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Solubility, distribution and transport of halothane in blood Pang, Yew Choi 1979

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SOLUBILITY, DISTRIBUTION AND TRANSPORT OF HALOTHANE IN BLOOD by YEW CHOI PANG B.Sc. (Hons) M c G i l l U n i v e r s i t y , 1972 M.Sc. U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1979 © Yew Choi Pang, 1979 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 representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS D a t e October 10, 1979 A B S T R A C T Halothane (1,1,1-trifluoro-2-bromo-2-chloroethane) i s a commonly employed general anaesthetic. A d i r e c t i n j e c t i o n g a s - l i q u i d chromatographic procedure was developed to q u a n t i t a t i v e l y estimate the halothane concentration of blood and other aqueous f l u i d s . This used a s p e c i a l l y designed e x t e r n a l i n j e c t i o n port which obviated a p r e l i m i n a r y separation of the anaesthetic from the aqueous phase. The method was extended to include q u a n t i t a t i v e e s t i m a t i o n of methoxyflurane ( 1 , l - d i c h l o r o - 2 , 2 - d i f l u o r o e t h y l - m e t h y l e t h e r ) , d i e t h y l e t h e r and ethanol over the approximate range 1-100 mg/100 ml. This a n a l y t i c a l method was used to i n v e s t i g a t e the q u a n t i t a t i v e i n t e r a c t i o n of halothane with major human blood components and the d i s t r i b u t i o n of halothane between c e l l s and plasma. The r e s u l t s obtained w i t h an e q u i l i b r i u m d i a l y s i s technique developed f o r t h i s study showed that haemoglobin, albumin, red c e l l membranes and t r i g l y c e r i d e - r i c h m i c e l l e s (chylomicrons and VLDL), but not y - g l o b u l i n , c o n t r i b u t e s i g n i f i c a n t l y to the s o l u b i l i t y , and thus the t r a n s p o r t , of halothane i n blood. A s i g n i f i c a n t amount of halothane i s a l s o d i s s o l v e d i n the aqueous phase. The r e s u l t s suggest that halothane i n t e r a c t s w i t h a f i n i t e number of surface s i t e s on haemoglobin and albumin. When the aqueous phase was saturated w i t h halothane, the average number of halothane molecules bound per haemoglobin and albumin molecule was approximately 5 and 20 r e s p e c t i v e l y . In the case of t r i g l y c e r i d e - r i c h m i c e l l e s and red c e l l membranes, the halothane molecules appeared to be located w i t h i n the hydrophobic core, since the amount of halothane s o l u b i l i z e d by the m i c e l l e s and membrane increased - i i i -w i t h i n c r e a s i n g free halothane concentration without showing evidence of s a t u r a t i o n of hydrophobic s i t e s . The r e s u l t s obtained from the e q u i l i b r i u m d i a l y s i s studies were used to c a l c u l a t e the d i s t r i b u t i o n of halothane between the c e l l s and plasma. This d i s t r i b u t i o n was a l s o experimentally determined by a n a l y s i s of the halothane concentration i n the plasma a f t e r c e n t r i f u g a t i o n of whole blood samples e q u i l i b r a t e d w i t h halothane. There was reasonable agreement between the r e s u l t s obtained by the two methods. The uptake and d i s t r i b u t i o n of halothane i n dog blood at d i f f e r e n t i n s p i r e d l e v e l s of halothane was studied by analysing the concentration of halothane i n whole blood and plasma of a r t e r i a l and mixed venous blood at d i f f e r e n t times a f t e r the i n d u c t i o n of anaesthesia. G e n e r a l l y , a steady s t a t e was reached approximately 2 hours a f t e r i n d u c t i o n . The time required for the d i s t r i b u t i o n of halothane between the plasma and c e l l s appeared to be much shorter than the time required to a t t a i n the steady s t a t e . This suggested that the d i s t r i b u t i o n of halothane between blood components c a l c u l a t e d from the r e s u l t s of the e q u i l i b r i u m d i a l y s i s s tudies i s a p p l i c a b l e to blood i n v i v o during anaesthesia. The a r t e r i a l blood halothane concentration c a l c u l a t e d by combining the experimentally determined e n d - t i d a l halothane p a r t i a l pressure and l i t e r a t u r e values f o r the blood gas p a r t i t i o n c o e f f i c i e n t are d i f f e r e n t from those determined experimentally. This suggested that halothane i n the a l v e o l i and halothane i n the a r t e r i a l blood were not i n thermodynamic e q u i l i b r i u m , as commonly accepted. - i v -TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS i x ABBREVIATIONS x GENERAL INTRODUCTION 1 PART I - ANALYTICAL METHOD 2 INTRODUCTION 2 MATERIALS AND METHODS 11 The e x t e r n a l i n j e c t i o n port 11 Design 11 Operation 16 Gas chromatography 17 RESULTS 21 DISCUSSION 27 PART I I - SOLUBILITY AND DISTRIBUTION OF HALOTHANE IN HUMAN BLOOD: A MODEL STUDY 29 INTRODUCTION 29 MATERIALS 40 METHODS 41 1. A n a l y t i c a l methods 41 2. Studies of the bindin g of halothane to blood components 41 a. S a t u r a t i o n concentration of halothane i n s a l i n e 41 b. Hemoglobin 42 - v -c. Albumin 42 d. y - g l o b u l i n 43 e. T r i g l y c e r i d e r i c h m i c e l l e s 43 f. Red c e l l ghosts 44 g. D i a l y s i s of blood components against halothane 44 3. D i s t r i b u t i o n of halothane between c e l l s and plasma 47 RESULTS 51 1. S a t u r a t i o n concentration of halothane i n s a l i n e 51 2. Adsorption of halothane to haemoglobin 51 3. Adsorption of halothane to albumin 55 4. y - g l o b u l i n 59 5. Absorption of halothane to red c e l l ghosts 62 6. Absorption of halothane to t r i g l y c e r i d e - r i c h m i c e l l e s 63 7. D i s t r i b u t i o n of halothane between the components of blood 67 8. D i s t r i b u t i o n of halothane between c e l l s and plasma 67 DISCUSSION 73 PART I I I - UPTAKE AND DISTRIBUTION OF HALOTHANE IN DOG BLOOD IN VIVO 82 INTRODUCTION 82 MATERIALS AND METHODS 88 RESULTS AND DISCUSSION 91 CONCLUSIONS 106 BIBLIOGRAPHY 107 - v i -LIST OF TABLES I I n h a l a t i o n a l general anaesthetic molecules 3 I I D i r e c t i n j e c t i o n methods f o r the q u a n t i t a t i v e a n a l y s i s of 5 i n h a l a t i o n general anaesthetics I I I D i s t i l l a t i o n methods for the q u a n t i t a t i v e a n a l y s i s of i n h a l a t i o n 6 general anaesthetics IV Solvent e x t r a c t i o n methods for the q u a n t i t a t i v e a n a l y s i s of 7 i n h a l a t i o n general anaesthetics V Head space method f o r the q u a n t i t a t i v e a n a l y s i s of i n h a l a t i o n 8 general anaesthetics VI Performance c h a r a c t e r i s t i c s of the flame i o n i z a t i o n d e t e c t o r , 23 electrometer and e l e c t r o n i c i n t e g r a t o r V I I G a s - l i q u i d chromatographic a n a l y s i s of whole blood samples 26 c o n t a i n i n g known concentrations of halothane, methoxyflurane, d i e t h y l e t h e r and ethanol V I I I Reported dependence of the s o l u b i l i t y of halothane on blood 31 components IX S a t u r a t i o n concentration of halothane i n s a l i n e at 4 and 37°C 52 X Time re q u i r e d to reach e q u i l i b r i u m f o r the d i a l y s i s of haemoglobin. 53 against halothane XI Time r e q u i r e d to reach e q u i l i b r i u m f or the d i a l y s i s of albumin 57 against halothane X I I D i a l y s i s of y - g l o b u l i n against halothane 61 X I I I Time required to reach e q u i l i b r i u m f o r the d i a l y s i s of red c e l l 62 ghosts against halothane XIV Time re q u i r e d to reach e q u i l i b r i u m f or the d i a l y s i s of 65 t r i g l y c e r i d e - r i c h m i c e l l e s against halothane XV D i s t r i b u t i o n of halothane i n blood c a l c u l a t e d from the r e s u l t s 69 of e q u i l i b r i u m d i a l y s i s XVI Time re q u i r e d to reach e q u i l i b r i u m f or the d i s t r i b u t i o n of 70 halothane between c e l l s and plasma - v i i -XVII D i s t r i b u t i o n of halothane between c e l l s and plasma 72 XVIII Blood/gas p a r t i t i o n c o e f f i c i e n t for dog at 37°C 87 XIX Comparison of a r t e r i a l blood halothane concentration determined 96 experimentally and that c a l c u l a t e d from the e n d - t i d a l p a r t i a l pressure assuming thermodynamic e q u i l i b r i u m at 1.0% i n s p i r e d l e v e l XX Comparison of a r t e r i a l blood halothane concentration determined 97 experimentally and that c a l c u l a t e d from the e n d - t i d a l p a r t i a l pressure assuming thermodynamic e q u i l i b r i u m at 1.5% i n s p i r e d l e v e l XXI Comparison of a r t e r i a l blood halothane concentration determined 98 experimentally and that c a l c u l a t e d from the e n d - t i d a l p a r t i a l pressure assuming thermodynamic e q u i l i b r i u m at 2.0% i n s p i r e d l e v e l XXII Comparison of a r t e r i a l blood halothane concentration determined 99 experimentally and that c a l c u l a t e d from the e n d - t i d a l p a r t i a l pressure assuming thermodynamic e q u i l i b r i u m at 2.5% i n s p i r e d l e v e l XXIII E f f e c t of i n v i t r o e q u i l i b r a t i o n on the i n v i v o d i s t r i b u t i o n of 101 halothane between plasma and c e l l s - v i i i -LIST