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Effects of acute moderate hypoxemia on the pharmacokinetics of metoclopramide and its metabolites in… Kim, John 1995

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EFFECTS OF ACUTE MODERATE HYPOXEMIA ON THE PHARMACOKINETICS OF METOCLOPRAMIDE AND ITS METABOLITES IN CHRONICALLY INSTRUMENTED SHEEP b y J O H N K I M B . S c , S i m o n F r a s e r U n i v e r s i t y , 1 9 9 1 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s ) ( D i v i s i o n o f B i o p h a r m a c e u t i c s a n d P h a r m a c o k i n e t i c s ) W e a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A J a n u a r y 1 9 9 5 © J o h n K i m , 1 9 9 5 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. The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T Hypoxemia is known to induce various physiological changes which can result in pharmacokinetic changes. To examine the effect of acute, moderate hypoxemia in metoclopramide (MCP) pharmacokinetics, a continuous infusion [14 hours] of MCP was administered during pre-hypoxemia (2hr), hypoxemia (6hr) and post-hypoxemia (6hr) in non-pregnant sheep. Hypoxemia was achieved by lowering the ewe's inspired O 2 concentration. During the experiment, arterial blood and urine samples were collected. MCP and its mono- and di-deethylated metabolites were measured in these fluid samples using a gas chromatography-mass selective detector (GC-MSD) method. Steady-state concentrations of MCP were achieved in each of the three periods. During hypoxemia, MCP plasma steady-state concentration increased significantly from 50.72 ± 1.06 to 63.62 ± 1.79 ng/mL, and later decreased to 55.83 ± 1.15 ng/mL during the post-hypoxemic recovery period. Plasma mdMCP concentration (32.78 ± 1.73 ng/mL) also increased, compared to the control group (21.20 ± 1.39 ng/mL), during hypoxemia and the subsequent normoxemic period. Renal excretion of MCP and its metabolites significantly decreased during hypoxemia. Increased urine flow with decreased urine osmolality was also observed. Thus the results indicate that acute, moderate hypoxemia affects MCP pharmacokinetics. Ul T A B L E O F C O N T E N T S ABSTRACT ii T A B L E OF CONTENTS iii LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS x LIST OF SCHEMES xiii ACKNOWLEDGMENTS xiv 1. INTRODUCTION 1 1.1. Pharmacology and Clinical Use 1 1.2. Pharmacokinetics of Metoclopramide 3 1.3. Hypoxemia and Associated Physiological Changes 7 1.3.1 Hypoxemia and hypoxia 7 1.3.2 Physiological changes during hypoxemia 8 1.4. Respiratory Disorder and Drug Disposition and Metabolism 13 1.4.1. Effects of acute and chronic hypoxemia on drug metabolism 14 1.4.2. Effects of hypoxemia on hepatic drug metabolism 15 1.4.3. Effects of hypoxemia on drug disposition and protein binding 16 1.4.4. Digoxin kinetics in hypoxemia 18 1.4.5. Theophylline kinetics and respiratory disorders 19 1.4.6. Lidocaine kinetics/metabolism during hypoxemia and similarity with MCP 21 1.5. Rationale 23 1.6. Objectives 24 iv 2. EXPERIMENTAL 25 2.1. Materials and Supplies 25 2.1.1. Chemicals 25 2.1.2. Reagents 25 2.1.3. Enzymes 26 2.1.4. Solvents 26 2.1.5. Gases 26 2.1.6. Supplies for animal studies 27 2.2. Stock and Reagent Solutions 27 2.3. Sample Preparation, Extraction and Derivatization 28 2.3.1. Sample extraction 28 2.3.2. Analysis of glucuronide and sulphate conjugates 31 2.4. Standard Curve Preparation 32 2.5. Instrument and Equipment 32 2.5.1. Gas chromatography 3 2 2.5.2. Operating conditions for the GC and MSD 33 2.6. Animal Preparation 34 2.6.1. Animal handling 34 2.6.2. Surgical preparation 34 2.7. Experiment Protocol 36 2.8. Recording Procedures and Analysis of Physiological Parameters 39 2.9. Data Analysis 40 2.9.1. Determination of steady-state drug concentration 40 2.9.2. Calculation of pharmacokinetic parameters 41 2.9.3. Statistical tests 42 V 3. RESULTS 43 3.1. Quantitative Analytical Assay Methods for Metoclopramide and Selective Metabolites 43 3.1.1. GC-MSD methods for the quantitative analysis of MCP and selective metabolites 43 3.1.2. Calibration (standard) curve 46 3.1.3. Enzyme incubation 46 3.2. Physiological Changes Associated with Hypoxemia 51 3.2.1 Arterial blood gas status and pH 52 3.2.2. Arterial blood lactate and glucose concentration 56 3.2.3. Plasma electrolyte concentration, urine flow, osmolality and pH and renal osmolal excretion rate 60 3.2.4. Mean arterial blood pressure and heart rate 66 3.3 Metoclopramide Pharmacokinetics following i.v. Infusion to Steady-State and Induction of Hypoxemia 67 3.3.1. Steady-state plasma MCP concentration and total body clearance (TBC) during normoxemia and hypoxemia 67 3.3.2. Plasma mdMCP concentration during hypoxemic and normoxemic MCP steady-state 75 3.4. Renal Excretion of MCP and its Metabolites Following the i.v. Infusion to Steady-state and Induction of Hypoxemia 77 3.4.1. Renal clearance of MCP and its metabolites during normoxemia and hypoxemia 77 3.4.2. Fractional renal excretion constants of MCP and its metabolites 82 4. DISCUSSION 87 4.1. GC-MSD Method of Analysis of Metoclopramide and Selected Metabolites 87 4.2. Physiological Changes associated with Hypoxemia 4.2.1. Induction of hypoxemia and blood gas changes 4.2.2. Blood pH and lactate concentration during hypoxemia 89 89 91 vi 4.2.3. Adrenosympathetic system and blood glucose level during hypoxemia 93 4.2.4. Urine flow and osmolality during hypoxemia and MCP infusion 95 4.3. Statistical and Practical Aspects of Experiment Design 99 4.3.1. Designof experimental protocol 99 4.3.2. Statistical and practical considerations for determining the steady-state drug concentration 103 4.3.3. Theoretical aspects of infusion with loading dose 106 4.4. Metoclopramide Pharmacokinetics following i.v. Infusion to Steady-State and Induction of Hypoxemia 111 4.4.1. Steady-state plasma MCP concentratioxi and total body clearance (TBC) during normoxemia and hypoxemia 111 4.4.2. Plasma mdMCP concentration during normoxemia and hypoxemia 114 4.4.3. Renal excretion of MCP and its metabolites during normoxemia and hypoxemia 116 5. S U M M A R Y AND CONCLUSIONS 120 6. REFERENCES 121 APPENDIX A 137 LIST O F T A B L E S Table 1. Weighted calibration curve data (mean peak area ratio ± SD) for ddMCP, mdMCP and MCP in plasma 49 Table 2. Weighted calibration curve data (slope, intercept and r2) for ddMCP, mdMCP and MCP of enzyme incubation study in urine 50 Table 3. Identification and weight of ewes, samples collected and nitrogen /MCP infusion. 51 Table 4. Mean (± SEM) arterial blood pH, gas partial pressure (PaCCte and PaCte), bicarbonate (HCCb") concentration and base excess. 54 Table 5. Mean (± SEM) blood lactate concentration (mmol/L). 59 Table 6. Mean (± SEM) blood glucose concentration (mmol/L). 59 Table 7. Mean (± SEM) plasma electrolyte concentration, haemoglobin content and urine flow, pH and osmolality. 65 Table 8. Mean (± SEM) heart rate (beats/min). 66 Table 9. Mean (± SEM) arterial blood pressure (mm Hg). 67 Table 10. Mean (± SEM) steady-state plasma MCP concentration (ng/mL). 69 Table 11. Plasma MCP total body clearance (TBC). . 74 Table 12. Mean (± SEM) plasma mdMCP concentrations during the MCP infusion (ng/mL). 76 Table 13a. MCP renal clearance (L/h)1 [CLrenai(Mcp)] 80 Table 13b. MCP renal clearance (L/h)2 [CLrenamCp)] 80 Table 14a. mdMCP renal clearance (L/h)1 [CLrenai(mdMcp)] 81 Table 14b. mdMCP renal clearance (L/h)2 [CLrenai(mdMCP)] 81 Table 15. MCP fractional renal excretion constants \fu(MCP)] 85 Table 16. mdMCP fractional renal excretion constants \fU(mdMCP)] 86 Table 17. ddMCP fractional renal excretion constants \fU(ddMCP)] 86 viii LIST OF FIGURES Figure 1. Representative total ion chromatograms (SIM mode) of 1 ng/mL of ddMCP, mdMCP and MCP, and 33.3 ng/mL of B M Y spiked in 0.3 mL of plasma. Superimposed chromatograms of blank plasma and urine are also shown. 44 Figure 2. Mass spectra of ddMCP (A), mdMCP (B), M C P (C) and the internal standard B M Y (D) obtained in S C A N mode, showing the molecular ions (M +) and the ions used for quantitative analysis. 45 Figure 3. Representative calibration (weighted) curves for MCP, mdMCP and ddMCP from spiked plasma, [mean ± SD] 48 Figure 4a . Mean (± SEM) arterial blood pH and gas partial pressure ( P a C 0 2 and Pa02) over the duration of the experiment [experimental group]. 55 Figure 4b. Mean (± SEM) arterial blood pH and gas partial pressure ( P a C 0 2 and Pa02) over the duration of the experiment [control group]. 56 Figure 5a. Mean (± SEM) arterial blood lactate and glucose (mmol/L) over the duration of the experiment [experimental group]. 58 Figure 5b. Mean (± SEM) arterial blood lactate and glucose (mmol/L) over the duration of the experiment [control group]. 58 Figure 6a. Mean (± SEM) urine flow (mL/h) and osmolality (mOsm/Kg) over the duration of the experiment [experimental group] 62 Figure 6b. Mean (± SEM) urine flow (mL/h) and osmolality (mOsm/Kg) over the duration of the experiment [control group] 62 Figure 7. Correlation of osmolality (mOsmol/Kg) and urine flow (mL/h). Correlation coefficient (r)=0.80 63 Figure 8a. Mean (± SEM) plasma M C P and mdMCP concentration (ng/mL) over the duration of the experiment [experimental group]. 70 Figure 8b. Mean (± SEM) plasma MCP and mdMCP concentration (ng/mL) over the duration of the experiment [control group]. 71 Figure 9. Representative plot of the accumulated drug (or metabolite) in urine (ZDu) versus A U C . The slope of the curve represents the renal clearance [L/h] 79 Figure 10. Representative plot of the accumulated drug (or metabolite) in urine (ZDu) versus time (h). The slope of the curve represents the product of the fractional renal excretion rate constant for MCP (or metabolite) and the infusion rate constant of MCP. 84 Figure 11. Simulation of Cp after simple i.v. infusion without loading dose using pharmacokinetic parameters from Riggs et al. (1989). 108 Figure 12. Simulation of Cp after i.v. infusion with loading dose (one-compartment model). 108 Figure 13. Simulation of Cp after i.v. infusion with loading dose (two-compartment model) using pharmacokinetic parameters from Riggs et al. (1989). 109 LIST OF ABBREVIATIONS <|> determining power rj 2 population variance A.R.E. amount remained to be excreted ACS American Chemical Society A N F atrial natriuretic factor A N O V A analysis of variance A U C area under the curve A V P arginine vasopressin B M Y 4-amino-5-chloro-2(2-butanone-3-yl)-oxy-N,N-diethylaminoethyl benzamide BSP sufobromophthalein Clt total body clearance COPD chronic obstructive pulmonary disease Css apparent arterial steady-state concentration C V coefficient of variation ddMCP N,N-dideethyl-metoclopramide Du accumulated drug/metabolite in urine EI electron impact fu fractional renal metabolite elimination constant fm metabolite formation fraction constant fmu renal metabolite elimination fraction constant /„ fractional renal elimination constant GC-ECD gas chromatography-electron capture detector GC-MSD gas chromatography-mass selective detector GFR glomerular filtration rate G X glycinexylidide HC1 hydrochloric acid HCO3" bicarbonate HFB heptafluorobutyryl HFBA heptafluorobutyric anhydride I.D. inner diameter ICG Indocyanine Green kio apparent drug elimination rate constant from the central compartment ki2, k2i apparent first order inter-compartmental distribution rate KE apparent first order drug elimination rate constant kf metabolite formation rate constant KmO*2 Michaelis affinity constant of oxygen kmu renal metabolite elimination rate constant ka infusion rate ku renal elimination rate constant LD i.v. bolus loading dose LOQ limit of quantitation M A maternal arterial blood sample MB amount of metabolite in the body MCP metoclopramide mdMCP N-monodeethyl-metoclopramide M E G X N-monoethyl-glycinexylidide MSD mass selective detector NaOH sodium hydroxide PaCG*2 arterial carbon dioxide partial pressure PaG"2 arterial oxygen partial pressure PO2 oxygen partial pressure PTFE polytetrafluorethylene r coefficient of determination r 2 coefficient of correlation SCAN mass scanning mode SD standard deviation S E M standard error of the mean SIM selective ion monitoring mode SMZ sulphamethazine TBC total body clearance T E A triethylamine TRIS 2-amino-2-hydroxymethyl-1,3-propanediol UHP ultra-high purity UR urine total collection V c apparent volume of distribution of the central compartment v d apparent volume of distribution X B amount of drug in the body xi i i L I S T O F S C H E M E S Scheme I Comparison of N-deethylation reactions of MCP and lidocaine. 22 Scheme II Extraction procedure for MCP and its metabolites. 30 Scheme LTJ Schematic diagrams of the experimental protocol 37 Scheme IV MCP infusion-hypoxemia sampling protocol 38 Scheme V Diagram of metabolic and renal elimination of a drug 82 Scheme VI . Diagrams of the compartmental model used for computer simulation. 107 X I V ACKNOWLEDGMENTS I would like to express my sincere appreciation and thanks to my research supervisors Drs. James E. Axelson and Dan W. Rurak, for their guidance, encouragement and support. Special thanks to Dr. Wayne Riggs for his warm support and friendship throughout the program. Thanks to Drs. Frank Abbott, Joan Douglas, Don Lyster, Keith McErlane for their advice and guidance throughout my study. I would like to thank to Mr. Eddie Kwan and Ms. Caroline Hall for their technical assistance in animal studies. I would also like to thank to Mr. George Tonn and Sanjeev Kumar for their critical evaluation, support and friendship. Thanks to all the graduate students in the laboratory, particularly Dr. Matthew Wright, Dr. Swamy Yeleswaram, Ms. Judit Orbay for their friendship and help. A special thanks to my family for their support and understanding. I greatly appreciate the financial support by Medical Research Council of Canada and British Columbia Medical Services Summer Scholarship. 1 1. INTRODUCTION 1.1 Pharmacology and Clinical Use M e t o c l o p r a m i d e ( M C P ) , 4 - a m i n o - 2 - m e t h o x y - 5 - c h l o r o - N - ( 2 - d i e t h y l a m i n o e t h y l ) b e n z a m i d e , i s a p o t e n t a n t i e m e t i c a n d g a s t r i c m o t i l i t y m o d i f i e r t h a t h a s b e e n u s e d t o t r e a t n a u s e a a n d v o m i t i n g a s s o c i a t e d w i t h u r e m i a a n d t o r e d u c e t h e i n c i d e n c e o f v o m i t i n g a n d p u l m o n a r y a s p i r a t i o n a s s o c i a t e d w i t h e m e r g e n c y a n a e s t h e s i a ( D a v i e s a n d H o w e l l s , 1 9 7 3 ; D u n d e e et al, 1 9 7 4 ) . M C P i s s t r u c t u r a l l y r e l a t e d t o p r o c a i n a m i d e , b u t i s d e v o i d o f a n t i a r r h y t h m i c o r l o c a l a n a e s t h e t i c a c t i v i t y a t a n t i e m e t i c c l i n i c a l d o s e s ( H a r r i n g t o n et al, 1 9 8 3 ) . H o w e v e r , M C P - e l i c i t e d p r o l a c t i n s e c r e t i o n i n c r e a s e a n d e x t r a p y r a m i d a l s y m p t o m s h a v e b e e n r e p o r t e d ( P i n d e r et al, 1 9 7 6 ) . T h e p h a r m a c o l o g i c a l a c t i o n s o f M C P a r e m o s t p r o n o u n c e d i n t h e g a s t r o i n t e s t i n a l t r a c t w h e r e g e n e r a l i z e d i n c r e a s e s i n m o t i l i t y a r e s e e n a f t e r o r a l a n d i . v . a d m i n i s t r a t i o n ( P i n d e r et al, 1 9 7 6 ; S c h u l t z e - D e l r i e u , 1 9 8 1 ) . / C 2 H 5 H 2 N >f J— C O - N H - C H 2 - C H 2 - N N X = < C 2 H 5 O C H 3 M e t o c l o p r a m i d e H2N—if V — C O - N H - C H 2 - C H 2 - N X W C 2 H 5 P r o c a i n a m i d e 2 M e t o c l o p r a m i d e i s e f f e c t i v e i n r e d u c i n g p o s t o p e r a t i v e v o m i t i n g ( L i n d a n d B r e i v i k , 1 9 7 0 ) . A t h i g h d o s e s ( 5 d o s e s o f 2 m g / K g a d m i n i s t e r e d i n t r a v e n o u s l y o v e r 8 h o u r s ) , M C P i s e f f e c t i v e i n r e d u c i n g t h e n a u s e a a n d v o m i t i n g a s s o c i a t e d w i t h t h e u s e o f c i s p l a t i n u m ( G r a l l a et al, 1 9 8 1 ) a n d o t h e r c h e m o t h e r a p e u t i c a g e n t s ( S t r u m et al, 1 9 8 4 ) . M C P a l s o i n c r e a s e s l o w e r o e s o p h a g e a l s p h i n c t e r t o n e , o e s o p h a g e a l p e r i s t a l s i s a n d g a s t r i c e m p t y i n g , a l l o f w h i c h w o u l d r e l i e v e p a t i e n t s w i t h g a s t r o - o e s o p h a g e a l r e f l u x ( B r i g h t -A s a r e a n d E l - B a s s o u s s i , 1 9 8 0 ; W i n n a n et al, 1 9 8 0 ) . M C P r e d u c e s t h e n a u s e a a s s o c i a t e d w i t h m i g r a i n e a n d i n c r e a s e s t h e r a t e o f a b s o r p t i o n o f o r a l a n a l g e s i c a g e n t s , i n c l u d i n g a s p i r i n ( M a t t s , 1 9 7 4 ; V o l a n s , 1 9 7 8 ) . M e t o c l o p r a m i d e a l s o s i g n i f i c a n t l y r e d u c e s t h e e m e s i s o f e a r l y p r e g n a n c y ( S i n g h a n d L e a n , 1 9 7 0 ) a n d d u r i n g l a b o u r ( M c G a r r y , 1 9 7 1 ; V e l l a et al, 1 9 8 5 ) . I t s i g n i f i c a n t l y i n c r e a s e s t h e g a s t r i c e m p t y i n g r a t e o f w o m e n i n l a b o u r ( H o w a r d a n d S h a r p , 1 9 7 3 ) a n d i n c r e a s e s t h e t o n e o f t h e l o w e r e s o p h a g e a l s p h i n c t e r , w h i c h i s i m p a i r e d d u r i n g p r e g n a n c y a n d l a b o u r ( B r o c k - U t n e et al, 1 9 7 8 ) . T h e s e a c t i o n s a n d t h e a b i l i t y t o d i m i n i s h t h e f r e q u e n c y o f v o m i t i n g d u r i n g l a b o u r h a v e l e d t o i t s m o r e f r e q u e n t u s e i n e l e c t i v e a n d e m e r g e n c y o b s t e t r i c a l a n a e s t h e s i a ( C o h e n et al, 1 9 8 4 ; S h a u g h n e s s y , 1 9 8 5 ) . M C P a l s o p r e v e n t s n a u s e a a n d v o m i t i n g a s s o c i a t e d w i t h e p i d u r a l a n a e s t h e s i a d u r i n g e l e c t i v e C a e s a r e a n s e c t i o n ( C h e s t n u t et al, 1 9 8 7 ) . T h e a n t i e m e t i c e f f e c t s o f M C P a r e t h o u g h t t o b e m e d i a t e d t h r o u g h b o t h p e r i p h e r a l ( g a s t r o i n t e s t i n a l ) a n d c e n t r a l s i t e s . T h e d r u g i s b e l i e v e d t o r a i s e t h e t h r e s h o l d o f t h e c h e m o r e c e p t o r t r i g g e r z o n e f h r o u g h a n t a g o n i s m o f d o p a m i n e r e c e p t o r s , a n d t o d e c r e a s e t h e s e n s i t i v i t y o f v i s c e r a l n e r v e s w h i c h t r a n s m i t 3 a f f e r e n t i m p u l s e s f r o m t h e g a s t r o i n t e s t i n a l t r a c t t o t h e e m e t i c c e n t r e i n t h e l a t e r a l r e t i c u l a r f o r m a t i o n ( P i n d e r et al, 1 9 7 6 ) . A d e c r e a s e i n r e n a l p l a s m a f l o w o f a b o u t 2 0 % h a s b e e n r e p o r t e d i n o n c o l o g y p a t i e n t s r e c e i v i n g h i g h d o s e s o f M C P ( 1 - 2 . 5 m g / k g ) ( I s r a e l et al, 1 9 8 6 ) , a n d a l s o a d e c r e a s e i n h e p a t i c b l o o d f l o w w a s n o t e d i n t h e r a t a t M C P d o s e s o f > 2 5 m g / k g , r e s u l t i n g i n d o s e - d e p e n d e n t k i n e t i c s ( T a r n et al, 1 9 8 1 a ) . M C P a l s o s t i m u l a t e s t h e r e l e a s e o f v a r i o u s h o r m o n e s i n m a n a n d i n a n i m a l s . P r o l a c t i n r e l e a s e w a s o b s e r v e d i n t h e r a t , h e a l t h y a d u l t s , p r e g n a n t w o m e n , c h i l d r e n ( H a r r i n g t o n et al, 1 9 8 3 ; A r v e l a et al., 1 9 8 3 ) , n e o n a t e s ( R u p p e r t et al, 1 9 8 6 ) a n d p r e g n a n t e w e s ( F i t z g e r a l d a n d C u n n i n g h a m , 1 9 8 2 ) . T h i s e f f e c t i s l i k e l y m e d i a t e d b y a n t a g o n i s m o f t h e d o p a m i n e - m e d i a t e d i n h i b i t i o n o f p r o l a c t i n s e c r e t i o n b y t h e p i t u i t a r y o r h y p o t h a l a m u s . A n i n c r e a s e d l e v e l o f p l a s m a a l d o s t e r o n e w a s r e p o r t e d i n r a t s , a n d m a n ( H a r r i n g t o n et al, 1 9 8 3 ; S o m m e r s et al, 1 9 8 8 ; V o n m o o s et al, 1 9 9 0 ) a s a r e s u l t o f i n h i b i t i o n o f c e n t r a l d o p a m i n e r e c e p t o r s ( A l b i b i a n d M c C a l l u m , 1 9 8 3 ) o r a c e t y l c h o l i n e r e l e a s e f r o m p o s t - g a n g l i o n i c c h o l i n e r g i c n e r v e t e r m i n a l s w i t h i n t h e a d r e n a l c o r t e x ( S o m m e r s et al, 1 9 8 8 ) . S t i m u l a t i o n o f v a s o p r e s s i n s e c r e t i o n h a s a l s o b e e n r e p o r t e d i n h e a l t h y h u m a n v o l u n t e e r s ( N o r b i a t o et al, 1 9 8 6 ) . 1.2. Pharmacokinetics of Metoclopramide A t w o - c o m p a r t m e n t m o d e l a d e q u a t e l y d e s c r i b e s t h e d i s p o s i t i o n o f M C P f o l l o w i n g i . v . a d m i n i s t r a t i o n i n h u m a n s ( B a t e m a n et al, 1 9 8 0 ) . T h e d r u g i s r a p i d l y d i s t r i b u t e d w i t h a r e l a t i v e l y h i g h v o l u m e o f d i s t r i b u t i o n i n h u m a n s ( 2 . 2 t o 3 . 4 L / K g ) i n d i c a t i n g e x t e n s i v e e x t r a v a s c u l a r d i s t r i b u t i o n , w h i c h m a y b e e x p e c t e d f o r a l i p i d - s o l u b l e , b a s i c d r u g . R a p i d p l a c e n t a l t r a n s f e r o c c u r s i n m a n ( B y l s m a - H o w e l l et al, 1 9 8 3 ; C o h e n et al, 1 9 8 4 ) a n d i n s h e e p ( R i g g s et al, 1 9 8 8 ; 1 9 8 9 ) . M C P i s e x t e n s i v e l y m e t a b o l i s e d via s u l p h a t e a n d g l u c u r o n i d e c o n j u g a t i o n i n r a b b i t a n d h u m a n s ( A r i t a et al, 1 9 7 0 ; C o w a n et al, 1 9 7 6 ; B a t e m a n et al, 1 9 8 0 ) , a n d b y O - d e m e t h y l a t i o n , N - d e e t h y l a t i o n a n d a m i d e h y d r o l y s i s i n r a b b i t , r a t , a n d d o g ( A r i t a et al, 1 9 7 0 ; B a k k e a n d S e g u r a , 1 9 7 6 ; C o w a n et al, 1 9 7 6 ) . T h e e l i m i n a t i o n h a l f - l i f e i s = 1 2 0 m i n i n t h e d o g ( B a t e m a n et al, 1 9 8 0 ) a n d = 5 0 m i n i n t h e r a t w i t h a d o s e d e p e n d e n t i n c r e a s e a t d o s e s e x c e e d i n g 1 5 m g / k g ( T a r n et al, 1 9 8 1 a ) . T h e d o s e d e p e n d e n t k i n e t i c s a t h i g h e r d o s e s a r e l i k e l y d u e t o a l t e r e d h e p a t i c b l o o d f l o w ( T a r n et al, 1 9 8 1 a ) , a n o b s e r v a t i o n c o n f i r m e d u s i n g t h e I n d o c y a n i n e G r e e n ( I C G ) c l e a r a n c e m e t h o d . T h e p l a s m a e l i m i n a t i o n h a l f - l i f e i n n o n - p r e g n a n t , m a t e r n a l , a n d f e t a l s h e e p i s 6 3 ± 1 4 , 8 8 ± 1 7 , a n d 1 1 3 ± 2 9 m i n , r e s p e c t i v e l y ( m e a n ± S E M ) , a f t e r a n i . v . b o l u s d o s e ( R i g g s et al, 1 9 8 8 ) . T h e v o l u m e o f d i s t r i b u t i o n i n s h e e p i s h i g h e r ( 5 . 5 t o 7 . 0 L / K g ) t h a n i n h u m a n s ( 2 . 2 t o 4 . 0 L / K g ) ( R i g g s et al, 1 9 8 8 ) . Hepatic clearance and metabolism : W i t h i n 2 4 h o u r s o f i . v . b o l u s M C P a d m i n i s t r a t i o n i n h u m a n s , a b o u t 8 0 % o f t h e d o s e w a s e x c r e t e d i n t h e u r i n e a s f r e e d r u g ( = 2 5 % o f t h e d o s e ) , g l u c u r o n i d e ( = 2 - 5 % o f t h e d o s e ) a n d s u l p h a t e ( = 5 0 % o f t h e d o s e ) c o n j u g a t e s ( T e n g et al, 1 9 7 7 ; B a t e m a n et al, 1 9 8 0 ) . T h e r a t i o o f c o n j u g a t e s e x c r e t e d f r o m h u m a n s a n d a v a r i e t y o f a n i m a l s i s s i g n i f i c a n t l y d i f f e r e n t , s u g g e s t i n g s i g n i f i c a n t s p e c i e s d i f f e r e n c e s i n M C P m e t a b o l i s m . S u l p h a t e c o n j u g a t i o n a n d g l u c u r o n i c a c i d c o n j u g a t i o n a r e t h e t w o m a j o r m e t a b o l i c p a t h w a y s f o r M C P i n h u m a n s a n d t h e r a b b i t ( C o w a n et al, 1 9 7 6 ) . H o w e v e r , t h e s u l p h a t e a n d g l u c u r o n i d e c o n j u g a t e s o f M C P h a v e n o t b e e n f o u n d i n t h e d o g a n d t h e r a t , w h e r e t h e d e e t h y l a t i o n o f M C P i s t h e d o m i n a n t m e t a b o l i c p a t h w a y ( T e n g et al, 5 1 9 7 7 ) . I n h u m a n s , t h e t o t a l b o d y c l e a r a n c e a p p r o x i m a t e s h e p a t i c p l a s m a f l o w ( 1 1 . 6 1 m L / m i n / K g ) , w i t h r e n a l c l e a r a n c e ( 2 . 6 m L / m i n / K g ) a c c o u n t i n g f o r = 2 0 % o f t o t a l b o d y c l e a r a n c e , s u g g e s t i n g t h e c l e a r a n c e i s f l o w - l i m i t e d r a t h e r t h a n m e t a b o l i c c a p a c i t y - l i m i t e d ( B a t e m a n et al, 1 9 8 0 ) . I n s h e e p , t h e t o t a l b o d y c l e a r a n c e i s c o n s i d e r a b l y h i g h e r ( 8 6 . 7 m L / m i n / K g ) c o m p a r e d t o h u m a n s , w h i c h c o i n c i d e s w i t h t h e h i g h e r h e p a t i c b l o o d f l o w i n s h e e p ( 0 . 5 - 3 . 0 L / m i n ) t h a n h u m a n s ( 0 . 5 - 1 . 5 L / m i n ) . T h e l i v e r i s t h o u g h t t o b e t h e m a j o r s i t e o f M C P e l i m i n a t i o n ( D e s m o n d a n d W a t s o n , 1 9 8 6 ) . T h e e l i m i n a t i o n h a l f - l i f e o f M C P i n h u m a n s i s 2 . 6 t o 4 . 6 h o u r s ( B a t e m a n et al, 1 9 8 0 ; G r a f f n e r et al, 1 9 7 9 ) , w h e r e a s i n s h e e p , i t i s m u c h s h o r t e r ( 1 . 1 - 1 . 6 h o u r s ) ( R i g g s et al, 1 9 8 8 ) . A h i g h t o t a l b o d y c l e a r a n c e i n s h e e p h a s a l s o b e e n o b s e r v e d w i t h d r u g s s u c h a s d i p h e n h y d r a m i n e ( Y o o et al, 1 9 8 6 ) , m e p e r i d i n e ( S z e t o et al, 1 9 7 8 ) , a n d l i d o c a i n e ( B l o e d o w et al, 1 9 8 0 ) . Renal failure : S t u d i e s o f M C P k i n e t i c s i n r e n a l f a i l u r e s h o w e d a s i g n i f i c a n t d e c r e a s e i n t o t a l b o d y c l e a r a n c e ( B a t e m a n et al, 1 9 8 1 ; T a r n et al, 1 9 8 1 b ; W r i g h t et al, 1 9 8 8 ) w i t h a n i n c r e a s e d t e r m i n a l h a l f - l i f e i n b o t h h u m a n s a n d r a t . H o w e v e r , t h e c o n t r i b u t i o n o f r e n a l c l e a r a n c e o f M C P a s i n t a c t d r u g a s a f r a c t i o n o f t o t a l b o d y c l e a r a n c e i s = 2 0 % , t h u s , i t i s n o t e a s y t o e x p l a i n t h e o b s e r v e d 2 - 4 f o l d d e c r e a s e i n t o t a l b o d y c l e a r a n c e n o t e d i n r e n a l f a i l u r e b o t h i n h u m a n s a n d t h e r a t s . A r e n a l f a i l u r e - i n d u c e d c h a n g e i n e x t r a h e p a t i c m e t a b o l i s m w a s p r o p o s e d t o e x p l a i n t h e d e c r e a s e d t o t a l b o d y c l e a r a n c e ( T a r n et al, 1 9 8 1 b ) , h o w e v e r , K a p i l et al. ( 1 9 8 4 ) r u l e d o u t s i g n i f i c a n t e x t r a h e p a t i c m e t a b o l i s m u s i n g t i s s u e m e t a b o l i s m s t u d i e s in vitro. D i m i n i s h e d h e p a t i c M C P m e t a b o l i s m s e c o n d a r y t o r e n a l f a i l u r e w a s a l s o s u g g e s t e d ( B a t e m a n . , 1 9 8 1 ; K a p i l et al, 1 9 8 4 ; W r i g h t et al, 1 9 8 8 ) . W r i g h t et al. ( 1 9 8 8 ) s p e c u l a t e d t h e p r e s e n c e o f a n u n i d e n t i f i e d s u b s t a n c e i n t h e p l a s m a o f 6 u r e m i c p a t i e n t s w h i c h m i g h t i n h i b i t M C P m e t a b o l i s m . A n o t h e r p o s s i b l e e x p l a n a t i o n c o u l d b e t h a t t h e a c c u m u l a t i o n o f m e t a b o l i t e s , i n c l u d i n g g l u c u r o n i d e a n d s u l p h a t e c o n j u g a t e s , m i g h t r e d u c e t h e a c t i v i t y o f t h e m e t a b o l i c e n z y m e s i n v o l v e d i n t h e e l i m i n a t i o n o f t h e d r u g t h r o u g h a n e g a t i v e f e e d b a c k m e c h a n i s m ( S t r y e r , 1 9 8 8 ) . Hepatic cirrhosis : I n c r e a s e d M C P p l a s m a c o n c e n t r a t i o n s a r e o b s e r v e d i n h e p a t i c c i r r h o s i s p a t i e n t s ( H e l l s t e r n et al., 1 9 8 7 ; M a g u e u r et al, 1 9 9 1 ; A l b a n i et ai, 1 9 9 1 ) . A r e d u c t i o n o f f u n c t i o n a l h e p a t i c b l o o d f l o w d u e t o i n t r a - a n d e x t r a - h e p a t i c s h u n t i n g i s t h e l i k e l y c a u s e o f t h e a l t e r e d M C P k i n e t i c s w i t h t h i s c o n d i t i o n ( M a g u e u r et al, 1 9 9 1 ) , a l t h o u g h r e d u c e d h e p a t i c m e t a b o l i s m c a n n o t b e r u l e d o u t . P l a s m a c o n c e n t r a t i o n s o f ox-a c i d g l y c o p r o t e i n , t h e m a j o r b i n d i n g p r o t e i n o f M C P , i s s t a b l e i n c i r r h o s i s ( K r e m e r et al, 1 9 8 8 ) a n d t h e p l a s m a b i n d i n g o f M C P i s r e l a t i v e l y l o w , i.e. = 4 0 % ( W e b b et al, 1 9 8 6 ) . T h e r e f o r e , t h e p r o t e i n b i n d i n g (i.e. f r e e f r a c t i o n ) a n d t i s s u e d i s t r i b u t i o n o f M C P a r e u n l i k e l y t o b e a f f e c t e d . P h a s e J J p a t h w a y s o f m e t a b o l i s m i.e. c o n j u g a t i o n r e a c t i o n s , w h i c h a c c o u n t f o r t h e m a j o r i t y o f M C P e l i m i n a t i o n i n h u m a n s , a r e n o t s i g n i f i c a n t l y a f f e c t e d b y l i v e r d y s f u n c t i o n ( M a g u e u r et al, 1 9 9 1 ) . T h e r e f o r e , f r o m t h e d a t a d e s c r i b i n g M C P k i n e t i c s a s s o c i a t e d w i t h r e n a l a n d h e p a t i c d y s f u n c t i o n , i t i s s u g g e s t e d t h a t d i s e a s e - i n d u c e d c h a n g e s i n M C P k i n e t i c s m a y b e d u e t o a l t e r e d b l o o d f l o w d i s t r i b u t i o n a n d c h a n g e s i n i n t r i n s i c c a p a c i t y f o r d r u g m e t a b o l i s m . 7 1.3. Hypoxemia and Associated Physiological Changes 1.3.1. Hypoxemia and hypoxia H y p o x e m i a i s a r e d u c t i o n i n b l o o d P 0 2 , w h e r e a s h y p o x i a i s a f a l l i n t i s s u e 0 2 u t i l i z a t i o n w h e n t i s s u e o x y g e n d e l i v e r y i s n o t s u f f i c i e n t t o m e e t n o r m a l m e t a b o l i c d e m a n d s . H y p o x i a c a n b e a c o n s e q u e n c e o f s e v e r e h y p o x e m i a , b u t c a n a l s o o c c u r w i t h o u t a r e d u c t i o n i n b l o o d P Q 2 , a s i n t h e c a s e o f c a r b o n m o n o x i d e p o i s o n i n g o r s e v e r e a n e m i a . H y p o x i a i s u s u a l l y a c c o m p a n i e d b y m e t a b o l i c / l a c t i c a c i d o s i s d u e t o a n a e r o b i c m e t a b o l i s m . W h e n h y p o x e m i a c a u s e s P 0 2 t o d r o p b e l o w t h e l e v e l t h a t i s r e q u i r e d t o m a i n t a i n t h e o x y g e n d i f f u s i o n g r a d i e n t f r o m c a p i l l a r y b l o o d t o m i t o c h o n d r i a , l a c t i c a c i d o s i s w i l l o c c u r d u e t o a n a e r o b i c g l y c o l y s i s ( D e n i s o n , 1 9 8 6 ) . O x i d a t i v e p h o s p h o r y l a t i o n a c c o u n t s f o r a b o u t 8 5 p e r c e n t o f t o t a l b o d y o x y g e n c o n s u m p t i o n ( W e i b e l , 1 9 8 4 ) . H o w e v e r , o x i d a t i v e p h o s p h o r y l a t i o n i s n o t l i k e l y a f f e c t e d b y l o c a l P 0 2 , s i n c e t h e M i c h a e l i s a f f i n i t y c o n s t a n t (Km02) o f c y t o c h r o m e a 3 o x i d a s e f o r o x y g e n i s b e l o w 1 m m H g . O t h e r e n z y m e s i n v o l v e d i n t h e o x y g e n - c o n s u m i n g p r o c e s s e s s u c h a s h y d r o x y l a s e s , o x y g e n a s e s , a n d o x i d a s e s ( e x c e p t c y t o c h r o m e a 3 ) , h a v e m u c h h i g h e r a n d w i d e l y d i f f e r i n g K m 0 2 , r a n g i n g f r o m 5 t o 2 5 0 m m H g , t h e r e f o r e t h e s e p r o c e s s e s a r e m u c h m o r e v u l n e r a b l e t o h y p o x e m i a . W h e n t h e b o d y i s d e p r i v e d o f o x y g e n p r o g r e s s i v e l y , q u a n t i t a t i v e l y m i n o r b u t q u a l i t a t i v e l y i m p o r t a n t p r o c e s s e s w i l l f a i l l o n g b e f o r e t h e r e i s a n o t i c e a b l e i m p a i r m e n t o f t o t a l b o d y o x y g e n u p t a k e o r o x i d a t i v e p h o s p h o r y l a t i o n ( D e n i s o n , 1 9 8 6 ) . T h u s , f o r e x a m p l e , s o m e o x i d a t i v e d r u g m e t a b o l i s m m a y b e a f f e c t e d i n r e l a t i v e m i l d h y p o x i a / h y p o x e m i a ( J o n e s , 1 9 8 1 ) . 8 1.3.2. Physiological changes during hypoxemia C o m p e n s a t o r y r e s p o n s e m e c h a n i s m s d u r i n g h y p o x e m i a c o n s i s t o f : 1. increased oxygen extraction and decreased oxygen demand in tissues. 2. increased cardiac output. 