 4 . 5 0 + 1 . 2 9 32 .89+0 .69 36 .15+0 .76 Whole b l o o d a l a n i n e (mM) l a c t a t e (mM) g l u c o s e (mM) 3 . 7 4 + 0 . 0 3 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 P l a s m a P l a s m a P r o t e i n (mg/%) 7 . 0 3 + 0 . 1 3 3 .34+0 .26 6 .02+0.11 3 .77+0 .13 T o t a l c a r b o n (2) 0 . 1 9 + 0 . 0 2 0 .42+0 .02 - -V a l u e s = mean (+ SEM) m o l e s c a r b o n / l i t e r 101 Specific activity-time curves for the amino acid mixture and alanine are presented in Fig. 11. The observed data are best described by a three exponential equation with a corresponding mean R2 value of 0.986 and 0.988 for [u- 1 4C] amino acid mixtureand alanine respectively. The kin e t i c parameters obtained from the single injection of a mixture of amino acids and alanine are presented in Table 9. The metabolic half l i f e of the slow component of the amino acid mixture was s i g n i f i c a n t l y (P<0.05) higher than those observed with alanine. The half l i f e representing the fast component 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 less (P< 0.05) than that of alanine, indicating a more rapid removal from the primary free amino acid pool. The alanine pool size was 7.67*0.39 mgC/kg and was not sign-i f i c a n t l y d ifferent from the plasma pool size 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 fe 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 acid 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 respectively. The to t a l entry rate for 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 alanine. • The extent of recycling of the amino acid mixture and alanine was 85.3±0.03 and 71.1±0.05% respectively (Table 10). No s i g n i f i c a n t differences were noted between the mean total residence time for the amino acid mixture and alanine; 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 in the maternal ci c u l a t i o n following the adminstration of these labelled substrates. 102 "D < O c "E < lOOL U 0> ^ o t/5 1 4 9 U14CJ Alanine Ul4cl Amino Acid Mixture 9 * " 9 4 ? T 20 60 100 Time (min) 140 180 Fig. 11. Semi logarithmic plot of C amino acids and l 4C 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). Table 9 Kinetic parameters of amino acid metabolism in the ovine fetus in. utero . Labelled substrate injected Pool size (Q) (mgC/kg) Irreversible rate of disposal (D.R.) (mgC/min/kg) Total entry rate (TER) (mgC/min/kg) Volume of dist r i b u t i o n (v) [% Bwt) Half l i f e (min) T l T 2 T 3 [U- 1 4C] amino acid mixture (n=4) 8 . 2 5 a +1.15 ? . . 3 0 1 a +0.277 1 5 . 8 2 a +1.30 l a 0 . 3 0a +0.03 1 . 4 2 8 3 +0.47 81 +2 8 a 77 [ u - 1 4 c ] alanine (n=10) 7 . 6 7 a +0.39 2 . 0 2 1 a +0.34 8 . 6 9 b +1.14 6 6 . 0 5 b +4.09 0 . 8 8 b +0.14 2 . 9 9 b +0.42 52 +1 . 8 8 b . 78 Values = mean (+ SEM) a ' b Supercripts with different alphabets in the column denote s t a t i s t i c a l significance (P < 0 . 0 5 ) 104 Table 10 Recycling of amino acids and alanine in the ovine fetus in utero'. Parameter [U-1 4C] Amino Acid Mixture (n=4) [U- 1 4C] Alanine (n=10) Fraction of total turnover irreversibly lost 0.146 + 0.021 a 0.271 ± 0.041b Fraction of total turnover recycled 0.853 ± 0.0303 0.711 ± 0.052b Mean total residence time (T) (min) 7.22 ± 0.89 a 6.13 ± 1.72a Number of cycles (v) 6.46 ± 0.93a 3.27 ± 0.74 b 1 Values = means (± SEM) a ' b Superscripts with different alphabets in the rows denote stat is t ica l significance (P < 0.05) 105. Table 11 Conversion of alanine to lactate and glucose in the ovine fetus i n utero . Parameter 2 3 Alanine—> Lactate (precursor) (product) 2 4 Alanine Glucose (precursor) (product) % product C derived from alanine C^  27.07 ±4.85 4.11 ±1.09 Rate of alanine C g going to product C (mgC/min/kg) 0.696 ±0.131 0.161 ±0.040 % of alanine i r r e v e r s i b l e disposal going to product C? 34.95 ±4.40 8.85 ±0.36 1 Values = means (± SEM) o 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 ± 0.16 (mgC/min/kg) 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 ^ 106. In experiments where [U-^C] alanine and [2- 3H] glucose were injected simultaneously into the fetus, attempts were made to quantitate the conver-sion of alanine carbon into lactate 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 th i s experi-ment was 4.05 + 0.16 mgC/min/kg and compared favourably with the resu l t obtained i n Experiment II a. A rapid transfer of alanine [^C] was observed in lactate and glucose, with peak a c t i v i t y occurring 5-10 minutes after the injection of alanine. The proportion of lactate C derived from alanine C was 27.07 + 4.85% (Table 11). The rate of conversion of alanine C going to lactate 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 rate. The fraction 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 of glucose determined simultaneously using [2- H] glucose, the conversion of alanine C going to glucose C occurred at 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 rate. Discussion In this 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 in utero using isotopic d i l u t i o n techniques. The single in j e c t i o n of a mixture of [U- 1 4C] amino acids was used to estimate the disposal rates from the plasma free amino acid pool. The simultaneous inje c t i o n of [u- 1 4C] alanine and [2- 3H] glucose was made to quantitate the conversion of.alanine carbon to other compounds and i t s potential contribu-tion to gluconeogenesis. 