OF FIGURES 1. Diagrammatic r e p r e s e n t a t i o n of the f r o n t view of the e x t e r n a l 12 i n j e c t i o n port 2. Engineering drawings of the e x t e r n a l i n j e c t i o n port 13 3. Schematic c a r r i e r gas flow diagram of the e x t e r n a l i n j e c t i o n port 15 4. Sample chromatograms from the a n a l y s i s of halothane, methoxyflurane, 24 d i e t h y l ether and ethanol 5. Diagrammatic r e p r e s e n t a t i o n of the e q u i l i b r i u m d i a l y s i s assembly 45 6. Adsorption of halothane to haemoglobin 54 7. Scatchard P l o t of halothane b i n d i n g to haemoglobin 56 8. Adsorption of halothane to albumin 58 9. Scatchard P l o t of halothane b i n d i n g to albumin 60 10. Absorption of halothane to red c e l l ghosts 64 11. Absorption of halothane to t r i g l y c e r i d e - r i c h m i c e l l e s at constant 66 t r i g l y c e r i d e concentration 12. Absorption of halothane to t r i g l y c e r i d e r i c h m i c e l l e s at constant 68 free halothane concentration 13. Blood and plasma halothane concentration i n a r t e r i a l and mixed 92 venous blood at constant i n s p i r e d l e v e l of 1.0% 14. Blood and plasma halothane concentration i n a r t e r i a l and mixed 93 venous blood at constant i n s p i r e d l e v e l of 1.5% 15. Blood and plasma halothane concentration i n a r t e r i a l and mixed 94 venous blood at constant i n s p i r e d l e v e l of 2.0% 16. Blood and plasma halothane concentration i n a r t e r i a l and mixed 95 venous blood at constant i n s p i r e d l e v e l of 2.5% - i x -ACKNOWLEDGEMENTS Dr. P.E. Reid and Dr. D.E. Brooks, my research s u p e r v i s o r s , have made t h i s work p o s s i b l e through t h e i r advice, support, t o l e r a n c e , and above a l l , t h e i r good sense of humour. I would l i k e to thank Jan Reid for her h o s p i t a l i t y , Professor C.F.A. C u l l i n g f o r h i s help, i n p a r t i c u l a r f o r showing me that ghosts are v i s i b l e , Dr. K.M. Leighton for h i s a c t i v e support of t h i s research p r o j e c t , C a r o l i n e Bruce f o r c a r r y i n g out the dog experiments, Dr. G. Gray f o r the haemoglobin analyses, Dr. W. Ramey for the arrangement of the "hot room" f a c i l i t i e s , Johan Janzen and Michael Shum f o r t e c h n i c a l i n f o r m a t i o n , Sandra Sturgeon f o r the expert preparation of the manuscript, Dr. D. Vance for a sample of o l e i c a c i d , and the f o l l o w i n g blood donors: Dr. P.E. Reid, Charles Ramey, Dr. R.H. Pearce, Amir A l - S u h a i l and Dorothy Emslie. The Red Cross a l s o k i n d l y s u pplied many u n i t s of outdated blood. This research p r o j e c t was supported by a grant from the Canadian Heart Foundation. The author i s g r a t e f u l to Dr. D. Applegarth f o r a Graduate A s s i s t a n t s h i p and to the U n i v e r s i t y of B.C. for a Summer Research Scholarship and U n i v e r s i t y Graduate Fell o w s h i p . R o l f Muelchen deserves much more than my s p e c i a l thanks. Not only d i d he design and construct the e x t e r n a l i n j e c t i o n p o r t , he a l s o rescued t h i s p r o j e c t more than once, i . e . trouble-shot e s s e n t i a l pieces of equipment which threatened to withdraw t h e i r s e r v i c e s from time to time. L a s t , but by no means l e a s t , I would l i k e to mention Charles Ramey, who p a r t i c i p a t e d i n many f r u i t f u l d i s c u s s i o n s , provided t e c h n i c a l help, and maintained an optimum management of the l a b o r a t o r y , a l l of which c o n t r i b u t e d to making research an enjoyable experience. - x -ABBREVIATIONS ESR — E l e c t r o n s p i n resonance G - Gibbs free energy G" - Chemical p o t e n t i a l GLC - G a s - l i q u i d chromatography MAC - Minimum a l v e o l a r concentration MAP - Minimum a l v e o l a r p a r t i a l pressure mg% - M i l l i g r a m s per 100 grams MW - Molecular weight n - Number of moles N - Avogradro's number NMR - Nuclear magnetic resonance P - Pressure PH - P a r t i a l pressure of halothane PBS - Phosphate b u f f e r s a l i n e R - U n i v e r s a l gas constant T - Absolute temperature TG - T r i g l y c e r i d e Ul - M i c r o l i t r e V - Volume V - P a r t i a l s p e c i f i c volume VLDL - Very Low Density L i p o p r o t e i n - xl -«&* % M fk & *fo AS* #3 £ *\\ ? in-fa-rt Mr 369-286 BC. - 1 -GENERAL INTRODUCTION Halothane i s the most commonly used i n h a l a t i o n a l general anaesthetic i n North America, and has a very high potency compared to other i n h a l a t i o n anaesthetics (Saidman et a l . 1967; S t o e l t i n g et a l . 1970; Eger et a l . 1965b). I t s s o l u b i l i t y i n blood i s much higher than that i n water or s a l i n e (Steward et a l . 1973). This c l e a r l y suggests that halothane, i n a d d i t i o n to being d i s s o l v e d i n the aqueous phase of blood, i s a l s o associated w i t h , or c a r r i e d by, one or more blood components. As yet there has been no c o n c l u s i v e evidence presented to show that the increased s o l u b i l i t y of halothane i n blood as compared to s a l i n e i s due to any major blood component. The present study was aimed at e l u c i d a t i n g which are the important blood components c o n t r i b u t i n g to the transport of halothane and how s i g n i f i c a n t i s t h i s c o n t r i b u t i o n i n human whole blood. The approaches commonly taken for the study and i n t e r p r e t a t i o n of the uptake and d i s t r i b u t i o n of i n h a l a t i o n anaesthetics are based on the concept of e q u i l i b r i u m thermodynamics. Since a l i v i n g organism does not n e c e s s a r i l y f u n c t i o n at or c l o s e to the e q u i l i b r i u m c o n d i t i o n , the use of an e q u i l i b r i u m thermodynamical concept i n the treatment of the k i n e t i c s of anaesthetic uptake may not be tenable. Therefore i n v i v o experiments were c a r r i e d out i n dogs to t e s t the v a l i d i t y of the e q u i l i b r i u m assumption. In a d d i t i o n , these experiments provided d e t a i l e d information on the blood l e v e l of anaesthetic during halothane anaesthesia. - 2 -PART 1 - A n a l y t i c a l Method INTRODUCTION I n h a l a t i o n general anaesthetics are e i t h e r gaseous or v o l a t i l e compounds commonly employed i n surgery to produce anaesthetic e f f e c t s . Table I shows the chemical s t r u c t u r e s of c l i n i c a l l y s u c c e s s f u l anaesthetics. These are very simple, e i t h e r short chain hydrocarbons (eg. cyclopropane) or ethers (eg. d i e t h y l e t h e r ) . The more r e c e n t l y discovered anaesthetics are a l l s u b s t i t u t e d by halogens. Halothane (1956) i s a hydrocarbon s u b s t i t u t e d by f l u o r i n e , c h l o r i n e and bromine w h i l e fluroxene (1953), methyoxyflurane and enflurane (1967) are ethers s u b s t i t u t e d by f l u o r i n e and c h l o r i n e . The p h y s i c a l , chemical and anaesthetic p r o p e r t i e s of these halogenated agents have been e x t e n s i v e l y reviewed by Rudo and Krantz (1974). C l i n i c a l l y the amount of anaesthetics administered i s monitored by i n f r a -red absorption w i t h a c c u r a t e l y c a l i b r a t e d flow meters (Wolfson 1968). This method i s u n s u i t a b l e f o r experimental research because only anaesthetic i n the gas phase can be analysed. Much e f f o r t has been d i r e c t e d to develop a method f o r analysing various anaesthetics i n blood and t i s s u e samples. Various methods, i n c l u d i n g autoradiography (Cohen et a l . 1972), mass spectroscopy (Jones et a l . 1953), and electroencephalography (Wolfson et a l . 1967) have been i n v e s t i g a t e d for t h i s purpose. G a s - l i q u i d chromatography (GLC) i s by f a r the most s a t i s f a c t o r y method because i t i s r e l a t i v e l y inexpensive and the theory underlying i t s operation i s w e l l understood. GLC procedures f o r the q u a n t i t a t i v e a n a l y s i s of i n h a l a t i o n anaesthetics i n blood and other f l u i d s are of two types: e i t h e r d i r e c t , i n which the gas chromatography i s performed on - 3 -Table I - I n h a l a t i o n general anaesthetic molecules H I Cl-C-Cl I C l Chloroform H H H H I 1 1 1 H-C-C-O-C-C-H H H Ether I I H H F C l I I F-C-C-H I I F Br Halothane C l F H I I I H-C-C-O-C-H I I I C l F H Methoxyflurane H-C >C-H H H Cyclopropane H C l F I I I F-C-C-O-C-CH-5 I I I F F F Enflurane F H H H II I / F-C-C-0-C=C II I \ F H H H Fluroxene - 4 -the sample i t s e l f , or i n d i r e c t , i n which the anaesthetic i s separated from the sample p r i o r to i n j e c t i o n . 1. D i r e c t Methods D i r e c t i n j e c t i o n methods are l i s t e d i n Table I I . They can be d i v i d e d f u r t h e r i n t o three general c l a s s e s : (a) those i n which the sample i s i n j e c t e d i n t o the i n j e c t i o n port of the chromatograph, (b) those i n which i n j e c t i o n i s made i n t o a removable glass l i n e r i n s e r t e d i n t o the i n j e c t i o n p o r t , and (c) those i n which i n j e c t i o n i s made i n t o a heated pre-column device i s o l a t e d from the columns and then, a f t e r a period of time, the v o l a t i l e s are swept onto the column by a stream of c a r r i e r gas v i a a switching v a l v e . D i r e c t i n j e c t i o n methods are r a p i d , apparently simple and can be used w i t h small samples of blood (Yamamura et a l . 