3. preferential redistribution of available oxygen supply. T h e r a t e a t w h i c h o x y g e n i s d e l i v e r e d t o t h e t i s s u e s b y t h e a r t e r i a l b l o o d i s t h e p r o d u c t o f c a r d i a c o u t p u t a n d t h e a r t e r i a l o x y g e n c o n c e n t r a t i o n . W h e n a r t e r i a l 0 2 c o n c e n t r a t i o n i s r e d u c e d a s i n h y p o x e m i a , o x y g e n d e l i v e r y c a n b e m a i n t a i n e d , o r a t l e a s t i t s f a l l c a n b e l i m i t e d , b y : A. increasing oxygen extraction and decreasing oxygen demand in tissues: C a i n a n d C h a p l e r ( 1 9 7 9 ) e x a m i n e d t h e o x y g e n e x t r a c t i o n (i.e. i n c r e a s i n g t h e a r t e r i a l - v e n o u s o x y g e n c o n c e n t r a t i o n d i f f e r e n c e ) b y t h e c a n i n e h i n d l i m b d u r i n g h y p o x i c h y p o x i a (i.e. h y p o x i a d u e t o r e d u c e d i n s p i r e d o x y g e n c o n t e n t ) . A s i g n i f i c a n t i n c r e a s e i n o x y g e n e x t r a c t i o n d u r i n g h y p o x e m i a w a s o b s e r v e d (i.e. r a n g i n g f r o m = 2 0 % i n n o r m o x e m i a t o = 6 0 % i n m o d e r a t e a n d t o = 8 0 % i n s e v e r e h y p o x e m i a ) . O x y g e n u p t a k e b y t i s s u e w a s m a i n t a i n e d d u r i n g m o d e r a t e h y p o x e m i a , t h e r e f o r e n o m e t a b o l i c v a s o d i l a t o r y e f f e c t w a s d e v e l o p e d . H o w e v e r , d u r i n g s e v e r e h y p o x e m i a , a s i g n i f i c a n t d e c r e a s e i n o x y g e n u p t a k e , a n i n c r e a s e i n c a r d i a c o u t p u t a n d l i m b b l o o d f l o w , a n d d e c r e a s e i n t o t a l a n d p e r i p h e r a l r e s i s t a n c e w e r e o b s e r v e d ( C a i n a n d C h a p l e r , 1 9 7 9 ) . T h i s s u g g e s t s t h a t p r o l o n g e d s e v e r e h y p o x e m i a ( > 2 0 m i n ) c a u s e s a n i n c r e a s e d b l o o d f l o w b y l o c a l m e t a b o l i c v a s o d i l a t i o n i n s p i t e o f a n y c e n t r a l l y m e d i a t e d c o n s t r i c t o r a c t i o n [ a u t o r e g u l a t o r y e s c a p e ] ( G r a n g e r et al, 1 9 7 5 ; 1 9 7 6 ) . A n i n c r e a s e i n 0 2 e x t r a c t i o n c a u s e s a f a l l i n v a s c u l a r P 0 2 . T h e r e f o r e , t h i s 9 c o m p e n s a t o r y m e c h a n i s m w i l l l i k e l y b e l i m i t e d b y t h e o n s e t o f a u t o r e g u l a t o r y e s c a p e i.e. l o c a l m e t a b o l i c v a s o d i l a t i o n , s i n c e a c r i t i c a l a r t e r i a l o x y g e n l e v e l ( P a 0 2 = - 1 0 - 1 2 m m H g ) i n t i s s u e s i s n e e d e d t o m a i n t a i n a n a d e q u a t e 0 2 d i f f u s i o n g r a d i e n t f r o m b l o o d t o m i t o c h o n d r i a ( C o n n e t t et al, 1 9 9 0 ) . B. increasing cardiac output: A n e l e v a t e d a o r t i c b l o o d f l o w i s a s s o c i a t e d p r i m a r i l y w i t h a n i n c r e a s e i n h e a r t r a t e w h i c h i s m e d i a t e d i n l a r g e p a r t b y e n h a n c e d s y m p a t h o a d r e n a l a c t i v i t y ( K r a s n e y , 1 9 6 7 ) , a n d a f a l l i n s y s t e m i c v a s c u l a r r e s i s t a n c e ( K o n t o s et al, 1 9 6 5 ; S y l v e s t e r et al, 1 9 7 9 ) . A n e t i n c r e a s e i n c a r d i a c o u t p u t a n d a r t e r i a l p r e s s u r e ( K o n t o s et al, 1 9 6 7 ) i s m e d i a t e d b y t h e p e r i p h e r a l c h e m o r e c e p t o r i n t e r a c t i o n s , s u c h a s a p u l m o n a r y v a s o d i l a t o r r e f l e x ( D a l y a n d R o b i n s o n , 1 9 6 8 ) , t h e l o c a l v a s o d i l a t i n g a c t i o n o f h y p o x i a ( D a u g h e r t y et al, 1 9 6 7 ) , a n d i n a d d i t i o n , b y t h e d i r e c t c e n t r a l n e r v o u s s y s t e m a c t i o n o f h y p o x i a ( D o w n i n g et al, 1 9 6 3 ) . T h e p r e f e r e n t i a l r e d i s t r i b u t i o n o f c a r d i a c o u t p u t d u r i n g h y p o x i a w a s r e p o r t e d i n t h e d o g ( K r a s n e y , 1 9 7 1 ; A d a c h i et al, 1 9 7 6 ) a n d i n t h e s h e e p ( N e s a r a j a h et al, 1 9 8 3 ) . R e d i s t r i b u t i o n o f b l o o d f l o w i s a c c o m p l i s h e d b y l o c a l e f f e c t s o f h y p o x i a , w h i c h p r o d u c e s v a s o d i l a t i o n i n c o r o n a r y a n d c e r e b r a l v e s s e l s , a n d b y c h e m o r e c e p t o r r e f l e x , w h i c h p r o d u c e s v a s o c o n s t r i c t i o n i n s k e l e t a l m u s c l e a n d t h e s p l a n c h n i c b e d a n d d i l a t i o n i n c o r o n a r y v e s s e l s ( H e i s t a d a n d A b b o u d , 1 9 8 0 ) . S u p e r i o r c a v a l f l o w w a s e n h a n c e d a t t h e e x p e n s e o f i n f e r i o r c a v a l f l o w d u r i n g h y p o x i a , b u t t h i s h y p o x i c r e d i s t r i b u t i o n o f s y s t e m i c f l o w w a s a b o l i s h e d b y c h e m o r e c e p t o r d e n e r v a t i o n ( K r a s n e y , 1 9 7 1 ; M a l o et al, 1 9 8 4 ) . C a r o t i d a n d a o r t i c c h e m o r e c e p t o r s p r o v i d e f o r r e f l e x v a s o c o n s t r i c t i v e s u p p o r t o f t h e c i r c u l a t i o n d u r i n g s y s t e m i c h y p o x i a ( C h a l m e r et al, 1 9 6 5 ; D a l y a n d S c o t t , 1 9 6 4 ) , w h i c h 10 l e a d s t o p r e f e r e n t i a l r e d i s t r i b u t i o n o f b l o o d f l o w t o t h e c e p h a l i c r e g i o n d u r i n g h y p o x i a , w i t h a d e c l i n e i n s p l a n c h n i c b l o o d f l o w . L o c a l f l o w i s a l t e r e d v a r i a b l y i n h y p o x i a d e p e n d i n g u p o n t h e r e l a t i v e b a l a n c e o f v a s o c o n s t r i c t o r a n d v a s o d i l a t o r c o m p o n e n t s , w h i c h i s i n f l u e n c e d b y t h e l e v e l o f o x y g e n i n t h e b l o o d ( A d a c h i et al., 1 9 7 6 ) . F o r e x a m p l e , t h e p u l m o n a r y c i r c u l a t i o n i s v e r y s e n s i t i v e t o t h e d e c r e a s e i n t h e P 0 2 , w h i c h a l t e r s t h e r e g i o n a l d i s t r i b u t i o n o f p u l m o n a r y a r t e r i a l b l o o d f l o w [ h y p o x i c v a s o c o n s t r i c t i o n ] ( B e n j a m i n a n d G o r l i n , 1 9 5 2 ; W e s t , 1 9 8 8 ) . I n c o n t r a s t , t h e c o r o n a r y c i r c u l a t i o n e x h i b i t s a v e r y s e n s i t i v e v a s o d i l a t o r r e s p o n s e t o t h e l o c a l d e c r e a s e i n P 0 2 ( G r e g g , 1 9 5 0 ; A d a c h i et ai, 1 9 7 6 ) . T h e m a i n d e t e r m i n a n t s o f t h e d i s t r i b u t i o n o f c a r d i a c o u t p u t a r e t h e r e l a t i v e a r t e r i a l f l o w r e s i s t a n c e a n d v e n o u s p r e s s u r e s ( M i t z r e a n d G o l d b e r g , 1 9 7 5 ) , a n d t h e s e c h a n g e s i n t h e d i s t r i b u t i o n o f c a r d i a c o u t p u t r e f l e c t a l t e r a t i o n i n t h e a r t e r i a l p r e s s u r e - f l o w r e l a t i o n s h i p o f e a c h v a s c u l a r b e d ( M a l o et al, 1 9 8 4 ) . T h e p r e f e r e n t i a l r e d i s t r i b u t i o n o f c a r d i a c o u t p u t a n d r e d u c e d o x y g e n d e m a n d i n n o n - e s s e n t i a l t i s s u e s (e.g. d i g e s t i v e s y s t e m a n d s k e l e t a l m u s c l e s ) w i l l t e m p o r a r i l y c o m p e n s a t e f o r a r e d u c e d o x y g e n s u p p l y u p t o a c e r t a i n l i m i t . H o w e v e r , t h i s m e c h a n i s m i s a l s o l i m i t e d b y t h e d e v e l o p m e n t o f s y s t e m i c a n d l o c a l l a c t i c a c i d o s i s a s a c o n s e q u e n c e o f t h e i n c r e a s e d a n a e r o b i c g l y c o l y s i s i n t i s s u e s , w h i c h i n t u r n c a u s e s l o c a l v a s o d i l a t i o n ( N e s a r a j a h et al, 1 9 8 3 ) . F u r t h e r m o r e , a c i d o s i s w i l l c a u s e a d e c r e a s e i n t h e h e m o g l o b i n G * 2 s a t u r a t i o n b y t h e B o h r e f f e c t , w h i c h i s f o l l o w e d b y r e d u c e d o x y g e n d e l i v e r y a n d a f u r t h e r d r o p i n p H , u l t i m a t e l y c o m p r o m i s i n g t h e o x y g e n d e l i v c y t o t h e v i t a l o r g a n s ( N e s a r a j a h et al, 1 9 8 3 ) 11 Hepatic haemodynamics and function: M o d e r a t e h y p o x e m i a i n c a t s ( L a r s e n et al, 1 9 7 6 ) a n d i n d o g s ( H u g h e s et al, 1 9 7 9 ; S c h o l t h o l t a n d S h i r a i s h i , 1 9 7 0 ) d o e s n o t s i g n i f i c a n t l y a l t e r e i t h e r h e p a t i c a r t e r i a l o r p o r t a l v e n o u s c o n d u c t a n c e . H o w e v e r , m o r e s e v e r e h y p o x e m i a i n t h e d o g s i g n i f i c a n t l y i n c r e a s e s s y s t e m i c a r t e r i a l p r e s s u r e a n d r e d u c e s h e p a t i c a r t e r i a l b l o o d f l o w , i n d i c a t i n g i n c r e a s e d h e p a t i c a r t e r i a l v a s c u l a r r e s i s t a n c e ( H u g h e s et al, 1 9 7 9 ) . P o r t a l v e n o u s b l o o d f l o w s e e m s t o b e u n a f f e c t e d b y h y p o x e m i a ( S c h o l t h o l t a n d S h i r a i s h i , 1 9 7 0 ; L a r s e n et al, 1 9 7 6 , I s h i k a w a et al, 1 9 7 4 ; H u g h e s et al, 1 9 7 9 ) , b u t M a t h i e a n d B l u m g a r t ( 1 9 8 3 ) s h o w e d a s m a l l b u t s i g n i f i c a n t i n c r e a s e i n p o r t a l v e n o u s f l o w . T h e s e d i f f e r e n t o b s e r v a t i o n s a r e l i k e l y r e l a t e d t o t h e p r e c i s e t i m i n g o f m e a s u r e m e n t a n d t h e l e v e l o f h y p o x e m i a c h o s e n f o r t h e i n v e s t i g a t i o n . O v e r a l l , t h e e f f e c t s o f h y p o x e m i a o n h e p a t i c h a e m o d y n a m i c s s e e m t o b e m i n o r . A l t e r n a t i v e l y , R o t h a n d R u b i n ( 1 9 7 6 ) s h o w e d a s i g n i f i c a n t d e c r e a s e i n t o t a l h e p a t i c a n d p o r t a l v e n o u s f l o w , w i t h a s l i g h t i n c r e a s e i n h e p a t i c a r t e r i a l f l o w i n a s t u d y o f h y p o x i c h y p o x i a . T h i s d i s c r e p a n c y i s l i k e l y d u e t o t h e h y p o c a p n i a w h i c h r e s u l t s f r o m h y p e r v e n t i l a t i o n . H y p e r c a p n i a a n d h y p o c a p n i a h a v e b e e n s h o w n t o a f f e c t t o t a l h e p a t i c f l o w s i g n i f i c a n t l y ( M a t h i e a n d B l u m g a r t , 1 9 8 3 ; H u g h e s et al, 1 9 7 9 ) . H y p e r c a p n i a t e n d s t o i n c r e a s e t o t a l h e p a t i c a n d p o r t a l v e n o u s f l o w , d u e t o d e c r e a s e d m e s e n t e r i c v a s c u l a r r e s i s t a n c e ( M a t h i e a n d B l u m g a r t , 1 9 8 3 ) . I t h a s b e e n s u g g e s t e d t h a t h e p a t i c f u n c t i o n i s m o r e s e n s i t i v e t o h y p o x e m i a t h a n o t h e r o r g a n s i n s p i t e o f m i n o r a l t e r a t i o n i n h a e m o d y n a m i c s , b e c a u s e = 7 0 % o f h e p a t i c b l o o d s u p p l y i s v e n o u s i.e. l o w b l o o d o x y g e n t e n s i o n ( P r e i s i g et al, 1 9 7 2 ) . O n t h e o t h e r 12 h a n d , t h e l i v e r r e c e i v e s n e a r l y 2 5 % o f t h e t o t a l c a r d i a c o u t p u t a n d h a s a l o w o x y g e n e x t r a c t i o n r a t i o i.e. = 1 5 - 2 0 % ( R o w e l l et al, 1 9 8 4 ) . S t u d i e s o n h u m a n s a n d o t h e r s p e c i e s ( R o w e l l et al, 1 9 6 8 ; 1 9 8 4 ) s h o w e d t h a t t h e l i v e r c a n e x t r a c t n e a r l y 1 0 0 % o f t h e a v a i l a b l e 0 2 a n d t h i s i n c r e a s e i n o x y g e n e x t r a c t i o n i s t h e m a j o r a d j u s t m e n t o b s e r v e d i n r e s p o n s e t o r e d u c e d h e p a t i c 0 2 d e l i v e r y ( L a r s e n et al, 1 9 7 6 ; T a s h k i n et al, 1 9 7 2 ) . D e s p i t e t h i s e f f i c i e n t 0 2 e x t r a c t i o n , a b n o r m a l i t i e s i n s u l f o b r o m o p h t h a l e i n ( B S P ) r e t e n t i o n ( S h o r e y et al, 1 9 6 9 ) a n d b i l e f l o w ( L a r s e n et al, 1 9 7 6 ) w e r e o b s e r v e d d u r i n g m o d e r a t e h y p o x e m i a . T h e e x t r a c t i o n o f I n d o c y a n i n e G r e e n w a s a l s o r e d u c e d d u r i n g b o t h m o d e r a t e a n d s e v e r e h y p o x e m i a ( B l a c k m o n a n d R o w e l l , 1 9 8 6 ; M a r l e a u et al, 1 9 8 7 ) . H o w e v e r , B l a c k m o n a n d R o w e l l ( 1 9 8 6 ) s h o w e d t h a t a c u t e h y p o x e m i a i n h u m a n s d i d n o t a f f e c t s p l a n c h n i c 0 2 u p t a k e i n s p i t e o f a f a l l i n h e p a t i c v e n o u s 0 2 c o n t e n t . I n a d d i t i o n , h e p a t i c g l u c o s e r e l e a s e a n d l a c t a t e u p t a k e w e r e n o t a f f e c t e d b y a c u t e h y p o x e m i a i n t h i s s t u d y . Renal function : H y p o x e m i a i n f l u e n c e s t h e r e n a l e x c r e t i o n o f s o d i u m a n d w a t e r . I t h a s b e e n l o n g k n o w n t h a t a c u t e m o d e r a t e h y p o x e m i a ( P a 0 2 > 4 0 m m H g ) c a u s e s a n i n c r e a s e i n u r i n e v o l u m e ( d i u r e s i s ) a n d r e n a l e x c r e t i o n o f s o d i u m a n d c h l o r i d e i n h e a l t h y v o l u n t e e r s ( B u r r i l l et al, 1 9 4 5 ; B e r g e r et al, 1 9 4 9 ; K i l b u r n et al, 1 9 7 1 ) . A c u t e m o d e r a t e h y p o x e m i a a l s o c a u s e s r e n a l v a s o d i l a t i o n , t h e r e b y d e c r e a s i n g r e n a l v a s c u l a r r e s i s t a n c e . T h u s , m e a n r e n a l b l o o d f l o w i s m a i n t a i n e d d e s p i t e r e d u c e d m e a n a r t e r i a l p r e s s u r e . T h e r e a r e s i g n i f i c a n t c h a n g e s i n i n t r a r e n a l h a e m o d y n a m i c s w i t h o u t s i g n i f i c a n t c h a n g e s i n e x c r e t o r y f u n c t i o n s s u c h a s g l o m e r u l a r f i l t r a t i o n r a t e a n d s o d i u m e x c r e t i o n ( Z i l l i g et al, 1 9 7 8 ) . D i u r e s i s d u r i n g m o d e r a t e h y p o x e m i a s e e m s t o b e r e l a t e d t o d e c r e a s e d p l a s m a a l d o s t e r o n e ( C o l i c e a n d R a m i r e z , 1 9 8 5 ) a n d a n g i o t e n s i n I I ( V o n m o o s et al, 1 9 9 0 ) l e v e l s , 13 a n d i n c r e a s e d a t r i a l n a t r i u r e t i c f a c t o r c o n c e n t r a t i o n s ( d u S o u i c h et al, 1 9 8 7 ) . B a s i c r e n a l m e t a b o l i s m i s a l s o m a i n t a i n e d i n m o d e r a t e h y p o x e m i a i n s p i t e o f r e d u c e d o x y g e n d e l i v e r y ( S i n a g o w i t z et al, 1 9 7 6 ) . I n p a t i e n t s w i t h c h r o n i c o b s t r u c t i v e p u l m o n a r y d i s e a s e , r e n a l p h y s i o l o g i c a l f u n c t i o n s s u c h a s r e n a l p e r f u s i o n , g l o m e r u l a r f i l t r a t i o n a n d s o d i u m e x c r e t i o n w e r e r e d u c e d a b r u p t l y d u r i n g s e v e r e h y p o x e m i a ( P a 0 2 < 4 0 m m H g ) o r h y p e r c a p n i a ( P a C 0 2 > 6 0 m m H g ) ( K i l b u r n et al, 1 9 7 1 ; B r u n s , 1 9 7 8 ) . 1.4. Respiratory Disorders and Drug Disposition and Metabolism T h e e f f e c t s o f r e s p i r a t o r y d i s o r d e r s i n c l u d i n g h y p o x e m i a o n d r u g d i s p o s i t i o n a n d m e t a b o l i s m a r e t h e s u b j e c t o f s e v e r a l r e v i e w s ( d u S o u i c h et al, 1 9 7 8 ; F a r r e l l , 1 9 8 7 ; J o n e s et al, 1 9 8 9 ; a n d T a b u r e t et al, 1 9 9 0 ) . T h e o n s e t o f h y p o x e m i a i s t h e c o m m o n p a t h w a y o f m o s t o f t h e f r e q u e n t l y o b s e r v e d r e s p i r a t o r y d i s o r d e r s s u c h a s a s t h m a a n d c h r o n i c o b s t r u c t i v e p u l m o n a r y d i s e a s e ( C O P D ) . C h a n g e s i n P a C 0 2 a n d b l o o d p H a r e a l s o a s s o c i a t e d w i t h h y p o x e m i a . I n s e v e r e p n e u m o n i a , p u l m o n a r y e d e m a a n d p u l m o n a r y e m b o l i s m , h y p o x e m i a i s a s s o c i a t e d w i t h h y p o c a p n i a a n d r e s p i r a t o r y a l k a l o s i s ( W e s t , 1 9 8 8 ) , w h e r e a s i n C O P D , h y p o x e m i a i s a s s o c i a t e d w i t h h y p e r c a p n i a a n d r e s p i r a t o r y a c i d o s i s . T h e i n c r e a s e d a d v e r s e e f f e c t s a n d t o x i c i t y a s s o c i a t e d w i t h c l i n i c a l d o s e a d m i n i s t r a t i o n o f d i g o x i n ( S m i t h , 1 9 7 5 ; D o h e r t y et al, 1 9 7 7 ) a n d t h e o p h y l l i n e ( Z w i l l i c h et al, 1 9 7 5 ; H e n d e l e s et al, 1 9 7 7 ) t o p a t i e n t s w i t h p u l m o n a r y d i s o r d e r s s u g g e s t s t h a t c h a n g e s i n d r u g c l e a r a n c e i s o f m a j o r s i g n i f i c a n c e . 14 1.4.1. Effects of acute and chronic hypoxemia on drug metabolism M e r r i t t a n d M e d i n a ( 1 9 6 8 ) o b s e r v e d t h a t h e x o b a r b i t a l s l e e p i n g t i m e i n m i c e w a s r e d u c e d a t a l t i t u d e . A l o w p l a s m a c o n c e n t r a t i o n o f h e x o b a r b i t a l a t a l t i t u d e w a s r e p o r t e d i n t h e s t u d y . T h e in vitro m e t a b o l i s m o f h e x o b a r b i t a l i n h e p a t i c m i c r o s o m e s w a s h i g h e r i n t h e p r e p a r a t i o n f r o m t h e r a t s a t a l t i t u d e t h a n t h o s e f r o m t h e r a t s a t g r o u n d l e v e l . S i m i l a r r e s u l t s w e r e s h o w n i n m a n y d r u g s u n d e r g o i n g o x i d a t i v e m e t a b o l i s m s u c h a s z o x a z o l a m i n e , p h e n o b a r b i t a l , a n d p h e n y l b u t a z o n e ( M e d i n a a n d M e r r i t t , 1 9 7 0 ) . I n c o n t r a s t , C u m m i n g a n d M a n n e r i n g ( 1 9 7 0 ) r e p o r t e d i n c r e a s e d h e x o b a r b i t a l h a l f -l i f e a t l o w P 0 2 ( = 4 5 r n m H g ) i n b o t h i s o l a t e d l i v e r a n d in vivo, a n d p r o v e d r e d u c e d h e p a t i c m e t a b o l i s m a t l o w e r P 0 2 . R o t h a n d R u b i n ( 1 9 7 6 ) f u r t h e r e x a m i n e d t h e r e l a t i o n s h i p b e t w e e n h e p a t i c b l o o d f l o w a n d h e x o b a r b i t a l m e t a b o l i s m i n h y p o x i c - h y p o x e m i a a n d c a r b o n m o n o x i d e ( C O ) i n d u c e d h y p o x e m i a . B o t h C O - i n d u c e d a n d h y p o x i c h y p o x e m i a d e c r e a s e d P a 0 2 a n d o x y g e n s a t u r a t i o n , w h e r e a s P a C 0 2 i s r e d u c e d o n l y i n h y p o x i c h y p o x e m i a ( h y p o c a p n i a d u e t o h y p e r v e n t i l a t i o n ) . A r e d i s t r i b u t i o n o f c a r d i a c o u t p u t i n d u c e d b y h y p o x e m i a (i.e. 3 3 % r e d u c t i o n i n h e p a t i c b l o o d f l o w ) w a s o b s e r v e d i n h y p o x i c h y p o x i a / h y p o c a p n i a , r e s u l t i n g i n r e d u c e d h e p a t i c m e t a b o l i s m o f h e x o b a r b i t a l . T h e s e e x p e r i m e n t s s e e m e d t o g i v e c o n t r a d i c t o r y r e s u l t s f r o m t h e p r e v i o u s s t u d i e s ( M e r r i t t a n d M e d i n a , 1 9 6 8 ; M e d i n a a n d M e r r i t t , 1 9 7 0 ) , h o w e v e r , w e s h o u l d c o n s i d e r t h a t r a t s a t a l t i t u d e i n t h e s t u d i e s o f M e d i n a a n d M e r r i t t w e r e e x p o s e d t o c h r o n i c h y p o x e m i a (i.e. 5 d a y s ) , w h e r e a s t h e s t u d i e s o f C u m m i n g a n d M a n n e r i n g ( 1 9 7 0 ) a n d R o t h a n d R u b i n ( 1 9 7 6 ) i n v o l v e d a c u t e h y p o x e m i a . 15 T h e s e s t u d i e s d e m o n s t r a t e d t h e d i f f e r e n t e f f e c t s o f a c u t e a n d c h r o n i c h y p o x e m i a . A c u t e h y p o x i a i s u s u a l l y a s s o c i a t e d w i t h i m p a i r e d h e p a t i c d r u g m e t a b o l i s m ( J o n e s , 1 9 8 1 ) , w h e r e a s c h r o n i c h y p o x i a m a y a c t u a l l y s t i m u l a t e o x i d a t i v e d r u g m e t a b o l i z i n g e n z y m e s y s t e m s [ i n d u c t i o n ] ( d u S o u i c h et al, 1 9 7 8 ) . T h e r e f o r e , t h e p h a r m a c o k i n e t i c s t u d y d u r i n g h y p o x e m i a m u s t t a k e i n t o a c c o u n t t h e d i f f e r e n t e f f e c t s o f a c u t e a n d c h r o n i c h y p o x e m i a o n d r u g m e t a b o l i s m a n d , i n t u r n , d r u g k i n e t i c s . 1.4.2. Effects of hypoxemia on hepatic drug metabolism I t i s v a l u a b l e t o e x a m i n e t h e s t u d i e s o n a n t i p y r i n e k i n e t i c s d u r i n g h y p o x e m i a , s i n c e a n t i p y r i n e e l i m i n a t i o n s e e m s t o b e p r o p o r t i o n a l t o t h e n o n - s p e c i f i c h e p a t i c m i c r o s o m a l e n z y m e a c t i v i t y ( K o l m o d i n e et al, 1 9 6 9 ) . I n a d d i t i o n , a n t i p y r i n e i s d i s t r i b u t e d e v e n l y t h r o u g h o u t b o d y w a t e r , a n d m e t a b o l i z e d i n t h e l i v e r , w i t h o u t a n y e x t e n s i v e e x c r e t i o n b y t h e k i d n e y i n a n u n c h a n g e d f o r m . ( L a y b o u m et al, 1 9 8 6 ) . C u m m i n g ( 1 9 7 6 ) o b s e r v e d t h a t p a t i e n t s w i t h l o w P a 0 2 ( l e s s t h a n 5 5 m m H g ) h a d a p r o l o n g e d a n t i p y r i n e h a l f - l i f e ( 1 8 . 4 h r ) c o m p a r e d w i t h t h o s e w h o s e P a 0 2 w e r e g r e a t e r t h a n 5 5 m m H g ( 8 . 4 h r ) . L a y b o u m et al. ( 1 9 8 6 ) a l s o f o u n d l o w e r a n t i p y r i n e c l e a r a n c e (i.e. = = 1 8 % ) i n p a t i e n t s w i t h p u l m o n a r y d i s e a s e t h a n i n h e a l t h y v o l u n t e e r s . I n c o n t r a s t , A g n i h o t r i et al ( 1 9 7 8 ) o b s e r v e d e n h a n c e d a n t i p y r i n e c l e a r a n c e i n p a t i e n t s w i t h C O P D . S o m e p a t i e n t s s h o w e d h y p e r c a p n i a , w h i c h c o u l d c a u s e a n i n c r e a s e i n h e p a t i c b l o o d f l o w ( H u g h e s et al, 1 9 7 9 ) a n d , i n t u r n , h e p a t i c d r u g c l e a r a n c e . P a t i e n t s w i t h C O P D a r e a l s o l i k e l y t o s u f f e r c h r o n i c h y p o x e m i a , t h u s l e a d i n g t o t h e i n d u c t i o n o f h e p a t i c e n z y m e 16 s y s t e m s ( d u S o u i c h et al, 1 9 7 8 ) . T h e r e f o r e , t h e s e c o n t r a d i c t i n g o b s e r v a t i o n s m a y h a v e r e s u l t e d f r o m d i f f e r e n t c l i n i c a l c o n d i t i o n s i n t h e s e s t u d i e s . 1.4.3. Effects of hypoxemia on drug disposition and protein binding D u S o u i c h et al. ( 1 9 7 8 ) s u g g e s t e d t h a t t h e e f f e c t s o f r e s p i r a t o r y d i s e a s e o n t h e e f f i c a c y o f m o s t l o n g t e r m a n t i b i o t i c u s a g e i s l i k e l y t o b e m i n i m a l , s i n c e t h e t h e r a p e u t i c i n d i c e s a r e w i d e . H o w e v e r , h y p o x e m i a c a u s e s s i g n i f i c a n t a l t e r a t i o n i n a m i n o g l y c o s i d e s e r u m p h a r m a c o k i n e t i c s . M y e r s et al. ( 1 9 7 7 ) s h o w e d a s i g n i f i c a n t i n c r e a s e i n h a l f - l i f e o f a m i k a c i n i n h y p o x e m i c ( P a 0 2 < 5 0 m m H g ) i n f a n t s ( 7 . 3 h r ) c o m p a r e d t o n o r m o x e m i c i n f a n t s ( 4 . 8 h r ) . T h e d e c r e a s e d c l e a r a n c e i n h y p o x e m i a i s l i k e l y d u e t o a r e d u c e d r e n a l b l o o d f l o w , w i t h a c o n c o m i t a n t d e c r e a s e i n g l o m e r u l a r f i l t r a t i o n r a t e f r o m r e n a l v a s o c o n s t r i c t i o n ( B r u n s , 1 9 7 8 ) . H y p o x i c s t i m u l a t i o n o f a o r t i c c h e m o r e c e p t o r s r e s u l t s i n r e n a l v a s o c o n s t r i c t i o n ( K o m e r , 1 9 6 3 ) . A r e d u c t i o n i n a m i k a c i n a n d g e n t a m i c i n c l e a r a n c e d u r i n g h y p o x e m i a w a s o b s e r v e d i n r a t a n d r a b b i t ( M i r h i j et al, 1 9 7 8 ) . T h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n w a s n o t a f f e c t e d b y h y p o x e m i a , t h u s t h e c h a n g e i n d r u g c l e a r a n c e i s d i r e c t l y r e l a t e d t o a c h a n g e i n d r u g e l i m i n a t i o n . S i n c e a m i n o g l y c o s i d e e x c r e t i o n i s a l m o s t e n t i r e l y m e d i a t e d b y g l o m e r u l a r f i l t r a t i o n ( L e v y et al. 1 9 7 5 ) , t h e a l t e r a t i o n i n r e n a l e x c r e t i o n d u r i n g h y p o x e m i a c o u l d a c c o u n t f o r t h e c h a n g e i n a m i k a c i n e l i m i n a t i o n . V e r y d i f f e r e n t r e s u l t s w e r e o b s e r v e d w i t h s u l p h a m e t h a z i n e ( d u S o u i c h a n d C o u t e a u , 1 9 8 4 ) . S i g n i f i c a n t i n c r e a s e s i n t h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n a n d n o n -r e n a l c l e a r a n c e w e r e o b s e r v e d i n r a b b i t s d u r i n g h y p o x e m i a a n d m e t a b o l i c a c i d o s i s . A n i n c r e a s e i n t h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n d u r i n g h y p o x e m i a i s p r o b a b l y r e l a t e d t o 17 d e c r e a s e d p r o t e i n b i n d i n g o f t h i s d r u g , t h u s l e a d i n g t o a n i n c r e a s e i n s u l p h a m e t h a z i n e f r e e f r a c t i o n . S i n c e t h e p K a o f s u l p h a m e t h a z i n e i s 7 . 4 , a c h a n g e i n p l a s m a p H a s s o c i a t e d w i t h p u l m o n a r y d i s o r d e r s w i l l a f f e c t t h e i o n i z a t i o n o f s u l p h a m e t h a z i n e . W i t h h y p e r c a p n i a a n d m e t a b o l i c a c i d o s i s , t h e p r o p o r t i o n o f n o n - i o n i z e d s u l p h a m e t h a z i n e i n c r e a s e d ( t h e r a t i o o f i o n i z e d t o n o n - i o n i z e d s u l p h a m e t h a z i n e d e c r e a s e d b y 4 2 % ) , a n d t h i s s u b s e q u e n t l y m a y a f f e c t t h e d i s t r i b u t i o n a n d c l e a r a n c e o f s u l p h a m e t h a z i n e . H o w e v e r , t h e c h a n g e s i n b l o o d p H a n d s u b s e q u e n t a l t e r a t i o n i n r e n a l e l i m i n a t i o n w i l l h a v e v a r i o u s r e s u l t s , s i n c e t h e i n c r e a s e i n n o n - i o n i z e d d r u g m a y n o t o n l y e n h a n c e d r u g d i f f u s i o n ( i n c r e a s i n g v o l u m e o f d i s t r i b u t i o n , g l o m e r u l a r f i l t r a t i o n r a t e a n d n o n - r e n a l c l e a r a n c e ) , b u t a l s o i n c r e a s e t u b u l a r r e - a b s o r p t i o n ( d e c r e a s i n g r e n a l c l e a r a n c e ) . S i n c e t h e s e c h a n g e s i n d i s p o s i t i o n c a n c o u n t e r b a l a n c e e a c h o t h e r , t h e c o n s e q u e n c e a p p e a r s t h a t t h e c h a n g e s i n p H a s s o c i a t e d w i t h h y p e r c a p n i a o n l y s l i g h t l y a f f e c t s s u l p h a m e t h a z i n e k i n e t i c s . I n c o n t r a s t , h y p o x e m i a , w h i c h h a s l i t t l e e f f e c t o n b l o o d p H , s i g n i f i c a n t l y i n c r e a s e d s u l p h a m e t h a z i n e f r e e f r a c t i o n , t h e r e b y s i g n i f i c a n t l y i n c r e a s i n g n o n - r e n a l c l e a r a n c e a n d e x e r t i n g a g r e a t e r e f f e c t o n d r u g k i n e t i c s ( d u S o u i c h et al, 1 9 7 8 ; d u S o u i c h a n d C o u t e a u , 1 9 8 4 ) . D r u g s s u c h a s p h e n o b a r b i t a l ( W a d d e l l a n d B u t l e r , 1 9 5 7 ) , t h e s a l i c y l a t e s ( H i l l , 1 9 7 1 ) a n d s u l p h a e t h i d o l e ( K o s t e n b a u d e r et al, 1 9 6 2 ; D e t t l i et al, 1 9 6 7 ) s h o w a n i n c r e a s e d v o l u m e o f d i s t r i b u t i o n a n d d e c r e a s e d r e n a l e l i m i n a t i o n d u r i n g h y p o x e m i a . 18 1.4.4. Digoxin kinetics in hypoxemia A n i n c r e a s e d s e n s i t i v i t y t o d i g i t a l i s i n p a t i e n t s w i t h C O P D / h y p o x e m i a ( B a u m et al., 1 9 5 6 ; B a u m et al., 1 9 5 9 ; M o r r i s o n a n d K i l l i p , 1 9 7 1 ) a n d i n d o g s w i t h h y p o x i a ( B e l l e r a n d S m i t h , 1 9 7 2 ; H a r r i s o n et al. 1 9 6 8 ) h a s b e e n r e p o r t e d . I t w a s s u g g e s t e d t h a t h y p o x e m i a d e c r e a s e s d i g o x i n p l a s m a c o n c e n t r a t i o n , b u t i n c r e a s e s m y o c a r d i a l d i g o x i n l e v e l s i n a n a e s t h e t i z e d a n d a r t i f i c i a l l y v e n t i l a t e d d o g s ( H a r r o n et al, 1 9 7 8 ) , h o w e v e r , t h i s o b s e r v a t i o n ( i n c r e a s e d m y o c a r d i a l d i g o x i n l e v e l ) w a s l a t e r c o n t r a d i c t e d ( S a i t o et al, 1 9 8 1 ; d u S o u i c h et al, 1 9 8 5 a , b ) . D u S o u i c h et al, ( 1 9 8 5 a ) s h o w e d p l a s m a c o n c e n t r a t i o n s o f d i g o x i n w e r e l o w e r i n h y p o x e m i c a n d h y p e r c a p n i c d o g s . T h i s d e c r e a s e d d i g o x i n p l a s m a c o n c e n t r a t i o n i s p r o b a b l y d u e t o a n i n c r e a s e i n t h e a p p a r e n t t o t a l b o d y c l e a r a n c e ( - 4 5 % ) a n d t h e v o l u m e o f d i s t r i b u t i o n ( - 3 6 % ) , m o s t l y i n t h e p e r i p h e r a l c o m p a r t m e n t s . D i g o x i n t i s s u e c o n c e n t r a t i o n s w e r e i n c r e a s e d d u r i n g h y p o x e m i a - h y p e r c a p n i a , e s p e c i a l l y i n l i v e r ( - 1 5 % ) , w h i c h s u g g e s t s t h a t t h e i n c r e a s e i n t o t a l b o d y c l e a r a n c e i s m a i n l y d u e t o a n i n c r e a s e i n h e p a t i c c l e a r a n c e . A n i n c r e a s e i n h e p a t i c b l o o d f l o w h a s b e e n r e p o r t e d d u r i n g h y p e r c a p n i a ( D u t t o n et al, 1 9 7 6 ; H u g h e s et al, 1 9 7 9 ) , f u r t h e r s u p p o r t i n g t h e s p e c u l a t i o n o f i n c r e a s e d h e p a t i c c l e a r a n c e , s i n c e m a n y d r u g s e x h i b i t b l o o d f l o w - l i m i t e d m e t a b o l i c r a t e . I n c r e a s e d d i g o x i n b i n d i n g t o e r y t h r o c y t e m e m b r a n e s w a s a l s o r e p o r t e d d u r i n g h y p o x e m i a / h y p e r c a p n i a ( d u S o u i c h et al, 1 9 8 5 a ) , a n d t h e r e s e e m s t o b e a d i r e c t r e l a t i o n s h i p b e t w e e n e r y t h r o c y t e d i g o x i n r e c e p t o r a n d c a r d i a c g l y c o s i d e r e c e p t o r ( A k e r a , 1 9 7 7 ) a n d v o l u m e o f d i s t r i b u t i o n ( A r o n s o n a n d G r a h a m - S m i t h 1 9 7 6 ; 1 9 7 7 ) . T h e r e f o r e , i t i s s p e c u l a t e d t h a t t h e c h a n g e i n t h e d i s t r i b u t i o n p a t t e r n i s p a r t l y d u e t o t h e c h a n g e i n r e c e p t o r b i n d i n g d u r i n g h y p o x e m i a a n d h y p e r c a p n i a . 19 T h e i n f l u e n c e o f h y p o x e m i a o n d i g o x i n k i n e t i c s a n d t i s s u e d i s t r i b u t i o n w a s f u r t h e r e x a m i n e d u s i n g t r i t i a t e d d i g o x i n b y d u S o u i c h et al. ( 1 9 8 5 b ) . S i m i l a r t o t h e h y p o x e m i a -h y p e r c a p n i a s t u d y ( d u S o u i c h et al, 1 9 8 5 a ) , a s i g n i f i c a n t c h a n g e i n v o l u m e o f d i s t r i b u t i o n w a s o b s e r v e d d u r i n g h y p o x e m i a ( d u S o u i c h et al, 1 9 8 5 b ) . H o w e v e r , t h i s i n c r e a s e i n t h e v o l u m e o f d i s t r i b u t i o n w a s l a r g e l y d u e t o a n i n c r e a s e i n t h e s i z e o f t h e c e n t r a l c o m p a r t m e n t r a t h e r t h a n t h e p e r i p h e r a l c o m p a r t m e n t . T h e r e f o r e , t h e i n c r e a s e i n t h e v o l u m e o f d i s t r i b u t i o n i s l i k e l y d u e t o a s i g n i f i c a n t i n c r e a s e i n p l a s m a p r o t e i n b i n d i n g o f d i g o x i n d u r i n g h y p o x e m i a . T o t a l b o d y c l e a r a n c e o f d i g o x i n w a s n o t s i g n i f i c a n t l y c h a n g e d , b u t a n i n c r e a s e i n r e n a l c l e a r a n c e a n d a d e c r e a s e i n n o n - r e n a l c l e a r a n c e o c c u r r e d d u r i n g h y p o x e m i a ( d u S o u i c h et al. 1 9 8 5 b ) . U n l i k e w i t h t h e h y p o x e m i a - h y p e r c a p n i a s t u d y ( d u S o u i c h et al, 1 9 8 5 a ) , n o h e p a t i c a c c u m u l a t i o n o f d i g o x i n w a s o b s e r v e d . T h e s e r e s u l t s o n h e p a t i c d i g o x i n a c c u m u l a t i o n a r e c o n s i s t e n t w i t h t h e f i n d i n g s r e g a r d i n g h e p a t i c b l o o d f l o w i n h y p o x e m i a ( L a r s e n et al, 1 9 7 6 ; R i c h a r d s o n a n d W i t h r i n g t o n , 1 9 8 1 ) a n d h y p o x e m i a / h y p e r c a p n i a ( H u g h e et al, 1 9 7 9 ; M a t h i e a n d B l u m g a r t , 1 9 8 3 ) . I n c r e a s e d h e p a t i c b l o o d f l o w d u r i n g h y p o x e m i a - h y p e r c a p n i a r e s u l t e d i n h e p a t i c a c c u m u l a t i o n o f d i g o x i n , w h e r e a s h y p o x e m i a a l o n e d i d n o t a l t e r h e p a t i c b l o o d f l o w o r h e p a t i c d i g o x i n a c c u m u l a t i o n . 1.4.5. Theophylline kinetics and respiratory disorders T h e r e h a s b e e n e x t e n s i v e s t u d y o f t h e o p h y l l i n e k i n e t i c s d u r i n g r e s p i r a t o r y d i s o r d e r s , b e c a u s e t h e o p h y l l i n e i s a p o t e n t b r o n c h o d i l a t o r u s e d i n t h e m a n a g e m e n t o f a s t h m a a n d c h r o n i c o b s t r u c t i v e a i r w a y d i s e a s e s ( H e n d e l e s et al, 1 9 8 3 , 1 9 8 5 , 1 9 8 6 ; 20 O g i l v i e , 1 9 7 8 ) . K o l b e c k et al ( 1 9 7 9 ) e x a m i n e d t h e i n f l u e n c e o f a c u t e r e s p i r a t o r y a c i d o s i s a n d a l k a l o s i s o n t h e v o l u m e o f d i s t r i b u t i o n a n d h a l f - l i f e i n d o g s . N o c h a n g e s i n v o l u m e o f d i s t r i b u t i o n w e r e n o t e d , b u t t h e r e w a s a r e d u c e d h a l f - l i f e (i.e. i n c r e a s e d e l i m i n a t i o n r a t e ) d u r i n g r e s p i r a t o r y a c i d o s i s . S i n c e o n l y 8 % o f a d o s e o f t h e o p h y l l i n e w a s r e c o v e r e d i n u r i n e a s t h e p a r e n t c o m p o u n d ( O g i l v i e , 1 9 7 8 ) , t h e c h a n g e i n c l e a r a n c e w a s l i k e l y d u e t o t h e c h a n g e i n m e t a b o l i c c l e a r a n c e . H o w e v e r , C l o z e l et al. ( 1 9 8 1 ) f o u n d n o s i g n i f i c a n t c h a n g e i n t h e o p h y l l i n e k i n e t i c s d u r i n g r e s p i r a t o r y a c i d o s i s i n d o g s , w h i c h i s c o n t r a r y t o t h e f i n d i n g o f K o l b e c k et al. ( 1 9 7 9 ) . A r e d u c e d t h e o p h y l l i n e c l e a r a n c e w a s o b s e r v e d i n h e p a t i c c i r r h o s i s p a t i e n t s ( M a n g i o n e et al. 1 9 7 8 ) , w h i l e s m o k e r s w e r e f o u n d t o h a v e i n c r e a s e d c l e a r a n c e ( P o w e l l et al, 1 9 7 7 ; 1 9 7 8 ) . T h e s e r e s u l t s f u r t h e r s u g g e s t t h a t t h e o p h y l l i n e i s m a i n l y e l i m i n a t e d b y h e p a t i c m i c r o s o m a l o x i d a t i v e e n z y m e s . L e t a r t e a n d d u S o u i c h ( 1 9 8 4 ) o b s e r v e d i n c r e a s e d t h e o p h y l l i n e s e r u m c o n c e n t r a t i o n s d u r i n g h y p o x e m i a a n d / o r h y p e r c a p n i a , b u t n o t w i t h m e t a b o l i c a c i d o s i s i n r a b b i t s . T h e d e c r e a s e d c l e a r a n c e w a s m a i n l y d u e t o a r e d u c t i o n i n n o n - r e n a l t h e o p h y l l i n e c l e a r a n c e , t h u s s u g g e s t i n g a r e d u c t i o n i n t h e o p h y l l i n e b i o t r a n s f o r m a t i o n . A d e c r e a s e d v o l u m e o f d i s t r i b u t i o n w a s a l s o o b s e r v e d w i t h h y p o x e m i a , t h e r e f o r e t h e e l i m i n a t i o n r a t e c o n s t a n t w a s n o t s i g n i f i c a n t l y a f f e c t e d d u r i n g h y p o x e m i a u n l i k e w i t h h y p e r c a p n i a a n d h y p o x e m i a / h y p e r c a p n i a . S a u n i e r et al. ( 1 9 8 7 ) f u r t h e r e x a m i n e d t h e e f f e c t s o f a c u t e a n d c h r o n i c h y p o x e m i a o n t h e o p h y l l i n e d i s p o s i t i o n i n c o n s c i o u s d o g s . I n c o n t r a s t t o t h e p r e v i o u s s t u d i e s , n o c h a n g e s i n t h e o p h y l l i n e d i s p o s i t i o n w e r e o b s e r v e d . F u r t h e r m o r e , t h e r e c o v e r y o f t h e o p h y l l i n e a n d i t s m e t a b o l i t e s , 1 , 3 - d i m e t h y l u r i c a c i d a n d 3 - m e t h y l x a n t h i n e , i n u r i n e w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l . 21 1.4.6. Lidocaine kinetics/metabolism during hypoxemia and similarity with MCP T h e a v a i l a b l e d a t a o n l i d o c a i n e k i n e t i c s d u r i n g h y p o x e m i a / h y p e r c a p n i a m a y p r o v i d e s o m e i n s i g h t a b o u t t h e e f f e c t s o f t h e s e p e r t u r b a t i o n s o n M C P k i n e t i c s , s i n c e t h e s e t w o d r u g s s h a r e s o m e s i m i l a r c h e m i c a l s t r u c t u r e a n d in vivo m e t a b o l i s m a n d p h a r m a c o k i n e t i c p r o p e r t i e s , d e s p i t e t h e i r r a t h e r d i f f e r e n t p h a r m a c o l o g i c a l e f f e c t s . M a r l e a u et al. ( 1 9 8 7 ) e x a m i n e d t h e e f f e c t s o f h y p o x e m i a a n d / o r h y p e r c a p n i a o n l i d o c a i n e a n d I n d o c y a n i n e G r e e n k i n e t i c s i n r a b b i t s . I n h u m a n s , l i d o c a i n e i s e x t e n s i v e l y a n d r a p i d l y m e t a b o l i s e d i n t h e l i v e r i.e. i t i s c h a r a c t e r i z e d b y a h e p a t i c e x t r a c t i o n r a t i o o f 7 0 % ( S t e n s o n et al, 1 9 7 1 ) , t h u s t h e h e p a t i c c l e a r a n c e i s l i k e l y t o b e a f l o w - l i m i t e d p r o c e s s , j u s t a s w i t h I C G a n d M C P . T h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n a n d t h e t o t a l b o d y c l e a r a n c e o f l i d o c a i n e w e r e n o t s i g n i f i c a n t l y a f f e c t e d a t l o w e r l i d o c a i n e d o s e s ( 1 3 0 m g / m i n / K g , i n f u s i o n ) . T h e s e r u m t o c e r e b r o s p i n a l f l u i d r a t i o o f l i d o c a i n e w a s n o t m o d i f i e d w i t h a l o w l i d o c a i n e d o s e , b u t s l i g h t d e c r e a s e s i n l i d o c a i n e c l e a r a n c e w e r e o b s e r v e d w i t h h i g h e r d o s e s ( i n f u s i o n , 2 6 0 m g / m i n / K g ) . A n i n c r e a s e d s e r u m l e v e l o f o n e m e t a b o l i t e , N - m o n o e t h y l - g l y c i n e x y l i d i d e ( M E G X ) , w a s o b s e r v e d w i t h h y p e r c a p n i a a n d h y p o x e m i a - h y p e r c a p n i a ( M a r l e a u et al, 1 9 8 7 ) . T h e i n c r e a s e d p l a s m a M E G X c o n c e n t r a t i o n i s m o r e l i k e l y d u e t o a d e c r e a s e i n t h e 3 - h y d r o x y l a t i o n o f M E G X , r a t h e r t h a n d u e t o a d e c r e a s e i n t h e d e e t h y l a t i o n o f M E G X t o g l y c i n e x y l i d i d e ( G X ) ( S u z u k i et al, 1 9 8 4 ) , t h e r e f o r e t h e e l i m i n a t i o n o f M E G X i s r e d u c e d w h i l e t h e G X p l a s m a c o n c e n t r a t i o n w a s s i m i l a r i n h y p e r c a p n i a a n d h y p o x e m i a - h y p e r c a p n i a . 