107. Amino acid metabolism A major concern with the single injection or continuous infusion r 14 - i of a mixture of [U- C] amino acids i s that there are some amino acids that are not included in the mixture and yet account for a s i g n i f i c a n t proportion of ninhydrin positive compounds in plasma (Wolff and Bergman, 1972). Although glutamine, arginine, c i t r u l l i n e , onithine, N- and 3-methyl his t i d i n e and carnosine are excluded from the 15 amino acids injected into the f e t a l c i r c u l a t i o n , i t can be calculated from t h e i r umbilical venous-arterial difference and blood flow (Lemons e t al_., 1976), that these amino acids con-tribute to only 8% of the umbilical amino acid uptake. Thus, i t was assumed that the radioactive mixture used in this experiment closely represents the mixture of amino acids that are transferred to the fetus from the umbilical c i r c u l a t i o n . Multicompartmental analysis yielded three exponents which best described the s p e c i f i c activity-time curves of amino acid mixture and r-14 - i alanine. Similar results have been reported with a single [ C] amino acid injection by Henriques et al_. (1955) in the rabbit or a mixture of [^4C] amino acids by R e i l l y and Green (1975) in the rat. Though the pool size of alanine and amino acid mixture was si 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 similar to the value of 2% that can be calculated 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 equivalent to the total body water of the fetus. 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 in equal concentrations throughout the body water pool but rather are 108 concentrated in certain organs to a greater extent than in the blood (Munro, 1970). The use of whole blood for alanine s p e c i f i c a c t i v i t y determinations and plasma for amino acid mixture could have also contributed, to some extent to the observed differences in 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 mgC/ min/kg) was s i m i l a r to the umbilical 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 Prior and Christenson (1977) using continuous infusion procedures and 30% greater than Lemons et a l . (1976) using umbilical venous a r t e r i a l differences. The exchange of alanine [ 1 4C] atoms with [^C] atoms of intermediates formed from alanine would result in isotope d i l u t i o n and thus overestimate the disposal rate of alanine. This may not necessarily lead to a veno-arterial difference. The extent of recycling of amino acids observed in this experi-ment (Table 10) i s greater than the estimates of 36% reported in adult rats ( R e i l l y and Green, 1975). Wolff and Bergman (1972) have indicated that amino acid recycling involves the i n t r a c e l l u l a r pool and tissue pro-tein pool. It i s unlikely that the large amount of recycling observed during the 3-hour experimental period could be attributed to the protein pool, the fastest of which has been reported to have a half l i f e of approxi-mately 24 hours (Young, 1979). I t , therefore, appears that recycling involves the i n t r a c e l l u l a r tissue pool, which has been shown to turnover very rapidly in order to sustain the rapid growth rate of the fetus (Young, 1979). This i s further 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 lactate or glucose (Experiment II a), though the tot a l residence time of these compounds i s similar (Table 10). 109 I t has been shown that the placenta delivers to the mother a s i g n i f i c a n t amount of nitrogen derived from amino acids in the form of urea and ammonia (Gresham et al_., 1972a; Holzman et al_., 1979). The f a i l u r e to detect amino acid carbon r a d i o a c t i v i t y in the maternal c i r c u l a t i o n 14 following the inj e c t i o n of C amino acids to the fetus raises the question whether amino acids of f e t a l o r i g i n are excreted across the placental barrier into 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 results 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 lactate and glucose The use of integral equations and r a t i o of the area under the precursor-product s p e c i f i c activity-time curves were used in th i s study as a measure of quantitating the conversion of alanine carbon to lactate and glucose carbon in the ovine fetus. The proportion of alanine C going to lactate C (27%) compares favourably with the result (23%) reported by Prior and Christenson (1977), using a continuous infusion technique. Foster et al_. (1980) have recently shown in dogs that carboxyl carbon of alanine exchanges rapidly with lactate. The rapid exchange between alanine and lactate 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 in f e t a l tissues (Stevenson et al_., 1976) and contribution of alanine to oxidative metabolism i n the fetus. The evidence for the existence of gluconeogenesis in the ovine fetus has been a controversial topic. The presence of gluconeogenic no. enzymes in feta l ruminant l i v e r (Ballard et al_., 1965) and the sharp increases in phosphoenol pyruvate carboxykinase (EC. 4.1.1.3.2.) a c t i v i t y between 130-140 days of gestation in the ovine fetus (Warnes et a l . , 1977b) prompted many workers to find the functional significance of these enzymes. Anand et aj_. (1980.) recently reported that no gluconeogenesis was observed in the fetus following induced f e t a l hypoglycemia with i n s u l i n infusions. This may not be surprising, 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. Prior (1980) recently has reported, using a continuous infusion of [U-^C] lactate, that 22% of the glucose turnover was derived from lactate. In this 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 Prior and Christenson (1977) in the fetus and 3.5% by Brockman and Berman (1975) in the adult ewe. Anand and Sperling (1978) have proposed that the apparent gluconeogenic a c t i v i t y originating from [U- 1 4C] alanine, in the experiments of Prior and Christenson (1977) was due to the the [ l 4C] 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 [^4C] glucose. The fact that no [^4C] a c t i v i t y was detected in the maternal c i r c u l a t i o n following the injection of [U- 1 4C] alanine to the fetus in t h i s experiment, i s s u f f i c i e n t evidence to contradict t h i s hypothesis. The proportion of alanine C (8.8%) going to glucose C in this study when