1966). They may, however, s u f f e r from problems a s s o c i a t e d w i t h contamination o f the columns with n o n - v o l a t i l e blood components ( B u t l e r et a l . 1967; Kolmer et a l . 1975a; Cousins and Mazze 1972) and subsequent b a s e l i n e d r i f t due to the slow e l u t i o n of such components ( B u t l e r et a l . 1967; Cousins and Mazze 1972), clogging of the syringes used for i n j e c t i o n (Kolmer et a l . 1975a; Cousins and Mazze 1972), d i s t o r t i o n and broadening of the peaks ( B u t l e r et a l . 1967; Kolmer et a l . 1975; Jones et a l . 1972; Cousins and Mazze 1972), ghost peaks (Kolmer et a l . 1975; Cousins and Mazze 1972) and poor r e p r o d u c i b i l i t y (Yamamura et a l . 1966; B u t l e r et a l . 1967). 2. I n d i r e c t Methods The i n d i r e c t GLC methods are l i s t e d i n Tables I I I , IV and V. They are: (a) d i s t i l l a t i o n , i n which the anaesthetics are d i s t i l l e d or vaporized from Table I I - D i r e c t i n j e c t i o n g a s - l i q u i d chromatographic methods for the q u a n t i t a t i v e e s timation of i n h a l a t i o n general anaesthetics i n blood Method Precolumn Anaesthetics Detector C a l i b r a t i o n S tationary Approximate Range Device Analysed Method Phase of s e n s i t i v i t y (mg/100 ml) Cyclopropane Lowe 1964 Ether None Methoxy flur a n e Flame Peak Height Chromosorb 3-40 Halothane I o n i z a t i o n C a l i b r a t i o n P Lowe and Beckham Chloroform 1964 T r i f l u o r o e t h y l -v i n y l ether Lassberg and E t s t e n Glass Cyclopropane Flame Peak Area DC550 10-70 1965 l i n e r I o n i z a t i o n C a l i b r a t i o n S i l i c o n e O i l Cousins and Mazze Glass Halothane Flame Peak Area OV101 2-40 1972 l i n e r Methoxyfluorane I o n i z a t i o n C a l i b r a t i o n Douglas et a l . Glass Halothane Flame Peak Height Chromosorb 20-50 1970 l i n e r I o n i z a t i o n C a l i b r a t i o n P Yokota et a l . "Vap o r i z i n g Cyclopropane Flame Peak Height ' Ethylene g l y c o l NG 1967 apparatus" Halothane I o n i z a t i o n C a l i b r a t i o n succinate Methoxyflurane and thermal D i e t h y l ether c o n d u c t i v i t y N i t r o u s oxide Cole et a l . "Precolumn Halothane Flame Anesthetic to FFAP 5-40 1975 Device" Methoxyflurane I o n i z a t i o n I n t e r n a l Standard Peak Height Ratio NG: not given Table I I I - D i s t i l l a t i o n Methods for the q u a n t i t a t i v e e stimation of i n h a l a t i o n general anaesthetics i n blood by g a s - l i q u i d chromatography Method Anaesthetic Analysed Detector C a l i b r a t i o n Method Statio n a r y Phase Approximate Range of s e n s i t i v i t y (mg/100 ml) Gadsden et a l . Halothane Thermal Peak Height d i - 2 - e t h y l h e x y l 9-55 1965 C o n d u c t i v i t y C a l i b r a t i o n sebacate Rackow et a l . D i e t h y l e t h e r Flame Peak Area NG NG 1966 I o n i z a t i o n C a l i b r a t i o n O N NG: not given i - 7 -Table IV - Solvent extraction methods for the qu.ntit.ti*. estimation of inhalation general anaesthetics in blood bv gas-liquid chromatography ' Anaesthetics Anelyead Calibration Method Stationary Phase Approximate range ef sensitivity (Kg/100 al) Butler and H i l l 1961 Rut ledge ct a l . 1963 Wolfeon at a l . 1966a Wolfson et a l . 1966b Hartley et a l . 1968 Cervenko 1968 Douglas et a l . 1970 Allotc et a l . 1971 Jones et a l . 1972 Flai Ion Thermal Conductivity Peak Beight Calibration Peak Height Calibration Methoxyflurane Plane lonisation Halothane Ether Attallah and Creddes Halothane 1972 Oavii et a l . 1975 E l l i a and Sto*1 ting Pluroxene 1975 Poobalaaingaa 1976 Halliday et a l . 1977 Halothane Methoxyflurane Toner et a l . Rnflurene 1977 Plaae Ionization Plane Ion ication Electros Capture Plane Ionization Methoxyflurane PIa Electron Capture Electron Capture Plane Ion ication Cyclopropane Tr i ch1oroethene Electron Capture Anaesthetic to Internal Standard (toluene) Peak Height katio Anaesthetic to Internal Standard (chloroform) Peak Height tatio Peak Height Calibration Anaesthetic to Internal Standard (diethylether) Peak Height Ratio Peak Area Calibration Anaesthetic to Internal Standard (chloroform) Peak Height tatio Peak Area Calibration Peak Beight Calibration Peak Height Calibration Peek Height Calibration n-fieptane n—Heptane Carbon disulphide Silicone Plaid MS 350 Tide SB 50 Carbon 8X30 tetrachloride n-Heptane Silicone Pluid KS 550 Carbon Silicone Oil tetrachloride MS 550 n-Heptane Carbon tetrachloride Silieooe Pluid KS 550 Silicone Pluid SZ30 MS 200/20 C.S. n-Heptane Tetraehloro-•thylene Anaesthetic to Internal Standard ( cr i ch 1 or oe t hy 1 en e ) Peak Height Ratio Anaesthetic to Internal Standard (chloroform, toluene) Peak Height Ratio Anaesthetic to Internal Standard (methoxyflarene) Peek Beight Ratio Carbon Disulphide Carbon tetrachloride Carbon Disulphide Silicone Pluid MS 550 Diiaodecyl phthalate Silicone Pluid MS 550 B-Beptaj Poropack Q 1-20 5-20 4-15 0.5-8 i 0.5-4 i ole/1 ole/I •C: not given - 8 -Table V - Head space methods for the quantitative estimation of inhalation general anaesthetics in blood by gas-1iquid chromatography Method Anaesthetic Analysed Detector Calibration Method Stationary Phase Approximate Range of S e n s i t i v i t y Noehren and Cudmore 1961 Diethylether NG Peak Height Calibration Tetraethylene glycol dimethylether 25-168 mg/100 ml Bowes 1964 Nitrous oxide Thermistor Peak Height Calibration Dimethylsulphoxide NG Yamamura et a l . 1966 Halothane Methoxyflurane Cyclopropane Ether Nitrous oxide Flame Ionization Thermal Conductivity Peak Height Calibration PEG DOP Activated charcoal 10-20 mg/100 ml 100-200 mg/100 ml 10-100 mg/100 ml Butler et a l . 1967 Halothane Flame Ionization Peak Height Calibration Silicone gum rubber NG Theye 1968 Halothane Thermal Conductivity Peak Height Calibration Amine 220 and Carbowax 400 0.3-3.82 (v/v) Fink and Morikawa 1970 Halothane Flame Ion ization Peak Height Calibration SE30 NG Wagner et a l . 1974 Halothane Ether Cyclopropane Flame I on iz at ion Peak Height Calibration Poropak T 10"4-0.1X (v/v) Kolmer et a l . 1975a Halothane Flame Ion ization Peak Height Calibration Porasil S Carbowax 400 lppm-52 (v/v) Heavner et a l . 1976 Halothane Flame Ionization Peak Height Calibration Poropak Q 0.02-2.5Z (v/v) Renzi and Waud 1977 Enflurane Ether Fluroxene Halothane Isothurane Methoxyflurane NC Peak Height Calibration NG NG NG: not given - 9 -the sample and subsequently t r a n s f e r r e d on to the column, (b) solvent e x t r a c t i o n , i n which the anaesthetics are extracted from the sample by d i r e c t mixing wit h an organic phase, an a l i q u o t of which i s subsequently i n j e c t e d i n t o the chromatograph and (c) head space, i n which the sample i s allowed to e q u i l i b r a t e w i t h a f i x e d amount of a i r and a f t e r e q u i l i b r i u m i s reached, a sample of the e q u i l i b r a t e d a i r i s i n j e c t e d i n t o the chromatograph. Such p r e l i m i n a r y procedures, designed to separate the anaesthetics from the sample, are used to circumvent the above mentioned d i f f i c u l t i e s encountered i n the d i r e c t i n j e c t i o n methods. However, these pretreatments al s o can produce d i f f i c u l t i e s . They are time consuming (Yamamura et a l . 1966; B u t l e r et a l . 1967; Kolmer 1975a; Yakota et a l . 1967; Cousins and Mazze 1972; Cole et a l . 1975), i n v o l v e a p o t e n t i a l l o ss of the agent (Cousins and Mazze 1972) and may be d i f f i c u l t to apply to small samples (Cousins and Mazze 1972). Furthermore, solvent e x t r a c t i o n of the anaesthetic r e s u l t s i n the concurrent e x t r a c t i o n of l i p i d m a t e r i a l s from the blood or t i s s u e samples. These w i l l be deposited onto the column upon i n j e c t i o n , p a r t i a l l y o f f s e t t i n g the intended advantage of the method ( B u t l e r 1967). Solvent e x t r a c t i o n a l s o leads to large solvent peaks which may i n t e r f e r e w i t h the e s t i m a t i o n of the anaesthetic peak and/or r e s u l t i n prolonged e l u t i o n times (Wolfson et a l . 1966a; Douglas et a l . 1970; Jones et a l . 