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The effects of the beta-adrenergic agonist, ritodrine, in the fetal lamb Van der Weyde, Marlene P. 1990

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THE EFFECTS OF THE BETA-ADRENERGIC AGONIST, RITODRINE, IN THE FETAL LAMB By MARLENE P. VAN DER WEYDE B.Sc, Simon Fraser University, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in. THE FACULTY OF GRADUATE STUDIES (Faculty of Medicine Department of Obstetrics and Gynaecology Human Reproductive Biology Programme) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1990 © Marlene P. van der Weyde, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date fop, 3 . mO DE-6 (2/88) ABSTRACT Ritodrine i s a beta-adrenergic agonist commonly used to i n h i b i t premature labor contractions i n women. The primary goal of r i t o d r i n e t o c o l y s i s i s to prolong gestation, however, other indications may include f e t a l d i s t r e s s . The purpose of the current study was to examine the metabolic and behavioral e f f e c t s of r i t o d r i n e i n the fetus, using the chronically instrumented pregnant sheep as an experimental model. Ritodrine was infused continuously into 11 f e t a l lambs at a rate of 2.6 ug/minute for a period of 8, 12 or 24 hours. Samples were taken simultaneously at predetermined time periods from a f e t a l femoral artery, umbilical vein, maternal femoral artery and uterine vein for the analysis of f e t a l and maternal a r t e r i a l and umbilical and uterine venous blood gases, acid-base status, hematocrit, r i t o d r i n e concentration, uterine and umbilical blood flow, and glucose, lactate and oxygen concentrations and fluxes. Cardiovascular and behavioral variables were monitored continuously. The average concentration of r i t o d r i n e i n f e t a l a r t e r i a l plasma was 20.0 ± 2.7 ng/ml (range 9.5 to 3 4.7 ng/ml) at the end of the infusion. This concentration i s within the range of cord lev e l s obtained i n r i t o d r i n e exposed human fetuses at b i r t h (7 to 79 ng/iriL) . Fetal a r t e r i a l plasma r i t o d r i n e l e v e l s at 8 hours post-infusion were s t i l l s u f f i c i e n t l y elevated to exert considerable f e t a l e f f e c t s . The apparent tolerance of the fetus to given plasma leve l s of drug varied considerably among animals. The infusion of r i t o d r i n e resulted i n many t y p i c a l beta-adrenergic receptor mediated responses i n the fetus. Fetal a r t e r i a l glucose l e v e l s rose to 79% above the control by the end of the infusion. This was associated with an increase i n f e t a l glucose delivery (70% above the contro l ) , a decrease i n the umbilical veno-arterial glucose concentration difference and a tendency for f e t a l glucose uptake to decline. Fetal a r t e r i a l plasma lactate concentrations rose more than f i v e -f o l d during the infusion of r i t o d r i n e . This was associated with a r i s e i n f e t a l lactate delivery (540% above the cont r o l ) , a s l i g h t increase i n the umbilical veno-arterial plasma lactate concentration difference and a tendency for f e t a l lactate uptake to r i s e . Fetal oxygen consumption rose progressively and s i g n i f i c a n t l y throughout the infusion of r i t o d r i n e and during the f i r s t 8 hours of post-infusion, reaching a maximum of 22% above the control by 8 hours post-infusion. Umbilical blood flow remained unchanged, therefore umbilical oxygen delivery was not increased to meet the additional oxygen demands of the i v f e t u s . The r i s e i n f e t a l oxygen consumption was hence accomplished through an i n c r e a s e i n f e t a l f r a c t i o n a l oxygen e x t r a c t i o n (from a c o n t r o l value of 3 2 . O i l . 1 % to a maximum of 51.6±1.8% by 1.5 hours of i n f u s i o n ) . The r i s e i n f e t a l oxygen e x t r a c t i o n r e s u l t e d i n c o n current d e c l i n e s i n f e t a l a r t e r i a l Po 2 (78% of the c o n t r o l ) and 0 2 content (55% of the c o n t r o l ) and a widening of the v e n o - a r t e r i a l oxygen content d i f f e r e n c e . By the end of the i n f u s i o n , u m b i l i c a l venous Po 2 and 0 2 content v a l u e s had a l s o f a l l e n s i g n i f i c a n t l y to 78% and 75% of the c o n t r o l r e s p e c t i v e l y . These l a t t e r changes r e s u l t e d i n a c o n c u r r e n t 25% d e c l i n e i n f e t a l oxygen d e l i v e r y which i n t u r n c o n t r i b u t e d to the r i s e i n f e t a l oxygen e x t r a c t i o n . F e t a l a r t e r i a l and u m b i l i c a l venous pH d e c l i n e d r a p i d l y and s i g n i f i c a n t l y from c o n t r o l v a l u e s of 7.370±0.004 and 7.401±0.005 t o 7.274±0.025 and 7.306±0.023 r e s p e c t i v e l y by the end of the i n f u s i o n . The acidemia was r e f l e c t e d by s i g n i f i c a n t d e c l i n e s i n base excess v a l u e s and appeared t o be e n t i r e l y m e t a b o l i c i n nature, r e s u l t i n g from e l e v a t e d blood l a c t a t e l e v e l s . The acidemia l i k e l y c o n t r i b u t e d t o the p r o g r e s s i v e f a l l i n f e t a l blood 0 2 content through a rightward s h i f t of the oxyhemoglobin d i s s o c i a t i o n curve (Bohr e f f e c t ) . The r i s e i n f e t a l oxygen consumption was r e f l e c t e d by a s i m i l a r (although n o n s i g n i f i c a n t ) i n c r e a s e i n u t e r i n e oxygen consumption. U t e r o p l a c e n t a l oxygen consumption appeared to V remain u n a l t e r e d . The r i s e i n u t e r i n e oxygen consumption was not accompanied by a corresponding i n c r e a s e i n u t e r i n e oxygen d e l i v e r y , hence u t e r i n e oxygen e x t r a c t i o n rose t o 23.8±1.9% (from a c o n t r o l v a l u e of 19.5±1.6%) by 1.5 hours po s t -i n f u s i o n . The r i s e i n u t e r i n e oxygen e x t r a c t i o n r e s u l t e d i n s i g n i f i c a n t d e c l i n e s i n u t e r i n e venous Po and Co v a l u e s which ^ 2 2 l i k e l y c o n t r i b u t e d t o the f a l l i n f e t a l oxygen d e l i v e r y . F e t a l h e a r t r a t e i n c r e a s e d s i g n i f i c a n t l y t o 21% (34 beats per minute, bpm) above the c o n t r o l (162±7 bpm) d u r i n g the f i r s t 1.5 hours of r i t o d r i n e i n f u s i o n . I t remained e l e v a t e d by an average of 16% (26 bpm) throughout the remainder of the i n f u s i o n and the f i r s t 8 hours of p o s t - i n f u s i o n , r e t u r n i n g t o the c o n t r o l by the end of the p o s t - i n f u s i o n p e r i o d . F e t a l a r t e r i a l p r e s s u r e remained unchanged from the c o n t r o l (46.2±1.5 mm Hg). The i n c i d e n c e of f e t a l b r e a t h i n g a c t i v i t y f e l l s i g n i f i c a n t l y from an o v e r a l l average c o n t r o l v a l u e of 43.2±2.6% t o an average of 28.1±6.8% d u r i n g the r i t o d r i n e i n f u s i o n p e r i o d . In most animals, b r e a t h i n g was most depressed near the end of the i n f u s i o n . The i n c i d e n c e of low v o l t a g e e l e c t r o c o r t i c a l (ECoG) a c t i v i t y a l s o f e l l s i g n i f i c a n t l y by an average of 7.5% while t h a t of h i g h v o l t a g e ECoG rose by 7.3%. A l t e r a t i o n s i n intermediate v o l t a g e a c t i v i t y were not observed. The i n c i d e n c e of f e t a l r a p i d eye movement also tended to f a l l by an average of 8.2% during the infusion of r i t o d r i n e . These behavioral changes may have resulted from the development of f e t a l hypoxemia, rather than as a d i r e c t e f f e c t of r i t o d r i n e . In conclusion, these data have demonstrated that r i t o d r i n e infusion to f e t a l lambs res u l t s in s i g n i f i c a n t physiological and behavioral changes i n the fetus. These e f f e c t s may put the fetus at r i s k , p a r t i c u l a r l y i n situations where f e t a l oxygen delivery i s already reduced, as i n various states of compromised pregnancy. v i i TABLE OF CONTENTS A b s t r a c t i i L i s t of Tables x i i i L i s t of F i g u r e s xv A b b r e v i a t i o n s x v i i Acknowledgements xx D e d i c a t i o n x x i 1. INTRODUCTION 1 1.1 Preterm Labor 1 A. D e f i n i t i o n 1 B. Incidence 1 C. M o r t a l i t y and M o r b i d i t y 2 D. E t i o l o g y 3 E. Management 3 1.2 C l i n i c a l Pharmacology of R i t o d r i n e 5 A. Mechanism of A c t i o n 6 i Mechanism of Myometrial 6 C o n t r a c t i o n i i Mechanism of A c t i o n of 8 Beta-Adrenergic A g o n i s t s B. E f f i c a c y 9 C. Pharmacodynamics and P l a c e n t a l 13 T r a n s f e r v i i i D. Maternal Side E f f e c t s 15 i C a r d i o v a s c u l a r E f f e c t s 16 i i Pulmonary E f f e c t s 17 i i i Renal E f f e c t s 17 i v M e t a b o l i c E f f e c t s 18 E. F e t a l Side E f f e c t s 19 i S t u d i e s i n Humans 2 0 i i S t u d i e s i n Sheep 21 a. C a r d i o v a s c u l a r E f f e c t s 21 b. Pulmonary E f f e c t s 2 2 c. M e t a b o l i c E f f e c t s 2 3 F. Neonatal Side E f f e c t s 25 1.3 R a t i o n a l e and S i g n i f i c a n c e 26 1.4 O v e r a l l O b j e c t i v e 28 1.5 S p e c i f i c O b j e c t i v e s 28 2. EXPERIMENTAL 30 2.1 M a t e r i a l s 30 A. Drugs and Chemicals 30 B. C a t h e t e r s and E l e c t r o d e s 31 2.2 P r e p a r a t i o n of S o l u t i o n s f o r I n f u s i o n 35 A. R i t o d r i n e 35 B. A n t i p y r i n e 3 5 C. S a l i n e 36 i x 2.3 P r e p a r a t i o n of S o l u t i o n s f o r Assays 36 A. Glucose Assay 36 B. L a c t a t e Assay 3 7 C. A n t i p y r i n e Assay 3 7 2.4 Animal P r e p a r a t i o n 38 A. Breeding 38 B. Surgery 39 2.5 Recording and Measurement 45 A. B i o p h y s i c a l V a r i a b l e s 45 B. Measurement of U t e r i n e and 46 U m b i l i c a l Blood Flow C. Blood Sample A n a l y s i s 47 i Blood Gases and Hematocrit 48 i i Glucose 48 a. P r e p a r a t i o n f o r A n a l y s i s 48 b. Assay 49 i i i L a c t a t e 50 a. P r e p a r a t i o n f o r A n a l y s i s 50 b. Assay 50 i v A n t i p y r i n e 51 a. P r e p a r a t i o n f o r A n a l y s i s 51 b. Assay 52 v R i t o d r i n e 53 a. P r e p a r a t i o n f o r A n a l y s i s 53 b. Assay 54 X 2.6 Experimental P r o t o c o l 54 A. Drug A d m i n i s t r a t i o n 55 i I n f u s i o n Rate and Route 55 i i I n f u s i o n D u r a t i o n 55 B. Sampling Schedule 55 C. C o n t r o l S a l i n e I n f u s i o n s 56 2.7 A n a l y s i s 57 A. C a l c u l a t i o n s 57 i Glucose, L a c t a t e and 0__ 57 d e l i v e r y t o the Fetus i i Glucose, L a c t a t e and 0 2 57 D e l i v e r y t o the Uterus i i i Glucose, L a c t a t e and 0 2 57 Uptake by the Fetus i v Glucose, L a c t a t e and 0 2 57 Uptake by the Uterus v F r a c t i o n a l 0 E x t r a c t i o n 57 2 v i U t e r o p l a c e n t a l Oa Consumption . . . . 58 B. E s t i m a t i o n of F e t a l Weight i n u t e r o . . . . 58 C. C a r d i o v a s c u l a r V a r i a b l e s 59 D. B e h a v i o r a l V a r i a b l e s 60 E. S t a t i s t i c a l A n a l y s i s 61 x i 3.0 RESULTS 62 3.1 Summary of Experimental Outcomes 62 3.2 O r g a n i z a t i o n of Time P e r i o d s i n 62 A n a l y s i s of R e s u l t s 3.3 F e t a l Data: R i t o d r i n e and C o n t r o l 68 S a l i n e I n f u s i o n s A. A r t e r i a l and U m b i l i c a l Venous 70 Blood Gases, pH, and Hematocrit B. A r t e r i a l and U m b i l i c a l Venous 76 Glucose and L a c t a t e L e v e l s C. U m b i l i c a l Blood Flow, O D e l i v e r y , . . . . 79 0 2 Consumption, V-A Co 2 D i f f e r e n c e and O E x t r a c t i o n 2 D. Glucose and L a c t a t e Fluxes 85 E. A r t e r i a l Pressure and Heart Rate 88 Data F. B r e a t h i n g A c t i v i t y 93 G. E l e c t r o c o r t i c a l and E l e c t r o o c u l a r 99 A c t i v i t y H. A r t e r i a l Plasma L e v e l s of R i t o d r i n e . . . 106 3.4 Maternal Data: R i t o d r i n e and C o n t r o l 106 S a l i n e I n f u s i o n s A. A r t e r i a l and U t e r i n e Venous Blood . . . . 110 Gases, pH, and Hematocrit B. A r t e r i a l and U t e r i n e Venous 113 Glucose and L a c t a t e L e v e l s C. U t e r i n e Blood Flow, 0 2 D e l i v e r y 113 0 2 E x t r a c t i o n and T o t a l U t e r i n e and U t e r o p l a c e n t a l 0 2 Consumption D. Glucose and L a c t a t e L e v e l s and Fluxes . . 120 E. A r t e r i a l Pressure and Heart Rate Data . . 120 x i i 4.0 DISCUSSION 126 4.1 Drug A d m i n i s t r a t i o n 126 4.2 F e t a l Plasma L e v e l s of R i t o d r i n e 127 4.3 Ontogeny of Beta-Receptor Mediated 131 Responses i n the F e t a l Lamb 4.4 U t e r i n e and U m b i l i c a l Blood Flow 133 4.5 F e t a l Glucose Metabolism . . . 141 4.6 F e t a l L a c t a t e Metabolism 145 4.7 F e t a l - M a t e r n a l Oxygen Metabolism 154 A. F e t a l Oxygen Consumption 156 B. F e t a l Oxygen D e l i v e r y 159 C. F e t a l Blood Gas Status 160 D. T o t a l U t e r i n e and U t e r o p l a c e n t a l 166 Oxygen Consumption E. U t e r i n e Blood Gas Status 168 F. F e t a l M e t a b o l i c Outcome 170 4.8 F e t a l C a r d i o v a s c u l a r F u n c t i o n 171 4.9 F e t a l B e h a v i o r a l A c t i v i t y 174 '4.10 C l i n i c a l Relevance 183 5.0 SUMMARY AND CONCLUSIONS 185 6.0 REFERENCES 191 x i i i LIST OF TABLES TABLE 1. L i s t of completed r i t o d r i n e infusion 64 experiments. TABLE 2. L i s t of completed saline infusion 65 experiments. TABLE 3. L i s t of excluded experiments 66 TABLE 4. L i s t of incomplete experiments 66 TABLE 5. Analysis of time periods 68 TABLE 6. Fetal a r t e r i a l pH and B.E. values at 69 C, EI and PF. TABLE 7. Fetal a r t e r i a l pH, B.E. and Po values 71 measured at C, EI and PF (averaged within groups which were c l a s s i f i e d according to the duration of r i t o d r i n e i n f u s i o n ) . TABLE 8. Fetal a r t e r i a l and umbilical venous blood . . 72 gas values, pH, hematocrit, and glucose and lactate levels before, during and af t e r r i t o d r i n e infusion to the fetus. TABLE 9. Fetal a r t e r i a l and umbilical venous blood . . . 77 gas values, pH, hematocrit, and glucose and lactate levels before, during and af t e r control saline infusion to the fetus. TABLE 10. Fetal umbilical blood flow, 0 2 delivery, . . . 81 0 2 consumption, V-A Co2 difference and f r a c t i o n a l 0 2 extraction before, during and a f t e r r i t o d r i n e infusion to the fetus. TABLE 11. Fetal umbilical blood flow, 0 2 delivery, . . . 84 0 2 consumption, V-A Co2 difference and f r a c t i o n a l 0 2 extraction before, during and a f t e r control saline infusion to the fetus. TABLE 12. Fetal glucose and lactate uptakes and . . . . 86 d e l i v e r i e s before, during and a f t e r r i t o d r i n e infusion to the fetus. TABLE 13. Fetal glucose and lactate uptakes and . . . . 89 d e l i v e r i e s before, during and afte r control saline infusion to the fetus. xiv TABLE 14. Fetal heart rate and a r t e r i a l pressure . . . . 90 before, during and afte r r i t o d r i n e infusion to the fetus. TABLE 15. Fetal heart rate and a r t e r i a l pressure . . . . 94 before, during and afte r control saline infusion to the fetus. TABLE 16. Average o v e r a l l incidence of f e t a l breathing 104 movements, e l e c t r o c o r t i c a l state and eye a c t i v i t y (EOG) before, during and a f t e r the infusion of r i t o d r i n e to the fetus. TABLE 17. Fetal a r t e r i a l plasma leve l s of r i t o d r i n e . . 107 TABLE 18. Maternal a r t e r i a l and uterine venous blood . I l l gas values, pH, hematocrit and glucose and lactate l e v e l s before, during and a f t e r r i t o d r i n e infusion to the fetus. TABLE 19. Maternal a r t e r i a l and uterine venous blood . 114 gas values, pH, hematocrit and glucose and lactate l e v e l s before, during and a f t e r control saline infusion to the fetus. TABLE 20. Uterine blood flow and uterine 0 2 delivery, . 115 0 2 consumption and 0 2 extraction before, during and afte r r i t o d r i n e infusion to the fetus. TABLE 21. Uterine blood flow and uterine 0 2 delivery, . 119 0 2 consumption and 0 2 extraction before, during and a f t e r control saline infusion to the fetus. TABLE 22. Uterine glucose and lactate uptakes and . . 121 d e l i v e r i e s before, during and a f t e r r i t o d r i n e infusion to the fetus. TABLE 23. Uterine glucose and lactate uptakes and . . 122 d e l i v e r i e s before, during and a f t e r control saline infusion to the fetus. TABLE 24. Maternal heart rate and a r t e r i a l pressure . 123 before, during and afte r r i t o d r i n e infusion to the fetus. TABLE 25. Maternal heart rate and a r t e r i a l pressure . . 125 before, during and afte r control saline infusion to the fetus. LIST OF FIGURES xv FIGURE 1. Chemical structure of r i t o d r i n e 5 FIGURE 2. Mechanism for the contraction of 7 smooth muscle. FIGURE 3. Catheters u t i l i z e d i n the chronic 32 pregnant sheep preparation. FIGURE 4. E l e c t r o c o r t i c a l and electroocular 34 electrodes u t i l i z e d i n the chronic pregnant sheep preparation. FIGURE 5. Summary of experiments 63 FIGURE 6. Sampling and infusion schedule 67 FIGURE 7. Fetal a r t e r i a l and umbilical venous . . . . 73 Po values and f e t a l a r t e r i a l and 2 umbilical venous Pco 2 values before, during and afte r r i t o d r i n e infusion to the fetus. FIGURE 8. Fetal a r t e r i a l pH and base excess 75 values before, during and afte r r i t o d r i n e infusion to the fetus. FIGURE 9. Fetal a r t e r i a l and umbilical venous . . . . 78 glucose concentrations and f e t a l a r t e r i a l and umbilical venous lactate concentrations before, during and af t e r r i t o d r i n e infusion to the fetus. FIGURE 10. Mean percent change from the control . . . . 82 of f e t a l f r a c t i o n a l oxygen extraction, oxygen consumption, umbilical blood flow, oxygen delivery and umbilical venous oxygen content before, during and af t e r r i t o d r i n e infusion to the fetus. FIGURE 11. Fetal glucose and lactate d e l i v e r i e s . . . . 87 and f e t a l glucose and lactate uptakes before, during and af t e r r i t o d r i n e infusion to the fetus. FIGURE 12 . Fetal heart rate and a r t e r i a l pressure . . . 91 before, during and afte r r i t o d r i n e infusion to the fetus. xvi FIGURE 13. Representative polygraph recordings of amniotic pressure, a r t e r i a l pressure, heart rate, tracheal pressure, electrocorticogram, and ele c t r o -oculargram i n the f e t a l lamb. 92 FIGURE 14. C y c l i c tendency of f e t a l breathing a c t i v i t y . FIGURE 15. Fetal breathing before, during and af t e r r i t o d r i n e infusion to the fetus. 95 97 FIGURE 16. Representative polygraph recording i l l u s t r a t i n g the three states of e l e c t r o c o r t i c a l a c t i v i t y i n the f e t a l lamb. 101 FIGURE 17. High, low and intermediate e l e c t r o - . . c o r t i c a l state before, during and afte r r i t o d r i n e infusion to the fetus. 102 FIGURE 18. Low and high e l e c t r o c o r t i c a l state, breathing and Po2 values before, during and afte r r i t o d r i n e infusion to the fetus. 105 FIGURE 19. Fetal a r t e r i a l plasma r i t o d r i n e . . . concentrations verses: f e t a l a r t e r i a l Po2, t o t a l f e t a l oxygen consumption, pHa and f e t a l heart rate. FIGURE 20. Fetal a r t e r i a l glucose concentration and heart rate as a function of f e t a l a r t e r i a l plasma r i t o d r i n e concentration. FIGURE 21. Uterine venous Po2 and Co2 before, . . during and afte r r i t o d r i n e infusion to the fetus. 108 109 112 FIGURE 22. Total uterine oxygen uptake and t o t a l uterine f r a c t i o n a l oxygen extraction before, during and af t e r r i t o d r i n e infusion to the fetus. 117 FIGURE 23. Total uterine and t o t a l f e t a l oxygen uptake and the uterine-umbilical Po2 difference before, during and af t e r r i t o d r i n e infusion to the fetus. 118 x v i i ABBREVIATIONS ANOVA a n a l y s i s of v a r i a n c e BE base excess bpm beats per minute C c o n t r o l p e r i o d °C degrees c e n t i g r a d e cm centimeter Co 2 oxygen content C0 2 carbon d i o x i d e gas DG1 glucose d e l i v e r y DLac l a c t a t e d e l i v e r y Do 2 oxygen d e l i v e r y ECoG e l e c t r o c o r t i c a l a c t i v i t y EI end of i n f u s i o n EOG e l e c t r o o c u l a r a c t i v i t y e xt e x t r a c t i o n FFA f e t a l femoral a r t e r y g gram Glupt glucose uptake Hct hematocrit hr hour 1.51 1.5 hours of i n f u s i o n i . d i n s i d e diameter i.m i n t r a m u s c u l a r I.U i n t e r n a t i o n a l u n i t i . v intravenous x v i i i kD k i l o d a l t o n kg k i l o g r a m L l i t e r Laupt . l a c t a t e uptake M molar ( m o l e s / l i t e r ) MFA maternal femoral a r t e r y mg m i l l i g r a m min minute mL m i l l i l i t e r MLCK myosin l i g h t c h a i n k i n a s e mm m i l l i m e t e r mM m i l l i m o l a r ( m i l l i m o l e s / l i t e r ) ng nanogram 0 2 oxygen gas o.d o u t s i d e diameter PI. 5 1.5 hours post i n f u s i o n P8 8 hours post i n f u s i o n Pco 2 p a r t i a l pressure of Co 2 i n blood PF post f i n a l i n f u s i o n pH negative l o g hydrogen i o n c o n c e n t r a t i o n Po 2 p a r t i a l pressure of 0 2 i n blood Qum u m b i l i c a l b l o o d flow Qut u t e r i n e blood flow REM r a p i d eye movement RTD r i t o d r i n e ug microgram xix uterine vein umbilical vein venoarterial oxygen consumption X X ACKNOWLEDGEMENTS I wish to sin c e r e l y thank Dr. Dan W. Rurak for providing me with the opportunity to join his research group. His supervision, guidance, encouragement and f i n a n c i a l support are very much appreciated. I would also l i k e to thank my co-supervisor, Dr. J.E. Axelson and the other members of my committee, Dr. S. E f f e r and Dr. J. Skala for t h e i r advice and in t e r e s t i n t h i s study. I wish to extend my sincere gratitude to Mrs. Sandy Jones for her expert technical assistance, steadfast encouragement, and most of a l l , her friendship. I would also l i k e to thank Mr. Eddie Kwan and Mr. Sun Dong Yoo for t h e i r assistance with animal preparations. Special thanks to Mr. Matthew Wright for the drug analysis, experimental assistance and advice. F i n a l l y , I would l i k e to thank the Research Div i s i o n of B.C.'s Children's Hospital for providing me with a two year Graduate Studentship. This project was supported i n part by the Medical Research Council of Canada. xxi To my husband Andy, for your love, encouragement and understanding. 1 1. INTRODUCTION 1.1 PRETERM LABOR A. D e f i n i t i o n Labor and delivery occurring before 3 7 completed weeks of gestation (calculated from the f i r s t day of the l a s t menstrual period) i s c l a s s i f i e d as preterm (World Health Organization, 1969, 1977). In the past, a b i r t h weight of <2 500 grams has been equated with prematurity, however, as many as one-third of these infants are born beyond the 37th week of gestation and are growth retarded (Creasy and Resnik, 1989) . B. Incidence Preterm labor continues to be a major cause of perinatal mortality and morbidity, accounting for the majority (>70%) of perinatal deaths i n otherwise normal newborns (Creasy and Resnik, 1989). In North America and i n the United Kingdom, 5 to 9 percent of a l l d e l i v e r i e s occur prematurely (Creasy and Resnik, 1989). Despite evolving management practices and improvements i n prenatal health care, a decrease in the incidence of perinatal mortality and morbidity r e s u l t i n g from preterm b i r t h has not been observed (Behrman, 1985; Leveno and 2 Cunningham, 1987; King et a l . , 1988). Showing the lowest proportional decline of a l l major causes of perinatal mortality and morbidity, preterm labor and delivery has become one of the most important o b s t e t r i c a l disorders to be overcome. C. Mortality and Morbidity Improving o b s t e t r i c and neonatal management has led to the s u r v i v a l of preterm infants of increasingly lower gestational age. Many t e r t i a r y care centers report s u r v i v a l rates of greater than 50, 90 and close to 100 percent at gestational ages of greater than 25, 29, and 32 weeks respectively (Main and Main, 1986). Although few, there have been reports of infants born as early as 2 3 weeks surviving to hospital discharge (survival rates at t e r t i a r y care centers are usually higher than i n general health care centers). While advances i n neonatal health care have improved the sur v i v a l rate of preterm infants, the cost i s high both i n s o c i a l and monetary terms. Preterm infants are at r i s k for s p e c i f i c diseases related to organ immaturity, which r e s u l t i n a large percentage of infants who require expensive care. Clearly, the optimal solution to the problem of preterm b i r t h i s prevention. 3 D. E t i o l o g y There are many r i s k f a c t o r s which predispose women to premature l a b o r and d e l i v e r y (Hoffman and Bakketeig, 1984; Creasy and Resnik, 1989). These can be s u b d i v i d e d i n t o a number of groups i n c l u d i n g : 1) socioeconomic f a c t o r s (eg. low socioeconomic s t a t u s , n u t r i t i o n a l d e p r i v a t i o n ) , 2) p e r s o n a l c h a r a c t e r i s t i c s (eg. b l a c k r a c e , low pre-pregnancy weight, maternal age of <20 years, or maternal age of >35 years a t f i r s t pregnancy o n l y ) , 3) l i f e s t y l e (eg. o c c u p a t i o n a l s t r e s s or f a t i g u e , c i g a r e t t e smoking), 4) p r e v i o u s medical h i s t o r y (eg. p r e v i o u s preterm b i r t h , p r i o r a b o r t i o n s , u t e r i n e anomalies) and 5) aspects of c u r r e n t pregnancy (eg. m u l t i p l e pregnancy, i n f e c t i o n , c e r v i c a l incompetence, surgery, or many o t h e r p o s s i b l e medical d i s o r d e r s ) . These f a c t o r s account f o r approximately h a l f of a l l preterm l a b o r s ; the r e s t remain i d i o p a t h i c i n o r i g i n ( C h a l l i s and Olson, 1988). E. Management P a r t u r i t i o n occurs as a r e s u l t of many complex i n t e r a c t i o n s between mother and f e t u s i n v o l v i n g the s e q u e n t i a l m aturation of an endocrine organ communication system ( C h a l l i s and Olson, 1988). A g r e a t d e a l of r e s e a r c h has been undertaken t o understand the p h y s i o l o g i c a l b a s i s of term l a b o r so t h a t the mechanisms which u n d e r l i e the i n i t i a t i o n of 4 preterm labor may be better understood and hence e f f e c t i v e l y managed. Therapeutic attempts to arrest labor contractions i s referred to as t o c o l y s i s . The word " t o c o l y s i s " comes from the Greek word roots "tokos," meaning c h i l d b i r t h and " l y s i s , " meaning d i s s o l u t i o n . The rationale for the pharmacologic intervention of preterm labor i s that the incidence of peri n a t a l mortality and morbidity i s s i g n i f i c a n t l y diminished with increasing gestation length ( C a r i t i s , 1983). The goals of t o c o l y s i s are to either delay labor u n t i l term, or to delay labor for a b r i e f period so that other measures may be taken (eg. enhancement of f e t a l lung maturity by c o r t i c o s t e r o i d administration or transfer of the mother to a center with intensive perinatal care f a c i l i t i e s ) . Clearly, however, the benefits of continued gestation must be balanced against the r i s k s of continued pregnancy and pharmacologic intervention. Several classes of t o c o l y t i c agents have been developed which a f f e c t one or more of the c o n t r a c t i l e processes i n the myometrium ( C a r i t i s et a l . . 1988). Tocolysis with beta-adrenergic agonists, however, remains the treatment of choice in many centers (Keirse, 1984 a,b; Taslimi et a l . , 1989). Of the numerous beta-adrenergic agents u t i l i z e d i n t o c o l y s i s , including hexoprenaline, salbutamol, isoxsuprine, fenoterol, terbutaline, and r i t o d r i n e , the l a t t e r two drugs are most 5 commonly employed. Other pharmacologic agents u t i l i z e d i n c l u d e magnesium s u l f a t e , p r o s t a g l a n d i n i n h i b i t o r s , and c a l c i u m channel b l o c k e r s . Ethanol and progesterone have p r e v i o u s l y been employed as t o c o l y t i c agents, however, due t o qu e s t i o n s of s a f e t y and e f f i c a c y , t h e i r use appears t o have been abandoned. 1.2 CLINICAL PHARMACOLOGY OF RITODRINE F i g u r e 1. Chemical S t r u c t u r e of R i t o d r i n e R i t o d r i n e h y d r o c h l o r i d e (Yutopar ) (erythro-p-hydroxy-c<-[ l - [ ( p - h y d r o x y p h e n e t h l y 1 ) a m i n o ] e t h y l ] b e n z y l a l c o h o l h y d r o c h l o r i d e ) i s a potent beta-2 s e l e c t i v e a d r e n e r g i c a g o n i s t which was developed s p e c i f i c a l l y f o r the treatment of preterm l a b o r ( P h i l i p s - D u p h a r , BV, The N e t h e r l a n d s ) . While r i t o d r i n e i s p r i m a r i l y s e l e c t i v e f o r the beta-2 r e c e p t o r s , i t does i n t e r a c t with the beta-1 r e c e p t o r s t o a c e r t a i n e x t e n t . R i t o d r i n e was the f i r s t t o c o l y t i c drug t o be approved by the 6 Food and Drug Administration (FDA) for the treatment of preterm labor i n the United States (Barden et a l . f 1980). As one of the most extensively studied t o c o l y t i c drugs, r i t o d r i n e was licensed for use i n the United Kingdom (1975) and i n the United States (1980) following more than ten years of c l i n i c a l research. A. Mechanism of Action i Mechanism of Myometrial Contraction Smooth muscle contraction (Figure 2) i s dependent upon the i n t e r a c t i o n of the c o n t r a c t i l e proteins, a c t i n and myosin (Roberts, 1984; C h a l l i s and Olson, 1988). Actin filaments consist of two strands of globular a c t i n molecules polymerized into a h e l i c a l formation. Myosin molecules are composed of two heavy chains, each containing an alpha-helical portion and a globular head at the amino terminal end. Each myosin globular head contains an a c t i n binding s i t e , ATPase a c t i v i t y , a 20 kD l i g h t chain with Ca 2 + and Mg2+ binding s i t e s and a second 17 kD l i g h t chain of unknown function. The energy required for contraction (to form the cross-l i n k i n g covalent bonds between the c o n t r a c t i l e proteins) i s provided by the hydrolysis of ATP to ADP and inorganic phosphate, a reaction which i s catalyzed by ATPase a c t i v i t y 1) Resting state. A c t i n My os Myosin i n h e a d —E J J l • 20 Kd l i g h t chain 2) Myosin l i g h t chain kinase (MLCK), activated through binding the calcium-calmodulin complex, phosphorylates the 20 kD myosin l i g h t chain on the myosin globular head. •—P MLCK ATP ADP+(p) 3) ATPase becomes activated, r e s u l t i n g i n the hydrolysis of ATP (to ADP + Pi) and the subsequent binding of the myosin head to a c t i n . ATP ADP + ® 4) The myosin head releases the products of ATP hydrolysis and undergoes a conformational change such that the myosin head p u l l s against the a c t i n filament (power stroke). ADP -f-5) The 20 Kd myosin l i g h t chain becomes dephosphorylated by myosin l i g h t chain phosphatase (MLCP) and an ATP molecule binds to the myosin head. These processes r e s u l t i n the release of myosin from a c t i n . 6) The cycle i s complete. • Figure Mechanism for the contraction of smooth muscle. 8 on the myosin globular head. Activation of ATPase a c t i v i t y , however, i s dependent upon the phosphorylation of a serine residue on the 20 kD myosin l i g h t chain by the enzyme myosin l i g h t chain kinase (MLCK). MLCK a c t i v i t y thus serves as a pi v o t a l step i n the control of myometrial a c t i v i t y . MLCK in turn i s activated by calmodulin, a calcium-sensitive regulatory protein which upon binding calcium, activates MLCK through phosphorylation. Thus any hormonal or neural s t i m u l i which increases i n t r a c e l l u l a r calcium (by release of calcium from membrane bound calcium v e s i c l e s or the sarcoplasmic reticulum) w i l l stimulate contraction. i i Mechanism of Action of Beta-Adrenergic Agonists Ritodrine and other beta-adrenergic agonists e l i c i t uterine relaxation through in t e r a c t i o n with uterine beta-2 adrenoreceptors located on the outer c e l l u l a r plasma membrane (Roberts, 1984; Nuwayhid and Rajabi, 1987). Receptor stimulation r e s u l t s i n the a c t i v a t i o n of the enzyme, adenylate cyclase, which catalyzes the conversion of ATP to c y c l i c AMP1. The subsequent r i s e i n i n t r a c e l l u l a r c y c l i c AMP (cAMP) le v e l s are believed to r e s u l t in myometrial relaxation v i a a number of mechanisms including: i ) acti v a t i o n of cAMP-dependent XA mechanism for the acti v a t i o n of adenylate cyclase, "The C o l l i s i o n Coupling Model," has been proposed by Alberts et a l , 1983 . 