expressed as a % of the alanine i r r e v e r s i b l e disposal rate i s similar to the value (7-9%) obtained in adult monogastrics (Chockinov et a l . , 1978; Foster et aj_., 1980), but i s almost half as much (15-20%) reported in adult ruminants (Brockman and Bergman, 1975). A si m i l a r estimate (7.3%) was reported by Prior and Christenson (1977) in the ovine fetus in t h e i r I l l 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 ^ 2C atoms with 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-^4C] rather than [U-^C] lactate in this experiment may partly explain the lack of labell ing in glucose. As shown in a subsequent experiment (Expt III, p. 128 ), approxi-mately 63% of [ll-^C] lactate was found to be oxidized. The randomization of lactate carbons during passage through the Krebs cycle would reduce the extent of labell ing in glucose even i f lactate conversion to glucose did take place. These di lution effects are l ike ly to be more pronounced in single injection than continuous infusion studies. Further work is needed to c lar i fy 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. The specif ic activity-time data were f i t ted to a three exponential curve. Based on the rates of total entry and irreversible 112 disposal, 85% of the amino acid turnover was recycled in t h i s study. The r e l a t i v e impermeability of the placenta to amino acids may account for the amount of recycling observed in this study. The frequency at which amino acids returned to the blood pool was shown to be greater than other metabolic compounds; however the mean total 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 lactate and glucose C was determined. Based on the area under the s p e c i f i c activity-time curves of the precursor and the product i t was estimated that 8 and 27% of the irrever-s i b l e disposal rate of alanine C were converted to glucose C and lactate C respectively. Though th i s conversion indicates that the ovine fetus was capable of gluconeogenesis, the transfer of C atoms from alanine to glucose may not be a quantitative measure of the rate of gluconegenesis due to the 12 14 metabolic exchange of C atoms with C atoms in the oxaloacetate pool. 113 . Experiment III Measurement of carbon dioxide production and substrate oxidation by the ovine fetus in utero using [' 4C]-1abel1ed compounds Introduction The metabolic fuels for oxidative metabolism of the fetus have been reviewed extensively by Battaglia and Meschia (1978). Using the elegant technique of umbilical catheterization, 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, lactate and amino acids have been reported to contribute approximately 46, 20 and 25% respectively to the to t a l oxygen consumed by the ovine fetus. To provide a direct 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; Prior and Christenson, 1977; Anand et al_., 1979, 1980; Hodgson et jH. , 1980; P r i o r , 1980). However, quantitative measurements of carbon dioxide output from labelled substrates in chronically cathe-terized conceptus have not been reported. In the post-natal l i f e the 14 14 excretion of CO2 in breath following the administration of 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 spe c i f i c r a d i o a c t i v i t y of CO2 in blood, urine or expired a i r (Whitelaw, 1974). A high correlation between the s p e c i f i c a c t i v i t y 14 14 of plasma CO2 and respiratory C02>during the oxidation of labelled substrates in man was reported recently by Clague and Keir (1979). This 114 coupled with the fact that f e t a l respiration 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 in f e t a l blood. The measurement of f e t a l C 0 2 production rates and the quantitation of the oxidation of substrates, by the ovine fetoplacental tissues in^utero were investigated in this experiment. Materials and Methods Animals The animals, surgery and the maintenance practices have been described previously. In experiments where [U- 1 4C] NaHCO^ was infused, two catheters were introduced into the f e t a l c i r c u l a t i o n . The catheter used for sampling f e t a l blood was introduced to a distance of 6 - 8 cm into the external saphenous vein in one of the hind limbs. Another catheter was passed deeply into the external saphenous vein in the other leg for infusing radioactive compounds. The t i p of this catheter was found at autopsy to l i e in 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 tracer experiments were undertaken. Cannulae were also placed i n the jugular vein of the ewes one day prior to the experiment. Metabolic Studies A) Irreversible 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 infusion technique. The priming dose consisted 115. 14 of 30 uCi of NaH CO^ followed by a continuous infusion at the rate of 14 0.5 uCi/min. Solutions of NaH C0 3 were prepared in s t e r i l e saline and made s l i g h t l y alkaline by addition of 0.1 N NaOH. Fetal and maternal blood samples for C0 2 and s p e c i f i c a c t i v i t y determination were taken at 30 minute intervals prior to and during the period of isotope equilibrium. The t o t a l C0 2 content of whole blood was calculated using the Henderson-Hasselbalch equa-tion from PC0 2 and pH values which were determined within 5 min after 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 C 0 2 in blood was deter-mined by adding 0.5 ml of 6 M perchloric acid from the side arm of Warburg 14 flasks to 0.5 ml of whole blood in the main chamber. The C0 2 which was released was trapped in hyamine hydroxide (Don M i l l s , Ont.) placed i n the central w e l l . After standing for 1 hour at 25°C an aliquot 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, Oakvil 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 1 4C0 3 added to blood (Appendix Table 4). To correct for the retention of 1 4 C 0 2 in slowly mixing pools such as bone, NaH^CO^ was 14 injected