22 Scheme I Comparison of the N-deethylation reactions of MCP and lidocaine ci H 2 N — ^ \ — CO - N H - i C H 2 - C H 2 - N ^ / C 2 H 5 OCH C 9 H 2 " 5 Metoclopramide Cl H 2 N — ^ CO - N H - C H 2 - C H 2 - N H - C 2 H 5 OCH , monodeethyl MCP Cl \ H 9N—ft V - C O - N H - C H 2 - C H 2 - N H 2 OCH 3 dideethyl MCP / C H 3 ^ NH - C O - C H 2 - N ^ / C 2 H 5 C,H 2 " 5 Lidocaine / C H 3 ^ ^ — N H - C O - C H j - N H - C ^ j CH 3 monomethylglycinexylidide (MEGX) . C H 3 ^ ^ — N H - C O - C H - NH 2 C H 3 glycinexylidide (GX) T h i s b i o t r a n s f o r m a t i o n i.e. d e e t h y l a t i o n o f l i d o c a i n e t o M E G X a n d G X m a y s h a r e s i m i l a r c h a r a c t e r i s t i c s w i t h t h e m e t a b o l i s m o f M C P t o m o n o - a n d d i - d e e t h y l M C P . D u S o u i c h et al. ( 1 9 9 2 ) o b s e r v e d i n c r e a s e d p l a s m a M E G X a n d G X c o n c e n t r a t i o n s d u r i n g h y p o x e m i a i n a l i d o c a i n e i n f u s i o n s t u d y i n d o g s , w h e r e a s p l a s m a l i d o c a i n e c o n c e n t r a t i o n s r e m a i n e d u n c h a n g e d . T h e r a d i o a c t i v e m i c r o s p h e r e m e t h o d w a s u s e d t o e s t i m a t e h e p a t i c , r e n a l a n d c e r e b r a l b l o o d f l o w s t h r e e h o u r s a f t e r t h e e n d o f l i d o c a i n e i n f u s i o n . W i t h a c u t e m o d e r a t e h y p o x e m i a , t h e r e w a s a n i n c r e a s e i n c e r e b r a l b l o o d f l o w , b u t r e n a l a n d h e p a t i c b l o o d f l o w s w e r e n o t a f f e c t e d . I t w a s c o n c l u d e d t h a t a c u t e m o d e r a t e h y p o x e m i a d e c r e a s e d t h e r a t e o f e l i m i n a t i o n o f b o t h a c t i v e m e t a b o l i t e s o f l i d o c a i n e w i t h o u t 23 m o d i f y i n g t h e p e r f u s i o n t o t h e o r g a n s r e s p o n s i b l e f o r t h e i r e l i m i n a t i o n ( d u S o u i c h et al, 1 9 9 2 ) . 1.5. Rationale T h e r e i s s u b s t a n t i a l e v i d e n c e t h a t p u l m o n a r y d y s f u n c t i o n c a u s i n g h y p o x e m i a i n f l u e n c e s d r u g d i s p o s i t i o n a n d m e t a b o l i s m . T h e s e c h a n g e s a r e l i k e l y e i t h e r a d i r e c t c o n s e q u e n c e o f t h e p e r t u r b a t i o n s i n b l o o d g a s a n d a c i d - b a s e s t a t u s (e.g. r e s p i r a t o r y a l k a l o s i s d u e t o h y p e r v e n t i l a t i o n a n d l a c t i c a c i d o s i s ) o r a r e s u l t o f t h e c a r d i o v a s c u l a r a n d m e t a b o l i c r e s p o n s e s t o h y p o x e m i a (e.g. r e d i s t r i b u t i o n o f c a r d i a c o u t p u t ) . T h e r e i s a l s o a p o s s i b i l i t y t h a t t h e r a p e u t i c a g e n t s a d m i n i s t e r e d w i l l i n t e r f e r e w i t h t h e c o m p e n s a t o r y r e s p o n s e t o h y p o x e m i a . A n u m b e r o f p h a r m a c o k i n e t i c p a r a m e t e r s w e r e a l t e r e d i n h y p o x e m i c c o n d i t i o n s . H o w e v e r , t h e c h a n g e s w e r e h i g h l y v a r i a b l e a n d d e p e n d e n t o n t h e c h e m i c a l c h a r a c t e r i s t i c s o f t h e s p e c i f i c d r u g , t h e p r e v a i l i n g b l o o d g a s / a c i d - b a s e c o n d i t i o n s , p r o t e i n b i n d i n g a n d t h e s p e c i e s u s e d i n t h e e x p e r i m e n t s . T h e s e c h a n g e s w e r e l i k e l y c a u s e d b y v a r i o u s p h y s i o l o g i c a l c h a n g e s i n d u c e d b y h y p o x e m i a / a c i d o s i s a s d e s c r i b e d i n S e c t i o n 1 . 4 . T h e s t u d y o f M C P k i n e t i c s a n d p h a r m a c o d y n a m i c s d u r i n g h y p o x e m i a w i l l r e s u l t i n a n i m p r o v e m e n t o f o u r u n d e r s t a n d i n g i n t h e f o l l o w i n g a r e a s : 1 . A p h a r m a c o k i n e t i c s t u d y d u r i n g h y p o x e m i a w i l l p r o v i d e i n f o r m a t i o n o n t h e c h a n g e s i n b o t h M C P d i s p o s i t i o n a n d m e t a b o l i s m . I 24 2 . A s t u d y o f m e t a b o l i t e k i n e t i c s w i l l p r o v i d e i n f o r m a t i o n o n a l t e r a t i o n i n d r u g m e t a b o l i s m d u r i n g h y p o x e m i a . T h e a s s e s s m e n t o f d r u g m e t a b o l i s m i s i m p o r t a n t , s i n c e t h e r e i s i n c r e a s i n g e v i d e n c e t h a t t h e m e t a b o l i t e s o f s o m e d r u g s (e.g. m o r p h i n e g l u c u r o n i d e s ( M u l d e r , 1 9 9 2 ) a n d m o n o - a n d d i - d e e t h y l a t e d l i d o c a i n e , M E G X a n d G X ( N a r a n g et ai, 1 9 7 8 ) ) h a v e t h e i r o w n p h a r m a c o l o g i c a l a c t i v i t y ( a g o n i s t i c a n d / o r a n t a g o n i s t i c ) a n d , i n s e l e c t e d i n s t a n c e s , t o x i c i t y . T h i s t o x i c o l o g i c a l r e s p o n s e m a y b e a l t e r e d d u r i n g h y p o x e m i a a n d h y p o x i a . T h e p a s t s t u d i e s o f M C P h a v e p r o v i d e d a s o l i d b a s e o f p h a r m a c o k i n e t i c , m e t a b o l i c a n d p h a r m a c o d y n a m i c i n f o r m a t i o n i n n o r m o x e m i c n o n - p r e g n a n t a n d p r e g n a n t s h e e p . T h e p r e s e n t s t u d y w i l l e x a m i n e o f t h e e f f e c t o f h y p o x e m i a / h y p o x i a o n a l l t h e s a l i e n t p a r a m e t e r s c h a r a c t e r i z i n g M C P i n n o n - p r e g n a n t s h e e p . S u c h a s t u d y w i l l p r o v i d e e s s e n t i a l g r o u n d w o r k f o r f u t u r e s t u d i e s o f t h e e f f e c t s o f h y p o x e m i a o n m a t e r n a l a n d f e t a l M C P d i s p o s i t i o n a n d m e t a b o l i s m d u r i n g p r e g n a n c y . 1.6. Objectives T h e o b j e c t i v e s o f t h e p r e s e n t s t u d y a r e : 1 . t o c o n f i r m t h e a p p l i c a b i l i t y i n a h y p o x e m i a s t u d y o f a n e x i s t i n g G C - M S D a s s a y m e t h o d f o r M C P a n d i t s m e t a b o l i t e s , m o n o - a n d d i - d e e t h y l M C P . 2 . t o d e t e r m i n e t h e e x t e n t t o w h i c h t h e p h a r m a c o k i n e t i c s o f M C P a n d i t s m e t a b o l i t e s a r e a l t e r e d b y r e d u c e d o x y g e n s u p p l y . 3 . t o e x a m i n e p h y s i o l o g i c a l c h a n g e s i n d u c e d b y h y p o x e m i a d u r i n g s t e a d y - s t a t e d r u g a d m i n i s t r a t i o n . 25 2. EXPERIMENTAL 2.1 Materials and Supplies 2.1.1 Chemicals 4 - a r m n o - 5 - c h l o r o - 2 - m m o n o h y d r o -c h l o r i d e m o n o h y d r a t e ( M C P » H C 1 » H 2 0 ) a n d M C P ' H C L 5 m g / m L ( R e g l a n ® I n j e c t a b l e , 2 a n d 5 m L a m p o u l e s ) w e r e o b t a i n e d f r o m A . H . R o b i n s R e s e a r c h L a b o r a t o r i e s ( R i c h m o n d , V A , U S A ) a n d A . H . R o b i n s C a n a d a I n c . ( M o n t r e a l , P Q ) . 4 - a m i n o - 5 - c h l o r o - 2 - m e t h o x y -N - e t h y l - a m i n o e t h y l - b e n z a m i d e m o n o h y d r o c h l o r i d e m o n o h y d r a t e ( m o n o d e e t h y l M C P » H C 1 » H 2 0 ) a n d 4 - a m i n o - 5 - c h l o r o - 2 - m e t h o x y - a r n i n o e t h y l - b e n z a m i d e m o n o -h y d r o c h l o r i d e m o n o h y d r a t e ( d i d e e t h y l M C P » H C 1 » H 2 0 ) w e r e s y n t h e s i z e d i n o u r l a b o r a t o r y ( R i g g s et al, 1 9 9 4 ) . T h e i n t e r n a l s t a n d a r d , 4 - a m i n o - 5 - c h l o r o - 2 ( 2 - b u t a n o n e - 3 -y l ) - o x y - N , N - d i e t h y l a m i n o e t h y l b e n z a m i d e m o n o h y d r o c h l o r i d e m o n o h y d r a t e ( B M Y « H C 1 » H 2 0 ) w a s s u p p l i e d b y B r i s t o l - M y e r s S q u i b b C o . ( W a l l i n g f o r d , C T , U S A ) . 2.1.2 Reagents S o d i u m a c e t a t e a n d 2 - a m i n o - 2 - h y d r o x y m e t h y l - l , 3 - p r o p a n e d i o l ( T R I S f r e e b a s e ) w e r e o b t a i n e d f r o m B D H C h e m i c a l s , T o r o n t o , O N . A m e r i c a n C h e m i c a l S o c i e t y ( A C S ) r e a g e n t g r a d e S o d i u m H y d r o x i d e p e l l e t s w e r e o b t a i n e d f r o m F i s h e r S c i e n t i f i c C o . , F a i r L a w n , N J . A C S r e a g e n t g r a d e H y d r o c h l o r i c a c i d 3 7 % w a s o b t a i n e d f r o m A m e r i c a n S c i e n t i f i c a n d C h e m i c a l , S e a t t l e , W A . A m m o n i a S o l u t i o n 2 5 % w a s o b t a i n e d f r o m F i s h e r 26 S c i e n t i f i c C o . H e p t a f l u o r o b u t y r i c a n h y d r i d e ( H F B A ) a n d t r i e t h y l a m i n e ( T E A ) s e q u a n a l G r a d e w e r e o b t a i n e d f r o m P i e r c e C h e m i c a l C o . , R o c k f o r d , I L , U S A . 2.1.3 Enzymes G l u c u r o n i d a s e ( G l u c u r a s e ® : P - D - G l u c u r o n i d e g l u c u r o n o s o h y d r o l a s e : E C 3 . 2 . 1 . 3 ( b o v i n e l i v e r ) a p p r o x . 5 0 0 0 S i g m a u n i t s / m L , a c e t a t e b u f f e r e d t o p H 5 . 0 a t 2 5 ° C ) a n d s u l f a t a s e ( A r y l s u l f a t a s e , a r y l - s u l f a t e s u l f o h y d r o l a s e , p h e n o l s u l f a t a s e ; E C 3 . 1 . 6 . 1 1 9 u n i t s / m L p a r t i a l l y p u r i f i e d e n z y m e i n 5 0 % g l y c e r o l - 0 . 0 1 M T r i s s o l u t i o n , p H 7 . 5 [ L o t 4 2 H 6 8 1 4 ] ) w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l C o . , S t . L o u i s , M O , U S A . 2.1.4 Solvents T o l u e n e , a n d d i c h l o r o m e t h a n e ( d i s t i l l e d i n g l a s s ) w e r e p u r c h a s e d f r o m C a l e d o n L a b o r a t o r y . I n c . , G e o r g e t o w n , O n t . . D e i o n i z e d w a t e r w a s p r o d u c e d o n s i t e u s i n g a M i l l i -Q ® s y s t e m , M i l l i p o r e C o r p . , B e d f o r d , M A . . A C S r e a g e n t g r a d e m e t h a n o l a n d a c e t o n e w e r e o b t a i n e d f r o m B D H C h e m i c a l s , T o r o n t o , O N . 2.1.5 Gases H y d r o g e n U l t r a H i g h P u r i t y ( U H P ) , h e l i u m U H P a n d a r g o n / m e t h a n e ( 9 5 : 5 ) w e r e o b t a i n e d f r o m M a t h e s o n G a s P r o d u c t s C a n a d a L t d . , E d m o n t o n , A B . 27 2.1.6. Supplies for animal studies T h e f o l l o w i n g s u p p l i e s w a s u s e d i n t h e a n i m a l s t u d i e s : n e e d l e s a n d p l a s t i c d i s p o s a b l e L u r e - L o k ® s y r i n g e s f o r d r u g a d m i n i s t r a t i o n a n d s a m p l e c o l l e c t i o n ( B e c t o n -D i c k i n s o n C a n a d a , M i s s i s s a u g a , O N ) ; h e p a r i n i z e d V a c u t a i n e r ® t u b e s ( V a c u t a i n e r S y s t e m s , R u t h e r f o r d , N J , U S A ) ; 1 5 m L P y r e x ® d i s p o s a b l e g l a s s c u l t u r e t u b e s ( C o m i n g G l a s s w o r k s , C o m i n g , N Y , U S A ) ; p o l y t e t r a f l u o r e t h y l e n e ( P T F E ) l i n e d s c r e w c a p s ( C a n l a b , V a n c o u v e r , B C ) ; s i l i c o n e r u b b e r t u b i n g f o r c a t h e t e r p r e p a r a t i o n ( D o w C o r n i n g , M i d l a n d , M I , U S A ) . 2.2. Stock and Reagent Solutions M e t o c l o p r a m i d e » H C l » H 2 0 ( = 1 1 . 8 2 m g o f M C P « H C 1 » H 2 0 i s e q u i v a l e n t t o = 1 0 m g o f M C P f r e e b a s e ) , m o n o d e e t h y l M C P » H C 1 » H 2 0 ( = 1 2 . 0 0 m g o f m d M C P « H C l » H 2 0 i s e q u i v a l e n t t o = 1 0 m g o f m d M C P f r e e b a s e ) a n d d i d e e t h y l M C P » H C 1 » H 2 0 ( = 1 2 . 2 4 m g o f d d M C P » H C l » H 2 0 i s e q u i v a l e n t t o = 1 0 m g o f d d M C P f r e e b a s e ) w e r e a c c u r a t e l y w e i g h e d a n d d i s s o l v e d i n H P L C g r a d e w a t e r i n i n d i v i d u a l v o l u m e t r i c f l a s k s . U s i n g s e r i a l d i l u t i o n , t h e s e s t o c k s o l u t i o n s w e r e a d d e d t o a c o m b i n e d s t o c k s o l u t i o n o f f i n a l c o n c e n t r a t i o n o f = 0 . 0 4 j i g / r n L e a c h . T h e i n t e r n a l s t a n d a r d , B M Y » H C 1 » H 2 0 ( = 1 1 . 0 3 m g o f B M Y * H C 1 » H 2 0 i s e q u i v a l e n t t o = 1 0 m g o f B M Y f r e e b a s e ) w a s a c c u r a t e l y w e i g h e d a n d d i s s o l v e d i n d e i o n i z e d w a t e r u s i n g s e r i a l d i l u t i o n t o a f i n a l c o n c e n t r a t i o n o f = 0 . 2 | i g / m L . T h e s e s t o c k s o l u t i o n s w e r e s t o r e d a t 4 ° C f o r u p t o t w o m o n t h s . S o d i u m h y d r o x i d e ( N a O H ) , a s I M a n d 5 M s o l u t i o n s , w a s p r e p a r e d b y d i s s o l v i n g N a O H p e l l e t s i n d e i o n i z e d w a t e r . A q u e o u s a m m o n i a ( 4 % ) s o l u t i o n w a s p r e p a r e d b y 28 d i l u t i n g a m m o n i a s o l u t i o n s t r o n g ( 2 7 % ) i n d e i o n i z e d w a t e r . T r i e t h y l a m i n e 0 . 0 1 2 5 M i n t o l u e n e w a s p r e p a r e d b y d i l u t i n g t r i e t h y l a m i n e w i t h t o l u e n e a n d s u b s e q u e n t l y a d d i n g f o u r o r f i v e N a O H p e l l e t s t o t h e r e s u l t i n g s o l u t i o n . A 0 . 2 M ( p H 5 . 0 ) s o d i u m a c e t a t e b u f f e r , f o r g l u c u r o n i d a s e i n c u b a t i o n , w a s p r e p a r e d b y d i s s o l v i n g s o d i u m a c e t a t e i n d e i o n i z e d w a t e r a n d a d j u s t i n g t h i s s o l u t i o n t o a f i n a l p H o f 5 . 0 u s i n g g l a c i a l a c e t i c a c i d . T R I S b u f f e r , 0 . 0 5 M ( p H 7 . 5 ) , f o r s u l p h a t a s e i n c u b a t i o n w a s p r e p a r e d b y d i s s o l v i n g T R I S f r e e b a s e i n d e i o n i z e d w a t e r a n d a d j u s t i n g t h e f i n a l p H o f t h i s s o l u t i o n t o 7 . 5 u s i n g a 1 M H C 1 s o l u t i o n . 2.3 Sample Preparation, Extraction and Derivatization T h e p r o c e d u r e f o r s a m p l e e x t r a c t i o n a n d d e r i v a t i z a t i o n r e m a i n e d t h e s a m e a s p u b l i s h e d b y R i g g s et al. ( 1 9 9 4 ) . A l l g l a s s w a r e u s e d i n t h e p r e p a r a t i o n o f s t o c k s o l u t i o n s a n d e x t r a c t i o n w a s p r e -w a s h e d w i t h d e t e r g e n t i n . a n a u t o m a t i c - d i s h w a s h e r a n d t h e n s o a k e d i n c h r o m i c a c i d f o r a m i n i m u m o f s i x h o u r s . T h e r e a f t e r , i t w a s t h o r o u g h l y r i n s e d w i t h t a p w a t e r o v e r 6 h o u r s u s i n g a m e c h a n i c a l r i n s e r a n d f i n a l l y w i t h d i s t i l l e d / d e i o n i z e d w a t e r b e f o r e b e i n g d r i e d o v e r n i g h t . 2.3.1 Sample extraction A l l e x t r a c t i o n s w e r e c a r r i e d o u t i n 1 5 m L a n d 1 0 r n L g l a s s c u l t u r e t u b e s w i t h P T F E - l i n e d s c r e w c a p s . P l a s m a ( 1 0 0 - 2 0 0 u L ) a n d u r i n e ( 1 0 0 - 2 0 0 j i L o f 5 0 x d i l u t i o n i n d e i o n i z e d w a t e r ) o b t a i n e d f r o m a n i m a l s t u d i e s w e r e a d d e d t o c l e a n 1 5 m L t u b e s 29 c o n t a i n i n g 0 . 5 m L I M N a O H ( p H = 1 4 ) a n d 0 . 3 m L o f B M Y i n t e r n a l s t a n d a r d s o l u t i o n ( = 0 . 2 j i g / m L ) . T h e a q u e o u s p h a s e w a s t h e n a d j u s t e d t o a f i n a l v o l u m e o f 2 . 2 m L w i t h d e i o n i z e d w a t e r . S i x m L o f d i c h l o r o m e t h a n e w a s a d d e d , t u b e s c a p p e d , a n d M C P a n d i t s s e l e c t i v e m e t a b o l i t e s a n d B M Y w e r e e x t r a c t e d i n t o t h e o r g a n i c l a y e r b y s h a k i n g f o r 2 0 m i n o n a r o t a r y s h a k e r ( L a b q u a k e ® m o d e l 4 1 5 - 1 1 0 , L a b I n d u s t r i e s , B e r k e l e y , C A , U S A ) . A f t e r s h a k i n g , t u b e s w e r e s t o r e d i n a f r e e z e r ( = - 2 0 ° C ) f o r 1 0 m i n . F o l l o w i n g c e n t r i f u g a t i o n a t 2 5 0 0 r p m f o r 5 m i n , t h e t u b e s w e r e r e m o v e d a n d s h a k e n l i g h t l y t o b r e a k a n y e m u l s i o n t h a t m a y h a v e f o r m e d d u r i n g t h e e x t r a c t i o n p r o c e s s . F o l l o w i n g a f u r t h e r 5 m i n o f c e n t r i f u g a t i o n , t h e a q u e o u s l a y e r w a s v a c u u m a s p i r a t e d a n d d i s c a r d e d . T h e r e m a i n i n g o r g a n i c l a y e r w a s t r a n s f e r r e d t o c l e a n 1 0 m L t u b e s u s i n g P a s t e u r p i p e t t e s a n d t a k e n t o d r y n e s s u s i n g a n A S 2 9 0 A u t o m a t i c S p e e d V a c ® c o n c e n t r a t o r ( S a v a n t I n s t r u m e n t s I n c . , F a r m i n g d a l e , N Y , U S A ) u n d e r c o n t r o l l e d v a c u u m . T h e d r i e d r e s i d u e w a s r e c o n s t i t u t e d w i t h 0 . 8 m L o f t o l u e n e c o n t a i n i n g 0 . 0 1 2 5 M T E A . A 2 0 u L v o l u m e o f h e p t a f l u o r o b u t y r i c a n h y d r i d e ( H F B A ) w a s a d d e d , t u b e s c a p p e d , v o r t e x - m i x e d ( V o r t e x -G e n i e ® , F i s h e r S c i e n t i f i c I n d u s t r i e s , S p r i n g f i e l d , M A , U S A ) a n d t h e s e m i x t u r e s w e r e p l a c e d i n a n o v e n a t 5 5 ° C f o r 6 0 m i n . A f t e r c o o l i n g t o r o o m t e m p e r a t u r e , t h e e x c e s s d e r i v a t i z i n g a g e n t ( H F B A ) w a s r e m o v e d b y h y d r o l y s i s w i t h 0 . 5 m L o f d e i o n i z e d w a t e r ( v o r t e x - m i x f o r 1 0 s e c ) a n d n e u t r a l i z e d w i t h 0 . 5 m L o f 4 % a m m o n i a s o l u t i o n ( v o r t e x - m i x f o r 1 0 s e c ) . F o l l o w i n g c e n t r i f u g a t i o n o f t h e t u b e s a t 2 5 0 0 r p m f o r 1 m i n , t h e t o l u e n e l a y e r w a s i m m e d i a t e l y t r a n s f e r r e d t o a c l e a n a u t o s a m p l e r v i a l w i t h a g l a s s i n s e r t a n d c a p p e d w i t h a P T F E - l i n e d a l u m i n u m s e a l . A 2 u L v o l u m e a l i q u o t w a s i n j e c t e d i n t o t h e G C - M S D f o r t h e f i n a l a s s a y m e a s u r e m e n t . 30 Scheme II Extraction procedure for MCP and its metabolites Plasma and urine samples (0.1-0.2 mL) Blank plasma or urine samples spiked with MCP/metabolite standard 1M NaOH, 0.5 mL BMY (I.S.), 0.3 mL Water q.s. 2.0 mL Dichloromethane, 6 mL Shake, 20 min Centrifuge, 2 X 5 min j under reduced pressure at 409C Dried extract 0.0125 M TEA in toluene, 0.8 mL HFBA, 20 uL 1 hour at 559C Cool to room temperature Water, 0.5 mL, vortex 10 sec 4% aq. ammonia, 0.5 mL, vortex 10 sec Centrifuge, 1 min Organic layer Aspirate aqueous layer Evaporate to dryness Discard aqueous layer Inject into GC/MSD (2 uL) 31 2.3.2 Analysis of glucuronide and sulphate conjugates T h e p r e s e n c e o f g l u c u r o n i d e a n d s u l p h a t e c o n j u g a t e s o f M C P , m d M C P a n d d d M C P i n u r i n e w a s d e t e r m i n e d b y e n z y m a t i c h y d r o l y s i s . T h e h y d r o l y s i s p r o c e d u r e u s e d i n t h e p r e s e n t s t u d y w a s a m o d i f i e d v e r s i o n o f B r a s h e r et al. ( 1 9 8 8 ) f o r t h e d e t e r m i n a t i o n o f r i t o d r i n e c o n j u g a t e s i n h u m a n s . U r i n e s a m p l e s w e r e d i v i d e d i n t o 3 a l i q u o t s o f 0 . 1 m L t o d e t e r m i n e t h e c o n c e n t r a t i o n s o f t h e c o m p o u n d s i n n o n - c o n j u g a t e d f o r m [ S e t I ] , g l u c u r o n i d e - c o n j u g a t e d f o r m [ S e t I I ] a n d s u l p h a t e - c o n j u g a t e d f o r m [ S e t I I I ] . Non-conjugated [Set I] : A v o l u m e o f 0 . 9 m L o f d e i o n i z e d w a t e r w a s a d d e d t o e a c h a l i q u o t . Glucuronide-conjugated [Set II] : V o l u m e s o f 0 . 4 m L 0 . 2 M s o d i u m a c e t a t e b u f f e r ( p H 5 . 0 ) a n d 0 . 5 m L o f g l u c u r o n i d a s e w e r e a d d e d t o e a c h a l i q u o t t o p r o v i d e a g l u c u r o n i d a s e a c t i v i t y o f 2 5 0 0 U / m L . Sulphate-conjugated [Set III] : A v o l u m e o f 0 . 9 m L o f d i l u t e d s u l p h a t a s e s o l u t i o n i n 0 . 0 5 M T R I S b u f f e r ( p H 7 . 5 ) w a s a d d e d e a c h a l i q u o t t o g i v e a f i n a l s u l p h a t a s e a c t i v i t y o f 0 . 2 5 U / m L . A l l t h e a l i q u o t s w e r e i n c u b a t e d o v e r n i g h t a t 3 7 ° C i n a w a t e r b a t h . F o l l o w i n g i n c u b a t i o n , t h e s a m p l e s w e r e c o o l e d t o r o o m t e m p e r a t u r e a n d e x t r a c t e d a s d e s c r i b e d i n S e c t i o n 2 . 3 . 1 . N o n - c o n j u g a t e d M C P / m d M C P / d d M C P w e r e m e a s u r e d f r o m t h e s a m p l e s i n S e t I . T h e c o n c e n t r a t i o n s o f g l u c u r o n i d e - c o n j u g a t e d M C P / m d M C P / d d M C P w e r e 32 c a l c u l a t e d b y d e d u c t i n g n o n - c o n j u g a t e d c o n c e n t r a t i o n s f r o m t h e s a m p l e s i n S e t I I . T h e c o n c e n t r a t i o n o f s u l p h a t e - c o n j u g a t e s w a s c a l c u l a t e d s i m i l a r l y f r o m t h e s a m p l e s i n S e t I I I . 2.4 Standard Curve Preparation V o l u m e s o f 0 . 0 2 , 0 . 0 4 , 0 . 0 8 , 0 . 1 6 , 0 . 3 2 , 0 . 6 4 a n d 0 . 8 0 m L o f m i x e d s t o c k s o l u t i o n o f M C P , m d M C P a n d d d M C P w e r e p i p e t t e d i n t o 1 5 m L t u b e s c o n t a i n i n g 0 . 5 m L 1 M N a O H , 0 . 3 m L o f i n t e r n a l s t a n d a r d B M Y s o l u t i o n ( 0 . 3 u , g / m L ) a n d c o r r e s p o n d i n g b l a n k b i o l o g i c a l f l u i d s . T h e a q u e o u s p h a s e w a s a d j u s t e d t o a f i n a l v o l u m e o f 2 . 2 m L w i t h d e i o n i z e d w a t e r a n d t h e n e x t r a c t e d a n d d e r i v a t i z e d a s d e s c r i b e d i n S e c t i o n 2 . 3 . A s t a n d a r d c u r v e f o r e a c h o f M C P , m d M C P a n d d d M C P w a s c a l c u l a t e d a n d p l o t t e d b y t h e p e a k a r e a r a t i o o f h e p t a f l u o r o b u t y r y l ( H F B ) d e r i v a t i v e s o f M C P , m d M C P a n d d d M C P o v e r B M Y v e r s u s t h e k n o w n c o n c e n t r a t i o n o f M C P , m d M C P a n d d d M C P f r e e b a s e . A w e i g h t i n g f a c t o r 1 / Y ( a r e a r a t i o ) w a s u s e d f o r l i n e a r r e g r e s s i o n o f t h e s t a n d a r d c u r v e s a c c o r d i n g t o t h e . m e t h o d u s e d i n R i g g s et al. ( 1 9 9 4 ) . 2.5 Instruments and Equipment 2.5.1 Gas chromatography (GC-MSD) A M o d e l 5 8 9 0 S e r i e s I I H e w l e t t - P a c k a r d g a s c h r o m a t o g r a p h e q u i p p e d w i t h a H P 5 9 7 1 A M a s s S e l e c t i v e D e t e c t o r ( M S D ) , s p l i t - s p l i t l e s s c a p i l l a r y i n l e t s y s t e m , a n d a W i n d o w s ® G C C h e m S t a t i o n o n a H P V e c t r a ® 2 5 T 4 8 6 c o m p u t e r ( H e w l e t t - P a c k a r d C o . , A v o n d a l e , P A ) w a s u s e d f o r t h e M S D a s s a y . A 2 5 m x 0 . 2 m m I . D . c r o s s - l i n k e d 5 % p h e n y l m e t h y l s i l i c o n e ( U l t r a - 2 ® ) 0 . 3 3 u \ m f i l m t h i c k n e s s f u s e d - s i l i c a c a p i l l a r y c o l u m n 33 ( H e w l e t t - P a c k a r d C o . , A v o n d a l e , P A ) , 7 8 m m x 2 m m I D . b o r o s i l i c a t e g l a s s i n l e t l i n e r s ( H e w l e t t - P a c k a r d C o . , A v o n d a l e , P A ) , a n d T h e r m o g r e e n ® L B - 2 s i l i c o n e r u b b e r s e p t a w e r e a l s o u s e d . 2.5.2 Operating conditions for the GC and MSD T h e o p e r a t i n g c o n d i t i o n s f o r t h e G C / M S D s y s t e m w e r e a s f o l l o w s : i n j e c t i o n p o r t t e m p e r a t u r e , 2 6 0 ° C ; p u r g e t i m e , 3 0 s e c ; s p l i t v e n t f l o w , 3 0 m L / r n i n ; i n i t i a l c o l u m n t e m p e r a t u r e , 1 0 0 ° C ; c o l u m n h e a d p r e s s u r e , 1 0 p . s . i . ; c a r r i e r g a s ( h e l i u m ) f l o w r a t e , 3 m L / m i n ; t e m p e r a t u r e p r o g r a m : 1 0 0 ° C h o l d f o r 0 . 8 m i n , t e m p e r a t u r e r a m p a t 4 0 ° C / m i n t o 2 7 0 ° C t h e n h o l d c o n s t a n t f o r 5 m i n , a n d t h e n a f u r t h e r i n c r e a s e t o 2 9 0 ° C a t 7 0 ° C / m i n a n d h o l d f o r 3 m i n , f o l l o w e d b y a f u r t h e r i n c r e a s e t o 3 0 0 ° C a t 7 0 ° C / m i n a n d a final h o l d f o r 3 . 5 m i n t o c l e a n o u t t h e c o l u m n a f t e r e a c h r u n ; T o t a l r u n t i m e , 1 6 . 9 8 m i n ; M S D t r a n s f e r l i n e a n d s o u r c e t e m p e r a t u r e , 3 1 0 ° C . A l i q u o t s o f 2 u L a r e i n j e c t e d o n t o t h e c o l u m n . A H P 5 9 7 1 A M a s s S e l e c t i v e D e t e c t o r w a s u s e d i n e l e c t r o n i m p a c t ( E I ) i o n i z a t i o n m o d e w i t h - 7 0 e V i o n i z a t i o n e n e r g y a n d 3 0 0 u A e m i s s i o n c u r r e n t . T h e M S D w a s m a n u a l l y t u n e d t o t h e i o n s o f m/e 1 0 0 , 2 6 4 a n d 4 1 4 u s i n g t h e s t a n d a r d p e r f l u o r o t r i b u t y l a m i n e . C h r o m a t o g r a m s w e r e g e n e r a t e d i n b o t h m a s s s c a n n i n g ( S C A N ) a n d s e l e c t i v e i o n m o n i t o r i n g ( S I M ) m o d e s i n t h e s t u d y . F o r t h e q u a n t i t a t i v e a s s a y , M C P a n d i t s m e t a b o l i t e s w e r e o p t i m a l l y d e t e c t e d i n S I M m o d e u s i n g t h e t o t a l i o n c u r r e n t o f m/e 3 8 0 f o r d d M C P a n d m d M C P ( G r o u p 1 ) , m/e 8 6 a n d 3 8 0 f o r M C P ( G r o u p 2 ) a n d m/e 8 6 a n d 3 6 6 f o r B M Y ( G r o u p 3 ) . T h e d w e l l t i m e w a s s e t a t 7 0 0 m s e c , p e r g r o u p , w h i c h p r o v i d e d 1 . 3 7 s c a n c y c l e / s e c f o r G r o u p 1 a n d 0 . 6 9 7 s c a n c y c l e / s e c f o r G r o u p s 2 a n d 3 . 34 2.6 Animal preparation 2.6.1 Animal handling T h e e w e s ( D o r s e t , S u f f o l k , o r c r o s s b r e d ) w e r e b r o u g h t t o t h e A n i m a l U n i t i n t h e C h i l d r e n ' s V a r i e t y R e s e a r c h C e n t r e a b o u t o n e w e e k p r i o r t o s u r g e r y t o a l l o w t h e m t o b e c o m e a c c l i m a t i z e d . T h e e w e s w e r e f a s t e d o v e r n i g h t b e f o r e s u r g e r y a n d e a c h r e c e i v e d a 3 - m g i . v . i n j e c t i o n o f a t r o p i n e s u l f a t e ( A s t r a P h a r m a c e t i c a l s I n c . , M i s s i s s a u g a , O N ) 1 0 - 1 5 m i n p r i o r t o i n d u c t i o n o f a n a e s t h e s i a w i t h P e n t o t h a l ( 1 m g / k g , i . v . ; A b b o t t L a b o r a t o r y , M o n t r e a l , Q u e . ) . F o l l o w i n g e n d o t r a c h e a l i n t u b a t i o n , a n a e s t h e s i a w a s m a i n t a i n e d b y v e n t i l a t i n g t h e e w e s t h r o u g h o u t t h e s u r g e r y w i t h 1 . 0 - 1 . 5 % h a l o t h a n e ( A y e r s t L a b o r a t o r y , M o n t r e a l , Q u e . ) a n d 6 0 - 7 0 % N 2 0 i n o x y g e n . 2.6.2. Surgical Preparation A s e p t i c t e c h n i q u e s w e r e e m p l o y e d t h r o u g h o u t s u r g e r y . S t e r i l e s i l i c o n e r u b b e r c a t h e t e r s ( D o w C o r n i n g , C o m i n g , N Y ) , f i l l e d w i t h h e p a r i n i z e d 0 . 9 % s a l i n e , w e r e i m p l a n t e d i n t h e f e m o r a l v e i n a n d a r t e r y . I n s o m e a n i m a l s , a n u l t r a s o u n d t r a n s i t - t i m e f l o w p r o b e ( T r a n s o n i c S y s t e m s I n c . , I t h a c a , N Y , U S A ) w a s i m p l a n t e d o n t h e l e f t f e m o r a l a r t e r y . T h e n , a n o b l i q u e i n c i s i o n w a s m a d e i n t h e a b d o m e n t o t h e r i g h t o f t h e u m b i l i c u s , a n d t h e l i v e r a n d b o w e l w e r e r e t r a c t e d t o e x p o s e t h e g a l l b l a d d e r . T h e g a l l b l a d d e r w a s h e l d s t e a d y w i t h a n a r t e r y f o r c e p s , a n d a s m a l l i n c i s i o n w a s m a d e i n t h e a p e x , t h r o u g h w h i c h a s i l i c o n e r u b b e r c a t h e t e r w a s i n s e r t e d . T h e c a t h e t e r w a s s e c u r e d b y a p u r s e s t r i n g s u t u r e w h i c h a l s o c l o s e d t h e g a l l b l a d d e r i n c i s i o n . E x c e p t d u r i n g e x p e r i m e n t s , t h i s 35 c a t h e t e r w a s k e p t s e a l e d t o a l l o w f o r t h e n o r m a l d r a i n a g e o f b i l e t h r o u g h t h e b i l e d u c t . H o w e v e r , t h e s e f l o w p r o b e s a n d b i l e c a t h e t e r s w e r e u s e d i n t h e o t h e r s t u d i e s . A l l c a t h e t e r s w e r e f i l l e d w i t h h e p a r i n i z e d s a l i n e ( 1 2 U / m L ) , t u n n e l e d s u b c u t a n e o u s l y , a n d e x t e r i o r i z e d t h r o u g h a s m a l l i n c i s i o n i n t h e r i g h t f l a n k o f t h e e w e w h e r e t h e y w e r e s t o r e d i n a c l o t h p o u c h . T h e a b d o m i n a l i n c i s i o n w a s c l o s e d i n l a y e r s . A c a t h e t e r f o r n i t r o g e n i n f u s i o n ( 1 0 . 0 m m o . d . ) ( F i s h e r S c i e n t i f i c , C a m b r i d g e , M A , U S A ) w a s t h e n i m p l a n t e d i n t h e t r a c h e a , b e t w e e n a d j a c e n t t r a c h e a l r i n g s , 5 - 6 c m b e l o w t h e l a r y n x , a n d i n s e r t e d f o r 4 - 5 c m ; t h i s c a t h e t e r d i d n o t a f f e c t n o r m a l b r e a t h i n g b y t h e e w e ( G l e e d et al., 1 9 8 6 ) . D u r i n g s u r g e r y , a n i . v . d r i p o f 5 % d e x t r o s e s o l u t i o n ( 5 0 0 m L ; B a x t e r C a n a d a , T o r o n t o , O N ) w a s a d m i n i s t e r e d . I m m e d i a t e l y f o l l o w i n g s u r g e r y , a m p i c i l l i n ( 5 0 0 m g ; N o v o p h a r m L t d . , T o r o n t o , O N ) a n d g e n t a m i c i n ( 4 0 m g ; G a r a m y c i n ® ; S c h e r i n g , P o i n t e C l a i r e , Q u e . ) w e r e a d m i n i s t e r e d i . m . t o t h e e w e . F o l l o w i n g s u r g e r y , t h e e w e s w e r e k e p t i n h o l d i n g p e n s w i t h o t h e r s h e e p a n d g i v e n f r e e a c c e s s t o f o o d a n d w a t e r . A m p i c i l l i n ( 5 0 0 m g ) a n d g e n t a m i c i n ( 4 0 m g ) i . m . w e r e a l s o g i v e n p r o p h y l a c t i c a l l y t o t h e e w e f o r 5 d a y s f o l l o w i n g s u r g e r y . A l l a n i m a l s w e r e a l l o w e d t o r e c o v e r f o r a m i n i m u m o f 3 d a y s b e f o r e e x p e r i m e n t s . 36 2 . 7 Experimental Protocol O n e x p e r i m e n t a l d a y s , t h e e w e w a s p l a c e d i n a m o n i t o r i n g c a g e a d j a c e n t t o t h e h o l d i n g p e n i n f u l l v i e w o f c o m p a n i o n e w e s a n d w i t h f r e e a c c e s s t o f o o d a n d w a t e r . A F o l e y ® c a t h e t e r ( B a r d U r o l o g i c a l D i v . , C R B a r d I n c . , C o n i n g t o n , G A , U S A ) w a s i n s e r t e d via t h e u r e t h r a f o r c o n t i n u o u s u r i n e c o l l e c t i o n . M e t o c l o p r a m i d e h y d r o c h l o r i d e ( R e g l a n ® i n j e c t a b l e 5 m g / m L , A . H . R o b i n s , M o n t r e a l , Q u e . ) w a s d i l u t e d w i t h s t e r i l e i s o t o n i c s a l i n e t o 3 m g / m L . T h e i n f u s i o n [ r a t e = 0 . 2 1 m g ( 0 . 0 7 m L ) / m i n ] w a s p r e c e d e d b y a l o a d i n g d o s e o f 1 5 m g ( 5 m L ) g i v e n via t h e f e m o r a l v e i n . A f t e r r e a c h i n g s t e a d y - s t a t e d u r i n g n o r m o x i a ( 2 h r s . ) , h y p o x e m i a w a s i n d u c e d b y g i v i n g 7 L / m i n o f n i t r o g e n t h r o u g h t h e i m p l a n t e d t r a c h e a l c a t h e t e r . T h e b l o o d g a s s t a t u s w a s m o n i t o r e d b y t a k i n g a r t e r i a l b l o o d s a m p l e s a t t h e i n t e r v a l s d e s c r i b e d i n t h e s a m p l i n g s c h e d u l e . T h e n i t r o g e n f l o w r a t e w a s a d j u s t e d t o m a i n t a i n t h e h y p o x e m i a a t t h e d e s i r e d l e v e l o f P a 0 2 (i.e. 5 5 - 6 0 m m H g ) . T h e h y p o x e m i c p e r i o d w a s c o n t i n u e d f o r 6 h o u r s . T h e n , t h e n i t r o g e n f l o w w a s s t o p p e d a n d t h e i n f u s i o n w a s c o n t i n u e d f o r a n o t h e r 6 h o u r s . T h e t o t a l i n f u s i o n t i m e o f M C P w a s t h u s 1 4 h o u r s . A r t e r i a l b l o o d ( 2 . 5 m L ) s a m p l e s f o r M C P d e t e r m i n a t i o n a l o n g w i t h b l o o d g a s s a m p l e s ( ~ 0 . 7 - 1 . 0 m L ) w e r e t a k e n a s s h o w n i n t h e p r o t o c o l [ S c h e m e I V ] . T h e b l o o d s a m p l e s f o r M C P d e t e r m i n a t i o n w e r e t r a n s f e r r e d i m m e d i a t e l y t o a h e p a r i n i z e d V a c u t a i n e r ® ( B e c t o n - D i c k i n s o n , R u t h e r f o r d , N J ) a n d c e n t r i f u g e d a t 3 5 0 0 r p m f o r 1 0 m i n . U r i n e s a m p l e s w e r e c o l l e c t e d a c c o r d i n g t o t h e p r o t o c o l . T h e v o l u m e a n d p H o f u r i n e w e r e m e a s u r e d w i t h a g r a d u a t e d c y l i n d e r a n d a F i s h e r A c c u m e t ® p H m e t e r m o d e l 6 2 0 ( F i s h e r S c i e n t i f i c I n c . , C a m b r i d g e , M A , U S A ) . P l a s m a a n d u r i n e s a m p l e s w e r e 37 t r a n s f e r r e d t o d i s p o s a b l e b o r o s i l i c a t e g l a s s c u l t u r e t u b e s ( C o r n i n g G l a s s , C o r n i n g , N Y ) w i t h T e f l o n o r P T F E - l i n e d s c r e w c a p s a n d s t o r e d a t -20 ° C u n t i l t h e t i m e o f a s s a y . C o n t r o l e x p e r i m e n t s w e r e a l s o c a r r i e d o u t , u s i n g t h e s a m e M C P i n f u s i o n a n d s a m p l i n g p r o t o c o l , b u t w i t h o u t h y p o x e m i a . T h e f o l l o w i n g a r e t h e s c h e m a t i c d i a g r a m s o f e x p e r i m e n t a l p r o t o c o l u s e d i n t h e p r e s e n t s t u d y . Scheme III Schematic diagrams of the experimental protocol 1. Experimental Group HourO 2 8 14 Pre-hypoxemic (normoxemic) Hypoxemic Post-hypoxemic (normoxemic) 2. Control Group HourO 2 8 14 Pre-hypoxemic Post-hypoxemic 3 V . , Normoxemic , ' . . (normoxemic) (normoxemic) 38 S c h e m e IV M C P infusion-hypoxemia sampling protocol [Pre-hypoxemic peroid (2 hours)] 0:05 BL BG 0:00 Loading dose (15 mg)/lnfusion (rate U 0:05 BL BG 0:15 BL 0:30 BL BG U 0:45 BL 1:00 BL BG U 1:15 BL 1:30 BL 2:00 BL BG U blood controls n.) fluid controls : hypoxemia starts [Hypoxemic period (6 hours)] * rate of N2 flow = 7 L/min. initially and increased to 11 L/min. 2:05 BL BG 2:30 BG 3:00 BL BG U 4:00 BL BG U 5:00 BL BG U 6:00 BL BG U 7:00 BL BG U 7:15 BL BG 7:30 BL BG 8:00 BL BG U hypoxemia ends [Post-hypoxemic period (6 hours)] 8:05 BL BG 8:30 BG 9:00 BL BG U 10:00 BL BG U 11:00 BL BG U 12:00 BL BG U 13:00 BL BG U 13:15 BL BG 13:30 BL BG 14:00 BL BG U : infusion ends * BL : plasma sample, BG: blood gas sample, U : urine sample. 