1972). The head space method, although f r e e of the t e c h n i c a l disadvantages of solvent e x t r a c t i o n , does not d i r e c t l y y i e l d the concentration of the anaes-t h e t i c i n the sample, but only the p a r t i a l pressure of the anaesthetic i n the gas phase i n e q u i l i b r i u m w i t h the sample. Thus, unless the anaesthetic p a r t i a l pressure of the sample i s the de s i r e d r e s u l t , another method should be used i f p o s s i b l e . - 10 -For the reasons discussed above i t was considered d e s i r a b l e to develop a simple and r e l i a b l e a n a l y t i c a l method f o r experimental anaesthetics research. The method was to be a p p l i c a b l e to small samples, give r e s u l t s d i r e c t l y i n concentration u n i t s , and i n v o l v e a minimum amount of pretreatment of the sample. To t h i s end, an e x t e r n a l i n j e c t i o n p o r t * , i e . a f i l t r a t i o n system placed i n the c a r r i e r gas stream before the column, was used which allowed d i r e c t i n j e c t i o n of a blood sample i n t o the preheated c a r r i e r gas stream of the gas chromatograph. Evaporation at a high temperature causes the n o n - v o l a t i l e components to be trapped i n the f i l t e r and only the v o l a t i l e components enter the column. This e l i m i n a t e s the contamination problem encountered i n other d i r e c t i n j e c t i o n methods and permits the r a p i d a n l y s i s of halothane, methoxyflurane, d i e t h y l ether and ethanol i n whole blood over the approximate range 1-100 mg/100 ml. *The e x t e r n a l i n j e c t i o n port was designed and constructed by Mr. R o l f Muelchen. MATERIALS AND METHODS Halothane (Hoechst Pharmaceuticals, Montreal, Canada), methoxyflurane (Abbott L a b o r a t o r i e s , Vancouver, Canada) and d i e t h y l e t h e r (spectroanalysed; F i s h e r S c i e n t i f i c , Montreal, Canada) were used without f u r t h e r p u r i f i c a t i o n . Isobutanol (reagent grade; M a l l i n c k r o d t , St. L o u i s , Mo., U.S.A.) was d r i e d over anhydrous potassium carbonate and p u r i f i e d by f r a c t i o n a l d i s t i l l a t i o n (Vogel 1954). Ethanol was p u r i f i e d by the use of a Grignard reagent procedure (Vogel 1954). Vacuum grease (Dow, Midland, Michigan) was obtained from F i s c h e r S c i e n t i f i c , Montreal, Canada. Swagelok f i t t i n g s (Crawford F i t t i n g Company, Solon, Ohio) were obtained from Columbia Valve & F i t t i n g Co., Vancouver. A l l other chemicals employed were of reagent grade or b e t t e r . S i l i c o n e rubber 0-rings 3/16" I.D. and 5/16" O.D. HT8 low bleed septa were obtained from A p p l i e d Science, State C o l l e g e , Pa., U.S.A. Chromosorb 101, 103, 105, and 107 were purchased from Johns M a n v i l l e , Denver, Colo., U.S.A. and R e a c t i - v i a l s (0.3 ml and 1 ml) and M i n i n e r t valves from P i e r c e , Rockford, 111., U.S.A. These v i a l s were supplied with g a s - t i g h t t e f l o n l i n e d seals and served as leak-proof containers f o r halothane. Each M i n i n e r t valve was equipped w i t h a septum to prevent loss of v o l a t i l e compounds when the valve was opened f o r sampling. Gas chromatographic syringes were obtained from Hamilton Co., Reno, Nevada, U.S.A. The e x t e r n a l i n j e c t i o n port  Design The design of the e x t e r n a l i n j e c t i o n port i s shown diagrammatically i n F i g . 1; F i g . 2 shows d e t a i l e d engineering drawings. The e x t e r n a l i n j e c t i o n - 12 -T O G L C F i g . 1. Diagrammatic r e p r e s e n t a t i o n of the f r o n t view of the e x t e r n a l i n j e c t i o n port. - 13 -F i g . 2. Engineering drawings of the e x t e r n a l i n j e c t i o n p o r t . - 14 -port c o n s i s t s e s s e n t i a l l y of a heat r e s e r v o i r and a r o t a r y valve both of which c o n t a i n channels for d i r e c t i n g the flow of c a r r i e r gas. Both the r e s e r v o i r and r o t a r y valve are constructed of br a s s , t h e i r r e s p e c t i v e plane face p l a t e s being separated by a 1/32" Teflon gasket against which the valve r o t a t e s . Mounted on the f r o n t of the r o t a r y valve are: (a) a sp r i n g which can be used to t i g h t e n the valve against the Teflon gasket to achieve a gas t i g h t s e a l , (b) a gas chromatographic i n j e c t i o n port of conventional design sealed w i t h a septum and (c) a p a i r of handles which allow r o t a t i o n of the v a l v e . The r e s e r v o i r i s maintained at 180°C with a heater set i n t o a w e l l d r i l l e d i n t o the body of the r e s e r v o i r ; a s i m i l a r w e l l contains a thermometer. The base of the port i s attached, by a gas t i g h t s e a l , to the i n j e c t i o n port system of the gas chromatograph and can be r e a d i l y modified f o r use with a v a r i e t y of instruments. The c a r r i e r gas enters the port through a Swagelok f i t t i n g sealed i n t o the side of the r e s e r v o i r ; t h i s r e q u i r e s a d i v e r s i o n of the c a r r i e r gas stream p r i o r to the point where i t enters the o r i g i n a l i n j e c t i o n port of the gas chromatograph. F i g . 3 i l l u s t r a t e s the flow paths of the c a r r i e r gas w i t h i n the e x t e r n a l i n j e c t i o n port when the r o t a r y valve i s i n the "on" o p e r a t i o n a l and the " o f f " non-operational p o s i t i o n s . In the "on" p o s i t i o n the c a r r i e r gas passes s u c c e s s i v e l y through the f i x e d channels a and b l o c a t e d , r e s p e c t i v e l y , i n the body, of the heat r e s e r v o i r and the r o t a r y v a l v e . I t then proceeds v i a the f i x e d channel c lo c a t e d i n the r e s e r v o i r , i n t o a glass U-tube (3/16" O.D.) connecting c with the f i x e d channel d located i n the body of the r e s e r v o i r . The c a r r i e r gas then passes through f i x e d channels e and f l o c a t e d , r e s p e c t i v e l y , i n the r o t a r y valve and the r e s e r v o i r and enters the gas chromatography column through the base of the i n j e c t i o n p o r t . The U-tube i s - 15 -F i g . 3. Schematic c a r r i e r gas flow diagram of the e x t e r n a l i n j e c t i o n port. - .16 -l o c a t e d on the l e f t side (see F i g . 2) and i s sealed i n t o p o s i t i o n w i t h s i l i c o n e rubber 0-rings l i g h t l y l u b r i c a t e d w i t h a high temperature vacuum grease. Before i n s e r t i o n the ends of the U-tube were a l s o l u b r i c a t e d with the same vacuum grease. A lever attached to the r e s e r v o i r (see F i g . 2) prevents the e j e c t i o n of the U-tube when the valve i s i n the "on" p o s i t i o n (approximate pressure 20-25 p . s . i . ) and als o serves to maintain a gas t i g h t s e a l . Holes s i t u a t e d i n the appropriate p o s i t i o n s i n the Tef l o n gasket a l l o w free passage of gases between the f i x e d channels i n the r e s e r v o i r and the r o t a r y v a l v e . When the valve i s i n the "on" p o s i t i o n the needle of a Hamilton gas chromatographic syringe w i l l pass through both the septum of the i n j e c t i o n port mounted on the f r o n t of the valve and the Te f l o n gasket and enter the U-tube p e r m i t t i n g i n j e c t i o n d i r e c t l y i n t o the pre-heated c a r r i e r gas stream. When the valve i s i n the " o f f " p o s i t i o n the U-tube i s by-passed and the c a r r i e r gas passes d i r e c t l y i n t o the gas chromatograph through channels a, b and f. This enables the U-tube to be changed simply and r a p i d l y without i n t e r f e r i n g w i t h the gas flows i n the chromatograph. Operation P r i o r to the f i r s t operation of the e x t e r n a l i n j e c t i o n port the apparatus o i s maintained at 180 C at a pressure on the Tef l o n gasket of 15-20 pounds for 24-36 hr. Under these c o n d i t i o n s the Te f l o n gasket softens and i s moulded i n t o the shape of the i n s i d e surfaces of the r o t a r y valve and heat r e s e r v o i r . This ensures a good s e a l and provided the r e s e r v o i r i s maintained at 180°C remoulding i s only necessary when a new gasket i s f i t t e d . With the r o t a r y valve i n the "on" p o s i t i o n a sample of blood (4-40 u l ) i s i n j e c t e d d i r e c t l y i n t o a loose glass wool f i l t e r plug i n s e r t e d i n t o the - 17 -U-tube. To avoid clogging of the needle of the syringe by coagulated blood components i n j e c t i o n was accomplished with a chaser technique i n which the blood sample i n the syringe b a r r e l was separated from a plug of water by a bubble of a i r . When the a n a l y s i s i s completed the r o t a r y valve i s switched to the " o f f " p o s i t i o n and the U-tube i s replaced. On switching to the "on" p o s i t i o n the equipment i s ready for another i n j e c t i o n . Changing the U-tube occupies between 15 and 60 sec. Gas chromatography Columns were prepared as f o l l o w s . A 6 f t x 4 mm I.D. s t a i n l e s s s t e e l column was packed w i t h Chromosorb 101 (80-100 mesh) and conditioned f o r 16 hr at 200°C with c a r r i e r gas flow. The conditioned Chromosorb 101 was then unpacked from the s t a i n l e s s s t e e l column and repacked i n t o two 6 f t x 2 mm I.D. glass columns. Gas chromatography was performed with these glass columns temperature programmed (temperature programming i s a technique i n gas chromatography i n which the temperature of the columns i s increased from an i n i t i a l to a f i n a l temperature at a predeterminated r a t e ) from 110-180°C at 6°C/min i n a Hewlett-Packard 7610A gas chromatograph f i t t e d w i t h dual flame i o n i z a t i o n d e t e c t o r s . The i n j e c t i o n port of the chromatograph was maintained at 200°C, the e x t e r n a l i n j e c t i o n port at 180°C and the flame i o n i z a t i o n detector at 250°C. Gas flow-rates employed were as f o l l o w s : c a r r i e r 25 ml/min; a u x i l i a r y 35 ml/min, hydrogen 50 ml/min and a i r 470 ml/min. Nitrogen was used as c a r r i e r gas. Range r e s i s t a n c e of the electrometer was set at 8 10 ohms. Peak areas were measured with a Hewlett-Packard 3370B e l e c t r o n i c i n t e g r a t o r operated at "Up" and "Down" slope s e n s i t i v i t i e s of 0.1 mV/min. I n j e c t i o n volumes were chosen to y i e l d a minimum anaesthetic peak area of 5 x 10 uV sec whenever p o s s i b l e , otherwise 40 u l samples were i n j e c t e d . A l l samples prepared f o r a n a l y s i s of anaesthetic concentrations were kept at 0-4°C during sampling. The syringe was r i n s e d once with chromic a c i d and then e x t e n s i v e l y w i t h water a f t e r each i n j e c t i o n . This prevented clogging of the syringe by n o n - v o l a t i l e blood components a f t e r repeated i n j e c t i o n s . To obviate d a i l y c a l i b r a t i o n of the gas chromatograph, an i n t e r n a l standard, i s o b u t a n o l , was used. The r a t i o of the detector response f o r the anaesthetic and the standard, d i v i d e d by the r a t i o of t h e i r weights i n a sample i s a constant f o r the type of detector used i n t h i s study, provided that the detector i s not saturated (Novak et a l . 