into 3 fetuses in separate experiments. Blood C0 2 r a d i o a c t i v i t y was determined at intervals 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 in the entire 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 plot of tota 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 di f f e r e n t 14 times was computed and expressed as a per cent of administered NaH CO^  14 appearing i n C0 2 > 116. Oxidation of substrates The oxidation of substrates was determined in conjunction 14 with experiments II a and b. Approximately 50 uCi of C labelled glucose, lactate, alanine, amino acid mixture and acetate were injected as a bolus 14 into the f e t a l c i r c u l a t i o n and the speci f i c r a d i o a c t i v i t y of blood C0 2 was monitored. The spec i f i c a c t i v i t y of the labelled substrates in blood was determined by ion-exchange chromatography as described in Experiment I I . Normalized s p e c i f i c radioactivity-time curves of labelled precursors 14 and C0 2 in blood were described by biexponential equations. I n i t i a l estimates of kinetic parameters were calculated using AUTOAN Fortran computer program and the f i n a l least squares f i t t i n g of the data was done using NONLIN (Michigan), as described in Experiment II a. Radiochemicals [U- 1 4C] acetate (51 mCi/mmole) was obtained from ICN Pharmaceu-t i c a l s , Montreal. NaH 1 4C0 3 (58 mCi/mmole) was supplied by Amersham 14 Corp., Oakville, Ont. The details of C-glucose, lactate, alanine and amino acid mixture are given in Experiment II a and b. Calculations The i r r e v e r s i b l e disposal rate of carbon dioxide was determined 14 using a continuous infusion of NaH CO^. Rate of NaH 1 4C0 3 infusion (nCi/min) 1. Irreversible disposal rate of C09= — . mj_ (mgC/min/kg) Blood I HC0 2 plateau s P e c i f l ^ n j ^ ^ y 117, The rate of oxidation of substrates was calculated by 2 procedures following single injections of labelled components: A) From the isotopic y i e l d i n the product (Heath and Barton, 1973). ( i ) Isotopic y i e l d in product (CO^) = Irreversible disposal rate of C0 2 x ( i i ) Fraction of substrate oxidized = Isotopic y i e l d in C0 2(uCi) substrate injected (uCi) ( i i i ) Rate of substrate oxidation (mgC/min/kg) Irreversible disposal rate of substrate x Fraction of substrate oxidized. where, Aps = Area under the s p e c i f i c radioactivity-time curve in the product (C0 2) following injection of labelled substrates. Dose = yXi of substrate injected. B) From carbon transfer quotient r a t i o (Kleiber et al_., 1956). ( i ) Rate of substrate oxidation Fraction of C0 2 from substrate (mgC/min/kg) oxidation x Irreversible dis-posal rate of C0 2 ( i i ) Fraction of C0 2 from substrate oxidation ( i i i ) Per cent C0 2 derived from substrate 1 4 C 0 9 s p e c i f i c a c t i v i t y (dt) £ 14 C-substrate s p e c i f i c a c t i v i t y o (dt) Rate of substrate oxidation Substrate Irreversible disposal rate -x 1 / 118. 3. Time (Tmax) for s p e c i f i c a c t i v i t y of blood '^ CG^  to reach a maximum following injection of labelled substrates was estimated as follows: T m a x ( 1 4 C 0 ? ) = • log 2 where • 14 a and B are formation and elimination rate constants of CO^ sp e c i f i c activity-time curves (Ritschel, 1976) Data were subjected to an analysis of variance and where a si g n i f i c a n t difference was notec means were compared using Newmann Keuls multiple range test. Linear regression lines and correlation coefficients were calculated by the method of least 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 in Experiment I I , several measurements of total CO^ concentration i n fe t a l whole blood were made. The total CO^ content of fe t a l venous whole blood ranged from 22 to 24 meq/liter in a l l experiments. Changes in the s p e c i f i c a c t i v i t y of fe t a l and maternal blood 14 14 C O 2 with time following primed dose-continuous infusion of NaH CO^ 14 are shown in Figure 12. The sp e c i f i c a c t i v i t y of fe t a l blood. C O 2 reached a plateau approximately 90 minutes after the infusion commenced and remained constant thereafter. The mean i r r e v e r s i b l e disposal rate Table 12 Substrate oxidation rates in the ovine fetus in utero . % Substrate Oxidized Rate of Oxidation (mgC/min/kg) % C0 2 from Substrate Substrate (min) Procedure I.Y. T.Q. Procedure I.Y. T.Q. Procedure I.Y. T.Q. Glucose (N = 8) 19.60 ± 0.15a 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 ± 0.85a 28.52 ± 3.99 26.93 ± 3.08 0.549 ± 0.09 0.511 ± 0.09 6.49 ± 1.4 6.82 ± 1.2 Amino acid mixture (N = 4) 9.30 ± 1.00c -23.55 ± 3.22 -0.59 ± 0.09 -8.19 ± 0.9 2 Acetate (N = 5) 10.30 ± 1.39c 15.82 ± 2 2.81 -0.10 ± 2 0.20 J 1.70 ± 0.2 -a' b' c Supercripts with different alphabets in the column denote s t a t i s t i c a l significance P< 0.05. 1 Values ± standard error 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—*—\ X J 30 60 90 120 150 TIME (minutes) 180 210 240 Fig. 12 Specific r a d i o a c t i v i t y of fetal and maternal blood , 4 C 0 2 following orimed dose-infusion of NaH'^CG^. 121. of carbon dioxide in 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 retention of CO2 in slowly mixing pools, i t was found that only 82.5 ± 5.4% of the adminis-14 tered r a d i o a c t i v i t y in ,NaH COg was recovered in f e t a l blood at the end of 3 h (Fig. 13). Correction factors were therefore applied to the values 14 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 CO2 when C-labelled substrates were 14 injected intravenously. The s p e c i f i c a c t i v i t y of maternal blood CO2 closely followed the increase in the fetus and reached a plateau after 90 minutes (Fig. 12). At steady state conditions, the r a t i o of the speci-14 f i c a c t i v i t y of maternal and f e t a l blood CO2 was 0.14 ± .01 indicating the 14% of maternal CO2 production can be accounted for by the placental transfer of CO2. 