39 2.8 Recording Procedures and Blood Gas Analysis F o r 2 4 h o u r s b e f o r e , d u r i n g a n d 2 4 h o u r s f o l l o w i n g M C P a d m i n i s t r a t i o n , a r t e r i a l p r e s s u r e w a s m e a s u r e d u s i n g a d i s p o s a b l e s t r a i n - g a u g e D T X t r a n s d u c e r ( S t a t h a m m o d e l P 2 3 D b , G o u l d I n c . , O x n a r d , C A ) . H e a r t r a t e w a s m e a s u r e d f r o m t h e a r t e r i a l p u l s e p r e s s u r e u s i n g a c a r d i o t a c h o m e t e r ( M o d e l 9 8 5 7 , S e n s o r m e d i c C o r p . , A n a h e i m , C A ) . T h e s e v a r i a b l e s w e r e r e c o r d e d o n a p o l y g r a p h r e c o r d e r ( B e c k m a n R 6 1 2 r e c o r d e r , B e c k m a n , S c h i k k e r P a r k , I L o r G o u l d T A 4 0 0 0 t h e r m a l a r r a y r e c o r d e r , G o u l d I n c . , V a l l e y V i e w , O H ) . T h e a n a l o g s i g n a l s o f a r t e r i a l p r e s s u r e a n d h e a r t r a t e w e r e c o n v e r t e d s i m u l t a n e o u s l y t o d i g i t a l d a t a u s i n g a n o n - l i n e c o m p u t e r s y s t e m c o n s i s t i n g o f a n A p p l e H e ® c o m p u t e r , I n t e r a c t i v e s y s t e m s ( D a i s y E l e c t r o n i c s , N e w t o n S q u a r e , P A ) , a n d A - D c o n v e r t e r a n d c l o c k c a r d ( M o u n t a i n S o f t w a r e , S c o t t ' s V a l l e y , C A ) w i t h a s a m p l i n g r a t e o f 1 5 H z a n d a v e r a g e d o v e r 1 m i n . T h e r e c o r d i n g s w e r e a n a l y s e d t o p r o v i d e a n e s t i m a t e o f a r t e r i a l p r e s s u r e a n d h e a r t r a t e a v e r a g e d o v e r 1 m i n i n t e r v a l s . S a m p l e s ( = 1 m L ) f o r b l o o d g a s a n a l y s i s a n d g l u c o s e / l a c t a t e m e a s u r e m e n t w e r e t a k e n s i m u l t a n e o u s l y w i t h t h o s e f o r d r u g a n a l y s i s . A r t e r i a l P a 0 2 , P a C 0 2 , b a s e e x c e s s a n d p H w e r e m e a s u r e d w i t h a n J L 1 3 0 6 p H / b l o o d g a s a n a l y z e r ( A l l i e d I n s t r u m e n t a t i o n L a b o r a t o r y , M i l a n , I t a l y ) s e t a t a t e m p e r a t u r e o f 3 7 . 0 ° C a n d c o r r e c t e d t o 3 9 . 0 ° C . O x y g e n s a t u r a t i o n a n d h e m o g l o b i n c o n t e n t w e r e m e a s u r e d , i n d u p l i c a t e , u s i n g a n O S M 2 H e m o x i m e t e r ® ( R a d i o m e t e r , C o p e n h a g e n , D e n m a r k ) . B l o o d g l u c o s e a n d l a c t a t e c o n c e n t r a t i o n s w e r e m e a s u r e d , i n t r i p l i c a t e , u s i n g a g l u c o s e / l a c t a t e 2 3 0 6 S T A T p l u s a n a l y z e r ( Y S I I n c . , Y e l l o w S p r i n g , O H , U S A ) . O s m o l a l i t y o f u r i n e w a s m e a s u r e d b y 40 f r e e z i n g - p o i n t d e p r e s s i o n m e t h o d u s i n g A d v a n c e d D i g i M a t i c ® O s m o m e t e r M o d e l 3 D 2 ( A d v a n c e d I n s t r u m e n t s , N o r w o o d , M A , U S A ) . 2.9 Data Analysis 2.9.1. Determination of steady-state drug concentration S t e a d y - s t a t e M C P c o n c e n t r a t i o n w a s d e t e r m i n e d a c c o r d i n g t o t h e f o l l o w i n g p r o c e d u r e s a n d c r i t e r i a : 1. Visual inspection : t h e p l o t o f p l a s m a c o n c e n t r a t i o n versus t i m e w a s v i s u a l l y i n s p e c t e d f o r a p l a t e a u p o r t i o n u s i n g a s t r a i g h t e d g e r u l e r . 2 . Coefficient of variance : t h e c o e f f i c i e n t o f v a r i a t i o n f o r t h e d a t a s e t o f M C P c o n c e n t r a t i o n s i n t h e p l a t e a u p o r t i o n w a s c a l c u l a t e d . W i t h a m a x i m u m c r i t e r i o n o f C V = 1 0 %, a l l e x t r e m e o u t l i e r ( s ) w e r e e l i m i n a t e d f r o m t h e d a t a s e t . 3. Student's t-test/Analysis of Variance (ANOVA) for the regression : t h e s l o p e o f l i n e a r r e g r e s s i o n l i n e f r o m t h e d a t a s e t o f p l a s m a M C P c o n c e n t r a t i o n i n t h e p l a t e a u p o r t i o n s w a s a n a l y z e d u s i n g a t w o - t a i l e d t - t e s t a n d A N O V A f o r t h e c o r r e l a t i o n w i t h t h e n u l l h y p o t h e s i s ( H o ) : s l o p e = 0 a g a i n s t t h e a l t e r n a t i v e h y p o t h e s i s ( H A ) : s l o p e * 0 w i t h a l p h a = 0 . 0 5 . I n t h e s e t e s t s , t h e r e j e c t i o n o f H o s u g g e s t s t h a t t h e p l a s m a d r u g c o n c e n t r a t i o n t e n d s t o e i t h e r i n c r e a s e o r d e c r e a s e i n a g i v e n p e r i o d (i.e. a s t e a d y - s t a t e w a s n o t a c h i e v e d ) . 41 2.9.2. Calculation of pharmacokinetic parameters T o t a l b o d y c l e a r a n c e (Ch) w a s e s t i m a t e d a s : CU = ko / Css w h e r e ko i s t h e i n f u s i o n r a t e a n d Css i s t h e a p p a r e n t a r t e r i a l s t e a d y - s t a t e c o n c e n t r a t i o n . R e n a l c l e a r a n c e v a l u e s o f M C P a n d m d M C P w e r e c a l c u l a t e d f r o m 1) d i v i d i n g t h e a c c u m u l a t e d d r u g / m e t a b o l i t e r e c o v e r e d i n u r i n e (Du) b y a r e a u n d e r t h e p l a s m a d r u g / m e t a b o l i t e c o n c e n t r a t i o n s c u r v e ( A U C ) a s a f u n c t i o n o f t i m e d u r i n g t h e h y p o x e m i c a n d n o r m o x e m i c p e r i o d s [Du(t2-t1)/AUC(t2-t1)] a n d 2) c a l c u l a t i o n u s i n g t h e s l o p e o f t h e a c c u m u l a t e d d r u g / m e t a b o l i t e i n u r i n e (Du) v e r s u s A U C ( s e e A p p e n d i x A f o r t h e e q u a t i o n d e r i v a t i o n ) . T h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t o f M C P (fu = ku / w a s c a l c u l a t e d b y d i v i d i n g t h e s l o p e o f t h e a s y m p t o t e o f t h e a c c u m u l a t e d M C P i n u r i n e v e r s u s t i m e c u r v e w i t h t h e i n f u s i o n r a t e , ko. Du = (k*ko/KE)t - (ku*k„/KE2) t h e s l o p e o f t h e a s y m p t o t e = ku*ko/KE w h e r e ku i s t h e r e n a l e x c r e t i o n r a t e , a n d Du i s t h e c u m u l a t i v e a m o u n t o f i n t a c t d r u g i n u r i n e . S i n c e ko i s g i v e n , t h e r a t i o (ku/ KE) c a n b e c a l c u l a t e d . T h e d e t a i l e d e q u a t i o n d e r i v a t i o n s a r e s h o w n i n a p p e n d i x A a n d t h e o r e t i c a l a n d p r a c t i c a l d i s c u s s i o n i s d e s c r i b e d i n t h e s e c t i o n 4.4.2. T h e f r a c t i o n a l r e n a l m e t a b o l i t e e x c r e t i o n c o n s t a n t s f o r m d M C P , d d M C P a n d c o n j u g a t e s w e r e a l s o c a l c u l a t e d i n t h e s t u d y . T h e s e p a r a m e t e r s a r e t h e p r o d u c t o f t w o 42 f r a c t i o n a l c o n s t a n t fm(metaboiite) a n d fmu(metaboiites). T h e m e t a b o l i t e f o r m a t i o n f r a c t i o n c o n s t a n t fm(metaboiue) i s t h e f r a c t i o n o f kflmetaboiue)/ K.E(Parent drug), w h i c h r e p r e s e n t s t h e m e t a b o l i c e l i m i n a t i o n p r o p o r t i o n ( t o a s p e c i f i c m e t a b o l i t e ) o f t h e t o t a l p a r e n t d r u g e l i m i n a t i o n . T h e s e c o n d f r a c t i o n a l c o n s t a n t , t h e r e n a l m e t a b o l i t e e x c r e t i o n f r a c t i o n fmu(metaboiite) i s t h e f r a c t i o n o f kmu(metabolite)/ Km(metabolite), w h i c h r e p r e s e n t s t h e r e n a l e x c r e t i o n p r o p o r t i o n o f t h e t o t a l m e t a b o l i t e e l i m i n a t i o n . T h e r e f o r e t h e c o m p o s i t e f r a c t i o n a l c o n s t a n t , t h e f r a c t i o n a l r e n a l m e t a b o l i t e e x c r e t i o n c o n s t a n t fu(metaboiite) fm(metaboiite)fmu(metaboiite), r e p r e s e n t s t h e p r o p o r t i o n o f r e n a l e x c r e t i o n o f a s p e c i f i c m e t a b o l i t e f r o m t h e t o t a l d r u g e l i m i n a t i o n . 2.9.3 Statistical tests S t a t i s t i c a l e v a l u a t i o n s w e r e p e r f o r m e d o n v a r i o u s p h a r m a c o k i n e t i c a n d p h y s i o l o g i c a l p a r a m e t e r s u s i n g e i t h e r S t u d e n t ' s t - t e s t ( p a i r e d o r u n p a i r e d ) , F - t e s t , A N O V A ( A n a l y s i s o f V a r i a n c e ) a n d T u k e y t e s t . T h e l e v e l o f s i g n i f i c a n c e w a s c h o s e n p < 0 . 0 5 . T h e o r y a n d f o r m u l a e u s e d f o r s t a t i s t i c a l - a n a l y s i s w e r e o b t a i n e d f r o m Z a r - ( 1 9 8 4 ) , a n d M i c r o s o f t E x c e l ® f o r W i n d o w s ® p r o g r a m ( M i c r o s o f t C o r p . , R e d m o n d , W A , U S A ) w i t h A n a l y s i s T o o l s ® a n d S l i d e W r i t e P l u s ® f o r W i n d o w s ® ( A d v a n c e d G r a p h i c s S o f t w a r e I n c . , C a r l s b a d , C A , U S A ) w e r e u s e d f o r d a t a p r o c e s s i n g / a n a l y s i s a n d g r a p h i c a l p r e s e n t a t i o n . T h e m e a n v a l u e s i n t h e t e x t a n d t a b l e s a r e p r e s e n t e d a s t h e m e a n ± s t a n d a r d e r r o r o f t h e m e a n ( S E M ) , u n l e s s o t h e r w i s e d e s c r i b e d . 43 3. RESULTS 3.1. Quantitative Analytical Assay Methods for Metoclopramide and Selected Metabolites. 3.1.1. GC-MSD method for the quantitative analysis of MCP and selected metabolites. F o r t h e q u a n t i t a t i v e a n a l y s i s o f M C P , m o n o d e e t h y l M C P ( m d M C P ) a n d d i d e e t h y l M C P ( d d M C P ) , a G C - M S D m e t h o d o f R i g g s et al. ( 1 9 9 4 ) w a s u s e d f o r i t s h i g h s e n s i t i v i t y , s e l e c t i v i t y a n d r e p r o d u c i b i l i t y . R e p r e s e n t a t i v e c h r o m a t o g r a m s o f M C P , m d M C P a n d d d M C P ( 1 n g / m L e a c h ) i n p l a s m a a n d b l a n k e x t r a c t s f r o m p l a s m a a n d u r i n e a r e s h o w n i n F i g u r e 1 . T h e s t e p - l i k e s h i f t i n g o f t h e b a s e l i n e o f t h e c h r o m a t o g r a m s a t 1 0 . 2 5 m i n r e p r e s e n t s a c h a n g e i n t h e b a c k g r o u n d i o n c u r r e n t a t t h e S I M g r o u p s w i t c h i n g (i.e. f r o m G r o u p 1 [m/e 3 8 0 ] t o G r o u p 2 [m/e 8 6 a n d 3 8 0 ] ) . A n a d d i t i o n a l g r o u p s w i t c h i n g o c c u r s w i t h G r o u p 3 [m/e 8 6 & 3 6 6 ] a t 1 1 . 5 0 m i n w i t h n o v i s i b l e c h a n g e i n b a s e l i n e . I n s p i t e o f t h e s e c h a n g e s i n t h e s e l e c t i v e i o n m o n i t o r i n g g r o u p , t h e b a s e l i n e r e m a i n e d s t a b l e t h r o u g h o u t t h e a s s a y . C h r o m a t o g r a m s f r o m u r i n e a n d b i l e a r e s i m i l a r t o t h o s e f r o m p l a s m a . T h e i n d i v i d u a l r e t e n t i o n t i m e s o f t h e H F B - d e r i v a t i v e s o f d d M C P , m d M C P , M C P a n d t h e i n t e r n a l s t a n d a r d B M Y w e r e 9 . 2 9 m i n , 1 0 . 1 3 m i n , 1 0 . 3 8 m i n a n d 1 2 . 3 3 m i n , r e s p e c t i v e l y . N o s i g n o f s i g n i f i c a n t i n t e r f e r e n c e f r o m t h e e n d o g e n o u s c o m p o u n d s w a s s h o w n i n a n y o f t h e b i o l o g i c a l f l u i d s c o l l e c t e d i n t h e s t u d y . F i g u r e 2 ( A - D ) s h o w s t h e m a s s s p e c t r a o f M C P , m o n o d e e t h y l M C P ( m d M C P ) , d i d e e t h y l M C P ( d d M C P ) a n d t h e i n t e r n a l s t a n d a r d , B M Y , r e s p e c t i v e l y . B o t h M C P a n d B M Y u n d e r g o e x t e n s i v e f r a g m e n t a t i o n r e s u l t i n g i n a b a s e p e a k o f m/e 8 6 ( F i g u r e 2 , C a n d D ) . I n a d d i t i o n t o t h e i o n o f m/e 8 6 , a l e s s a b u n d a n t i o n o f m/e 3 8 0 a n d 3 6 6 , 44 r e s p e c t i v e l y , w a s a l s o m o n i t o r e d f o r M C P a n d B M Y , t o e n h a n c e t h e s e l e c t i v i t y . T h e b a s e p e a k o f i o n m/e 3 8 0 w a s u s e d f o r d d M C P a n d m d M C P . [Abundance 7000 6000 5000 4000 3000 2000 1000 0 (Time ->9. 12 . 33 MCP ddMCP 9 . 29 A mdMCP 10.38 A -10.13 BMY T 1 1 1 1 1 1 1 1~| 1 1 1 I J 1 00 9.50 10.00 10.50 1 1 1 1 1 1 I 1 1 1 1 I 1 1 1 1 i ' 11.00 11.50 12.00 12.50 2000 A 1000 Blank plasma i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i i ' i i 1 1 1 i 1 time ->9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 2 0 0 0 1 0 0 0 Blank urine i — i —i—i—|— i —i— i — i —|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i ' ' i i 1 [Time ->9 .00 9 .50 10 .00 10 .50 11 .00 1 1 . 5 0 1 2 . 0 0 1 2 . 5 0 F i g u r e 1 . R e p r e s e n t a t i v e t o t a l i o n c h r o m a t o g r a m s ( S I M m o d e ) o f 1 n g / m L d d M C P , m d M C P , M C P a n d 3 3 . 3 n g / m L B M Y a d d e d t o 0 . 3 m L o f p l a s m a . S u p e r i m p o s e d c h r o m a t o g r a m s o f b l a n k p l a s m a a n d u r i n e a r e a l s o s h o w n . 45 2 c S .2 5 J3 -5 — c c c3 CD C y o CQ cn § < c oo < -o o- .S U 2 15 •£> O ed e c u c 3 T 3 C O 0) ca U DO — tn C C | B S cn ' CN 3 60 46 3.1.2. Calibration (standard) curve C a l i b r a t i o n ( s t a n d a r d ) c u r v e s w e r e o b t a i n e d b y a n a l y z i n g b l a n k p l a s m a , u r i n e o r b i l e s p i k e d w i t h v a r y i n g a m o u n t s o f M C P , m d M C P a n d d d M C P ( 1 , 2 , 4 , 8 , 1 6 , 2 4 , 3 2 a n d 4 0 n g / m L o f e a c h ) a n d p l o t t i n g t h e a r e a r a t i o o f t h e h e p t a f l u o r o b u t y r i c ( H F B ) d e r i v a t i v e s o f M C P / B M Y , m d M C P / B M Y a n d d d M C P / B M Y a g a i n s t t h e i n d i c a t e d M C P , m d M C P a n d d d M C P c o n c e n t r a t i o n s , r e s p e c t i v e l y [ F i g u r e 3 ] . T h e d a t a f o r a r e p r e s e n t a t i v e c a l i b r a t i o n u s e d i n t h e q u a n t i t a t i o n o f M C P , m d M C P a n d d d M C P f r o m p l a s m a a r e p r e s e n t e d i n T a b l e 1 . L i n e a r i t y i s o b s e r v e d o v e r t h e c o n c e n t r a t i o n r a n g e s t u d i e d ( 1 - 4 0 n g ) w i t h a c o e f f i c i e n t o f v a r i a t i o n ( C V ) < 1 5 % f o r a l l t h e d a t a p o i n t s c o m p o s i n g t h e c u r v e s o f d d M C P , m d M C P a n d M C P . T h e l i m i t o f q u a n t i t a t i o n ( L O Q ) w a s 1 n g / m L f o r a l l t h r e e c o m p o u n d s i n t h e b i o l o g i c a l f l u i d s c o l l e c t e d i n t h e s t u d y . T h e c o e f f i c i e n t o f v a r i a t i o n w a s r e l a t i v e l y l o w e r i n t h e m i d - r a n g e o f t h e c a l i b r a t i o n c u r v e a n d h i g h e r a t e i t h e r t h e l o w - o r h i g h - e n d r a n g e o f t h e c u r v e . T h e c o e f f i c i e n t s o f r e g r e s s i o n a n d t h e l i n e o f b e s t - f i t t h r o u g h t h e d a t a p o i n t s w a s d e t e r m i n e d f r o m l i n e a r r e g r e s s i o n w i t h a w e i g h t i n g f a c t o r o f 1 / Y . 3.1.3. Enzyme incubation T h e e n z y m e a c t i v i t i e s o f g l u c u r o n i d a s e a n d s u l p h a t a s e w e r e t e s t e d f o l l o w i n g t h e s t a n d a r d p r o c e d u r e s p r o v i d e d b y t h e s u p p l i e r u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s d e s c r i b e d i n S e c t i o n 2 . 3 . I n o r d e r t o e v a l u a t e t h e e f f e c t o f g l u c u r o n i d a s e a n d s u l p h a t a s e i n c u b a t i o n o n t h e q u a n t i t a t i o n o f M C P a n d i t s m e t a b o l i t e s , 3 s e t s o f c a l i b r a t i o n c u r v e s ( n o e n z y m e i n c u b a t i o n , g l u c u r o n i d a s e i n c u b a t i o n a n d s u l p h a t a s e i n c u b a t i o n ) w e r e p r e p a r e d a n d e x t r a c t e d u s i n g t h e s a m e p r o c e d u r e s l i s t e d i n S e c t i o n 2 . 3 . S i n c e b i o l o g i c a l s a m p l e s s p i k e d w i t h s t a n d a r d d r u g s 47 d o n o t c o n t a i n a n y c o n j u g a t e s o f M C P , m d M C P a n d d d M C P , t h e c a l i b r a t i o n c u r v e s f r o m e a c h s e t w o u l d n o t s h o w a n y d i f f e r e n c e u n l e s s e n z y m e i n c u b a t i o n s h a v e a f f e c t e d t h e s a m p l e s . T a b l e 2 l i s t s t h e r e s u l t s o f t h e w e i g h t e d l i n e a r r e g r e s s i o n o f 3 s e t s . T h e s l o p e s a n d i n t e r c e p t s f r o m e n z y m e i n c u b a t i o n o f g l u c u r o n i d a s e a n d s u l p h a t a s e ( S e t J J a n d 1 3 1 ) w e r e a n a l y z e d a g a i n s t S e t I w h i c h w a s i n c u b a t e d w i t h o u t a n y e n z y m e u s i n g S t u d e n t ' s t - t e s t f o r l i n e a r r e g r e s s i o n . N o s i g n i f i c a n t d i f f e r e n c e i n s l o p e s o r i n t e r c e p t s w a s o b s e r v e d a m o n g t h e s e c a l i b r a t i o n c u r v e s . T h e r e f o r e , t h e s e r e s u l t s d e m o n s t r a t e t h a t t h e e n z y m e i n c u b a t i o n p r o c e d u r e d o e s n o t a f f e c t t h e q u a n t i t a t i o n a n d l i n e a r i t y o f t h e a s s a y m e t h o d . P l a s m a a n d u r i n e s a m p l e s f r o m t h e e x p e r i m e n t s w e r e i n c u b a t e d w i t h g l u c u r o n i d a s e a n d s u l p h a t a t e t o d e t e r m i n e t h e e x i s t e n c e o f t h e s e c o n j u g a t e s o f M C P , m d M C P a n d d d M C P , h o w e v e r , n o s i g n i f i c a n t a m o u n t o f c o n j u g a t e w a s d e t e c t e d . 48 4.00 3.20 2.40 h 1.60 0.80 0.00 o ddMCP mdMCP MCP 16 24 32 40 Standard Drug (ng/mL) F i g u r e 3. R e p r e s e n t a t i v e c a l i b r a t i o n ( w e i g h t e d ) c u r v e s f o r M C P , m d M C P a n d d d M C P f r o m s p i k e d p l a s m a , [ m e a n ± S D ] 49 Table 1. Weighted calibration curve data (mean peak area ratio ± SD) for ddMCP, mdMCP and MCP in plasma.* Cone. (ng/mL) ddMCP mdMCP MCP 40 3.95 ±0.32 (8.11%) 2.23 ±0.25 (11.03%) 1.65 ± 0.07 (4.48%) 32 2.86 ±0.21 (7.31%) 1.75 ±0.17 (9.69%) 1.26 ±0.04 (2.93%) 24 2.20 ± 0.20 (9.09%) 1.25 ± 0.08 (6.00%) 0.93 ± 0.05 (4.95%) 16 1.47 ±0.12 (8.09%) 0.85 ± 0.07 (8.32%) 0.62 ± 0.05 (8.39%) 8 0.70 ± 0.02 (3.50%) 0.38 ± 0.04 (9.58%) 0.32 ± 0.03 (7.78%) 4 0.34 ± 0.03 (8.99%) 0.18 ±0.02 (10.38%) 0.16 ±0.01 (4.43%) 2 0.18 ±0.01 (3.10%) 0.10 ±0.01 (12.07%) 0.08 ± 0.01 (11.48%) 1 0.10 ±0.01 (5.95%) 0.06 ± 0.01 (6.64%) 0.05 ±0.01 (12.38%) slope 0.0934 0.0541 0.0397 intercept -0.0068 -0.0099 0.0042 r2 0.9932 0.9930 0.9976 Average CV 6.77% 9.21% 7.10% The values shown in the parenthesis are the coefficient of variance. 50 Table 2. Weighted calibration curve data (slope, intercept and r2) for ddMCP, mdMCP and MCP with enzyme incubation study in urine. ddMCP mdMCP MCP Set l slope 0.0976 0.0539 0.0411 (no enzyme) intercept -0.0176 -0.0252 -0.0073 r2 0.9969 0.9937 0.9995 Set II slope 0.0903 0.0524 0.0393 (glucuronidase) intercept -0.0082 -0.0073 0.0001 r2 0.9978 0.9941 0.9998 Set III slope 0.0937 0.0489 0.0396 (sulphatase) intercept -0.0071 -0.0085 0.0030 r2 0.9980 0.9914 0.9996 3.2 Physiological Changes Associated with Hypoxemia T a b l e 3 l i s t s t h e i d e n t i f i c a t i o n a n d t h e b o d y w e i g h t o f e w e s o n w h i c h e x p e r i m e n t s w e r e p e r f o r m e d , a n d t h e t y p e s o f s a m p l e s c o l l e c t e d a n d t h e d u r a t i o n o f t h e i n f u s i o n . T h e b o d y w e i g h t o f t h e e x p e r i m e n t a l g r o u p a v e r a g e d 6 1 . 4 ± 3 . 5 K g ( M e a n ± S E M ) a n d t h e c o n t r o l g r o u p a v e r a g e d 6 1 . 3 ± 2 . 1 K g . A r t e r i a l p l a s m a s a m p l e s f o r d r u g a s s a y a n d b l o o d g a s / p H / l a c t a t e / g l u c o s e m e a s u r e m e n t , w e r e c o l l e c t e d a c c o r d i n g t o t h e e x p e r i m e n t p r o t o c o l s h o w n i n a p p e n d i x A . I n a d d i t i o n , u r i n e a n d b i l e s a m p l e s w e r e a l s o c o l l e c t e d a c c o r d i n g t o t h e a n i m a l e x p e r i m e n t a l p r o t o c o l . Table 3. Identification and weight of ewes, samples collected and nitrogen/MCP infusion. Ewe No. Weight Sample* Nitrogen Infusion M C P Infusion* (Kg) (L/min) Change (hr) Experimental 102 69.5 MA.UR Initial 7 to 10 at hr 7.5 14 139 60.4 MA.UR Initial 7 to 8 at hr 6 14 989 69.5 MA,UR 7 14 1154 53.1 MA,UR 7 14 1158 54.5 MA,UR 7 14 Mean 61.4 S E M 3.53 Control 220 61.7 MA, UR 0 14 327 55.4 MA, UR 0 14 328 63.6 MA, UR 0 15** 1159 64.5 MA, UR 0 Mean 61.3 p > 0.05 # S E M 2.05 * M A : maternal arterial blood sample, UR: urine total collection. ** M C P infusion was temporary disrupted. *** M C P infusion was discontinued at hour 9. A M C P infusion rate (0.21 mg/min) with 15 mg loading dose at hour 0. * weight compared between the two groups according to Student's t-test. 52 3.2.1. Arterial blood gas status and pH T h e m e a n ( ± S E M ) a r t e r i a l b l o o d g a s p a r t i a l p r e s s u r e o f C 0 2 a n d 0 2 ( P a C 0 2 a n d P a 0 2 ) a n d b l o o d p H o f t h e e x p e r i m e n t a l a n d c o n t r o l g r o u p s a r e s h o w n i n F i g u r e 4 a a n d 4 b , r e s p e c t i v e l y . M e a n ( ± S E M ) v a l u e s f o r a r t e r i a l b l o o d g a s p r e s s u r e , p H , o x y g e n s a t u r a t i o n , b a s e e x c e s s a n d b i c a r b o n a t e c o n c e n t r a t i o n a r e p r e s e n t e d i n T a b l e 4 . Pa02 and 02 saturation : T h e i n i t i a t i o n o f n i t r o g e n i n f u s i o n t h r o u g h t h e i n t r a - t r a c h e a l c a t h e t e r a t h o u r 2 i n t h e e x p e r i m e n t a l g r o u p d e c r e a s e d a r t e r i a l P a 0 2 , i n l e s s t h a n 5 m i n . M e a n P a 0 2 w a s s i g n i f i c a n t l y d e c r e a s e d ( = 4 8 % ) f r o m 1 1 6 . 3 5 ± 2 . 8 3 t o 6 0 . 3 1 ± 1 . 5 7 m m H g ( p < 0 . 0 0 0 1 ) d u r i n g t h e h y p o x e m i c p e r i o d . 0 2 s a t u r a t i o n w a s a l s o s i g n i f i c a n t l y d e c r e a s e d ( = 1 8 % ) f r o m 1 0 0 . 1 6 ± 0 . 4 0 % t o 8 2 . 7 8 ± 1 . 5 3 % d u r i n g h y p o x e m i a . I n s o m e a n i m a l s ( e w e n o s . 1 0 2 a n d 1 3 9 ) , P a 0 2 i n c r e a s e d s l i g h t l y ( = 7 0 - 8 0 m m H g ) d u r i n g t h e l a t e h y p o x e m i c p e r i o d , a t w h i c h p o i n t t h e n i t r o g e n f l o w w a s i n c r e a s e d t o 9 - 1 1 L / m i n t o m a i n t a i n t h e P a 0 2 c l o s e t o 6 0 m m H g . A f t e r 6 h o u r s o f h y p o x e m i a , t h e i n t r a - t r a c h e a l n i t r o g e n i n f u s i o n w a s s t o p p e d . P a 0 2 a n d 0 2 s a t u r a t i o n s h a v e r e t u r n e d t o 1 1 6 . 3 8 ± 1 . 2 6 m m H g a n d 1 0 0 . 1 5 ± 0 . 2 0 %, r e s p e c t i v e l y , l e v e l s w h i c h a r e s i m i l a r t o t h e p r e - h y p o x e m i c l e v e l ( p > 0 . 0 5 ) . N o c h a n g e s i n P a 0 2 o r 0 2 s a t u r a t i o n w a s o b s e r v e d t h r o u g h o u t t h e p e r i o d s i n t h e c o n t r o l g r o u p . PaC02, bicarbonate concentration and base excess : D u r i n g h y p o x e m i a , t h e m e a n P a C 0 2 a l s o s i g n i f i c a n t l y d e c r e a s e d ( = 1 0 % ) f r o m 4 1 . 7 1 ± 0 . 3 9 t o 3 7 . 3 5 ± 0 . 4 8 m m H g ( p < 0 . 0 0 0 5 ) , a r e d u c t i o n o f a m u c h l e s s e r d e g r e e t h a n P a 0 2 . B a s e e x c e s s w a s d e c r e a s e d s i g n i f i c a n t l y f r o m 3 . 4 ± 0 . 2 9 ( p r e - h y p o x e m i c ) t o 2 . 5 ± 0 . 2 9 m e q / L ( p o s t - h y p o x e m i c p e r i o d ) . I n i t i a l l y , t h e b a s e e x c e s s w a s s l i g h t l y i n c r e a s e d a t t h e o n s e t o f h y p o x e m i a , f r o m 3 . 9 ( a t h o u r 53 2 ) t o 4 . 8 m e q / L ( a t 2 : 0 5 ) , a n d g r a d u a l l y d e c r e a s e d . B l o o d b i c a r b o n a t e ( H C 0 3 ~ ) c o n c e n t r a t i o n w a s a l s o d e c r e a s e d d u r i n g h y p o x e m i a f r o m 2 6 . 5 1 ± 0 . 3 2 m e q / L t o 2 5 . 2 1 + 0 . 2 5 m e q / L , b u t w i t h o u t a n i n i t i a l i n c r e a s e a s s h o w n i n b a s e e x c e s s . A f t e r n i t r o g e n i n f u s i o n w a s s t o p p e d , t h e P a C 0 2 r e t u r n e d t o 4 0 . 8 2 ± 0 . 5 2 m r n H g , a l e v e l s i m i l a r t o t h e p r e - h y p o x e m i c p e r i o d ( p > 0 . 0 5 ) . H o w e v e r , a f t e r c e s s a t i o n o f t h e n i t r o g e n i n f u s i o n , b a s e e x c e s s a n d b i c a r b o n a t e ( H C 0 3 ~ ) c o n c e n t r a t i o n s r e m a i n e d l o w e r t h a n t h e p r e - h y p o x e m i c l e v e l s . I n t h e c o n t r o l g r o u p , n o s i g n i f i c a n t c h a n g e w a s o b s e r v e d i n P a C 0 2 , b i c a r b o n a t e c o n c e n t r a t i o n o r b a s e e x c e s s . Blood pH and hemoglobin concentration : A r t e r i a l b l o o d p H s h o w e d a v e r y s l i g h t a p p a r e n t i n c r e a s i n g t r e n d f r o m 7 . 4 2 4 ± 0 . 0 0 5 1 d u r i n g t h e p r e - h y p o x e m i c p e r i o d ( n o r m o x e m i a ) t o 7 . 4 4 2 ± 0 . 0 0 5 7 d u r i n g t h e h y p o x e m i c p e r i o d , a l t h o u g h t h i s d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e . D u r i n g t h e p o s t - h y p o x e m i c p e r i o d , b l o o d p H d e c r e a s e d t o 7 . 4 0 9 ± 0 . 0 0 5 3 ( p < 0 . 0 0 0 5 ) , a l e v e l s i g n i f i c a n t l y l o w e r t h a n t h o s e o f t h e p r e - h y p o x e m i c a n d h y p o x e m i c p e r i o d s . T h i s d e l a y e d a c i d o s i s a p p e a r s t o c o i n c i d e w i t h t h e d e l a y e d l a c t i c a c i d a c c u m u l a t i o n i n t h e b l o o d , a s s h o w n i n T a b l e 5 . D u r i n g t h e h y p o x e m i c p e r i o d , t h e h e m o g l o b i n c o n c e n t r a t i o n a l s o i n c r e a s e d s l i g h t l y ( s t a t i s t i c a l l y n o t s i g n i f i c a n t ; p = 0 . 0 5 8 ) f r o m 7 . 9 7 ± 0 . 2 3 g % ( p r e - h y p o x e m i c p e r i o d ) t o 8 . 4 0 ± 0 . 1 5 g % ( h y p o x e m i c p e r i o d ) , a n d w a s r e s t o r e d t o 7 . 8 3 ± 0 . 1 8 g % d u r i n g t h e p o s t - h y p o x e m i c p e r i o d . 54 Table 4. Mean (± SEM) arterial blood pH, gas partial pressure (PaC0 2 and Pa0 2 ) , bicarbonate (HCGy) concentration and base excess.1 pre-hypoxemia hypoxemia post-hypoxemia pH Experimental Control 7.424 ± 0.0053 7.501 ± 0.0076 7.442 ± 0.0057 7.495 ± 0.0068 7.409 ± 0.0053 * 7.492 ± 0.0063 PaCG-2 (mm Hg) Experimental Control 41.27 ±0.39 37.14 ±0.41 37.35 ± 0.48 " 37.70 ± 0.65 40.83 ± 0.58 37.22 ± 0.50 Pa02 (mmHg) Experimental Control 116.9 ± 1.15 117.7 ± 1.90 60.7 ± 1.57 *"# 116.1 ±1.88 116.5 ±0.71 120.0 ±1.91 O2 sat (%) Experimental Control 100.16 ±0.40 97.07 ±0.17 82.78 ± 1.53 *"# 96.60 ± 0.22 100.15 ±0.20 96.59 ±0.14 HCO3- (meq/L) Experimental Control 26.51 ±0.32 29.29 ± 0.38 25.21 ±0.25 28.86 ± 0.40 25.57 ±0.25 28.35 ± 0.46 Base Excess (meq/L) Experimental Control 3.4 ± 0.27 7.3 ±0.46 2.5 ±0.29 6.7 ± 0.42 2.1 ±0.28 6.3 ± 0.47 Hemoglobin (g %) Experimental Control 7.95 ± 0.23 7.71 ±0.13 8.40 ±0.15 7.76 ±0.13 7.83 ±0.18 7.75 ±0.17 * Significantly lower than pre-hypoxemia and hypoxemia according to A N O V A (p < 0.0025) and Tukey test (p < 0.005). ** Significantly lower than pre-hypoxemia and post-hypoxemia according to A N O V A ( p < 0.0005) and Tukey test (p < 0.001). *** Significantly lower than pre-hypoxemia and post-hypoxemia according to A N O V A (p < 0.0001) and Tukey test (p < 0.0005). **** Significantly higher than hypoxemia and post-hypoxemia according to A N O V A and Tukey test (p < 0.05). # Significantly lower than control group according to Student's t-test (p < 0.0001). 1 n = 5 for the experimental group, n = 4 for the control group 55 130 -£ 104 JL TI.LT [5f 1T 78 52 26 Hypoxemia' T T X J > O < 1 ^ - 0 1 T T T5._ • o j _ \ 0 — o — 2 i -IVJ-o o i l XT±-+ - ± + T - T T T T -+ V-X X 8.00 7.86 7.72 7.58 7.44 7.30 x GL -o O O m I— i < 10 12 14 Time (hr) Figure 4a. Mean (± S E M ) arterial blood p H and gas partial pressure ( P a C 0 2 and Pa0 2 ) over the duration of the experiment [experimental group, n = 5]. —H— pH, - o - PaQ 2 , - • - P a C 0 2 . 56 X E CD =J co to CD co CO co a. 140 -S 112 h 8.00 7.88 7.76 7.64 O O m .5 Q) < Time (hr) F i g u r e 4b. M e a n ( ± S E M ) a r t e r i a l b l o o d p H a n d g a s p a r t i a l p r e s s u r e ( P a C C » 2 a n d P a C « 2 ) o v e r t h e d u r a t i o n o f t h e e x p e r i m e n t [ c o n t r o l g r o u p , n = 4] - I - p H , - o - P a Q 2 ) - • - P a C Q 2 . 3.2.2. Arterial blood lactate and glucose concentration during hypoxemia T a b l e s 5 a n d 6 l i s t t h e m e a n ( ± S E M ) l a c t a t e a n d g l u c o s e c o n c e n t r a t i o n s o f t h e e x p e r i m e n t a l a n d c o n t r o l g r o u p s . V i s u a l i n s p e c t i o n o f t h e p l o t s o f a r t e r i a l b l o o d l a c t a t e c o n c e n t r a t i o n v e r s u s t i m e f o r t h e e x p e r i m e n t a l g r o u p ( F i g u r e 5a) s h o w s t h a t t h e r e w e r e n o t i c e a b l e c h a n g e s i n l a c t a t e a n d g l u c o s e c o n c e n t r a t i o n w i t h i n t h e h y p o x e m i c a n d p o s t -57 h y p o x e m i c p e r i o d s . E a c h o f t h e h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s w e r e s u b d i v i d e d i n t o t h r e e 2 - h o u r i n t e r v a l s f o r t h e s t a t i s t i c a l a n a l y s i s . I n t h e e x p e r i m e n t a l g r o u p , t h e a r t e r i a l b l o o d l a c t a t e c o n c e n t r a t i o n s g r a d u a l l y i n c r e a s e d ( = 6 0 % b y h o u r 8 ) d u r i n g t h e h y p o x e m i c p e r i o d ( s t a t i s t i c a l l y s i g n i f i c a n t l y a t t h e l a t e h y p o x e m i c p e r i o d i.e. h o u r 6 : 0 0 - 8 : 0 0 ) a n d r e m a i n e d e l e v a t e d a f t e r c e s s a t i o n o f t h e n i t r o g e n i n f u s i o n . T h e l a c t a t e l e v e l s h a d r e t u r n e d t o t h e p r e - h y p o x e m i c l e v e l b y t h e l a t e p o s t - h y p o x e m i c p e r i o d ( h o u r 1 2 : 0 0 - 1 4 : 0 0 ) . T h u s , t h e l a c t a t e c o n c e n t r a t i o n s f r o m 6 : 0 0 t o 1 2 : 0 0 (i.e. d u r i n g l a t e h y p o x e m i a a n d s u b s e q u e n t r e c o v e r y p e r i o d ) w e r e s i g n i f i c a n t l y ( p < 0 . 0 5 ) h i g h e r t h a n t h e r e m a i n i n g e x p e r i m e n t p e r i o d s [ b a s e l i n e - l e v e l ] . I n t h e c o n t r o l g r o u p , l a c t a t e c o n c e n t r a t i o n r e m a i n e d a t a l e v e l s i m i l a r t o t h a t d u r i n g t h e p r e - h y p o x e m i c p e r i o d t h r o u g h t h e e n t i r e e x p e r i m e n t ( p > 0 . 8 5 ) [ F i g u r e 5 b ] . A r t e r i a l b l o o d g l u c o s e c o n c e n t r a t i o n a l s o i n c r e a s e d , t o a l e s s e r d e g r e e ( = 1 0 % h i g h e r a t h o u r 8 ) , i n t h e l a t e h y p o x e m i c p e r i o d , i n a m a n n e r s i m i l a r t o t h e i n c r e a s e s e e n i n t h e l a c t a t e c o n c e n t r a t i o n . D u r i n g t h e p o s t - h y p o x e m i c p e r i o d , t h e g l u c o s e l e v e l r e m a i n e d h i g h e r t h a n t h a t o f t h e p r e - h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p , I n c o n t r a s t , i n t h e c o n t r o l g r o u p , t h e g l u c o s e c o n c e n t r a t i o n r e m a i n e d r e l a t i v e l y c o n s t a n t . F i g u r e 5 b s h o w s a d e c r e a s i n g t r e n d t h r o u g h t h e h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s , b u t t h i s d e c r e a s e w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t . 58 1.35 1.15 0.95 3.25 if 3.00 2.75 0.75 h 0.55 2.50 o E E o o F i g u r e 5a. M e a n ( ± S E M ) a r t e r i a l b l o o d l a c t a t e a n d g l u c o s e c o n c e n t r a t i o n ( m m o l / L ) o v e r t h e d u r a t i o n o f t h e e x p e r i m e n t [ e x p e r i m e n t a l g r o u p , n = 5]. - o - b l o o d l a c t a t e a n d - • - g l u c o s e . F i g u r e 5b. M e a n ( ± S E M ) a r t e r i a l b l o o d l a c t a t e a n d g l u c o s e c o n c e n t r a t i o n ( m m o l / L ) o v e r t h e d u r a t i o n o f t h e e x p e r i m e n t [ c o n t r o l g r o u p , n = 4]. - o - b l o o d l a c t a t e a n d - • - g l u c o s e . Table 5. Mean (± SEM) blood lactate concentration (mmol/L).1 Period Experimental Control Pre-hypoxemia 0:00-2:00 0.558 ± 0.024 0.524 ± 0.047 Hypoxemia 2:00-4:00 4:00-6:00 6:00-8:00 0.578 ± 0.035 0.644 ± 0.054 0.849 ± 0.044* 0.476 ± 0.035 0.504 ± 0.062 0.565 ± 0.052 Post-Hypoxemia 8:00-10:00 10:00-12:00 12:00-14:00 0.879 ± 0.049* 0.804 ± 0.064* 0.615 ±0.025 0.614 ±0.043 0.533 ± 0.069 0.446 ± 0.029 Significantly higher than baseline group (pre-hypoxemia, 2:00-6:00 and 12:00-14:00) according A N O V A (p < 1E-10) and Tukey test (p < 0.05). n = 5 for the experimental group and n = 4 for the control group. Table 6. Mean (± SEM) blood glucose concentration (mmol/L).1 Period Experimental Control Pre-hypoxemia 0:00-2:00 2.74 ± 0.05 3.24 ±0.14 Hypoxemia 2:00-4:00 4:00-6:00 6:00-8:00 2.81 ±0.05 2.81 ±0.07 2.91 ±0.04* 3.23 ±0.17 3.09 ± 0.23 3.17±0.18 Post-Hypoxemia 8:00-10:00 10:00-12:00 12:00-14:00 3.03 ± 0.04* 2.97 ± 0.09* 2.98 ±0.05* 2.92 ±0.16 2.78 ±0.17 2.80 ±0.11 Significantly higher than baseline group (pre-hypoxemia and 2:00-6:00) according to A N O V A (p < 3E-05) and Tukey test (p < 0.05). n = 5 for the experimental group and n = 4 for the control group. 60 3.2.3. Plasma electrolyte concentration, urine flow, osmolality and pH and renal osmolal excretion rate. T a b l e 7 l i s t s m e a n ( ± S E M ) p l a s m a e l e c t r o l y t e ( N a + , K + a n d C l " ) c o n c e n t r a t i o n s , h e m o g l o b i n c o n t e n t a n d u r i n e f l o w ( v o l u m e c o l l e c t e d p e r h o u r ) , o s m o l a l i t y a n d p H . T h e m e a n u r i n e f l o w i n t h e e x p e r i m e n t a l g r o u p w a s i n c r e a s e d s i g n i f i c a n t l y d u r i n g h y p o x e m i a , f r o m a n a v e r a g e o f 8 0 . 2 0 ± 1 2 . 6 5 m L / h r d u r i n g t h e p r e - h y p o x e m i c p e r i o d t o 1 3 1 . 9 0 ± 1 9 . 5 3 m L / h r d u r i n g t h e h y p o x e m i c p e r i o d , a n d l a t e r w a s r e s t o r e d t o 7 8 . 3 0 ± 7 . 5 8 m L / h r d u r i n g t h e p o s t - h y p o x e m i c p e r i o d ( p < 0 . 0 2 5 ) . A s s h o w n i n F i g u r e 6 a , h y p o x e m i a - i n d u c e d d i u r e s i s w a s o b s e r v e d i n t h e e a r l y t o m i d d l e h y p o x e m i c p e r i o d ( b e t w e e n h o u r 2 : 0 0 t o 6 : 0 0 ) a n d r e t u r n e d t o t h e v a l u e s c l o s e t o t h e n o r m o x e m i c l e v e l d u r i n g t h e l a t e h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s . H o w e v e r , i n t w o o f t h e a n i m a l s i n t h e e x p e r i m e n t a l g r o u p ( e w e n o . 1 1 5 4 a n d 1 1 5 8 ) , h y p o x e m i a - i n d u c e d d i u r e s i s w a s n o t o b s e r v e d . I n t h e c o n t r o l g r o u p , t h e r e w a s n o s i g n i f i c a n t c h a n g e i n u r i n e f l o w t h r o u g h o u t t h e e n t i r e e x p e r i m e n t a l p e r i o d [ F i g u r e 6 b ] . I n a d d i t i o n , u r i n e o s m o l a l i t y w a s a l s o s i g n i f i c a n t l y d e c r e a s e d f r o m 1 1 4 1 . 1 5 ± 3 7 . 2 2 m O s m o l / K g ( p r e - h y p o x e m i c ) t o 8 0 8 . 1 3 ± 9 2 . 9 9 m O s m o l / K g d u r i n g h y p o x e m i a , a n d r e s t o r e d t o 1 1 7 2 . 0 3 ± 4 0 . 7 7 m O s m o l / K g a f t e r h y p o x e m i a . A g a i n , s i m i l a r t o u r i n e f l o w , t h e u r i n e o s m o l a l i t y i n e w e n o . 1 1 5 4 a n d 1 1 5 8 d i d n o t c h a n g e d u r i n g h y p o x e m i a . T h e s e r e s u l t s d e s c r i b i n g u r i n e f l o w a n d o s m o l a l i t y s u g g e s t t h a t t h e r e m a y b e s o m e c o r r e l a t i o n b e t w e e n t h e s e t w o p a r a m e t e r s . T h u s , t h e p l o t o f u r i n e o s m o l a l i t y v e r s u s u r i n e f l o w w a s p l o t t e d a s s h o w n i n F i g u r e 7 . A s s e e n i n t h e p l o t , t h e r e i s a n a p p a r e n t i n v e r s e - r e l a t i o n s h i p b e t w e e n t h e s e t w o p a r a m e t e r s ( r = 0 . 8 0 ) . I n a d d i t i o n , t h e r e n a l o s m o l a l e l i m i n a t i o n r a t e c o n s t a n t , t h e p r o d u c t o f u r i n e o s m o l a l i t y a n d u r i n e f l o w , w a s a l s o s i g n i f i c a n t l y d e c r e a s e d f r o m 8 8 . 2 3 61 m O s m o l / h i n t h e p r e - h y p o x e m i c p e r i o d t o 6 1 . 3 2 m O s m o l / h d u r i n g t h e h y p o x e m i c p e r i o d , a n d l a t e r r e s t o r e d t o 8 9 . 