1970). Hence t h i s r a t i o , or response f a c t o r , was employed to determine the anaesthetic concentrations. Response f a c t o r i s defined by the equation: Response f a c t o r = peak area agent x wt. isobutanol [1] peak area i s o b u t a n o l wt. agent Response f a c t o r s were determined as f o l l o w s . Isobutanol was added to h a l f f i l l a preweighed 3 ml R e a c t i - v i a l equipped with a magnetic s t i r r e r and a M i n i n e r t v a l v e . This was weighed. The anaesthetic was then added to com-p l e t e l y f i l l the v i a l , which was again weighed. A f t e r thorough mixing, 2 u l of the mixture was t r a n s f e r r e d , using a Hamilton 10 u l s y r i n g e , i n t o another s i m i l a r l y equipped 3 ml R e a c t i v i a l f i l l e d w i t h whole blood a n t i c o a g u l a t e d with EDTA. This was thoroughly mixed f o r 2 hr at 0-4°C. Samples from t h i s blood mixture were analysed by gas chromatography. Analyses of blood samples co n t a i n i n g anaesthetics were performed on EDTA anticoagulated blood using 0.3 ml R e a c t i - v i a l s ( a c t u a l volume approximately - 19 0.5 ml) each equipped with a magnetic s t i r r i n g bar and a M i n i n e r t v a l v e . Water was added to the v i a l such that the remaining volume, when the v i a l was f i l l e d to the r i m , was 0.5 ml. A standard s o l u t i o n (50 u l ) c o n t a i n i n g an a c c u r a t e l y known q u a n t i t y of i s o b u t a n o l i n water (approximately 260 mg per 100 g) was added and the v i a l was completely f i l l e d w i t h the blood sample to be analysed (450 u l ) . The q u a n t i t i e s of blood and i n t e r n a l standard employed were e s t a b l i s h e d by weighing the v i a l at the appropriate times. The samples were mixed and gas chromatography was performed on a l i q u o t s of 4-40 u l . The concentration of the anaesthetic was c a l c u l a t e d from the formula: Anaesthetic concentration = peak area anaesthetic x wt. I.S. x c o n c e n t r a t i o n I.S. peak area I.S. x wt. blood sample x response f a c t o r [2] where I.S. = i n t e r n a l standard s o l u t i o n . This gives the anaesthetic concentration i n weight of anaesthetic per u n i t weight of blood. I t can be converted to a volume concentration by m u l t i p l y i n g by the d e n s i t y of blood, which i s e s s e n t i a l l y a constant (Diem and Lentner, 1970). S i m i l a r l y the volume concentration of an anaesthetic i n any medium can be obtained by m u l t i p l y i n g the weight concentration c a l c u l a t e d from equation [2] w i t h the density of the medium. When the r e q u i r e d d e n s i t y was not a v a i l -a ble from the l i t e r a t u r e , i t was experimentally determined. In most cases the density found was s u f f i c i e n t l y c l o s e to u n i t y and was not a s i g n i f i c a n t cor-r e c t i o n f a c t o r . The e f f e c t of storage on the halothane concentrations of blood samples was tested as f o l l o w s . Standard s o l u t i o n s of halothane i n blood were prepared by d i r e c t weighing of halothane i n EDTA anticoagulated blood. These were mixed for 2 hrs at 4°C. R e a c t i - v i a l s (1.0 ml), c o n t a i n i n g a glass bead, were - 20 -f i l l e d with these blood samples i n such a manner that no a i r bubble was trapped i n the v i a l . To achieve t h i s , the v i a l was o v e r f i l l e d w i t h blood, the glass bead was dropped i n t o the v i a l , then the t e f l o n l i n e d s e a l was s l i d h o r i z o n t a l l y i n t o p l a c e , d i s p l a c i n g the excess blood, and the cap was then screwed on f i n g e r - t i g h t . The samples were stored at 4° f o r 7 and 14 days, thoroughly mixed and analysed by gas chromatography. - 21 -RESULTS An e x t e r n a l i n j e c t i o n port temperature of 180"C was found to be neces-sary to maintain a gas t i g h t s e a l between the T e f l o n gasket and the face p l a t e s of the r o t a r y valve and the heat r e s e r v o i r . Under these c o n d i t i o n s the glass wool plug served the dual f u n c t i o n of p r o v i d i n g a l a r g e , heated surface area for evaporation of the v o l a t i l e s and of a trap for the n o n - v o l a t i l e components. The rate of evaporation was presumably c o n t r o l l e d by both the pre-heating of the c a r r i e r gas stream i n the body of the heat r e s e r v o i r and the d i s p e r s i o n of the i n j e c t e d blood sample. When l i q u i d was deposited on a p o r t i o n of the U-tube free of glass wool the f l u i d beaded and evaporated s l o w l y , a phenomenon accompanied by peak broadening on gas chromatography. Successive i n j e c t i o n s of volumes of blood up to a t o t a l of 10 u l could be made before i t was necessary to i n s e r t a f r e s h U-tube but use of samples of between 10 and 40 u l n e c e s s i t a t e d the i n s e r t i o n of a f r e s h U-tube a f t e r each i n j e c t i o n . The 0-rings used to s e a l the U-tube i n t o the port gave some blee d i n g problems when new. These could be e l i m i n a t e d , however, by heating the r i n g s f o r 16 h at 160°C before i n s e r t i o n i n t o the p o r t . The e f f e c t i v e l i f e of the r i n g s was improved by l i g h t l u b r i c a t i o n with a high-temperature vacuum grease (Dow). Rou t i n e l y the septum of the i n j e c t i o n assembly, mounted to the f r o n t of the r o t a r y v a l v e , was changed once a week; i t could, i n any case, be used f o r at l e a s t 35-40 i n j e c t i o n s . P r e l i m i n a r y experiments were conducted to evaluate the use of methanol, ethanol, n- and isopropanol, n-, sec-, t e r t - and isobutanol and isoamyl a l c o h o l as i n t e r n a l standards for the e s t i m a t i o n of halothane and methoxy-- 22 -fluran e on one or more of the column packings Chromosorb 101, 103, 105, or 107 under e i t h e r isothermal or temperature-programmed c o n d i t i o n s . Isobutanol was found to be a cheap and convenient i n t e r n a l standard since i t could be r e a d i l y p u r i f i e d , was s o l u b l e i n blood at the concentrations employed and had a r e t e n t i o n time intermediate between that of halothane and methoxyflurane; i t al s o proved to be a good i n t e r n a l standard f or both ethanol and d i e t h y l ether. The use of a temperature programme r e s u l t e d i n the separation of the water peak from the halothane peak p e r m i t t i n g accurate measurements of peak area. Chromosorb 101 was the p r e f e r r e d packing m a t e r i a l since i t gave l i t t l e or no bleed and was very s t a b l e ; a column has been used with s a t i s f a c t o r y r e s u l t s f o r more than 1,000 i n j e c t i o n s over 2 years. Severe " t a i l i n g " of chromatographic peaks i s a sign of degradation of the column packing. New columns were used when t h i s occurred. Furthermore Chromosorb 101 can be employed, under the same operating c o n d i t i o n s , f or the r a p i d a n a l y s i s of halothane, methoxyflurane, ethanol and d i e t h y l e t h e r . Using the c o n d i t i o n s s p e c i f i e d above the system e x h i b i t e d l i t t l e or no b a s e l i n e d r i f t . Such d r i f t when i t occurred could be correct e d by a s i n g l e blank program run or by a short period of c o n d i t i o n i n g at 180°C. Table VI l i s t s the s e n s i t i v i t y of the flame i o n i s a t i o n detector and the performance c h a r a c t e r i s t i c s of the electrometer and i n t e g r a t o r when halothane, methoxyflurane, ethanol, d i e t h y l ether and isobutanol were analysed as described above. F i g . 4 shows sample chromatograms obtained from these analyses. Both heparin and disodium EDTA can be used as the antico a g u l a n t . The l a t t e r was used because i t was le s s expensive and more s t a b l e . Whole blood obtained from the Red Cross, when i n j e c t e d d i r e c t l y i n t o the gas chromato-Table VI Performance c h a r a c t e r i s t i c s of the flame i o n i z a t i o n detector, electrometer and e l e c t r o n i c i n t e g r a t o r Performance c h a r a c t e r i s t i c Isobutanol Halothane Methoxyflurane D i e t h y l ether Ethanol Concentration (mg%) 11 98 52 60 56 Volume of i n j e c t i o n ( u l ) 15 15 40 10 10 Detector output at maximum (A) 2.56 x 10-1° 3. .44 x 10-10 4. .18 x 10-10 6, .50 x 10-10 9, .61 x 10-10 Int e g r a t o r input at maximum (mV) (=electrometer output) 2.56 3. .44 4. ,18 6, .50 9, .61 Peak area (uV.sec) 7.422 x 1 0 4 1 .363 x 10 4 9. .543 x 10 4 1. .222 x 10 5 2. ,354 x 105 These performance c h a r a c t e r i s t i c data correspond to peaks A-E of the sample chromatograms i n F i g . 