9 protein kinase A which subsequently phosphorylates MLCK (at a s i t e d i f f e r e n t from the calmodulin phosphorylation act i v a t i o n s i t e ) and hence inactivates MLCK by reducing i t s a f f i n i t y for the calcium-calmodulin complex, i i ) decreasing i n t r a c e l l u l a r calcium lev e l s through stimulation of calcium uptake and extrusion, i i i ) membrane hyperpolarization and iv) decreasing the permeability of gap junctions. Another c y c l i c AMP mediated mechanism, i n which uterine relaxation i s e l i c i t e d through beta-mimetic induced increases i n placental progesterone production, has also been suggested ( C a r i t i s and Zeleznic, 1980; C a r i t i s et a l . , 1982). B. E f f i c a c y Tocolysis with r i t o d r i n e and other beta-mimetic drugs has been a subject of controversy for many years. Their widespread use i n the treatment of preterm labor would appear paradoxical i n view of the fac t that a conclusive statement on e f f i c a c y has not yet been made. Despite varying opinions on safety, benefit and e f f i c a c y , beta-mimetic t o c o l y s i s remains the treatment of choice, in d i c a t i n g that many c l i n i c i a n s w i l l resort to a treatment with unknown e f f i c a c y and r i s k s i n order to avoid the known r i s k s of preterm b i r t h (Keirse, 1984 a,b). The fundamental lack of agreement on the ris k - b e n e f i t r a t i o for the use of beta-mimetics i n to c o l y s i s i s due primarily to the inadequacy of most t r i a l s addressing 10 t h i s issue. In an attempt to eliminate some of the confusion, King et a l . , (1988), conducted a pooled analysis of sixteen methodologically acceptable con t r o l l e d t r i a l s i n which beta-mimetic agents were compared with either placebo or no active treatment i n the management of preterm labor. From t h i s analysis i t was concluded that beta-mimetic t o c o l y s i s i s more ef f e c t i v e than both placebo and standard treatment i n the management of preterm labor, as r e f l e c t e d by a reduction i n the frequency of preterm b i r t h and low b i r t h weight. The e f f e c t was most marked i n delaying delivery for 24 hours. The treatment did not, however, r e s u l t i n a reduction i n peri n a t a l mortality, nor a lowered incidence of severe respiratory disorders i n the newborn. The uncertainty over the e f f i c a c y of beta-mimetic t o c o l y s i s arises primarily from the fa c t that labor i s often arrested for an i n s u f f i c i e n t period of time, with contractions returning despite continued therapy. The persistence of labor may be due to an overwhelming influence of labor enhancing factors (eg. oxytocin and prostaglandins) which overcome the labor i n h i b i t i n g e f f e c t of the drug ( C a r i t i s et a l . , 1987). A more l i k e l y explanation, however, i s that the myometrium becomes desensitized to the t o c o l y t i c drug. Desensitization i s a common c e l l u l a r adaptive mechanism denoted as a state of 11 diminished responsiveness following p r i o r exposure to an agonist, hormone, or drug (Harden, 1983). Studies i n humans and i n sheep have shown that continuous exposure of myometrium to beta-adrenergic agonists both in v i t r o and i n vivo results i n i n i t i a l myometrial relaxation followed by desensitization with the return of myometrial contractions (Anderson et a l . , 1980; Berg et a l . , 1982; Ke et a l . , 1984; Casper and Lye, 1986; C a r i t i s et a l . , 1987). In view of the fa c t that women are normally exposed to r e l a t i v e l y high concentrations of drug (25-250 ng/mL) for extended periods of time (12-36 hours), desensitization may serve as a l i k e l y explanation for the decreased responsiveness of the uterus to beta-mimetic agents over time ( C a r i t i s et a l . , 1987). While the mechanism for myometrial desensitization i s unclear, studies indicate that i t may involve both post-receptor-cAMP-mediated mechanisms (Berg et a l . , 1982) as well as hormone receptor s p e c i f i c events such as decreased receptor density (Berg et e l . r 1985; C a r i t i s , 1987; Michel et a l . , 1989). Recent reports indicate that desensitization may be prevented by intermittent, as opposed to continuous infusion of beta-adrenergic agonists. This phenomena has been demonstrated for isoproterenol both in v i t r o , using human myometrial s t r i p s , (Ke et a l . , 1984) and in vivo, using a non-pregnant sheep model, (Casper and Lye, 1986). Recently, Spatling et a l . , (1989) found that i n comparison with continuous drug infusion, p u l s a t i l e administration of fenoterol (beta-mimetic drug) required a lower t o t a l drug dosage and a reduced length of therapy for therapeutic success. An increase i n b i r t h weight was also observed. Ritodrine has also been employed i n the treatment of f e t a l d i s t r e s s , a condition associated with reduced f e t a l oxygen supply (Sheybany and Murphy, 1982; Hutchon, 1982; Mendez-Bauer et a l . , 1987; Cabero et a l . r 1988). Fetal d i s t r e s s often occurs as a r e s u l t of the ischemic e f f e c t which uterine contractions may have on an already compromised maternal-placental c i r c u l a t i o n (eg. cord complications, preeclampsia, placental lesions, intrauterine growth retardation e t c . ) . The c l i n i c a l manifestations of f e t a l d i s t r e s s include low f e t a l blood pH, f e t a l heart rate abnormalities and meconium stained amniotic f l u i d . The treatment for f e t a l d i s t r e s s normally involves emergency cesarian section. Short term t o c o l y s i s (bolus dose or <2 hours of infusion) with r i t o d r i n e and other beta-mimetic drugs (Lipshitz et a l . . 1986) are also employed to improve f e t a l condition. This improvement i s thought to occur through a reduction i n uterine a c t i v i t y which enhances placental blood flow, r e s u l t i n g i n a more e f f i c i e n t interchange i n the i n t e r v i l l o u s spaces. The purpose of t o c o l y s i s during f e t a l d i s t r e s s i s generally to lessen the urgency of cesarean section and to allow for f e t a l recovery before b i r t h . The e f f i c a c y of beta-mimetic t o c o l y s i s i n the treatment of f e t a l d i s t r e s s has not yet been elucidated. While most studies report favorable r e s u l t s , further research i s c l e a r l y warranted. C. Pharmacodynamics and Placental Transfer Ritodrine t o c o l y s i s i n humans i s normally i n i t i a t e d by i . v . administration at a rate of 50 to 100 ug/min. The dosage i s then increased i n 50 ug/min steps u n t i l any of the following occur: labor contractions cease, unacceptable side e f f e c t s develop, or labor progresses despite administration of the maximum dosage (300 ug/min) (Bardon e t _ a l . , 1980). If t o c o l y s i s i s achieved, the infusion i s continued for a further 6 to 24 hours a f t e r which i t i s replaced by two consecutive 24 hour periods of intramuscular (or subcutaneous) inje c t i o n s followed by oral administration. The therapeutic serum concentration of r i t o d r i n e i n women undergoing t o c o l y s i s has not yet been established owing to the v a r i a b i l i t y i n not only drug d i s p o s i t i o n , but i n the c e r v i c a l status and degree of uterine a c t i v i t y at the commencement of therapy (Creasy and Resnik, 1989). Serum l e v e l s of r i t o d r i n e r i s e with increasing infusion rate and the concentration may vary by as much as 100% among patients at low infusion rates. In h i b i t i o n of labor contractions has been achieved at serum r i t o d r i n e concentrations ranging from 15 to 123 ng/ml ( C a r i t i s et a l . , 1983; Smit et a l . f 1984; Holleboom et a l . , 19871 . These values overlap considerably with the concentration range found i n women i n whom toco l y s i s has f a i l e d (60 to 146 ng/ml just before the infusion i s terminated) ( C a r i t i s et a l . f 1983; Kuhnert et a l . , 1986). The half l i f e of r i t o d r i n e i n pregnant women has been reported to be 2 hours (Gross et a l . , 1987). In humans, r i t o d r i n e i s metabolized to inactive glucuronide and sulf a t e conjugates and i s excreted i n the urine as both unchanged drug and i t s metabolites (Barden et a l . , 1980). Approximately 76% of the drug i s excreted as a conjugate (Kuhnert et a l . , 1986). I t has been reported that at l e a s t 93% of r i t o d r i n e i s excreted i n the urine during the f i r s t 36 hours following the cessation of t o c o l y s i s . Low le v e l s of the drug are detectable i n plasma for up to 80 hours post infusion (Kuhnert et a l . , 1986). Human studies indicate rapid and appreciable transfer of r i t o d r i n e to the fetus (Gander et a l . , 1980; Van Lierde and Thomas, 1982; Gross et a l . , 1985; Fujimoto et a l . , 1986). Fetal umbilical venous r i t o d r i n e concentrations at delivery have been reported to vary from 7 to 79 ng/mL and i n some cases approach, or exceed maternal lev e l s (Gander et a l . , 1980; Van L i e r d e and Thomas, 1982; Fujimoto e t a l . , 1986; Kuhnert e t a l . , 1986). U n f o r t u n a t e l y , these data p r o v i d e l i t t l e i n f o r m a t i o n on the degree t o which the f e t u s i s exposed t o r i t o d r i n e . Extreme t e c h n i c a l and e t h i c a l c o n s t r a i n t s l i m i t human p l a c e n t a l t r a n s f e r s t u d i e s t o the extent t h a t o n l y s i n g l e f e t a l u m b i l i c a l blood samples are taken a t the time of d e l i v e r y . T h i s type of study does not enable one t o determine a c c u r a t e l y the extent of f e t a l drug exposure as the va l u e s o b t a i n e d vary depending on the exact time a t which the sample i s taken (Levy and Hayton, 197 3; Anderson e t a l . . 1980). The extent t o which r i t o d r i n e c r o s s e s the sheep p l a c e n t a has not been e x t e n s i v e l y s t u d i e d and methodological problems i n s t u d i e s c a r r i e d out thus f a r preclu d e d e f i n i t i v e c o n c l u s i o n s . N e v e r t h e l e s s , the a v a i l a b l e evidence suggests t h a t the p l a c e n t a l t r a n s f e r of r i t o d r i n e i n sheep i s low (K l e i n h o u t and Veth, 1975; Fugimoto e t a l . , 1984 ). The apparent d i f f e r e n c e between sheep and human p l a c e n t a l p e r m e a b i l i t y t o r i t o d r i n e may be r e l a t e d t o p l a c e n t a l s t r u c t u r a l or metabolic d i f f e r e n c e s . D. Maternal Side E f f e c t s B e ta-adrenergic agents u t i l i z e d i n t o c o l y s i s e l i c i t many e f f e c t s i n a d d i t i o n t o u t e r i n e r e l a x a t i o n and t h i s i s due p r i m a r i l y t o two f a c t o r s . F i r s t l y , while r i t o d r i n e i s s e l e c t i v e f o r beta-2 r e c e p t o r s , i t a l s o i n t e r a c t s w i t h b e t a -1 r e c e p t o r s t o a c e r t a i n extent. Secondly, b e t a - a d r e n e r g i c r e c e p t o r s are d i s t r i b u t e d i n a u b i q u i t o u s f a s h i o n such t h a t r i t o d r i n e e f f e c t s a whole host of organs r e s u l t i n g i n many u n d e s i r a b l e s i d e e f f e c t s i n the mother (reviews: B e n e d e t t i , 1986; Nuwayhid and R a j a b i , 1987; C a r i t i s e t a l . , 1988; Graber, 1989; Creasy and Resnik, 1989). i C a r d i o v a s c u l a r E f f e c t s R i t o d r i n e a d m i n i s t r a t i o n r e s u l t s i n a dose r e l a t e d c a r d i o a c c e l e r a t i o n of 19 t o 40 beats per minute a t the i n f u s i o n range recommended f o r t o c o l y s i s ( C a r i t i s , 1983). The r i s e i n heart r a t e i s a r e s u l t o f both d i r e c t m y o c a r d i a l s t i m u l a t i o n ( b e t a - l - r e c e p t o r mediated) and r e f l e x c o r r e c t i o n i n response t o a decrease i n p e r i p h e r a l v a s c u l a r r e s i s t a n c e (beta-2-receptor mediated). The p o t e n t i a l f o r hypotension tends t o be o f f s e t by a 50% or more i n c r e a s e i n c a r d i a c output r e s u l t i n g from both i n o t r o p i c and c h r o n o t r o p i c s t i m u l a t i o n ( b e t a - l - r e c e p t o r mediated) of the h e a r t . While mean a r t e r i a l p ressure g e n e r a l l y remains unchanged, t h e r e i s u s u a l l y a f a l l i n d i a s t o l i c p r e s s u r e coupled with a r i s e i n s y s t o l i c p r e s s u r e r e s u l t i n g i n a widened pulse p r e s s u r e . Other p o t e n t i a l l y s e r i o u s c a r d i o v a s c u l a r c o m p l i c a t i o n s of r i t o d r i n e therapy, i n c l u d i n g myocardial ischemia and c a r d i a c arrhythmias, occur i n 1 t o 5 percent of p a t i e n t s . U t e r o p l a c e n t a l b l o o d flow has been reported to increase i n complicated pregnancy (preeclampsia, hypertension and growth retardation) and to decrease, increase or remain unchanged i n normal pregnancy (Brettes et a l . f 1976; Sunio et a l . , 1978; Brotenek and Brotanek, 1981; Sunio, 1982; Jouppila et a l . , 1985). i i Pulmonary Effects Hyperventilation, dyspnea, chest pain and tightness are often associated with r i t o d r i n e therapy, however, pulmonary edema i s probably the most serious of a l l beta-mimetic complications. Occurring i n approximately 5% of patients, pulmonary edema i s thought to r e s u l t primarily as a consequence of increased hydrostatic pressure i n lung c a p i l l a r i e s secondary to a combination of f l u i d overload, decreased c o l l o i d oncotic pressure, and the physiologic a l t e r a t i o n s produced by pregnancy and to c o l y s i s (Pisani et a l . , 1989). If not recognized and treated early, pulmonary edema may lead to adult respiratory d i s t r e s s syndrome and possibly death. i i i Renal Effects Ritodrine administration predisposes the patient to f l u i d overload through a number of mechanisms including: reduced renal blood flow, decreased glomerular f i l t r a t i o n rate, 18 act i v a t i o n of the renin-angiotensin system, increased plasma aldosterone and a n t i d i u r e t i c hormone, increased sodium and water reabsorption and decreased urinary output. i v Metabolic Effects Stimulation of l i v e r and muscle beta-2 adrenergic receptors increases the rates of gluconeogenesis ( l i v e r ) and glycogenolysis ( l i v e r and muscle), both of which r e s u l t i n the development of hyperglycemia. Hyperinsulinemia follows as a consequence of both the increase i n plasma glucose l e v e l s and the d i r e c t stimulation of pancreatic adrenergic receptors. Stimulation of beta-1 receptors on f a t c e l l s increases the rate of l i p o l y s i s , r e s u l t i n g i n the mobilization of free f a t t y acids and g l y c e r o l . If the products of these metabolic pathways ( i . e . pyruvate, g l y c e r o l , f a t t y acids) are formed i n excess of t h e i r oxidation rates, they begin to accumulate and are subsequently converted into lactate and ketone bodies. The accumulation of lactate and ketones, coupled with a decline i n serum bicarbonate may r e s u l t i n the development of maternal acidosis. L a c t i c a c i d o s i s and ketoacidosis are r a r e l y problems of r i t o d r i n e therapy, however, they have been reported, most often i n patients with impaired glucose tolerance (Desir et a l . , 1978; Mordes et a l . , 1982; Richards et a l . . 1983). A f e t a l death associated with severe r i t o d r i n e induced maternal ketoacidosis occurred in a 28 year old, well controlled, i n s u l i n - r e q u i r i n g d i a b e t i c patient (Schilthuis and Aarnoudse, 1980). Hypokalemia commonly occurs during r i t o d r i n e therapy, as a r e s u l t of potassium i n f l u x from e x t r a c e l l u l a r into i n t r a c e l l u l a r spaces. Elevations i n serum transaminase and thyroid hormone concentrations have also been reported (Lotgering et a l . , 1986; Essed et a l . , 1987)). Extended intravenous r i t o d r i n e , over 24 hours r e s u l t s i n a normalization of various cardiovascular and metabolic parameters. Heart rate and serum concentrations of glucose, la c t a t e , and potassium have a l l been reported to return to baseline values over 24 hours. A desensitization of l i v e r , muscle, pancreas, and heart, s i m i l a r to that observed i n the uterus has been suggested (Richards et a l . f 1983; Hendrichs et a l . , 1986). Other common, but less serious side effects include tremor, nervousness, headache, anxiety, epigastric d i s t r e s s , i l e u s , bloating, diarrhea, nausea and vomiting. E. Fetal Side Effects The administration of r i t o d r i n e to the mother may a f f e c t the fetus both d i r e c t l y and i n d i r e c t l y (Unbehaun, 1974). Ind i r e c t l y , maternal cardiovascular changes may influence the uteroplacental or f e t a l c i r c u l a t i o n . In addition, maternal 20 metabolic perturbations may disturb f e t a l homeostasis through altered placental transfer of nutrients and metabolites. Ritodrine may also i n t e r a c t with placental beta-adrenergic receptors, r e s u l t i n g i n alterations i n placental metabolism and nutrient transfer. Direct e f f e c t s i n the fetus may r e s u l t through placental transfer of r i t o d r i n e and subsequent stimulation of f e t a l beta-adrenergic receptors. The presence of beta-adrenergic receptors i n the fetus i s well established, however, the s e n s i t i v i t y of the fetus to agonists such as ri t o d r i n e i s related to gestational age and the a b i l i t y of the fetus to metabolize the drug. i Studies i n Humans The extreme l i m i t a t i o n s of human pregnancy-related research have precluded the a c q u i s i t i o n of s u f f i c i e n t data regarding the e f f e c t s of r i t o d r i n e on the human fetus. While newer techniques have provided a greater means for f e t a l investigation (eg. doppler ultrasound, cordocentesis), studies thus far have been limited primarily to the observation of f e t a l heart rate patterns. Most human fetuses appear to be able to tolerate r i t o d r i n e , however, there have been many reports of f e t a l and neonatal cardiac complications (Katz and Seeds, 1989). While increases i n f e t a l heart rate are a common e f f e c t of beta-mimetic t o c o l y s i s , t h i s i n i t s e l f does not present a threat to the fetus. Possible compromising cardiovascular complications of r i t o d r i n e in the fetus include: supraventricular tachycardia, a t r i a l f l u t t e r , cardiac f a i l u r e , hydrops f e t a l i s , and myocardial i n f a r c t i o n . i i Studies i n Sheep The pregnant ewe model has proven to be extremely useful i n studies addressing the ef f e c t s of drugs i n the fetus. The introduction of techniques which permit d i r e c t monitoring of the fetus i n utero, allow for experimentation under conditions that are r e l a t i v e l y undisturbed (Meschia et a l . f 1966; Nathanielsz et a l . , 1980). a. Cardiovascular Effects Ritodrine infusion to f e t a l lambs (at rates employed i n human tocolysis) has been shown to r e s u l t in elevations i n f e t a l heart rate (Siimes et a l . , 1978; Basset et a l . , 1989). In the Basset et a l . , (1989) study, f e t a l tachycardia persisted for 12 to 24 hours, but f e l l to the control by 48 hours despite continued infusion. A desensitization of the f e t a l myocardium to prolonged r i t o d r i n e infusion was suggested. Ritodrine infusion (similar rate) to the ewe or fetus (<1 hour in duration) has also been shown to e l i c i t a r e d i s t r i b u t i o n of cardiac output i n the fetus (Siimes et a l . , 1978). Elevations i n blood flow to the adrenal glands and 22 myocardium were observed and the percentage of cardiac output delivered to these organs was increased as well. Mean cardiac output remained unchanged (Siimes et a l . , 1978; Creasy and Siimes, 1979). Studies investigating the e f f e c t s of r i t o d r i n e on uterine and umbilical blood flow i n pregnant sheep have shown the former to f a l l , while the l a t t e r appears to remain unaffected (Ehrenkranz et a l . , 1976; Siimes and Creasy, 1979). b. Pulmonary Effects Long term infusion of r i t o d r i n e (24 hours) to f e t a l lambs (at rates employed during human to c o l y s i s ) has been shown to r e s u l t i n a 6.9 f o l d decrease i n tracheal f l u i d flow, an increase i n the surfactant content of lung lavage, and an improvement i n f e t a l lung s t a b i l i t y as measured from pressure volume curves (Warburton et a l . . 1987a). An association between r i t o d r i n e infusion, accelerated pulmonary glycogen depletion and increased a v a i l a b i l i t y of surfactant phospholipid i n a l v e o l i has also been reported (Warburton, et a l . , 1987b). The authors have hypothesized that pulmonary glycogen deposits, through beta-2 stimulation, become a source of energy to drive the active processes of surfactant production. On the basis of these re s u l t s and others, a possible b e n e f i c i a l e f f e c t of r i t o d r i n e in the prevention of f e t a l respiratory d i s t r e s s syndrome has been suggested. 23 c. Metabolic Effects Infusion of r i t o d r i n e to the ewe Short (1 hour) and long (48-120 hours) term infusion of r i t o d r i n e to pregnant ewes (at rates employed i n human toc o l y s i s ) have been shown to r e s u l t i n increased maternal and f e t a l plasma leve l s of glucose, i n s u l i n , l a c t a t e and pyruvate (Simmes and Creasy, 1980; Basset et a l . , 1985). Basset et a l . . (1985) reported that with the exception of lac t a t e , f e t a l metabolite concentration changes p a r a l l e l e d the maternal changes reaching peak le v e l s a f t e r approximately 6 hours of r i t o d r i n e infusion. Control values were restored within 72 hours despite continued drug administration (with the exception of lactate which remained s u b s t a n t i a l l y elevated throughout the infusion). Fetal alpha-amino acid nitrogen concentrations f e l l for six to eight hours aft e r the s t a r t of the infusion while glucagon lev e l s remained unaltered. There were no changes i n f e t a l blood gases aside from a slow decline i n f e t a l Pao af t e r 48 hours of infusion. Block et a l . , 2 ' (1989) have recently reported decreases i n f e t a l umbilical venous and descending a o r t i c oxygen contents during r i t o d r i n e infusion to ewes. 24 I n f u s i o n of r i t o d r i n e t o the f e t u s Basset e t a l . , (1989) has shown t h a t long term (48-80 hours) i n f u s i o n of r i t o d r i n e d i r e c t l y t o the f e t u s r e s u l t s i n g l u cose, l a c t a t e and i n s u l i n changes s i m i l a r to those observed d u r i n g maternal i n f u s i o n . Again, a l l m e tabolic parameters r e t u r n e d t o c o n t r o l v a l u e s w i t h i n 72 hours d e s p i t e c o n t i n u e d i n f u s i o n , i n d i c a t i n g an a t t e n u a t i o n of b e t a - a d r e n e r g i c responsiveness. F e t a l alpha-amino a c i d n i t r o g e n l e v e l s were not a l t e r e d . An important d i f f e r e n c e between the maternal verse s f e t a l r i t o d r i n e i n f u s i o n experiments was t h a t i n the l a t t e r case, the d e c l i n e i n f e t a l Pao 2 was much g r e a t e r . R i t o d r i n e i n f u s i o n t o the f e t u s r e s u l t e d i n severe hypoxemia which was maintained d u r i n g the f i r s t 48-72 hours of i n f u s i o n . T h i s p e r t u r b a t i o n was probably one the most compromising of the observed f e t a l e f f e c t s . S i m i l a r r i t o d r i n e - i n d u c e d f e t a l m e t a bolic changes, i n c l u d i n g a f a l l i n a r t e r i a l Pao 2 (by 5.1 mmHg), have been r e p o r t e d elsewhere (Warburton e t a l . , 1987). The mechanism f o r the hypoxemia has not y e t been e l u c i d a t e d , however, the mechanisms governing the changes i n f e t a l m e t a b o l i t e l e v e l s , are l i k e l y s i m i l a r t o those which occur i n the ewe. 25 F. Neonatal Side Effects Adaptation or attenuation of the f e t a l response to beta-mimetic infusion appears to occur i n a manner s i m i l a r to that observed i n the mother. At f i r s t glance, t h i s may seem desirable i n protecting the fetus from prolonged exposure to drug induced cardiovascular and metabolic perturbations. However, since beta-adrenergic mechanisms regulate many processes which are c r u c i a l to early neonatal s u r v i v a l , such as the mobilization of l i p i d and glycogen stores, infants delivered shortly a f t e r long term beta-mimetic t o c o l y s i s may be at r i s k due to attenuation of beta-adrenergic responsiveness (Basset et a l . , 1985). Metabolic and cardiovascular complications including hypoglycemia, heart rate and rhythm disturbances, thickening of the i n t r a v e n t r i c u l a r septum, myocardial i n f a r c t i o n and other complications have been reported i n neonates exposed to r i t o d r i n e i n utero ( Kazzi et a l . f 1987; Katz and Seeds, 1989). The degree to which the infant i s affected i s dependent upon the extent of drug exposure and the length of time elapsed between termination of drug infusion and delivery of the infant. Neonatal complications are generally more severe i f the drug f a i l s and the infant i s born shortly thereafter. Long term ef f e c t s i n children who have been exposed to r i t o d r i n e i n utero have not been observed (Freysz et a l . , 26 1977; Polowczyk e t a l . , 1984; Hadders-Algra e t a l . , 1986). 1.3 RATIONALE AND SIGNIFICANCE The unchanging i n c i d e n c e of preterm l a b o r and d e l i v e r y , and i t s s i g n i f i c a n t c o n t r i b u t i o n t o p e r i n a t a l m o r t a l i t y and m o r b i d i t y have pressured many h e a l t h c a r e workers i n t o the use of pharmacologic agents whose s a f e t y and e f f i c a c y have not y e t been u n e q u i v o c a l l y proven. Beta-mimetic t o c o l y s i s i s commonly employed i n the treatment of preterm l a b o r and r i t o d r i n e i s one of the most w i d e l y used of the b e t a -a d r e n e r g i c agents. The use of r i t o d r i n e i n t o c o l y s i s , i n s p i t e of i t s unknown e f f i c a c y (but known a b i l i t y t o c r o s s the human p l a c e n t a and e x e r t e f f e c t s on the f e t u s ) , i s an i n d i c a t i o n of the present m o t i v a t i o n t o a d m i n i s t e r some s o r t of treatment to i n h i b i t preterm l a b o r . C l e a r l y , f u r t h e r data on the e f f e c t s of r i t o d r i n e on the f e t u s are r e q u i r e d so t h a t the r i s k - b e n e f i t r a t i o may be more c l e a r l y d e f i n e d . Data r e g a r d i n g the e f f e c t s of r i t o d r i n e on the human f e t u s are g e n e r a l l y l a c k i n g . T h i s i s due to the extreme t e c h n i c a l and e t h i c a l c o n s t r a i n t s p l a c e d upon human pregnancy-r e l a t e d r e s e a r c h . The obvious importance of a c q u i r i n g knowledge on the f e t a l e f f e c t s of r i t o d r i n e has n e c e s s i t a t e d the use animal experimentation. The c h r o n i c a l l y instrumented pregnant sheep model has proven t o be extremely u s e f u l i n t h i s 27 regard as i t allows for continued observation and experimentation under conditions that are r e l a t i v e l y undisturbed. The a p p l i c a b i l i t y to humans of information obtained from f e t a l lambs may be questioned, however, s i m i l a r i t i e s which exi s t between the two species indicate that the pregnant ewe i s an appropriate model for study. Fetal lambs and humans are sim i l a r i n cardiovascular and behavioral parameters, metabolic rate, and blood gas values. Important differences, however, occur i n placental structure and permeability, and i n the mechanisms for the onset of labor. In the current study, r i t o d r i n e was administered to the fetus as opposed to the ewe for two reasons. F i r s t l y , the placental transfer of r i t o d r i n e i n sheep i s lim i t e d which i s in contrast to humans where placental transfer appears to occur more r e a d i l y . Secondly, f e t a l infusion allows for the observation of the d i r e c t e f f e c t s that r i t o d r i n e has i n the fetus. Drug infusion to the ewe would have resulted in maternal cardiovascular and metabolic perturbations which could have i n d i r e c t l y effected the fetus by a l t e r i n g the placental transfer of nutrients and metabolites. There have been a number of studies investigating the metabolic and cardiovascular e f f e c t s of r i t o d r i n e i n ewes and f e t a l lambs. While recent studies have reported f e t a l hypoxemia during r i t o d r i n e administration, a mechanism for 28 these changes has not y e t been e l u c i d a t e d . A p r i n c i p a l g o a l of the c u r r e n t study was to c o n f i r m t h i s change and t o e l u c i d a t e the p o s s i b l e mechanism. 1.4 OVERALL OBJECTIVE The o v e r a l l o b j e c t i v e of the c u r r e n t study was t o i n f u s e r i t o d r i n e d i r e c t l y t o the f e t u s f o r a p e r i o d of up t o 24 hours ( a t t a i n i n g plasma r i t o d r i n e l e v e l s comparable t o those observed i n human f e t u s e s ) and to examine the e f f e c t s of t h i s i n f u s i o n on v a r i o u s f e t a l and maternal p h y s i o l o g i c a l parameters u s i n g the c h r o n i c a l l y instrumented pregnant sheep p r e p a r a t i o n . 1.5 SPECIFIC OBJECTIVES i ) t o examine the e f f e c t s of r i t o d r i n e on f e t a l and maternal a r t e r i a l and u m b i l i c a l and u t e r i n e venous blood gases, acid-base s t a t u s , hematocrit and glu c o s e , l a c t a t e and oxygen c o n c e n t r a t i o n s and f l u x e s . i i ) t o v e r i f y p r e v i o u s l y r e p o r t e d r i t o d r i n e - i n d u c e d d e c l i n e s i n f e t a l a r t e r i a l and u m b i l i c a l venous Po and Co 2 2 (oxygen content) val u e s and to e l u c i d a t e a p o s s i b l e mechanism f o r these changes. 29 i i i ) to examine the effects of r i t o d r i n e on f e t a l and maternal cardiovascular parameters including heart rate, a r t e r i a l pressure, and umbilical and uterine blood flows. iv) to study the e f f e c t s of r i t o d r i n e on f e t a l behavioral states including e l e c t r o c o r t i c a l and electroocular a c t i v i t i e s and breathing movements. v) to correlate the observed f e t a l e f f e c t s with f e t a l a r t e r i a l plasma l e v e l s of r i t o d r i n e . 30 2. EXPERIMENTAL 2.1 MATERIALS A. Drugs and Chemicals Ritodrine hydrochloride, (Yutopar® Injectable, 10 mg/mL) was purchased from Bristol-Myers Pharmaceutical Group, (Ottawa, Ontario). Other injectables u t i l i z e d i n sheep preparations including atropine sulphate (0.6 mg/mL, Astra Pharmacy Inc., Mississauga, Ontario), a m p i c i l l i n (Penbritin®, 500 mg, Ayrest Laboratories, Montreal, Quebec), heparin sodium (Heparin Leo®, 1000 I.U./mL, Leo Laboratories Canada Ltd., Pickering, Ontario), 5.0% dextrose (Travenol Canada Inc., Mississauga, Ontario), 0.9% sodium chloride (Astra Pharmacy Inc., Travenol Canada Inc.) and s t e r i l e water (Squibb Canada Inc., Montreal, Quebec) were obtained from Shaughnessy Hospital Pharmacy, Vancouver B.C. The anesthetics, halothane (Fluothane®, Ayrest Laboratories) and thiopental sodium injectable (Pentathol®, Abbott Laboratories, Montreal, Quebec), were purchased from Shaughnessy Hospital and the University of B r i t i s h Columbia Pharmacies, B.C. respectively. Reagents required for the glucose and lactat e sigma assay k i t s , including glucose standard solution (1.0 mg/mL), 0.3 N zinc sulphate, 0.3 N barium hydroxide, O-dianisidine dihydrochloride powder, PGO capsules (containing 500 I.U. glucose oxidase, 100 Purpruogallin units peroxidase and buffer s a l t s ) , l a c t a t e dehydrogenase, glycine buffer, and nicotinamide adenine dinucleotide were purchased from Sigma Chemical Co., St. Louis, Missouri. Perchloric acid (0.8%), required for l a c t a t e assay was d i l u t e d from a 70.0 - 72.0 % sol u t i o n purchased from Fisher S c i e n t i f i c Co. , F a i r Lawn, New Jersey. Antipyrine (2,3-dimethyl-l-phenyl-3-pyrazolin-5-l; Phenazone) was purchased from Sigma Chemical Company. Reagents required for antipyrine assay including sodium n i t r a t e (ACS grade) , zinc sulphate (ZnS04-7H20, ACS reagent grade) , s u l f u r i c acid (4N & 6N d i l u t e d from ACS grade concentrated s u l f u r i c acid), sodium hydroxide (ACS grade NaOH pel l e t s ) were a l l purchased from Fisher S c i e n t i f i c Co., F a i r Lawn, New Jersey. B. Catheters and Electrodes Catheters (Figure 3) implanted into maternal and f e t a l vessels were composed of 12 5 cm lengths of s i l i c o n e rubber tubing (Silastic® medical grade tubing, Dow Corning Corporation, Midland, Michigan). Maternal a r t e r i a l , f e t a l tracheal and amniotic catheters had inside (i.d.) and outside (o.d.) diameters of 0.040" and 0.085" respectively. Uterine 32 a) Basic common design of the maternal femoral a r t e r i a l , uterine venous, and f e t a l femoral a r t e r i a l , t a r s a l venous and tracheal catheters. See text (page 31) for d e t a i l s and s p e c i f i c catheter measurements. Catheter s p e c i f i c length & diameter Anchoring suture S i l i c o n e rubber tubing b) Amniotic Catheter Additional perforations c) Umbilical Venous Catheter Narrow inse r t of s i l i c o n e rubber tubing Figure 3. Catheters u t i l i z e d in the chronic pregnant sheep preparation (not drawn to s c a l e ) . 