3 7 m O s m o l / h i n t h e p o s t - h y p o x e m i c p e r i o d . T h e r e f o r e , t h e n e g a t i v e c o r r e l a t i o n b e t w e e n u r i n e o s m o l a l i t y a n d f l o w a n d h y p o x e m i a - i n d u c e d r e d u c t i o n i n o s m o l a l e x c r e t i o n s u g g e s t s t h a t t h e i n c r e a s e d u r i n e f l o w d u r i n g h y p o x e m i a i s d u e t o i n c r e a s e d w a t e r e x c r e t i o n r a t h e r t h a n a s e c o n d a r y u r i n e f l o w i n c r e a s e b y e l e v a t e d e l e c t r o l y t e / o s m o l a l e x c r e t i o n ( W a l k e r , 1 9 8 2 ; C o l i c e et al, 1 9 9 1 ) . S i m i l a r t o t h e f i n d i n g s w i t h u r i n e v o l u m e a n d o s m o l a l i t y , t h e u r i n e p H s i g n i f i c a n t l y d e c r e a s e d f r o m 7 . 5 3 2 ± 0 . 0 2 1 8 i n t h e p r e - h y p o x e m i c p e r i o d t o 7 . 4 6 8 ± 0 . 0 1 4 4 i n t h e h y p o x e m i c p e r i o d . L a t e r , t h e u r i n e p H w a s r e s t o r e d t o 7 . 5 1 9 ± 0 . 0 1 0 2 i n t h e p o s t - h y p o x e m i c p e r i o d , w h i c h i s a l e v e l s i m i l a r t o t h e p r e - h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p . I n c r e a s e d r e n a l l a c t a t e e x c r e t i o n m a y b e r e l a t e d t o t h e u r i n a r y a c i d o s i s d u r i n g h y p o x e m i a , b u t n o l a c t a t e m e a s u r e m e n t w a s c o n d u c t e d d u r i n g t h e e x p e r i m e n t . T h e r e f o r e , n o d i r e c t c o r r e l a t i o n c o u l d b e m a d e i n t h e p r e s e n t s t u d y . I n t h e c o n t r o l g r o u p , t h e p h y s i o l o g i c a l p a r a m e t e r s s u c h a s u r i n e f l o w , p H a n d o s m o l a l i t y r e m a i n e d f a i r l y c o n s t a n t t h r o u g h o u t t h e e x p e r i m e n t a l p e r i o d s ( p » 0 . 0 5 ) . H o w e v e r , u r i n e f l o w ( p < 0 . 0 5 ) , u r i n e p H a n d o s m o l a l i t y ( p « 0 . 0 5 ) i n t h e e x p e r i m e n t a l g r o u p w e r e s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p d u r i n g t h e h y p o x e m i c p e r i o d ( h o u r 2 t o 8 ) . E 500 400 300 200 100 Hypoxemia' •T T T I 1500 1200 900 600 300 2 4 6 8 10 12 14 Time (hr) O E E O Figure 6a. Mean (± SEM) urine flow (mL/hr) and osmolality (mOsm/Kg) over the duration of the experiment [experimental group, n = 5]. - o - urine osmolality and - • - urine flow. E 500 400 300 200 100 1500 1200 900 600 300 Figure 6b. Mean (± SEM) urine flow (mL/hr) and osmolality (mOsm/Kg) over the duration of the experiment [control group, n = 4] - O - urine osmolality and - • - urine flow. 63 CD 2£ o E J5 o E V) o CD c "C 3 1800 1440 1080 720 360 80 160 240 320 400 Urine flow (mL/hr) F i g u r e 7 . C o r r e l a t i o n o f u r i n e o s m o l a l i t y ( m O s m / K g ) a n d u r i n e f l o w ( m L / h r ) . C o r r e l a t i o n c o e f f i c i e n t ( r ) = 0 . 8 0 . P l a s m a s o d i u m ( N a + ) c o n c e n t r a t i o n w a s n o t s i g n i f i c a n t l y c h a n g e d d u r i n g h y p o x e m i a . N a + c o n c e n t r a t i o n t e n d s t o i n c r e a s e s l i g h t l y a s t h e i n f u s i o n p r o c e e d s , f r o m 1 4 6 . 6 ± 1 . 0 2 m M d u r i n g t h e p r e - h y p o x e m i c p e r i o d t o 1 5 0 . 1 ± 0 . 5 9 m M d u r i n g t h e h y p o x e m i c p e r i o d . H o w e v e r , t h i s a p p a r e n t t e n d e n c y t o w a r d s a n i n c r e a s e w a s o b s e r v e d i n b o t h t h e e x p e r i m e n t a l a n d c o n t r o l g r o u p , a n d y e t , n o d i f f e r e n c e w a s f o u n d b e t w e e n t h e t w o g r o u p s . T h e r e f o r e , i t s u g g e s t s t h a t h y p o x e m i a d o e s n o t a f f e c t t h e p l a s m a N a + c o n c e n t r a t i o n , i n s p i t e o f c h a n g e s i n u r i n e f l o w a n d o s m o l a l i t y . H o w e v e r , p l a s m a p o t a s s i u m ( K + ) c o n c e n t r a t i o n w a s i n c r e a s e d s i g n i f i c a n t l y d u r i n g h y p o x e m i a f r o m 4 . 1 2 ± 0 . 0 3 0 m M ( p r e - h y p o x e m i c ) t o 4 . 2 4 ± 0 . 0 2 2 m M ( h y p o x e m i c ) [ p < 64 0 . 0 5 ] , a n d r e t u r n e d t o 4 . 1 5 ± 0 . 0 3 4 m M d u r i n g t h e p o s t - h y p o x e m i c p e r i o d . I n c o n t r a s t , p l a s m a K + c o n c e n t r a t i o n i n t h e c o n t r o l g r o u p r e m a i n e d c o n s t a n t d u r i n g t h e h y p o x e m i c p e r i o d , 4 . 1 1 ± 0 . 0 3 0 m M a n d 4 . 1 7 ± 0 . 0 2 5 m M , a n d s i g n i f i c a n t l y i n c r e a s e d t o 4 . 4 1 ± 0 . 0 3 7 m M d u r i n g t h e p o s t - h y p o x e m i c p e r i o d . P l a s m a c h l o r i d e i o n ( C l " ) c o n c e n t r a t i o n w a s s i g n i f i c a n t l y i n c r e a s e d d u r i n g t h e h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p , f r o m 1 1 1 . 4 ± 0 . 4 8 m M ( p r e - h y p o x e m i c ) t o 1 1 4 . 1 ± 0 . 5 3 m M ( h y p o x e m i c ) , a n d r e m a i n e d e l e v a t e d t h r o u g h o u t t h e p o s t - h y p o x e m i c p e r i o d ( 1 1 3 . 4 ± 0 . 5 7 m M ) . I n t h e c o n t r o l g r o u p , p l a s m a C l " c o n c e n t r a t i o n w a s a l s o i n c r e a s e d d u r i n g t h e h y p o x e m i c p e r i o d f r o m 1 1 0 . 7 ± 0 . 2 4 m M t o 1 1 2 . 0 ± 0 . 1 8 m M , b u t t o a l e s s e r d e g r e e ( s i g n i f i c a n t l y l o w e r , p < 0 . 0 1 ) t h a n t h e e x p e r i m e n t a l g r o u p . 65 Table 7. Mean (± SEM) plasma electrolyte concentration, hemoglobin content and urine flow, urine pH and osmolality.1 pre-hypoxemia hypoxemia post-hypoxemia Urine Flow (ml_/hr) Experimental 80.20 ± 12.65 131.90 ± 19.53** 78.30 ± 7 . 5 8 Control 76.00 ± 15.38 92.58 ± 10.54 94.96 ± 10.52 Urine pH Experimental 7.532 ± 0 . 0 2 1 8 7.468 ± 0 . 0 1 1 2 " * * 7.535 ± 0 . 0 1 4 8 Control 7.551 ± 0 . 0 1 3 4 7.514 ± 0 . 0 1 4 4 7.519 ± 0 . 0 1 0 2 Osmolality (mOsmol/Kg) Experimental 1141.15 ± 37.22 808.13 ± 92.99**** 1172.03 ± 40.77 Control 1241.69 ± 63.64 1239.88 ± 46.28 1256.92 ± 29.08 Plasma N a + (mmol/L) Experimental 146.6 ± 1.02 150.1 ± 0 . 5 9 149.1 ± 0 . 9 8 Control 148.4 ± 0 . 2 5 150.2 ± 0 . 2 7 • 150.1 ± 0 . 1 8 Plasma K + (mmol/L) Experimental 4.12 ± 0.030 4.24 ± 0.022A 4.15 ± 0.034 Control 4.11 ± 0.030 4.17 ± 0.025 4.41 ± 0.037™*** Plasma Cl" (mmol/L) Experimental 111.4 ± 0 . 4 8 ™ 114.1 ± 0;53* # # # 113.4 ± 0 . 5 7 Control 110.7 ± 0 . 2 4 ™ 112.0 ± 0 . 1 8 112.5 ± 0 . 1 9 * Significantly higher; p < 0.025 according to A N O V A and p < 0.025 from post-hypoxemia and p < 0.10 from pre-hypoxemia in Tukey test. ** Significantly lower; p < 0.001 according to A N O V A and p < 0.001 from post-hypoxemia and p < 0.06 from pre-hypoxemia in Tukey test. # Significantly lower; p < 0.05 from control group according to Student's t-test. ** Significantly lower; p < 0.005 from control group according to Student's t-test. ### Significantly higher; p < 0.0001 from experiment group according to Student's t-test. #### Significantly higher; p < 0.01 from control group according to Student's t-test. A Significantly higher; p < 0.05 from control group according to Student's t-test. A A Significantly higher; p < 0.0001 from pre-hypoxemic and hypoxemic period according to A N O V A and Tukey test. A A A Significantly lower; p < 0.005 from hypoxemic and post-hypoxemic periods according to A N O V A and Tukey test. 1 n = 5 for the experimental group and n = 4 for the control group. 66 3.2.4. Mean arterial blood pressure and heart rate. M e a n a r t e r i a l b l o o d p r e s s u r e a n d h e a r t r a t e w e r e a l s o m e a s u r e d i n s o m e o f t h e a n i m a l s i n t h e s t u d y . T a b l e s 8 a n d 9 l i s t m e a n h e a r t r a t e a n d a r t e r i a l b l o o d p r e s s u r e d u r i n g t h e p r e - h y p o x e m i c , h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s . D u r i n g t h e h y p o x e m i c p e r i o d , m e a n h e a r t r a t e a n d a r t e r i a l b l o o d p r e s s u r e w e r e s i g n i f i c a n t l y i n c r e a s e d ( p < 0 . 0 5 ) t o 1 2 2 . 4 9 ± 9 . 1 5 b e a t s / m i n a n d 9 6 . 9 9 ± 1 0 . 2 6 m m H g f r o m 1 0 8 . 9 5 ± 6 . 6 1 b e a t s / m i n a n d 9 0 . 3 2 ± 1 0 . 6 3 m m H g ( t h e p r e - h y p o x e m i c p e r i o d ) i n t h e e x p e r i m e n t a l g r o u p . A f t e r t h e c e s s a t i o n o f n i t r o g e n i n f u s i o n , b o t h a r t e r i a l p r e s s u r e a n d h e a r t r a t e r e t u r n e d t o t h e l e v e l s c l o s e t o t h o s e o f t h e p r e - h y p o x e m i c p e r i o d s ( p > 0 . 0 5 ) . M e a n a r t e r i a l p r e s s u r e i n t h e c o n t r o l g r o u p r e m a i n e d c o n s t a n t t h r o u g h o u t t h e e n t i r e e x p e r i m e n t . M e a n h e a r t r a t e i n t h e c o n t r o l g r o u p a p p e a r e d t o d e c r e a s e g r a d u a l l y t h r o u g h t h e e x p e r i m e n t p e r i o d , b u t i t w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t . Table 8. Mean (± SEM) heart rate (beats/min) Ewe # pre-hypoxemia hypoxemia post-hypoxemia Experimental 139 100.0 ±1.3 109.3 ±0.7 89.3 ±0.9 989 105.0 ±1.0 118.1 ±0.5 93.4 ±0.4 1158 121.9 ±0.8 140.1 ±0.7 129.7 ±0.6 Mean ± SEM 109.0 ±6.6 122.5 ±9.2* 104.2 ±12.8 Control 327 112.7 ±1.7 110.6 ±0.8 96.4 ±0.6 220 119.6 ±0.7 112.1 ±0.4 101.1 ±0.4 1159 125.0 ±0.5 123.3 ±0.3 121.0 ±0.3 Mean ± SEM 119.1 ±3.6 115.3 ±4.0 106.1 ±7.5 Significantly higher than the pre-HO and post-HO periods according to paired t-tests (p < 0.05) 67 Table 9. Mean (± SEM) arterial blood pressure (mm Hg) Ewe # pre-hypoxemia hypoxemia post-hypoxemia Experimental 139 70.8 ±0.4 77.6 ±0.2 72.5 ±0.25 989 107.3 ±0.3 112.6 ±0.3 96.0 ±0.27 1158 92.8 ±0.5 100.8 ±0.3 100.3 ±0.33 Mean ±SEM 90.3 ± 10.6 97.0 ±10.3* 89.6 ±8.62 Control 327 85.9 ±1.0 85.4 ±0.6 73.5 ±0.5 220 76.3 ±0.3 78.2 ±0.2 80.6 ±0.3 1159 94.6 ±0.3 95.3 ±0.2 94.4 ±0.2 Mean ±SEM 85.6 ±5.3 86.3 ±5.0 82.8 ±6.1 Significantly higher than the pre-hypoxemic period according to paired t-tests (p < 0.01). 3.3 Metoclopramide Pharmacokinetics Following Lv. Infusion to Steady-state and Induction of Hypoxemia. 3.3.1. Steady-state plasma MCP concentration and total body clearance (TBC) during normoxemia and hypoxemia. I n f u s i o n p a r a m e t e r s f o r t h e s t u d y w e r e c h o s e n b a s e d o n r e s u l t s o b t a i n e d d u r i n g t h e p r e v i o u s m e t o c l o p r a m i d e s t u d y i n s h e e p ( R i g g s , 1 9 8 9 ) . S i m i l a r t o t h e p r e v i o u s s t u d y , a v i s u a l i n s p e c t i o n o f p l a s m a M C P c o n c e n t r a t i o n s a n d s t a t i s t i c a l a n a l y s i s i n d i c a t e d t h a t t h e M C P s t e a d y - s t a t e c o n c e n t r a t i o n s w e r e a t t a i n e d b e t w e e n 3 0 m i n a n d 7 5 m i n o f t h e i n f u s i o n d u r a t i o n ( i n f u s i o n r a t e a t 0 . 2 1 m g / m i n ) a f t e r a n i n i t i a l 1 5 m g i . v . b o l u s l o a d i n g d o s e i n 8 o f 9 68 a n i m a l s ( 4 i n t h e e x p e r i m e n t a l g r o u p a n d 4 i n t h e c o n t r o l g r o u p ) u s e d i n t h e s t u d y . O n e a n i m a l i n t h e e x p e r i m e n t a l g r o u p ( E w e N o . 1 1 5 4 ) d i d n o t r e a c h t h e s t a t e - s t a t e c o n c e n t r a t i o n w i t h i n 2 h o u r s o f i n f u s i o n , d e s p i t e t h e a d m i n i s t r a t i o n o f a l o a d i n g d o s e . D a t a f r o m t h i s a n i m a l w a s e x c l u d e d f r o m t h e f i n a l p h a r m a c o k i n e t i c d a t a a n a l y s i s , s i n c e t h e h y p o x e m i c a n d p o s t - h y p o x e m i c s t e a d y - s t a t e d r u g c o n c e n t r a t i o n s c o u l d n o t b e c o m p a r e d t o t h e p r e -h y p o x e m i c s t e a d y - s t a t e c o n c e n t r a t i o n s . S e m i - l o g a r i t h m i c p l o t s o f m e a n ( ± S E M ) p l a s m a M C P a n d m d M C P c o n c e n t r a t i o n v e r s u s t i m e p r o f i l e s o b t a i n e d f o l l o w i n g t h e i n i t i a l i . v . l o a d i n g d o s e a n d 1 4 - h o u r i n f u s i o n a r e s h o w n i n F i g u r e 8 a f o r t h e e x p e r i m e n t a l g r o u p a n d F i g u r e 8 b f o r t h e c o n t r o l g r o u p . T h e p l a s m a d d M C P c o n c e n t r a t i o n w a s b e l o w t h e a s s a y q u a n t i t a t i o n l i m i t i n t h e p l a s m a s a m p l e s a s s a y e d , t h u s , i t w a s n o t a p p r o p r i a t e t o a t t e m p t s t a t i s t i c a l a n a l y s i s a n d p l o t t i n g o f t h e p l a s m a d d M C P d a t a . T a b l e 1 0 l i s t s m e a n ( ± S E M ) p l a s m a m e t o c l o p r a m i d e c o n c e n t r a t i o n s i n t h e e x p e r i m e n t a l a n d c o n t r o l g r o u p s . P l a s m a m e t o c l o p r a m i d e r e a c h e d a v e r a g e s t e a d y - s t a t e c o n c e n t r a t i o n s o f 5 0 . 7 2 ± 1 . 0 6 n g / m L f o r t h e e x p e r i m e n t a l g r o u p a n d 5 0 . 8 4 ± 0 . 9 9 n g / m L f o r t h e c o n t r o l g r o u p i n t h e p r e - h y p o x e m i c p e r i o d ( p > 0 . 0 5 ) . S t e a d y - s t a t e c o n c e n t r a t i o n s w e r e o b t a i n e d 3 0 - 4 5 m i n a f t e r i n i t i a t i o n o f i n f u s i o n i n 3 e w e s a n d b y 1 t o 1 . 2 5 h o u r i n 5 e w e s . D u r i n g t h e h y p o x e m i c p e r i o d , t h e m e a n M C P s t e a d y - s t a t e c o n c e n t r a t i o n i n c r e a s e d s i g n i f i c a n t l y f r o m 5 0 . 7 2 ± 1 . 0 6 t o 6 3 . 6 2 ± 1 . 7 9 n g / m L ( p < 0 . 0 0 0 1 ) i n t h e e x p e r i m e n t a l g r o u p . I n t h e c o n t r o l g r o u p , p l a s m a M C P s t e a d y - s t a t e c o n c e n t r a t i o n r e m a i n e d c o n s t a n t ( p > 0 . 0 5 ) . T h e e l e v a t e d M C P s t e a d y - s t a t e c o n c e n t r a t i o n d u r i n g h y p o x e m i a i n t h e e x p e r i m e n t a l g r o u p i s s i g n i f i c a n t l y h i g h e r t h a n t h a t o f t h e c o n t r o l g r o u p ( p < 0 . 0 0 5 ) . 69 Table 10. Mean (± SEM) steady-state plasma MCP concentration (ng/mL) Ewe# pre-hypoxemia hypoxemia post-hypoxemia Experimental 102 45.80 ±1.95* 65.39 ±1.61** 56.58 ±1.79*** 139 54.75 ±2.12* 73.31 ±3.14 72.68 ±1.81 989 68.26 ±2.68 74.71 ±4.03**** 59.31 ±0.91 1158 30.95 ±1.03 43.69 ± 1.94** 33.64 ±1.11 Mean ± SEM 1 49.94 ±7.83 64.28 ±7.16* 55.55 ±8.11 Mean ±SEM2 50.72 ± 1.06 63.62 ±1.79**# 55.83 ±1 .15 # # Control 327 65.07 ±3.41 78.39 ±4.01 75.47 ±3.05 328 57.65 ±3.81 55.02 ±1.19 59.99 ±1.88 220 43.91 ±1.86 38.48 ± 3.02 42.42 ±1.58 1159 33.08 ±1.50 44.41 ±1.30 41.46 ±2.64 Mean ± S E M 1 49.93 ±7.12 54.08 ±8.80 54.84 ±8.09 Mean ±SEM2 50.84 ±0.99 54.23 ±2.15 51.74 ±1.59 * Significantly lower than hypoxemic and post-hypoxemic period according to ANOVA/Tukey test (p< 0.05). ** Significantly higher than pre- and post-hypoxemic period according to ANOVA/Tukey test (p < 0.05). *** Significantly higher than pre-hypoxemia and lower than hypoxemic period according to A N O V A and Tukey tes (p < 0.05). **** Significantly higher than post-hypoxemic period according to ANOVA/Tukey tests (p < 0.005). # # Significantly higher than control group according to Student's t-test (p < 0.005). Significantly higher than control group according to Student's t-test (p < 0.05). A Significantly higher than pre- and post-hypoxemic periods according to paired t-tests (p < 0.05). 1 mean ± sem calculated from the set of individual plasma M C P steady-state concentrations and analyzed by serie; of paired t-tests among the periods. ( n = 4) 2 mean ± sem calculated from the mean plasma M C P concentration curve and analyzed by A N O V A and Tukey test. 70 F i g u r e 8a. M e a n ( ± S E M ) p l a s m a M C P a n d m d M C P c o n c e n t r a t i o n ( n g / m L ) o v e r t h e d u r a t i o n o f t h e e x p e r i m e n t [ e x p e r i m e n t a l g r o u p , n = 4]. - o - M C P a n d -•- m d M C P . 71 100 / .0—6- •° „ o o . _ ; • T l 1 10 10 12 14 Time (hr) F i g u r e 8 b . M e a n ( ± S E M ) p l a s m a M C P a n d m d M C P c o n c e n t r a t i o n ( n g / m L ) o v e r t h e d u r a t i o n o f t h e e x p e r i m e n t - [ c o n t r o l g r o u p , n = 4 ] . - o - M C P a n d -•- m d M C P . 72 D u r i n g t h e p o s t - h y p o x e m i c p e r i o d , t h e a r t e r i a l p l a s m a M C P s t e a d y - s t a t e c o n c e n t r a t i o n d e c r e a s e d t o a c o n c e n t r a t i o n c l o s e t o t h a t s e e n i n t h e p r e - h y p o x e m i c p e r i o d ( 5 5 . 8 3 ± 1 . 1 5 n g / m L ) i n t h e e x p e r i m e n t a l g r o u p . H o w e v e r , t h i s c o n c e n t r a t i o n w a s s i g n i f i c a n t l y h i g h e r t h a n t h a t o f t h e e q u i v a l e n t p e r i o d i n t h e c o n t r o l g r o u p ( p = 0 . 0 4 ) . T h e m e a n M C P s t e a d y - s t a t e c o n c e n t r a t i o n s i n t h e c o n t r o l g r o u p r e m a i n e d t h e s a m e t h r o u g h o u t t h e e n t i r e e x p e r i m e n t a l p e r i o d ( p > 0 . 0 5 ) . T h e r e w a s s o m e i n d i v i d u a l v a r i a b i l i t y i n p h a r m a c o k i n e t i c r e s p o n s e t o t h e h y p o x e m i c s t r e s s . I n g e n e r a l , h y p o x e m i a a p p e a r s t o h a v e i n d u c e d a r e d u c t i o n i n d r u g e l i m i n a t i o n (i.e. i n c r e a s e d p l a s m a s t e a d y - s t a t e c o n c e n t r a t i o n ) , b u t t h e r e w a s a n o t i c e a b l e d i f f e r e n c e i n M C P k i n e t i c s d u r i n g t h e p o s t - h y p o x e m i c r e c o v e r y p e r i o d i n t h e e x p e r i m e n t a l g r o u p . I n e w e n u m b e r 9 8 9 a n d 1 1 5 8 , t h e M C P s t e a d y - s t a t e c o n c e n t r a t i o n s i n c r e a s e d s i g n i f i c a n t l y d u r i n g h y p o x e m i a a n d r e t u r n e d t o t h e p r e - h y p o x e m i c l e v e l d u r i n g t h e s u b s e q u e n t p o s t - h y p o x e m i c r e c o v e r y p e r i o d . I n e w e n u m b e r 1 0 2 , t h e M C P c o n c e n t r a t i o n a l s o i n c r e a s e d s i g n i f i c a n t l y d u r i n g h y p o x e m i a , b u t d i d n o t r e t u r n t o a p r e - h y p o x e m i c l e v e l . T h e M C P c o n c e n t r a t i o n d u r i n g t h e p o s t - h y p o x e m i c p e r i o d i n t h i s a n i m a l r e m a i n e d b e t w e e n t h e p r e - h y p o x e m i c a n d h y p o x e m i c l e v e l ( b u t s i g n i f i c a n t l y d i f f e r e n t f r o m b o t h ) . A l t e r n a t i v e l y , t h e M C P c o n c e n t r a t i o n i n c r e a s e d s i g n i f i c a n t l y d u r i n g h y p o x e m i a i n e w e n u m b e r 1 3 9 , a n d r e m a i n e d h i g h t h r o u g h o u t t h e p o s t - h y p o x e m i c p e r i o d . T a b l e 1 1 l i s t s t h e p l a s m a M C P t o t a l b o d y c l e a r a n c e ( T B C ) i n b o t h t h e e x p e r i m e n t a l a n d c o n t r o l g r o u p s . I n t h e e x p e r i m e n t a l g r o u p , T B C d e c r e a s e d s i g n i f i c a n t l y f r o m 2 7 4 . 2 2 ± 4 7 . 9 9 L / h ( 4 . 4 7 ± 1 . 0 4 L / h / K g ) t o 2 0 5 . 4 0 ± 2 8 . 1 7 L / h ( 3 . 3 3 ± 0 . 6 6 L / h / K g ) d u r i n g 73 h y p o x e m i a . D u r i n g t h e p o s t - h y p o x e m i c p e r i o d , T B C w a s i n c r e a s e d t o 2 4 5 . 7 8 ± 4 4 . 2 4 L / h ( 4 . 0 0 ± 0 . 9 6 L / h / K g ) . I n c o n t r a s t , t h e T B C o f c o n t r o l g r o u p r e m a i n e d r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e e x p e r i m e n t p e r i o d ( p > 0 . 0 5 ) . T h u s , t h e s e r e s u l t s f r o m c r o s s c o m p a r i s o n e x p e r i m e n t s s u g g e s t t h a t t h e t o t a l b o d y c l e a r a n c e o f m e t o c l o p r a m i d e i s r e d u c e d d u r i n g m o d e r a t e h y p o x e m i a ( P a O * 2 = 6 0 r r i m H g ) a n d r e s t o r e d d u r i n g t h e s u b s e q u e n t r e c o v e r y p e r i o d ( p o s t - h y p o x e m i a ) . 74 Table 11. Plasma MCP total body clearance (TBC)§. Ewe No. pre-hypoxemia hypoxemia post-hypoxemia change* Experimental 102 275.11 192.70 222.70 -30% (3.96) (2.77) (3.20) 139 230.12 171.87 173.36 -25% (3.81) (2.84) (2.87) 989 184.58 168.64 212.43 -9% (2.66) (2.42) (3.06) 1158 407.08 288.40 374.61 -29% (7.47) (5.29) (6.87) Mean ± SEM (L/h) 274.22 ± 47.99 205.40 ±28.17* 245.78 ± 44.24 Mean ± SEM (L/h/Kg) 4.47 ± 1.04 3.33 ± 0.66* 4.00 ± 0.96 Control 327 193.64 160.73 166.96 -16% (3.50) (2.90) (3.01) 328 218.57 228.76 210.04 +5% (3.44) (3.60) (3.30) 1159 380.87 283.70 303.94 -25% (5.90) (4.40) (4.71) 220 286.95 327.45 297.03 +14% (4.65) (5.31) (4.81) Mean ± SEM (L/h) 270.01 ±41.89 250.16 ±36.00 244.49 ± 35.53 Mean ± SEM (L/h/Kg) 4.37 ± 0.58 4.05 ± 0.52 3.96 ± 0.47 Numbers in parentheses show the total body clearance normalized to ewe body weight (L/h/Kg). [n = 4] Significantly lower than pre-hypoxemic period (p < 0.05) according to paired t-test. Relative change during hypoxemic period compared to pre-hypoxemic period. 75 3.3.2. Plasma mdMCP concentration during hypoxemic and normoxemic MCP steady-state. T h e m e a n ( ± S E M ) a r t e r i a l p l a s m a c o n c e n t r a t i o n o f m d M C P d u r i n g M C P s t e a d y - s t a t e i s l i s t e d i n T a b l e 1 2 . P l a s m a m d M C P c o n c e n t r a t i o n ( m e t a b o l i t e ) i n c r e a s e d s i g n i f i c a n t l y ( p < 0 . 0 0 5 ) f r o m t h e p r e - h y p o x e m i c t o t h e h y p o x e m i c p e r i o d s i n b o t h e x p e r i m e n t a l a n d c o n t r o l g r o u p s . T h i s i n c r e a s e i n p l a s m a m d M C P c o n c e n t r a t i o n , u n l i k e t h a t o f p l a s m a M C P c o n c e n t r a t i o n , d o e s n o t a p p e a r t o b e r e l a t e d t o t h e i n d u c t i o n o f h y p o x e m i a , s i n c e t h e e l e v a t i o n o f p l a s m a m d M C P c o n c e n t r a t i o n w a s g r a d u a l t h r o u g h o u t t h e p r e - h y p o x e m i c p e r i o d a n d o b s e r v e d i n b o t h e x p e r i m e n t a l a n d c o n t r o l g r o u p s . T h e r e f o r e , i t i s l i k e l y t o b e a r e s u l t o f g r a d u a l m e t a b o l i t e a c c u m u l a t i o n d u r i n g t h e d r u g i n f u s i o n . V i s u a l i n s p e c t i o n o f t h e p l a s m a m d M C P c o n c e n t r a t i o n - t i m e p r o f i l e s ( F i g u r e s 8 a a n d 8 b ) s u g g e s t s t h a t m d M C P c o n c e n t r a t i o n a l s o r e a c h e d a n a p p a r e n t s t e a d y - s t a t e i n t h e e w e s i n b o t h t h e e x p e r i m e n t a l a n d c o n t r o l g r o u p s , h o w e v e r , t h e t i m e t o a n a p p a r e n t s t e a d y - s t a t e w a s l o n g e r t h a n f o r M C P , r a n g i n g f r o m 4 t o 5 h o u r s . P l a s m a m d M C P c o n c e n t r a t i o n w a s n o t s i g n i f i c a n t l y c h a n g e d d u r i n g t h e p o s t -h y p o x e m i c p e r i o d . T h e m e a n m d M C P c o n c e n t r a t i o n s d u r i n g t h e h y p o x e m i c ( 3 2 . 7 8 ± 1 . 7 3 n g / m L ) a n d p o s t - h y p o x e m i c p e r i o d s ( 3 3 . 0 7 ± 1 . 0 8 n g / m L ) w e r e s i m i l a r ( p » 0 . 0 5 ) . H o w e v e r , w h e n t h e p l a s m a m d M C P c o n c e n t r a t i o n s o f t h e h y p o x e m i c a n d p o s t -h y p o x e m i c p e r i o d s i n t h e e x p e r i m e n t g r o u p w e r e c o m p a r e d t o t h o s e o f t h e c o n t r o l g r o u p , m d M C P c o n c e n t r a t i o n s o f e x p e r i m e n t a l g r o u p w e r e s i g n i f i c a n t l y h i g h e r ( p < 0 . 0 0 0 1 ) , s u g g e s t i n g a h i g h e r p l a s m a m d M C P a c c u m u l a t i o n d u r i n g h y p o x e m i a . 76 Table 12. Mean (± SEM) arterial plasma mdMCP concentrations during the MCP infusion (ng/mL) Ewe # pre-hypoxemia hypoxemia post-hypoxemia Experimental 102 22.99 ± 1.72 30.35 ±2.94 27.20 ±1.68 139 13.12 ±1.13* 26.63 ±0.51 30.20 ±1.13 989 21.89 ±1.37* 39.03 ±2.44 38.47 ±1.51 1158 18.33 ±3.36* 40.01 ±1.55 34.99 ±2.01 Mean ± SEM 18.62 ±1.30* 32.78 ± 1.73* 33.07 ± 1.08# Control 327 13.17 ±1.75* 23.18 ±1.97 25.59 ±1.41 328 24.01 ±0.61** 26.89 ±0.63 27.42 ± 1.23 220 13.38 ±0.53 13.72 ±0.39 16.37 ±0.59*** 1159 5.40 ±0.17* 19.31 ±1.94 26.84 ±1.12 Mean ± SEM 14.26 ±0.68* 21.20 ±1.39 22.27 ±0.81 Significantly lower than hypoxemic and post-hypoxemic period according to ANOVA/Tukey test (p< 0.005) Significantly lower than hypoxemic and post-hypoxemic period according to ANOVA/Tukey test (p< 0.05). Significantly higher than pre-hypoxemia and hypoxemic period according to ANOVA/Tukey test (p < 0.05) Significantly higher than control group according to Student's t-test (p < 0.0001). 77 3.4. Renal Excretion of Metoclopramide and its Metabolites Following the Lv. Infusion to Steady-state and Induction of Hypoxemia. 3.4.1. Renal clearance of MCP and its metabolites during hypoxemia and normoxemia. R e n a l c l e a r a n c e v a l u e s o f M C P a n d m d M C P w e r e c a l c u l a t e d f r o m 1 ) d i v i d i n g t h e a m o u n t r e c o v e r e d (Dw) b y a r e a u n d e r d r u g p l a s m a c o n c e n t r a t i o n s c u r v e (AUC„~) a s a f u n c t i o n o f t i m e d u r i n g t h e h y p o x e m i c a n d n o r m o x e m i c p e r i o d s [Du(t2-t])/AUC(t2-tl)] a n d 2 ) c a l c u l a t i o n u s i n g t h e s l o p e o f t h e a c c u m u l a t e d d r u g i n u r i n e (Du~) v e r s u s (AUC„~) ( s e e F i g u r e 9 a n d A p p e n d i x A f o r t h e e q u a t i o n d e r i v a t i o n ) . S i n c e t h e p l a s m a c o n c e n t r a t i o n o f d d M C P a p p e a r s t o b e b e l o w t h e q u a n t i t a t i o n l i m i t , i t s r e n a l c l e a r a n c e v a l u e c o u l d n o t b e c a l c u l a t e d . A s s h o w n i n T a b l e s 1 3 a n d 1 4 , t h e r e n a l c l e a r a n c e v a l u e s o b t a i n e d b y b o t h m e t h o d s w e r e r e l a t i v e l y c o n s i s t e n t . S o m e d i s c r e p a n c i e s b e t w e e n t h e c a l c u l a t e d c l e a r a n c e v a l u e s w e r e o b s e r v e d w h e n t h e r e w e r e d a t a p o i n t ( s ) n o t i c e a b l y d e v i a t i n g f r o m t h e r e g r e s s i o n l i n e i n t h e p l o t o f Du" v e r s u s AUC0". T a b l e s 1 3 a a n d 1 3 b s h o w t h e M C P r e n a l c l e a r a n c e c a l c u l a t e d b y b o t h m e t h o d 1 a n d 2 d u r i n g t h e h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s . D u e t o t h e s h o r t d u r a t i o n o f t h e p r e -h y p o x e m i c p e r i o d {i.e. 2 h o u r s w i t h 3 d a t a p o i n t s ) , t h e r e n a l c l e a r a n c e d u r i n g t h i s p e r i o d c o u l d o n l y b e a p p r o x i m a t e d , a n d t h e v a l u e d u r i n g h y p o x e m i a w a s c o m p a r e d t o t h e p o s t -h y p o x e m i c v a l u e s . I n t h e e x p e r i m e n t a l g r o u p , r e n a l M C P c l e a r a n c e d u r i n g h y p o x e m i a ( 2 . 7 5 ± 0 . 2 2 L / h ) w a s s i g n i f i c a n t l y l o w e r ( b y = 6 4 % ) t h a n d u r i n g t h e p o s t - h y p o x e m i c p e r i o d ( 8 . 1 5 ± 1 . 8 6 L / h ) . I n c o n t r a s t , t h e r e n a l c l e a r a n c e r e m a i n e d c o n s t a n t ( 7 . 0 1 ± 0 . 7 2 L / h a n d 6 . 7 3 ± 0 . 5 9 L / h r e s p e c t i v e l y ) d u r i n g t h e e q u i v a l e n t t i m e s i n t h e c o n t r o l g r o u p . I n a d d i t i o n , r e n a l c l e a r a n c e i n t h e e x p e r i m e n t a l g r o u p w a s s i g n i f i c a n t l y l o w e r t h a n i n t h e c o n t r o l g r o u p d u r i n g 78 t h e h y p o x e m i c p e r i o d , b u t n o t a f t e r w a r d s . T h e r e w a s a c o n s i d e r a b l e i n d i v i d u a l v a r i a t i o n i n t h e r e d u c t i o n o f r e n a l c l e a r a n c e d u r i n g h y p o x e m i a . F o r e x a m p l e , e w e n u m b e r 1 1 5 8 s h o w e d a n a p p a r e n t 7 5 % r e d u c t i o n i n t h e r e n a l c l e a r a n c e d u r i n g h y p o x e m i a , w h e r e a s e w e n u m b e r 1 3 9 s h o w e d a 3 3 % d e c r e a s e . T a b l e s 1 4 a a n d 1 4 b l i s t t h e m d M C P r e n a l c l e a r a n c e d u r i n g h y p o x e m i a a n d p o s t -h y p o x e m i a . T h e v a l u e s o f m d M C P r e n a l c l e a r a n c e w e r e c o n s i d e r a b l y h i g h e r ( = 6 - 7 f o l d ) t h a n t h o s e o f M C P . H i g h e r u r i n a r y a c c u m u l a t i o n a n d , t o a l e s s o r d e g r e e , l o w e r p l a s m a m d M C P c o n c e n t r a t i o n c o n t r i b u t e d t o h i g h e r r e n a l c l e a r a n c e v a l u e s f o r m d M C P . S i m i l a r t o t h e M C P r e n a l c l e a r a n c e i n t h e e x p e r i m e n t a l g r o u p , t h e m d M C P r e n a l c l e a r a n c e d u r i n g h y p o x e m i a ( 1 4 . 6 0 ± 2 . 5 6 L / h ) w a s s i g n i f i c a n t l y l o w e r ( = 6 2 % ) t h a n d u r i n g p o s t - h y p o x e m i a ( 3 8 . 5 7 ± 9 . 0 9 L / h ) . I n t h e c o n t r o l g r o u p , t h e r e n a l c l e a r a n c e r e m a i n e d c o n s t a n t ( 4 3 . 9 0 ± 9 . 4 1 L / h a n d 3 8 . 3 9 ± 7 . 1 2 L / h , r e s p e c t i v e l y ) . C o n s e q u e n t l y , a s w i t h M C P r e n a l c l e a r a n c e , t h e v a l u e f o r m d M C P i n t h e e x p e r i m e n t a l g r o u p w a s s i g n i f i c a n t l y l o w e r t h a n t h a t i n t h e c o n t r o l g r o u p d u r i n g t h e h y p o x e m i c p e r i o d , b u t n o t d u r i n g p o s t - h y p o x e m i a . T h e r e w a s a l s o a c o n s i d e r a b l e i n d i v i d u a l v a r i a t i o n i n t h e r e d u c t i o n o f m d M C P r e n a l c l e a r a n c e d u r i n g h y p o x e m i a , a l t h o u g h l e s s t h a n t h a t s e e n f o r M C P r e n a l c l e a r a n c e . F o r e x a m p l e , w i t h e w e n o . 1 1 5 8 , t h e r e w a s a n a p p a r e n t 6 9 % r e d u c t i o n i n r e n a l c l e a r a n c e d u r i n g h y p o x e m i a w h i l e e w e n o . 1 3 9 s h o w e d a 3 9 % d e c r e a s e . 79 cn o E o . o •a to E O o < 6000 5000 g 4000 3000 2000 1000 HO = 2.89 L/h; post-HO = 8.99 L/h / ' / / / A / /'+ ,. — „ + / + / / , i 180 360 540 AUC (mcg*h/L) 720 900 F i g u r e 9 R e p r e s e n t a t i v e p l o t o f t h e a c c u m u l a t e d d r u g ( o r m e t a b o l i t e ) i n u r i n e ( Z D u ) v e r s u s A U C . S l o p e o f t h e c u r v e r e p r e s e n t s t h e r e n a l c l e a r a n c e [ L / h ] ( e w e n o . 1 0 2 i n t h e e x p e r i m e n t a l g r o u p ) . H O = h y p o x e m i a , p o s t - H O = p o s t - h y p o x e m i a . Table 13a. MCP renal clearance (L/h)1 [CLmnaifMCPJ] CLrenal(MCP) ewe no. hypoxemia post-hypoxemia Experimental 102 3.20 8.25 Group 139 2.09 2.92 989 3.33 7.21 1158 2.94 11.07 Mean 2.89* # 7.36 ± S E M 0.28 1.69 Control 220 6.58 6.97 Group 327 4.96 5.78 328 7.59 8.01 1159 7.73 7.41 Mean 6.72 7.04 ± S E M 0.64 0.47 * Significantly lower (p<0.03) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. 1 renal clearance calculated using time-averaged urinary excretion [Du(t2-tl)/AUC(t2-tl)]. Table 13b. MCP renal clearance (L/h)2 [CLrenai(MCPJ] CLrenal(MCP) ewe no. hypoxemia post-hypoxemia Experimental 102 2.89 8.99 Group 139 2.09 3.13 989 2.95 8.39 1158 3.05 12.08 Mean 2.75* # 8.15 ± S E M 0.22 1.86 Control 220 6.83 6.21 Group 327 5.11 5.44 328 7.66 7.06 1159 8.45 8.19 Mean 7.01 6.73 ± S E M 0.72 0.59 * Significantly lower (p<0.025) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. 2 renal clearance calculated using the slope of Du(t-0)/AUC(t-0) plot. Table 14a. mdMCP renal clearance (L/h)1 [CLrenai(mdMCP)] CLrenal(mdMCP) ewe no. hypoxemia post-hypoxemia Experimental 102 12.38 26.67 Group 139 12.14 20.90 989 21.79 53.68 1158 11.44 40.66 Mean 14.44** 35.48 ± S E M 2.46 7.35 Control 220 65.96 59.97 Group 327 35.75 36.23 328 44.25 52.12 1159 30.88 22.03 Mean 44.21 42.59 ± S E M 7.76 8.45 * Significantly lower (p<0.02) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. 1 renal clearance calculated using time-averaged urinary excretion [Du(t2-tl)/AUC(t2-tl)]. Table 14b. mdMCP renal clearance (L/h)2 [CLrenai(mdMCP)] CLrenal(mdMCP) ewe no. hypoxemia post-hypoxemia Experimental 102 9.84 26.72 Group 139 13.20 21.69 989 21.85 61.70 1158 13.49 44.17 Mean 14.60** 38.57 ± S E M 2.56 9.09 Control 220 70.91 55.85 Group 327 31.65 33.64 328 42.58 42.10 1159 30.44 21.99 Mean 43.90 38.39 ± S E M 9.41 7.12 * Significantly lower (p<0.02) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. 2 renal clearance calculated using the slope of Du(t-0)/AUC(t-0) plot. 82 3.4.2. Fractional renal excretion constants of MCP and its metabolites T h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t s w e r e a p p r o x i m a t e d f o r M C P a n d t w o o f i t s m e t a b o l i t e s , m d M C P a n d d d M C P , i n t h e p r e s e n t s t u d y . T h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t s (e.g. fu(MCP) = ku/KE) w e r e c a l c u l a t e d b y d i v i d i n g t h e s l o p e o f t h e a s y m p t o t e o f t h e a c c u m u l a t e d d r u g / m e t a b o l i t e s i n u r i n e v e r s u s t i m e c u r v e w i t h t h e i n f u s i o n r a t e , ko ( F i g u r e 10), a n d t h e s e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t s a r e d i m e n s i o n l e s s (i.e. t h e y r e p r e s e n t a p r o p o r t i o n o f t h e t o t a l e x c r e t i o n c o n s t a n t i n t e r m s o f a f r a c t i o n ) . T h e d e t a i l e d e q u a t i o n d e r i v a t i o n s a r e s h o w n i n a p p e n d i x A a n d t h e o r e t i c a l a n d p r a c t i c a l d i s c u s s i o n i s d e s c r i b e d i n s e c t i o n 4.4. T h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t w a s u s e d t o d e t e r m i n e r e l a t i v e c h a n g e s i n t h e r e n a l e x c r e t i o n o f t h e p a r e n t d r u g a n d i t s m e t a b o l i t e (i.e. t h e p r o p o r t i o n o f t h e r e n a l e x c r e t i o n t o t h e t o t a l b o d y e l i m i n a t i o n o f t h e d r u g / m e t a b o l i t e ) . Scheme V Diagram of metabolic and renal elimination of a drug kf kmu where X B and M B represent the amount of drug and metabolite in the body, respectively, X u and M u represent the drug and metabolite excreted in urine, respectively. k u and K f are renal and metabolite elimination rate constants and the sum of these two rate constants is equal to K E , apparent first order drug elimination rate constant, kmu represents renal elimination rate constant of the metabolite. 83 T a b l e 1 5 l i s t s t h e f r a c t i o n a l r e n a l M C P e x c r e t i o n c o n s t a n t (fu(incp) = ku/KE). D u e t o t h e s h o r t d u r a t i o n o f t h e p r e - h y p o x e m i c p e r i o d (i.e. 2 h o u r s w i t h 3 d a t a p o i n t s ) , t h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t , fu(MCP), w a s n o t c a l c u l a t e d d u r i n g t h e p r e - h y p o x e m i c p e r i o d . A s w i t h t h e M C P a n d m d M C P r e n a l c l e a r a n c e v a l u e s , t h e /U(MCP> v a l u e d u r i n g h y p o x e m i a w a s c o m p a r e d t o t h e p o s t - h y p o x e m i c e s t i m a t e . D u r i n g t h e h y p o x e m i c p e r i o d , t h e /U(MCP> i s s i g n i f i c a n t l y l o w e r b y 5 8 % , ( 0 . 0 1 3 8 ± 0 . 0 0 1 9 ) t h a n d u r i n g t h e p o s t - h y p o x e m i c p e r i o d ( 0 . 0 3 2 9 ± 0 . 0 0 5 3 ) i n t h e e x p e r i m e n t a l g r o u p . I n c o n t r o l g r o u p , t h e /U(MCP> r e m a i n e d f a i r l y c o n s t a n t t h r o u g h o u t t h e s e p e r i o d s , 0 . 0 2 9 6 ± 0 . 0 0 3 2 a n d 0 . 0 2 8 2 ± 0 . 