4 - 24 -1 1 I F W -A—A B V 1 i — . c w 1 1 1 1 I G 1 • i i i D 1 1 1 1 H l l l l l 1 i i i i i i 0 2 4 6 8 10 0 2 4 6 8 12 14 Time in Minutes F i g . 4. Sample chromatograms from the a n a l y s i s of halothane (A), methoxy-flura n e (B), d i e t h y l ether (c) and ethanol (D) usi n g , i n each case, i s o b u t a n o l (E, F, G, H) as i n t e r n a l standard. The water peak i n each chromatogram i s l a b e l l e d W. The v e r t i c a l l i n e s c r o s s i n g the base l i n e s are event markers a r i s i n g from the i n t e g r a t o r . - 25 -graph, gave rise to peaks which interfered with the isobutanol peak. Table VII shows the results obtained by analysis of blood samples containing halothane, methoxyflurane, diethylether and ethanol over the approximate concentration range 1-100 mg%. The anaesthetic concentrations of these samples were accurately known from the weight of the anaesthetics directly added. As can be seen excellent recoveries were obtained over the entire concentration range for a l l the compounds investigated. Reacti-vials proved to be efficient for storing blood samples containing halothane for periods as long as two weeks at 4°C. Such storage permits efficient staging of analyses when the experimental design requires multiple samplings. - 26 -Table VII Gas-liquid chromatographic analysis of whole blood samples containing known concentrations of halothane, methoxyflurane, diethyl ether and ethanol Compound added Halothane Methoxyflurane Diethyl ether Ethanol Response factor 0.207 + 0.008 0.242 + 0.009 0.924 + 0.001 0.797 + 0.024 (isobutanol • 1) ~~ Retention time (min)* 6.21 +0.20 12.31 +0.13 5.36 +0.15 3.21 +0.08 Relative retention time 0.697 1.397 0.608 0.382 (isobutanol • 1) Added Found Added Found Added Found Added Found (mgX) (agZ) (mg*) (mgl) (ngZ) (mgX) (mgX) (mgZ) 102.7 105.2 110.4 111.3 111.6 115.3 119.7 121.7 102.3 103.6 94.1 90.9 93.5 97.3 92.8 90.4 54.7 53.1 54.7 54.2 67.4 66.6 62.2 63.6 15.3 15.0 24.4 23.5 14.9 13.8 14.2 14.3 13.2 12.2 11.4 12.1 10.3 10.7 12.7 11.9 11.5 11.7 4.7 4.3 5.0 4.9 5.2 5.5 1.2 1.0 0.7 0.8 1.0 0.7 0.26 0.69 0.5 0.9 d.f.*** 12 4 4 3 N.S. N.S. N.S. N.S. After 1 week storage 18.8 17.8 After 2 weeks storage 18.4 18.6 *In these studies the retention times for isobutanol were 8.91 + 0.56, 8.81 + 0.56, 8.81 + 0.56 and 8.40 + 0.48 for analysis of halothane, methoxyflurane, diethyl ether and ethanol respectively. **Paired t test (Henry et a l . 1974). Differences between quantities added and found were not s i g n i f i c a n t l y different from rero. ***d.f. * degrees of freedom. N.S. • not si g n i f i c a n t DISCUSSION Comparison of the a n a l y t i c a l system described here with other d i r e c t i n j e c t i o n methods (see Table I I ) , i n d i c a t e s that i t appears to combine a l l the advantages of these systems without any of t h e i r disadvantages. Thus i t permitted the r a p i d , d i r e c t , q u a n t i t a t i v e a n a l y s i s of the blood concentrations of four v o l a t i l e compounds on a s t a b l e , r e a d i l y a v a i l a b l e column packing. None of the d i f f i c u l t i e s a s s o ciated w i t h b a s e l i n e d r i f t ( B u t l e r et a l . 1967; Cousins and Mazze 1972), ghost peaks (Kolmer et a l . 1975a; Cousins and Mazze 1972), i n t e r f e r e n c e from water (Kolmer et a l . 1975a; Cousins and Mazze 1972), poor r e p r o d u c i b i l i t y (Yamamura 1966; B u t l e r 1967), contamination of the columns w i t h n o n - v o l a t i l e components ( B u t l e r et a l . 1967; Kolmer et a l . 1975a; Cousins and Mazze 1972), and non-uniform evaporation which n e c e s s i t a t e d a preheating p e r i o d of the sample w i t h i n the pre-column device (Yokota et a l . 1967; Cole et a l . 1975) were encountered. The use of an i n t e r n a l standard coupled wit h the measurement of peak areas, rather than peak h e i g h t s , obviated the need f o r c a l i b r a t i o n curves and e l i m i n a t e d problems a s s o c i a t e d w i t h measurements of broadened or d i s t o r t e d peaks. The accuracy of the i n t e r n a l standard method i s v i r t u a l l y independent of the r e p r o d u c i b i l i t y of the a n a l y t i c a l c o n d i t i o n s , and i s recommended f o r q u a n t i t a t i v e a n a l y s i s at low concentrations ( E t t r e , 1967). Reference to Table VII demonstrates t h a t , w i t h the procedure described here accurate analyses can be performed over a wide range of concentrations. The e x t e r n a l i n j e c t i o n port can be e a s i l y constructed from inexpensive m a t e r i a l s and has proved to be r e l i a b l e and simple to operate f o r r o u t i n e purposes. I n s t a l l a t i o n r e q u i r e s a minimum of m o d i f i c a t i o n of e x i s t i n g gas chromatographs and the port can be r e a d i l y adapted f o r use w i t h other gas chromatographs. Furthermore the U-tube assembly appears to o f f e r d i s t i n c t advantages over previous systems (Yokota et a l . 1967; Cole et a l . 1975) since i t i s e a s i l y a c c e s s i b l e and can be r a p i d l y changed without i n t e r r u p t i n g the c a r r i e r gas flow. This i s a valuable feature when large numbers of samples have to be analysed, as exemp l i f i e d by the experiments described i n Parts I I and I I I of t h i s work. The range of s e n s i t i v i t y of t h i s method i s equal to or b e t t e r than other reported methods ( l i s t e d i n Tables I I , I I I and IV) i n which a flame i o n i z a t i o n detector was used f o r analysing anaesthetic concentrations i n blood. The recovery at approximately 10 mg/100 ml and 100 mg/100 ml was 92-106% and 97-104% r e s p e c t i v e l y . - 29 -PART I I - S o l u b i l i t y and D i s t r i b u t i o n of Halothane i n Human Blood: A Model  Study INTRODUCTION There i s l i t t l e known about the mechanism of the transport of i n h a l a t i o n general anaesthetic agents i n blood. Although there have been model studies of the i n t e r a c t i o n of anaesthetic agents w i t h both a r t i f i c i a l membranes ( H i l l 1974; M e t c a l f e and Burger 1968; T r u d e l l and Hubbell 1976; Koehler et a l . 1977) and haemoglobin and other p r o t e i n s ( M i l l a r et a l . 1971; Laasberg and Hedley-Whyte 1971; Schulman et a l . 1970; Hanisch et a l . 1969; Barker et a l . 1975), they do not provide q u a n t i t a t i v e i n f o r m a t i o n on the c o n t r i b u t i o n of the var i o u s components of blood to the transport of ana e s t h e t i c s . The mechanism of a c t i o n of general anaesthetics i s being i n t e n s i v e l y i n v e s t i g a t e d . Examination of the chemical s t r u c t u r e s of i n h a l a t i o n general anaesthetics (Table I ) showed that they l a c k a common s t r u c t u r a l s p e c i f i c i t y . This suggests that there i s no s p e c i f i c receptor at the s i t e of a c t i o n . M i l l e r et a l . (1972) found that the potency of i n h a l a t i o n anaesthetics was d i r e c t l y c o r r e l a t e d w i t h t h e i r s o l u b i l i t y i n an o l i v e o i l phase, and i t was suggested that the s i t e of a c t i o n i s at the l i p i d region of the membrane of the neuron. Using a l c o h o l s and erythrocyte membrane as models, i t was shown that anaesthetics caused membrane expansion (Seeman, 1972, 1974; Seeman et a l . 1969, 1971). A l s o , n-alkanes and n-alkanols were shown to increase the hydro-phobic thickness of an a r t i f i c i a l membrane (Haydon et a l . 1977). Expansion of the membrane may cause an increase i n the di s o r d e r of the membrane s t r u c t u r e , - 30 -which then a f f e c t s the p r o t e i n or l i p o p r o t e i n r esponsible f o r the transmission of nerve impulse. In the l a t e r a l phase separation hypothesis ( T r u d e l l , 1977), anaesthetics act by f l u i d i z i n g nerve membrane so that c r i t i c a l l i p i d regions no longer c o n t a i n the phase separations required to f a c i l i t a t e the conform-a t i o n a l changes of p r o t e i n s or l i p o p r o t e i n s necessary f o r membrane e x c i t a t i o n or the re l e a s e of t r a n s m i t t e r s to occur. F l u i d i z a t i o n of membrane i n the presence of anaesthetic molecules has been observed with d i f f e r e n t probes, i n c l u d i n g f i r e f l y luminescence (Ueda and Kamaya, 1973; Ueda et a l . 1974), NMR (Shieh et a l . 1975) and ESR ( T r u d e l l and Cohen, 1975). In a d d i t i o n , the con-cept of membrane expansion as a primary cause for anaesthesia i s supported by observations that a high ambient pressure was capable of r e v e r s i n g anaesthesia (Seeman, 1977). Presumably compression of the membrane reduces the increase i n f l u i d i t y due to the presence of anaesthetic molecules. Since current t h e o r i e s of the a c t i o n of anaesthetics favor an a n a e s t h e t i c -membrane i n t e r a c t i o n (Schneider 1968; M i l l e r et a l . 1973; Richards 1976; Halsey 1974; T r u d e l l 1974; Eger et a l . 1965; Saidman et a l . 1967; Hansch et a l . 1975; Haydon et a l . 