33 venous, f e t a l a r t e r i a l and t a r s a l venous catheters had i . d and o.d values of 0.025" and 0.047" respectively. The umbilical venous catheter was s i m i l a r i n size to the maternal a r t e r i a l catheter but had an additional piece of s i l a s t i c tubing ( i . d . 0.020", o.d. 0.037") inserted into one end (for implantation). S i l k sutures (Davis & Geek® Cyanamid Canada Inc., Montreal, Quebec) were securely fastened to each catheter at the implantation end (to anchor the catheter to the vessel and t i s s u e ) . The c o r t i c a l electrodes (Figure 4) were prepared by p u l l i n g three 155 cm lengths of insulated multistranded s t a i n l e s s s t e e l wire (Biomedical Wire, Cooner Wire Co. , Chatsworth, Ca l i f o r n i a ) through a 125 cm long length of s i l i c o n e rubber tubing ( i . d . 0.058", o.d. 0.077"). A single knot was t i e d on both ends at the points at which the wires exited the tube (exit knots). On one end (for implantation), 2 p l a s t i c disks (1 mm thick, 5 mm diameter) were s l i d onto two of the wires (1 disk per wire) and secured 4 cm beyond the e x i t knot. A knot was made below each disk and the i n s u l a t i o n removed from the remaining portion (5 mm) of the wire. The t h i r d wire was used as a ground electrode; a single knot was t i e d 2 cm beyond the e x i t knot (10.5 cm length of ground wire e x i t s tube) and a 5 mm segment of i n s u l a t i o n was removed immediately beyond i t . On the other end of the electrode (for recording) a knot was placed on each wire 34 a) E l e c t r o c o r t i c a l electrode P l a s t i c disk S i l i c o n e rubber tubing sulated s t a i n l e s s steel multistranded wire Figure 4. E l e c t r o c o r t i c a l and electroocular electrodes u t i l i z e d i n the chronic pregnant sheep preparation (not drawn to scale). See text (page 33) for d e t a i l s and s p e c i f i c electrode measurements. 35 (2 knots on ground) at a distance of 12 cm beyond the e x i t knot and the in s u l a t i o n removed immediately beyond them. The ocular electrodes (Figure 4) were prepared s i m i l a r l y to the c o r t i c a l electrodes with the following differences. F i r s t l y , only two wires were pulled through the s i l a s t i c tube. On one end (for implantation), a 5 mm length of in s u l a t i o n was removed from each of the two wires, at a distance of 8 cm beyond the e x i t knot (16 cm of wire exits the tube). On the other end of the electrode (for recording), single knots were placed on each wire at a distance of 7.5 cm beyond the e x i t knot with the ins u l a t i o n being removed from the remaining portion of the wire (8 cm). 2.2 PREPARATION OF SOLUTIONS FOR INFUSION A. Ritodrine Ritodrine solution for infusion (40 ug/mL) was prepared by di s s o l v i n g 0.2 mL stock solution (10 mg/mL) into 0.9% isotonic saline to a f i n a l volume of 50 mL. B. Antipyrine Antipyrine stock solution (150 mg/mL) was prepared by diss o l v i n g 37.5 g antipyrine (phenazone) into deionized, d i s t i l l e d water to a f i n a l volume of 250 mL. The solut i o n was then f i l t e r e d i n 25 mL aliquots through 0.22 um f i l t e r s (Millex®-GS, M i l l i p o r e Corp., Bedford Massachusetts) into s t e r i l e glass v i a l s (Miles Laboratories Inc., Spokane, Washington) and stored at 4°C. For experiments, the antipyrine solution was further d i l u t e d to a f i n a l concentration of 75 mg/mL (25 mL antipyrine d i l u t e d i n isot o n i c s aline to a f i n a l volume 50 mL). C. Saline Heparinized saline (12.0 I.U./mL) u t i l i z e d i n flushing catheters was prepared by in j e c t i n g 3.0 mL sodium heparin into a 2 50 mL bag of 0.9% sodium chloride. 2.3 PREPARATION OF SOLUTIONS FOR ASSAYS A. Glucose Assay The entire contents of one v i a l containing O-dianisidine dihydrochloride (50 mg/vial) was reconstituted i n 2 0 mL deionized, d i s t i l l e d water. One PGO capsule (containing 500 I.U. glucose oxidase, 100 purpruogallin units of peroxidase and buffer salts) was dissolved i n 100 mL deionized, d i s t i l l e d water to which 1.6 mL of the above O-dianisidine dihydrochloride solution had previously been added. These 37 solutions were stored at 4°C for up to one month. B. Lactate Assay To 50.0 mg of nicotinamide adenine dinucleotide were added: 20 mL deionized, d i s t i l l e d water, 10 mL glycine buffer and 0.5 mL lactate dehydrogenase. This solution was stored at 4°C for up to two days. C. Antipyrine Assay Antipyrine standard solution for assay (50 mg/L) was prepared by dis s o l v i n g 0.25 g of antipyrine powder into 50 mL deionized d i s t i l l e d water to y i e l d a stock solution concentration of 5 mg/mL. A 2 mL aliquot of t h i s solution was further d i l u t e d i n deionized, d i s t i l l e d water to a t o t a l of 200 mL to y i e l d the desired concentration of 50 mg/L. Aqueous sodium n i t r a t e (.024 M) was prepared by di s s o l v i n g 0.2 g of sodium n i t r a t e powder into a t o t a l of 100 mL deionized, d i s t i l l e d water. Zinc sulfate solution was prepared by dis s o l v i n g 100.0 g of zinc sulfate powder into 250 mL deionized, d i s t i l l e d water (to which 40 mL of 6 N s u l f u r i c acid had previously been added) and d i l u t i n g the entire mixture to a t o t a l volume of 1 L. Four and six normal s u l f u r i c acid were dil u t e d from concentrated s u l f u r i c acid. Aqueous sodium hydroxide (0.75 N) was prepared by di s s o l v i n g 38 sodium hydroxide p e l l e t s into deionized, d i s t i l l e d water. 2.4 ANIMAL PREPARATION A. Breeding Time dated pregnant sheep of Suffolk and Dorset breed were obtained throughout the year (September through June) by breeding estrus synchronized groups of ewes (2 or 3 ewes per group) on predetermined dates at the UBC Department of Animal Sciences. Estrus synchronization was accomplished though intravaginal implantation of Veramix® Sheep Sponges (Tuco Products Co. , Orangeville, Ontario) which release medroxyprogesterone acetate, a progestogen which r e s u l t s i n anovulation upon absorption into the ewe. Immediately upon removal of the sponge (14 days l a t e r ) , ovulation was induced by intramuscular i n j e c t i o n of 500 I.U. Pregnant Mares' Serum Gonadotropin (Ayrest Laboratories) , a product which stimulates f o l l i c u l a r development i n functional ovaries. Ewes were expected to go into estrous approximately 36 to 48 hours l a t e r and hence were immediately placed with a ram. Ewes whom did not become pregnant during the f i r s t estrus cycle, remained with the ram to be bred during the second cycle which was expected to occur approximately two weeks af t e r the f i r s t . Pregnancy was determined by measurement of serum progesterone l e v e l s i n the ewe at approximately 17 days post-ovulation and 39 was confirmed by real-time ultrasound p r i o r to surgery. B. Surgery The ewes were brought into the unit at least one week pr i o r to surgery to enable them to become accustomed to the environment. They were placed together i n a holding pen and were fed a die t s i m i l a r to that which they had received at the UBC Department of Animal Science. A l f a l f a cubes and mixed grains were provided twice d a i l y while water and s a l t blocks were always available. The ewes were s u r g i c a l l y prepared at 116-125 days (mean 123 ± 2 days) gestation. A l l animals were fasted for approximately 18 hours pr i o r to surgery, having access to water only. Atropine su l f a t e (3 mg) was administered v i a the jugular vein of the ewe roughly 10 minutes before surgery to reduce s a l i v a r y secretions. Anaesthesia was induced through i . v . i n j e c t i o n of 1 g thiopental sodium and was maintained (following intubation) with 1.5 to 2.0% (v/v) halothane and nitrous oxide (70%) i n oxygen. Approximately 500 mL of 5% dextrose solution was administered to the ewe v i a an i . v . drip into a jugular vein which had been catheter!zed by transcutaneous puncture. The abdomen of the ewe was prepared for surgery by shaving and then s t e r i l i z i n g the abdominal area with providone-iodine t o p i c a l solution (Rougier Inc., Chambly, Quebec). The remaining areas of the ewe were draped with s t e r i l e sheets. The ent i r e surgical procedure was conducted under aseptic s u r g i c a l conditions. Access to the fetus was gained though a midline abdominal i n c i s i o n followed by two small uterine i n c i s i o n s . The f i r s t uterine i n c i s i o n afforded access to the f e t a l head and neck. The trachea was exposed through a small i n c i s i o n i n the skin and subsequent blunt dissection. A small puncture was made between two adjacent rings of c a r t i l a g e , approximately 1-2 cm below the larynx. The catheter was inserted, advanced approximately 4 to 5 cm, and a f f i x e d by applying a drop of Krazy Glue® (Feature Products Inc., Mississauga, Ontario) to the i n s e r t i o n s i t e . The catheter was secured to the skin overlying the i n c i s i o n using the anchoring sutures ( s i l k sutures attached to the catheter). The i n c i s i o n was then closed. Free flow of tracheal f l u i d was not obstructed by the catheterization. C o r t i c a l electrodes (to record e l e c t r o c o r t i c a l a c t i v i t y ) were implanted into the fetus by making a transverse i n c i s i o n on the dorsal s k u l l , just r o s t r a l to the ears. The scalp was folded back and secured, exposing the p a r i e t a l region of the s k u l l . The periosteum was scraped o f f the bone and two 1 mm 41 diameter holes (approximately 3.0 cm on e i t h e r side of the midline) were hand d r i l l e d over the p a r i e t a l cortex ( d r i l l b i t diameter 0.0625 inch, F u l l e r Tool Co. Ltd., PteClaire, Quebec). The electrode wires were inserted into the holes such that they rested on the p a r i e t a l dura mater and were a f f i x e d by gluing the p l a s t i c disks to the s k u l l with Krazy Glue®. The ground electrode was sutured under the scalp. The i n c i s i o n was closed and the electrodes were anchored to the skin near the base of the s k u l l . The ocular electrodes (to detect eye movements) were implanted by making a small v e r t i c a l i n c i s i o n on the outer canthus overlying the o r b i t a l ridge of the zygomatic bone of each eye. The electrode wire (one for each eye) was guided (using a small cutting needle) underneath the o r b i t a l ridge and upon e x i t i n g the l a t e r a l side, was t i e d such that the stripped section of the wire was knotted adjacent to the bone. The electrode was a f f i x e d by applying a drop of Krazy Glue® to the knot. The ocular electrodes were then anchored to the e l e c t r o c o r t i c a l electrodes near the base of the s k u l l . The f i r s t of two amniotic catheters was subsequently secured to the skin of the f e t a l neck. The fetus was then placed back into the uterus and the i n c i s i o n closed. The second uterine i n c i s i o n provided access to the f e t a l hind limbs and umbilical vein. The r i g h t and l e f t femoral a r t e r i e s were generally the f i r s t vessels to be catheterized. The femoral a r t e r i a l pulse was located i n the groin by palpation and a small i n c i s i o n was made above i t . The artery was exposed by blunt dissection and three s i l k sutures were passed beneath the vessel. The d i s t a l suture was t i e d o f f (to permanently c o n s t r i c t the vessel at the d i s t a l end) while loose single knots were placed i n the middle and proximal sutures. While maintaining temporary proximal vessel c o n s t r i c t i o n (by placing tension on the proximal suture) a hole was placed between the d i s t a l and middle sutures. The catheter was inserted into the vessel, advanced into the descending aorta (approximately 5 to 6 cm), and secured by tying the d i s t a l and middle sutures around the catheter. The catheter was a f f i x e d by a drop of Krazy Glue® and further secured by tying the catheter's anchoring sutures to muscle tissue on either side of the vessel. The i n c i s i o n was then closed. The r i g h t and l e f t f e t a l t a r s a l veins were located by v i s u a l observation (the wool from the l a t e r a l aspect of the lower limbs was shaved to aid i n vessel location and catheterization) . A small i n c i s i o n was made d i r e c t l y above the vessel and the vein was exposed by blunt dissection. Two s i l k sutures were placed beneath the vessel and loose single knots were t i e d . While maintaining tension on both sutures, a hole was made between them. The catheter was inserted, 43 advanced into the i n f e r i o r vena cava (approximately 11 to 12 cm) , and secured by tying the sutures around the catheter. Krazy Glue® was applied to the i n s e r t i o n s i t e and the two anchor sutures were t i e d to the skin overlying the vessel. The i n c i s i o n was then closed. The umbilical vein was normally the f i n a l f e t a l vessel to be catheterized. The fetus was further delivered u n t i l the umbilicus was exposed. An i n c i s i o n was made above one of the two umbilical veins at the point at which they entered the fetus through the umbilicus. The vein was exposed (within the umbilicus) by blunt dissection and two s i l k sutures were placed within the vessel wall i n a transverse fashion (approximately 3 mm from one another). A small puncture was made between these sutures (using an 18 gauge needle) and the catheter was inserted (directed towards f e t a l body). The sutures were t i e d i n a manner so as to form a figure-eight suture around the catheter i n s e r t i o n s i t e . The catheter was then a f f i x e d with Krazy Glue®. The proximal anchoring suture was secured to the skin close to the i n c i s i o n s i t e while the d i s t a l anchoring suture was fastened to the lower abdomen. The i n c i s i o n was then closed. The umbilical venous cathet e r i z a t i o n was non-occlusive and did not impair blood flow to the fetus. Before returning the fetus to the uterus, the second amniotic catheter was secured to the lower abdomen of the fetus. The uterine i n c i s i o n was then closed. 44 Catheterization of the maternal femoral artery and uterine vein were accomplished i n a manner sim i l a r to that of the f e t a l femoral artery and l a t e r a l t a r s a l vein respectively. A l l catheters and leads were tunnelled subcutaneously and exteriorized through a small i n c i s i o n i n the r i g h t flank of the ewe where they were stored i n a denim pouch. The midline abdominal i n c i s i o n was closed i n layers and the i n c i s i o n on the r i g h t flank of the ewe was c a r e f u l l y closed (as were a l l incisions) so as to maintain patency of the catheters. Amniotic f l u i d l o s t during surgery was replaced with i r r i g a t i o n s a line (Travenol Canada Inc., Mississauga, Ontario) through the amniotic catheter. Upon completion of surgery, catheters were flushed with s t e r i l e heparinized s a l i n e (0.9% NaCl solution containing 12 I.U./mL) and thereafter on a d a i l y basis. A n t i b i o t i c prophylaxis i n the ewe consisted of two separate doses of a m p i c i l l i n on the day of surgery (500 mg i . v , 500 mg i.m.) followed by 500 mg a m p i c i l l i n i.m. on each of the f i r s t 3 post-surgical days. The fetus received two separate doses of a m p i c i l l i n on the day of surgery (500 mg i . v , 500 mg into amniotic c a v i t y ) , followed by 500 mg a m p i c i l l i n into the amniotic cavity on a d a i l y basis for the duration of the preparation. Following surgery, the ewes were kept i n holding pens i n the company of other sheep and were allowed a minimum of three days to recover before experimentation. S t e r i l e techniques were 45 exercised continually i n order to maintain a f e t a l environment which was as s t e r i l e as possible. 2.5 RECORDING AND MEASUREMENT A. Biophysical Variables The following variables were recorded continuously on an 8 channel (Beckman R612, Beckman Instruments Inc., S c h i l l e r Park, I l l i n o i s ) or 12 channel (Sensormedics R711, Sensormedics, Anaheim, Ca l i f o r n i a ) dynograph recorder using appropriate Beckman or Sensormedic input couplers: f e t a l a r t e r i a l , tracheal, and amniotic pressures, maternal a r t e r i a l pressure, maternal and f e t a l heart rates, f e t a l electrocorticogram (ECoG), and f e t a l electrooculogram (EOG). A r t e r i a l , tracheal and amniotic pressures were measured with Gould Statham P23Db or P23ID strain-gage transducers or Gould DTX disposable transducers (Spectramed Inc., Oxnard, C a l i f o r n i a ) whose signals were received by 9872 strain-gage couplers (Beckman Instruments Inc. and Sensormedics). Maternal and f e t a l heart rates were measured with 9875 cardiotachometers (Beckman Instruments Inc. and Sensormedics) triggered by the a r t e r i a l pulse pressure. Fetal ECoG and EOG were measured with 9806A high-gain AC/DC amplifiers (Beckman Instruments Inc. and Sensormedics). The analog signals from amniotic and a r t e r i a l pressures and heart rates were d i g i t i z e d and processed on-line with an Apple H e computer system (Apple Computer Inc., Cupertino, Ca l i f o r n i a ) containing an analog to d i g i t a l conversion board (AI-13 Analog Input System, Daisi Electronics Inc., Newton Square, Pennsylvania). D i g i t i z e d information from each variable was averaged at one minute in t e r v a l s and the mean one minute values were stored every 3 0 minutes on floppy diskettes. Fetal a r t e r i a l pressure was corrected for intrauterine pressure. B. Measurement of Uterine and Umbilical Blood Flow Umbilical and uterine blood flows were measured using the steady-state antipyrine d i f f u s i o n technique developed by Meschia et a l . . (1967). Antipyrine stock solution (150 mg/mL) was d i l u t e d i n isotonic saline to 75 mg/mL and was infused into a f e t a l t a r s a l vein at a constant rate of 4.9 mg/min (volume flow, 0.0648 mL/min) using a Harvard Apparatus Infusion pump (Harvard Apparatus, M i l l i s , Massachusetts). Samples were taken simultaneously from the f e t a l femoral artery (FFA), umbilical vein (UV), maternal femoral artery (MFA) and a main uterine vein (UTV) roughly 90 minutes a f t e r the s t a r t of the infusion, at which time steady state had been attained. Umbilical blood flow was calculated as the quotient of the antipyrine infusion rate and the umbilical arteriovenous concentration difference. Uterine blood flow was calculated s i m i l a r l y as the quotient of the antipyrine infusion rate and the uterine venoarterial concentration difference. C. Blood Sample Analysis Blood samples were taken simultaneously from the FFA, UV, MFA, and UTV catheters at precise time periods (see Section 2. 6B for sampling schedule). Before withdrawing a sample, the catheter was cleared of saline by withdrawing three times the catheter volume into another syringe. Subsequent to sampling, t h i s mixture of blood and saline was returned to the animal, and the catheter flushed with heparinized saline (approximately 2 mL) . A t o t a l volume of 3.5 mL of blood was taken from each catheter during each sample for analysis of blood gases, oxygen content and hematocrit (0.5 mL blood required) and plasma lactate, glucose, antipyrine, and r i t o d r i n e (0.3, 0.2, 1.0, and 1.5 mL blood required re s p e c t i v e l y ) . Blood gas samples were c o l l e c t e d i n preheparinized blood gas syringes (Marquest Medical Products Inc., Englewood, Colorado) and analyzed immediately. Blood for the remaining measurements were c o l l e c t e d i n 3 mL syringes (Beckton Dickinson and Co.) and immediately emptied into c h i l l e d , heparinized polystyrene tubes (Evergreen S c i e n t i f i c International Inc., Los Angles, C a l i f o r n i a ) for the i n i t i a l stages of analysis (see below). 48 i Blood Gases and Hematocrit Fetal and maternal blood Po2, Pco 2, pH, base excess, and bicarbonate were measured with an IL 1306 pH/blood gas analyzer ( A l l i e d Instrumentation Laboratory, Milano, I t a l y ) . Oxygen content was measured on a Lexington Instruments Lex-02-Con-K oxygen analyzer (Lexington Instruments Corporated, Waltham, Massachusetts). Oxygen content was measured i n duplicate or u n t i l two sequential measurements were within 0.2 v o l % of one another and the average was taken as the f i n a l value. Hematocrit (also measured i n duplicate) was measured by micro centrifugation of whole blood i n c a p i l l a r y tubes ( S c i e n t i f i c Products, McCaw Park, I l l i n o i s ) which were subsequently read on a micro-capillary reader (International Equipment Co., Needham Hts, Massachusetts). i i Glucose a. Preparation for Analysis Samples for glucose determination were prepared for analysis by adding 0.2 mL whole blood to 0.9 mL deionized, d i s t i l l e d water. To t h i s mixture were added 0.55 mL of 0.3 N zinc sulfate followed 10 minutes l a t e r by 0.55 mL barium hydroxide. Samples were vortexed a f t e r each step. Glucose samples were then centrifuged at 4000 r.p.m for 15 minutes and 49 the supernatant re f r i g e r a t e d at 4 °C u n t i l assayed, b. Assay Glucose samples were assayed within two weeks using a Sigma glucose assay k i t (Sigma Chemical Co., St. Louis, Missouri). The assay i s based on the following reactions: 1) Glucose, i n the presence of water, oxygen and glucose oxidase i s oxidized to gluconic acid and peroxide. 2) 0-d i a n i s i d i n e , i n the presence of peroxidase and the peroxide produced i n reaction 1 becomes oxidized. The number of moles of oxidized O-dianisidine produced i s equivalent to the number of moles of glucose o r i g i n a l l y present. The concentration of oxidized O-dianisidine i s determined by spectrophotometric analysis (absorbs at a wavelength of 450 nm) . Preparation of the reagents required for t h i s assay were described i n Section 2.3 A. A l l analyses were done i n duplicate. Glucose standards were made to 0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 75.0, and 100.0 mg% by adding 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15 and 0.20 mL of a glucose standard solution (1.0 mg/mL) to polystyrene tubes and bringing the volume of each tube to 1.1 mL with deionized, d i s t i l l e d water. The following method was used i n the analysis of both the glucose standards and plasma samples. To glass t e s t tubes, each containing 5.0 mL PGO reagent (see Section 2.3 50 A), were added 0.5 mL of sample. The glass tubes were then sealed, vortexed, incubated in a water bath at 37°C for 25 minutes and placed i n the dark at room temperature for 10 minutes. Absorbance readings were measured at 450 nm on eithe r a Pye Unicam SP8-400 UV/VIS spectrophotometer (Pye Unicam Ltd, Cambridge, England) or a G i l f o r d Instrument model Stasar II spectrophotometer ( G i l f o r d Instrument Laboratories Inc., Oberlin, Ohio). A standard glucose concentration verses absorbance curve was constructed for each analysis and the plasma glucose concentrations were calculated. i i i Lactate a Preparation for Analysis Samples for lactate determination were deproteinized by adding 0.3 mL whole blood to 0.6 mL of 8% perchloric acid. Samples were then centrifuged at 4000 r.p.m. for 15 minutes and the supernatent refrigerated at 4°C u n t i l analysis. b Assay Lactate samples were assayed within 3 weeks using a Sigma lac t a t e assay k i t (Sigma Chemical Co., St. Louis, Missouri). The assay i s based on the following reaction: L a c t i c acid i n the presence of excess nicotinamide adenine dinucleotide (NAD) 51 and lactate dehydrogenase i s converted to equimolar quantities of pyruvic acid and NADH. The NADH concentration i s determined by spectrophotometric analysis (absorbs at a wavelength of 340 nm). The number of moles of NADH produced i s equivalent to the number of moles of lactate o r i g i n a l l y present. A l l samples were analyzed i n duplicate. Preparation of the reagents required f or t h i s assay are described i n Section 2.3 B. The following procedure was used i n the analysis of lactate concentrations. To polystyrene tubes, each containing 1.4 mL of NAD/lactate dehydrogenase solution (see Section 2.3 B) were added 0.1 mL of sample. The tubes were incubated i n a water bath at 37 °C for 45 minutes and then at room temperature for a further 5 minutes. Absorbance values were measured spectrophotometrically at 340 nm. A standard curve provided by the Sigma Chemical Company was used i n c a l c u l a t i n g lactat e concentrations (slope of curve used i n t h i s c a l c u l a t i o n i s highly reproducible). i v Antipyrine a. Preparation for Analysis Blood for antipyrine measurement was centrifuged at 4000 r.p.m. for 15 minutes and the supernatant frozen u n t i l 52 analysis, b. Assay Antipyrine concentrations were measured using the method developed by Davidson and Mclntyre (1956). The assay i s based on the following reaction: Antipyrine, in the presence of sodium n i t r i t e and s u l f u r i c acid forms an equimolar quantity of 4-nitrosoantipyrine. The concentration of 4-nitroso-antipyrine i s determined by spectrophotometric analysis (absorbs at 3 50 nm) . The number of moles of nitrosoantipyrine i s equal to the number of moles of antipyrine o r i g i n a l l y present. The readings are stable for approximately 20 minutes. Preparation of the reagents required for t h i s assay have been described i n Section 2.3 C. A l l samples were analyzed in duplicate. Antipyrine standards were made to 2.5, 5.0, 7.5, 10.0, 12.5, 15, and 20.0 mg/L by adding 0.15, 0.30, 0.45, 0.60, 0.75, 0.90 and 1.20 mL antipyrine standard solution (50 mg/L) to polystyrene tubes and bringing the volume of each tube to 3.0 mL with deionized, d i s t i l l e d water. The following method was used i n the analysis of both the antipyrine standards and plasma samples. To polystyrene tubes, each containing 0.6 mL d i s t i l l e d , deionized water and 0.4 mL zinc s u l f a t e solution (see Section 2.3C), were added 200 uL of sample. The tubes were vortexed and l e f t standing at room temperature for 10 minutes. To each tube were further added 0.4 mL of 0.75 N NaOH and following vortex, samples were incubated at 4°C for a minimum of 2 hours. Subsequent to incubation, samples were centrifuged at 3000 r.p.m for 20 minutes and 1 mL aliquots of supernatant were transferred to new polystyrene tubes. Each standard and plasma sample then received 50 and 20 uL of 4N s u l f u r i c acid respectively. After being vortexed, each standard and plasma sample received 100 and 40 uL of 0.04 N sodium n i t r a t e respectively. A l l samples were sealed with parafilm, vortexed and incubated i n a water bath at 30 °C for 20 minutes. Immediately following incubation, absorbance readings were measured at 350 nm on a spectrophotometer. A standard antipyrine concentration verses absorbance curve was constructed and the plasma antipyrine concentrations were calculated. v Ritodrine a. Preparation for Analysis Blood for r i t o d r i n e measurement (1.5 mL) was centrifuged at 4000 r.p.m. for 15 minutes and the supernatent stored i n glass culture tubes (with polytetrafluoroethylene-lined screw caps) at -20 °C u n t i l analysis. 54 b. Assay A s e n s i t i v e and s e l e c t i v e assay f o r the q u a n t i t a t i o n of r i t o d r i n e c o n c e n t r a t i o n s i n plasma was developed by Mr. M.R. Wright of the Department of Pharmaceutical Sciences a t the U n i v e r s i t y of B r i t i s h Columbia. The method i n v o l v e s the d e r i v a t i z a t i o n of r i t o d r i n e and i n t e r n a l standard (5-hydroxy propafenone) with h e p t a f l u o r o b u t y r i c anhydride (HBFA). The d e r i v a t i z e d compounds were measured with f u s e d - s i l i c a gas chromatography u s i n g e l e c t r o n - c a p t u r e d e t e c t i o n ( s p l i t l e s s i n j e c t i o n t e c h n i q u e ) . Plasma r i t o d r i n e c o n c e n t r a t i o n s were obt a i n e d u s i n g a c a l i b r a t i o n curve which was c o n s t r u c t e d by p l o t t i n g the area r a t i o s of d e r i v a t i z e d r i t o d r i n e and i n t e r n a l s tandard a g a i n s t a known amount of r i t o d r i n e . R i t o d r i n e c o n c e n t r a t i o n s as low as 1 ng/ml from sample volumes of between 100 to 500 uL were d e t e c t a b l e . Mr. Wright measured the r i t o d r i n e c o n c e n t r a t i o n s i n a l l f e t a l and maternal blood samples. 2.6 EXPERIMENTAL PROTOCOL Ewes were t r a n s f e r r e d to a s m a l l monitoring pens ad j a c e n t to the l a r g e r h o l d i n g pens, remaining i n f u l l view of companion ewes. T h i s served to minimize a n x i e t y i n the ewe w h i l e being monitored. The ewe had f r e e access to food and water. A. Drug A d m i n i s t r a t i o n i I n f u s i o n Rate and Route R i t o d r i n e (10 mg/mL) was d i l u t e d i n i s o t o n i c s a l i n e t o 40 ug/mL (0.2 mL st o c k r i t o d r i n e s o l u t i o n d i l u t e d t o a t o t a l of 50 mL) and was i n f u s e d c o n t i n u o u s l y i n t o a f e t a l t a r s a l v e i n a t a r a t e of 2.6 ug/min (1.3 ug/min.kg assuming a f e t a l weight of 2 kg) u s i n g a Harvard Apparatus I n f u s i o n Pump (Harvard Apparatus, M i l l i s , Massachusetts). i i I n f u s i o n D u r a t i o n R i t o d r i n e was i n f u s e d t o the f e t u s f o r a p e r i o d of 8, 12, or 24 hours. The d u r a t i o n of the i n f u s i o n was determined by the changes i n f e t a l a c i d base s t a t u s . The i n f u s i o n of r i t o d r i n e was stopped before the 24 hour maximum ( i . e . a t 8 or 12 hours) i n those cases were f e t a l pH and BE had f a l l e n below 7.30 and -1.0 r e s p e c t i v e l y by e i t h e r of the two e a r l i e r time p e r i o d s ; otherwise the i n f u s i o n was continued t o 24 hours. B. Sampling Schedule Samples were taken from the f e t a l femoral a r t e r y (FFA), u m b i l i c a l v e i n (UV), maternal femoral a r t e r y (MFA) and main uterine vein (UTV) at predetermined time periods (see Figure 6, Section 3.2). Control samples were taken at -48, -24, and -1 hour before the infusion of r i t o d r i n e . Upon commencement of drug administration, samples were taken at 1.5, 8, 12, and 24 hours of infusion (depending on the duration of the infusion) and at +1.5, +8, +24, and +48 hours post-infusion. Samples sets were taken i n duplicate at each time period, one each being taken i n high and low f e t a l ECo2G state. In those cases where f e t a l ECoG was not monitored, sample sets were separated by approximately 15 minutes. A t o t a l volume of 3.5 mL was taken from each catheter during each sample for analysis as explained i n Section 2.5 C. Thus a t o t a l volume of 14 mL of blood was taken each from the fetus and ewe during each duplicate sampling session [(3.5 mL blood) (2 catheters, A/V) (2 samples, duplicate) = 14 mL blood)]. Fetal blood was replaced by either maternal blood or blood from other ewes (where possible). C. Control Saline Infusions Control experiments were car r i e d i n the same manner as the r i t o d r i n e experiments with the exception that r i t o d r i n e was replaced by isotonic saline. A l l saline infusions were 2 4 hours i n duration. ANALYSIS  Calculations Glucose, Lactate and Oxygen Delivery (DF) to the Fetus Umbilical Venous X Umbilical Blood Flow = DF Concentration Glucose, Lactate and Oxygen Delivery (DU) to the Uterus Maternal Femoral A r t e r i a l X Uterine Blood Flow = DU Concentration Glucose, Lactate and Oxygen Uptake (UPF) by the Fetus (Umbilical Venous - Femoral A r t e r i a l ) X Umbilical = UPF (Concentration Concentration ) Blood Flow Glucose, Lactate, and Oxygen Uptake (UPU) by the Uterus (Maternal Femoral - Uterine Venous) X Uterine = UPU ( A r t e r i a l Cone. Concentration ) Blood Flow Fractional Oxygen Extraction (EX) ( f e t a l or uterine) (Oxygen Consumption / Oxygen Delivery) X 100 = EX (%) v i Uteroplacental oxygen consumption (C) 58 S i n g l e Pregnancy: U t e r i n e 0 , 2 Consumption F e t a l 0 2 , Consumption C i Twin Pregnancy: U t e r i n e 0 2 - (0 2 Consumption + 0 2 Consumption) = C 2 Consumption ( F i r s t Fetus Second Fetus) Since the second f e t u s was not c a t h e t e r i z e d , i t ' s oxygen consumption was c a l c u l a t e d as f o l l o w s : 0 2 consumption 2nd f e t u s = (0 2 consumption 1st fetus/kg) X (weight of the 2nd f e t u s i n kg). **** Unless otherwise i n d i c a t e d , the above c a l c u l a t e d v a r i a b l e s are normally expressed as umol/min or umol/min.kg. B. E s t i m a t i o n of F e t a l Weight i n u t e r o The f e t a l v a r i a b l e s l i s t e d i n S e c t i o n 2.7 A were expressed on a per kg f e t a l weight b a s i s and were c a l c u l a t e d d a i l y over the s i x day span of the experiment. In order t o account f o r the changing weight of the growing f e t u s over time, the f e t a l weight i n utero was estimated on each experimental day using the method developed by Koong et a l . , (1975). Assuming a normal growth curve, the i n utero f e t a l weight can be calculated using the following equation: log IU WT = log BW + 0.00165 ( 2 x d x G A + d ) - 0.0556 d where WT = f e t a l weight IU = intrauterine BW = birthweight GA = gestational age d = # days between b i r t h and the GA for which the weight i s being calculated C. Cardiovascular Variables Fetal and maternal heart rates and a r t e r i a l pressures were monitored continuously on a polygraph recorder (see Section 2.5 A). A t o t a l of t h i r t y readings (1 reading/min) immediately before and afte r each sample were averaged to give an o v e r a l l value for each variable at each time period. Fetal a r t e r i a l pressure was corrected for amniotic pressure by subtraction of the l a t t e r from the former. 