0 0 4 0 , r e s p e c t i v e l y . C o n s e q u e n t l y , t h e fu(MCP) v a l u e d u r i n g t h e h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p w a s s i g n i f i c a n t l y l o w e r t h a n c o r r e s p o n d i n g e s t i m a t e i n t h e c o n t r o l g r o u p . T h e f r a c t i o n a l r e n a l m e t a b o l i t e e x c r e t i o n c o n s t a n t s f o r m d M C P a n d d d M C P w e r e a l s o c a l c u l a t e d i n t h e s t u d y . B e c a u s e t h e i n d i v i d u a l p a r a m e t e r s s u c h a s m e t a b o l i t e f o r m a t i o n a n d e l i m i n a t i o n r a t e c o n s t a n t s c a n n o t b e d e t e r m i n e d w i t h t h e p r e s e n t s t u d y d e s i g n , u n l e s s t h e m e t a b o l i t e s a r e i n j e c t e d i n d i v i d u a l l y , t h e s e p a r a m e t e r s a r e t h e p r o d u c t o f t w o f r a c t i o n a l constants fmfmetaboiite) a n d fmu(metaboiites). T h e m e t a b o l i t e f o r m a t i o n f r a c t i o n " c o n s t a n t fm(metaboiite) i s t h e f r a c t i o n o f kflmetaboiite)/ K.E(Parent drug), w h i c h r e p r e s e n t s t h e m e t a b o l i c p r o p o r t i o n ( t o a s p e c i f i c m e t a b o l i t e ) o f t h e t o t a l p a r e n t d r u g e l i m i n a t i o n . T h e s e c o n d f r a c t i o n a l c o n s t a n t , t h e r e n a l m e t a b o l i t e e l i m i n a t i o n f r a c t i o n fmu(metaboiite) i s t h e f r a c t i o n o f kmu(metaboiite)/ Km(metaboiite), w h i c h r e p r e s e n t s t h e r e n a l e x c r e t i o n p r o p o r t i o n o f t h e t o t a l m e t a b o l i t e e l i m i n a t i o n . T h e r e f o r e , t h e c o m p o s i t e f r a c t i o n a l c o n s t a n t , t h e r e n a l m e t a b o l i t e e x c r e t i o n f r a c t i o n fu(metaboiite) = fm(metaboiite) xfmu(metaboiite), r e p r e s e n t s t h e p r o p o r t i o n o f r e n a l e x c r e t i o n o f a s p e c i f i c m e t a b o l i t e f r o m t h e t o t a l d r u g e l i m i n a t i o n . F o r e x a m p l e , i f t h e v a l u e o f fU(metaboiite A> = 84 fm(metaboiite A) x fmu(metaboiite A) w a s 0 . 0 1 = 0 . 0 5 * 0 . 2 , t h e n i t w o u l d m e a n t h a t 5 % o f p a r e n t d r u g w a s b i o - t r a n s f o r m e d i n t o t h e m e t a b o l i t e A a n d 2 0 % o f t h e m e t a b o l i t e w a s e x c r e t e d i n t h e u r i n e (i.e. o v e r a l l 1 % o f t h e p a r e n t d r u g i s e x c r e t e d i n t h e u r i n e i n t h e f o r m o f m e t a b o l i t e A ) . 4000 8 > 3200 h E C D-o -o •o TJ o < 2400 1600 800 HO = 159, 58; post-HO = 320.20 A-J Z L 6 8 Time (hr) 10 12 14 F i g u r e 1 0 . R e p r e s e n t a t i v e p l o t o f t h e a c c u m u l a t e d d r u g ( o r m e t a b o l i t e ) i n u r i n e ( S D u ) v e r s e s t i m e ( h ) . T h e s l o p e o f t h e c u r v e r e p r e s e n t s t h e p r o d u c t o f t h e f r a c t i o n a l r e n a l e x c r e t i o n r a t e c o n s t a n t f o r M C P ( o r m e t a b o l i t e ) a n d t h e i n f u s i o n r a t e c o n s t a n t o f M C P . ( E w e n o . 1 0 2 i n t h e e x p e r i m e n t a l g r o u p ) . H O = h y p o x e m i a , p o s t - H O = p o s t - h y p o x e m i a . T h e f r a c t i o n a l r e n a l m e t a b o l i t e e x c r e t i o n c o n s t a n t s o f m d M C P a n d d d M C P a r e l i s t e d i n T a b l e s 1 6 a n d 1 7 . T h e fu(mdMCP) w a s s i g n i f i c a n t l y l o w e r ( - 6 5 % ) d u r i n g h y p o x e m i a ( 0 . 0 3 7 8 ± 0 . 0 0 9 8 ) c o m p a r e d t o t h a t o f t h e p o s t - h y p o x e m i c p e r i o d ( 0 . 1 0 8 0 ± 0 . 0 3 3 9 ) i n t h e e x p e r i m e n t a l g r o u p . I n t h e c o n t r o l g r o u p , t h e fu(mdMCP) r e m a i n e d c o n s t a n t , 0 . 0 7 1 9 ± 0 . 0 0 8 5 a n d 0 . 0 7 1 1 ± 0 . 0 1 2 5 , r e s p e c t i v e l y . T h u s , t h e fu(mdMCP) v a l u e d u r i n g h y p o x e m i a i n t h e 85 e x p e r i m e n t a l g r o u p w a s a l s o s i g n i f i c a n t l y l o w e r t h a n t h a t o f t h e c o n t r o l g r o u p . S i m i l a r t o t h e r e n a l c l e a r a n c e v a l u e s f r o m t h e p r e v i o u s s e c t i o n , t h e fu(mdMCP) v a l u e s w e r e a b o u t 2 t o 4 f o l d h i g h e r t h a n t h o s e offu(MCP). I n s o m e c a s e s ( e w e n u m b e r s 9 8 9 a n d 1 1 5 8 a n d 3 2 8 ) , t h i s p r o c e s s h a s a c c o u n t e d f o r m o r e t h a n 1 0 % o f t h e t o t a l d r u g e l i m i n a t i o n . The f*U(ddMCP) w a s a l s o s i g n i f i c a n t l y l o w e r ( - 6 0 % ) d u r i n g h y p o x e m i a ( 0 . 0 2 2 3 ± 0 . 0 5 6 ) c o m p a r e d t o t h a t o f t h e p o s t - h y p o x e m i a ( 0 . 0 5 6 2 ± 0 . 0 1 5 2 ) i n t h e e x p e r i m e n t a l g r o u p . I n t h e c o n t r o l g r o u p , t h e fr(mdMCP) r e m a i n e d c o n s t a n t , 0 . 0 2 5 9 ± 0 . 0 0 5 3 a n d 0 . 0 2 5 9 ± 0 . 0 0 5 2 , r e s p e c t i v e l y . H o w e v e r , u n l i k e fu(MCP) andfu(mdMCP), t h e fu(ddMCP) v a l u e d u r i n g h y p o x e m i a i n t h e e x p e r i m e n t a l g r o u p w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e c o n t r o l g r o u p . Table 15. MCP fractional renal excretion constants [fu(MCPJ\-fu(MCP) ewe no. hypoxemia post-hypoxemia Experimental 102 0.0151 0.0401 Group 139 0.0124 0.0182 989 0.0183 0.0410 1158 0.0094 0.0325 Mean 0.0138** 0.0329 ±SEM 0.0019 0.0053 Control 220 0.0205 0.0214 Group 327 0.0312 0.0250 328 0.0349 0.0399 1159 0.0319 0.0266 Mean 0.0296 0.0282 ±SEM 0.0032 0.0040 * Significantly lower (p<0.02) than the post-hypoxemic period according to paired t-test. # Significantly lower (p<0.005) than the control group according to t-test. a Renal elimination fraction constant fu(MCP) denotes the ratio of ku/KE of M C P . Table 16. mdMCP fractional renal excretion constants* [fu(mdMCPJ\-f*u(mdMCP) ewe no. hypoxemia post-hypoxemia Experimental 102 0.0248 0.0560 Group 139 0.0273 0.0530 989 0.0668 0.1955 1158 0.0323 0.1274 Mean 0.0378** 0.1080 ±SEM 0.0098 0.0339 Control 220 0.0766 0.0758 Group 327 0.0638 0.0614 328 0.0933 0.1032 1159 0.0539 0.0442 Mean 0.0719 0.0711 ±SEM 0.0085 0.0125 * Significantly lower (p<0.05) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.05) than the control group according to t-test. a f*u(mdMCP) denotes the composite fraction constant fm(mdMCP) x fmu(mdMCP), the product Of the ratio o f kflmdMCP)/KE(MCP) and kmu(mdMCP)/Km(mdMCP). Table 17. ddMCP fractional renal excretion constants* [fu(ddMCP)]-f*u(ddMCP) ewe no. hypoxemia post-hypoxemia Experimental 102 0.0127 0.0262 Group 139 0.0188 0.0369 989 0.0384 0.0926 1158 0.0194 0.0693 Mean 0.0223* 0.0562 ±SEM 0.0056 0.0152 Control 220 0.0365 0.0377 Group 327 0.0153 0.0157 328 0.0181 0.0190 1159 0.0335 0.0314 Mean 0.0259 0.0259 ±SEM 0.0053 0.0052 * Significantly lower (p<0.02) than post-hypoxemia according to paired t-test. a fu(ddMCP) denotes the composite fraction constant fm(ddMCP) x fmu(ddMCP), the product of the ratio of kf(ddMCP)/KE(MCP) and kmu(ddMCP)/Km<ddMCP). 87 4. DISCUSSION 4.1. GC-MSD Method of Analysis of Metoclopramide and its Metabolites A G C - M S D m e t h o d f o r t h e a s s a y o f M C P , m d M C P a n d d d M C P , p r e v i o u s l y d e v e l o p e d a n d v a l i d a t e d i n o u r l a b o r a t o r y ( R i g g s et al, 1 9 9 4 ) , w a s c h o s e n f o r i t s h i g h s e n s i t i v i t y a n d r e p r o d u c i b i l i t y f o r a p p l i c a t i o n t o t h e p r e s e n t s t u d y . T h e G C - M S D m e t h o d h a s a n u m b e r o f i n h e r e n t a d v a n t a g e s o v e r t h e G C - E C D m e t h o d p r e v i o u s l y d e v e l o p e d i n o u r l a b o r a t o r y ( R i g g s et al, 1 9 8 3 ; 1 9 9 0 ) , d u e t o t h e s e l e c t i v e n a t u r e o f m a s s s p e c t r o m e t r y . T h e s i m u l t a n e o u s d e t e c t i o n a n d q u a n t i f i c a t i o n o f M C P a n d i t s m e t a b o l i t e s a t h i g h e r ( = 2 x ) s e n s i t i v i t y w a s m a d e p o s s i b l e u s i n g M S D t e c h n o l o g y . M o r e o v e r , t h e e x t r a c t i o n p r o c e d u r e s a r e s i m p l e r t h a n w i t h t h e G C - E C D m e t h o d , t h e r e b y r e d u c i n g t h e s a m p l e p r e p a r a t i o n t i m e s i g n i f i c a n t l y . T h e u s e o f n a r r o w b o r e f u s e d s i l i c a c a p i l l a r y c o l u m n s a n d G C - M S D w i t h s e l e c t i v e i o n m o n i t o r i n g e n a b l e d u s t o s e p a r a t e a n d q u a n t i t a t i v e l y a n a l y z e t h e d e e t h y l a t e d m e t a b o l i t e s o f M C P i n t h e p r e s e n c e o f M C P i n t h e b i o l o g i c a l f l u i d s c o l l e c t e d i n t h e s t u d y . T w o d e e t h y l a t e d m e t a b o l i t e s o f M C P , m d M C P a n d d d M C P , w e r e a n a l y s e d f r o m v a r i o u s b i o l o g i c a l f l u i d s . T h e u s e o f n a r r o w b o r e ( 0 . 2 m m I D . ) f u s e d s i l i c a c a p i l l a r y c o l u m n s i n t h e p l a c e o f t h e c o n v e n t i o n a l 0 . 3 3 m m I . D . c o l u m n s a n d t h e s o l v e n t t r a p p i n g e f f e c t ( R i g g s et al, 1 9 9 4 ) h a s p r o v i d e d b a s e l i n e s e p a r a t i o n a m o n g t h e m e t a b o l i t e s a n d M C P . T h i s i n c r e a s e d s e p a r a t i o n , e s p e c i a l l y b e t w e e n M C P a n d m d M C P ( a m e t a b o l i t e o f t e n p r e s e n t i n q u a n t i t i e s g r e a t e r t h a n t h e i n t a c t M C P ) w a s e s s e n t i a l f o r t h e a c c u r a t e q u a n t i t a t i o n o f t h e s e c o m p o u n d s i n u r i n e . S i g n i f i c a n t a m o u n t s o f m d M C P w e r e r e c o v e r e d i n u r i n e d u r i n g t h e i n f u s i o n s t u d y , t h e r e f o r e t h e r e i s a p o s s i b i l i t y o f o v e r - e s t i m a t i o n o f M C P i n t h e s e s a m p l e s i f t h e a n a l y s i s p r o v i d e s i n s u f f i c i e n t s e p a r a t i o n , p a r t i c u l a r l y w h e n G C - E C D i s t h e m e t h o d o f c h o i c e . I n c o n t r a s t , t h e u s e o f s e l e c t i v e i o n m o n i t o r i n g m e t h o d i n G C - M S D h a s e n a b l e d t h e d e t e c t i o n o f t h e a n a l y t e s w i t h m i n i m a l i n t e r f e r e n c e f r o m e n d o g e n o u s c o m p o u n d s . A s s h o w n i n F i g u r e 1 , t h e i n t e r f e r e n c e f r o m c o n t a m i n a n t s w a s m i n i m a l e v e n w i t h r e d u c e d e x t r a c t i o n p r o c e d u r e s . L i n e a r i t y o v e r a 1 - 4 0 n g / m L c o n c e n t r a t i o n r a n g e w a s o b s e r v e d f o r M C P , m d M C P a n d d d M C P e x t r a c t e d f r o m p l a s m a a n d u r i n e w i t h i n d i v i d u a l s t a n d a r d c u r v e s f r o m e a c h m a t r i x h a v i n g a c o e f f i c i e n t o f d e t e r m i n a t i o n ( r 2 ) o f a t l e a s t 0 . 9 8 . T h e m e t h o d h a s b e e n f o u n d t o b e r e l i a b l e w i t h a n o v e r a l l a v e r a g e c o e f f i c i e n t o f v a r i a t i o n l e s s t h a n 1 0 % a n d t h e l i m i t o f q u a n t i t a t i o n o f 1 n g / m L . T h e s t a n d a r d c u r v e s w e r e l i n e a r , h o w e v e r , s l i g h t l y g r e a t e r v a r i a b i l i t y (i.e. s l i g h t l y h i g h e r C V v a l u e s ) w a s o b s e r v e d i n t h e c o r r e s p o n d i n g d r u g c o n c e n t r a t i o n b e l o w 4 n g / m L . S i n c e a s l i g h t v a r i a t i o n i n t h e p e a k a r e a r a t i o a t t h e h i g h e r c o n c e n t r a t i o n s c a n g r e a t l y a f f e c t t h e y - i n t e r c e p t o f t h e l i n e a r r e g r e s s i o n c u r v e , t h e l o w e r d r u g c o n c e n t r a t i o n s i n t h e c u r v e w i l l b e a f f e c t e d m o r e b y t h i s v a r i a t i o n . T h e u s e o f t h e w e i g h t i n g f a c t o r o f l / ( p e a k a r e a r a t i o ) i m p r o v e d t h e l i n e a r i t y o v e r t h e w h o l e r a n g e , e s p e c i a l l y a t t h e l o w e r c o n c e n t r a t i o n r a n g e . E v e n t h o u g h i t w a s n o t c r u c i a l t o t h e i n f u s i o n s t u d y , t h e G C -M S D m e t h o d p e r m i t t e d a t t a i n m e n t o f a m i n i m u m q u a n t i t a t i o n l i m i t o f 1 n g / m L , r e p r e s e n t i n g 2 . 5 p g a t t h e d e t e c t o r , a r e a s o n a b l e i m p r o v e m e n t o v e r t h e q u a n t i t a t i o n l i m i t o f 2 n g / m L o b t a i n e d w i t h t h e p r e v i o u s G C - E C D m e t h o d . T h u s , t h e G C - M S D m e t h o d w i t h s e l e c t i v e i o n m o n i t o r i n g p r o v i d e s a m o r e s t a b l e , s t r a i g h t f o r w a r d a n d r e l i a b l e m e a n s f o r m e a s u r e m e n t o f M C P a n d i t s m e t a b o l i t e s t h a n p r e v i o u s m e t h o d s o f M C P m e a s u r e m e n t . F u r t h e r m o r e , i t i s a n t i c i p a t e d t h a t , w h e n t h e p r e s e n t 89 w o r k i s c o m p l e t e d , t h e G C - M S D m e t h o d w i l l e n a b l e u s f u r t h e r i n v e s t i g a t i o n s o f s t a b l e i s o t o p e l a b e l e d M C P i n p r e g n a n t h y p o x e m i c a n i m a l s u n d e r a n o t h e r a n i m a l r e s e a r c h p r o t o c o l . 4.2. Physiological Changes Associated with Hypoxemia 4.2.1. Induction of hypoxemia and Blood gas changes S e v e r a l e x p e r i m e n t a l m e t h o d s o f i n d u c i n g h y p o x e m i a h a v e b e e n p r e v i o u s l y u s e d , s u c h a s r e d u c e d i n s p i r e d o x y g e n c o n t e n t a n d c a r b o n m o n o x i d e ( C O ) a d m i n i s t r a t i o n ( R o t h et al., 1 9 7 6 ) . T h e r e a r e s e v e r a l r e p o r t e d m e t h o d s f o r r e d u c i n g i n s p i r e d 0 2 c o n t e n t , i n c l u d i n g a n i s o l a t e d g a s c h a m b e r ( M e d i n a a n d M e r r i t t , 1 9 7 0 ) , a h y p o b a r i c c h a m b e r ( J a c o b s et ai, 1 9 8 8 ) , a P l e x i g l a s ® c h a m b e r e n c l o s i n g t h e h e a d a n d n e c k o f t h e e w e ( R u r a k et ai, 1 9 9 0 ) o r p l a s t i c b a g o v e r t h e h e a d ( d u S o u i c h et al., 1 9 8 4 ) i n t o w h i c h a g a s m i x t u r e c a n b e d e l i v e r e d t o c a u s e h y p o x e m i a , a n d l a s t l y , a n i n t r a - t r a c h e a l n i t r o g e n i n f u s i o n ( G l e e d et ai, 1 9 8 6 ) . T h e l a t t e r m e t h o d i n v o l v e s i n f u s i o n o f n i t r o g e n ( = 4 - 1 0 L / m i n ) via a s m a l l n o n - o c c l u s i v e c a t h e t e r c h r o n i c a l l y i m p l a n t e d i n t h e t r a c h e a . T h e n i t r o g e n m i x e s w i t h t h e a i r n o r m a l l y i n s p i r e d b y t h e a n i m a l , r e s u l t i n g i n a l o w e r i n g o f 0 2 c o n c e n t r a t i o n . V a r i o u s d e g r e e s o f h y p o x e m i a c a n b e a c h i e v e d b y v a r y i n g t h e n i t r o g e n f l o w r a t e . A r a p i d P a 0 2 e q u i l i b r a t i o n t i m e (=3 m i n ) c a n b e o b t a i n e d a f t e r c h a n g i n g t h e N 2 f l o w . I n t r a - t r a c h e a l n i t r o g e n i n f u s i o n w a s a n e f f e c t i v e m e t h o d o f l o w e r i n g P a 0 2 i n t h e s h e e p . T h e l o w e r e d P a 0 2 w a s r e a c h e d w i t h i n 5 m i n . o f t h e o n s e t o f h y p o x e m i a ( f i r s t s a m p l i n g a f t e r n i t r o g e n i n f u s i o n ) . M o r e o v e r , t h e a n i m a l d i d n o t a p p e a r t o b e d i s t u r b e d b y n i t r o g e n i n f u s i o n . T h e i m p l a n t a t i o n o f a n i n t r a - t r a c h e a l c a t h e t e r w a s a m i n o r s u r g i c a l p r o c e d u r e a n d p e r f o r m e d a t t h e s a m e t i m e w i t h o t h e r s u r g i c a l p r o c e d u r e s d u r i n g t h e g e n e r a l a n a e s t h e s i a . O t h e r m e t h o d s o f i n d u c i n g h y p o x e m i a i n t h e 90 e x p e r i m e n t a l a n i m a l , s u c h a s a n i s o l a t i o n g a s c h a m b e r a n d p l a c e m e n t o f t h e p l a s t i c b a g o v e r t h e h e a d , c o u l d p o t e n t i a l l y c a u s e u n d u e r e s t r i c t i o n a n d s t r e s s t o t h e a n i m a l s . T h e i n t r a -t r a c h e a l i n f u s i o n m e t h o d s e e m s t o m i n i m i z e s o m e o f t h e s i d e e f f e c t s o f o t h e r e x p e r i m e n t a l m e t h o d s u s e d i n t h e s h e e p . A l l a n i m a l s u n d e r t h e s t u d y h a v e f r e e a c c e s s t o w a t e r a n d f o o d i n t h e i r n o r m a l h o u s i n g b e f o r e a n d d u r i n g e x p e r i m e n t s . F u r t h e r m o r e , a l l e w e s a r e m a i n t a i n e d i n a r e l a t i v e l y c l o s e c o n t a c t w i t h o t h e r c o m p a n i o n e w e s d u r i n g e a c h e x p e r i m e n t a l p e r i o d , a n d , t h u s , e n v i r o n m e n t a l a l t e r a t i o n s d u r i n g a n d b e t w e e n e x p e r i m e n t s w e r e m i n i m i z e d . I n a d d i t i o n , t h e i n t r a - t r a c h e a l n i t r o g e n i n f u s i o n p r o c e d u r e c o n s u m e s l e s s n i t r o g e n (i.e. « 7 L / m i n v s . > 4 0 L / m i n f o r t h e c h a m b e r m e t h o d s ) , t h u s r e s u l t i n g i n a s u b s t a n t i a l c o s t s a v i n g s . I n t r a - t r a c h e a l n i t r o g e n i n f u s i o n ( 7 t o 1 1 L / m i n ) r a p i d l y i n d u c e d a m o d e r a t e h y p o x e m i a ( P a 0 2 ~ 6 0 m m H g ) i n t h e s h e e p , w i t h i n 5 m i n o f t h e o n s e t o f n i t r o g e n i n f u s i o n . T h i s i s s i m i l a r t o t h e r e s u l t r e p o r t e d f o r p r e g n a n t e w e s i n t h e s t u d y t h a t f i r s t d e s c r i b e d t h e n i t r o g e n i n f u s i o n t e c h n i q u e ( G l e e d et al, 1 9 8 6 ) . A t t h e s a m e t i m e , a s i g n i f i c a n t d e c r e a s e i n P a C 0 2 ( h y p o c a p n i a ) a n d a t e n d e n c y o f i n c r e a s e d b l o o d p H ( r e s p i r a t o r y a l k a l o s i s ) w a s o b s e r v e d i n t h e e x p e r i m e n t a l g r o u p . I n f u s e d n i t r o g e n m i x e d w i t h n o r m a l l y i n s p i r e d r o o m a i r w i l l r e s u l t i n a r e d u c t i o n o f o x y g e n c o n t e n t i n t h e i n s p i r e d g a s m i x t u r e i n t h e l u n g t h u s r e s u l t i n g i n r e d u c t i o n o f P a 0 2 . I n a d d i t i o n , h y p o c a p n i a i s c a u s e d b y h y p e r v e n t i l a t i o n , s i n c e a t y p i c a l r e s p o n s e t o r e d u c e d P a 0 2 i s i n c r e a s e d v e n t i l a t i o n ( h y p e r v e n t i l a t i o n ) ( D a l y , 1 9 6 3 ) . T h i s l o w e r e d P a 0 2 w a s m a i n t a i n e d t h r o u g h i n t r a - t r a c h e a l n i t r o g e n i n f u s i o n ( r a t e = 7 L / m i n ) . H o w e v e r , i n s o m e a n i m a l s , n i t r o g e n i n f u s i o n w a s i n c r e a s e d t o m a i n t a i n P a 0 2 a r o u n d 6 0 m m H g . A v a r i a t i o n i n i n d i v i d u a l r e s p o n s e t o h y p o x e m i a w a s o b s e r v e d , b u t c o r r e l a t i o n b e t w e e n h y p o x e m i c r e s p o n s e a n d p h y s i o l o g i c a l v a l u e s s u c h a s u r i n e f l o w a n d l a c t a t e w a s n o t o b v i o u s f r o m a v i s u a l s u r v e y . 91 4.2.2. Blood pH and lactate concentration during hypoxemia I n c r e a s e d b l o o d l a c t a t e c o n c e n t r a t i o n w a s o b s e r v e d i n t h e l a t e h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p a n d i t r e m a i n e d r e l a t i v e l y h i g h t h r o u g h t h e e a r l y a n d m i d d l e p o s t -h y p o x e m i c r e c o v e r y p e r i o d s . T h u s , t h e r e s e e m s t o b e a t r e n d o f a g r a d u a l i n c r e a s e i n t h e b l o o d l a c t a t e c o n c e n t r a t i o n d u r i n g h y p o x e m i a . H o w e v e r , t h i s a c c u m u l a t i o n o f l a c t a t e i s n o t l i k e l y t h e r e s u l t o f i n a d e q u a t e o x y g e n s u p p l y t o t h e t i s s u e s , s i n c e t h e 0 2 s a t u r a t i o n d u r i n g h y p o x e m i a i n t h e s t u d y r e m a i n e d a r o u n d 7 0 t o 8 0 %. I n a d d i t i o n , t h e " e x c e s s l a c t a t e " l e v e l , a n i n d i c a t i o n o f t i s s u e 0 2 a d e q u a c y , i s e s t i m a t e d t o b e c l o s e t o z e r o a t t h e l e v e l o f P a 0 2 o b t a i n e d i n t h e s t u d y ( = 6 0 m m H g ) ( C a i n , 1 9 6 5 ) , t h u s t h e c h a n g e i n t h e l a c t a t e c o n c e n t r a t i o n i n t h e p r e s e n t s t u d y i s n o t l i k e l y d u e t o 0 2 d e f i c i t o f b o d y . R a t h e r , i t i s m o r e l i k e l y r e l a t e d t o h y p o c a p n i a a n d t h e r e s u l t i n g c h a n g e s i n a c i d - b a s e b a l a n c e d u r i n g h y p o x e m i a ( H u c k a b e e , 1 9 5 8 ) . T h e i n c r e a s e d g l u c o s e l e v e l d u r i n g l a t e h y p o x e m i a a n d h y p o c a p n i a d u r i n g h y p o x e m i c p e r i o d m a y a l s o , i n p a r t , e x p l a i n t h e l a c t a t e a c c u m u l a t i o n . I t h a s b e e n s u g g e s t e d t h a t t h e l a c t a t e p r o d u c t i o n i s n o t c o n t r o l l e d e x c l u s i v e l y b y t h e a d e q u a c y o f c e l l u l a r o x y g e n a t i o n , a n d i s a f f e c t e d t o a v e r y s i g n i f i c a n t e x t e n t b y t h e p y r u v a t e c h a n g e s , h y p e r v e n t i l a t i o n o r p H a l t e r a t i o n o f t h e b o d y a n d c h a n g e s i n b l o o d g l u c o s e c o n c e n t r a t i o n ( H u c k a b e e , 1 9 5 7 ) a n d s y m p a t h e t i c a c t i v i t y . T a k a n o ( 1 9 6 8 ) h a s d e m o n s t r a t e d t h a t p l a s m a l a c t a t e a c c u m u l a t i o n i n d o g s w a s l e s s f o r i s o c a p n i c h y p o x i a c o m p a r e d t o h y p o c a p n i c h y p o x i a . C a i n ( 1 9 6 9 ) a l s o s h o w e d d i m i n i s h e d l a c t a t e a c c u m u l a t i o n d u r i n g h y p o x e m i a w i t h P - a d r e n e r g i c b l o c k a d e a n d h y p e r c a p n i a . F u r t h e r m o r e , C a i n ( 1 9 7 3 ) o b s e r v e d l a c t a t e a c c u m u l a t i o n i n d u c e d b y P - s t i m u l a t i o n w i t h o u t o x y g e n d e f i c i t . Z b o r o w s k a - S l u i s a n d 92 D o s s e t o r ( 1 9 6 7 ) o b s e r v e d i n c r e a s e d b l o o d l a c t a t e c o n c e n t r a t i o n i n h y p e r v e n t i l a t e d d o g s w i t h n o i n c r e a s e i n l a c t a t e p r o d u c t i o n a c r o s s m a j o r o r g a n s y s t e m s ( n o c h a n g e s i n t h e A - V l a c t a t e c o n c e n t r a t i o n d i f f e r e n c e a c r o s s l i v e r , m u s c l e , a n d g u t ) . I n t h i s s t u d y , a s m a l l d e c r e a s e i n h e p a t i c l a c t a t e u t i l i z a t i o n w a s o b s e r v e d , b u t i t w a s e s t i m a t e d t o a c c o u n t f o r o n l y a s m a l l p o r t i o n o f t h e l a c t a t e i n c r e a s e . A s t i m u l a t i o n o f r e d b l o o d c e l l g l y c o l y s i s b y l o w P a C C t e o r r e s p i r a t o r y a l k a l o s i s w a s a l s o o b s e r v e d ( M u r p h y , 1 9 6 0 ; Z b o r o w s k a - S l u i s a n d D o s s e t o r , 1 9 6 7 ) . W i t h n o e f f e c t i v e a e r o b i c m e t a b o l i c s y s t e m p r e s e n t , e n e r g y p r o d u c t i o n i n t h e r e d b l o o d c e l l i s a l m o s t b y a n a e r o b i c g l y c o l y s i s , a n d t h u s i n d e p e n d e n t o f c h a n g e s i n o x y g e n a v a i l a b i l i t y . T h e r e f o r e , t h e s e s t u d i e s s u g g e s t t h a t l a c t a t e a c c u m u l a t i o n d u r i n g a m o d e r a t e h y p o x e m i a i s a s s o c i a t e d w i t h a l t e r a t i o n i n b l o o d a c i d - b a s e b a l a n c e , a n d / o r g l u c o s e c o n c e n t r a t i o n a n d s y m p a t h e t i c a c t i v a t i o n . A c i d o s i s w a s a l s o o b s e r v e d i n t h e p o s t - h y p o x e m i c p e r i o d , b u t n o t i n t h e h y p o x e m i c p e r i o d . T h i s d e l a y e d r e s p o n s e s u g g e s t s t h a t a c i d o s i s i s l i k e l y a s e c o n d a r y r e s p o n s e t o h y p o x e m i a , r a t h e r t h a n a d i r e c t r e s p o n s e . T h e d e c r e a s e i n b l o o d p H s e e m s t o c o r r e s p o n d w i t h a n i n c r e a s e i n l a c t a t e l e v e l i n t h e l a t e h y p o x e m i c a n d e a r l y / m i d d l e p o s t - h y p o x e m i c p e r i o d ( h o u r 6 : 0 0 t o 1 2 : 0 0 ) . A n i n c r e a s e i n h y d r o g e n i o n c o n c e n t r a t i o n (i.e. a c i d o s i s d u r i n g p o s t - h y p o x e m i a ) i n t h e b l o o d i s l i k e l y a s e c o n d a r y r e a c t i o n t o t h e a c c u m u l a t i o n o f l a c t i c a c i d ( F r o m m e r , 1 9 8 3 ) . 93 4.2.3. Adrenosympathetic system and blood glucose during hypoxemia I n c r e a s e d h e a r t r a t e a n d m e a n a r t e r i a l b l o o d p r e s s u r e d u r i n g t h e h y p o x e m i c p e r i o d w a s o b s e r v e d i n t h e p r e s e n t s t u d y . S i n c e h y p o x e m i a i s k n o w n t o c a u s e a c t i v a t i o n o f t h e a d r e n o s y m p a t h e t i c s y s t e m a n d , a l s o , t o i n c r e a s e c i r c u l a t i n g c a t e c h o l a m i n e c o n c e n t r a t i o n s , t h e i n c r e a s e d h e a r t r a t e a n d a r t e r i a l b l o o d p r e s s u r e i n t h e s t u d y m a y b e a s s o c i a t e d w i t h t h i s a d r e n o s y m p a t h e t i c a c t i v a t i o n . S t i m u l a t e d a d r e n o s y m p a t h e t i c a c t i v i t y a n d i n c r e a s e d p l a s m a c a t e c h o l a m i n e c o n c e n t r a t i o n d u r i n g h y p o x i a h a s b e e n o b s e r v e d i n m a n y p r e v i o u s s t u d i e s . S i n c e C a n n o n a n d H o s k i n s ( 1 9 1 1 ) d e m o n s t r a t e d s e c r e t i o n o f e p i n e p h r i n e i n t o a d r e n a l v e n o u s b l o o d d u r i n g a s p h y x i a , i t h a s b e e n g e n e r a l l y a c c e p t e d t h a t h y p o x i a s t i m u l a t e s a s y m p a t h o a d r e n a l d i s c h a r g e . C l a u s t r e a n d P e y r i n ( 1 9 8 2 ) a n d C l a u s t r e et al. ( 1 9 8 5 ) r e p o r t e d a m i l d a d r e n o s y m p a t h e t i c a c t i v a t i o n a n d a s i g n i f i c a n t i n c r e a s e i n p l a s m a f r e e c a t e c h o l a m i n e ( e p i n e p h r i n e , n o r e p i n e p h r i n e a n d d o p a m i n e ) c o n c e n t r a t i o n d u r i n g a m o d e r a t e h y p o x i a i n r a t s a n d c a t s . T h e i n c r e a s e i n p l a s m a e p i n e p h r i n e l e v e l i n t h e s e s t u d i e s w a s e s p e c i a l l y p r o f o u n d d u r i n g h y p o x i a , a n d t h i s i n c r e a s e d e p i n e p h r i n e c o n c e n t r a t i o n m a y c o n t r i b u t e t o p h y s i o l o g i c a l c h a n g e s a s s o c i a t e d w i t h h y p o x i a . J o h n s o n et al. ( 1 9 8 3 ) a l s o o b s e r v e d e l e v a t e d a d r e n a l m e d u l l a r y a c t i v i t y a n d u r i n a r y e p i n e p h r i n e e x c r e t i o n d u r i n g a c u t e m o d e r a t e h y p o x e m i a i n d e p e n d e n t o f c a r d i a c s y m p a t h e t i c a c t i v i t y i n r a t s , t h u s s p e c u l a t i n g t h a t t h e r e s p o n s e d u r i n g a c u t e m o d e r a t e h y p o x e m i a i s p r i m a r y b y a d r e n a l m e d u l l a r y a c t i v a t i o n . I n a d d i t i o n , t h e a c t i v a t i o n o f t h e a d r e n o m e d u l l a r y s y s t e m b y h y p o x e m i a m a y h a v e s o m e r e s i d u a l e f f e c t e v e n a f t e r r e s t o r a t i o n o f P a C h . C r i t c h l e y et al. ( 1 9 8 0 ) s h o w e d t h a t t h e r e l e a s e o f c a t e c h o l a m i n e f r o m t h e a d r e n a l m e d u l l a , i n r e s p o n s e t o p r o l o n g e d c a r o t i d b o d y h y p o x e m i a , o u t l a s t e d t h e s t i m u l u s b y m o r e 94 t h a n 3 0 m i n . T h e y a l s o f o u n d t h a t a m o d e r a t e h y p o x e m i a w i t h c a r o t i d a r t e r i a l P a C h o f 5 0 - 6 0 m m H g w a s s u f f i c i e n t t o e v o k e t h e r e l e a s e o f c a t e c h o l a m i n e . A s m a l l , b u t s t a t i s t i c a l l y s i g n i f i c a n t , i n c r e a s e i n g l u c o s e c o n c e n t r a t i o n d u r i n g h y p o x e m i a w a s a l s o o b s e r v e d i n t h e p r e s e n t s t u d y . T h e r e l a t i v e l y s m a l l i n c r e a s e i n b l o o d g l u c o s e c o n c e n t r a t i o n i n t h e p r e s e n t s t u d y i s l i k e l y d u e t o a m o d e r a t e l e v e l o f h y p o x e m i a . T h e d e v e l o p m e n t o f a m o d e s t d e g r e e o f h y p e r g l y c e m i a u n d e r s t r e s s s u c h a s h y p o x i a a n d h y p o t h e r m i a m a y r e p r e s e n t a n a d a p t i v e r e s p o n s e t o s t r e s s , h e l p i n g t o p r o t e c t a n d m a i n t a i n t h e s u p p l y o f a n i m p o r t a n t f u e l f o r t h e b r a i n a n d o t h e r t i s s u e ( B a u m a n d P o r t e , 1 9 8 0 ) . I n c r e a s e d b l o o d g l u c o s e c o n c e n t r a t i o n d u r i n g s t r e s s ( s t r e s s h y p e r g l y c e m i a ) i s c o m m o n i n a c u t e h y p o x e m i a ( B a u m a n d P o r t e , 1 9 8 0 ) . E a r l i e r s t u d i e s h a v e s u g g e s t e d t h a t t h e e l e v a t e d b l o o d g l u c o s e c o n c e n t r a t i o n w a s a r e s u l t o f i n c r e a s e d h e p a t i c g l y c o g e n o l y s i s a n d t h e d i r e c t h o r m o n a l e f f e c t s o f h y p o x i a ( B r i t t o n a n d K l i n e , 1 9 4 5 ; H i m w i c h et al, 1 9 4 3 ; S h e l l e y , 1 9 6 1 ) . T h e r e i s a n i n c r e a s e d e p i n e p h r i n e s e c r e t i o n b y t h e a d r e n a l m e d u l l a i n c o m b i n a t i o n o f s e c o n d a r y g l u c a g o n r e l e a s e b y a - c e l l s o f p a n c r e a t i c i s l e t s d u r i n g h y p o x e m i a . T h i s , i n t u r n , s t i m u l a t e s g l y c o g e n b r e a k d o w n i n m u s c l e s a n d l i v e r , t h u s r e s u l t i n g i n t h e e l e v a t i o n o f b l o o d s u g a r l e v e l . C a t e c h o l a m i n e s c a n d i r e c t l y s t i m u l a t e g l u c o s e p r o d u c t i o n b y t h e l i v e r ( R i z z a et al, 1 9 8 0 ) . I n a d d i t i o n , t h e y c a n i n t e r f e r e w i t h i n s u l i n - m e d i a t e d g l u c o s e u p t a k e ( C h i a s s o n et al, 1 9 8 1 ; D e i b e r t a n d D e F r o n z o , 1 9 8 0 ) . T h e r e f o r e , i n c r e a s e d p l a s m a c a t e c h o l a m i n e l e v e l s d u r i n g h y p o x e m i a r e s u l t i n a n i n c r e a s e d b l o o d g l u c o s e l e v e l a n d s u b s e q u e n t s h u n t i n g o f b l o o d g l u c o s e t o i n s u l i n - i n d e p e n d e n t t i s s u e s , s u c h a s t h e b r a i n . T h e r e i s a l s o e v i d e n c e f o r a s t i m u l a t o r y e f f e c t o f c a t e c h o l a m i n e o n g l u c a g o n s e c r e t i o n m e d i a t e d via a p - a d r e n e r g i c a n d , i n 95 a l e s s o r d e g r e e , o c - a d r e n e r g i c m e c h a n i s m ( G e r i c h et al, 1 9 7 3 ; G e r i c h et al, 1 9 7 4 ; S a m o l s a n d W e i r , 1 9 7 9 ) . W h e n a n a e s t h e t i z e d d o g s w e r e m a d e a c u t e l y h y p o x i c , a v e r y l a r g e i n h i b i t i o n o f i n s u l i n s e c r e t i o n a n d i n c r e a s e d p l a s m a g l u c a g o n l e v e l s w e r e o b s e r v e d ( B a u m a n d P o r t e , 1 9 8 0 ) . T h e s y m p a t h e t i c n e r v o u s s y s t e m , a c t i v a t e d a t l o w o x y g e n t e n s i o n ( C o m l i n e a n d S i l v e r , 1 9 6 6 ; C u n n i n g h a m et al, 1 9 6 5 ) , h a s a n i m p o r t a n t r o l e i n m e d i a t i n g t h e h y p o x i c i n h i b i t i o n o f i n s u l i n r e l e a s e ( B a u m a n d P o r t e , 1 9 6 9 ) . a - a d r e n e r g i c r e c e p t o r s t i m u l a t i o n , k n o w n t o i n h i b i t i n s u l i n s e c r e t i o n , i s a m a j o r f a c t o r i n t h i s p r o c e s s ( B a u m a n d P o r t e , 1 9 7 2 ) . A l t h o u g h b o t h a- a n d p - a d r e n e r g i c r e c e p t o r s a r e s t i m u l a t e d w i t h s y m p a t h e t i c a c t i v a t i o n , a-a d r e n e r g i c s u p p r e s s i o n o f i n s u l i n p r e d o m i n a t e s i n h y p o x i a b e c a u s e p - a d r e n e r g i c e n h a n c e m e n t i s i m p a i r e d ( B a u m a n d P o r t e , 1 9 7 6 ) . T h i s s u p p r e s s i o n o f i n s u l i n i n c o m b i n a t i o n w i t h h e p a t i c g l y c o g e n o l y s i s m e d i a t e d b y e p i n e p h r i n e a n d g l u c a g o n d u r i n g h y p o x i a m a y e x p l a i n t h e e l e v a t e d l e v e l o f b l o o d g l u c o s e d u r i n g h y p o x e m i a a n d s u b s e q u e n t p o s t - h y p o x e m i a i n t h e p r e s e n t s t u d y . 4.2.4. Urine flow and osmolality during hypoxemia and MCP infusion. I n t h e p r e s e n t s t u d y , u r i n e f l o w w a s s i g n i f i c a n t l y i n c r e a s e d d u r i n g h y p o x e m i a , a t t h e s a m e t i m e , u r i n e o s m o l a l i t y w a s s i g n i f i c a n t l y d e c r e a s e d i n t h e e x p e r i m e n t a l g r o u p . D i u r e s i s d u r i n g a c u t e h y p o x e m i a , e i t h e r h y p o c a p n i c o r i s o c a p n i c , w a s d e m o n s t r a t e d i n t h e c o n s c i o u s d o g ( W a l k e r , 1 9 8 2 ) a n d r a t ( C o l i c e et al, 1 9 9 1 ) . S t u d i e s p r e v i o u s t o t h e s e , h o w e v e r , h a v e r e p o r t e d a v a r i e t y o f u r i n e f l o w r e s p o n s e s ( d i u r e s i s a n d a n t i d i u r e s i s ) t o a c u t e h y p o x e m i a ( S e l k u r t , 1 9 5 3 ; S t i c k n e y et al, 1 9 4 6 ; A n d e r s o n et al, 1 9 7 8 ) . H o w e v e r , m a n y o f t h e s e e a r l i e r 96 s t u d i e s u s e d a n a e s t h e t i z e d a n d / o r e x t e n s i v e l y m a n i p u l a t e d a n i m a l p r e p a r a t i o n s , w h i c h m a y h a v e c o m p l i c a t e d r e s u l t s a n d m a d e i n t e r p r e t a t i o n d i f f i c u l t . A c u t e h y p o x e m i a i n h u m a n s u s u a l l y h a s r e s u l t e d i n a d i u r e t i c r e s p o n s e ( B e r g e r et al, 1 9 4 9 ; B u r r i l l et al, 1 9 4 5 ; C u r r i e a n d U l l m a n , 1 9 6 1 ; U l l m a n , 1 9 6 1 ) . C u r r e n t l y , t h e m e c h a n i s m o f d i u r e s i s d u r i n g h y p o x e m i a i s n o t c l e a r l y u n d e r s t o o d . A n u m b e r o f m e c h a n i s m s s u c h a s i n c r e a s e d G F R s e c o n d a r y t o i n c r e a s e d r e n a l b l o o d f l o w a n d a r t e r i a l b l o o d p r e s s u r e , a n d h y p o x e m i a - i n d u c e d h o r m o n a l c h a n g e s i n p l a s m a a t r i a l n a t r i u r e t i c f a c t o r ( A N F ) , a l d o s t e r o n e a n d v a s o p r e s s i n l e v e l s h a v e b e e n p r o p o s e d . T h e d i u r e t i c r e s p o n s e d u r i n g h y p o x e m i a m a y b e , i n p a r t , r e l a t e d t o a h e m o d y n a m i c a l t e r a t i o n a s s o c i a t e d w i t h h y p o x e m i a . I n d o g s , h y p o c a p n i c h y p o x e m i a c a u s e d a n i n c r e a s e i n G F R a n d r e n a l b l o o d f l o w s e c o n d a r y t o t h e a r t e r i a l b l o o d p r e s s u r e a n d c a r d i a c o u t p u t i n c r e a s e ( w i t h a l e s s e r d e g r e e i n i s o c a p n i c h y p o x e m i a ) , w h i l e r e n a l v a s c u l a r r e s i s t a n c e r e m a i n e d u n a l t e r e d ( K o e h l e r et al, 1 9 8 0 ; W a l k e r , 1 9 8 2 ) . I n a d d i t i o n , g l o m e r u l a r f i l t r a t i o n r a t e ( G F R ) w a s a l s o i n c r e a s e d d u r i n g h y p o x e m i a i n t h e s e s t u d i e s . H o w e v e r , a c u t e h y p o x e m i a - i n d u c e d d i u r e s i s i n t h e r a t h a s b e e n a s s o c i a t e d w i t h a f a l l i n s y s t e m i c a r t e r i a l b l o o d p r e s s u r e a n d c a r d i a c o u t p u t ( C o l i c e et al, 1 9 9 1 ; O u et al, 1 9 8 9 ) , t h e r e b y s u g g e s t i n g t h e i n v o l v e m e n t o f o t h e r d i u r e t i c m e c h a n i s m ( s ) . I n t h e p r e s e n t s t u d y i n s h e e p , t h e r e w a s a s m a l l , b u t s t a t i s t i c a l l y s i g n i f i c a n t , i n c r e a s e i n m e a n a r t e r i a l b l o o d p r e s s u r e d u r i n g h y p o x e m i a i n t h e e x p e r i m e n t a l g r o u p . B u t , i t i s n o t c l e a r w h e t h e r t h e h y p o x e m i a - i n d u c e d d i u r e s i s i s , i n f a c t , d i r e c t l y r e l a t e d t o t h e i n c r e a s e d m e a n a r t e r i a l b l o o d p r e s s u r e , d u e t o l a c k o f i n f o r m a t i o n r e g a r d i n g r e n a l b l o o d f l o w a n d G F R . H o w e v e r , d i u r e s i s d u r i n g h y p o x e m i a m a y b e , a t l e a s t i n p a r t , d u e t o e l e v a t e d g l o m e r u l a r f i l t r a t i o n r a t e d u e t o a n i n c r e a s e i n r e n a l b l o o d f l o w s e c o n d a r y t o a r t e r i a l b l o o d p r e s s u r e i n c r e a s e . 