14 The changes in the s p e c i f i c a c t i v i t y of blood CO2 following 14 the injection of C-labelled substrates are shown in Fig. 14. The speci-14 f i c a c t i v i t y of blood CO2 reached a maximum within the f i r s t 3 minutes (Tmax) of lactate 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 in 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 after the in j e c t i o n of labelled glucose, alanine, acetate and amino acid mix-14 ture respectively. Secondly, the peak s p e c i f i c a c t i v i t y of blood CO2 was also higher (P< 0.05) in lactate than in a l l other substrates studied. The rates of substrate oxidation as well as t h e i r contribution to tot a l carbon dioxide production calculated 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). I t i s noteworthy that, on an average only 30.60 and 36.48% of glucose and lactate carbons respectively appeared in CO2. The extent of oxidation of alanine or amino acid carbons was 27.73 and 23.55% respectively. The 1-22. 90 80 60 •o w s O V tr o 40 20 1 30 60 __i —i •— 90 120 150 Time (minutes) 180 Fig. 13 Recovery of r a d i o a c t i v i t y of blood 1 4 C 0 2 after single injection of NaH^CC^. •032 r L T I M E Imin) Fig. 14 Specific r a d i o a c t i v i t y (S.A.) of blood 1 4C0? after injection of 1 4C-labe11ed ^ b s t r a t e s into f e t a l c i r c u l a t i o n . (L=l- l 4C-lactate; A=U-14C-acetate; Al=U-14 C-alamne; G=U-^C-glucose; AA=U-14C-amino acid mixture. The curves are reproduced from a Calcomp plotter. 1-8 r Irreversible disposal rate.(mgC/min/Kg) Pia is Relationship between rates of oxidation and ir r e v e r s i b l e disposal rates of glucose and lactate (•, 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 oxidation 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 contributing to 1.70% of t o t a l C0 2 production. The linear regression of rates of glucose and lactate oxidation on the i r r e v e r s i b l e rates of disposal i s shown in Fig. 15. Discussion C0 2 Disposal in 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 con-tents reported by Setchell et (1972) using isotopic tracers and 14.2 ml/min/kg by Graham (1964) using calorimetric techniques. In this study attempts were not made to cannulate the umbilical 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 contribution 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 single injection techniques was found to be unsatisfactory. 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 intercept describing t h i s pool were sub-jected to considerable error. The C0 2 production rates obtained from the 14 continuous infusion of NaH C0 3 are s l i g h t l y higher than the value of 11.19 ml/min/kg for the fetus plus the utero-placental tissues e s t i -mated from uterine and umbilical arterio-venous differences coupled with blood flowUMeschia et j i l _ . , 1980). The l a t t e r procedure does not take into account the retention and u t i l i z a t i o n of metabolic C0 ? by the tissues 126. and i s l i k e l y to underestimate true production. In this study, the C0 2 disposal rates have been corrected for these factors which results in s l i g h t l y 14 higher values. The retention of 17.5% of C0 2 in f e t a l tissues during 14 the administration of NaH C0 3 closely resembles the values of 17-20% in adult sheep (Bergman and Hogue, 1967; Annison et a]_., 1967). On the other hand, in 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 by calorimetric techniques (Whitelaw, 1974). This has been ascribed to low s p e c i f i c a c t i v i t i e s resulting from slowly mixing pools and inadequate length of infusion. Though a plateau 14 14 of C0 2 s p e c i f i c a c t i v i t y was reached within 90 min, the NaH CO3 infus-ions were continued for 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 indicating that the duration of infusion i s not a major factor for 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 2 may be obtained from the r a t i o (0.14) 14 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 after a plateau has been reached (Fig. 12). Assuming a maternal C0 2 pro-duction rate of 362 ml/min during late pregnancy (Whitelaw et aj_., 1972), the rate of placental transfer 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 calculated from the f e t a l com-partment 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 labelled lactate than from labelled glucose are si m i l a r to the findings of Shambaugh et jil.(1977a) in the tissues of f e t a l rats under in v i t r o 127. conditions. In one experiment where DJ- C]-lactate was injected instead of D-^C]-lactate, the rate of oxidation was 1.367 mgC/min/kg contributing to 18.94% of C0 2 production. This indicates that the higher rate of C0 2 production from lactate than from glucose i s not merely due to preferen-t i a l oxidation of C-l of lactate. The absence of hepatic glucokinase in the fetus (Ballard e_t al_., 1969) and the oxidation of glucose through an intermediary pool such as lactate having more carbon than the glucose pool (Table 5 ; Warnes et j f L , 1977a) may explain the slower C0 2 release from glucose than from lactate. Two methods of calculation were used to quantitate the transfer of substrate carbon to CG^-carbon. In the isotopic y i e l d procedure (Heath and Barton, 1973) the tot a l recovery of labelled carbon i n the product i s measured. This would give an estimation of the t o t a l f r action of the substrate oxidized by direct and indirect 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 oxidized, the rate of oxidation and contribution to C0 2 production are calculated. The transfer quotient r a t i o expresses the transfer of carbon between the precursor and the product but i s independent on the number and size of intermediary pools and rates of intermediary pathways (Kleiber et al_., 1956; Searle et a l . , 1975). I t i s of interest