60 D. Behavioral Variables Fetal behavioral variables which were measured include e l e c t r o c o r t i c a l a c t i v i t y (ECoG), electroocular a c t i v i t y (EOG) and breathing movements. These variables were a l l monitored continuously on a polygraph recorder (see Section 2.5 A) and v i s u a l l y analyzed. Fetal ECoG was d i f f e r e n t i a t e d into low (range of 25 to 50 microvolts), intermediate (range of 80 to 110 microvolts, and high (range of 125 to 175 microvolts) voltage amplitude. Fetal EOG was d i f f e r e n t i a t e d into periods of rapid eye movement (REM) and the absence of rapid eye movement (non-REM). Episodes of f e t a l breathing were i d e n t i f i e d by amplitude changes i n the tracheal pressure trace. Breathing episodes were detected when the breath amplitude reached a minimum of 1 mmHg and the episode was maintained for at least 10 seconds. A l l f e t a l behavioral variables were analyzed continuously from the beginning (-48 hrs) to the end (+48 hrs) of each experiment (as opposed to the cardiovascular variables which were analyzed for only 60 minutes at each time period) . Readings for each variable were then combined and averaged i n consecutive four hour intervals i n order to i d e n t i f y any c y c l i c a l patterns. Consecutive four hour averages were tabulated and presented graphically. 61 E. S t a t i s t i c a l Analysis Unless otherwise spec i f i e d , s t a t i s t i c a l analysis was performed using two-way analysis of variance (ANOVA) for repeated measures. In those cases where ANOVA indicated s t a t i s t i c a l s i g n i f i c a n c e at the l e v e l of p<.05, the Fisher's l e a s t s i g n i f i c a n t difference for multiple comparisons t e s t was used to compare ind i v i d u a l means. A l l values are expressed as mean ± S.E. 62 3.0 RESULTS 3.1 SUMMARY OF EXPERIMENTAL OUTCOMES A t o t a l of 25 animals were set up for experimentation. Included i n t h i s t o t a l are those animals which were s u r g i c a l l y prepared for study, connected to a recorder, and subjected to the i n i t i a l stages of experimentation. Of these 25 animals, a t o t a l of 15 successful experiments were conducted. These consisted of 11 r i t o d r i n e and 4 control saline infusions. There were 6 unsuccessful preparations where experiments had to be discontinued due to low f e t a l a r t e r i a l pH, intrauterine f e t a l death, catheter f a i l u r e or early delivery. Low f e t a l a r t e r i a l pH necessitated the exclusion of a further 4 completed experiments from the f e t a l data pool. Two of these 4 excluded experiments, where f e t a l acidosis was much less severe, were used i n maternal but not f e t a l data analysis. Detailed accounts of the experimental outcomes are given i n figure 5 and tables 1, 2, 3, and 4. 3.2 ORGANIZATION OF TIME PERIODS IN ANALYSIS OF RESULTS The infusion and sampling schedule was explained i n Sections 2.6 A and B. A diagrammatic representation of t h i s scheme i s given i n Figure 6. The time periods were analyzed as explained i n table 5 below. Figure 5. SUMMARY OF EXPERIMENTS 63 ANIMAL PREPARATIONS n = 25 Incomplete Expts (n=6) Excluded Expts (n=4) SUCCESSFUL EXPERIMENTS n = 15 RITODRINE INFUSIONS n = 11 SALINE INFUSIONS n = 4 Fet a l Metabolic Data (n=ll) Fetal Behavioral Data Br. (n=6) ECoG(n=5) EOG (n=3) Maternal Uteroplac. A r t e r i a l Metabolic Data Data (n=9) (n=6) Fetal Metabolic Data (n=4) Fetal Behavioral Data Br. (n=2) ECoG(n=0) EoG (n=0) Maternal A r t e r i a l Data *(n=6) Uteroplac. Metabolic Data *(n=5) * two control experiments were maternal data analysis; Expts, ECoG,electrocortical a c t i v i t y ; uteroplac., uteroplacental. excluded from f e t a l but not experiments; Br., breathing; EOG, electroocular a c t i v i t y 64 Table 1. L i s t of completed r i t o d r i n e infusion experiments. Ewe# Breed GA @ Sur. GA @ Exp. GA @ Bir t h #F BW Op.F BW F2 Fate 239 Dorset 123 129 135 1 2824 - iud § 3d PE 251 Dorset 123 128 140 1 3872 - iud @ 4d PEa 274 Dorset 123 127 141 1 3702 - del a l i v e 270 Suffolk 138 143 147 2 3450 3468 pp death 271 Dorset 125 134 137 1 2801 - del a l i v e 256 Dorset 118 123 139 1 3735 - del a l i v e 261b Dorset 125 130 139 2 2800 3532 iud @ 4d PEa 74b Suffolk 122 129 140 1 3120 - del a l i v e 77 Suffolk 123 134 144 1 4500 - del a l i v e 72b Suffolk 125 130 142 1 5802 - del a l i v e 165 Suffolk 118 123 142 1 4522 — iud @ 9d PE GA, gestational age; Sur.,surgery; Exp.,experiment; BW, b i r t h weight; #F, number of fetuses; Op.F, operated fetus; F2, un-operated twin fetus; iud, intrauterine death; d PE, days post experiment; del, delivered; pp, postpartum. a fetus infected PE. b ewe subjected to subsequent experiments ( i . e . ewe 261, diphen-hydramine amniotic bolus i n j e c t i o n and amniotic f l u i d volume; ewe 74, hypoxia experiment; ewe 72, diphenhydramine and meto-clopramide amniotic bolus i n j e c t i o n s ) . 65 Table 2. L i s t of completed saline infusion experiments. Ewe# Breed GA @ GA @ GA @ #F BW BW Fate Sur. Exp. Bi r t h Op.F F2 269a Dorset 122 129 142 2 2402 4156 iud @ 6d PE 102 Dorset 123 129 139 1 4210 - iud @ 4d PE 62a Dorset 125 131 142 2 2370 2908 del a l i v e 145a Dorset 116 122 135 1 2364 - del a l i v e 273b Dorset 116 122 ' 136 2 1692 3025 iud @ 4d PE 164b Suffolk 125 130 147 2 1837 4.13 0 iud @ 2d PE GA, gestational age; Sur., surgery; Exp., experiment; BW, b i r t h weight; #F, number of fetuses; Op.F, operated fetus; F2, un-operated twin fetus; iud, intrauterine death; d e l , delivery. a ewe subjected to subsequent experiments ( i . e . ewe 269, metoclopramide amniotic bolus i n j e c t i o n ; ewe 62, diphen-hydramine maternal infusion; ewe 145, l a b e t a l o l maternal i n j e c t i o n ) . b these animals were included i n maternal but not f e t a l data analysis. Table 3. L i s t of excluded experiments. 66 Ewe# Breed GA @ Sur. GA § Exp. Infus. Type Reason for Exclusion Fate 264 Dorset 122 128 Rtd. acidemia del a l i v e 227 Dorset 130 137 Rtd. acidemia pp death 273a Dorset 116 122 Sal. acidemia iud @ 4d PE 164a Suffolk 125 130 Sal. acidemia iud @ 2d PE GA, gestational age (days); Sur., surgery; Exp., experiment; Infus. Type, infusion type; Rtd., r i t o d r i n e ; Sal., s a l i n e ; pp, postpartum; iud, intrauterine death; d PE, days post experiment. a these animals were excluded from f e t a l but not maternal data analysis. Table 4. L i s t of incomplete experiments. Ewe# Breed GA @ GA @ Last Reason for Delivery Sur. Exp. Samp. Incompletion 23 2 Dorset 125 132 1.5 hr Catheter f a i l u r e A l i v e 253 Dorset 125 129 -24 hr Intrauterine death Dead 260 Dorset 123 129 -24 hr Intrauterine death Dead 258 Dorset 125 127 -24 hr Acidemia A l i v e 263 Dorset 122 130 - Intrauterine death Dead 52 Dorset 129 136 -1 hr Premature delivery A l i v e GA, gestational age (days); Sur., surgery; Exp., experiment; Last Samp., l a s t sample taken before experiment terminated. 67 Figure 6. Sampling and infusion schedule CONTROL PERIOD samples -48 hr -24 hr -1 hr INFUSION PERIOD samples 1.5 hr *8 hr *12 hr 24 hr POST INFUSION PERIOD samples +1.5 hr +8 hr +24 hr +48 hr * Ritodrine infusions were terminated at these points i n those cases where pH and B.E. f e l l below 7.30 and -1.0 meq/L respec t i v e l y . 68 Table 5. A n a l y s i s of time p e r i o d s Time P e r i o d Symbol Sample Composition C o n t r o l C mean of -48, -24 & -1 hr samples 1.5 hrs i n f u s i o n 1.51 1.5 hrs a f t e r i n f u s i o n begun End of i n f u s i o n E I a f i n a l sample before i n f u s i o n ended Post 1.5 hrs PI. 5 1.5 hrs post i n f u s i o n Post 8 hrs P8 8 hrs post i n f u s i o n Post F i n a l PF mean of +24 and +48 hr samples aEI sample taken a t 8, 12, or 24 hours a f t e r i n f u s i o n begun. 3.3 FETAL DATA: RITODRINE AND CONTROL SALINE INFUSIONS R i t o d r i n e and c o n t r o l s a l i n e i n f u s i o n experiments were begun a t 130±2 (range 129-134) and 128±2 (range 122-131) days g e s t a t i o n r e s p e c t i v e l y . The animals were gi v e n approximately 6 days t o recover from surgery b e f o r e experiments commenced (surgery f o r r i t o d r i n e and c o n t r o l s a l i n e experiments were performed a t 124±2 and 122±2 days g e s t a t i o n r e s p e c t i v e l y ) . The i n f u s i o n of r i t o d r i n e was continued f o r 8, 12 and 24 hours i n 4, 3 and 4 f e t u s e s r e s p e c t i v e l y . The i n f u s i o n d u r a t i o n f o r each animal i s given i n Table 6 along with the pH and BE v a l u e s measured at C, EI and PF. The i n f u s i o n of T a b l e . 6 F e t a l a r t e r i a l pH and B.E. v a l u e s a t C, E I a and PF. Ewe# In f . C EI PF C EI PF Dur. pH pH pH B. E. B.E. B.E 239 8 7.31 7. 25 7 . 30 0. 7 -4 . 4 -1.8 251 24 7.38 7 . 34 7 . 45 2. 5 0.8 7.2 274 12 7.36 7.29 7.34 2 . 9 -1. 8 2 . 3 270 b 12 7.35 7. 20 7.34 1. 8 -7.7 1.4 271 c 8 7 .35 7 .16 7 . 23 0. 8 -11.7 -6 . 7 256 24 7.40 7 .32 7.40 3 . 6 1.0 3 , 2 261 8 7.40 7.11 7.40 -0. 3 -14. 2 1.8 74 8 7 . 38 7 . 25 7 . 39 2 . 8 -6.3 4 . 3 77 12 7. 39 7 . 32 7 . 39 4. 0 -1.1 4.2 72 24 7 . 37 7 .32 7. 38 1. 9 -0.7 3 .1 165 24 7. 36 7 . 32 7 . 38 3 . 2 -0.7 3 . 9 I n f . Dur., i n f u s i o n d u r a t i o n ; C, c o n t r o l (mean of -48, -24, and -1 hour samples); EI, end i n f u s i o n ( f i n a l sample be f o r e r i t o d r i n e i n f u s i o n ended); PF, post f i n a l (mean of +24 and +48 hour samples); B.E., base excess (meq/L). a the i n f u s i o n of r i t o d r i n e was terminated before the 24 hour maximum ( i . e . a t 8 or 12 hours) i n those cases where the pH and B.E. had f a l l e n below 7.30 and -1.0 r e s p e c t i v e l y by e i t h e r of the two e a r l i e r time p e r i o d s . b ewe d e l i v e r e d a t 27 hours post i n f u s i o n thus PF r e p r e s e n t s the +24 hour sample o n l y . c ewe d e l i v e r e d a t 21 hours post i n f u s i o n thus PF r e p r e s e n t s the +8 hour sample o n l y . 70 r i t o d r i n e was stopped before the 24 hour maximum ( i . e . at 8 or 12 hours) i n those cases where f e t a l pH and BE f e l l below 7.30 and -1.0 respectively by either of the two e a r l i e r time periods. Fetal a r t e r i a l pH, BE, and Po 2 values at C, EI, and PF were grouped and averaged according to the duration of r i t o d r i n e infusion (Table 7). The fetuses i n the 24 hour infusion group were better able to cope with r i t o d r i n e than the 12 hour group, who i n turn were more tolerant to the drug than the 8 hour group, as indicated by the degree of acidemia. In contrast, the degree to which f e t a l Po2 values declined was greater i n the 12 and 24 hour r i t o d r i n e infusion groups (as opposed to the 8 hour group). A l l saline infusions were 24 hours i n duration. A. A r t e r i a l and Umbilical Venous Blood Gases, pH, and  Hematocrit Fetal a r t e r i a l and umbilical venous blood gas data, pH and hematocrit before, during and a f t e r r i t o d r i n e infusion to the fetus are presented i n Table 8. Fetal a r t e r i a l and umbilical venous Po2 values (Figure 7) f e l l s i g n i f i c a n t l y below the control by 1.5 hours of infusion and continued to decline to a t o t a l f a l l of 4.9 and 8.4 mm Hg respectively by the end of the infusion. The t o t a l percentage f a l l of 71 TABLE 7. Average f e t a l a r t e r i a l pH, B.E. and Po 2 v a l u e s measured a t C, EI and PF (averaged w i t h i n groups which were c l a s s i f i e d a c c o r d i n g t o the d u r a t i o n of r i t o d r i n e i n f u s i o n ) . ID Var. C EI PF 8 a pH 7.36±0.02 7.19±0.04 7.33±0.04 12 b 7.37±0.01 7.27±0.04 7.3610.02 24 c 7.38±0.01 7.33±0.01 7.40±0.02 8 BE 0.7±0.8 -9.1±2.3 -0.6±2.4 12 2.9±0.6 -3.512.1 2.610.8 24 2.810.4 0.110.5 4.410.1 8 PO 12 24 21.511.3 22.011.0 23.010.6 19.010.4 16.511.0 16.510.5 19.611.5 19.312.0 23.210.6 ID, i n f u s i o n d u r a t i o n (hours); Var., v a r i a b l e ; C, c o n t r o l (mean of -48, -24 and -1 hour samples); EI, end i n f u s i o n ( f i n a l sample b e f o r e r i t o d r i n e i n f u s i o n ended); PF, post f i n a l (mean of +24 and +48 hour samples); B.E., base excess (meq/L). a n=4 b n=3 c n=4 ( f o r each v a r i a b l e ) Table 8. Fetal a r t e r i a l and umbilical venous blood gas values, pH, hematocrit and glucose and lactate values before, during and af t e r r i t o d r i n e infusion to the fetus. FEMORAL ARTERY C 1.51 EI PI. 5 P8 PF Po 2 (mmHg) 21.9±0 . 6 19.911. 0* 17.010. 5* 17.310. 5* 17.7+0. 7* 20.811. 0 Pco 2 (mmHg) 47.6±1 . 0 48.310. 9 48.610. 9 48.510. 9 48 . 311. 1 47.610. 9 pH 7.370±0 .004 7.33910. 009 7.27410. 025* 7.27310. 033* 7.36310. 017 7.38110. 011 BE (meq/L) 2.6±. 43 0.310. 7* -3.4+1. 7* -3.412. 0* 2.411. 5 3.510. 6 Co 2 (mM) 3.7±0 . 2 3.1+0. 2* 2.110. 1* 2.110. 1* 2.5+0. 2* 3.110. 2* Glucose (mM) 0.72±0 .07 1.0410. 11* 1.2910. 18* 1.1610. 13* 1.1410. 13* 0.95+0. 09* Lactate (mM) 1.54±0 . 11 2.9210. 26 8.6711. 12* 9.2011. 54* 5.3411. 07* 2.9010. 60 Hct (%) 34.7±1 . 4 34.311. 4 33.611. 2 32.911. 2 34.011. 3 34.411. 3 UMBILICAL VEIN Po 2 (mmHg) 3 7.2±1 . 9 32.812. 1* 28.811. 5* 30.611. 5* 32.3+1. 6* 36.012. 2 Pco 2 (mmHg) 42.2±0 . 8 43.310. 6 42.210. 9 42.510. 7 41.810. 7 42.010. 6 pH 7.40110 . 005 7.36410. 009 7.306+0. 023* 7.30110. 034* 7.39810. 019 7.414+0. 010 BE (meq/L) 2.210 . 5 0.110. 8* -3.911. 6* -3.812. 0* 2.111. 4 3.210. 6 Co 2 (mM) 5.510 . 2 4.910. 2* 4.110. 2* 4.210. 2* 4.710. 2* 4.810. 2* Glucose (mM) 0.8310 . 07 1.0410. 11* 1.2810. 16 1.2210. 13* 1.2510. 10* 1.0210. 09* Lactate (mM) 1.7110 . 10 3.1310. 28 8.9411. 08 9.4411. 34* 5.5411. 07* 3.0410. 59 Values are mean 1 SE; n=9 for a l l time periods; BE, base excess; Co 2, 0 2 content; Hct, hematocrit; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours a f t e r r i t o d r i n e infusion begun; EI, end infusion ( f i n a l sample before r i t o d r i n e infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. CO cn X E CN O Q_ 40 35 -30 25 -20 15 -10 Figure 7 1.51 El P1.5 50 45 40 35 P8 PF o o 3 3 X Fetal arterial ( o ) and umbilical venous ( • ) Pc^ values and fetal arterial ( A ) and umbilical venous ( A ) Pco2 values before, during and after ritodrine infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (El) infusion, and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion. n=»9 for all time periods. Values are mean ± SEM. • Significant difference ( P < 0.05 ) from the control. a r t e r i a l (78% of c o n t r o l ) and u m b i l i c a l venous (79% of c o n t r o l ) Po 2 was s i m i l a r , however, r e c o v e r y of the former was slower. Both a r t e r i a l and u m b i l i c a l venous Po 2 v a l u e s remained s i g n i f i c a n t l y below p r e - i n f u s i o n v a l u e s u n t i l the f i n a l p o s t - i n f u s i o n p e r i o d , at which time they were o n l y s l i g h t l y below the c o n t r o l . Pco 2 v a l u e s were not a l t e r e d . A r t e r i a l and u m b i l i c a l venous Oz content (Co 2) ( F i g u r e 10) f e l l s i g n i f i c a n t l y below c o n t r o l v a l u e s (3.7±0.2 mM and 5.5±0.2 mM r e s p e c t i v e l y ) d u r i n g the f i r s t 1.5 hours of i n f u s i o n . By the end of the i n f u s i o n , u m b i l i c a l venous Co 2 had d e c l i n e d t o 75% of the c o n t r o l w h i l e a r t e r i a l Co 2 had f a l l e n more r a p i d l y and to a g r e a t e r extent (57% of the c o n t r o l ) . P a r t i a l Co 2 recovery was e v i d e n t a t 8 hours p o s t -i n f u s i o n , however, both a r t e r i a l and u m b i l i c a l venous Co 2 v a l u e s remained s i g n i f i c a n t l y below the c o n t r o l throughout the remainder of the experiment. A r t e r i a l and u m b i l i c a l venous pH ( F i g u r e 8) d e c l i n e d r a p i d l y and s i g n i f i c a n t l y from c o n t r o l v a l u e s of 7.370±0.004 and 7.401±0.005 r e s p e c t i v e l y , t o f i n a l i n f u s i o n v a l u e s of 7.274±0.025 and 7.306±0.023 r e s p e c t i v e l y . There was no improvement i n pH d u r i n g the f i r s t 1.5 hours of p o s t - i n f u s i o n , however, almost complete recovery was a t t a i n e d by 8 hours post i n f u s i o n . X CL 7.400 r 7.350 7.300 7.250 -7.200 Figure 8 1.51 El P1.5 P8 PF - 6 . 0 Fetal arterial pH ( o ) and base excess (BE, A ) values before, during and after ritodrine infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (El) infusion, and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion. n«*9 for all time periods. Values are mean ± SEM. • Significant difference ( P < 0.05 ) from the control. LTJ m 3 (Ji 76 Associated with the decline i n pH was a f a l l i n base excess (BE) (Figure 8). A r t e r i a l and umbilical venous BE declined s i g n i f i c a n t l y from control values (2.610.43 and 2.210.5 meq/L respectively) during the f i r s t 1.5 hours of infusion reaching minimum values (-3.4+1.7 and -3.911.6 meq/L respectively) by the end of the infusion. Similar to pH, BE showed no improvement during the f i r s t 1.5 hours of post-infusion, but returned to control values by 8 hours post-in f u s i o n . Hematocrit was not altered from the control value of 34.711.4%. Fetal a r t e r i a l and umbilical venous blood gas data, pH and hematocrit before, during and a f t e r control saline infusion to the fetus are presented i n Table 9. There were no s i g n i f i c a n t alterations observed i n any of these variables. B. A r t e r i a l and Umbilical Venous Plasma Glucose and Lactate  Levels Fetal a r t e r i a l and umbilical venous plasma glucose and lactate l e v e l s before, during and a f t e r r i t o d r i n e infusion to the fetus are presented i n Table 8 and are shown graphically i n Figure 9. A r t e r i a l and umbilical venous glucose lev e l s rose s i g n i f i c a n t l y above the control (0.72+0.07 and 0.8310.07 mM respectively) by 1.5 hours of infusion, attaining maximal Table 9. F e t a l a r t e r i a l and u m b i l i c a l venous blood gas v a l u e s , pH, hematocrit and glucose and l a c t a t e values before, d u r i n g and a f t e r c o n t r o l s a l i n e i n f u s i o n to the f e t u s . FEMORAL ARTERY C 1. 51 EI PI. 5 P8 PF Po 2 (mmHg) 24.7±1. 5 24.3±1. 0 23.610. 7 24.011 .1 24.411. 1 24.011. 0 Pc o 2 (mmHg) 4 7.5±1. 1 48.6±0. 9 49.411. 2 48.411 .0 47.410. 5 48.910. 7 pH 7.377±0. O i l 7.365±0. 009 7.36810. 015 7 . 37810 .019. 7.37510. 018 7.36510. 020 BE (meq/L) 2.5±0. 3 2.7±0. 3 3.211. 2 3 .311 .1 2.711. 3 2.811. 3 Co 2 (mM) 3.8±0. 2 3.6±0. 2 3.414. 2 3 . 510 .1 3.210. 1 3.210. 1 Glucose (mM) 0.88±0. 11 0.84±0. 10 0.8710. 14 0.7610 .15 0.7010. 8 0.7410. 08 L a c t a t e (mM) 1.26±0. 10 1.27±0. 11 1.3810. 12 1.3710 . 13 1.5810. 16 1.4410. 11 Hct (%) 31.4±1. 2 30.7±1. 6 31.211. 6 31.111 .6 30.711. 3 32.411. 0 UMBILICAL VEIN Po 2 (mmHg) 41.3±4. 9 41.0±2. 7 40.112. 9 41.413 .9 40.412. 2 40.012. 5 P c o 2 (mmHg) 43.4±1. 2 43.511. 0 43.811. 0 42.910 . 5 42.311. 1 43 . 210. 7 pH 7.39510. 010 7.39810. 011 7.40010. 016 7.41010 .015 7.40510. 019 7.39210. 018 BE (meq/L) 2.2±0. 3 2.510. 4 2.911. 1 3 . 211 .0 3 .411. 1 2.211. 3 Co 2 (mM) 5.1±0. 2 5.110. 2 5.010. 2 5.010 . 2 4.710. 1 4.910. 2 Glucose (mM) 1.0110. 13 0.9610. 11 1.1510. 13 0.8810 .13 0.8410. 09 0.8310. 11 L a c t a t e (mM) 1.39±0. 11 1.3810. 14 1.5010. 14 1.4410 . 11 1.7510. 17 1.5710. 11 Values are mean 1 SE; n=4 f o r a l l time p e r i o d s ; BE, base excess; Co 2, 0 2 content; Hct, hematocrit; C, c o n t r o l (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours a f t e r s a l i n e i n f u s i o n begun; EI, end i n f u s i o n ( f i n a l sample before s a l i n e i n f u s i o n ended); PI.5, 1.5 hours post i n f u s i o n ; P8, 8 hours post i n f u s i o n ; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the c o n t r o l v a l u e . 78 2 E © in o o o 2.00 1.50 -1.00 -0.50 -0.00 1.51 El P1.5 P8 PF 12.00 8.00 v O -4-» u o 4.00 0.00 ^ C 1.51 El P1.5 P8 PF Figure 9. Fetal arterial (o ) and umbilical venous (• ) glucose concentrations (top panel) and fetal arterial ( * ) and umbilical venous ( * ) lactate concentrations (lower panel) before, during and after ritodrine infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (•) infusion, and 1.5 hour (P1.5). 8 hour (P8) and final (PF) post infusion. n=9 for all time periods. Values are mean ± SEM. * Significant difference ( P < 0.05 ) from the control. values (79% and 55% above the control respectively) by the end of the infusion. Plasma glucose concentrations declined upon infusion termination, however, leve l s remained s i g n i f i c a n t l y elevated throughout the post-infusion period. A r t e r i a l and umbilical venous lactate l e v e l s rose more than f i v e f o l d during the infusion of r i t o d r i n e , attaining maximum level s by 1.5 hours post-infusion (Figure 8). Lactate l e v e l s rose s i g n i f i c a n t l y from a r t e r i a l and umbilical venous control values of 1.54±0.11 and 1.71±0.10 mM respectively to 8.67±1.12 and 8.9411.08 mM respectively by the end of the infusion. Lactate concentrations continued to r i s e during the f i r s t 1.5 hours of post infusion but declined thereafter. Levels were no longer s i g n i f i c a n t l y elevated by the f i n a l post infusion period. Fetal a r t e r i a l and umbilical venous plasma glucose and lactate l e v e l s before, during and a f t e r control saline infusion to the fetus are presented i n Table 9. There were no s i g n i f i c a n t a l t e r a t i o n s i n these variables. C. Umbilical Blood Flow, 0^  Delivery, 0 2 Consumption. V-A Co Difference and 0 Extraction 2 2 Umbilical blood flow, 0 2 delivery, 0 2 consumption, the V-A Co difference and 0 extraction data before, during and 80 a f t e r r i t o d r i n e infusion to the fetus are presented i n Table 10 and i n Figure 10. Umbilical blood flow was unaltered from the control (206120 mL/min.kg) during the infusion of r i t o d r i n e , however, a nonsignificant f a l l i n flow occurred during the post-infusion period. 0 2 delivery f e l l progressively and s i g n i f i c a n t l y during the infusion of r i t o d r i n e , from a control value of 1115197 umol/min/kg to a f i n a l infusion value of 838168 umol/min/kg. A minimum i n 0 2 delivery (71% of control) was attained at 1.5 hours post-infusion but delivery increased slowly thereafter (although remaining s i g n i f i c a n t l y below the control throughout the post-infusion period). During the infusion period, the decline i n 0 2 delivery was due s o l e l y to the concurrent f a l l i n umbilical venous 0 2 content (Figure 10). However, the slow recovery of 0 2 delivery during the post-infusion period was l i k e l y due not only to the low a r t e r i a l 0 2 content, but to the concurrent, nonsignificant f a l l i n umbilical blood flow as well. Fetal 0 2 consumption increased progressively and s i g n i f i c a n t l y to 19% above the control (342125 umol/min.kg) by the end of the infusion. Consumption continued to r i s e during the f i r s t 8 hours of post-infusion (to 22% above the control) but returned to the control by the end of the post-infusion period. Table 10. F e t a l u m b i l i c a l blood flow, d e l i v e r y , 0 2 consumption, V-A Co 2 d i f f e r e n c e and f r a c t i o n a l 0 2 e x t r a c t i o n Before, d u r i n g and a f t e r r i t o d r i n e i n f u s i o n to the f e t u s . c 1.51 EI PI. 5 P8 PF Qum 206±20 218120 207116 190119 187116 182110 (mL/min.kg) Cvo 2 5.5±0.2 4.9+0.2* 4.110.2* 4.210.2* 4.7+0.2* 4.810.2* (mM) Do 2 1115±97 10241101 838168* 789178* 868159* 888167* (umol/min.kg) Vo 2 342125 389136 407130* 406141* 416134* 318115 (umol/min.kg) V-A Co 2 1.710.1 1.9+0.1 2.010.1 2.210.2* 2.110.2* 1.810.1 (mM) 0~ ext 32.011.1 (?) 38.7+2.9* 49.011.7* 51.611.8* 48.112.7* 37.212.8* Values are mean 1 SE; n=9 f o r a l l time p e r i o d s ; Qum, u m b i l i c a l blood flow; Cvo 2, u m b i l i c a l venous 0 2 content; Do 2, 0 2 d e l i v e r y ; Vo 2, 0 2 consumption; V-A Co 2 (veno-a r t e r i a l Co 2 d i f f e r e n c e ) ; 0 2 ext, 0 2 e x t r a c t i o n ; 6, c o n t r o l (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours a f t e r r i t o d r i n e i n f u s i o n begun; EI, end i n f u s i o n ( f i n a l sample before r i t o d r i n e i n f u s i o n ended); PI.5, 1.5 hours post i n f u s i o n ; P8, 8 hours post i n f u s i o n ; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s -t i c a l l y s i g n i f i c a n t change (P < 0.05) from the c o n t r o l v a l u e . 00 H o c o o H— o Figure 10. 1.51 El P1.5 P8 PF Mean percent change from the control of fetal fractional oxygen extraction ( • ). oxygen consumption ( O ), umbilical blood flow ( * ), oxygen delivery ( • ) and umbilical venous oxygen content ( o ) before, during and after ritodrine Infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (El) infusion and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion, n-9 for all time periods. Values are mean ± SEM. • Significant difference ( P < 0.05) from the control. CD 83 The r i s e i n f e t a l 0 2 consumption coupled with the concurrent f a l l i n 0 2 delivery resulted i n an increase i n f e t a l f r a c t i o n a l 0 2 extraction. From a control value of 32.0±1.2%, f r a c t i o n a l 0 2 extraction rose to 49.0±1.7% by the end of the infusion. Extraction continued to r i s e during the post-infusion period reaching a peak of 51.611.8% by 1.5 hours post-infusion. While values declined slowly thereafter, they remained s i g n i f i c a n t l y elevated throughout the remainder of the post-infusion period. The r i s e i n f e t a l f r a c t i o n a l 0 2 extraction was a d i r e c t consequence of an increased umbilical V-A (venoarterial) Co2 difference ( f e t a l a r t e r i a l Co2 f e l l to a greater extent than did umbilical venous Co ). The umbilical venoarterial Co 2 ' 2 difference rose s i g n i f i c a n t l y above the control (1.710.1) to a maximum value of 2.210.1 by 1.5 hours post infusion. The difference f e l l thereafter and was no longer s i g n i f i c a n t l y elevated by the f i n a l post-infusion sample. Umbilical blood flow, 02 delivery, 0 2 consumption, the umbilical V-A Co2 difference and Oz extraction data before, during and a f t e r control saline infusion to the fetus are presented i n Table 11. The only observed change was a s i g n i f i c a n t f a l l i n 0 2 delivery (20% below the control) during the 8 hour and f i n a l post-infusion periods. This f a l l i n 0 2 delivery was due primarily to a concurrent, non-significant Table 11. F e t a l u m b i l i c a l blood flow, d e l i v e r y , 0 2 consumption, V-A C o 2 d i f f e r e n c e and f r a c t i o n a l 0 2 e x t r a c t i o n Before, during and a f t e r c o n t r o l s a l i n e i n f u s i o n to the f e t u s . c 1.51 EI PI. 5 P8 PF Qum 242128 253129 218134 231134 207120 199115 (mL/min.kg) Cvo 2 5.110.2 5.110.2 5.010.2 5.010.2 4.710.1 4.910.2 (mMJ Do 2 1213195 12531107 10631150 11391154 962185* 967179* (umol/min.kg) V o 2 317137 362133 343147 326141 297111 326122 (umol/min.kg) V-A Co 2 1.310.1 1.510.1 1.610.1 1.510.1 1.510.1 1.710.1 (mM) 0 9 ext 25.811.3 (?) 29.311.8 32.412.3 30.611.9 31.812.8 34.311.2* Values are mean 1 SE; n=4 f o r a l l time p e r i o d s ; Qum, u m b i l i c a l blood flow; Cvo 2, u m b i l i c a l venous 0 2 content; Do 2, 0 2 d e l i v e r y ; Vo 2, 0 2 consumption; V-A C o 2 (veno-a r t e r i a l Co 2 d i f f e r e n c e ) ; 0 2 ext, 0 2 e x t r a c t i o n ; C, c o n t r o l (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours a f t e r s a l i n e i n f u s i o n begun; EI, end i n f u s i o n ( f i n a l sample before s a l i n e i n f u s i o n ended); PI.5, 1.5 hours post i n f u s i o n ; P8, 8 hours post i n f u s i o n ; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the c o n t r o l v a l u e . 85 f a l l i n umbilical blood flow (15% below control at P8 and 18% below control at PF). The contribution of a depressed umbilical venous Co to the f a l l i n O delivery was minimal. 2 2 J The concurrent umbilical venous Coz values were only 8% and 4% below the control respectively. D. Glucose and Lactate Levels and Fluxes Fetal glucose and lactate uptakes and d e l i v e r i e s before, during and a f t e r r i t o d r i n e infusion to the fetus are presented in Table 12 and Figure 11. Glucose delivery rose s i g n i f i c a n t l y to 70% above the control (163±14 umol/min.kg) by the end of the infusion. Glucose delivery f e l l thereafter and was no longer s i g n i f i c a n t l y elevated at the f i n a l post-infusion sample. Lactate delivery increased f i v e - f o l d from a control value of 347±33 umol/min.kg to a f i n a l infusion value of 1875±274 umol/min.kg. Lactate delivery declined slowly thereafter and was no longer s i g n i f i c a n t l y elevated at the f i n a l post-infusion sample. A trend towards a decline i n glucose uptake during the infusion of r i t o d r i n e suggested that glucose was being exported to the placenta v i a the umbilical artery. Conversely, lactate uptake tended to increase. These changes Table 12. Fetal glucose and lactate uptakes and d e l i v e r i e s before, during and after r i t o d r i n e infusion to the fetus. C 1.51 EI PI. 5 P8 PF DG1 163114 . 215132 278152* 236138* 233128* 187122 (umol/min.kg) Glu.upt. 1518 -5113 -8121 1114 1817 13110 (umol/min.kg) DLac 347133 678178 18751274* 18161286* 10471232* 5421102 (umol/min.kg) Lac.upt. 3414.3 4618.6 52115 47113 3717 2515 (-umo l/min. kg) Values are mean 1 SE; n=9 for a l l time periods; DG1, glucose delivery; Glu.upt., glucose uptake; DLac, lactate delivery; Lac.upt. lactate uptake; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours after r i t o d r i n e infusion begun; EI, end infusion ( f i n a l sample before ritodrine infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. CO 87 c E o E CD > 13 Q CD CO O O _3 o 400 x 350 300--250 200 150 2200 1800 1400 --1000 600 200 a o r*-a ro O CD_ <" CD 3 3 p' I d 1.51 El P1.5 P8 PF cn _* c "E E o CL Z3 co cn o o a o 55 45 35 25 15 5 -5 -15 T70 60 --50 --40 --30 1.51 El P1.5 P8 PF 20 Figure 11. a o i-*-a r-f n cz XI O a 3 o_ \ 3 p' Fetal glucose ( ° ) and lactate (a ) deliveries (top panel) and fetal glucose ( • ) and lactate ( • ) uptakes (lower panel) before, during and after ritodrine infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (El) infusion, and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion. n=9 for all time periods. Values are mean ± SEM. * Significant difference ( P < 0.05 ) from the control. 