97 A c u t e h y p o x i a i n a n a e s t h e t i z e d d o g s h a s b e e n r e p o r t e d t o s t i m u l a t e a r g i n i n e v a s o p r e s s i n ( A V P ) r e l e a s e a n d a n a n t i d i u r e t i c r e s p o n s e ( A n d e r s o n et al, 1 9 7 8 ) , b u t a c u t e h y p o x i a i n c o n s c i o u s h u m a n s a n d r a t s d o e s n o t a f f e c t p l a s m a A V P l e v e l s ( d u S o u i c h et al, 1 9 8 7 ; A s h a c k et al, 1 9 8 5 ; J o n e s et al, 1 9 8 1 ) . A l d o s t e r o n e l e v e l s f a l l w i t h a c u t e h y p o x i a i n h u m a n s , b u t o n l y a f t e r 3 0 t o 6 0 m i n o f e x p o s u r e ( C o l i c e a n d R a m i r e z , 1 9 8 5 ) . T h u s , t h e s e s t u d i e s s u g g e s t e d t h a t A V P a n d a l d o s t e r o n e m a y n o t b e c l o s e l y r e l a t e d t o a c u t e h y p o x i a -i n d u c e d d i u r e s i s . T h e i n v o l v e m e n t o f A N F i n h y p o x e m i a - i n d u c e d d i u r e s i s w a s a l s o s u g g e s t e d ( C o l i c e et al, 1 9 9 1 ; K o l l e r et al, 1 9 9 0 ; S h i m i z u a n d N a k a m u r a , 1 9 8 6 ) . I t h a s b e e n r e p o r t e d t h a t a c u t e h y p o x e m i a i s a p o t e n t s t i m u l u s f o r a t r i a l n a t r i u r e t i c f a c t o r ( A N F ) r e l e a s e ( d u S o u i c h et al, 1 9 8 7 ; R a m i r e z et al, 1 9 8 8 ; L a w r e n c e et al, 1 9 9 0 ) i n h u m a n s , i n i s o l a t e d r a t a n d r a b b i t h e a r t s ( B a e r t s c h i et al, 1 9 8 6 ) a n d i n r a b b i t s ( B a e r t s c h i et al, 1 9 8 8 ) . A n i m m e d i a t e A N F r e l e a s e f r o m t h e h e a r t a t t h e o n s e t o f h y p o x i a w a s r e p o r t e d ( B a e r t s c h i et al, 1 9 8 6 ) , w h i c h c o i n c i d e s w i t h t h e t r a n s i e n t d i u r e s i s i n a c u t e h y p o x e m i a . A c o r r e l a t i o n b e t w e e n p l a s m a A N F c o n c e n t r a t i o n a n d a r t e r i a l b l o o d p r e s s u r e w a s s u g g e s t e d ( d u S o u i c h et al, 1 9 8 7 ) . H o w e v e r t h e i n c r e a s e d p l a s m a A N F l e v e l s d u r i n g h y p o x e m i a a r e m o r e l i k e l y a s s o c i a t e d w i t h r i g h t a t r i a l d i s t e n t i o n s e c o n d a r y t o p u l m o n a r y h y p e r t e n s i o n ( O u et al, 1 9 8 6 ) a n d h y p e r v e n t i l a t i o n ( C o l i c e et al, 1 9 9 1 ) , s i n c e t h e d i s t e n t i o n o f c a r d i a c a t r i a i s t h e p r i m a r y p h y s i o l o g i c a l s t i m u l u s f o r t h e r e l e a s e a n d s y n t h e s i s o f A N F ( L a n g e et al, 1 9 8 7 ) . T h e r e w a s a s i g n i f i c a n t d e c r e a s e i n u r i n e o s m o l a l i t y d u r i n g h y p o x e m i a , u n l i k e t h e a c u t e m o d e r a t e h y p o x e m i a - i n d u c e d d i u r e s i s i n t h e s t u d i e s o f W a l k e r ( 1 9 8 2 ) a n d C o l i c e et al. ( 1 9 9 1 ) w h i c h s h o w e d n o c h a n g e s i n u r i n e o s m o l a l i t y d u r i n g h y p o x e m i a . T h e a v e r a g e r e n a l o s m o l a l e x c r e t i o n r a t e , t h e p r o d u c t o f u r i n e f l o w a n d u r i n e o s m o l a l i t y , w a s a l s o d e c r e a s e d 98 s i g n i f i c a n t l y d u r i n g h y p o x e m i a . T h e o s m o l a l e x c r e t i o n p a r a m e t e r w a s u s e d i n t h e s t u d y a n d c o u l d b e r e g a r d e d a s a r o u g h i n d i c a t o r o f t h e q u a n t i t y o f s a l t a n d o t h e r s o l u b l e c o m p o u n d s e x c r e t e d i n u r i n e i n a g i v e n t i m e , s i n c e u r i n e e l e c t r o l y t e a n a l y s i s c o u l d n o t b e p e r f o r m e d s a t i s f a c t o r i l y i n a l l t h e u r i n e s a m p l e s i n t h e p r e s e n t s t u d y . U r i n e e l e c t r o l y t e a n a l y s i s o f t h e t w o a n i m a l s i n t h e e x p e r i m e n t a l g r o u p w a s p e r f o r m e d , a n d t h e r e a p p e a r t o b e n o s i g n i f i c a n t c h a n g e s i n e l e c t r o l y t e e x c r e t i o n (i.e. n o n a t r i u r e s i s o r k a l i u r e s i s ) d u r i n g h y p o x e m i a i n c o n t r a s t t o t h e f i n d i n g o f W a l k e r ( 1 9 8 2 ) a n d C o l i c e et al. ( 1 9 9 1 ) . F i g u r e 7 s h o w s t h e r e l a t i o n s h i p b e t w e e n u r i n e o s m o l a l i t y a n d u r i n e f l o w . T h e r e w a s a n e g a t i v e c o r r e l a t i o n w i t h t h e c o e f f i c i e n t o f d e t e r m i n a t i o n ( r ) o f 0 . 8 0 , w h i c h m e a n s t h e i n c r e a s e d u r i n e f l o w w a s a c c o m p a n i e d b y l o w e r e d u r i n e o s m o l a l i t y . T h e s e r e l a t i o n s h i p s s u g g e s t t h a t t h e i n c r e a s e d u r i n e f l o w d u r i n g h y p o x e m i a i s n o t l i k e l y t h e r e s u l t o f t h e i n c r e a s e d u r i n e v o l u m e e x c r e t i o n s e c o n d a r y t o i n c r e a s e d e l e c t r o l y t e e x c r e t i o n b y t h e k i d n e y , a s s h o w n . i n t h e p r e v i o u s s t u d i e s ( W a l k e r , 1 9 8 2 ; C o l i c e et al, 1 9 9 1 ) . T h e r e f o r e , t h e d i u r e s i s i n t h e c u r r e n t s t u d y m a y b e d u e t o e n h a n c e d w a t e r e x c r e t i o n i n d e p e n d e n t o f u r i n a r y e l e c t r o l y t e e x c r e t i o n . S i n c e h y p o x e m i a w a s i n d u c e d d u r i n g t h e c o n t i n u o u s M C P i n f u s i o n , t h e p r e s e n c e o f M C P c o u l d b e a s s o c i a t e d w i t h t h e l o w u r i n e o s m o l a l i t y . S i n c e b o t h t h e a d m i n i s t r a t i o n o f M C P a n d a c u t e h y p o x e m i a a r e k n o w n t o a f f e c t r e n a l h a e m o d y n a m i c s a n d u r i n e f l o w i n a n o p p o s i n g d i r e c t i o n , t h e p r e s e n t s t u d y w h i c h c o m b i n e s t h e s e t w o f a c t o r s r a i s e s a n i n t e r e s t i n g p e r s p e c t i v e . M C P i s r e p o r t e d t o d e c r e a s e r e n a l b l o o d f l o w a n d u r i n e f l o w i n h u m a n s ( I s r a e l et al, 1 9 8 6 ) . N o r b i a t o et al. ( 1 9 8 6 ) r e p o r t e d t h a t M C P d e c r e a s e d f r e e w a t e r e x c r e t i o n i n m a n b y i n c r e a s e d v a s o p r e s s i n s e c r e t i o n . D u r i n g a c u t e m o d e r a t e h y p o x e m i a s i m i l a r t o t h e p r e s e n t s t u d y ( o x y g e n s a t u r a t i o n = 7 5 - 8 0 % ) , t h e p l a s m a a l d o s t e r o n e c o n c e n t r a t i o n ( e i t h e r n o r m a l o r e l e v a t e d b y M C P a d m i n i s t r a t i o n ) w a s 99 s i g n i f i c a n t l y r e d u c e d a n d A N F l e v e l s w e r e s l i g h t l y i n c r e a s e d i n h u m a n s ( V o n m o o s et al, 1 9 9 0 ; L a w r e n c e et al, 1 9 9 0 ) . T h e h y p o x e m i a - i n d u c e d i n c r e a s e i n p l a s m a A N F l e v e l s c a u s e d a p r o m i n e n t s u p p r e s s i o n o f p l a s m a a l d o s t e r o n e w i t h o u t n a t r i u r e s i s ( L a w r e n c e et al, 1 9 9 0 ; L a w r e n c e a n d S h e n k e r , 1 9 9 1 ) . I n a d d i t i o n , a l o w - d o s e A N F i n f u s i o n , w h i c h i s a s i m i l a r l e v e l t o a m o d e r a t e h y p o x e m i a - i n d u c e d A N F i n c r e a s e , h a s l i t t l e o r n o e f f e c t o n n a t r i u r e s i s ( S h e n k e r et al, 1 9 9 2 ) . T h e s e r e s u l t s m a y e x p l a i n t h e a b s e n c e o f n a t r i u r e s i s d u r i n g h y p o x e m i a i n t h e p r e s e n t s t u d y . U n f o r t u n a t e l y , t h e l a c k o f i n f o r m a t i o n o f p l a s m a h o r m o n e l e v e l s (e.g. A N F a n d a l d o s t e r o n e ) d o e s n o t a l l o w u s t o a d d r e s s t h i s s u b j e c t i n t h e p r e s e n t s t u d y , a n d t h u s f u r t h e r s t u d i e s m a y b e r e q u i r e d . 4.3. Statistical and Practical Aspects of Experiment Design 4.3.1. Design of experimental protocol T h e i n f u s i o n e x p e r i m e n t a l p r o t o c o l w i t h 3 p h a s e s ( p r e - h y p o x e m i c , h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s ) i n t h e s t u d y w a s d e v e l o p e d i n o r d e r t o a d d r e s s s o m e o f p r o b l e m s c o m m o n l y a s s o c i a t e d w i t h s t u d y i n g p h a r m a c o k i n e t i c s d u r i n g h y p o x e m i a . M o s t p r e v i o u s p h a r m a c o k i n e t i c s t u d i e s d u r i n g h y p o x e m i a h a v e b e e n b a s e d o n t h e i . v . b o l u s d e s i g n w i t h d r u g a d m i n i s t r a t i o n t o t h e s e p a r a t e h y p o x e m i c a n d c o n t r o l ( n o r m o x e m i c ) g r o u p s [ D e s i g n I ] ( d u S o u i c h et al, 1 9 8 5 a , b ) . I n s o m e s t u d i e s , p a i r e d e x p e r i m e n t s w e r e p e r f o r m e d o n t h e s a m e s u b j e c t s [ D e s i g n J J ] ( S a u n i e r et al, 1 9 8 7 ; C l o z e l et al, 1 9 8 1 ) . S i n c e t h e r e a r e c o n s i d e r a b l e i n d i v i d u a l v a r i a t i o n s i n t h e p h y s i o l o g i c a l a n d p h a r m a c o k i n e t i c r e s p o n s e s t o h y p o x e m i a ( d u S o u i c h et al, 1 9 7 8 ; T a b u r e t et al, 1 9 9 0 ) , c o n d u c t i n g a c o m p a r a t i v e s t u d y o f p h a r m a c o k i n e t i c s o n s e p a r a t e h y p o x e m i c a n d c o n t r o l g r o u p s [ D e s i g n I ] p o s e s s o m e p o t e n t i a l p r o b l e m s . T h i s i s p a r t i c u l a r l y t r u e w h e n t h e s a m p l e s i z e i s s m a l l a n d t h e v a r i a n c e i n t h e 100 g r o u p i s l a r g e , a s h a s b e e n e n c o u n t e r e d i n p r e v i o u s s t u d i e s . A l a r g e r s a m p l e s i z e w o u l d b e r e q u i r e d t o t e s t a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t w o g r o u p s w i t h a h i g h e r p o p u l a t i o n v a r i a n c e a n d s m a l l m i n i m u m d e t e c t a b l e d i f f e r e n c e b e t w e e n p o p u l a t i o n m e a n s ( Z a r , 1 9 8 4 ) . T h u s , t h i s d e s i g n m a y b e s u i t a b l e f o r c o n d u c t i n g e x p e r i m e n t s i n a l a r g e p o p u l a t i o n o f s m a l l a n i m a l s p e c i e s s u c h a s t h e r a t a n d r a b b i t w h e r e m i n i m a l s u r g i c a l p r e p a r a t i o n s a r e i n v o l v e d . T h i s d e s i g n [ D e s i g n I ] w a s r e j e c t e d f r o m c o n s i d e r a t i o n d u e t o t h e p r a c t i c a l l i m i t a t i o n o f c o n d u c t i n g a l a r g e n u m b e r o f c o m p l i c a t e d s u r g e r i e s a n d p h a r m a c o k i n e t i c e x p e r i m e n t s o n l a r g e r a n i m a l s l i k e s h e e p . T w o e x p e r i m e n t a l p r o t o c o l s w e r e i n i t i a l l y c o n s i d e r e d . O n e p r o t o c o l w a s a p a i r e d i . v . b o l u s s t u d y [ D e s i g n II], w h e r e t w o s e p a r a t e i . v . b o l u s d o s e s o f M C P w o u l d b e g i v e n o n s e p a r a t e d a y s u n d e r h y p o x e m i c a n d n o r m o x e m i c c o n d i t i o n s , s i m i l a r t o S a u n i e r et al. ( 1 9 8 7 ) . T h e o t h e r w a s c o n t i n u o u s M C P i n f u s i o n w i t h n o r m o x e m i c c o n t r o l a n d h y p o x e m i c p e r i o d s [ D e s i g n H I ] . T h e i . v . b o l u s m e t h o d o f f e r s c o m p l i m e n t a r y i n f o r m a t i o n t o t h a t o b t a i n e d w i t h t h e p r e s e n t i . v . i n f u s i o n m e t h o d , i.e. t h e e l i m i n a t i o n r a t e c o n s t a n t a n d t h e v o l u m e o f d i s t r i b u t i o n . H o w e v e r , t h e p a i r e d i . v . b o l u s m e t h o d p o s e s s e v e r a l p r a c t i c a l p r o b l e m s i n t h e p h a r m a c o k i n e t i c s t u d y o f h y p o x e m i a . T h e p r e s e n t s t u d y r e q u i r e s t h e q u a n t i t a t i v e c o m p a r i s o n o f p h a r m a c o k i n e t i c p a r a m e t e r s b e t w e e n h y p o x e m i c a n d n o r m o x e m i c c o n d i t i o n s , i n o r d e r t o e v a l u a t e t h e i m p a c t o f h y p o x e m i a o n d r u g d i s p o s i t i o n . A c e r t a i n t i m e d e l a y (i.e. a t l e a s t 2 - 3 d a y s ) b e t w e e n p a i r e d e x p e r i m e n t s w o u l d b e r e q u i r e d t o e n s u r e t o t a l e l i m i n a t i o n o f d r u g a n d r e l a t e d m e t a b o l i t e s f r o m t h e b o d y b e t w e e n s u c c e s s i v e e x p e r i m e n t s a n d a l l t h e c o n d i t i o n s e x c e p t i n s p i r e d g a s c o n t e n t s h o u l d b e i d e n t i c a l f o r b o t h e x p e r i m e n t s . T h e i . v . i n f u s i o n m e t h o d [ D e s i g n I I I ] h a s e l i m i n a t e d t h e s e r e q u i r e m e n t s o f a p a i r e d e x p e r i m e n t a n d r e d u c e s t h e t i m e f o r e x p e r i m e n t s . P h y s i o l o g i c a l a n d p h a r m a c o k i n e t i c c h a n g e s i n d u c e d b y h y p o x e m i a 101 c o u l d b e d e m o n s t r a t e d w i t h i n a s i n g l e e x p e r i m e n t . I n a d d i t i o n , i t w o u l d b e p o s s i b l e t o o b s e r v e d y n a m i c p h y s i o l o g i c a l a n d p h a r m a c o k i n e t i c c h a n g e s d u r i n g a n d a f t e r h y p o x e m i a . A n o t h e r r e a s o n f o r d e v e l o p i n g t h e i n f u s i o n m e t h o d w a s t o e s t a b l i s h i n t r a - s u b j e c t c o n t r o l s , s i n c e i t h a s b e e n r e p o r t e d t h a t i n d i v i d u a l t o l e r a n c e t o w a r d h y p o x e m i c s t r e s s a n d t h e d e g r e e o f p h a r m a c o k i n e t i c c h a n g e s a r e h i g h l y v a r i a b l e ( d u S o u i c h et al, 1 9 7 8 ; T a b u r e t et al, 1 9 9 0 ) . T h e u s e o f a n i n t r a - s u b j e c t c o n t r o l m e t h o d i n t h e p r e s e n t p r o t o c o l h a s g r e a t l y i m p r o v e d t h e s t a t i s t i c a l a n a l y s i s o f p h a r m a c o k i n e t i c a n d p h y s i o l o g i c a l d a t a . F o r e x a m p l e , t h e d a t a s e t o f t h e p l a s m a M C P s t e a d y - s t a t e c o n c e n t r a t i o n f r o m p r e - h y p o x e m i c , h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s f r o m a s i n g l e e x p e r i m e n t w a s u s e d a s t h e p o p u l a t i o n a n d t h e m e a n v a l u e s f o r e a c h t i m e p e r i o d w e r e c o m p a r e d u s i n g a n a l y s i s o f v a r i a n c e ( A N O V A ) . W h e r e a s , i n o t h e r m e t h o d s [ D e s i g n I a n d J J ] , i t w o u l d b e n e c e s s a r y t o c o m p a r e t h e h y p o x e m i c a n d c o n t r o l g r o u p v a l u e s o f p h a r m a c o k i n e t i c p a r a m e t e r s s u c h a s r a t e o f e l i m i n a t i o n , w h i c h h a v e c o n s i d e r a b l y h i g h e r v a r i a n c e . T h i s p r o c e s s o f c o m p a r i s o n o f t h e p h a r m a c o k i n e t i c a n d p h y s i o l o g i c a l p a r a m e t e r s f r o m t h e h y p o x e m i c a n d n o r m o x e m i c p e r i o d s w i t h i n a s i n g l e a n i m a l , i n s t e a d o f t h e h y p o x e m i c a n d c o n t r o l g r o u p s , g r e a t l y d e c r e a s e s a [ t h e p o p u l a t i o n v a r i a n c e ] a n d t h u s r e s u l t i n g i n a h i g h e r § v a l u e [ t h e d e t e r m i n i n g p o w e r ] o f t h e t e s t i n a g i v e n n u m b e r o f e x p e r i m e n t s ( Z a r , 1 9 8 4 ) . T h i s m e a n s t h a t t h e p o w e r o f t h e t e s t i n a g i v e n n u m b e r o f t h e e x p e r i m e n t s u b j e c t s w a s i m p r o v e d i n t h e p r e s e n t s t u d y . T h e r e f o r e , t h e i n t r a - s u b j e c t c o m p a r i s o n s h o u l d p r o v i d e a m o r e r e l i a b l e i n t e r p r e t a t i o n o f t h e e f f e c t o f h y p o x e m i a a n d a l s o r e d u c e t h e n u m b e r o f a n i m a l s r e q u i r e d f o r t h e s t u d y . I n a d d i t i o n t o t h e i n t r a - s u b j e c t c o n t r o l p e r i o d s ( S c h e m e J J I ) , t h e c o n t r o l g r o u p e x p e r i m e n t s w e r e c o n d u c t e d i n f o u r o f t h e e w e s . I n t h e c o n t r o l g r o u p , t h e i n f u s i o n s t u d y w a s 102 c o n d u c t e d a c c o r d i n g t o t h e s a m e p r o t o c o l a s i n t h e e x p e r i m e n t a l g r o u p ( S c h e m e I V ) , e x c e p t f o r t h e n i t r o g e n i n f u s i o n - i n d u c e d h y p o x e m i a . T h e d a t a s e t s c o r r e s p o n d i n g t o t h e s a m e p e r i o d s b e t w e e n e x p e r i m e n t a l a n d c o n t r o l g r o u p s w e r e q u a l i t a t i v e l y c o m p a r e d a n d s t a t i s t i c a l l y a n a l y s e d . F o r e x a m p l e , a p a r a l l e l c o m p a r i s o n o f c h a n g e s i n M C P s t e a d y - s t a t e c o n c e n t r a t i o n s b e t w e e n t h e e x p e r i m e n t a l a n d c o n t r o l g r o u p s e n a b l e s u s t o c o n f i r m t h a t a n y c h a n g e s o b s e r v e d a r e n o t a n a r t i f a c t o f t h e e x p e r i m e n t , s u c h a s m i g h t h a v e a r i s e n d u e t o t h e p r o l o n g e d i n f u s i o n . T h u s , t h e c o m p a r i s o n o f d a t a f r o m t h e e x p e r i m e n t a l w i t h t h e c o n t r o l g r o u p e l i m i n a t e s a n y p o s s i b i l i t y t h a t t h e r e s u l t s w e r e a f f e c t e d b y f a c t o r s n o t r e l a t e d t o h y p o x e m i a , s u c h a s o b s c u r e t i m e - d e p e n d e n t e f f e c t s . S t a t i s t i c a l c o m p a r i s o n s u s i n g S t u d e n t ' s t - t e s t w e r e p e r f o r m e d b e t w e e n t w o g r o u p s , h o w e v e r , i n s o m e p a r a m e t e r s w i t h h i g h e r v a r i a t i o n , t h e s e s t a t i s t i c a l a n a l y s i s c o u l d n o t a d e q u a t e l y p e r f o r m e d d u e t o l o w 0 v a l u e [ t h e d e t e r m i n i n g p o w e r o f t h e t e s t ] (i.e. h i g h v a r i a n c e a n d l o w s a m p l e n u m b e r ) . T h e s e c o m p a r i s o n s , i n a s e n s e , a r e s i m i l a r t o t h e c o m p a r i s o n i n t h e D e s i g n I t y p e e x p e r i m e n t , w h i c h c o m p a r e s t h e d a t a f r o m t w o d a t a s e t s r a n d o m l y c h o s e n f r o m t h e t w o p o p u l a t i o n s . T h e r e f o r e , t h e c o n t r o l g r o u p i n t h e p r e s e n t s t u d y p r o v i d e s t h e a d d i t i o n a l c o m p a r i s o n w i t h t h e p h y s i o l o g i c a l a n d p h a r m a c o k i n e t i c d a t a g a t h e r e d d u r i n g t h e h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t g r o u p . 103 4.3.2. Statistical and practical considerations for determining the steady-state drug concentration. P r e v i o u s s t u d i e s o n m e t o c l o p r a m i d e p h a r m a c o k i n e t i c s i n s h e e p ( R i g g s et al, 1 9 8 8 : 1 9 9 0 ) r e p o r t e d t h a t t h e h a l f - l i f e o f M C P i n n o n - p r e g n a n t s h e e p w a s a b o u t 5 0 m i n a n d t h a t a s t e a d y - s t a t e c o n c e n t r a t i o n c o u l d b e a c h i e v e d w i t h i n 1 h o u r u s i n g a n a p p r o p r i a t e l o a d i n g d o s e . T h e o r e t i c a l l y , i t w o u l d r e q u i r e 4 - 5 h o u r s t o r e a c h a s t e a d y - s t a t e w i t h o u t a l o a d i n g d o s e . H o w e v e r , t h e t i m e t o s t e a d y - s t a t e c a n b e g r e a t l y r e d u c e d w i t h a n a p p r o p r i a t e l o a d i n g d o s e ( s e e S e c t i o n 4 . 3 . 3 . ) . S i n c e t h e p r o t o c o l u s e d i n t h e p r e s e n t s t u d y r e q u i r e s t h e d e t e r m i n a t i o n o f t h r e e s t e a d y - s t a t e d r u g c o n c e n t r a t i o n s w i t h i n a s i n g l e e x p e r i m e n t ( o n e f o r e a c h p r e - h y p o x e m i c , h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s i n t h e e x p e r i m e n t a l g r o u p a n d t h e e q u i v a l e n t p e r i o d s i n t h e c o n t r o l g r o u p ) , i t w a s e s s e n t i a l t o s e t u p a n a p p r o p r i a t e a s s e s s m e n t p r o c e d u r e f o r t h e s t e a d y - s t a t e d r u g c o n c e n t r a t i o n s . F o r t h e a s s e s s m e n t o f s t e a d y - s t a t e , t h e t h r e e f o l l o w i n g c r i t e r i a w e r e u s e d . T h e s e c r i t e r i a w e r e d e s i g n e d s o t h a t t h e s t e a d y - s t a t e c o n c e n t r a t i o n c o u l d b e e x p e r i m e n t a l l y d e t e r m i n e d i n a p r a c t i c a l a n d s t a t i s t i c a l l y s o u n d m a n n e r . I n a d d i t i o n , i t w a s d e s i g n e d t o m i n i m i z e b i a s d u r i n g t h e d a t a p r o c e s s i n g p r o c e d u r e : 1 . t h e p l a s m a M C P c o n c e n t r a t i o n v e r s u s t i m e p l o t w a s v i s u a l l y i n s p e c t e d . P l a t e a u s e c t i o n s i n t h e p r e - h y p o x e m i c , h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s (i.e. t h e s e c t i o n w h e r e p l a s m a c o n c e n t r a t i o n s w e r e r e m a i n e d r e l a t i v e l y c o n s t a n t ) w e r e c h o s e n f o r f u r t h e r s t a t i s t i c a l a n a l y s i s . 2 . t h e d a t a s e t s f r o m t h e s e p l a t e a u s e c t i o n s w e r e a n a l y s e d f o r t h e c o e f f i c i e n t o f v a r i a t i o n ( C V ) . T h e m a x i m u m C V l i m i t o f 1 0 % w a s u s e d a s c r i t e r i a f o r t h e d e v i a t i o n f r o m m e a n s t e a d y - s t a t e c o n c e n t r a t i o n . T h e d a t a s e t f r o m t h e p r e - h y p o x e m i c p e r i o d t e n d s t o e x h i b i t 104 h i g h e r C V v a l u e s d u e t o t h e s h o r t e r t i m e a l l o w e d t o a t t a i n s t e a d y - s t a t e a n d t h e s m a l l e r n u m b e r o f d a t a p o i n t s g a t h e r e d . C o n s e q u e n t l y , a C V v a l u e o f 1 5 % w a s u s e d a s t h e l i m i t i n t w o o f t h e a n i m a l s . D u r i n g t h e d a t a p r o c e s s i n g p r o c e d u r e , t h e e x t r e m e o u t l i e r s ( m o r e e x t r e m e t h a n 2 S D f r o m t h e m e a n ) f r o m e a c h d a t a s e t w e r e e l i m i n a t e d . I n t h i s c a s e t h e t e r m i n a l d a t a p o i n t s f r o m t h e l a t e r t i m e p e r i o d w e r e g i v e n p r e f e r e n c e t o r e m a i n i n t h e s e t o v e r e a r l i e r d a t a p o i n t s , s i n c e t h e s t e a d y - s t a t e d r u g c o n c e n t r a t i o n i s l i k e l y a t t a i n e d d u r i n g t h e l a t e r t i m e p e r i o d . I n p r a c t i c e , a n y e x t r e m e o u t l i e r ( s ) w a s c a r e f u l l y e x a m i n e d f o r p o s s i b l e e r r o r ( s ) a n d i n s o m e c a s e s r e - a s s a y e d . A f t e r t h i s s t e p , a t e r m i n a l d a t a p o i n t f r o m t h e e a r l i e s t t i m e p e r i o d w a s r e m o v e d u n t i l t h e C V v a l u e i s e q u a l o r s m a l l e r t h a n t h e l i m i t , t h e s l o p e (P) o f t h e r e g r e s s i o n l i n e w a s a l s o e x a m i n e d . T h e s l o p e o f t h e r e g r e s s i o n l i n e c o m p u t e d f r o m t h e s a m p l e d a t a e x p r e s s e d q u a n t i t a t i v e l y t h e s t r a i g h t - l i n e d e p e n d e n c y o f Y o n X (i.e. p l a s m a d r u g c o n c e n t r a t i o n o n t i m e ) i n t h e s a m p l e ( Z a r , 1 9 8 4 ) . I f t h e s l o p e o f t h e r e g r e s s i o n l i n e o f t h e p l a t e a u s e c t i o n i s s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o , t h e n i t w o u l d s u g g e s t t h a t p l a s m a d r u g c o n c e n t r a t i o n h a s a t r e n d o f e i t h e r a n i n c r e a s e o r d e c r e a s e o v e r t h e g i v e n t i m e (i.e. s t e a d y - s t a t e w a s n o t r e a c h e d ) . T h e r e f o r e , t h e s l o p e w a s a n a l y s e d t o d e t e r m i n e d w h e t h e r i t w a s d i f f e r e n t f r o m z e r o u s i n g a n a l y s i s o f v a r i a n c e ( A N O V A ) a n d t - t e s t f o r l i n e a r r e g r e s s i o n . I n s t a t i s t i c a l t e r m s , t h e l i n e a r r e g r e s s i o n o f t h e d a t a s e t w a s p e r f o r m e d w i t h t h e n u l l - h y p o t h e s i s ( H o : P = 0 ) t h a t t h e s l o p e o f t h e l i n e a r r e g r e s s i o n l i n e i s z e r o a n d t h e a l t e r n a t e h y p o t h e s i s , HA : P ^ 0 . T h e a n a l y s i s o f v a r i a n c e ( A N O V A ) f o r l i n e a r r e g r e s s i o n a n d S t u d e n t ' s t - t e s t w e r e u s e d w i t h t h e l e v e l o f s i g n i f i c a n c e , a = 0 . 0 5 . T h i s p r o c e d u r e w a s c h o s e n t o e n s u r e t h a t t h e r e i s n o t e n d e n c y f o r a g r a d u a l i n c r e a s e o r d e c r e a s e i n t h e d r u g c o n c e n t r a t i o n s o v e r t i m e . T h e r e j e c t i o n o f t h e n u l l - h y p o t h e s i s ( H o ) w o u l d m e a n t h a t s t e a d y - s t a t e w a s n o t r e a c h e d . 105 F r o m t h i s s t e a d y - s t a t e a s s e s s m e n t , i n m o s t o f a n i m a l s , t h e M C P s t e a d y - s t a t e c o n c e n t r a t i o n w a s f o u n d t o b e r e a c h e d b e t w e e n 3 0 m i n a n d 7 5 m i n w i t h a 1 5 m g l o a d i n g d o s e i n t h e p r e - h y p o x e m i c p e r i o d . O n e a n i m a l s ( e w e n o . 1 1 5 4 ) w a s f o u n d n o t t o r e a c h s t e a d y - s t a t e w i t h i n 2 h o u r s o f i n f u s i o n w i t h a l o a d i n g d o s e . D a t a f r o m t h i s a n i m a l w a s e x c l u d e d f r o m t h e s u b s e q u e n t p h a r m a c o k i n e t i c d a t a a n a l y s i s , s i n c e t h e h y p o x e m i c a n d p o s t -h y p o x e m i c s t e a d y - s t a t e c o n c e n t r a t i o n c o u l d n o t b e c o m p a r e d t o t h e p r e - h y p o x e m i c s t e a d y -s t a t e c o n c e n t r a t i o n . A f t e r o n s e t o f h y p o x e m i a , a n o t h e r s t e a d y - s t a t e ( h y p o x e m i c ) w a s r e a c h e d b e t w e e n 2 t o 4 h o u r s f r o m t h e o n s e t o f h y p o x e m i a . T h e p o s t - h y p o x e m i c s t e a d y - s t a t e w a s r e a c h e d b e t w e e n 2 t o 3 h o u r s a f t e r s t o p p i n g n i t r o g e n i n f u s i o n . T h i s a p p a r e n t s h o r t e r t i m e t o s t e a d y - s t a t e d u r i n g t h e p o s t - h y p o x e m i c p e r i o d , a s c o m p a r e d t o t h a t o f t h e h y p o x e m i c p e r i o d , i s l i k e l y d u e t o t h e f a c t t h a t t h e h a l f - l i f e o f M C P d u r i n g t h e p o s t - h y p o x e m i c p e r i o d i s s h o r t e r ( = 2 0 % ) t h a n t h a t o f t h e h y p o x e m i c p e r i o d . 106 4.3.3. Theoretical aspects of infusion with loading dose. T h e s t e a d y - s t a t e d r u g c o n c e n t r a t i o n ( C p ° ° i n f ) a f t e r a c o n t i n u o u s d r u g i n f u s i o n i s t h e p o i n t w h e r e t h e r a t e o f i n p u t o f d r u g ( i n f u s i o n r a t e ) i s e q u a l t o t h e r a t e o f d r u g l e a v i n g t h e b o d y ( e l i m i n a t i o n r a t e ) . S i n c e t h e i n f u s i o n r a t e i s c o n s t a n t , t h e t i m e t o r e a c h s t e a d y - s t a t e i s p r i m a r i l y d e p e n d e n t u p o n t h e e l i m i n a t i o n r a t e c o n s t a n t . F o r t h e p r e s e n t s t u d y , t h e o n e - c o m p a r t m e n t o p e n m o d e l a n d t h e t w o - c o m p a r t m e n t o p e n m o d e l w i t h d r u g e l i m i n a t i o n f r o m t h e c e n t r a l o r " w e l l p e r f u s e d " c o m p a r t m e n t ( s e e t h e s c h e m a t i c d i a g r a m s o n t h e n e x t p a g e ) , w e r e u s e d f o r c o m p u t e r - a i d e d s i m u l a t i o n , t h e o r e t i c a l i n t e r p r e t a t i o n a n d c a l c u l a t i o n o f p h a r m a c o k i n e t i c p a r a m e t e r s . O t h e r c o n c e p t s p r o v i d e d u t i l i t y i n d a t a i n t e r p r e t a t i o n , i n c l u d i n g p h y s i o l o g i c a l m o d e l i n g , m o m e n t a n a l y s i s a n d t h e n o n -c o m p a r t m e n t a l c l e a r a n c e c o n c e p t . U s i n g p h a r m a c o k i n e t i c p a r a m e t e r s d e t e r m i n e d b y R i g g s et al. ( 1 9 8 9 ) i n n o n - p r e g n a n t s h e e p , t h e c o m p u t e r a i d e d s i m u l a t i o n s o f i . v . i n f u s i o n b a s e d o n t h e o n e - a n d t w o - c o m p a r t m e n t m o d e l s w e r e p e r f o r m e d a s s h o w n i n t h e F i g u r e 1 1 . I t w o u l d t a k e a p p r o x i m a t e l y 3 . 4 h o u r s t o r e a c h a s t e a d y - s t a t e ( 9 5 % ) a n d 5 . 2 h o u r s f o r 9 9 % f r o m t h e o n e -c o m p a r t m e n t m o d e l w i t h o u t a l o a d i n g d o s e . I n t h e t w o - c o m p a r t m e n t m o d e l , t h e t i m e t o r e a c h s t e a d y - s t a t e w a s s l i g h t l y l o n g e r ( 4 . 0 h o u r f o r 9 5 % a n d 6 . 4 h o u r f o r 9 9 % ) t h a n t h a t o f t h e o n e - c o m p a r t m e n t m o d e l . T h e l o n g e r t i m e t o r e a c h s t e a d y - s t a t e i n t h e t w o - c o m p a r t m e n t i s l i k e l y t o b e a c o n s e q u e n c e s o f t h e d e l a y e d t r a n s f e r o f d r u g b e t w e e n t h e c e n t r a l a n d p e r i p h e r a l c o m p a r t m e n t s , w h e n c o m p a r e d t o i m m e d i a t e d r u g d i s t r i b u t i o n i n t h e o n e -c o m p a r t m e n t m o d e l . 107 Scheme V Diagrams of the compartment models used for computer simulation. One-compartment Open Model ko (+ LD) Two-compartment Open Model with central drug elimination ko (+ LD) Central Compartment Vc 12 21 Peripheral Compartment Vr 10 No-where ko represents the infusion rate constant of MCP, LD is the i.v. bolus loading dose, Vd is the apparent volume of distribution in one-compartment model, Vc and Vr is apparent volume of the central and peripheral compartment, respectively, Ke is the apparent first order MCP elimination rate constant, k 1 2 and k 2 i are the apparent first order inter-compartmental distribution rate constants for MCP and k 1 0 is the apparent MCP elimination rate constant from the central compartment. 108 70 - r 60 --2-ccrrp - - - 1-ocrrp Ia\er (95%) upper (105%) 0 1 1 i i i i i I I 1 1 i i i i i i i i i i i i i 1 1 1 i 1 1 i i i i i i 1 1 1 i i i i 1 1 1 i i 1 1 1 1 i i i i i i i i i i i I I 1 1 1 i i i i i i i i i 1 1 i i i 11 i i i i i i i i i i i 111 i i i O T — C N C O T L £ > < O I ^ C O O O T ime (hour) F i g u r e 1 1 . S i m u l a t i o n o f C p a f t e r s i m p l e i . v . i n f u s i o n w i t h o u t l o a d i n g d o s e u s i n g p h a r m a c o k i n e t i c p a r a m e t e r s f r o m R i g g s et al. ( 1 9 8 9 ) O 0 . 6 Q . o 0.4 0.2 4- . i u 1 1 1 1 1 1 1 1 CSI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I - NoLD - 100% LD - 90% LD - 80% LD - 110% LD - 120% LD — Lower (95%) — Upper (105%) T imeAidf-life F i g u r e 1 2 . S i m u l a t i o n o f C p a f t e r i . v . i n f u s i o n w i t h l o a d i n g d o s e ( o n e - c o m p a r t m e n t m o d e l ) 109 tS-30 T ime (hour) F i g u r e 1 3 . S i m u l a t i o n o f C p a f t e r i . v . i n f u s i o n w i t h l o a d i n g d o s e ( t w o - c o m p a r t m e n t m o d e l ) u s i n g p h a r m a c o k i n e t i c p a r a m e t e r s f r o m R i g g s et al. ( 1 9 8 9 ) I n t h e p r e s e n t s t u d y , a l o a d i n g d o s e w a s c o m b i n e d w i t h t h e i n f u s i o n t o r e d u c e t i m e r e q u i r e d t o r e a c h t h e s t e a d y - s t a t e d r u g c o n c e n t r a t i o n . T h e r e f o r e , f u r t h e r c o m p u t e r s i m u l a t i o n s o f t h e i n f u s i o n w i t h a l o a d i n g d o s e w e r e p e r f o r m e d u s i n g t h e o n e - a n d t w o -c o m p a r t m e n t m o d e l a s s h o w n i n F i g u r e s 1 2 a n d 1 3 . A n i d e a l ( o p t i m a l ) l o a d i n g d o s e w i t h t h e o n e - c o m p a r t m e n t m o d e l i s LD = Css * Vd, w h i c h t h e o r e t i c a l l y r e s u l t s i n i n s t a n t a n e o u s a r r i v a l a n d m a i n t e n a n c e o f a s t e a d y - s t a t e a s s h o w n i n F i g u r e 1 2 . H o w e v e r , i n t h e a c t u a l e x p e r i m e n t a l a n d t h e r a p e u t i c e n v i r o n m e n t , i t i s n e i t h e r p r a c t i c a l n o r p o s s i b l e t o c a l c u l a t e a n d a d j u s t a l o a d i n g d o s e f o r e a c h i n d i v i d u a l s u b j e c t , t h u s t h e l o a d i n g d o s e c a l c u l a t e d f r o m t h e m e a n v a l u e s o f Css a n d Vd w o u l d b e u s e d f o r a l l t h e e x p e r i m e n t s u b j e c t s . T h e r e f o r e , t h i s l o a d i n g d o s e r e g i m e n c o u l d r e s u l t i n e i t h e r o v e r - o r u n d e r - e s t i m a t i o n o f t h e o p t i m a l l o a d i n g d o s e . F i g u r e 1 2 s h o w s a s i m u l a t e d p l o t o f p l a s m a d r u g c o n c e n t r a t i o n ( i n t e r m s o f p l a s m a 110 s t e a d y - s t a t e d r u g c o n c e n t r a t i o n ) v e r s u s t i m e ( i n t e r m s o f h a l f - l i f e ) , w h i c h b a s e d o n t h e o n e -c o m p a r t m e n t m o d e l w i t h f i r s t - o r d e r k i n e t i c s . A l o a d i n g d o s e o f 9 0 % o r 1 1 0 % o f t h e o p t i m a l d o s e w i l l t a k e a p p r o x i m a t e l y 1 h a l f - l i f e t o r e a c h t h e r a n g e o f s t e a d y - s t a t e . A l o a d i n g d o s e o f 8 0 % o r 1 2 0 % o f t h e o p t i m a l d o s e w i l l t a k e a p p r o x i m a t e l y 2 h a l f - l i v e s . T h e r e f o r e , e v e n w i t h s o m e d e v i a t i o n f r o m t h e o p t i m a l l o a d i n g d o s e , t h e t i m e t o r e a c h s t e a d y - s t a t e w i l l b e g r e a t l y r e d u c e d w i t h a n a p p r o p r i a t e l o a d i n g d o s e . I n t h e t w o - c o m p a r t m e n t m o d e l , t h e u s e o f a l o a d i n g d o s e i s m o r e c o m p l i c a t e d d u e t o t h e d i s t r i b u t i o n p h a s e t o t h e p e r i p h e r a l c o m p a r t m e n t . U n l i k e t h e o n e - c o m p a r t m e n t m o d e l , t w o l o a d i n g d o s e p r o t o c o l s a r e p o s s i b l e f o r d r u g s b e s t d e s c r i b e d b y a t w o - c o m p a r t m e n t p h a r m a c o k i n e t i c c h a r a c t e r i s t i c s . A l o a d i n g d o s e o f LD = Css * Vc w i l l r e s u l t i n a n i m m e d i a t e p l a s m a d r u g c o n c e n t r a t i o n e q u a l t o t h e s t e a d y - s t a t e c o n c e n t r a t i o n , h o w e v e r t h e p l a s m a d r u g c o n c e n t r a t i o n w i l l b e d e c r e a s e d b e l o w t h e s t e a d y - s t a t e c o n c e n t r a t i o n a n d g r a d u a l l y i n c r e a s e d a s s h o w n i n F i g u r e 1 3 . A n a l t e r n a t i v e l o a d i n g d o s e o f LD = Css * Vss w i l l i n i t i a l l y g i v e a h i g h e r p l a s m a c o n c e n t r a t i o n t h a n t h e s t e a d y - s t a t e c o n c e n t r a t i o n , b u t v e r y r a p i d l y d e c r e a s e d t o t h e s t e a d y - s t a t e c o n c e n t r a t i o n . T h e t i m e t o s t e a d y - s t a t e u s i n g t h e s e c o n d r e g i m e n i s u s u a l l y m u c h s h o r t e r t h a n t h e f o r m e r . T h e r e f o r e , a l o a d i n g d o s e o f 1 5 m g o f M C P w a s u s e d i n t h e s t u d y a s s u g g e s t e d b y R i g g s ( 1 9 8 9 ) , w h i c h r e p r e s e n t s t h e l o a d i n g d o s e c l o s e t o t h e s e c o n d p r o t o c o l . T h e u s e o f a l o a d i n g d o s e a l s o g r e a t l y r e d u c e d t h e t i m e t o a t t a i n s t e a d y - s t a t e i n t h e t w o c o m p a r t m e n t m o d e l . T h i s m a y b e a b e t t e r a p p r o x i m a t i o n f o r M C P , s i n c e t h e M C P p h a r m a c o k i n e t i c s f o l l o w i n g t h e i . v . b o l u s d o s e e x h i b i t e d t w o - c o m p a r t m e n t c h a r a c t e r i s t i c s ( R i g g s et al, 1 9 8 8 ) . W i t h a n o p t i m a l l o a d i n g d o s e o f LD = Css * Vss, i t w i l l t a k e a b o u t 3 0 m i n u t e s t o a t t a i n t h e r a n g e o f s t e a d y - s t a t e ( 9 5 - 1 0 5 % o f C s s ) . A l o a d i n g d o s e o f 8 0 % a n d 1 2 0 % o f t h e o p t i m a l d o s e w i l l t a k e a p p r o x i m a t e l y 1 . 