that the rate of substrate oxidation and c o n t r i -bution to C0 2 production calculated by the two procedures (Table 12) were very close to each other. A p a r t i c u l a r l y s i g n i f i c a n t finding in t h i s study pertains to the extent of substrate oxidation and contribution to C0 2 production. Oxidation accounts only for 15.80 - 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 single experiment 128. where LU-l4C>lactate was injected'62.3% was found to be oxidized. Even these values may be considered to be a maximum since oxidation rates based on 1 4C02 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 tissues i s u t i l i z e d for anabolic purposes than for oxidative metabolism. The positive relationship between oxidation rates and i r r e v e r s i b l e rates of disposal (Fig. 15) indicates 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 tissues depends largely on the materno-fetal transfer confirm-ing previous reports (Boyd e_t _al_. , 1973). The fact that lactate may be formed from glucose in the placenta (Experiment II a; Warnes et al_., 1977a) may explain the s i m i l a r i t y i n the oxidative metabolism of these compounds in r e l a t i o n to th e i r i r r e v e r s i b l e disposal rates. The fraction of total CO^ derived from the oxidation of glucose (15.0%) i s simi l a r to the value of 22% obtained by Setchell et al_. (1972) in f e t a l sheep using tracer techniques. Kronfeld and Van Soest (1976) have summarized available data on substrate contribution to C0 2 production and have concluded that approximately 8-11% of CO2 was derived from oxida-tion of glucose i n ruminants and 10-50% i n non-ruminants. The mean value of 15% contribution by glucose to to t a l C0 2 production i s within the range reported for adult monogastric animals. Though the re l a t i v e importance of various substrates to overall f e t a l oxidative metabolism would depend on the metabolic fuel mixture a v a i l -able (Shambaugh et £l_., 1977a,b) contribution of lactate to CO2 production at a level equal to that of glucose indicates that the fetus r e l i e s on other 129. substrates, in addition to glucose, for oxidation. A major proportion of glucose carbon i s therefore used for alternative purposes. A similar explanation would hold good for the small contribution (8.19%) of amino acid carbon to CO^ production. It i s also noteworthy that, based on the direct oxidation of the substrate used in this study, approximately 40% of total CO2 production by the fetoplacental tissues can be detected. This suggests that, in addition to glucose, lactate, amino acids and acetate, other substrate(s) may be involved in f e t a l metabolism. Though the present experiment have been designed to study the oxidative metabolism of the fetoplacental unit as a whole i t i s possible to obtain quantitative informa-tion on the unidirectional metabolism of the fetus and placenta separately by the simultaneous use of isotopic tracers and umbilical venoarterial concentration differences coupled with blood flow. This approach has been used in the metabolism of l i v e r (Brockman and Bergman, 1975). Conclusion Experiment III 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 in the ovine fetus . in utero. The rate of CO2 production noted in this 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 labelled bicarbonate infused into the f e t a l c i r c u l a t i o n was collected in the mother. It was noted that placental transfer of C0 2 resembled the C0 2 production rate from the f e t a l placental compartment alone. 130. From the results of the oxidation of sp 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 for by oxidation. 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 available for synthetic purposes than was hitherto reported. Using both the isotopic y i e l d and transfer quotient procedures to estimate the per cent of CO2 produced from these substrates, i t was concluded that other substrate(s) contribute to the total f e t a l oxidative metabolism. Although the procedure used in th i s study estimates the oxidative metabolism of the entire fetoplacental unit rather than the fetus i t s e l f , the fact 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 quantitative contribution of substrates to total oxidative metabolism should be reassessed. 131. General Conclusions Surgical techniques were developed and standardized for introducing vascular catheters into the fetus so that the metabolism of substrates may be studied in vivo without stress. 14 3 Using single injections of [U- C] and [2- H] glucose the irre v e r -s i b l e disposal rates, pool s i z e , volume of d i s t r i b u t i o n and other ki 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 related 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 this 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 in fe t a l body mass. Glucose label-recirculated 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 in the maternal c i r c u l a t i o n following injection into the fetus, indicating a low permeability of lactate in the ovine placenta. Recycling of glucose and lactate in the fetus occurred to an extent of 70-80% of the total turnover of these compounds and i t was concluded that this was an important feature of fe t a l glucose and lactate metabolism. • Studies on the metabolism of a mixture, of amino acids and alanine were performed to quantitate further the u t i l i z a t i o n of amino acids by the fetoplacental unit. The rate of i r r e v e r s i b l e disposal of [U- l 4C] amino acid mixture was si m i l a r to that of [U- 1 4C] glucose and [1-^C] lacta t e ; how-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- 1 4C] alanine, i t was calculated 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 circum-stances under which gluconeogenesis is manifested. Carbon dioxide rates were determined by a primed