1977), one might expect that a s i g n i f i c a n t p r o p o r t i o n of the anaesthetic agent would be transported by the red c e l l membrane. However, there has been no conclusive evidence f o r or against t h i s hypothesis, and as shown i n Table V I I I i t has been v a r i o u s l y reported that the s o l u b i l i t y of halothane i n blood has e i t h e r a p o s i t i v e or negative dependence on, or i s independent of, haematocrit. These apparently c o n t r a d i c t o r y r e s u l t s are probably due to the lack of both a systematic approach and of a p r e c i s e knowledge of the composition of the system examined. For example, i n most i n v e s t i g a t i o n s the concentrations - 3 1 -Table V I I I - Reported dependence of the s o l u b i l i t y of halothane on blood components Authors Species Blood Components Tested Results Mapleson et a l . 1972 Cowles et a l . 1971a Steward et a l . 1975 Larson et a l . 1962 Hans and H e l r i c h 1966 Lowe and Hagler 1969 Laasberg and Hedley-Whyte 1970 Cowles et a l . 1971a E l l i s and S t o e l t i n g 1975 Saraiva et a l . 1977a Munson et a l , 1978 Rabbit Hematocrit Dog Hemoglobin Hematocrit Dog Hematocrit Human Hemoglobin Human Hemoglobin Hematocrit Human Hematocrit Human Albumin y - g l o b u l i n Hemoglobin Human Hemoglobin Hematocrit Human Hematocrit Human Hemoglobin Albumin T o t a l serum p r o t e i n T r i g l y c e r i d e C h o l e s t e r o l G l o b u l i n Albumin/globulin r a t i o A l bumin/total p r o t e i n r a t i o Hematocrit Human T r i g l y c e r i d e p o s i t i v e dependence negative dependence* negative dependence* negative dependence* p o s i t i v e dependence p o s i t i v e dependence negative dependence negative dependence p o s i t i v e dependence negative dependence p o s i t i v e and negative dependence+ negative dependence negative dependence p o s i t i v e dependence negative negative negative p o s i t i v e p o s i t i v e p o s i t i v e negative negative dependence* dependence* dependence* dependence dependence* dependence* dependence* dependence* negative dependence* no dependence * S t a t i s t i c a l l y i n s i g n i f i c a n t +Maximum s o l u b i l i t y at normal when hemoglobin con c e n t r a t i o n hemoglobin concentration and lower s o l u b i l i t y i s higher or lower than normal of the plasma p r o t e i n s were unknown, although they are known (Chiou and Hsiao 1974) to i n f l u e n c e the s o l u b i l i t y of halothane i n plasma. Further, none of the studies d i s t i n g u i s h e d between absorption to the red c e l l membrane and adsorption to haemoglobin, and many were made on e i t h e r whole blood or plasma and do not provide i n f o r m a t i o n on the l o c a t i o n of anaesthetics w i t h i n the blood. Most of these studies are d i f f i c u l t to evaluate because they d i d not give s u f f i c i e n t d e t a i l s on the time taken to reach e q u i l i b r i u m , w i t h the exception of Laasberg and Hedley-Whyte (1970). The study of Saraiva et a l . (1977a) i s the most complete i n terms of the number of blood components i n v e s t i g a t e d and probably more r e l i a b l e due to the l a r g e r number of blood samples used. A l l of these studies (Table V I I I ) made use of the blood/gas p a r t i t i o n c o e f f i c i e n t or Ostwald S o l u b i l i t y C o e f f i c i e n t as an index of the s o l u b i l i t y of halothane i n the aqueous phase being examined. The blood/gas p a r t i t i o n coef-f i c i e n t of halothane i s defined as the r a t i o of the c o n c e n t r a t i o n of halothane i n blood to that i n a gas phase of f i x e d volume i n e q u i l i b r i u m w i t h the blood phase. P a r t i t i o n c o e f f i c i e n t of halothane = [Halothane] i n blood [Halothane] i n gas phase ^2] Thus a high blood/gas p a r t i t i o n c o e f f i c i e n t means a high s o l u b i l i t y of the anaesthetic i n blood. P a r t i t i o n c o e f f i c i e n t s are not l i m i t e d to gas and blood but extend to any 2 phases. Blood/gas, t i s s u e / b l o o d and o i l / g a s p a r t i t i o n c o e f f i c i e n t s are often used i n the theory of the uptake and d i s t r i b u t i o n of anaesthetics i n the body, and i n t h e o r i e s of the molecular mechanism of anaes-t h e s i a . As w i t h the blood/gas p a r t i t i o n c o e f f i c i e n t the t i s s u e / b l o o d and o i l / gas p a r t i t i o n c o e f f i c i e n t s describe the r e l a t i v e c apacity per u n i t volume of - 33 -each phase f o r the anaesthetic. Thus a p o s i t i v e (or negative) dependence of the s o l u b i l i t y of halothane on a blood component means that the p a r t i t i o n c o e f f i c i e n t of halothane increases (or decreases) when the conc e n t r a t i o n of that blood component i s increased (or decreased). Other terms describe the same or nearly the same e q u i l i b r i u m d i s t r i b u t i o n of anaesthetic between gas and l i q u i d phases. The Ostwald S o l u b i l i t y Coef-f i c i e n t i s defined as the volume of gas absorbed per u n i t volume of solvent at the temperature of the measurement when the p a r t i a l pressure of the d i s s o l v e d gas equals 1 atm. (760 mm Hg). The Ostwald S o l u b i l i t y C o e f f i c i e n t presumes that one phase i s pure gas, thus g i v i n g a f r a c t i o n a l c oncentration of 1. Since the gas phase i s always the denominator of the r a t i o and assigned a value of u n i t y , the Ostwald S o l u b i l i t y C o e f f i c i e n t n u m e r i c a l l y equals the p a r t i t i o n c o e f f i c i e n t . The t h e o r e t i c a l b a s i s for the p a r t i t i o n c o e f f i c i e n t i s Henry's Law which states t h a t , at e q u i l i b r i u m , the p a r t i a l pressure of a d i s s o l v e d gas i s p r o p o r t i o n a l to i t s concentration i n the solvent i n which i t i s d i s s o l v e d (Moore, 1962). Thus i f Henry's Law a p p l i e s to anaest h e t i c s , the p a r t i t i o n c o e f f i c i e n t would be a constant. O r i g i n a l l y Henry's Law was developed f o r the treatment of the behaviour of gas molecules d i s s o l v e d i n a homogeneous l i q u i d phase at i n f i n i t e d i l u t i o n . Since a l l the gas molecules d i s s o l v e d i n the homogeneous l i q u i d phase experience the same physicochemical environment at i n f i n i t e d i l u t i o n , the p a r t i a l pressure i s a measure of the a c t i v i t y of a l l gas molecules d i s s o l v e d i n the solvent (Moore, 1962). The concept of p a r t i a l pressure has gained widespread acceptance i n anaesthesiology. - 34 -The use of p a r t i a l pressure arose from the thermodynamic treatment of a s o l u t i o n c o n t a i n i n g a d i s s o l v e d v o l a t i l e substance. Chemical thermodynamic e q u i l i b r i u m at constant temperature and pressure i s defined i n terms of the fu n c t i o n G (Gibb's free energy). For a simple two-phase system, the d i f f e r e n c e i n the value of G f o r each phase i s a measurement of how f a r apart the two phases are from e q u i l i b r i u m . When the two phases are at e q u i l i b r i u m , the d i f f e r e n c e of the value of G f o r the two phases, AG, i s zero. G a p p l i e s to a phase or a system as a whole. Another f u n c t i o n derived from G, c a l l e d the chemical p o t e n t i a l , a p p l i e s to a s i n g l e chemical species. The chemical p o t e n t i a l G^ for the i t h chemical species, i s defined as f o l l o w s : where n. = number of moles of the i t h chemical species 1 When chemical e q u i l i b r i u m between two phases A and B i s reached at constant temperature and pressure, i . e . when AG = 0, i t can be proven (Moore, 1962) that the chemical p o t e n t i a l of any given chemical species i n the two phases are equal, or where G. = chemical p o t e n t i a l of the i t h chemical species i n phase A = chemical p o t e n t i a l of the i t h chemical species i n phase B Thus, while G i s used to describe the e q u i l i b r i u m between two macroscopic phases, G. i s the corresponding parameter for e q u i l i b r i u m between the molecules of a p a r t i c u l a r chemical species i n the two d i f f e r e n t phases. The d i f f e r e n c e i n the value of the chemical p o t e n t i a l G^ between the two phases i s a measure of how f a r apart the chemical species i n the two phases are from - 35 -e q u i l i b r i u m with each other. I f the chemical p o t e n t i a l of the i t h species i n ~A phase A, i . e . G^ i s l a r g e r than the corresponding chemical p o t e n t i a l -B . . . G^ i n phase B, there w i l l be a net flow of the i t h chemical species from phase A to phase B u n t i l the two chemical p o t e n t i a l s are equal. A chemical p o t e n t i a l as such cannot be e a s i l y measured. Therefore i t i s necessary to r e l a t e i t to another e a s i l y measurable q u a n t i t y . Such a r e l a t i o n s h i p i s a v a i l a b l e for a gas phase. I t can be shown (Moore 1962) t h a t , for a vapor such as halothane, i t s chemical p o t e n t i a l i s r e l a t e d to i t s p a r t i a l pressure as i n the f o l l o w i n g equation: % " ? H + R T l n PH where G„ = chemical p o t e n t i a l of halothane i n the gas phase H G° = standard state chemical p o t e n t i a l of halothane