5 h o u r t o r e a c h t h e r a n g e . I l l T h e r e f o r e , t h e s e c o m p u t e r - a i d e d s i m u l a t i o n s t h e o r e t i c a l l y d e m o n s t r a t e t h a t t h e t i m e t o t h e s t e a d y - s t a t e c a n b e g r e a t l y r e d u c e d b y a n a p p r o p r i a t e l o a d i n g d o s e . T h i s c o n f i r m s t h e f i n d i n g o f R i g g s et al. ( 1 9 8 9 ) t h a t s t e a d y - s t a t e c o n d i t i o n s f o r M C P w e r e a c h i e v e d w i t h i n a p p r o x i m a t e l y 2 h a l f - l i v e s u s i n g a l o a d i n g d o s e o f 1 5 m g . I n t h e p r e s e n t s t u d y , M C P s t e a d y -s t a t e c o n c e n t r a t i o n w a s a c h i e v e d b e t w e e n 0 . 5 t o 1 . 2 5 h o u r s a f t e r i n i t i a t i o n o f t h e i n f u s i o n a n d a d m i n i s t r a t i o n o f a l o a d i n g d o s e , w h i c h i s i n g o o d a g r e e m e n t w i t h t h e p r e s e n t t h e o r e t i c a l d e r i v a t i o n a n d c o m p u t e r - a i d e d s i m u l a t i o n . 4.4. Metoclopramide Pharmacokinetics Following the Lv. Infusion to Steady-state and Induction of Hypoxemia 4.4.1. Steady-state plasma MCP concentration and total body clearance of MCP during normoxemia and hypoxemia T h e p r e s e n t s t u d y s h o w s t h a t a c u t e m o d e r a t e h y p o x e m i a a f f e c t s p l a s m a m e t o c l o p r a m i d e p h a r m a c o k i n e t i c s i n c h r o n i c a l l y i n s t r u m e n t e d c o n s c i o u s s h e e p . T h e s t e a d y -s t a t e p l a s m a M C P c o n c e n t r a t i o n s i g n i f i c a n t l y i n c r e a s e d a n d t o t a l b o d y c l e a r a n c e d e c r e a s e d d u r i n g a c u t e m o d e r a t e h y p o x e m i a . D u r i n g t h e p o s t - h y p o x e m i c p e r i o d , t h e s t e a d y - s t a t e p l a s m a M C P c o n c e n t r a t i o n d e c r e a s e d t o a l e v e l s i m i l a r t o t h a t o f t h e p r e - h y p o x e m i c p e r i o d . T h e m e a n p l a s m a M C P c o n c e n t r a t i o n d u r i n g t h e p o s t - h y p o x e m i c p e r i o d w a s s l i g h t l y h i g h e r t h a n t h e m e a n p l a s m a M C P c o n c e n t r a t i o n d u r i n g t h e p r e - h y p o x e m i c p e r i o d , b u t w a s n o t s i g n i f i c a n t l y d i f f e r e n t ( p > 0 . 0 5 ) . T h e r e f o r e , t h e s e r e s u l t s s u g g e s t e d t h a t a c u t e m o d e r a t e h y p o x e m i a r e d u c e s t o t a l b o d y c l e a r a n c e o f M C P (i.e. r e s u l t i n g i n a h i g h e r p l a s m a M C P s t e a d y - s t a t e c o n c e n t r a t i o n ) . 112 A r e d u c t i o n i n t o t a l b o d y d r u g c l e a r a n c e d u r i n g a c u t e h y p o x e m i a w a s o b s e r v e d w i t h a n u m b e r o f d r u g s s u c h a s t h e o p h y l l i n e ( S a u n i e r et al, 1 9 8 7 ) , a m i n o g l y c o s i d e s ( M i r h i j et al, 1 9 7 8 ) , p h e n y t o i n ( B a b i n i a n d d u S o u i c h , 1 9 8 6 ) a n d p r o p o f o l ( A u d i b e r t et al, 1 9 9 2 ) . D r u g s s u c h a s h e x o b a r b i t a l a n d a n t i p y r i n e , w h i c h u n d e r g o o x i d a t i v e b i o t r a n s f o r m a t i o n b y t h e h e p a t i c m i x e d o x y g e n a s e s , a r e d i r e c t l y a f f e c t e d b y r e d u c e d o x y g e n t e n s i o n ( J o n e s , 1 9 8 1 ) . C o n j u g a t i o n r e a c t i o n s s u c h a s g l u c u r o n i d a t i o n a n d s u l p h a t i o n , w h i c h a r e t h e m a j o r e l i m i n a t i o n p a t h w a y o f m a n y d r u g s , a r e a l s o a f f e c t e d b y l o w o x y g e n t e n s i o n ( A w a n d J o n e s , 1 9 8 1 ) , b u t t h e f o r m a t i o n o f t h e s e c o n j u g a t e s d o e s n o t a p p e a r t o b e a s i g n i f i c a n t p r o p o r t i o n o f M C P e l i m i n a t i o n i n s h e e p . I n a d d i t i o n , a c u t e h y p o x e m i a d e c r e a s e s t h e a c t i v i t y o f s e v e r a l i s o e n z y m e s o f t h e c y t o c h r o m e P - 4 5 0 ( S r i v a s t a v a et al, 1 9 8 0 ; J o n e s et al, 1 9 8 9 ; d u S o u i c h et al, 1 9 9 0 ) . T h e r e a r e a l s o o b s e r v a t i o n s t h a t m o d e r a t e h y p o x i a c o u l d p r o m o t e t h e f o r m a t i o n o f o x y g e n f r e e - r a d i c a l s ( P r o u l x a n d d u S o u i c h , 1 9 9 0 ; B r a s s et al, 1 9 9 1 ) t h a t a r e a b l e t o d i m i n i s h t h e a c t i v i t y o f c e r t a i n i s o z y m e s o f t h e c y t o c h r o m e P - 4 5 0 ( P r o u l x a n d d u S o u i c h , 1 9 9 0 ) . T h e s e r e p o r t s s u g g e s t t h a t a c u t e h y p o x e m i a r e d u c e s d r u g e l i m i n a t i o n p r o c e s s e s , i n g e n e r a l , w h i c h i s i n a g r e e m e n t w i t h t h e f i n d i n g s o f t h e p r e s e n t s t u d y . A l t e r n a t i v e l y , n o c h a n g e i n p h a r m a c o k i n e t i c s d u r i n g a c u t e h y p o x e m i a w a s o b s e r v e d w i t h d r u g s s u c h a s f u r o s e m i d e ( d u S o u i c h et al, 1 9 8 5 ) a n d d i l t i a z e m ( d u S o u i c h et al, 1 9 9 3 ) . I n s o m e c a s e s , t h e r e i s a n i n c r e a s e i n t h e a p p a r e n t t o t a l b o d y c l e a r a n c e d u r i n g a c u t e h y p o x e m i a w i t h s o m e d r u g s s u c h a s s u l p h a m e t h a z i n e ( d u S o u i c h et al, 1 9 8 4 ) a n d d i g o x i n ( d u S o u i c h et al, 1 9 8 5 a ; 1 9 8 5 b ) . H o w e v e r , t h i s i n c r e a s e i n t o t a l b o d y c l e a r a n c e w a s m a i n l y d u e t o a n i n c r e a s e i n t h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n (i.e. i n c r e a s e d t i s s u e d i s t r i b u t i o n a n d c h a n g e s i n p l a s m a p r o t e i n b i n d i n g ) , r a t h e r t h a n t o a n a c t u a l i n c r e a s e i n d r u g e l i m i n a t i o n f r o m t h e b o d y . 113 N o s t u d i e s o n t h e e n z y m a t i c p a t h w a y f o r t h e o x i d a t i v e N - d e e t h y l a t i o n o f M C P t o m d M C P a n d d d M C P h a v e b e e n r e p o r t e d , h o w e v e r t h e N - d e e t h y l a t i o n o f a s t r u c t u r a l l y s i m i l a r c o m p o u n d , l i d o c a i n e , a p p e a r s t o b e m e t a b o l i z e d b y t h e h e p a t i c P - 4 5 0 e n z y m e s y s t e m ( S u z u k i et al, 1 9 8 4 ; B a r g e t z i et al, 1 9 8 9 ) a n d t h e r e a r e m a n y r e p o r t s t h a t N - d e a k y l a t i o n r e a c t i o n s s u c h a s d e m e t h y l a t i o n , d e e t h y l a t i o n a n d d e p r o p y l a t i o n a r e m e d i a t e d b y t h e h e p a t i c P - 4 5 0 e n z y m e s y s t e m ( T e s t a a n d J e n n e r , 1 9 7 6 ) . I n a d d i t i o n , t h e l i v e r i s t h o u g h t t o b e t h e m a j o r m e t a b o l i c s i t e f o r M C P ( K a p i l et al, 1 9 8 4 ; D e s m o n d a n d W a t s o n , 1 9 8 6 ) . T h e r e f o r e i t i s p r o b a b l e t h a t t h e N - d e e t h y l a t i o n o f M C P i s a l s o m e d i a t e d b y t h e h e p a t i c P - 4 5 0 e n z y m e s y s t e m . In vitro s t u d i e s h a v e d e m o n s t r a t e d t h a t a l o w p a r t i a l p r e s s u r e o f o x y g e n r e d u c e d t h e Km v a l u e o f t h e d e m e t h y l a t i o n o f e t h y l - m o r p h i n e ( H o l t z m a n et al, 1 9 8 3 ; E r i c k s o n et al, 1 9 8 2 ) a n d o f t h e h y d r o x y l a t i o n o f p h e n y t o i n ( T s u r u et al, 1 9 8 2 ) , i n d i c a t i n g t h a t a d e q u a t e o x y g e n p r e s s u r e m a y b e e s s e n t i a l f o r t h i s r o u t e o f b i o t r a n s f o r m a t i o n . I n c o n s c i o u s r a b b i t s , h y p o x e m i a r e d u c e d t h e d e m e t h y l a t i o n a n d / o r t h e h y d r o x y l a t i o n o f t h e o p h y l l i n e ( L e t a r t e et al, 1 9 8 4 ) a n d t h e h y d r o x y l a t i o n o f p h e n y t o i n ( d u S o u i c h et al, 1 9 8 6 ) . I n a d d i t i o n , a c u t e h y p o x e m i a a p p e a r s t o d e c r e a s e t h e a c t i v i t y o f t h e c y t o c h r o m e P - 4 5 0 i n r a t l u n g a n d l i v e r ( S r i v a s t a v a et al, 1 9 8 0 ) . T h e s e s t u d i e s s u g g e s t e d t h a t h y p o x e m i a m a y a f f e c t s e v e r a l c y t o c h r o m e P - 4 5 0 i s o z y m e s , a n d t h u s m a y e x p l a i n , i n p a r t , t h e a c c u m u l a t i o n i n p l a s m a o f M C P a n d m d M C P d u e t o r e d u c e d m e t a b o l i c e l i m i n a t i o n o f t h e d r u g . T h e e f f e c t o f h y p o x e m i a o n p l a s m a d d M C P c o n c e n t r a t i o n w a s n o t d e t e r m i n e d , d u e t o t h e l o w p l a s m a c o n c e n t r a t i o n o f t h e m e t a b o l i t e . A l t e r n a t i v e l y , c h a n g e s i n h e p a t i c b l o o d f l o w d u r i n g a c u t e h y p o x e m i a - h y p o c a p n i a c o u l d e x p l a i n t h e r e d u c t i o n o f M C P c l e a r a n c e . A m o d e r a t e h y p o x e m i a a l o n e a p p e a r s t o e x e r t o n l y m i n i m a l e f f e c t i n t o t a l h e p a t i c b l o o d f l o w ( L a s e n et al, 1 9 7 6 ; H u g h e s et al, 1 9 7 9 ; d u 114 S o u i c h et al, 1 9 9 2 ) . H o w e v e r , a h y p o x e m i a i n c o m b i n a t i o n w i t h e i t h e r h y p o c a p n i a o r h y p e r c a p n i a m a y a f f e c t t o t a l h e p a t i c b l o o d f l o w m o r e t h a n h y p o x e m i a a l o n e ( H u g h e s et al, 1 9 7 9 ; M a t h i e a n d B l u m g a r t , 1 9 8 3 ) . S i n c e h e p a t i c e l i m i n a t i o n o f M C P i s e s s e n t i a l l y m o d u l a t e d b y t h e b l o o d f l o w t o t h e l i v e r [ f l o w - l i m i t e d ] ( B a t e m a n et al, 1 9 8 0 ) , t h e e f f e c t o f a c u t e h y p o x e m i a - h y p o c a p n i a o n M C P c l e a r a n c e m a y b e e x p l a i n e d b y t h e r e d u c t i o n i n t o t a l h e p a t i c b l o o d f l o w . 4.4.2. Plasma mdMCP concentration during normoxemia and hypoxemia D e a l k y l a t i o n o f s e c o n d a r y a n d t e r t i a r y a m i n e g r o u p s t o y i e l d p r i m a r y a n d s e c o n d a r y a m i n e g r o u p s , r e s p e c t i v e l y , i s o n e o f t h e m o s t i m p o r t a n t a n d m o s t f r e q u e n t l y e n c o u n t e r e d r e a c t i o n s i n d r u g m e t a b o l i s m . B i o l o g i c a l N - d e a l k y l a t i o n o c c u r s w i t h o u t a p p a r e n t c h a n g e s i n t h e s t a t e o f o x i d a t i o n o f t h e n i t r o g e n a t o m , b u t t h e r e m o v e d a l k y l g r o u p i s o x i d i s e d ( T e s t a a n d J e n n e r , 1 9 7 6 ) . I n t h e p r e s e n t s t u d y , c o n s i d e r a b l e p l a s m a m d M C P c o n c e n t r a t i o n s ( o n a v e r a g e = 3 0 - 5 0 % o f p l a s m a M C P c o n c e n t r a t i o n ) w e r e f o u n d . P l a s m a m d M C P c o n c e n t r a t i o n a l s o a p p e a r e d t o r e a c h a n a p p a r e n t s t e a d y - s t a t e i n b o t h e x p e r i m e n t a l a n d c o n t r o l g r o u p s . H o w e v e r , i t i s n o t p o s s i b l e t o c o n f i r m t h a t s t e a d y - s t a t e , i n a c o n v e n t i o n a l s e n s e , w a s a c h i e v e d i n t h e s t u d y , s i n c e t h e i n p u t r a t e c o n s t a n t o f m d M C P {i.e. a p r o d u c t t o i n f u s i o n r a t e o f M C P a n d kf(MCP->mdMCP)) c o u l d n o t b e e v a l u a t e d i n t h e c u r r e n t i n f u s i o n d e s i g n . P l a s m a m d M C P c o n c e n t r a t i o n w a s s i g n i f i c a n t l y h i g h e r i n t h e e x p e r i m e n t a l g r o u p a s c o m p a r e d t o t h e c o n t r o l g r o u p . T h e i n c r e a s e d m d M C P c o n c e n t r a t i o n i n t h e e x p e r i m e n t a l g r o u p a p p e a r s t o b e r e l a t e d t o t h e i n d u c t i o n o f h y p o x e m i a , h o w e v e r , m d M C P c o n c e n t r a t i o n d i d n o t d e c r e a s e d u r i n g t h e p o s t - h y p o x e m i c p e r i o d . T h e i n t e r p r e t a t i o n o f m e t a b o l i t e k i n e t i c s , m a i n l y m d M C P i n t h i s s t u d y , i s m o r e c o m p l i c a t e d t h a n t h e p a r e n t d r u g , s i n c e p l a s m a 115 m d M C P c o n c e n t r a t i o n s d e p e n d o n : ( 1 ) t h e r a t e o f N - d e e t h y l a t i o n o f M C P t h a t y i e l d s m d M C P ; ( 2 ) t h e r a t e o f d e e t h y l a t i o n o f m d M C P p r o d u c i n g d d M C P ; ( 3 ) r e n a l e l i m i n a t i o n r a t e o f m d M C P a n d ( 4 ) o t h e r b i o t r a n s f o r m a t i o n r a t e s f o r m d M C P . T h e i n p u t r a t e o f m d M C P ( t h e r a t e o f N - d e e t h y l a t i o n o f M C P ) c o u l d n o t b e d i r e c t l y d e t e r m i n e d i n t h e s t u d y . H o w e v e r , i t i s u n l i k e l y i n c r e a s e d d u r i n g h y p o x e m i a , s i n c e t o t a l b o d y c l e a r a n c e o f M C P w a s d e c r e a s e d , a n d , a s m e n t i o n e d a b o v e , t h e r e a r e r e p o r t s t h a t t h e c y t o c h r o m e P - 4 5 0 s y s t e m w h i c h m e d i a t e s N -d e e t h y l a t i o n r e a c t i o n i s r e d u c e d d u r i n g a c u t e h y p o x e m i a ( S r i v a s t a v a et al, 1 9 8 0 ) . T h e r e f o r e , t h e r a t e s o f m d M C P e l i m i n a t i o n p r o c e s s e s , s u c h a s r e n a l e x c r e t i o n a n d m d M C P m e t a b o l i s m , a r e l i k e l y d e c r e a s e d d u r i n g h y p o x e m i a . R e n a l e l i m i n a t i o n o f m d M C P w a s s i g n i f i c a n t l y d e c r e a s e d d u r i n g h y p o x e m i a a s s h o w n i n S e c t i o n 3 . 4 . S i m i l a r a c c u m u l a t i o n o f M E G X a n d G X m e t a b o l i t e s o f l i d o c a i n e w a s a l s o o b s e r v e d d u r i n g a c u t e h y p o x e m i a ( M a r l e a u et al, 1 9 8 7 ; d u S o u i c h etal, 1 9 9 2 ) . 4.4.3. Renal elimination of MCP and its metabolites during normoxemia and hypoxemia R e n a l e l i m i n a t i o n o f M C P a n d i t s m e t a b o l i t e s w a s e x a m i n e d i n t h e p r e s e n t s t u d y . S i n c e t h e i n f u s i o n p r o t o c o l w a s u s e d , t h e c o n v e n t i o n a l m e t h o d s o f d e t e r m i n i n g u r i n a r y e x c r e t i o n p a r a m e t e r s s u c h a s t h e p l o t s o f A . R . E . ( a m o u n t r e m a i n e d t o b e e x c r e t e d ) vs. t i m e o r u r i n a r y e x c r e t i o n r a t e vs. t i m e c o u l d n o t b e a p p l i e d i n t h i s s t u d y . I n a d d i t i o n , c h a n g e s i n d r u g e l i m i n a t i o n d u r i n g t h e h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d s c o m p l i c a t e d t h e e s t i m a t i o n o f r e n a l d r u g e l i m i n a t i o n p a r a m e t e r s . T h e r e f o r e , t w o m o d i f i e d m e t h o d s o f d e t e r m i n i n g u r i n a r y d r u g e x c r e t i o n d u r i n g t h e p h a s e d i n f u s i o n w e r e d e v e l o p e d a n d a p p l i e d i n t h e s t u d y . T h e f i r s t 116 m e t h o d w a s b a s e d o n t h e e s t i m a t i o n o f r e n a l c l e a r a n c e f r o m u r i n a r y d r u g a c c u m u l a t i o n a n d A U C [ M e t h o d l a a n d l b ] . T h e o t h e r m e t h o d w a s b a s e d o n t h e d e t e r m i n a t i o n o f f r a c t i o n a l r e n a l d r u g e x c r e t i o n c o n s t a n t , w h i c h w a s d e r i v e d a n d m o d i f i e d f r o m t h e c o n c e p t o f u r i n a r y e x c r e t i o n d e t e r m i n a t i o n d u r i n g i n f u s i o n b y G i b a l d i a n d P e r r i e r ( 1 9 8 2 ) [ M e t h o d 2 ] . F i r s t l y , t h e r e n a l c l e a r a n c e o f M C P a n d m d M C P w a s c a l c u l a t e d i n t h e s t u d y e i t h e r f r o m a ) d i v i d i n g t h e a m o u n t o f d r u g / m e t a b o l i t e r e c o v e r e d i n u r i n e (Du) b y t h e a r e a u n d e r p l a s m a d r u g / m e t a b o l i t e c o n c e n t r a t i o n c u r v e ( A U C ) i n a g i v e n t i m e p e r i o d [ M e t h o d l a ] , a n d b ) u s i n g t h e s l o p e o f t h e a c c u m u l a t e d d r u g / m e t a b o l i t e i n u r i n e (Du) v e r s u s A U C [ M e t h o d l b ] . A s s e e n i n S e c t i o n 3 . 4 . 1 . , b o t h m e t h o d s a p p e a r t o g i v e a s i m i l a r e s t i m a t i o n o f r e n a l c l e a r a n c e . T h e s l o p e f r o m t h e p r e - h y p o x e m i c p e r i o d c o u l d n o t b e d e t e r m i n e d , s i n c e t h e r e w e r e o n l y 3 d a t a p o i n t s i n t h e p e r i o d , a n d a l s o o n l y 1 o r 2 d a t a p o i n t s r e p r e s e n t i n g a u r i n e c o l l e c t i o n d u r i n g M C P s t e a d y - s t a t e . A s a r e s u l t , i n t h e e x p e r i m e n t a l g r o u p , t h e c l e a r a n c e v a l u e s o b t a i n e d d u r i n g t h e h y p o x e m i c p e r i o d w e r e c o m p a r e d w i t h t h o s e d e t e r m i n e d d u r i n g t h e s u b s e q u e n t p o s t - h y p o x e m i c s e g m e n t ( t o t a l i n g 1 2 o f t h e 1 4 h o u r t o t a l e x p e r i m e n t d u r a t i o n ) . I n a d d i t i o n , r e n a l c l e a r a n c e p a r a m e t e r s f r o m t h e e q u i v a l e n t p e r i o d i n t h e c o n t r o l g r o u p w e r e a l s o c o m p a r e d w i t h t h e h y p o x e m i c p e r i o d . S i n c e n o s i g n i f i c a n t c h a n g e i n r e n a l c l e a r a n c e i n t h e c o n t r o l g r o u p w a s o b s e r v e d , i t i s s u p p o r t e d t h a t t h i s a p p r o a c h i s v a l i d . S e c o n d l y , t h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t s f o r M C P , m d M C P a n d d d M C P , w e r e c a l c u l a t e d b y d i v i d i n g t h e s l o p e o f t h e a s y m p t o t e o f t h e Du v e r s u s t i m e c u r v e w i t h t h e i n f u s i o n r a t e o f M C P . I n t h e c a s e o f p a r e n t d r u g , t h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t (fu) i s t h e p r o p o r t i o n o f t h e r e n a l e l i m i n a t i o n r a t e c o n s t a n t o v e r t h e a p p a r e n t t o t a l d r u g e l i m i n a t i o n r a t e c o n s t a n t (ku/KE), w h i c h i n d i c a t e s t h e f r a c t i o n o f t o t a l d r u g e l i m i n a t i o n d u e t o r e n a l d r u g 117 e x c r e t i o n . I n a d d i t i o n , t h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t s f o r m e t a b o l i t e s w e r e a l s o c a l c u l a t e d . T h e t h e o r e t i c a l d e r i v a t i o n o f t h e s e p a r a m e t e r s w a s s i m i l a r t o t h o s e o f t h e p a r e n t d r u g . H o w e v e r , t h e f r a c t i o n a l r e n a l e x c r e t i o n p a r a m e t e r s d e t e r m i n e d f r o m t h e a c c u m u l a t i o n p l o t r e p r e s e n t h y b r i d f r a c t i o n a l c o n s t a n t s (i.e. a p r o d u c t o f f r a c t i o n a l r e n a l e l i m i n a t i o n c o n s t a n t o f a m e t a b o l i t e a n d f r a c t i o n a l m e t a b o l i c e l i m i n a t i o n c o n s t a n t o f t h e p a r e n t d r u g ) [ s e e S e c t i o n 3 . 4 . 2 . a n d A p p e n d i x A ] . T h e s e p a r a m e t e r s m a y a p p e a r t o b e h i g h l y c o m p l i c a t e d , b u t , i n s i m p l e t e r m s , r e p r e s e n t t h e f r a c t i o n / p r o p o r t i o n o f r e n a l e x c r e t i o n i n t h e f o r m o f a s p e c i f i c m e t a b o l i t e o u t o f t h e t o t a l d r u g e l i m i n a t i o n . D u e t o t h e c o n s t r a i n t s i m p o s e d b y t h e i n f u s i o n d e s i g n , i t w a s n o t f e a s i b l e t o d e t e r m i n e d a b s o l u t e (i.e. n o t f r a c t i o n a l c o n s t a n t s ) r e n a l e l i m i n a t i o n a n d m e t a b o l i c p a r a m e t e r s i n t h e s t u d y . S i n c e t h e p r e s e n t s t u d y w a s d e s i g n e d t o d e t e c t t h e c h a n g e s i n t h e d r u g k i n e t i c s d u r i n g h y p o x e m i a , i t i s c r i t i c a l t o d e t e r m i n e t h e r e n a l d r u g e l i m i n a t i o n p a r a m e t e r s d u r i n g b o t h n o r m o x e m i c a n d h y p o x e m i c p e r i o d w i t h i n a s i n g l e e x p e r i m e n t ( p h a s e d i n f u s i o n ) . A s s h o w n i n S e c t i o n 4 . 3 . , a n y i n f u s i o n c a n b e t h e o r e t i c a l l y c o n s i d e r e d a s a s e r i e s o f i n f u s i o n s w i t h a p p r o p r i a t e l o a d i n g d o s e s . T h e r e f o r e , e a c h p r e - h y p o x e m i c , h y p o x e m i c a n d p o s t - h y p o x e m i c p e r i o d i n t h e s t u d y c o u l d b e c o n s i d e r e d a s " s e p a r a t e " i n f u s i o n s w i t h a p p r o p r i a t e l o a d i n g d o s e s , a n d r e n a l e x c r e t i o n p a r a m e t e r s f r o m e a c h p e r i o d s c o u l d b e t r e a t e d i n d e p e n d e n t l y . P r e v i o u s s t u d i e s o n r e n a l c l e a r a n c e d u r i n g a c u t e h y p o x e m i a h a v e r e p o r t e d v a r i o u s r e s p o n s e s . N o c h a n g e s i n r e n a l c l e a r a n c e d u r i n g a c u t e h y p o x e m i a w e r e o b s e r v e d w i t h m a n y d r u g s s u c h a s t h e o p h y l l i n e ( L e t a r t e a n d d u S o u i c h , 1 9 8 4 ) , f u r o s e m i d e ( B a b i n i a n d d u S o u i c h , 1 9 8 6 ) , l i d o c a i n e ( d u S o u i c h et al, 1 9 9 2 ) a n d d i l t i a z e m ( d u S o u i c h et al, 1 9 9 3 ) . R e n a l c l e a r a n c e o f p h e n y t o i n d e c r e a s e d d u r i n g a c u t e h y p o x e m i a ( f r o m 0 . 2 t o 0 . 0 3 m L / m i n / K g ) , b u t 118 i t a p p a r e n t l y w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t ( d u S o u i c h et al, 1 9 8 6 ) . A l t e r n a t i v e l y , r e n a l c l e a r a n c e o f s u l f a m e t h a z i n e a p p e a r s t o b e i n c r e a s e d , b u t s t a t i s t i c a l l y n o t s i g n i f i c a n t , d u r i n g a c u t e h y p o x e m i a ( d u S o u i c h a n d C o u r t e a u , 1 9 8 4 ) . R e n a l c l e a r a n c e o f d i g o x i n w a s s i g n i f i c a n t l y i n c r e a s e d d u r i n g a c u t e h y p o x e m i a ( d u S o u i c h et al, 1 9 8 5 ) . I n o n e o f t h e s t u d i e s , r e n a l c l e a r a n c e o f m e t a b o l i t e s w a s m e a s u r e d . N o s i g n i f i c a n t c h a n g e s i n r e n a l c l e a r a n c e s o f l i d o c a i n e m e t a b o l i t e s , M E G X a n d G X , w e r e o b s e r v e d ( d u S o u i c h et al, 1 9 9 2 ) . T h e r e w a s s i g n i f i c a n t r e d u c t i o n i n r e n a l c l e a r a n c e o f M C P a n d m d M C P d u r i n g t h e h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p a s s h o w n i n S e c t i o n 3 . 4 . 1 . T h e r e d u c t i o n i n M C P r e n a l c l e a r a n c e ( a b o u t 2 . 7 5 L / h d u r i n g h y p o x e m i a a n d 8 . 1 5 L / h d u r i n g n o r m o x e m i a ) a r e m u c h m o r e e x t e n s i v e c o m p a r e t o t h e r e d u c t i o n i n t o t a l b o d y c l e a r a n c e ( 2 0 5 L / h d u r i n g h y p o x e m i a a n d 2 4 5 L / h d u r i n g n o r m o x e m i a ) . E v e n t h o u g h t h e r e n a l c l e a r a n c e o f M C P a c c o u n t s f o r o n l y a b o u t 5 % o f t o t a l b o d y c l e a r a n c e d u r i n g n o r m o x e m i a ( a b o u t 8 L / h o u t o f 2 4 5 L / h ) , t h e r e d u c t i o n o f r e n a l c l e a r a n c e d u r i n g h y p o x e m i a a c c o u n t s f o r a b o u t 1 5 % o f t h e c h a n g e i n t o t a l b o d y c l e a r a n c e d u r i n g h y p o x e m i a ( c h a n g e o f a b o u t 5 . 5 L / h o u t o f 4 0 L / h ) . T h e r e f o r e , t h e r e d u c t i o n i n r e n a l c l e a r a n c e o f M C P c o n t r i b u t e s s i g n i f i c a n t l y t o t h e r e d u c t i o n o f t o t a l b o d y c l e a r a n c e o f M C P . S i m i l a r l y , t h e r e n a l c l e a r a n c e o f m d M C P w a s s i g n i f i c a n t l y a f f e c t e d b y h y p o x e m i a ( 1 4 . 6 L / h d u r i n g h y p o x e m i a a n d 3 8 . 6 L / h d u r i n g n o r m o x e m i a ) . H o w e v e r , t h e t o t a l b o d y c l e a r a n c e o f m d M C P c o u l d n o t b e d e t e r m i n e d i n t h i s s t u d y , t h u s q u a n t i t a t i v e a s s e s s m e n t o f t h e c o n t r i b u t i o n o f t h e m d M C P r e n a l e l i m i n a t i o n t o t o t a l b o d y c l e a r a n c e o f M C P c o u l d n o t b e e v a l u a t e d . R e n a l e l i m i n a t i o n o f d d M C P w a s a l s o s i g n i f i c a n t l y a f f e c t e d b y a c u t e 119 h y p o x e m i a . T h e r e f o r e , a c u t e h y p o x e m i a i n t h e s t u d y a p p e a r s t o r e d u c e r e n a l e l i m i n a t i o n o f M C P a n d i t s d e e t h y l m e t a b o l i t e s . O v e r a l l , t h e p r e s e n t s t u d y o f t h e p h a r m a c o k i n e t i c s o f M C P a n d i t s d e e t h y l a t e d m e t a b o l i t e , m d M C P a n d d d M C P , d u r i n g a c u t e h y p o x e m i a h a s s h o w n t h a t t h e r e i s a r e d u c e d e l i m i n a t i o n o f M C P a n d i t s m e t a b o l i t e s d u r i n g a c u t e h y p o x e m i a , a n d t h i s r e d u c t i o n i n s y s t e m i c c l e a r a n c e i s a s s o c i a t e d w i t h a r e d u c e d r e n a l e l i m i n a t i o n o f t h e c o m p o u n d s . 120 5. SUMMARY AND CONCLUSIONS A c a p i l l a r y G C - M S D m e t h o d o f R i g g s et al. ( 1 9 9 4 ) f o r M C P , m d M C P a n d d d M C P w a s a d a p t e d a n d a p p l i e d t o s t u d y t h e e f f e c t o f a c u t e m o d e r a t e h y p o x e m i a o n t h e p h a r m a c o k i n e t i c s o f M C P a n d i t s d e e t h y l a t e d m e t a b o l i t e s i n p l a s m a a n d u r i n e f r o m n o n -p r e g n a n t e w e s f o l l o w i n g i . v . i n f u s i o n t o s t e a d y - s t a t e a n d i n d u c t i o n o f h y p o x e m i a via i n t r a -t r a c h e a l n i t r o g e n i n f u s i o n . A . A c u t e h y p o x e m i a i n d u c e s s e v e r a l p h y s i o l o g i c a l c h a n g e s i n n o n - p r e g n a n t e w e s d u r i n g c o n t i n u o u s M C P i n f u s i o n : 1 . R e d u c e s a r t e r i a l b l o o d P a C > 2 ( h y p o x e m i a ) a n d P a C 0 2 ( h y p o c a p n i a ) d u r i n g t h e h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p . 2 . E l e v a t e s a r t e r i a l b l o o d l a c t a t e a n d g l u c o s e l e v e l d u r i n g t h e l a t e h y p o x e m i c p e r i o d a n d f o r a t i m e a f t e r r e s t o r a t i o n o f n o r m o x i a . 3 . I n c r e a s e s u r i n e f l o w a n d d e c r e a s e s u r i n e o s m o l a l i t y a n d p H d u r i n g t h e h y p o x e m i c p e r i o d . B . H y p o x e m i a a f f e c t s t h e p h a r m a c o k i n e t i c s o f M C P a n d i t s d e e t h y l a t e d m e t a b o l i t e s , m d M C P a n d d d M C P . 1 . T o t a l b o d y c l e a r a n c e ( T B C ) w a s d e c r e a s e d d u r i n g h y p o x e m i a , a n d r e s t o r e d d u r i n g t h e p o s t - h y p o x e m i c r e c o v e r y p e r i o d . 2 . P l a s m a m d M C P c o n c e n t r a t i o n w a s i n c r e a s e d d u r i n g t h e h y p o x e m i c a n d p o s t -h y p o x e m i c p e r i o d a s c o m p a r e d t o t h e c o n t r o l g r o u p . 3 . 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T h e o r e t i c a l D e r i v a t i o n o f P h a r m a c o k i n e t i c E q u a t i o n s u s e d i n t h e s t u d y P a r t I : O n e - c o m p a r t m e n t O p e n M o d e l T h e f o l l o w i n g a r e s c h e m a t i c r e p r e s e n t a t i o n o f o n e - c o m p a r t m e n t o p e n m o d e l w i t h m e t a b o l i s m . ko (+ LD) Parent Drug Compartment Xp, Cp, Vp Metabolite Compartment Xm, Cm, Vm oth six T h e a b b r e v i a t i o n u s e d i n t h e f o l l o w i n g e q u a t i o n d e r i v a t i o n a n d t h e m a i n t e x t a r e : kf = metabolite/conjugate formation rate constant for the parent drug koth = non-metabolic elimination rate constant for the parent drug kr = renal elimination rate constant of the parent drug kmr = renal elimination rate constant of the metabolite kmnr - non-renal elimination rate of the metabolite KE = kf+ koth = apparent elimination rate of the parent drug km — kmr + kmnr = apparent elimination rate of the metabolite fm = kf/KE = metabolic fraction of the parent drug elimination fmr = kmr/km = renal fraction of the metabolite elimination fr = kr/ KE"= renal fraction of the parent drug elimination ko = infusion rate constant Do = loading dose Xp = amount of the parent drug in the body Cp = concentration of the parent drug in the body Vp = apparent volume of distribution of the parent drug in the body Xm = amount of the metabolite in the body Cm = concentration of the metabolite in the body Vm = apparent volume of distribution of the metabolite in the body ti = Do/ko = infusion time equivalent loading dose 138 T h e o r e t i c a l D e r i v a t i o n : T h e r a t e o f d r u g d i s p o s i t i o n i n t h e p a r e n t d r u g c o m p a r t m e n t d u r i n g a n i n f u s i o n d e f i n e d a s b e l o w dXp/dt = ko - KE * XP (1) w i t h L a p l a c e t r a n s f o r m a t i o n (£(X) = X), E q . 1 b e c o m e s sXP - XP(0) = ko/s - KeXp (2) C a s e 1 : a s i m p l e i n f u s i o n w i t h o u t l o a d i n g d o s e s i n c e XP(0) = 0, E q 2 . b e c o m e s sXp + KE* Xp = ko/s (3) XP = ko/(s(s + KE)) (4) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , XP = ko/KE * ( 1 - exp(-KE * t)) (5) CP = ko/KE/Vp *( 1- exp(-KE * t)) (6) C a s e 2 : a n i n f u s i o n w i t h a l o a d i n g d o s e s i n c e XP(0) = Do, E q 2 . b e c o m e s sXp + KE* XP = ko /s +Do (7) XP = ko/ (s(s + KE)) + DO/(S + KE) (8) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , XP = ko/KE * ( 1 - exp(-KE *t)) + Do*( 1- exp(-KE * t)) (9) CP = ko/KE/Vp * ( 1 - exp(-KE * t)) + Do/VP*(l- exp(-KE * t)) (10) U r i n a r y e x c r e t i o n dXu/dt = kr*XP (11) w i t h L a p l a c e t r a n s f o r m a t i o n , E q . 1 1 b e c o m e s sXu-Xu(0) = kr*Xp (12) C a s e 1 : a s i m p l e i n f u s i o n w i t h o u t l o a d i n g d o s e ( G i b a l d i a n d P e r r i e r , 1 9 8 4 ) s i n c e Xu(0) = 0, E q . 1 2 b e c o m e s sXu =kr*ko/S/(s+KE) (13) 139 Xu =kr*ko/s2/(s+KE) (14) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , Xu = kr* ko * t/KE - kr* ko/KE2 * (1 - exp(-KE * t)) (15) F r o m t h e p l o t o f Xu vs. t, w h e n t h e d r u g i s i n f u s e d t o a s t e a d y - s t a t e , t h e t e r m exp(-KE * t) a p p r o a c h e s z e r o . T h e a s y m p t o t e b e c o m e s Xu = kr* ko * t/KE - kr* ko/KE2 (16) T h e u r i n a r y e x c r e t i o n f r a c t i o n c o n s t a n t kr/KE c a n b e d e t e r m i n e d f r o m t h e s l o p e o f a s y m p t o t e , kr* ko/KE, b y d i v i d i n g ko. C a s e 2 : a n i n f u s i o n w i t h a l o a d i n g d o s e s i n c e XP(0) = Do , E q . 12 b e c o m e s sXu = kr* ko/s/(s+KE) + DO/(S+KE) (17) Xu =kr*ko/s2/(s+KE) +DO/S/(S+KE) (18) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , Xu = h * ko * t/KE - kr * ko/KE2 * ( 1 - exp( - KE * t)) + Do /KE*( 1 - exp( - KE * t)) (19) F r o m t h e p l o t o f Xu vs. t, w h e n t h e d r u g i s i n f u s e d t o a s t e a d y - s t a t e , t h e t e r m exp( - KE * t) a p p r o a c h e s z e r o . T h e a s y m p t o t e b e c o m e s Xu = kr* ko * t/KE + kr* ko /KE2 + DO/KE (20) T h e u r i n a r y e x c r e t i o n f r a c t i o n c o n s t a n t kr/KE c a n b e d e t e r m i n e d f r o m t h e s l o p e o f a s y m p t o t e , kr* ko /KE, b y d i v i d i n g ko. B y u s i n g t h e t - i n t e r c e p t , KE c a n b e d e t e r m i n e d , h o w e v e r t h e e r r o r a s s o c i a t e d w i t h t h e i n t e r c e p t i s a c c u m u l a t i v e i.e. a n y e r r o r i n e a c h d a t a p o i n t w i l l b e d i r e c t l y r e f l e c t e d i n t h e t -i n t e r c e p t o f t h e a s y m p t o t e u n l i k e t o t h e s l o p e , t h u s d o e s n o t w a r r a n t i t s u s e . T h e r a t e o f m e t a o b o l i t e d i s p o s i t i o n i n t h e m e t a b o l i t e c o m p a r t m e n t d u r i n g a n i n f u s i o n d e f i n e d a s b e l o w 140 dXm/dt = kf*Xp-Km*Xm (21) w i t h L a p l a c e t r a n s f o r m a t i o n (£(X) = X), E q . 2 1 b e c o m e s sXm - Xm( 0) = kf *Xp - Km Jim (22) C a s e 1 : a s i m p l e i n f u s i o n w i t h o u t l o a d i n g d o s e s i n c e Xm(0) = 0, E q 2 2 . b e c o m e s sXm = kf* ko/( s (s +KE)) - km * Xm (23) Xm = kf* ko /(s (s +KE)(S + km)) (24) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , Xm = kfkof 1/km /KE+exp(-Km t)/km/(km-KE)+exp(-kE t)/kE/(kE-Km)] (25) Cm = kfko/Vm [ 1/km /KE+exp(-Km t)/km/(km-KE) +exp(-kEt)AE/(kE-Km)] (26) C a s e 2 : a n i n f u s i o n w i t h a l o a d i n g d o s e sXm = kfko/(s(s+KE)) - kmXm + kf Do/(s + KE) (27) Xm = kf ko /(s(s +KE)(S + km))+ kf Do /((s +KE)(S + km)) (28) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , Xm = kf*ko [1/km /KE+exp(-Km *t)/km/(km-KE) +exp(-kE*t)/kE/(kE-Km) ] + kfDo /(s+KE)/(s+km) (29) Cm = kf*ko/Vm [ 1/km /KE+exp(-Km *t)/km/(km-KE) +exp(-kE*t)/kE/(kE-Km)] + kfDo /Vm/(s+KE)/(s+km) (30) U r i n a r y e x c r e t i o n dXmu/dt = kmr * Xm (31) w i t h L a p l a c e t r a n s f o r m a t i o n , E q . 3 1 b e c o m e s sXmu = kmr Xm (32) C a s e 1 : a s i m p l e i n f u s i o n w i t h o u t l o a d i n g d o s e sXmu — kmr ko kf/s/(s+KE)/(s+km) (33) Xmu — kmr ko kf/s2/(s+KE)/(s+km) (34) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , 141 Xmu — kmrkfko[t/kJKE -(KE+krm)/km2/KE2 + C * exp(-KE t)+ D * exp(-km t))] (35) where C = (km+2KE)/KE2/km2 - 1/(KE km (KE + kj) D = - KE/KE2/km2 + 1/(KE km (KE + km)) F r o m t h e p l o t o f Xmu vs. t, w h e n t h e d r u g i s i n f u s e d t o a s t e a d y - s t a t e , t h e t e r m s exp(-KE t) a n d exp(-km t) a p p r o a c h z e r o . T h e a s y m p t o t e b e c o m e s Xmu = kmr kfko[t /kJKE -(KE+krm)/km2/KE2J (36) T h e m e t a b o l i t e u r i n a r y e x c r e t i o n f r a c t i o n c o n s t a n t kmr kr/KE/km c a n b e d e t e r m i n e d f r o m t h e s l o p e o f a s y m p t o t e , ko kmr kr/KE/km, b y d i v i d i n g ko. C a s e 2 : a n i n f u s i o n w i t h a l o a d i n g d o s e sJXmu — kmr ko kf/s/(s+KE)/(s+km)+ kmr kfD0 /(s+KE)/(s+km) (37) Xmu — kmr ko kf/s2/(s+KE)/(s+km) + kmr kf DQ /s/(s+KE)/(s+km) (38) b y r e v e r s e L a p l a c e t r a n s f o r m a t i o n , Xmu = kmr kf ko [t /kJKE -(KE+krm)/km2/KE2 + C exp(-KE t)+ D * exp(-km t))] + kmr kf (D0 KE km - k0 (KE + km)) /KE2/km2 (39) where C = (km+2KE)/KE2/km2 - 1/(KE km (KE + km)) D = - KE/KE2/km2 + 1/(KE km (KE + kj) F r o m t h e p l o t o f Xmu vs. t, w h e n t h e d r u g i s i n f u s e d t o a s t e a d y - s t a t e , t h e t e r m s exp(-KE t) a n d exp(-km t) a p p r o a c h z e r o . T h e a s y m p t o t e b e c o m e s Xmu = kmr kfko[t AJKE -(KE+krm)/km2/KE2] + kmr kf (DaKE km " k0 (KE + km))/KE2AM2 (40) T h e m e t a b o l i t e [ u r i n a r y e x c r e t i o n f r a c t i o n c o n s t a n t kmr kr/KE/km c a n b e d e t e r m i n e d f r o m t h e s l o p e o f a s y m p t o t e , ko kmr kr/KE/km, b y d i v i d i n g ko. 

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