dose, continu-ous infusion technique. It was concluded from the ratio of fetal to maternal 14 C0 2 specif ic act iv i ty , measured at plateau specif ic act iv i ty that 14% of the maternal C0 2 production rate could be accounted for by fetal and placental metabolism. In studies where substrate oxidation was performed in conjunc-tion with the determination of the irreversible disposal rates, i t was shown that only 30, 6.2, 24, 15 and 26% of [U- 1 4C] glucose, lactate, amino acids, acetate and alanine were oxidized to CO,,. Substrate oxidation observed in this study accounts for only .40% of the total C0 2 production of the feto-placental t issues. The total inflow and outflow of carbon in the fetoplacental unit were estimated from the rates of irreversible disposal, oxidation and interconversion among metabolites and are presented in Fig. 16. Using the values indicated in Fig.16 with respect to oxidation and interconversion, the amount of substrate carbon used for anabolic purposes may be estimated. For the purpose of these calculat ions, the following substrates were assumed to be the major ones involved in fetal metabolism (Battaglia and Meschia, 1978). 133. Fig. 16 Composite picture of f e t a l substrate metabolism. Values inside brackets denote ml/min/Kg and those outside mgC/min/Kg. 134. Carbon balance in the fetus Total Carbon Glucose Lactate Amino Acids mgC/min/kg Irreversible disposal rate (mgC/min/kg) (A) 3.541 2.171 2.301 8.013 Oxidation rate (mgC/min/kg) Other disposals (mgC/min/kg) (B) 1.087 1.353 to fructose 3 0.576 to mother 1.771 0.543 to urea 0.085 2.983 2.432 Used for synthesis (mgC/min/kg) (C = A-B) 0.110 0.821 1.674 2.598 or 3.741 gC/day/kg 1 [U- C] lactate 2 1 mole amino acid yields 0.69mole urea (Schulz, 1978) Warnes et a l . (1977a) The net retension of carbon in the fetal 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 for synthesis of fetal t issues. 135. It i s also pertinent to discuss the contribution of substrate oxidation to the overall c a l o r i c requirement of the fetus. Depending on the technique employed, heat production in the fetus has been reported to be 42-52 (James et al_., 1972; Meschia et al_. ,1967a, oxygen consumption), 65 (Abrams et a]_., 1970., d i f f e r e n t i a l spirometry) and 90 kcal/day/kg (Graham, 1964, in d i r e c t calorimetry). The minimum oxygen consumption needed for the proportions of substrates found to be oxidized in this 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 cal/ml, t h i s would result in the production of 45 kcal/day/kg from oxidative metabolism. In addition energy i s also stored in the form of new tissues to the extent of 32 kcal/day/kg (Rattray et al_., 1974). Thus, the tota 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 in the middle of the range reported in the l i t e r a t u r e . This may suggest that oxidative metabolism alone i s not s u f f i c i e n t to account for the tota l c a l o r i c requirement of the fetus. On the other hand, the oxygen uptake of the fetoplacental unit i s approximately 11-12 ml/min/kg (Meschia et a l . , 1980) and would resu l t in the production of 78-85 kcal/day/kg from oxidative 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 fetus. The oxygen consumption of 6.41 ml/min/kg used i n the present calculations represents a minimal value to oxidize only those substrates studied in this investigation. Therefore, i t i s suggestive that other substrate(s) may be involved in f e t a l oxidative metabolism. In spite of the f a i l u r e to id 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 interest that, in t h e i r review of f e t a l metabolism, Battaglia and Meschia (1978) have concluded that "only a negligible amount of heat liberated 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 tual ly a l l the energy used to fuel fetal oxygen consumption is ultimately dissipated as heat". 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 differentiat ion and maintenance in the prenatal period may be dissipated as heat (Brody, 1945) i t does not imply that v i r tual ly 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 ut i l izat ion 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 relat ively very high and increases with net increase in tissue mass. Conclusions based on the Fick principle tend to underestimate the true ut i l izat ion of metabolites by the fetus. On the other hand, tracer techniques, part icular ly the single injection procedure, may overestimate disposal rates of substrates. Therefore isotopic tracer techniques con-ducted in conjunction with umbilical veno-arterial differences of metabolites and blood gases would appear to be more appropriate that either technique alone in assessing fetal metabolic requirements. 137. Bibliography Abrams, R., Caton, D., CIapp, J. and Barron, D.H. Thermal and metabolic features of l i f e in utero. C l i n . Obstet. Gynecol. 13:549-564, 1970. 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On the calculation of turnover time and turnover rate from experiments involving the use of label l ina agents. J . Gen. Physiol. 26:325-332, 1943. 158. Appendix Validation of Methods The following procedures were used for the separation and quantitation of labelled metabolites: 1. Isolation and recovery of glucose. Glucose s p e c i f i c a c t i v i t y measurements have routinely been performed by the formation of glucose derivatives. However, due to the nonspecificity of these procedures and the r e l a t i v e l y high concentration of fructose in f e t a l blood, anion exchange chromatographic procedure followed by the conversion of glucose to gluconic acid enzymatically was standardized. Glucose, in the presence of other neutrally charged metabolites was converted to gluconic acid by an excess of glucose oxidase and catalase and the resulting mixture separated by anion exchange chromatography. To test the purity and recovery of glucose i n t h i s procedure, aliquots of the neutral and acidic fractions were applied to paper chromatograms (descending) and developed in a solvent consisting of phenol:H20::NH3; 40:40:1; w/v/v for 18 hours. The separation of labelled glucose from fructose i s given in Appendix Fig. l . The recovery of labelled compounds added to the column i s given in Appendix Table 1 . The modification of the glucose oxidase procedure was as follows: 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 units/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 units/ mg, Sigma, St. Louis) in 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 ed 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 lactate. 