88 were not, however, s i g n i f i c a n t . Fetal glucose and lactate uptakes and d e l i v e r i e s before, during and a f t e r control saline infusion to the fetus are presented i n Table 13. Lactate delivery and glucose and la c t a t e uptakes were not s i g n i f i c a n t l y altered from the co n t r o l . Glucose delivery, however, f e l l below the control (242±32) at the 8 hour post (172±22) and f i n a l post (157±20) infusion periods. The f a l l i n glucose delivery at these time periods was due to the concurrent decline i n both umbilical blood flow and umbilical venous glucose concentrations. E. A r t e r i a l Pressure and Heart Rate Data Fetal heart rate and a r t e r i a l pressure data before, during and a f t e r the infusion of r i t o d r i n e to the fetus are presented i n Table 14 and i n Figure 12. Representative traces of these variables are shown in Figure 13. Fetal heart rate and a r t e r i a l pressure values were obtained at each i n d i v i d u a l time period as explained i n Section 2.7 C and were further grouped and analyzed according to Table 5 i n Section 3.2. During the f i r s t 1.5 hours of infusion, f e t a l heart rate increased s i g n i f i c a n t l y by 21% (34 beats per minute, bpm) , from a control value of 162 bpm to 196 bpm. Throughout the remainder of the infusion and the f i r s t 8 hours of post-Table 13. Fetal glucose and lactate uptakes and d e l i v e r i e s before, during and afte r control saline infusion to the fetus. 1. 51 EI PI. 5 P8 PF DG1 242±32 ( mol/min.kg) Glu.upt. 34±11 ( mol/min.kg) DLac 339±50 ( mol/min.kg) Lac.upt. 3 4±9 ( mol/min.kg) 240140 2812 359173 30110 217123 51116 316151 2916 200134 33110 330151 1818 172122* 157120* 3113 2318 361153 314141 3416 2616 Values are mean 1 SE; n=4 for a l l time periods; DG1, glucose delivery; Glu.upt., glucose uptake; DLac, lactate delivery; Lac.upt. lactate uptake; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours after saline infusion begun; EI, end infusion ( f i n a l sample before saline infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. CO Table 14. Fetal heart rate and a r t e r i a l pressure before, during and afte r r i t o d r i n e infusion to the fetus. C 1.51 EI PI. 5 P8 PF Heart Rate 16217 196±7* 189±6* 186±7* 188±9* 168±5 (beats/min) A r t e r i a l P. 46.211.5 46.912.0 46.112.1 46.712.0 47.111.6 49.212.1 (mmHg) Values are mean 1 SE; n=9 for a l l time periods. P., pressure; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours aft e r r i t o d r i n e infusion begun; EI, end infusion ( f i n a l sample before r i t o d r i n e infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. O 230 r 215 -200 185 170 -155 -1 52 - 48 - 44 - 40 36 12. 1.51 El P1.5 P8 PF Fetal heart rate ( • ) and arterial pressure ( • ) before during and after ritodrine Infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (El) Infusion, and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post Infusion. n=9 for all time periods. Values are mean ± SEM. * Significant difference ( P < 0.05 ) from the control. 92 5 min A m n i o t i c P r e s s u r e (mmHg). [ ^ U -4——c~ F A P (mmHg) FHR (BPM) 75 25 240 60 T r a c h e a l P r e s s u r e (mmHg) 25 ECoG 200fiV EOG 2 OOfiV Figure 13. Representative polygraph recordings of amniotic pressure, a r t e r i a l pressure, heart rate, tracheal pressure, electrocorticogram and electrooculargram in the f e t a l lamb. infusion, f e t a l heart rate remained s i g n i f i c a n t l y elevated by an average of 16% (26 bpm). Heart rate returned to the control during the f i n a l post-infusion period. A r t e r i a l pressure was not s i g n i f i c a n t l y altered during the experiment. Fetal heart rate and a r t e r i a l pressure data before, during and a f t e r control saline infusion to the fetus are presented i n Table 15. There were no s i g n i f i c a n t a l t e r a t i o n s i n e i t h e r of these variables. F. Breathing A c t i v i t y The incidence of f e t a l breathing movements was studied i n 6 r i t o d r i n e infused f e t a l lambs and the data analyzed as explained i n Section 2.7 D. Episodes of breathing movements were generally associated with low voltage e l e c t r o c o r t i c a l a c t i v i t y . A representative trace of f e t a l breathing a c t i v i t y was shown i n Figure 13. Fetal breathing a c t i v i t y was analyzed i n sequential four hour blocks to i d e n t i f y any c y c l i c a l patterns. A c y c l i c a l pattern of breathing was detected and i s shown in Figure 14. Figure 14 shows four consecutive 24 hour breathing cycles obtained from the 2 control and 2 post-infusion days. Also shown, as a repeating cycle, i s the mean 24 hour breathing c y c l e . This cycle was derived by averaging corresponding time Table 15. Fetal heart rate and a r t e r i a l pressure before, during and af t e r saline infusion to the fetus. C 1.51 EI PI.5 P8 PF Heart Rate 169±10 161112 162+14 14619 15019 158112 (beats/min) A r t e r i a l P. 47.411.2 46.411.5 47.811.1 48.311.3 47.811.0 49.811.9 (mmHg) Values are mean 1 SE; n=4 for a l l time periods. P., pressure; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours after saline infusion begun; EI, end infusion ( f i n a l sample before saline infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. VO XI o D_ i _ O X a; a. cn Ic •+-> D <U l _ m <u E c * 55 50 45 40 35 30 -25 / \ i A i / \ i / \ ° 7 " O \ / O \o , 7 1 . 0 / ' \ 7 • .'• W > /• • A 1 / * \ a \ i r • - 6 V 7 v.' 6 \ 1 r '•V / V \ • 7 \ J 7 V/ o Control Period • i i i i i i i i i i i • i i § i Post Infusion Period i i i i i i i i i o o o o o CN O O O O O CM CN O o o o o CN CN O O a o o CN CN Figure u. Time of Day Cyclic tendency of fetal breathing activity (n=6). Each point represents the mean % breathing over a 4 hour period (|.e 1000 represents 1000 - 1400 hours). • — • represents breathing over the two consecutive control and two consecutive post infusion days (ritodrine Infusion day not shown), o • • -o represents the average 24 hour breathing cycle derived as the mean of all control and post Infusion days. The average cycle Is repeated 4 times on the graph. u i periods from each of the two control and two post-infusion days and was then repeated four times on the graph. The incidence of f e t a l breathing a c t i v i t y was most frequent during late afternoon and evening ( i . e . from 1400 to 2200 hours) and occurred with a frequency of 45.8±3.0 %. Breathing a c t i v i t y f e l l to a trough (36.5±3.3%) i n the early morning (0600 to 1000 hours). The difference between the peak and trough periods was s t a t i s t i c a l l y s i g n i f i c a n t (P<0.05, unpaired two t a i l e d t - t e s t ) . In between the peak and trough periods (at 0300 to 0600 hours), there arose a second breathing peak having a frequency of 45.1± 3.2%. Breathing a c t i v i t y before, during and a f t e r r i t o d r i n e infusion to the fetus i s shown i n Figure 15. Data from only the f i r s t 12 hours of r i t o d r i n e infusion are presented ( i . e . 12 to 24 hours not shown) as the subsequent 12 hours are represented by only one animal. Also shown i s the average 24 hour breathing cycle which was derived as explained i n Figure 14. The average 24 hour breathing cycle shown in Figure 15 depicts the pattern of breathing which would l i k e l y have resulted had r i t o d r i n e not been infused. During the infusion of r i t o d r i n e , the incidence of breathing f e l l during a period of time when i t would otherwise have risen (assuming a s i m i l a r c y c l e ) . Breathing decreased s i g n i f i c a n t l y (P<.05, paired t-test) from an o v e r a l l average control value of 43.2±2.6% to an o v e r a l l average r i t o d r i n e infusion value of O *i_ <D D_ i_ O X a. C o a) DQ E 65 55 -45 35 25 -15 Control Period Post Infusion Period o o o o o CN CN o o o o o M CN o o o o o CN CN O o o o o CN CN O O O O O CN CN Figure 15. Time of Day ( • • ) Fetal breathing before, during and after ritodrine (RTD) infusion to the fetus. Each point represents the mean breathing over a 4 hr period (i.e. 1000 represents 1000—1400 hrs). Shown is the first 12 hours of a maximum 24 hr infusion. Not shown Is the 12-24 hr period of RTD infusion (n—1). The n values fall as the infusion duration rises. Control, n-6 each point; RTD, n-6 (1000-1800 hrs), n-3 (1800-2200 hrs); Post infusion, n-5 for first 12 hrs and n—6 thereafter. ( A A ) represents the average 24 hr breathing cycle (mean of 2 control and 2 post infusion days). 98 28.116.8% (Table 16). In most animals, breathing was most depressed near the end of the i n f u s i o n . 2 Breathing a c t i v i t y recovered completely during the post-infusion period (to 43.1+2.1%). Because of the apparent c y c l i c a l nature of f e t a l breathing a c t i v i t y , i t i s l i k e l y more appropriate to compare each consecutive 4 hour r i t o d r i n e infusion period with each corresponding 4 hour control period (the two control days were averaged for t h i s comparison) as opposed to analyzing o v e r a l l averages. In t h i s way, the changes observed during the infusion of r i t o d r i n e would not be obscured by the natural v a r i a t i o n i n breathing. A s i g n i f i c a n t difference i n the incidence of breathing was found between the second 4 hour r i t o d r i n e infusion (25.919.3%) period ( i . e . 5 to 8 hours of infusion) and the corresponding 4 hour control (48.214.1%) period (P < 0.05, paired t - t e s t ) . Further s i g n i f i c a n t changes were not observed, l i k e l y due to the f a l l i n n values as the infusion duration increased (see Figure 15 legend). Since the r i t o d r i n e infusion duration varied among animals, the time of day at which the post-infusion period began was also variable. Hence the n value during the i n i t i a l 2Breathing f e l l continually i n the 24 hour r i t o d r i n e infused animal, however, the f i n a l 12 hours of t h i s infusion are not shown in Figure 15. post-infusion period varied such that i t increased as more infusions were terminated. Breathing improved during the early post-infusion period and by 12 hours of post-infusion, breathing was not d i f f e r e n t from the control. Breathing a c t i v i t y was analyzed i n two saline infusion experiments. These experiments were not analyzed for c y c l i c a l patterns because the n value (n=2) was low and there were sections where the tracheal pressure trace was r e l a t i v e l y poor and hence d i f f i c u l t to read. The o v e r a l l incidence of f e t a l breathing a c t i v i t y during the infusion of saline (31.1±1.7%) was not d i f f e r e n t from that of the control (31.7±3.8%) or the post infusion period (28.8±2.4%). G. E l e c t r o c o r t i c a l and Electroocular A c t i v i t y Fetal e l e c t r o c o r t i c a l (ECoG) and electroocular a c t i v i t y (EOG) were analyzed i n f i v e and three r i t o d r i n e infused f e t a l lambs respectively as explained i n Section 2.7 D. Representative ECoG and EOG traces were shown i n Figure 13. Fetal ECoG and EOG a c t i v i t i e s were analyzed i n sequential four hour blocks i n order to i d e n t i f y any c y c l i c a l patterns, however, none were observed. During the control period, f e t a l lambs exhibited regular successive alternations between high and low e l e c t r o c o r t i c a l 100 a c t i v i t y (Figure 16). Brief episodes of intermediate voltage patterns were also observed, generally during periods of t r a n s i t i o n between episodes of high and low voltage a c t i v i t y . Fetal e l e c t r o c o r t i c a l a c t i v i t y (Table 16) before, during and a f t e r r i t o d r i n e infusion to the fetus are shown i n Figure 17. Data from only the f i r s t 12 hours of r i t o d r i n e infusion are presented ( i . e . 12 to 24 hours not shown) as the subsequent 12 hours are represented by only one animal. The o v e r a l l average incidence of high voltage ECoG a c t i v i t y rose s i g n i f i c a n t l y (P < 0.05, paired t-test) by an average of 7.3% (from a control value of 34.3±2.0% to 41.6±3.8%) during the infusion of r i t o d r i n e . High voltage ECoG a c t i v i t y returned to the control during the post infusion period (43.1±2.1%). Low voltage ECoG a c t i v i t y f e l l by an average of 7.5% (from a control value of 55.1±2.2% to 47.6±4.3%) during the infusion of r i t o d r i n e . Low voltage ECoG a c t i v i t y returned to the control during the post-infusion period (56.2±3.1%). There was no change i n the incidence of intermediate ECoG a c t i v i t y . The EOG pattern (Table 16) was analyzed i n only three animals, hence s t a t i s t i c a l analysis on t h i s data was not f e a s i b l e . A tendency, however, towards a f a l l i n rapid eye movement (REM) was apparent. The incidence of REM f e l l by an average of 8.2% from a control value of 49.1±2.2% to 40.9±7.0% during the infusion of r i t o d r i n e . EOG a c t i v i t y returned to 101 10 sec High I H Intermediate F i g u r e 16. R e p r e s e n t a t i v e polygraph r e c o r d i n g i l l u s t r a t i n g the t h r e e s t a t e s of e l e c t r o c o r t i c a l a c t i v i t y i n the f e t a l lamb. o V CL i_ •3 o X a. <u -M O -4-' CO o o o LU E Figure 17. 75 65 55 45 35 25 15 5 Control Period RTD 1^4 Poet Infusion Period 4*1 J I I I L J I I I I I I I I I I I I I I I I I I I I I I l_ o o o o o CM CM o o O O O CM CM O O O O O CM CM O O O O O CM CM O O O o o CM CM Time of Day High ( • ), low ( o ) and intermediate ( A ) electrocortical state (ECoG) before, during, and after ritodrine (RTD) Infusion to the fetus. Each point represents the mean % of time spent in an ECoG state over a 4 hour period. (I.e. 1000 represents 1000-1400 hrs).Shown Is the first 12 hours of a maximum 24 hour Infusion. Not shown is the 12-24 hr period of RTD infusion (n=1). The n values fall as the infusion duration rises. Control n=»5 each point; RTD, n-»5 (1000—1800) and n=3 (1800-2200 hrs); Post Infusion, n-4 for first 12 hrs and n-5 thereafter. 103 the control during the post-infusion period (48.1±2.9%). The average values of a l l behavioral variables before, during and a f t e r r i t o d r i n e infusion are presented i n Table 16. A summary of the r i t o d r i n e induced f e t a l behavioral changes (breathing and ECoG) as compared to the metabolic change (represented by Po2) i s presented i n Figure 18. During the infusion of r i t o d r i n e , the incidence of high ECoG a c t i v i t y rose while that of low ECoG a c t i v i t y and breathing movements f e l l . Since breathing movements are normally associated with low voltage a c t i v i t y , i t follows that both of these variables should f a l l together. Concurrent with the behavioral a l t e r a t i o n s were various metabolic perturbations, represented i n Figure 18, by the progressive f a l l i n Po2. A l l variables returned to pre-infusion values during the post-infusion period, however, the metabolic perturbations were slower to recover than were the behavioral changes. Unfortunately, the e l e c t r o c o r t i c a l and electroocular data obtained from the r i t o d r i n e infusion experiments could not be compared to corresponding control saline infusion data. E l e c t r o c o r t i c a l and electrooccular traces were obtained i n only two saline experiments and poor traces precluded the analysis of the data. 104 Table 16. Average o v e r a l l incidence of f e t a l breathing movements, e l e c t r o c o r t i c a l state and electroocular a c t i v i t y before, during and a f t e r the infusion of r i t o d r i n e to the fetus. n Control Ritodrine Post Period Infusion Infusion Breathing 6 4 3 . 2 ± 2 . 6 2 8 . 1 ± 6 . 8 * 43.1 ± 2.1 High ECoG 5 3 4 . 3 ± 2 . 0 41.6 ± 3.8* 3 4 . 3 ± 2 . 4 Low ECoG 5 55.1 ± 2.2 4 7 . 6 ± 4 . 3 * 56.2 ± 3.1 Int.ECoG 5 10.7 ± 0.8 10.7 ± 1.6 9.4 ± 1.0 EOG 3 49.1 ± 2.2 4 0 . 9 ± 7 . 0 48.1 ± 2.9 Values are mean ± SE; ECoG, e l e c t r o c o r t i c a l a c t i v i t y ; Int., intermediate; EOG, electroocular a c t i v i t y . * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control (paired t - t e s t ) . <a -4-< o •+-> (n O o O UJ \ C o E P 70 60 50 40 30 20 10 0 Control Period 35 A 30 25 A 20 15 o o o o o CM CM o o o o o CM CM O O O o o CM CM O O O O O CM CM o o o o o CM CM Time of Day Figure 18. Low ( • ) and high ( o ) ECoG state, breathing ( A ) and P 0 2 ( • ) values before, during and after ritodrine (RTD) Infusion to the fetus. Each ECoG and breathing point represents the mean % of time over a 4 hr period (i.e. 1000 represents 1000—1400 hrs). Each Po2 point represents one sampling period and is plotted within the appropriate 4 hr interval. Shown is the first 12 hrs of a maximum 24 hr infusion. See Figures 7, 15 and 17 for n values. H. A r t e r i a l Plasma Levels of Ritodrine 106 Fetal a r t e r i a l plasma r i t o d r i n e concentrations (Table 17) increased s i g n i f i c a n t l y throughout the infusion. Subsequent to infusion termination, r i t o d r i n e l e v e l s remained s i g n i f i c a n t l y elevated for a further 8 hours. The average r i t o d r i n e concentration at the time of infusion termination was 20.0±2.7 ng/ml. Fetal a r t e r i a l plasma r i t o d r i n e l e v e l s and f e t a l a r t e r i a l Po2, pH, 0 2 consumption and heart rate were compared graphically i n Figure 19. Fetal a r t e r i a l plasma glucose concentration and heart rate were also plotted as a function of r i t o d r i n e concentration (Figure 20). Both f e t a l heart rate and glucose concentration were s i g n i f i c a n t l y correlated (P<0.001) with the concentration of r i t o d r i n e . 3.4 MATERNAL DATA: RITODRINE AND CONTROL SALINE INFUSIONS Maternal a r t e r i a l data (femoral a r t e r i a l catheter implanted) were obtained from 9 and 6 r i t o d r i n e and control s a l i n e infusion experiments respectively. Uteroplacental data (femoral a r t e r i a l and uterine venous catheters implanted) were obtained from 6 and 5 r i t o d r i n e and control s a l i n e infusion experiments respectively. A summary of these experiments was presented i n Section 3.1. Table 17. Fetal a r t e r i a l plasma r i t o d r i n e concentration. c 1.51 EI PI. 5 P8 PF [RTD] (ng/ml) 0.00 n value 9 12.15±3.24* 8 20.0212.74* 9 11.1112.29* 9 7.0912.10* 8 1.2310.49 9 Values are mean 1 SE; [RTD], r i t o d r i n e concentration; n, number of animals; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours aft e r r i t o d r i n e infusion begun; EI, end infusion ( f i n a l sample before r i t o d r i n e infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. (The average r i t o d r i n e concentration at 8, 12 and 24 hours of r i t o d r i n e infusion were 15.9112.98, 13.9612.07 and 20.0915.20 ng/ml respectively.) 1.51 El PI .5 PB PF 7.400 • 1 24 j 1 • 20--(ng/ml) 7.350 (ng/ml) 16--•o X [RTD] 12--7.300 ierial 8--3 4--7.250 0--215 C 1.51 El PI.5 P8 PF Figure 19. Fetal arterial plasma ritodrine concentration verses: fetal arterial P02 (top left), total fetal oxygen consumption (top right), fetal pHQ(bottom left) and fetal heart rate (bottom right). .Time periods are control (C), 1.5 hour (1.51) and end (El) infusion, and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion. Values are mean ± SEM. n=9. • Significant 00 difference ( P < 0.05 ) from the control. H O 109 Fetal arterial glucose concentration (top panel) and heart rate (lower panel) as a function of fetal arterial plasma ritodrine concentration. n=8 animals. Regression equation, R and P values shown. 110 A. A r t e r i a l and Uterine Venous Blood Gases, pH, and  Hematocrit Maternal a r t e r i a l and uterine venous blood gases, pH and hematocrit before, during and a f t e r r i t o d r i n e infusion to the fetus are presented i n Table 18. Maternal a r t e r i a l Po 2 values were not s i g n i f i c a n t l y altered. In contrast, uterine venous Po2 (Figure 21) f e l l s i g n i f i c a n t l y below the control (58.8±1.1 mm Hg) by 1.5 hours of infusion and continued to decline to a minimum by 1.5 hours post-infusion (55.5±0.8 mm Hg) . Uterine venous Po 2 recovered thereafter and had returned to the control by the f i n a l post-infusion period. Uterine venous Co2 (Figure 21) f e l l s i g n i f i c a n t l y below the control (5.2±0.3 mM) by 1.5 hours of r i t o d r i n e infusion (4.810.2 mM) and remained s i g n i f i c a n t l y depressed thereafter. Maternal a r t e r i a l Co 2 did not f a l l below the control (6.410.3) u n t i l the f i n a l post-infusion sample (5.810.2 mM). Maternal a r t e r i a l hematocrit f e l l s i g n i f i c a n t l y below the control (29.611.5%) at 1.5 hours of infusion (27.711.0%) and remained at t h i s value thereafter. Maternal a r t e r i a l and uterine venous pH, Pco 2 and base excess remained unchanged throughout the experiment. •Maternal a r t e r i a l and uterine venous blood gas data, pH and hematocrit before, during and aft e r control saline Table 18. Maternal a r t e r i a l and uterine venous blood gas values, pH, hematocrit and glucose and lactate values before, during and af t e r r i t o d r i n e infusion to the fetus. FEMORAL ARTERY C 1.51 EI PI. 5 P8 PF Po 2 (mmHg) 132.014. 6 126.914. 3 124.412. 7 127.114. 8 131.819. 1 129.514. 8 Pco~ (mmHg) 33 .811. 1 34.5+0. 5 34.910. 9 - 34 . 010. 9 34.410. 7 34.610. 9 PH 2 7.46210. 015 7.46010. 007 7.47010. 009 7.47010. 008 7.45310. 009 7.47310. 008 BE (meq/L) 2.011. 3 2.410. 8 3.010. 7 2.610. 6 1.810. 8 3.410. 8 Co 2 (mM) 6.410. 3 6.210. 2 6.310. 2 6.310. 3 6.110. 2 5.810. 2* Glucose (mM) 2.4210. 14 2.5910. 17 2.7210. 28 2.7310. 22 2.7110. 11 2.7510. 10 Lactate (mM) 1.0710. 26 1.0310. 15 0.8810. 08 1.0210. 15 1.0110. 07 1.0310. 11 Hct (%) 29.611. 5 27.711. 0* 28.011. 2* 27.811. 3* 27.711. 1* 26.310. 8* UTERINE VEIN Po 2 (mmHg) 58.811. 1 56.411. 0* 56.110. 8* 55.510. 8* 57.910. 9 60.010. 7 Pco 0 (mmHg) 38.311. 0 38.310. 6 38.910. 9 38.510. 8 37.6+0. 6 37.910 . 8 PH 2 7.42510. 012 7.43210. 008 7.43510. 006 7.43510. 005 7.45310. 009 7.44710. 007 BE (meq/L) 2.111. 2 2.710. 7 2.810. 7 2.510. 7 2. 210. 9 3.510. 8 Co 2 (mM) 5. 210. 3 4.810. 2* 4.910. 1* 4.810. 3* 4.910. 2* 4.810. 2* Glucose (mM) 2.1710. 11 2.3110. 17 2.6410. 27 2.6110. 23 2.5110. 08 2.55+0. 11 Lactate (mM) 1.1510. 27 1.0710. 16 1.0010. 12 1.0310. 13 1.0410. 08 1.0710. 11 Values are mean 1 SE; n=6 for a l l time periods; BE, base excess; Co 2, 0 2 content; Hct, hematocrit; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours aft e r r i t o d r i n e infusion begun; EI, end infusion ( f i n a l sample before r i t o d r i n e infusion ended) PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. 64 Uterine venous Po £ ( ° ) and Co £( • ) before, during and after ritodrine Infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (El) infusion, and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion. n=8 for all time periods. Values are mean ± SEM. • Significant differences (P<0.05) from the control. infusion to the fetus are presented i n Table 19. Maternal a r t e r i a l and uterine venous Po2, Pco z, pH and BE remained unaltered throughout the experiment. Maternal a r t e r i a l Co2, f e l l s i g n i f i c a n t l y below the control (6.3±0.07 mM) to 5.710.1 mM at the 8 hour post and f i n a l post infusion periods. Uterine venous Co2 also f e l l at these time periods, however, the change was not s i g n i f i c a n t . Maternal hematocrit f e l l s i g n i f i c a n t l y below the control (28.4±0.5%) at 1.5 hours post-infusion (26.6±0.6%) and was stable thereafter. B. A r t e r i a l and Uterine Venous Plasma Glucose and Lactate  Levels Maternal a r t e r i a l and uterine venous plasma glucose and la c t a t e l e v e l s before, during and a f t e r r i t o d r i n e infusion to the fetus are given i n Table 18. Corresponding control saline infusion data are presented i n Table 19. There were no s i g n i f i c a n t changes observed in these variables. C. Uterine Blood Flow. Delivery, Extraction and Total  Uterine and Uteroplacental Consumption Uterine blood flow, Oz delivery, 0 2 consumption and 0 2 extraction data are given i n Table 20. Uterine blood flow and 0 2 delivery to the uterus were not s i g n i f i c a n t l y altered. Uterine O consumption, however, tended to r i s e to an average Table 19. Maternal a r t e r i a l and uterine venous blood gas values, pH, hematocrit and glucose and lactate values before, during and a f t e r control saline infusion to the fetus. FEMORAL ARTERY C 1. 51 EI PI. 5 P8 PF Po 2 (mmHg) 122 . 2±2. 6 122.9±5. 0 129.1±9. 6 125.715. 8 127.215. 3 124.816. 4 Pco- (mmHg) 35.3±0. 8 36.Oil. 3 34.9±1. 5 35.210. 8 34.111. 0 35.311. 8 PH 7.474±0. 012 7.464±0. 010 7.476±0. 017 7.47410. 007 7.47610. 005 7.46810. 022 BE (meq/L) 3.4±0. 6 3.3±0. 3 3.5±0. 6 3.510. 6 3.310. 6 3.410. 7 Co 2 (mM) 6.3±0. 1 6.3±0. 3 6.2±0. 3 6.210. 2 5.710. 1* 5.710. 1* Glucose (mM) 2.72±0. 07 2.81±0. 37 2.86±0. 11 2.8610. 18 2.8510. 05 2.7010. 08 Lactate (mM) 0.73±0. 05 0.74±0. 11 0.71±0. 12 0.6910. 08 0.9610. 14 0.8010. 10 Hct (%) 28.4±0. 5 29.311. 2 26.7±0. 6 26.610. 6* 25.910. 7* 25.910. 6* UTERINE VEIN Po 2 (mmHg) 58.9±2. 5 58.7±2. 3 60.412. 9 59.712. 3 60.813. 2 59.112. 7 Pco- (mmHg) 39 . 3±0 . 7 39.2±0. 5 39.110. 7 39.011. 2 37.911. 2 38.810. 9 PH 7.428±0. 012 7.436±0. 005 7.44010. 011 7.44210. 008 7.44610. 010 7.43610. 10 BE (meq/L) 3.3±0. 5 3.1±0. 3 3.310. 5 3.410. 6 3.010. 6 3.110. 7 Co 2 (mM) 5.0±0. 2 5.1±0. 3 5.010. 1 4.910. 2 4.610. 1 4.610. 1 Glucose (mM) 2.56±0. 10 2.50±0. 30 2.8010. 13 2.6910. 15 2.8110. 08 2.4910. 09 Lactate (mM) 0.76±0. 06 0.79±0. 11 0.7510. 13 0.7210. 08 0.9910. 15 0.8510. 10 Values are mean 1 SE; n=5 for a l l time periods; BE, base excess; Co 2, 0 2 content; Hct, hematocrit; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours a f t e r saline infusion begun; EI, end infusion ( f i n a l sample before saline infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. H Table 20. Uterine blood flow, and uterine 0 2 delivery, 0 2 consumption and f r a c t i o n a l 0 2 extraction before, during and after r i t o d r i n e infusion to the fetus. C 1.51 EI PI. 5 P8 PF Qut 1055±131 1234±157 10891123 10351139 12791184 11421103 (mL/min) Do 2 66021701 76071900 67731781 64271795 769011091 65081511 (umol/min) Vo 2 12361128 16451183 14991227 15191212 15271203 11341129 (umol/min) 0 ? ext 19.511.6 21.811.2 21.711.4 23.811.9* 20.512.1 17.311.4 ( ? ) Values are mean 1 SE; n=6 for a l l time periods; Qut, uterine blood flow; Do 2 0 2 delivery to the uterus; Vo 2, t o t a l uterine 0 2 consumption; 0 2 ext, uterine 0 2 extrac-tion; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours a f t e r r i t o -drine infusion begun; EI, end infusion ( f i n a l sample before r i t o d r i n e infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. 116 of 25% above the control (1236±128) throughout the infusion and f i r s t 8 hours of post-infusion (Figure 22). Uterine 0 2 consumption returned to the control by the f i n a l post-infusion period. These changes were not s t a t i s t i c a l l y s i g n i f i c a n t , but l i k e l y r e f l e c t the concurrent 18% r i s e i n f e t a l oxygen consumption (Figure 23). The uterine-umbilical venous Po 2 difference also tended to r i s e , remaining elevated u n t i l 8 hours post infusion (Figure 23). Oxygen extraction across the uterus (Figure 21) increased during the infusion of r i t o d r i n e , becoming s i g n i f i c a n t l y elevated above the control (19.5±1.6% ) by 1.5 hours post-infusion (23.8±1.9%). Fetal oxygen extraction had s i m i l a r l y reached a maximum at 1.5 hours post-infusion. Uterine oxygen extraction returned to the control by 8 hours post-infusion. Uteroplacental 0 2 consumption was calculated as the difference between t o t a l uterine and t o t a l f e t a l 0 2 uptake. The values obtained were extremely variable and often i n the negative range, hence these re s u l t s were determined to be unsuitable for analysis and w i l l not be presented. Uterine blood flow, 0 2 delivery, 0 2 extraction and t o t a l uterine 0 2 consumption before, during and a f t e r control saline infusion to the fetus are given i n Table 21. Uterine blood 'E o E D -t-> Q. Z> a -t-< o Figure 22. 2200 2000 1800 1600 1400 1200 1000 - 25 - 20 10 1.51 El P1.5 P8 PF Total uterine oxygen uptake ( o ) and total uterine fractional oxygen extraction ( • ) before, during and after ritodrine infusion to the fetus. Time periods are control (C), 1.5 hour (1.51) and end (El) infusion and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion. n=6 for all time periods. Values are mean ± SEM. *. Significant differences (P<0.05) from the control. O c r-t-CD - 15 t& m x i-t--1 Q O r+ O * -0 2000 c *E \ o E 3 CO o a. ZD CN O "5 ->-> o Figure 23. 1800 1600 1400 1200 1000 800 600 1.51 El P1.5 P8 PF Total uterine ( • ) and total fetal ( o ) oxygen uptake and the uterine—umbilical P02 difference ( A ) before, during and after ritodrine infusion to the fetus. Time periods are control (C), 1.5 hour (1.51), and end (El) infusion and 1.5 hour (P1.5), 8 hour (P8) and final (PF) post infusion. n«»6 for maternal values and n=9 for fetal values. Values are mean ± sem. • Significant difference ( P < 0.05 ) from the control. Table 21. Uterine blood flow, and uterine 0 2 delivery, 0 2 consumption and f r a c t i o n a l 0 2 extraction before, during and after control s a l i n e infusion to the fetus C 1.51 EI PI.5 P8 PF Qut 876±127 9661169 7791110 12801250* 8161122 10291166 (mL/min) Do 2 54881773 614211235 48151721 791311595* 46691745 58981969 (umol/min) Vo 2 10381101 12261204 9591263 15231284* 8731168 11171164 (umol/min) 0 2 ext 20.012.3 19.013.0 19.012.6 20.812.1 19.112.2 19.812.0 (*) Values are mean 1 SE; n=5 for a l l time periods; Qut, uterine blood flow; Do 2, 0 2 delivery to the uterus; Vo 2, t o t a l uterine 0 2 consumption; 0- ext, uterine 0 2 extrac-t i o n ; C, control (mean of -4 8, -24 and -1 hour samples); 1.51, 1.5 hours a f t e r s a l i n e infusion begun; EI, end infusion ( f i n a l sample before saline infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. 120 flow was s i g n i f i c a n t l y elevated above the control (876±127 umol/min.kg) at 1.5 hours post infusion (1280±250 umol/min.kg). This r i s e i n flow resulted i n s t a t i s t i c a l l y s i g n i f i c a n t elevations i n 0 2 delivery and 0 2 consumption during the same time period. 0 2 extraction was not s i g n i f i c a n t l y a l t e r e d from the control. D. Glucose and Lactate Levels and Fluxes Uterine glucose and lactate uptakes and d e l i v e r i e s before, during and a f t e r r i t o d r i n e infusion to the fetus are presented i n Table 22. The d e l i v e r i e s of glucose and lactate to the uterus were not s i g n i f i c a n t l y altered from the cont r o l . Trends, however, toward a decline i n uterine glucose uptake and a r i s e i n lactate uptake were apparent during the infusion. Corresponding control saline data are given i n Table 23. There were no s t a t i s t i c a l l y s i g n i f i c a n t changes observed. E . A r t e r i a l Pressure and Heart Rate Data Maternal a r t e r i a l pressure and heart rate data before, during and a f t e r r i t o d r i n e infusion to the fetus are presented in Table 24. Throughout the experiment, maternal a r t e r i a l pressure and heart rate remained unchanged from the control Table 22. Uterine glucose and lactate uptakes and d e l i v e r i e s before, during and after r i t o d r i n e infusion to the fetus. C 1. 51 EI PI. 5 P8 PF DG1 2655±342 3254±551 3018+580 2784+350 3466178 31571300 (umol/min) Glu.upt. 298196 283190 102+55 169171 291193 261121 (umol/min) DLac 951±105 12541232 9451158 976175 12451129 11281121 (umol/min) Lac.upt. -82±30 -55119 -28117 -6121 -24141 -61138 (umol/min) Values are mean 1 SE; n=6 for a l l time periods; DG1, glucose delivery to the uterus; Glu.upt., uterine uptake of glucose; DLac, lactate delivery to the uterus; Lac.upt., uterine uptake of lactate; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours aft e r r i t o d r i n e infusion begun; EI, end infusion ( f i n a l sample before r i t o d r i n e infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. Table 23. Uterine glucose and lactate uptakes and d e l i v e r i e s before, during and after control saline infusion to the fetus. C 1.51 EI PI. 5 P8 PF DG1 2386±368 27981629 22851319 37691840* 22951346 27711462 (umol/min) Glu.upt. 102145 268167 65+88 252181 110121 203 + 40' (umol/min) DLac 6541134 7701212 545198 9571271* 7801156 8171160 (umol/min) Lac.upt. -3815 -32120 -43117 -28114 -16116 -49113 (umol/min) Values are mean 1 SE; n=5 for a l l time periods; DG1, glucose delivery to the uterus; Glu.upt., uterine uptake of glucose; DLac, lactate delivery to the uterus; Lac. upt., uterine uptake of lactate; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours after saline infusion begun; EI, end infusion ( f i n a l sample before saline infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. Table 24. Maternal heart rate and a r t e r i a l pressure before, during and a f t e r r i t o d r i n e infusion to the fetus. 