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 particular 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 latter through a cation exchange column. Results are given in Appendix Table 3. 4. Recovery of 1 4C02-The oxidation of labelled substrates was monitored by measuring 1 4C02 in whole blood. Whole blood was ac idi f ied with perchloric acid and the liberated l^COg was collected in Hyamine Hydroxide. Results are given in Appendix Table 4. 5. Amino acid mixture (15 L-amino acids, in same proportions as a typical algal protein hydrolysate) - glycine, alanine, serine, threonine, proline, valine, isoleucine, leucine, phenylalanine, tyrosine, aspartic acid, glutamic acid and lysine. Table 1 : Per cent recovery of relabelled compounds after treatment with glucose oxidase and anion-exchange chromatography! 14 C-metabolite Init ia l Activity (Dpm) Neutral Fraction (Dpm) Recovery Acidic Fraction (Dpm) Recovery (%) l- 1 4C]gluconic acid (N = 6) 19.432 ± 241 - -19,093 ±259 98.11 ±0.91 14 2 U- C]glucose (N = 10) . 161,120 ± 398 2,509 ±167 1.55 ±0.34 148,484 ±1196 92.09 ±0.71 2-3H]glucose (N = 6) 42,180 ±11.82 210 ±21 .50 ±0.02 39,793 ±665.06 93.72 ±1.56 U- 1 4C]fructose 2 (N = 6) 78,388 ±618 77,119 ±667 98.38 ±0.46 - -U- 1 4C]glycerol (N = 6) 59,702 ±8.84 56,564 ±1,431 96.99 ±2.47 -» Values = means (± SEM) Rf values are shown in Appendix Fig 1 161. Table 2: Per cent recovery of 1- C lactate following anion exchange chromatography and thin layer chromatography'. Procedure I n i t i a l A c t i v i t y Dpm Recovery (%) Contamination from glucose (%) Anion Exchange chromatography 15,669 ±252 93.09 ±0.50 N.D. Thin Layer Chromatography (n-propanol:acetone:H 20 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. 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 lactate 1 . Procedure Neutral Recovery in Fraction Basic Acidic Cation exchange chromatography 1) Amino Acid mixture (N = 12) N.D. 99.1 ±0.96 N.D. 2) Alanine (N = 12) N.D. 90.3 ±2.46 N.D. Cation exchange and enzymatic conversion of alanine to lactate (N = 12) N.D. 1.2 ±0.09 82.4 ±0.64 Values = means (± SEM) 163. Table 4: Per cent recovery [ C] NaHCCu in saline and whole blood. I n i t i a l A c t i v i t y A c t i v i t y recovered % Recovered Vehicle (Dpm) in Hyamine Hydroxide (Dpm) Saline 168,000 ± 281 154,768 ± 201 91.4 ± 0.8 (n = 8) Whole 92.3 ± 1.4 Blood 171,080 ± 310 159,446 ± 240 (n = 8) Values = mean (± SEM) Rf=0.39 Rf s Q 5 6 Appendix f i g . 1 Separation of metabolites by descending paper chromatography g (phenol:water:NH3; 40/40/1; w/v/v.) 1 6 5 . 166. 200 600 1000 Total Carbon Cone. (mg./L) Appendix f i g . 3 Standard curve for total organic carbon determined by infra-red carbon analyzer. Appendix table 5 . Mean P 0 2 , P C O 2 and pH in maternal and fetal blood of conscious ewes during and f o i l owing surgery 1 Days Afte r Surgery Parameter 0 1 2 3 4 5 6 7 8 9 pH M 7.25S3 ±0.02 (5) 7 .452 B ±0.02 (7) 7.439 ±0.02 (11) 7.438 +0.01 (11) 7.451 ±0.01 ( I D 7.450 ±0.01 ( I D 7.445 ±0.01 (9) 7.415 ±0.01 (10) 7.401 ±0.03 (5) 7.467 ±0.021 (7) F 7.277° ±0.02 (5) 7 .348 D ±0.02 (8) 7.371 ±0.02 (11) 7.338 ±0.02 (11) 7.358 ±0.02 (11) 7.353 ±0.02 (13) 7.310 ±0.02 (9) 7.349 ±0.02 (10) 7.311 ±0.01 (4) 7.337 ±0.02 (6) M 39.18* ±2.20 (5) 3 5 . 8 5 F ±0.625 (8) 32.76 ±1.40 (11) 32.37 ±2.91 (11) 30.10 ±1.29 ( I D 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) PCO 2 (mm Hg) F 35.409 ± 0.9 (5) 4 1 . 7 5 H ±1.05 (7) 39.99 +1.03 (10) 41.57 ±1.02 (11) 40.58 ±0.77 (11) 40.51 +1.10 (13) 42.76 ±0.27 (9) 42.78 ±0.58 (10) 41.61 ±2.17 (6) M 104.670I ±8.20 (5) 42 .072 J ±1.85 (7) 40.334 ±2.11 (11) 42.993 ±1.12 (9) 43.676 +1.95 (10) 41.552 ±1.33 (12) 43.727 11.50 (8) 47.754 ±1.40 (8) 45.301 ±1.22 (4) 43.917 ±3.64 (6) PO 2 (mm Hg) F 25 .800 K ±0.76 • (5) 20.183 1 +0.78 (8) 22.061 ±0.624 (11) 22.037 ±0.94 (9) 21.808 ±0.76 (10) 21.866 ±1.07 (12) 22.292 ±1.153 (8) 17.480 ±0.623 (8) 19.341 ±1.29 (4) 20.294 ±0.844 (6) Data presented as means ± S.E.M. Values in parenthesis are number of observations T a b l e 6 : Mean hematocrit, blood glucose, lactate, 3-hydroxybutyrate and alpha amino nitrogen in fetal blood of conscious ewes.1 Days After Surgery 0 1 2 3 4 5 6 7 8 9 Hematocrit (%) 38.2 r 1.02 "(5) 37.83 r 1.3 "(8). 35.10 ^ 1.10 "(11) 33.72 r 1.00 "(ID 34.80 ^ 1.12 "(11) 34.05 r 1.12 "(13) 34.50 i- 1.25 "(9) 33.50 + 1.25 (10) 34.64 + 1.26 "(4) 34.00 + 1.50 16) Glucose * (mM) + 0.602 0.08 (3) + 0.602 0.02 (5) + 0.710 0.02 (4) + 0.731 0.02 (5) + 0.901 0.02 (5) . + 0.810 0.02 (4) + 0.880 0.13 (4) - 0.901 + 0.02 " (4) 0.870 + 0.02 " (4) * Lactate (mM) + 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) - 1.66 + 0.03 ~ (4) 1.70 + 0.04 " (4) * 8-Hydroxy-butyrate (mM) + 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) — 0.173 + 0.002 " (4) 0.159 + 0.001 ~ (4) * Alpha amino nitrogen (mg/100 ml) + 15.76 3.20 (3) + 14.40 0.71 (4) + 12.43 0.27 (4) + 11.02 0.45 (5) + 12.11 0.16 (5) + 11.39 0.94 (4) + 10.98 0.39 (4) — 10.48 + 0.10 " (4) 10.57 + 1.06 " (4) Data presented as means ± S.E.M. Values in parenthesis are number of observations * Differences between days 1-4 and 5-9 are s ta t is t i ca l l y significant (P < 0.05). 

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