1.51 EI PI. 5 P8 PF Heart Rate 114±7 111±8 114±7 110±7 112±5 114±6 (beats/min) A r t e r i a l P. 99.6±0.6 99.2±3.7 96.3±2.6 97.612.2 96.813.2 94.212.7 (mmHg) Values are mean 1 SE; n=4 for a l l time periods. P., pressure; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours after r i t o d r i n e infusion begun; EI, end infusion ( f i n a l sample before ri t o d r i n e infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i c a n t change (P < 0.05) from the control value. to oo 124 (99.6±0.6 mm Hg and 114±7 bpm re s p e c t i v e l y ) . Corresponding control saline data are shown i n Table 25. S t a t i s t i c a l l y s i g n i f i c a n t a l t e r a t i o n s were not observed. Table 25. Maternal heart rate and a r t e r i a l pressure before, during and a f t e r control saline infusion to the fetus. 1.51 EI PI.5 P8 PF Heart Rate 103±7 105±10 11016 10616 11813 11216 (beats/min) A r t e r i a l P. 97.013.3 99.213.9 97.415.8 97.416.0 99.715.4 94.815.8 (mmHg) Values are mean 1 SE; n=4 for a l l time periods. P., pressure; C, control (mean of -48, -24 and -1 hour samples); 1.51, 1.5 hours after saline infusion begun; EI, end infusion ( f i n a l sample before saline infusion ended); PI.5, 1.5 hours post infusion; P8, 8 hours post infusion; PF, post f i n a l (mean of +24 and +48 hour samples). * S t a t i s t i c a l l y s i g n i f i -cant change (P < 0.05) from the control value. 4.0 DISCUSSION 4.1 DRUG ADMINISTRATION The relevance of the current study to the human s i t u a t i o n was dependent upon the attainment of drug lev e l s i n the f e t a l lamb, that were comparable to those of the human fetus during r i t o d r i n e t o c o l y s i s . Since a r i t o d r i n e assay was not available at the onset of experimentation, the infusion rate was chosen on the basis of the physiological perturbations which i t produced. The chosen infusion rate (2.6 ug/min) had previously been consistently found to raise f e t a l heart rate i n sheep by 10 beats per minute for a period of at least two hours (Warburton et a l . , 1987a). Since human fetuses experience a s i m i l a r increase i n heart rate during r i t o d r i n e t o c o l y s i s , i t was assumed that the degree of drug exposure between the two species would l i k e l y have been s i m i l a r . The chosen infusion rate (2.6 ug/min) was at the lower end of the recommended dosing range for human t o c o l y s i s (50 to 350 ug/min or 0.8 to 5.4 ug/kg/min assuming a maternal weight of 65 kg (Barden et a l . , 1980) accounting for the fact that r i t o d r i n e l e v e l s i n the fetus are generally less than they are i n the mother (57 ± 30% ) (Gander et a l . , 1980). The rationale for d i r e c t f e t a l , as opposed to maternal drug infusion was discussed i n Section 1.3 of the Introduction. Other studies examining the e f f e c t s of r i t o d r i n e i n the f e t a l lamb have 127 employed similar infusion rates of both short (less than 1 hour) (Siimes et a l . , 1978; Siimes and Creasy, 1980) and long (2 to 4 days) (Basset et a l . , 1985; Basset et a l . , 1989) term duration. Since human fetuses are often exposed to prolonged r i t o d r i n e infusion (6 to 24 hours) during t o c o l y s i s , i t seemed more appropriate to conduct long term (24 hour) infusion experiments in the current study. 4.2 FETAL PLASMA LEVELS OF RITODRINE The primary purpose for measuring f e t a l a r t e r i a l plasma l e v e l s of r i t o d r i n e was to v e r i f y that the drug l e v e l s achieved were comparable to those observed i n human fetuses during t o c o l y s i s . A limited amount of pharmacodynamic information was also obtained, however, the study was not designed for in-depth pharmacokinetic analysis of r i t o d r i n e d i s p o s i t i o n i n the fetus. Such an analysis would have required a more r i g i d protocol with frequent sampling. Fetal a r t e r i a l plasma leve l s of r i t o d r i n e increased progressively and s i g n i f i c a n t l y over time (Figure 19), however, le v e l s varied considerably among animals of s i m i l a r infusion duration. This phenomenom has also been observed i n women during r i t o d r i n e t o c o l y s i s and has been attributed to possible differences i n drug metabolism or renal clearance ( C a r i t i s et a l . . 1983). Drug elimination from the f e t a l compartment may be influenced by a number of additional factors including 128 placental clearance, drug accumulation (eg. i n amniotic, a l l a n t o i c or tracheal f l u i d ) and drug r e c i r c u l a t i o n . The average plasma r i t o d r i n e concentration at the time of infusion termination (EI) was 20.0 ± 2.7 ng/ml (range 9.5 to 34.7 ng/ml). This concentration i s within the range of cord l e v e l s obtained i n r i t o d r i n e exposed human fetuses at b i r t h (7 to 79 ng/mL) (Gander et a l . , 1980; Van Lierde and Thomas, 1982; Fujimoto et a l . , 1986; Kuhnert et a l . , 1986). Ritodrine l e v e l s exceeded 15 ng/mL (n=5) at EI i n a l l animals exposed to less than 24 hours of r i t o d r i n e infusion (range 15.8 to 29.2 ng/ml). Infusion termination was not warranted at lower drug leve l s except i n cases where fetuses had pre-experimental acidemia ( i . e n=l excluded animal) or were i n labor (n=2). A l l 24 hour r i t o d r i n e infused animals appeared r e l a t i v e l y uncompromised at EI regardless of r i t o d r i n e concentration (range 9.5 to 34.7 ng/ml). While the infusion affected a l l fetuses to a cer t a i n extent, i t appeared that some fetuses were more tolerant to the drug than others. A s i m i l a r phenomenon! was apparent i n women ( C a r i t i s et a l . , 1983). Metabolically compromised fetuses or fetuses i n labor may be more susceptible to the e f f e c t s of r i t o d r i n e . Ritodrine concentration and f e t a l Po2, Vo2, pH and heart rate were compared as a function of time (Figure 19). These plots indicate that a rel a t i o n s h i p exists between the 129 concentration of r i t o d r i n e i n f e t a l plasma and the degree of e f f e c t . The plasma r i t o d r i n e concentration at 8 hours post-infusion was s t i l l s u f f i c i e n t l y elevated (7.1 ± 2.1 ng/mL) to exert considerable f e t a l e f f e c t s . The persistence of r i t o d r i n e i n f e t a l plasma may r e f l e c t slow clearance of the drug from deep tissu e compartments or drug r e c i r c u l a t i o n . Inadequacies of the Figure 19 plots preclude further in t e r p r e t a t i o n for a number of reasons. F i r s t l y , there are i n s u f f i c i e n t data points (note also that the time scale i s not l i n e a r ) . Secondly, the t o t a l drug dosage varied among animals depending upon the duration of the infusion. EI therefore represents an average of values obtained a f t e r v a r i a b l e degrees of drug exposure. In addition, many of the observed perturbations did not r e s u l t from d i r e c t beta-receptor stimulation, hence a d i r e c t r e l a t i o n s h i p to r i t o d r i n e concentration would not be expected. For instance the r i s e i n f e t a l oxygen consumption l i k e l y occurred secondary to increases i n glucose and i n s u l i n concentration. In contrast, elevations i n f e t a l heart rate and glucose concentration are mediated through d i r e c t beta-receptor stimulation. Positive correlations were obtained for both of these variables when plotted as a function of r i t o d r i n e concentration (Figure 20) . However, the response of both f e t a l heart rate and glucose concentration to a given 130 r i t o d r i n e l e v e l varied considerably. Similar phenomena have been observed i n humans ( C a r i t i s et a l . , 1983). Such differences may i n part r e f l e c t natural inter-animal v a r i a b i l i t y e s p e c i a l l y given the wide range of values obtained during the control period. Precise relationships between f e t a l plasma r i t o d r i n e concentrations and f e t a l heart rate and glucose levels (Figure 20) were not i d e n t i f i e d . For instance while f e t a l heart rate c l e a r l y r i s e s with elevations i n plasma r i t o d r i n e concentration, the point at which heart rate plateaus (despite increasing drug concentration) was not i d e n t i f i e d . A number of confounding factors may e x i s t within these plots. F i r s t l y , previous studies have shown that both glucose and heart rate responses to r i t o d r i n e become attenuated over time (Basset et a l . , 1989). Hence the response to given plasma levels of r i t o d r i n e may f a l l as the infusion duration i s increased. In addition, although elevations i n glucose concentration are mediated d i r e c t l y through beta-receptor stimulation, the l e v e l s of glucose i n f e t a l plasma are further influenced by c e l l u l a r uptake and u t i l i z a t i o n . Therefore a d i r e c t r e l a t i o n s h i p between glucose and r i t o d r i n e levels may in f a c t not e x i s t . 131 4.3 ONTOGENY OF BETA-RECEPTOR MEDIATED RESPONSES IN THE  FETAL LAMB The development and functioning of adrenergic responsiveness i n f e t a l lambs has been extensively studied (Barrett et a l . , 1972; Joelsson et a l . f 1972; Vapaavouri et a l . . 1973; Nawayhid et a l . , 1975; A s s a l i et a l . , 1977; Jones and Ritchie, 1978 a,b; Harris and Van Petten, 1978; Nakamura, 1987; Rawashdeh, 1988). Cardiovascular responses (eg. tachycardia and blood flow changes) to adrenergic stimulation are present by as early as 60 days gestation i n f e t a l lambs (Barrett et a l . , 1972). Different tissues begin to respond to adrenergic stimulation at varying points i n gestation and a d i f f e r e n t i a l maturation pattern for beta-receptor mediated responses i n the f e t a l lamb has been suggested (Rawashdeh et a l . , 1988). Once established, however, organ s e n s i t i v i t y to adrenergic stimulation appears to increase with gestation. This has been demonstrated by the increasing responsiveness of the f e t a l c i r c u l a t o r y system to various sympathetic agonists and antagonists with gestation (Vapaavouri et a l . , 1973; A s s a l i et a l . . 1977; Harris and Van Petten, 1978; Rawashdeh et a l . , 1988). The presence of beta-receptor responsiveness i n the f e t a l lamb during the l a s t t h i r d of gestation has been demonstrated through d i r e c t infusion of adrenergic agonists and antagonists of varying receptor s p e c i f i c i t i e s (RS). Infusion of epinephrine (RS: alpha = beta-1 = beta-2) norepinephrine (RS: alpha = beta-1 >> beta-2) and isoproterenol (RS: beta-1 = beta-2) to f e t a l lambs a l l r e s u l t i n increases in f e t a l heart rate (Jones and Ritchie, 1978b). The e f f e c t , i n each case, i s completely blocked by concurrent administration of propranolol (beta-blocker) but not by phentolamine (alpha-blocker). Infusion of methoxamine (alpha-agonist) or propranolol alone to f e t a l lambs has no e f f e c t on f e t a l heart rate. These studies c l e a r l y demonstrate the presence and s e n s i t i v i t y of f e t a l myocardial beta-1 adrenoceptors to adrenergic agonists and antagonists. Beta-receptor mediated alt e r a t i o n s i n f e t a l metabolic variables can be induced by at l e a s t by 115 days gestation (Jones and Ritchie, 1978a). Short term infusion (1 hour) of isoproterenol at physiologic rates (1.3 ug/min) res u l t s i n increases i n f e t a l glucose, lactate, alpha-amino nitrogen, i n s u l i n and plasma free f a t t y acids (Jones and Ritchie, 1978). These e f f e c t s are blocked by simultaneous infusion of propranolol, i n d i c a t i n g that they are at least i n part mediated by beta-adrenergic mechanisms. Glucose and lactate elevations have also been shown to be mediated by alpha-adrenergic mechanisms in the fetus. Other beta-receptor mediated responses observed i n the f e t a l lamb include renal vasodilation (Nakamura et a l . , 1987) and renal renin secretion 133 (Rawashdeh et a l . , 1988). 4.4 UTERINE AND UMBILICAL BLOOD FLOW The f e t a l lamb receives nutrient r i c h blood from the placenta v i a two umbilical veins which enter the f e t a l body through the umbilicus and fuse immediately thereafter to become the common umbilical vein (Faber and Thornburg, 1983). The l a t t e r vessel subsequently branches into the ductus venosus and the veins that enter the portal c i r c u l a t i o n . Approximately 50% of incoming umbilical venous blood perfuses the l i v e r while the remainder bypasses t h i s organ and enters d i r e c t l y into the i n f e r i o r vena cava v i a the ductus venosus (Edelstone et a l . , 1978). Blood from the f e t a l body returns to the placenta (approximately 40% of f e t a l cardiac output) v i a two umbilical a r t e r i e s which originate from the common umbilical artery (Heymann, 1989). The l a t t e r vessel comprises the terminal segment of the d i s t a l descending aorta, a r i s i n g just caudal to the i l i a c b i f u r c a t i o n (Berman et a l . , 1978; Rudolph and Heymann, 1980). Blood supply to the uteroplacenta occurs v i a the middle uterine a r t e r i e s which arise from the umbilical branch of the in t e r n a l i l i a c artery (Carter, 1975). The middle uterine a r t e r i e s branch a number of times, eventually forming the arcuate a r t e r i e s which send smaller branches to the myometrium 134 and larger r a d i a l branches ( r a d i a l arteries) to the endometrium and placentomes. Blood i s drained v i a veins which contribute to larger uterine veins i n the submucosa. The maternal vessels which supply and drain the placenta cannot be r e a d i l y divided from the vasculature of the rest of the uterus. Hence the c i r c u l a t i o n s are referred to c o l l e c t i v e l y as the uteroplacental c i r c u l a t i o n . In the l a s t month of pregnancy, approximately 84% of t o t a l uterine blood flow can be ascribed to placental flow (Makowski et a l . , 1968). The remainder i s d i s t r i b u t e d to the endometrium (13%) and myometrium (3%). A number of techniques have been developed to measure uterine and umbilical blood flow. The microsphere technique (Rudolph and Heymann, 1967a) accurately measures blood flow to any organ or region of the fetus or placenta. However, the number of flow measurements which can be made in a given animal i s li m i t e d (due to a limited number of available isotopes) rendering the method unsuitable to the current study where many flow estimates were required. Furthermore, the method may not accurately represent flow at the time of substrate measurement since blood samples for plasma metabolite l e v e l s and blood gases and those for microsphere determination are withdrawn sequentially (Battaglia and Meschia, 1986). Electromagnetic or ultrasonic flow transducers have also been employed for the measurement of 135 blood flow (Berman et a l . , 1975; Nimrod et a l . , 1989). The advantages of these techniques over the previous method are that radioisotopes are not required and continuous measurements of flow can be made. However, a considerably greater degree of maternal and f e t a l s u r g i c a l intervention i s required and there i s a dependence of the measurement on the maintenance of a precise alignment of the probe. Accurate c a l i b r a t i o n of the probes i s also often d i f f i c u l t . Uterine and umbilical blood flow i n the current study were measured using the steady state antipyrine d i f f u s i o n technique developed by Meschia et a l . , (1966). The investigators chose antipyrine as the t e s t substance for a number of reasons including: 1) i t i s not metabolized at appreciable rates, 2) i t i s not produced by the mother or fetus, 3) i t remains largely unbound i n the blood, 4) i t i s highly d i f f u s i b l e across the placenta (exhibits flow limited d i f f u s i o n ) and 5) i t exerts minimal e f f e c t s on the fetus at low concentrations. The technique involves the infusion of antipyrine at a constant rate into the f e t a l i n f e r i o r vena cava. I n i t i a l l y , the concentration of antipyrine i n the fetus r i s e s rapidly establishing a transplacental concentration gradient which promotes the d i f f u s i o n antipyrine from fetus to mother. After approximately 40 minutes of infusion, a steady state i s attained where the arteriovenous antipyrine concentration difference across the uterine and umbilical 136 c i r c u l a t i o n s are constant. In other words, the transplacental d i f f u s i o n rate of antipyrine becomes constant and equals approximately 90% of the infusion rate (at t h i s point, f e t a l l e v e l s tend to plateau and accumulation of antipyrine i n the fetus i s reduced to a minimum). Umbilical and uterine blood flow may then be calculated as the quotient of the antipyrine infusion rate ( i . e . transplacental d i f f u s i o n rate) and the arteriovenous concentration difference across the umbilical and uterine c i r c u l a t i o n s respectively. This method has been validated by simultaneous measurement with electromagnetic flow meters (Rudolph and Heymann, 1967b; Rurak and Gruber, 1983a). One of the advantages of the steady state antipyrine d i f f u s i o n technique i s that a r e l a t i v e l y large number of blood flow measurements can be made i n a single animal. In addition, measurements of both umbilical and uterine blood flows can be made simultaneously. Furthermore, minimal s u r g i c a l intervention i s required and animals do not have to be s a c r i f i c e d . A possible disadvantage, however, i s that antipyrine has been shown to have ef f e c t s i n the fetus. Studies have reported declines i n the level s of a number of prostaglandins i n the f e t a l and maternal c i r c u l a t i o n s during the infusion of antipyrine to the fetus, suggesting that antipyrine may be an i n h i b i t o r of prostaglandin synthesis (El Baldry et a l . . 1984; Pimental et a l . , 1986; Cashner et a l . , 1986; Andrianakis et a l . , 1989). Fetal prostaglandins appear to be produced by the placenta and f e t a l vasculature and normally c i r c u l a t e i n f e t a l blood at r e l a t i v e l y high concentrations ( C h a l l i s et a l . f 1978; C h a l l i s and Patrick, 1980). The relevance of changes i n f e t a l prostaglandin lev e l s become obvious when t h e i r influences on various f e t a l p hysiological parameters are considered including: ductus arteriosus patency (Clyman, 1987), f e t a l breathing a c t i v i t y (Kitterman et a l . , 1984), uterine contractions (Rankin and Phernetton, 1976; E l Badry, 1984) i n s u l i n response to glucose (Philipps et a l . , 1984), pulmonary blood flow (Mott and Walker, 1983; Cassin, 1987), and uterine and umb i l i c a l -placental blood flow (Novy et a l . , 1974; Rankin and Phernetton, 1976; Berman et a l . . 1978; Rankin and McLaughlin, 1979; Mott and Walker, 1983). In assessing the potential e f f e c t s of antipyrine i n the fetus, the rate and duration of administration must be considered. Reports indicate that f e t a l prostaglandin l e v e l s are reduced at f e t a l antipyrine infusion rates ranging from 4 to 30 mg/min (El Badry et a l . , 1984; Cashner et a l . f 1986; Pimental et a l . , 1986; Andrianakis et a l . , 1989; Reid et a l . , 1989). The lower infusion l i m i t at which uterine venous prostaglandin l e v e l s appear to be unaffected i s 1 mg/min (Pimental et a l . , 1986). In the current study, antipyrine was infused at 4.9 mg/min, therefore some decline i n prostaglandin 138 l e v e l s may have occurred. I t i s un l i k e l y , however, that t h i s resulted i n any s i g n i f i c a n t physiological e f f e c t s i n the fetus as r e l a t i v e l y high doses of antipyrine are required to exert a number of f e t a l physiologic changes. Studies have shown that antipyrine infusion rates as high as 10 mg/mL do not induce any changes i n umbilical or uterine blood flow as measured by electromagnetic flow meters (Cashner et a l . , 1986). Even higher infusion rates (30 mg/mL) have no e f f e c t on placental or regional blood flows although f e t a l ECoG patterns change s l i g h t l y (Reid et a l . , 1989). Fetal and maternal heart rate and a r t e r i a l pressure also appear to be unaffected by antipyrine infusion (Sunderji, S.G., 1984; Cashner et a l . . 1986). The eff e c t s of antipyrine on f e t a l metabolic status appear somewhat c o n f l i c t i n g . Some studies report declines i n f e t a l Po_, and pH a f t e r 3 hours of infusion at rates ranging from 15 to 30 mg/min ( E l Badry, 1984; Sunderji, et a l . , 1984; Reid, 1989). Others, (Pinmental et a l . , 1986 and Cashner et a l . , 1986) were unable to f i n d any change i n f e t a l metabolic status a f t e r 3 hours of antipyrine infusion at rates ranging from 1 to 15 mg/min. Alterations i n blood flow, breathing patterns and metabolic status, as a re s u l t of antipyrine infusion, were not observed i n the control experiments of the present study. These r e s u l t s and those of the studies mentioned above indicate that i t i s unl i k e l y that antipyrine had any s i g n i f i c a n t f e t a l e f f e c t s at the infusion rate employed. 139 The average umbilical blood flow i n the ch r o n i c a l l y instrumented f e t a l lamb i s approximately 200 mL/min.kg f e t a l body weight (Walker, 1984). The values obtained for control umbilical blood flow i n the current r i t o d r i n e (206±20 mL/min) and saline (242 ± 28 mL/min) infusion experiments were s i m i l a r to those obtained by others who have used both s i m i l a r (Meschia et a l . f 1966; Meschia e t _ a l . , 1980; P h i l l i p s , et a l . , 1984; Hay and Meznarich, 1986) and alternate (Clapp I I I , 1978; Faber and Thornburg, 1983) measurement techniques. Ritodrine infusion had no e f f e c t on umbilical blood flow. However, progressive, nonsignificant declines i n umbilical blood flow were observed during the post-infusion periods of both the r i t o d r i n e and saline infusion experiments. This f a l l may have resulted from an overestimation of d a i l y increases i n f e t a l body weight (since umbilical blood flow i s reported per kg f e t a l weight). A l t e r n a t i v e l y , umbilical blood flow (per kg f e t a l weight) may have f a l l e n progressively with gestation (Rurak, unpublished r e s u l t s ) . Siimes et a l . f (1978) reported that absolute umbilical blood flow did not change i n response to short term (< 1 hour) infusion (1.2 ug/kg.min) of r i t o d r i n e to the fetus. However, variable increases i n umbilical blood flow have been observed (Ehrenkranz, 1976) at f e t a l r i t o d r i n e infusion rates (25 ug/min) much higher than those employed i n the current study (2.6 ug/min). 140 Uterine blood flow i n pregnant sheep increases during gestation from roughly 30 mL/min i n the non-pregnant state to approximately 1000 to 2000 mL/min i n the l a s t two weeks of pregnancy (Meschia, 1980; Resnik, 1989). Control uterine blood flow i n the current r i t o d r i n e (1055±131 mL/min) and sa l i n e (876±127 mL/min) infusion experiments were within the range of those obtained by others who have used similar (Meschia et a l . , 1966; Meschia et a l . f 1980; Sunderji e t _ a l . , 1984) and alternate (Rosenfeld, 1974; Clapp, 1978) measurement techniques. Comparison of uterine blood flow measurements among d i f f e r e n t studies revealed a great deal of v a r i a b i l i t y , due i n part to inter-animal v a r i a b i l i t y as well as to va r i a t i o n s i n measurement technique (Faber and Thornburg, 1983). Other contributing factors include spontaneous d a i l y fluctuations i n uterine blood flow and progressive increases i n flow with gestation (Battaglia and Meschia, 1986). Siimes and Creasy (1979) infused r i t o d r i n e (1.4 ug/min.kg) d i r e c t l y to pregnant ewes (75 minutes) and observed no change i n absolute uterine blood flow, however, t o t a l uterine blood flow as a f r a c t i o n of cardiac output f e l l s i g n i f i c a n t l y . Using a s i m i l a r infusion protocol, Ehrenkranz et a l . , (1976) reported a 10% decline i n uterine blood flow. The infusion of r i t o d r i n e to the fetus i n the current study had no e f f e c t on uterine blood flow. 141 4.5 FETAL GLUCOSE METABOLISM Glucose i s the major nonprotein oxidative substrate i n f e t a l lambs and i t s oxidation accounts for approximately 28% of t o t a l f e t a l oxygen uptake (Hay et a l . , 1983; Hay et a l . , 1989). The continuous net flux of glucose from the uteroplacenta to the fetus occurs by f a c i l i t a t e d d i f f u s i o n and i s associated with a posit i v e maternal-to-fetal glucose concentration gradient (Battaglia and Meschia, 1988; Hay et a l . , 1990). The glucose gradient (which governs the glucose transfer rate) i s determined primarily by the rate of uteroplacental glucose consumption which i n turn i s dependent upon f e t a l plasma glucose concentration (Hay et a l . , 1990; DiGiacomo and Hay, 1990). Approximately one t h i r d of the glucose taken up by the uterus i s transferred to the fetus i n d i c a t i n g that the placenta consumes a large portion of maternally delivered glucose (Meschia et a l . , 1980). The majority of f e t a l glucose comes from exogenous supply ( i . e . maternal-to-fetal transfer) as opposed to endogenous sources (Battaglia and Meschia, 1988). Gluconeogenesis does not normally occur i n the f e t a l lamb (Warnes and Seamark, 1977; Hay et a l . , 1981; Menon and Sperling, 1988) except i n cases of chronic stress such as maternal or f e t a l hypoglycemia (DiGiacomo and Hay, 1989; DiGiacomo and Hay, 1990). In the current study, f e t a l a r t e r i a l and umbilical venous plasma glucose l e v e l s rose progressively and s i g n i f i c a n t l y throughout the infusion of r i t o d r i n e a t t a i n i n g maximal levels (79% and 55% above the control respectively) by the end of the infusion. Plasma glucose concentrations declined upon infusion termination, but remained s i g n i f i c a n t l y elevated throughout the post-infusion period. Elevations i n plasma glucose le v e l s have been observed i n other short and long term r i t o d r i n e infusion studies (Ehrenkranz et a l . , 1976; Siimes and Creasy, 1980; Warburton et a l . , 1987a; Warburton et a l . , 1988; Basset et a l . , 1989). The observed r i s e i n f e t a l a r t e r i a l plasma glucose concentration was associated with an increase i n f e t a l glucose delivery and a decrease i n the umbilical veno-arterial glucose concentration difference. In other words, the extent to which glucose le v e l s rose i n the umbilical artery exceeded that of the umbilical vein. These data suggest either a f a l l i n f e t a l glucose u t i l i z a t i o n or a r i s e i n f e t a l glucose production as eit h e r would r a i s e umbilical a r t e r i a l levels of t h i s substrate. A f a l l i n f e t a l glucose u t i l i z a t i o n , as implied by the tendency for uptake to decline would appear contrary to what i s expected since elevated plasma glucose levels have previously been associated with increased rates of glucose u t i l i z a t i o n i n the f e t a l lamb (Hay et a l . , 1983; Hay et a l . , 1989). It i s important to note, however, that uptake i s equal 143 to the product of umbilical blood flow and the substrate arteriovenous concentration difference. Uptake does not represent u t i l i z a t i o n f o r any substrate which may be produced or stored within the fetus. The tendency f o r glucose uptake to f a l l i n the current study does not necessarily imply that glucose u t i l i z a t i o n f e l l , but more l i k e l y , that f e t a l glucose production rose. This would rais e umbilical a r t e r i a l , r e l a t i v e to umbilical venous glucose concentrations r e s u l t i n g i n a smaller veno-arterial concentration difference and a f a l l i n uptake. This phenomenom has been observed i n fetuses made hyperglycemic by f e t a l glucose infusion (Simmons e t _ a l . , 1979). The suggested r i s e i n endogenous f e t a l glucose production may have occurred through beta-2 receptor mediated increases i n hepatic and s k e l e t a l muscle glycogenolysis (both tissues contain beta-2-receptors). Glycogenolysis occurs v i a a c y c l i c AMP mediated mechanism which has been described i n d e t a i l by Alberts et a l . . (1983). A beta-receptor mediated r i s e i n i n t r a c e l l u l a r cAMP l e v e l s r e s u l t s i n the sequential a c t i v a t i o n of the enzymes protein kinase, phosphorylase kinase and glycogen phosphorylase, the l a t t e r of which catalyzes the breakdown of glycogen to glucose. Elevations i n the hepatic a c t i v i t i e s of these enzymes, a concurrent increase i n plasma glucose concentration and a 50% reduction i n hepatic glycogen content occurred a f t e r 24 hours of r i t o d r i n e infusion i n f e t a l 144 lambs (Warburton et a l . , 1988). These re s u l t s confirm that r i t o d r i n e induced hyperglycemia occurs at least i n part, through accelerated hepatic glycogenolysis i n the f e t a l lamb. The contribution of s k e l e t a l muscle glycogenolysis to the development of hyperglycemia i s not known. Subsequent hypoglycemia i n the neonatal period due to depleted f e t a l glycogen stores may be a s i g n i f i c a n t r i s k of r i t o d r i n e t o c o l y s i s . Neonatal hypoglycemia following antenatal exposure to r i t o d r i n e have been reported i n some (Kazzi et a l . . 1987) but not a l l human studies (Leake et a l . , 1983). Insulin l e v e l s were not measured i n the current study, however, hyperinsulinemia i n f e t a l lambs i n response to r i t o d r i n e infusion has been reported (Warburton et a l . . 1988; Basset et a l . , 1989). Numerous studies have shown that moderate hyperglycemia stimulates i n s u l i n secretion i n f e t a l lambs (Basset and Thornburn, 1971; Basset, 1974; Philipps e t _ a l . , 1978; Houghton et a l . , 1989) and the response i s present by at least 110 days gestation (Houghton et a l . , 1989). However, the degree to which i n s u l i n release occurs i n response to d i r e c t stimulation of pancreatic adrenergic receptors i s not c l e a r . Basset and Thornburn (1971) have suggested that t h i s d i r e c t adrenergic response may be immature i n the f e t a l lamb, however, i t appears to be present i n 4 day o l d neonatal lambs (Tenenbaum and Cowett, 1985). 4.6 FETAL LACTATE METABOLISM Lactate i s the second major carbohydrate u t i l i z e d by the f e t a l lamb and i t s oxidation accounts for approximately 22% of f e t a l oxygen uptake (Hay et a l . , 1983; Battaglia and Meschia, 1986). Fetal lactate l e v e l s exceed those of the mother, a phenomenom once thought to r e f l e c t a chronic state of f e t a l hypoxia with anaerobic metabolism. However, f e t a l t issues are adequately oxygenated and the fetus i s a net consumer (as opposed to net producer) of lactate (Sparks et a l . , 1982; Bat t a g l i a and Meschia, 1986, pp 88-95). Approximately 24% of the lactate consumed by the fetus i s derived from the placenta (Sparks et a l . , 1982). As yet unresolved, i s the guestion of why larger guantities of lactate are delivered into the f e t a l as opposed to the maternal c i r c u l a t i o n , despite higher lactate concentrations i n the fetus (Sparks et a l . , 1982; Battaglia and Meschia, 1986, pp 88-95). The permeability of the placenta to lactate i s r e l a t i v e l y low and a carrier-mediated transfer mechanism has been suggested (Sparks et a l . , 1982; Battaglia and Meschia, 1986, pp 88-95). In the l a s t 20% of gestation, f e t a l l a c t a t e u t i l i z a t i o n exceeds umbilical uptake by three-fold such that 76% of f e t a l lactate uptake comes from endogenous production (derived from glucose and non-glucose precursors) (Sparks et a l . , 1982). Lactate i s produced i n the f e t a l carcass (Singh et a l . , 1984) and lung (Simmons and Charlton, 146 1988) while i t i s consumed by the f e t a l l i v e r (Gleason et a l . , 1985), kidney (Iwamoto and Rudolph, 1985), heart (Fischer et a l . , 1982) and p o r t a l l y drained viscera (Charlton et a l . , 1979) . In the current study, f e t a l a r t e r i a l and umbilical venous lac t a t e l e v e l s rose more than f i v e - f o l d during the infusion of r i t o d r i n e . Levels continued to r i s e during the f i r s t 1.5 hours of post-infusion but declined slowly thereafter. Elevations i n lactate levels have been observed i n other short and long term r i t o d r i n e infusion studies (Siimes and Creasy, 1980; Basset et a l . , 1989). Lactate delivery also increased s i g n i f i c a n t l y to 540% above the control by the end of the infusion. Lactate uptake tended to r i s e which i s consistent with previous studies reporting increases i n lactate uptake and u t i l i z a t i o n with r i s i n g plasma concentrations of t h i s substrate (Sparks et a l . , 1982; Hay et a l . , 1983). However, as explained e a r l i e r , information regarding substrate u t i l i z a t i o n cannot be inferred from the current study. Studies have shown that during hypoxemia, increases i n f e t a l plasma lactate levels are associated with declines i n both lactate uptake and the umbilical veno-arterial lactate concentration difference; sometimes to the extent that umbilical a r t e r i a l lactate lev e l s exceed those of the umbilical vein (Sparks et a l . , 1982; Milley, 1988). In the 147 current study, the opposite was observed; the r i s e i n f e t a l a r t e r i a l plasma lactat e concentration was associated with a s l i g h t increase i n the umbilical veno-arterial plasma l a c t a t e concentration difference with a tendency for l a c t a t e uptake to increase. The data suggest that the r i s e i n umbilical a r t e r i a l l a c t a t e concentration may have been balanced by a concurrent r i s e i n the placental production and de l i v e r y of lac t a t e to the fetus. The r i s e i n f e t a l a r t e r i a l plasma la c t a t e concentrations may therefore have resulted from increases i n both f e t a l endogenous and placental l a c t a t e production. The presence of beta-2 adrenergic receptors has been well established i n both human (Whitsett, 1980; Falkay and Kovacs, 1982) and sheep placenta (Whitsett et a l . , 1980; Padbury, 1981). Studies suggest that r i t o d r i n e i n the f e t a l c i r c u l a t i o n may have access to placental beta-adrenergic receptors since 1) beta-adrenergic receptors occur on f e t a l as well as maternal placental tissue i n sheep (Padbury et a l . , 1981) and 2) beta-adrenergic receptors appear to be more cl o s e l y related to the f e t a l than maternal c i r c u l a t i o n i n humans (Whitsett et a l . , 1980). Stimulation of placental beta-adrenergic receptors results i n numerous metabolic e f f e c t s including increased cAMP synthesis and glycogenolysis (Whitsett et a l . , 1980). Epinephrine has been shown to increase l a c t a t e production i n fresh human placental tissue 148 s l i c e s (Ginsburg and Jeacock, 1964; Ginsburg and Jeacock, 1968). These studies as well as the data presented herein support the p o s s i b i l i t y of r i t o d r i n e induced increases i n placental production and delivery of lactate to the f e t a l c i r c u l a t i o n . Recently, i t has been shown that uteroplacental glucose metabolism occurs primarily on placental tissues which have d i r e c t access to glucose molecules being c a r r i e d i n the umbilical c i r c u l a t i o n (Hay et a l . , 1990). In fa c t , the f e t a l glucose pool normally contributes roughly 40% of the glucose that i s metabolized by the placenta (Hay et a l . , 1984). The rate at which the placenta u t i l i z e s glucose i s d i r e c t l y r e l a t e d to f e t a l plasma concentrations of t h i s substrate (Hay et_ _ a l . , 1990; DiGiacomo and Hay, 1990). Therefore the elevated f e t a l glucose levels observed i n the current study may have resulted i n an increase i n the uteroplacental uptake of t h i s substrate from the f e t a l c i r c u l a t i o n ; perhaps i n an attempt to maintain the maternal-fetal glucose gradient. A r i s e i n uteroplacental glucose uptake may i n turn have resulted i n an increase i n the rate at which glucose was converted to lactate. Studies by Ginsburg and Jeacock (1968) indicate that both exogenous and endogenous sources of glucose appear to be important for placental lactate production. The authors found that lactate production i n human placental t i s s u e s l i c e s was greater (and glycogen depletion lower) when 149 incubated i n glucose as opposed to non-glucose media. Also, glucose uptake and lactate production increased proportionately with dose of epinephrine when glucose was present. Hence accelerations i n placental glucose uptake and conversion to lactate may have been stimulated by both glucose and r i t o d r i n e . The accumulation of lactate i n f e t a l plasma may also r e f l e c t slow clearance of t h i s substrate from the f e t a l c i r c u l a t i o n as the placental transfer of lactate i s low (Sparks et a l . r 1982). However, a number of other mechanisms involving the endogenous production of lactate by the fetus may have contributed to the development of f e t a l hyperlactacidemia. An understanding of basic lactate metabolism i s e s s e n t i a l to a discussion of these mechanisms (review: Buchalter, 1989). Lactate production and u t i l i z a t i o n occur i n the cytosol of c e l l s i n v i r t u a l l y a l l tissues. Lactate i s i n equilibrium with pyruvate, a revers i b l e reaction which i s catalyzed by the enzyme lactate dehydrogenase (LDH). The reduction of pyruvate to lactate involves the oxidation of the reduced form of nicotinamide adenine dinucleotide (NAD+) as shown i n equation one below. 150 Equation 1 Pyruvate + NADH + H+ — • Lactate + NAD+ The equilibrium strongly favors the formation of lactate and the concentration of lactate i s nearly 10 times that of pyruvate. Equation 1 can be rearranged as follows: Equation 2 [Lactate] = K [pyruvate] [NADH] [H+] [ NAD+ ] where K i s the equilibrium constant for the reduction of pyruvate to lac t a t e . From equation 2 i t i s clear that l a c t a t e concentration i s d i r e c t l y related to 1) the pyruvate concentration, 2) the NADH/NAD+ r a t i o (referred to as the c e l l u l a r redox state) and 3) the hydrogen ion concentration. Hence i t i s clear that excess lactate production does not necessarily imply tissue hypoxia (Huckabee, 1957). Lact i c acid i s almost completely dissociated at body pH (pKa 3.8) hence each mi l l i e q u i v a l e n t of metabolically produced l a c t i c a cid w i l l y i e l d one mil l i e q u i v a l e n t each of lactate anion and hydrogen ion. The res u l t i n g acidemia can be neutralized to a c e r t a i n extent by bicarbonate and non-bicarbonate buffers of the blood. Accelerated g l y c o l y s i s may have been one endogenous mechanism through which f e t a l lactate l e v e l s became elevated since the rate of glucose oxidation (and the f r a c t i o n of glucose oxidized) i s d i r e c t l y related to f e t a l blood glucose and i n s u l i n l e v e l s (Hay et a l . f 1983; Hay et a l . , 1989). A r i s e i n g l y c o l y s i s would have been functional i n counteracting the development of hyperglycemia and would have spared other substrates from oxidation. An accelerated rate of g l y c o l y s i s would also have increased pyruvate production which according to equation 2, would have raised lactate l e v e l s . Increases i n both lactate and pyruvate l e v e l s i n response to hyperglycemia (induced by glucose infusion) have previously been reported (Huckabee, 1957). Increases i n both pyruvate and lactate l e v e l s have also been observed during r i t o d r i n e infusion to f e t a l lambs (Siimes and Creasy, 1980). The degree to which s k e l e t a l muscle glycogenolysis and g l y c o l y s i s contributed to development of hyperlactacidemia i s unclear. However, since lactate cannot be metabolized by s k e l e t a l muscle, any lactate produced by t h i s tissue would have been released into the blood stream to be metabolized by the l i v e r . Accelerated l i p o l y s i s may have been another endogenous mechanism through which f e t a l lactate l e v e l s became elevated. L i p o l y s i s occurs v i a a c y c l i c AMP mediated mechanism and has been described i n d e t a i l by Stryer (1981). A beta-adrenergic receptor mediated r i s e in fat i n t r a c e l l u l a r cAMP levels r e s u l t s i n the sequential activation of the enzymes protein kinase and l i p a s e . Lipase hydrolyses t r i a c y l g l y c e r o l s to g l y c e r o l and f a t t y acids. Since both pyruvate and free f a t t y acids are converted to acetyl CoA before entering the c i t r i c acid cycle, an accelerated conversion of free f a t t y acids to acetyl CoA may r e s u l t i n an overwhelming of the c i t r i c acid cycle, thereby i n h i b i t i n g the conversion of pyruvate to acetyl CoA. This i n h i b i t i o n may r e s u l t i n the accumulation of pyruvate, and hence lactate. Fetal plasma free f a t t y acids were not measured i n the current study, however, elevations i n these substrates have been observed i n women undergoing r i t o d r i n e t o c o l y s i s (Lenz et a l . , 1979). In addition, Jones and Ritchie (1978) have reported that short term (1 hour) infusion of isoprenaline to f e t a l lambs at physiologic rates (1.3 ug/min) r e s u l t s i n large increases i n f e t a l plasma free f a t t y acids. This apparent increase i n f a t oxidation was blocked by propranolol supporting a beta-receptor mediated mechanism. The duration of isoprenaline infusion was short, however, and given that fat content i n f e t a l lambs i s r e l a t i v e l y low (2.8 to 4.0 % of t o t a l f e t a l tissue weight) (Body and Shorland, 1964; Body et a l . , 1966), i t i s u n l i k e l y that l i p o l y s i s would have continued over a prolonged period of time. Hence while f a t t y acid oxidation may have occurred in the current study, i t s contribution to the r i s e i n f e t a l l a c t a t e levels was l i k e l y minimal. 153 It i s also possible that the r i s e i n f e t a l l a c t a t e l e v e l s resulted from tiss u e hypoxia. When the supply of oxygen to mitochondria f a l l s below the amount required to sustain metabolic needs, the c e l l u l a r oxidation-reduction systems s h i f t to a more reduced state; that i s , the NADH/NAD+ r a t i o r i s e s (Huckabee, 1957; Buchalter et a l . , 1989). According to equation 2 (page 150), a r i s e i n the NADH/NAD+ r a t i o w i l l increase lactate l e v e l s . In other words, as the oxidation of NADH by molecular oxygen (through the electron transport chain) f a l l s due to oxygen deficiency, oxidation of NADH through the lactate dehydrogenase system r i s e s . In t h i s way, the NAD+ required f o r gl y c o l y s i s i s regenerated, despite oxygen deficiency. Since lactate i s a "dead end" product i t accumulates r e s u l t i n g i n hyperlactacidemia. The metabolism of pyruvate i s also affected by reduced oxygen supply, the r e s u l t of which i s an accumulation of pyruvate and hence la c t a t e . Analysis of the degree to which f e t a l blood Po 2 and Co2 values f e l l i n the current study suggest that the f e t a l t i s sues did not become hypoxic. Po2 values of approximately 10 to 13 mm Hg are normally associated with tissue hypoxia. In the current study the minimum observed Po 2 value was 17 mm Hg. Peeters et a l . (1979) have c l a s s i f i e d 4 states of hypoxia based on a r t e r i a l oxygen content. The minimum f e t a l a r t e r i a l oxygen content value observed in the current study (2.1 mM) f e l l into t h e i r class of "mild hypoxia" (0 2 content of 2 to 4 mM) where oxygen supply to some tissues was below normal, but adequate to meet oxidative requirements. In addition, hypoxia re f e r s to a state i n which f e t a l oxygen consumption i s reduced (Richardson, 1989; Rurak et a l . , 1990a). In the current study, oxygen consumption was elevated. Increases i n lactate l e v e l s have been observed i n other studies of prolonged (24 hours) induced, mild hypoxemia (Po 2 decline of 5 mm Hg) (Towell et a l . . 1987). I t has been suggested that lactate elevations under these conditions may r e s u l t at least i n part from a r i s e i n catecholamine lev e l s (Rurak et a l . , 1990a). Increases i n lactate l e v e l s were achieved through epinephrine infusion to f e t a l lambs (Jones and Ritchie, 1978) and t h i s e f f e c t was p a r t i a l l y blocked by concurrent administration of propranolol (Jones and Ritchie, 1983). Jacobs et a l . , (1988) have suggested that catecholamines may induce a r e d i s t r i b u t i o n of peripheral blood flow which may i n turn r e s u l t i n l o c a l anaerobic production of lactate i n non-vital t i s s u e s . 4.7 FETAL-MATERNAL OXYGEN METABOLISM Oxygen transport from the atmosphere to the fetus has been described as a series of steps which alternate between bulk and d i f f u s i o n a l transportation (Meschia, 1979). Oxygen pressure decreases progressively with each step such that umbilical venous oxygen tension (approximately 35 mm Hg i n f e t a l lambs) l i e s far below that of the maternal artery (approximately 90 mm Hg i n sheep) (Meschia, 1989). The transport of oxygen across the sheep placenta occurs by d i f f u s i o n and the involvement of an oxygen c a r r i e r system i s debatable (Faber and Thornburg, 1983, pp 73-74). The ovine placenta functions as an "imperfect venous e q u i l i b r a t o r " or a "concurrent exchanger" (Battaglia and Meschia, 1986, pp 28-48; Meschia, 1989). Within t h i s system, maternal and umbilical vascular streams (separated by vessel membranes) flow i n the same d i r e c t i o n within the exchange s i t e and e q u i l i b r a t e such that oxygen i s transferred along a maternal-f e t a l oxygen gradient. F i n a l e q u i l i b r a t i o n occurs between uterine venous and umbilical venous blood, hence the Po of ' 2 umbilical venous blood depends on, and can never be higher than that of uterine venous blood. A d i r e c t r e l a t i o n s h i p between uterine and umbilical venous Po values exists over 2 a wide range of uterine oxygen tensions i n sheep (Rankin et a l . , 1971; Wilkening and Meschia, 1983). However, the e f f i c i e n c y of oxygen transfer across the sheep placenta i s below that of an i d e a l concurrent exchange system r e s u l t i n g i n umbilical 0 2 tensions (approximately 35 mm Hg) that are normally 12 to 20 mm Hg below uterine venous 0 2 tensions (approximately 50 mmHg) (Wilkening and Meschia, 1983). There are many factors which may contribute to the uterine-umbilical Po2 difference including: 1) uneven maternal/fetal placental perfusion, 2) the configuration of maternal and f e t a l 156 placental vessels 3) placental vascular shunts, 4) a high rate of placental oxygen consumption 5) limited oxygen d i f f u s i n g capacity and 6) high oxygen a f f i n i t y of f e t a l blood (Wilkening and Meschia, 1983; Meschia, 1989). I t has also been hypothesized that the uterine-umbilical Po2 difference may be determined by the oxygen demand of the fetus ( B e l l e t _ a l . , 1987; Wilkening et a l . , 1988). A. Fetal Oxygen Consumption The normal range of oxygen consumption i n the f e t a l lamb i s 260 to 360 umol/min.kg (Battaglia and Meschia, 1986, pp 64-67). In the current study control oxygen consumption averaged 342±25 and 317±37 umol/min.kg i n the r i t o d r i n e and saline infusion experiments respectively. During the infusion of r i t o d r i n e , f e t a l oxygen consumption rose progressively and s i g n i f i c a n t l y a t t a i n i n g a maximum of 416134 umol/min.kg (22% above the control) by 8 hours post-infusion. Consumption returned to the control value by the end of the post-infusion period. The current study appears to be the f i r s t to have measured f e t a l oxygen consumption during r i t o d r i n e infusion. However, increases i n f e t a l oxygen consumption have been observed i n response to other adrenergic agonists including norepinephrine ( L o r i j n and Longo, 1980) and clenbuterol ( i n c a l f s ) (Eisemann et a l . , 1988). 157 The r i s e i n f e t a l oxygen consumption i n the current study may r e f l e c t an accelerated rate of f e t a l oxidative metabolism induced by elevated plasma leve l s of glucose, lactate and i n s u l i n i n the f e t a l lamb. Previous studies have shown a d i r e c t r e l a t i o n s h i p between f e t a l a r t e r i a l plasma glucose and lactate l e v e l s and t h e i r respective oxidation rates (Hay et a l . . 1983; Hay et a l . , 1989). Philipps et a l . (1984) reported a s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between the degree of hyperglycemia and elevations i n oxygen consumption i n f e t a l lambs. A doubling of f e t a l a r t e r i a l plasma glucose concentration (after 1 day of f e t a l glucose infusion) was associated with a 16% r i s e i n f e t a l oxygen consumption. In the current study, f e t a l glucose l e v e l s rose to 1.8 times the control by the end of the infusion and t h i s was associated with a 19% r i s e i n f e t a l oxygen consumption. Hyperinsulinemia also r e s u l t s i n elevations i n f e t a l oxygen consumption (Milley et a l . , 1984; Hay and Meznarich, 1986; Hay et a l . , 1989; Mi l l e y and Papacostas, 1989). Hay and Meznarich (1986) found that a 2 hour r i s e i n f e t a l i n s u l i n l e v e l s (glucose lev e l s kept constant using the glucose clamp technique) resulted i n a 13% r i s e i n f e t a l oxygen consumption. Although hyperglycemia and hyperinsulinemia both stimulate increases i n f e t a l oxygen consumption, Hay and Meznarich (1986) found that the r i s e i n oxygen consumption was not proportional to the larger r i s e i n glucose oxidation rate suggesting that other substrates had been spared from oxidation. Recent 158 reports suggest that hyperinsulinemia-induced elevations i n f e t a l oxygen consumption may be attributed s o l e l y to increases i n non-visceral t i s s u e oxygen consumption (Milley and Papacostas, 1989). Studies investigating the r e l a t i v e importance of glucose and i n s u l i n i n r a i s i n g f e t a l glucose u t i l i z a t i o n and oxygen consumption rates have indicated that t h e i r e f f e c t s are independent, but additive (Hay et a l . , 1988, 1989). While hyperglycemia increases glucose u t i l i z a t i o n , i n s u l i n promotes the entry of glucose into f e t a l c e l l s thereby increasing the rate at which glucose i s u t i l i z e d through oxidative and non-oxidative pathways ( i . e . f a t , glycogen or protein synthesis). Hence the combined ef f e c t s of both hyperglycemia and hyperinsulinemia may have played an important role i n the elevation of f e t a l oxygen consumption i n the current study. Whether the r i s e i n f e t a l lactate l e v e l s had any e f f e c t on f e t a l oxygen consumption i s not c l e a r . Studies have shown that the rate of lactate oxidation i s d i r e c t l y related to plasma leve l s of t h i s substrate (Hay et a l . , 1983). Furthermore, lactate i s a major nutrient of the f e t a l lamb which appears to be u t i l i z e d primarily through oxidation (Sparks et a l . , 1982). Fetal a r t e r i a l lactate l e v e l s and uptake rose i n the current study (although uptake did not r i s e s i g n i f i c a n t l y ) , hence an increase i n lactate oxidation may have contributed to the r i s e i n f e t a l oxygen consumption. However, the combined ef f e c t s of hyperglycemia and hyperinsulinemia were probably far more important i n elevating f e t a l oxygen consumption. Moreover, hyperinsulinemia does not e f f e c t lactate uptake (Milley and Papacostas, 1989). B. Fetal Oxygen Delivery Fetal oxygen delivery i s the product of umbilical blood flow and umbilical venous oxygen content. In the current study, oxygen delivery f e l l progressively and s i g n i f i c a n t l y during the infusion of r i t o d r i n e reaching i t s lowest l e v e l (29% below the control) by 1.5 hours post-infusion. Oxygen delivery increased thereafter, however, i t remained s i g n i f i c a n t l y below the control throughout the post-infusion period. The decline i n oxygen delivery observed during the r i t o d r i n e infusion period was the r e s u l t of a f a l l i n umbilical venous oxygen content. However, the slow recovery of oxygen delivery during the post-infusion period was l i k e l y due not only to the low umbilical oxygen content, but to the concurrent, nonsignificant f a l l i n umbilical blood flow (discussed i n Section 4.4). The mechanisms responsible for the decline i n umbilical venous oxygen content are proposed below. 160 C. Fetal Blood Gas Status Umbilical venous oxygen content values declined to 75% of the control by the end of the r i t o d r i n e infusion period while a r t e r i a l oxygen content values f e l l more rapidl y and to a greater extent becoming only 57% of the control during the same time period. Hence the venoarterial oxygen content difference increased s i g n i f i c a n t l y . Both a r t e r i a l and umbilical venous oxygen content values showed p a r t i a l recovery by 8 hours post-infusion, but remained s i g n i f i c a n t l y below the control throughout the remainder of the experiment. A r t e r i a l and umbilical venous Po2 values also f e l l (to 78% and 79% respectively) below the control by the end of the infusion but recovered slowly thereafter. Others have s i m i l a r l y reported declines i n f e t a l a r t e r i a l Po 2 values during prolonged (24 hours or longer) infusion of r i t o d r i n e to f e t a l lambs (Warburton et a l . , 1987a; Basset et a l . , 1989). Small decreases i n f e t a l a r t e r i a l Po2 have also been observed during maternal r i t o d r i n e infusion (Basset et a l . , 1985). Fetal blood gases remain unaltered, however, during short term infusion of r i t o d r i n e (1 to 2 hours) to the mother or fetus (Ehrenkranz et a l . . 1976; Siimes and Creasy, 1980) . The deterioration i n f e t a l blood gas status l i k e l y occurred as a r e s u l t of at le a s t three factors. F i r s t l y , the r i s e i n f e t a l oxygen consumption was not associated with a r i s e i n f e t a l oxygen delivery ( i n f a c t delivery f e l l ) , therefore f e t a l f r a c t i o n a l oxygen extraction increased s i g n i f i c a n t l y from a control value of 32.0% to a maximum of 51.6% by the 1.5 hour post-infusion period. This r i s e i n extraction resulted i n a f a l l i n f e t a l a r t e r i a l oxygen content (and Po2) hence the veno-arterial oxygen content difference rose. These l a t t e r changes may be explained as follows. Fetal oxygen extraction i s defined as the f r a c t i o n of delivered oxygen that i s consumed by the fetus: Equation 3) Fetal Oa extraction = Vo2/Do2 where Vo 2 and Do2 represent f e t a l oxygen consumption and delivery respectively. Since Vo 2 = Qum (Cvo 2 - Cao2) and Do2 = Qum (Cvo2) where Qum, Cvo2, and Caoz represent umbilical blood flow and umbilical venous and a r t e r i a l oxygen contents respectively, equation 3 can be rearranged to the following: Equation 4) Fetal Cao2 = Cvo2 (1 - Vo2/Do2) As indicated by equation 4, both the r i s e i n f e t a l oxygen extraction as well as the f a l l i n umbilical venous oxygen content contributed to the decline i n a r t e r i a l oxygen content. I t i s i n fact the f a l l i n a r t e r i a l oxygen content which l i m i t s the degree to which increases i n oxygen extraction can compensate for i n s u f f i c i e n t delivery of oxygen to the fetus. The second factor which l i k e l y contributed to the deter i o r a t i o n i n f e t a l blood gases was that the increase i n f e t a l oxygen consumption was not associated with a concurrent r i s e i n uterine blood flow or uterine oxygen delivery. Analogous to the f e t a l case, the observed r i s e i n uterine oxygen consumption ( r e f l e c t i n g the r i s e i n f e t a l oxygen consumption) was not associated with a r i s e i n uterine oxygen delivery, hence uterine oxygen extraction increased. The r i s e i n uterine oxygen extraction resulted i n a f a l l i n uterine venous Po and Co values. Since uterine and umbilical venous 2 2 Po 2 are d i r e c t l y r e l a t e d (Rankin et a l . , 1971; Wilkening and Meschia, 1983), the f a l l i n uterine venous Po 2 may have contributed to the decline i n umbilical venous Po . The 2 observed f a l l i n umbilical venous oxygen delivery necessitated even further increases i n f e t a l oxygen extraction. As discussed above, the uterine-umbilical venous Po2 difference (as well as the umbilical venous Po2 values) may i n part be determined by f e t a l oxygen demand (Wilkening et a l . , 1989). Reductions i n f e t a l oxygen consumption (induced through neuromuscular blockade) have been associated with 163 decreases i n the transplacental Po2 gradient and higher umbilical venous Po 2 values (Rurak and Gruber, 1983b; Wilkening et a l • , 1989). In contrast, increases i n f e t a l oxygen consumption (as during episodes of breathing) have been associated with decreases i n umbilical venous Po 2 values (Rurak and Gruber, 1983a). In the current study, the r i s e i n f e t a l oxygen demand was associated with an increase i n the transplacental oxygen gradient and a decrease i n umbilical venous Po values. 2 The t h i r d factor which l i k e l y contributed to the de t e r i o r a t i o n of f e t a l blood gases was the development of acidemia i n the fetus. Fetal a r t e r i a l pH f e l l s i g n i f i c a n t l y from a control value of 7.370±0.004 to 7.274±0.025 by the end of the infusion. The acidemia was r e f l e c t e d by s i g n i f i c a n t declines i n base excess values and appeared to be e n t i r e l y metabolic i n nature, r e s u l t i n g from the accumulation of l a c t i c acid i n f e t a l blood. The acidemia presumably lowered f e t a l vascular Po 2 l e v e l s through a rightward s h i f t of the oxyhemoglobin d i s s o c i a t i o n curve. Fetal a r t e r i a l hypoxemia has also been observed during induced hyperglycemia and hyperinsulinemia i n f e t a l lambs (Carson et a l . , 1980; Philipps et a l . , 1982; P h i l l i p s e t _ a l . , 1984; M i l l e y , 1984; Hay and Meznarich, 1986). The mechanism for these changes appeared to involve an increase i n f e t a l oxygen extraction secondary to a r i s e i n f e t a l oxygen consumption (without a concurrent r i s e i n umbilical oxygen d e l i v e r y ) . Alterations i n f e t a l blood oxygen a f f i n i t y or placental oxygen transport were not observed ( P h i l l i p s , 1984). In contrast, other studies indicate that under certa i n circumstances, compensatory mechanisms are e l i c i t e d during periods of elevated oxygen consumption which prevent declines i n f e t a l blood oxygen content and Po 2 values. Increases i n f e t a l oxygen consumption of 25% and 28%, evoked by norepinephrine ( L o r i j n and Longo, 1980a) and thyroxine infusions ( L o r i j n and Longo, 1980b) respectively to f e t a l lambs did not r e s u l t i n any s i g n i f i c a n t changes i n f e t a l blood oxygen status. The absence of e f f e c t on f e t a l oxygen status i n these studies could be explained by the concurrent r i s e i n umbilical blood flow which was s i m i l a r i n magnitude to the r i s e i n f e t a l oxygen consumption. In e f f e c t , the r i s e i n f e t a l oxygen consumption was matched by a sim i l a r increase i n f e t a l oxygen delivery, therefore f e t a l oxygen extraction d i d not increase and hypoxemia did not r e s u l t . In contrast, r i t o d r i n e does not increase umbilical blood flow, hence there i s a mismatch between f e t a l oxygen consumption and f e t a l oxygen delivery. Consequently f e t a l oxygen extraction increases and hypoxemia r e s u l t s . The studies by L o r i j n and Longo (1980 a,b) suggest that changes i n umbilical blood flow (as opposed to uterine blood gases) may be more important i n 165 determining the consequences of increased f e t a l oxygen consumption. The converse e f f e c t s of norepinephrine and r i t o d r i n e on umbilical blood flow presumably r e f l e c t the opposite e f f e c t s which these agents have on f e t a l systemic a r t e r i a l pressure; which i n turn r e f l e c t s t h e i r d i f f e r i n g receptor s p e c i f i c i t i e s (RS) 3. Umbilical blood flow i s effected p o s i t i v e l y by increases i n f e t a l systemic a r t e r i a l pressure ( i . e . through increases i n cardiac output or systemic vascular resistance) and negatively by increases i n umbilical vascular resistance (Heymann et a l . , 1975; Berman et a l . , 1978). Through stimulation of vascular alpha-adrenergic receptors, nor-epinephrine (RS: alpha = beta-1 » beta-2) increases systemic (but not umbilical-placental) vascular resistance r e s u l t i n g i n increases i n f e t a l systemic a r t e r i a l pressure; the r e s u l t being a r i s e i n umbilical-placental perfusion pressure and a r i s e i n umbilical blood flow (Berman et a l . , 1978; L o r i j n and Longo, 1980a). In contrast, r i t o d r i n e (RS: beta-2 > beta-1 » alpha) through i n t e r a c t i o n with vascular beta-2 receptors tends to reduce (Siimes et a l . , 1978) or not to change (Basset et a l . , 1989) (current study) f e t a l a r t e r i a l pressure, hence umbilical blood flow remains unaltered. Ritodrine does not 3The thyroxine induced increase i n umbilical blood flow i n the L o r i j n and Longo (1980b) study were reported to have occurred through a decline i n the umbilical-placental vascular resistance. 166 increase cardiac output (Siimes et a l . , 1978) hence the drug induced tachycardia does not r e s u l t i n a r i s e umbilical blood flow. D. Total Uterine and Uteroplacental Oxygen Consumption Control uterine oxygen consumption i n the r i t o d r i n e and saline infusion studies averaged 1236±128 and 1038±101 umol/min respectively which are within the range of values reported by others (400-2200 umol/min) (Meschia et a l . , 1966; Clapp, 1978; Meschia et a l . , 1980). Data regarding uteroplacental oxygen consumption was not reported as the v a r i a b i l i t y associated with these re s u l t s precluded int e r p r e t i v e analysis. Other published values for t o t a l uterine and uteroplacental oxygen consumption also appear to vary considerably, perhaps due to: i n t e r - and intra-animal v a r i a b i l i t y , m u l t i p l i c a t i o n of error associated with both flow and oxygen content measurements, d i f f e r i n g f e t a l weight and varying gestational age. The v a r i a b i l i t y associated with uteroplacental oxygen consumption i s even greater, l i k e l y because i t s c a l c u l a t i o n encompasses the v a r i a b i l i t y associated with both t o t a l uterine and t o t a l f e t a l oxygen consumption. As discussed above, t o t a l uterine oxygen consumption tended to r i s e to an average of 25% above the control throughout the infusion of r i t o d r i n e and during the f i r s t 8 hours of post-infusion. While uteroplacental oxygen consumption was not calculated for each i n d i v i d u a l animal, i t was possible to approximate these values by comparing t o t a l f e t a l and t o t a l uterine oxygen consumption data given i n Tables 10 and 20. Total uterine oxygen consumption rose by 263, 283 and 291 umol/min at EI, PI. 5 and P8 respectively. These values r e f l e c t s i m i l a r , concurrent elevations i n t o t a l f e t a l oxygen consumption of 251, 257, and 294 umol/min above the c o n t r o l . Both uterine and f e t a l oxygen consumption had returned to the control by the f i n a l post-infusion period. The s i m i l a r changes i n magnitude of uterine and f e t a l oxygen consumption imply that uteroplacental oxygen consumption remained unaltered. Hence the r i s e i n t o t a l uterine oxygen consumption appears to s o l e l y r e f l e c t the r i s e i n f e t a l oxygen consumption. Philipps et a l . (1984) reported that glucose infusion to f e t a l lambs resulted i n a 30% r i s e i n f e t a l oxygen consumption and a concurrent, non-significant r i s e i n t o t a l uterine oxygen consumption. The degree to which uterine and f e t a l oxygen consumption increased were s i m i l a r , hence, i t was concluded that hyperglycemia also had l i t t l e e f f e c t on uteroplacental consumption. 168 E. Uterine Blood Gas Status There were no s i g n i f i c a n t changes i n maternal a r t e r i a l blood gas values or i n the delivery of oxygen to the uterus during the infusion of r i t o d r i n e . However, as previously discussed, both uterine venous oxygen content and Po2 values f e l l s i g n i f i c a n t l y below the control by 1.5 hours of r i t o d r i n e infusion and continued to decline to a minimum by 1.5 hours of post-infusion (the f a l l i n uterine venous oxygen content was l i k e l y underestimated given that the uterine venous blood c o l l e c t e d i n t h i s study represented both placental and myometrial venous flow). Uterine blood gases returned to the control by the end of the post infusion period. Analogous to the f e t a l case, the observed r i s e i n uterine oxygen consumption was not associated with a r i s e i n uterine oxygen delivery, hence uterine oxygen extraction rose to a maximum of 22% above the control by 1.5 hours of infusion ( f e t a l oxygen extraction also reached a maximum at t h i s time). The r i s e i n uterine oxygen extraction resulted i n a f a l l i n uterine venous Po 2 and Co2 values which may have contributed to the f a l l i n umbilical oxygen delivery. Both maternal a r t e r i a l and uterine venous hematocrit f e l l s i g n i f i c a n t l y at 1.5 hours of r i t o d r i n e infusion and again at the end of the post-infusion period (hematocrit remained stable between these two time periods). The second drop i n hematocrit ( i . e . at the end of the post-infusion period) may have i n h i b i t e d the recovery of uterine venous Co2 during the l a t t e r post-infusion period. However, the f i r s t drop i n hematocrit was l i k e l y not responsible for the f a l l i n uterine venous Co2 which occurred during the infusion of r i t o d r i n e , as indicated by a number of factors. F i r s t l y , although maternal a r t e r i a l hematocrit was s i g n i f i c a n t l y depressed throughout the r i t o d r i n e infusion experiment (3.3% t o t a l f a l l ) , a r t e r i a l Co2 did not f a l l u n t i l the end of the post-infusion period. Hence the s i m i l a r f a l l i n uterine venous hematocrit (3.3% t o t a l f a l l ) would not l i k e l y have lowered uterine venous Co2 before the end of the post-infusion period. Furthermore, although hematocrit f e l l s i g n i f i c a n t l y during the s a l i n e infusion experiments (2.8% t o t a l venous f a l l ) , a s i g n i f i c a n t decline i n uterine venous Co 2 or Po2 was not observed. The cause of the decline i n maternal hematocrit i s not c l e a r . In many cases (but not a l l ) , blood taken from the fetus was replaced by maternal blood. However, the t o t a l amount of blood taken from the ewe each day was minimal and could not account for the entire decline i n maternal hematocrit (although i t may have been a contributing f a c t o r ) . F. Fetal Metabolic Outcome In summary, the development of f e t a l hypoxemia during the infusion of r i t o d r i n e l i k e l y occurred as a r e s u l t of at least three factors. F i r s t l y , f e t a l oxygen delivery did not react homeostatically to compensate for the increase i n f e t a l oxygen demands ( i n fac t oxygen delivery f e l l ) . Therefore f e t a l oxygen extraction increased which resulted i n a decline i n f e t a l a r t e r i a l oxygen content and Po 2 values. Secondly, the r i s e i n lactate l e v e l s resulted i n the development of s i g n i f i c a n t metabolic acidemia which l i k e l y further lowered f e t a l blood oxygen content and Po2 values through a rightward s h i f t of the oxyhemoglobin d i s s o c i a t i o n curve (Bohr e f f e c t ) . F i n a l l y , uterine oxygen delivery d i d not increase, hence uterine oxygen extraction rose and uterine venous Po2 and oxygen content values f e l l . The r e l a t i v e importance of t h i s l a t t e r change to the f a l l i n umbilical oxygen delivery i s not cl e a r given that increases i n umbilical blood flow may f u l l y compensate for increases i n f e t a l oxygen consumption ( L o r i j n and Longo, 1980 a,b). Progressive declines i n f e t a l a r t e r i a l oxygen content may eventually r e s u l t i n hypoxia, anaerobic metabolism, further acidemia and additional decreases i n oxygen content (Rurak et a l . , 1990a). The degree to which f e t a l oxygen extraction can r i s e and a r t e r i a l Po2 can f a l l before tissue oxygen supply becomes inadequate i s referred to as the "oxygen margin of safety" (Richardson, 1989) and i s an i n d i c a t i o n of the f e t a l oxygen reserve. Studies have shown that under normal conditions, the supply of oxygen to the fetus exceeds the minimum l e v e l necessary to sustain f e t a l l i f e by a factor of two (Meschia, 1985). Although the "oxygen margin of safety" provides the fetus with a degree of protection, t h i s becomes limited by the development of metabolic acidemia (Richardson, 1989; Rurak et a l . , 1990a). The development of metabolic acidemia i n the current study did appear to reduce the amount of oxygen that was available to the fetus. As discussed i n Section 4.6, tissue hypoxia was not indicated, however, whether the degree to which f e t a l oxygen consumption increased was s u f f i c i e n t to meet the increased metabolic requirements of the fetus i s not known. 4.8 FETAL CARDIOVASCULAR FUNCTION Fetal cardiovascular function and control have been extensively reviewed (Mott, 1982; Walker, 1984; Battaglia and Meschia, 1986, pp 189-211; Heymann, 1989). The primary functional c h a r a c t e r i s t i c s of the f e t a l cardiovascular system are a high cardiac output (almost maximal), high heart rate, high regional blood flows, low a r t e r i a l pressure and a low vascular resistance. These c h a r a c t e r i s t i c s are e f f e c t i v e i n counteracting low f e t a l a r t e r i a l Po 2 l e v e l s . Control of the f e t a l cardiovascular system i s achieved through regulation of a r t e r i a l pressure and l o c a l tissue blood flow, which i n turn are dependent upon cardiac output and peripheral vascular resistance. Reflex control (eg. baroreceptor and chemoreceptor reflexes) are also functional i n the fetus. Fetal cardiac output appears to be influenced by both sympathetic and parasympathetic innervation (which mature at d i f f e r e n t rates) and c i r c u l a t i n g epinephrine. Sympathetic control i s established as early as 60 days gestation. While re s t i n g f e t a l heart rate appears to be set by i n t r i n s i c mechanisms, i t i s influenced by the cardioaccelerating tone of the sympathetic system. As gestation continues, the parasympathetic system exerts an increasing influence on f e t a l heart rate which may account for the gradual f a l l i n heart rate over time. Peripheral vascular resistance appears to be under the control of l o c a l regulation (eg. l o c a l response to changes i n 02, H+, or C0 2 l e v e l s ) , autoregulation, sympathetic innervation (parasympathetic system i s less important except i n the pulmonary c i r c u l a t i o n ) , c i r c u l a t i n g epinephrine, hormones (eg. angiotensin and vasopressin) and prostaglandins. Control of the f e t a l cardiovascular system becomes more f i n e l y tuned with gestation. Control f e t a l heart rate and a r t e r i a l pressure values during the current r i t o d r i n e and s a l i n e infusion experiments averaged 162±7 and 169±10 beats per minute (bpm) and 46.2±1.5 and 47.4±1.2 mm Hg respectively. These values are within the normal range found i n f e t a l lambs during the l a s t t h i r d of gestation. The infusion of r i t o d r i n e did not r e s u l t i n any s i g n i f i c a n t changes i n f e t a l a r t e r i a l pressure, however, s i g n i f i c a n t elevations i n f e t a l heart rate were observed. Fetal heart rate was elevated by 34 bpm at 1.5 hours of infusion and remained elevated by an average of 26 bpm u n t i l 8 hours post-infusion. Heart rate returned to the control by the f i n a l post-infusion period. Other long and short term r i t o d r i n e infusion studies have noted s i m i l a r increases i n f e t a l heart rate at comparable drug infusion rates (Ehrenkranz, 1976; Siimes et a l . , 1978; Warburton et a l . , 1987; Basset et a l . , 1989). Similar to the current study, Basset et a l . , (1989) did not observe any s i g n i f i c a n t changes i n f e t a l a r t e r i a l pressure. The r i s e i n f e t a l heart rate presumably occurred through d i r e c t stimulation of myocardial beta-1 adrenergic receptors. A large body of evidence has demonstrated the s e n s i t i v i t y of the f e t a l myocardium to adrenergic agonists as was discussed i n Section 4.3. Cardiac output was not measured i n the current study, however, other r i t o d r i n e infusion studies have reported that t h i s variable remains unaltered (Siimes et a l . , 1978; Creasy and Siimes, 1979). This finding i s not surprising given that the f e t a l heart operates at the top of i t s v e n t r i c u l a r function curve and hence has a limited a b i l i t y to increase cardiac output (Rudolph and Heymann, 1976; Thornburg and Morton, 1983). Siimes et a l . (1978) have observed a r e d i s t r i b u t i o n of cardiac output to the myocardium and adrenal gland during r i t o d r i n e infusion. Similar organ flow changes occur during f e t a l hypoxia and hypoxemia (Cohn et a l . . 1974; Sheldon et a l . . 1979; Peeters et a l . . 1979; Lampe et a l . , 1988; Jansen, 1989; Rurak et a l . . 1990b). The advantage of t h i s r e d i s t r i b u t i o n i s to maintain oxygen del i v e r y to c r i t i c a l organs (heart, brain, adrenal gland and placenta) although at the expense of adequate delivery to other organs ( i . e . s k e l e t a l muscle). The mechanisms involved i n these organ flow changes appear to be complex and whether any beta-adrenergic mechanisms are involved i s not c l e a r . 4.9 FETAL BEHAVIORAL ACTIVITY The existence of spontaneous breathing movements i n the fetus gained acceptance a f t e r being described i n chr o n i c a l l y catheterized f e t a l sheep preparations (Merlet et a l . . 1970; Dawes et a l . . 1972). Fetal breathing a c t i v i t y has since been described i n many species (reviews: Kitterman, 1983; Jansen and Chernick, 1983; Harding, 1984; Dawes, 1984; Patrick and Gagnon, 1989) and appears to be a phenomenon of mammalian f e t a l development. Breathing movements i n f e t a l lambs have been observed at as early as 40 days gestation. These movements are described as rhythmic contractions of the diaphragm (and i n t e r c o s t a l muscles at times) and are detected as negative deflections on the tracheal pressure trace. Fetal breathing a c t i v i t y i s associated with the inward and outward movement of small volumes of tracheal f l u i d . The f l u i d i s produced by the f e t a l lung and the net outward flow (approximately 4.5 ml/min.kg) either enters the amniotic f l u i d , or i s swallowed. Episodes of f e t a l breathing a c t i v i t y may l a s t from several minutes to approximately one hour and are separated by periods of apnea of approximately equal duration. Breathing rates are i r r e g u l a r and occur at an average rate of approximately 40 to 60 per minute (range 10-250 breaths/min). The function of f e t a l breathing movements i s not clear, but experimental abolishment of t h i s a c t i v i t y r e s u l t s i n pulmonary hypoplasia. I t has thus been suggested that f e t a l breathing movements are important for stimulating growth and development of the f e t a l lung. The association of breathing movements with low voltage e l e c t r o c o r t i c a l a c t i v i t y (ECoG) and rapid eye movement sleep (REM) i n the f e t a l lamb was an i n t r i g u i n g observation which provided early evidence for the existence of f e t a l behavioral patterns (Dawes et a l . , 1972; Ruckebusch et a l . , 1972). Before 110 days gestation, f e t a l ECoG i s undifferentiated and f e t a l breathing and electroocular a c t i v i t y occur almost continuously (Clewlow et a l . . 1983). From 110 to 115 days gestation, there a r i s e short bursts of high voltage ECoG superimposed upon e x i s t i n g low voltage ECoG a c t i v i t y . During 176 t h i s period, f e t a l breathing i s d i s t i n c t l y episodic and i s associated with rapid eye movement a c t i v i t y (but not necessarily with low ECoG a c t i v i t y ) . By 12 0 days gestation, ECoG a c t i v i t y i s c l e a r l y d i f f e r e n t i a t e d and three behavioral states emerge within the fetus including: non-REM (quiet) sleep, REM (active) sleep and an aroused state (Ruckebusch, 1972; Jansen, 1983; Harding, 1984). Quiet sleep represents an unresponsive f e t a l state and i s characterized by the presence of high voltage ECoG a c t i v i t y , sluggish body movements and a lack of both REM and breathing a c t i v i t y (occurs approximately 55% of the time). REM sleep i s characterized by the presence of low voltage ECoG a c t i v i t y , REM, breathing movements and abrupt twitching body movements (occurs approximately 40% of the time). The aroused behavioral state i s characterized by low voltage ECoG, eye movements (not rapid), breathing movements, swallowing, and vigorous body movements (occurs approximately 5% of the time). A diurnal rhythm i n the incidence of f e t a l breathing a c t i v i t y and low voltage ECoG has been observed i n f e t a l lambs (Boddy et a l . , 1973). Breathing a c t i v i t y , as characterized by changes i n tracheal f l u i d flow and the tracheal pressure trace was found to peak during the l a t e evening (1900 to 2200 hours) and to trough during the early morning hours (0800 to 0900 hours) (Boddy et a l . , 1973). The peak period was 177 associated with an increase i n the rate and depth of breathing movements with a 2.0 to 2.8 f o l d increase i n tracheal f l u i d flow over the trough. The investigators also reported a complete association of breathing movements with low ECoG a c t i v i t y , the l a t t e r of which peaked during the l a t e evening (1900-2000 hours) and f e l l to a trough during the early morning hours (0400-0500). In the current study, a s i m i l a r diurnal rhythm i n f e t a l breathing a c t i v i t y was apparent. The peak incidence i n f e t a l breathing movements occurred during the l a t e afternoon and evening (1400 to 2200 hours) while a trough occurred during the early morning hours (0600-1000). Similar to the study by Boddy et a l . (1973), the difference between peak and trough breathing frequency was s t a t i s t i c a l l y s i g n i f i c a n t . A second smaller peak i n breathing a c t i v i t y was apparent from 0300 to 0600 hours, however, the s i g n i f i c a n c e of t h i s peak ( i f any) i s not known. A diurnal rhythm i n ECoG a c t i v i t y was not detected which may be due to the f a c t that only 5 animals were analyzed for ECoG (verses 8 animals analyzed for breathing a c t i v i t y ) . Unfortunately additional ECoG and breathing data from control saline experiments were not available. The incidence of f e t a l breathing a c t i v i t y i n the current study f e l l s i g n i f i c a n t l y from an o v e r a l l average control value of 43.2±2.6% to an average of 28.1±6.8% during the r i t o d r i n e infusion period. In most animals, breathing was maximally 178 depressed near the end of the infusion. The incidence of low voltage ECoG a c t i v i t y f e l l s i g n i f i c a n t l y by an average of 7.5% while that of high voltage ECoG a c t i v i t y rose by 7.3%. Since breathing i s generally associated with low voltage ECoG a c t i v i t y i t follows that both variables should decline concurrently. Alterations i n intermediate voltage a c t i v i t y were not observed. The incidence of REM tended to f a l l by an average of 8.2% during the infusion of r i t o d r i n e , however, t h i s variable was measured i n only 3 animals, hence s t a t i s t i c a l analysis was not possible. I t may not have been appropriate to compare the entire control period with the whole r i t o d r i n e infusion period i n the above analysis of f e t a l breathing a c t i v i t y as the diurnal rhythm associated with t h i s variable was not considered. Ritodrine induced breathing al t e r a t i o n s may have been obscured by the natural diurnal cycle, therefore the following analysis may be more appropriate. Since the infusion of r i t o d r i n e was begun at the same time of day i n each animal ( i . e . 1000 hours), i t was possible to compare the average breathing incidence i n each consecutive four hour r i t o d r i n e infusion i n t e r v a l with each corresponding 4 hour control i n t e r v a l (ECoG a c t i v i t y was not analyzed i n t h i s fashion as a diurnal rhythm was not i d e n t i f i e d ) . From t h i s analysis i t was c l e a r that r i t o d r i n e resulted i n a reduction i n breathing a c t i v i t y during a period of time when i t would otherwise have r i s e n (Figure 179 15). A s i g n i f i c a n t difference in breathing a c t i v i t y was found between the second 4 hour r i t o d r i n e infusion i n t e r v a l ( i . e . 5 to 8 hours of infusion) and the corresponding 4 hour control i n t e r v a l ( i . e . 1400-1800 hours). Since the t o t a l number of animals f e l l with continued infusion, s i g n i f i c a n c e could not be shown for reduced breathing a c t i v i t y beyond 8 hours of drug administration. The mechanisms c o n t r o l l i n g f e t a l behavioral a c t i v i t y under normal conditions are r e l a t i v e l y unclear (reviews: Jansen and Chernick, 1983; Dawes, 1984) hence, i t d i f f i c u l t to propose mechanisms for the observed r i t o d r i n e induced changes. Furthermore, numerous f e t a l metabolic perturbations were observed during the infusion of r i t o d r i n e , a l l of which may have influenced f e t a l behavioral a c t i v i t y . Studies investigating the e f f e c t s of glucose on breathing a c t i v i t y i n the f e t a l lamb indicate that hypoglycemia reduces breathing movements while hyperglycemia appears to have no e f f e c t (Boddy and Dawes, 1975; Richardson et a l . f 1982). In contrast, glucose does appear to have a stimulatory e f f e c t on breathing a c t i v i t y i n the human fetus (Natale et a l . . 1978). Acidemia produced by HC1 or NH Cl infusion to f e t a l lambs r e s u l t s i n stimulation of f e t a l breathing movements (Molteni et a l . , 1980; Hohimer and Bissonette, 1981). However, the degree and duration of acidosis required to i n i t i a t e t h i s change are not c l e a r . The e f f e c t s of epinephrine on f e t a l breathing a c t i v i t y 180 are extremely variable. Some studies report variable increases i n f e t a l breathing a c t i v i t y (Boddy and Dawes, 1975; Jones and Ritchie, 1978) while others do not (Jansen et a l . f 1986). Isoproterenol appears to be a potent stimulator of f e t a l breathing a c t i v i t y i n f e t a l lambs (Boddy and Dawes, 1975), however, Jansen et a l . (1986) found that the response could only be evoked during REM sleep. Jansen et a l . , (1986) also reported that while bolus doses of isoproterenol provided a powerful stimulus to breathing a c t i v i t y , infusion of isoproterenol (1 ug/min) resulted i n only inconsistent and unsustained increases i n breathing a c t i v i t y . An attenuation of response was suggested for the l a t t e r case. The r i s e i n f e t a l breathing a c t i v i t y i n response to bolus isoproterenol administration was abolished by propranolol blockade, suggesting the involvement of a beta-receptor mediated mechanism (Jansen et a l . , 1986). None of the above mentioned studies suggest that f e t a l breathing a c t i v i t y should f a l l during beta-adrenergic stimulation with r i t o d r i n e , i n f a c t the opposite e f f e c t would seem more l i k e l y . However, the development of hypoxemia may have been p a r t l y responsible for the observed behavioral changes. Hypoxemia i n the f e t a l lamb i s associated with a reduction i n the incidence of f e t a l breathing movements (Boddy et a l • , 1974; Maloney et a l . , 1975; Clewlow et a l . , 1983; Sameshima and Koos, 1986; Koos e t _ a l . , 1987 a&b; Moore et a l . , 181 1989). Hypoxia may (Boddy, 1974; Clewlow, 1983; Moore et a l . , 1989) or may not (Adamson, 1984; Sameshima and Koos 1986; Koos et a l . , 1987 a&b) reduce the incidence of low voltage e l e c t r o c o r t i c a l a c t i v i t y . Hypoxia also may (Boddy et a l . , 1974; Clewlow et a l . r 1983; ) or may not (Adamson et a l . , 1984; Sameshima and Koos, 1986; Koos et a l . , 1987 a&b) increase the frequency at which f e t a l ECoG a c t i v i t y switches from the low to high voltage state. The c o n f l i c t i n g r e s u l t s may r e f l e c t a dependency of these behavioral changes on the severity of the hypoxemia and on whether or not metabolic acidemia i s present. The incidence of REM (Natale et a l . , 1981; Sameshima and Koos, 1986; Koos et a l . , 1987 a&b) and f e t a l body movements (Natalie et a l . f 1981) are also reduced during hypoxemia. Studies investigating the e f f e c t s of long term moderate hypoxemia on f e t a l behavior indicate that r e s u l t i n g behavioral changes are reversed over time suggesting an a b i l i t y of the fetus to adapt to long term reductions i n blood oxygen content (Bocking et a l . f 1988; Koos et a l . , 1988). Studies investigating the effects of isocapnic hypoxemia in f e t a l lambs have shown that reductions i n f e t a l breathing and eye a c t i v i t y occur when a r t e r i a l Po 2 or Co2 values f a l l by greater than 5.9 mm Hg or 0.92 mM respectively (Koos et a l . , 1987a). Studies investigating the e f f e c t s of anemia i n f e t a l lambs (induced by isovolemic exchange of f e t a l blood 182 with plasma) have shown that breathing and eye a c t i v i t y f a l l when Co 2 (Po 2 constant) decline by 1.14 mM (Koos et a l . , 1987b). In the current study, f e t a l a r t e r i a l Po2 and Co 2 f e l l by 4.9 mmHg and 1.6 mM respectively. Based on the threshold values reported by Koos et a l . (1989 a&b) the decline i n f e t a l breathing a c t i v i t y observed i n the current study may have occurred as a r e s u l t of hypoxemia. The i n h i b i t o r y e f f e c t s of hypoxemia on f e t a l breathing a c t i v i t y may have overwhelmed any other factors which would normally have stimulated breathing a c t i v i t y (eg. acidemia or beta-adrenergic stimulation). This explanation i s supported by the observations of Jansen et a l . (1986) who found that the stimulatory e f f e c t s of isoproterenol on f e t a l breathing a c t i v i t y could not be evoked during hypoxemia. They concluded that the i n h i b i t o r y e f f e c t s of hypoxemia on f e t a l breathing a c t i v i t y were more powerful than the stimulatory e f f e c t s of isoproterenol. Hypoxemia-induced declines i n f e t a l breathing movements, low ECoG a c t i v i t y and REM sleep may occur through reduced brain oxygenation (Koos et a l . , 1987b). While the i n i t i a l response to hypoxemia i s to increase blood flow to the v i t a l organs (brain, heart and adrenal gland) (Cohn et a l . , 1974; Peeters et a l . , 1979; Sheldon e t _ a l . , 1979; Lampe et a l . , 1988; Jansen, 1989; Rurak et a l . , 1990b), there i s a l i m i t to which increases i n blood flow can compensate for decreases i n blood Po and oxygen content. Eventually the fetus resorts to 183 other adaptive measures to conserve oxygen, including a reduction i n oxygen consumption. This i s accomplished i n part, through reduced physical a c t i v i t y (eg. reducing breathing a c t i v i t y , body movements, REM sleep and low voltage ECoG a c t i v i t y ) (Boddy et a l . , 1974; Maloney et a l . , 1975; Natale et a l . , 1981; Clewlow et a l . , 1983; Sameshima and Koos, 1986; Koos et a l . , 1987 a&b; Moore et a l . , 1989). 4.10 C l i n i c a l Relevance The purpose of the current study was to provide information on the ri s k - b e n e f i t r a t i o for the use of r i t o d r i n e i n human t o c o l y s i s . Data regarding the eff e c t s of r i t o d r i n e i n the human fetus are lacking and t h i s i s large l y due to the technical and e t h i c a l constraints associated with pregnancy related research. The a p p l i c a b i l i t y of the current study to the human s i t u a t i o n may be questionable due to species differences. However, lack of an alternative to animal studies suggest that the information provided be considered seriously, but objectively. The current study was designed to be as relevant to the human s i t u a t i o n as possible. A chronic, r e l a t i v e l y undisturbed animal preparation was employed and r i t o d r i n e was infused i n a manner so as to obtain f e t a l plasma leve l s that were comparable to those of human fetuses exposed to r i t o d r i n e t o c o l y s i s . 184 The current study has demonstrated that r i t o d r i n e i n f u s i o n to f e t a l lambs r e s u l t s i n s i g n i f i c a n t p h y s i o l o g i c a l and behavioral changes the fetus. Similar d e t e r i o r a t i o n i n human fetuses exposed to r i t o d r i n e t o c o l y s i s are r a r e l y reported which may indicate that human fetuses are better able to cope with r i t o d r i n e than f e t a l lambs. However, assessment of the human neonate at b i r t h may not adequately represent the in utero state of the fetus. The profound cardiovascular and respiratory changes which occur at b i r t h may i n f a c t mask any e a r l i e r ritodrine-induced perturbations. Overall, the e f f e c t s of r i t o d r i n e would appear to put the fetus at r i s k , p a r t i c u l a r l y i n situations where f e t a l oxygen de l i v e r y i s already reduced, as i n various states of compromised pregnancy. Previous studies have indicated that the response to r i t o d r i n e i n f e t a l lambs becomes attenuated a f t e r prolonged infusion (Basset et a l . . 1989). Such adaptation may appear b e n e f i c i a l i n protecting the fetus from harmful r i t o d r i n e -induced perturbations. However, many adaptive responses c r u c i a l to early neonatal survival occur through beta-receptor mediated mechanisms (eg. l i p i d and glycogen mobilization). I n h i b i t i o n of these processes through an attenuation of beta-adrenergic responsiveness may put the newborn at r i s k at b i r t h , e s p e c i a l l y when the infant i s delivered shortly a f t e r t o c o l y s i s . 185 5.0 SUMMARY AND CONCLUSIONS Ritodrine was infused continuously into a f e t a l t a r s a l vein at a rate of 2.6 ug/minute for a period of 8, 12 or 24 hours. Samples were taken simultaneously at predetermined time periods from a f e t a l femoral artery, umbilical vein, maternal femoral artery and uterine vein for the analysis of f e t a l and maternal a r t e r i a l and umbilical and uterine venous blood gases, acid-base status, hematocrit, r i t o d r i n e concentration, uterine and umbilical blood flow, and glucose, lactate and oxygen concentrations and fluxes. Cardiovascular and behavioral variables were also monitored continuously. The infusion of r i t o d r i n e to the fetus resulted i n a number of f e t a l and maternal physiological perturbations. 1. The average concentration of r i t o d r i n e i n f e t a l a r t e r i a l plasma was 20.0 ± 2.7 ng/ml (range 9.5 to 34.7 ng/ml) at the end of the infusion. This concentration i s within the range of cord levels obtained i n r i t o d r i n e exposed human fetuses at b i r t h (7 to 79 ng/mL). 2. While a l l f e t a l lambs were affected by the infusion of r i t o d r i n e , some fetuses appeared to be more tolerant to a given drug l e v e l than others. 186 3. Fetal heart rate and glucose lev e l s were p o s i t i v e l y correlated with r i t o d r i n e concentration, however, the response to a given drug l e v e l varied considerably. 4. Fetal a r t e r i a l plasma concentrations of r i t o d r i n e at 8 hours post-infusion were s t i l l s u f f i c i e n t l y elevated to exert considerable f e t a l e f f e c t s . 5. Ritodrine infusion resulted i n many t y p i c a l beta-adrenergic receptor mediated responses i n the fetus, including elevations i n f e t a l a r t e r i a l plasma glucose and l a c t a t e concentrations. 6. The r i s e i n f e t a l a r t e r i a l plasma glucose concentration was associated with an increase i n f e t a l glucose d e l i v e r y , a decrease i n the umbilical veno-arterial glucose concentration difference and a tendency for f e t a l glucose uptake to decline. The data suggest that f e t a l endogenous glucose production rose, perhaps through beta-2 receptor mediated increases i n hepatic and s k e l e t a l muscle glycogenolysis. 7. The r i s e i n f e t a l a r t e r i a l plasma lactat e concentration was associated with an increase i n f e t a l lactate d e l i v e r y , a s l i g h t r i s e i n the umbilical veno-arterial plasma lactate concentration difference and a tendency for f e t a l l a c t a t e uptake to increase. The data suggest that the r i s e i n 187 umbilical a r t e r i a l lactate concentration was balanced by a concurrent r i s e i n the placental production and de l i v e r y of lactate to the fetus. Possible endogenous mechanisms through which f e t a l lactate l e v e l s rose include increased g l y c o l y s i s , elevated l i p o l y s i s , trapping of lactate within the fetus, and reduced tissue oxygen supply. 8. Fetal oxygen consumption increased progressively during the infusion of r i t o d r i n e , remaining s i g n i f i c a n t l y elevated beyond 8 hours post-infusion. The r i s e i n f e t a l oxygen consumption l i k e l y r e f l e c t e d an accelerated rate of f e t a l oxidative metabolism induced by elevated plasma l e v e l s of glucose, lactate and i n s u l i n i n the f e t a l lamb. 9. The r i s e i n f e t a l oxygen consumption was not compensated for by a r i s e i n umbilical blood flow. Therefore umbilical oxygen delivery was not increased to meet the additional oxygen demands of the fetus. The increase i n f e t a l oxygen consumption was thus accomplished through a r i s e i n f e t a l oxygen extraction. 10. The progressive increase i n f e t a l oxygen extraction resulted i n a decline i n f e t a l a r t e r i a l Po, and C» content 2 2 values. Umbilical venous Po and O content values also f e l l 2 2 which may i n part r e f l e c t the decline i n uterine venous Po . 188 11. The progressive decline i n umbilical venous 0 2 content resulted i n a f a l l i n 0 2 delivery. This led to further increases i n f e t a l oxygen extraction which resulted i n additional declines i n f e t a l a r t e r i a l 0 2 content and Po2 values. 12. The development of acidemia i n the fetus also l i k e l y contributed to the progressive f a l l i n f e t a l blood 0 2 content and Po2 values through a rightward s h i f t of the oxyhemoglobin d i s s o c i a t i o n curve. The acidemia appeared to be e n t i r e l y metabolic i n nature, r e s u l t i n g from the accumulation of l a c t i c acid i n f e t a l blood. 13. Uterine oxygen consumption tended to r i s e throughout the infusion of r i t o d r i n e , however, t h i s change was not s i g n i f i c a n t . The elevation l i k e l y r e f l e c t e d the concurrent r i s e i n f e t a l oxygen consumption. Since the r i s e i n uterine and f e t a l oxygen consumption were of s i m i l a r magnitude, i t appeared that uteroplacental oxygen consumption had remained unaltered. 14. The r i s e i n uterine oxygen consumption was not accompanied by a corresponding r i s e i n uterine oxygen delivery, hence uterine oxygen extraction increased s i g n i f i c a n t l y . The r i s e i n uterine oxygen extraction resulted i n a decline i n uterine venous Po and Co values. This was 189 l i k e l y partly responsible for the f a l l i n umbilical venous Po2. 15. Ritodrine infusion did not r e s u l t i n any s i g n i f i c a n t changes i n f e t a l a r t e r i a l pressure, however, s i g n i f i c a n t elevations i n heart rate were observed. The r i s e i n f e t a l heart rate probably occurred through d i r e c t stimulation of myocardial beta-1 adrenergic receptors. These ef f e c t s persisted beyond 8 hours of post-infusion. 16. The incidence of f e t a l breathing movements f e l l s i g n i f i c a n t l y during the infusion of r i t o d r i n e . This f a l l may have occurred i n response to the decline i n f e t a l a r t e r i a l Po 2 and Co2 values. A diurnal rhythm for breathing was also i d e n t i f i e d . 17. The incidence of high voltage ECoG a c t i v i t y rose while that of low voltage ECoG f e l l . These changes were of s i m i l a r magnitude. The incidence of REM also tended to decline, however, t h i s change was not s i g n i f i c a n t . These behavioral changes may also have resulted from the decline i n f e t a l a r t e r i a l Po and Co values. 2 2 18. Fetal hypoxia ( i . e . reduced oxygen consumption) was not evident i n the current study. However, the development of progressive hypoxemia and acidemia could put the fetus at 190 r i s k , p a r t i c u l a r y i n situations where f e t a l oxygen delivery i s already reduced, as i n various states of compromised pregnancy. An attenuation of beta-adrenergic reponsiveness through prolonged r i t o d r i n e infusion may o f f e r the fetus protection from the e f f e c t s of r i t o d r i n e . 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