i n the gas phase. H This i s a constant, defined as the value of the chemical p o t e n t i a l when the halothane p a r t i a l pressure i s 1 atmosphere. P = P a r t i a l pressure of halothane. I t i s the pressure exerted by H halothane molecules i n the gas phase, independent of other molecules which may also be present. The above equation assumes the v a l i d i t y of the i d e a l gas law: PV = nRT where P = pressure V = volume n = number of moles of the gas or vapor R = u n i v e r s a l gas constant T = absolute temperature This i d e a l gas law i s accurate under common ambient c o n d i t i o n s . For example, the d e v i a t i o n of carbon d i o x i d e from i d e a l behaviour at 40°C and 1 atmosphere i s 0.1%, and at 10 atmosphere i s 1% (Moore, 1962). I d e a l l y , there should also be a corresponding equation r e l a t i n g the chemical p o t e n t i a l of halothane d i s s o l v e d i n blood or any other f l u i d s to a qu a n t i t y d i r e c t l y measurable i n the f l u i d . U n f o r t u n a t e l y , there has yet been no s a t i s f a c t o r y t h e o r e t i c a l treatment g i v i n g r i s e to such an equation. Thus, the chemical p o t e n t i a l of halothane i n s o l u t i o n has to be i n d i r e c t l y r e l a t e d to the chemical p o t e n t i a l of halothane vapor i n a gas phase i n e q u i l i b r i u m w i t h that i n s o l u t i o n . Since, as discussed above, the chemical p o t e n t i a l i s the same f o r a chemical species ( i n t h i s case halothane) i n any two phases ( i n t h i s case a gas phase and the aqueous phase) i n thermodynamic e q u i l i b r i u m , the chemical p o t e n t i a l c a l c u l a t e d from the p a r t i a l pressure of halothane i n the gas phase i s nu m e r i c a l l y equal to the chemical p o t e n t i a l of halothane i n the aqueous phase. This i s the b a s i s f or the use of p a r t i a l pressure to describe the behaviour of i n h a l a t i o n anaesthetics because there i s a one to one correspondence between the chemical p o t e n t i a l and the p a r t i a l pressure i n the gas phase. Chemical p o t e n t i a l i s a fundamental q u a n t i t y a p p l i c a b l e to a chemical species whether or not the system i s i n thermodynamic e q u i l i b r i u m . However, the use of p a r t i a l pressure as a p r a c t i c a l a l t e r n a t i v e to describe the behaviour of i n h a l a t i o n anaesthetics i n aqueous s o l u t i o n (or i n any medium for that matter) n e c e s s a r i l y implies the s o l u t i o n i s i n thermodynamic e q u i l i b r i u m w i t h a gas phase i n which the p a r t i a l pressure i s measured. I t i s meaningless to use p a r t i a l pressure under non-equilibrium c o n d i t i o n s . - 37 -Anaesthetic concentrations i n blood and other b i o l o g i c a l media are t r a d -i t i o n a l l y expressed i n terms of p a r t i a l pressures of the anaesthetic vapor i n e q u i l i b r i u m w i t h a f l u i d , r a t h e r than i n concentration u n i t s and the p a r t i t i o n c o e f f i c i e n t s are c a l c u l a t e d from these p a r t i a l pressure measurements. Attempts to c o r r e l a t e the p a r t i t i o n c o e f f i c i e n t with the conc e n t r a t i o n of d i f f e r e n t blood components give r i s e to the r e s u l t s summarized i n Table V I I I . However, when the solvent i s a multiphase, heterogeneous mixture, e.g. whole blood, the i n t e r p r e t a t i o n of the p a r t i a l pressure becomes more d i f f i c u l t . The same p a r t i a l pressure of an anaesthetic i s by d e f i n i t i o n i n e q u i l i b r i u m w i t h a l l the phases even when the concentrations of the anaesthetic i n the d i f f e r e n t phases are not the same. Thus the p a r t i a l pressure per se i s i n s u f f i c i e n t to describe the q u a n t i t a t i v e d i s t r i b u t i o n of an anaesthetic i n the d i f f e r e n t phases of blood. Therefore, to study the q u a n t i t a t i v e c o n t r i b u t i o n s of the d i f f e r e n t blood components to the s o l u b i l i t y of halothane i n blood and to construct a model fo r the mode of i t s t r a n s p o r t , a d i f f e r e n t methodology i s necessary. E q u i l -i b r i u m d i a l y s i s was chosen f o r t h i s purpose. In the technique to be described, an aqueous s o l u t i o n of the blood component was placed i n one compartment and an aqueous s o l u t i o n of halothane was placed i n another compartment. The two compartments were separated by a semi-permeable membrane which allowed a small molecule (such as halothane) but not a macromolecule (such as the blood components used i n t h i s study) to pass through. During the d i a l y s i s , there i s a net d i f f u s i o n of halothane molecules i n t o the compartment w i t h the blood component u n t i l the chemical p o t e n t i a l s of halothane i n the two compartments are equal ( i . e . at e q u i l i b r i u m ) . When e q u i l i b r i u m i s reached, the number of - 38 -halothane molecules bound per molecule of the blood component i s given by the f o l l o w i n g equation ( M a r s h a l l , 1978, Sophianopoulos et a l . 1978): Number of halothane molecules = Number of molecules of blood component [Halothane].; - [Halothane] 0 x MW of blood component [Blood component] MW of halothane where [Halothane] = halothane concentration (g/ml) i n the compartment o without the blood component [Halothane]^ = halothane concentration (g/ml) i n the compartment with the blood component [Blood Component] = concentration of the blood component (g/ml) I f the concentration of the blood component and concentration of halothane i n the two compartments are known, the number of halothane molecules bound per molecule of the blood component can be c a l c u l a t e d . This equation does not take i n t o account the volume occupied by the blood component. To take t h i s i n t o account, l e t Q be the f r a c t i o n of the volume occupied by the blood component i n i t s compartment. Then 1-Q i s the e f f e c t i v e f r a c t i o n of the volume a v a i l a b l e f o r halothane i n the compartment with the blood component. The experimentally determined concentrations i n the compartment with the blood component are correct e d by d i v i d i n g w i t h 1-Q, and the above equation becomes: Number of halothane molecules  Number of molecules of blood component = [Halothane] n- - [ H a l o t h a n e ] Q 1-Q x MW of blood component [Blood component] MW of halothane 1-Q The a n a l y t i c a l method developed i n Part I of t h i s work was used for the q u a n t i t a t i v e determination of halothane concentrations i n these d i a l y s i s - 39 -experiments. D e t a i l e d and unambiguous r e s u l t s were obtained f o r the q u a n t i t a t i v e i n t e r a c t i o n s of halothane w i t h haemoglobin, red c e l l ghosts, albumin (both f a t t y a c i d f r e e and i n the presence of o l e i c a c i d ) , y - g l o b u l i n and t r i g l y c e r i d e - r i c h m i c e l l e s (chylomicrons and very low d e n s i t y l i p o p r o t e i n s ) . These r e s u l t s were a l s o used to c a l c u l a t e the d i s t r i b u t i o n of halothane between c e l l s and plasma and were compared to those obtained by an independent method, i n which whole blood samples c o n t a i n i n g d i s s o l v e d halothane, and the corresponding plasma samples obtained by c e n t r i f u g a t i o n , were analysed f o r halothane concentration. Comparison of the r e s u l t s obtained by these two d i f f e r e n t methods provides a t e s t for the proposed model of d i s t r i b u t i o n and t r a n s p o r t . - 40 -MATERIALS S i l i c o n e rubber septa and 25 ml screw cap v i a l s were obtained from P i e r c e Chemical Co., Rockford, 111., U.S.A. These v i a l s were supplied w i t h g a s - t i g h t t e f l o n l i n e d seals and served as leak proof containers f o r halothane. Gas-t i g h t syringes were obtained from Hamilton Co., Reno, Nevada, U.S.A. Amicon CF50 and CF25 membrane u l t r a f i l t e r s were obtained from Amicon Corp., Lexington, Mass. A l l the reagents f o r t r i g l y c e r i d e assay were obtained from Hycel Inc., Houston, Texas, U.S.A. 3,3',5,5 1-tetrabromo-m-cresolsulfonphthalein were obtained from American Monitor Corp., I n d i a n a p o l i s , Indiana, U.S.A. Human Y - g l o b u l i n , f a t t y a c i d free human albumin ( l e s s than 0.005% f a t ) and d i a l y s i s tubings were obtained from Sigma Chemical Co., St. L o u i s , Mo., U.S.A. A l l other organic and ino r g a n i c chemicals used were of reagent grade from F i s c h e r S c i e n t i f i c Co., Montreal, Canada. - 41 -METHODS 1. A n a l y t i c a l Methods Haemoglobin was assayed by the cyanohaemoglobin procedure (Van Kampen and Z i j l s t r a 1961), albumin by the method of Miyada et a l . (1972) and t r i g l y c e r i d e by the technique of Schmidt and Von Dahl (1968). Since anticoagulants may i n t e r f e r e w i t h the t r i g l y c e r i d e assay, e s t i m a t i o n of albumin and t r i g l y c e r i d e was performed on plasma samples prepared from blood samples taken with a p l a s t i c syringe and without added anticoagulant. The blood sample so obtained was immediately c e n t r i f u g e d i n p l a s t i c Eppendorf c e n t r i f u g e tubes at 10,000 g for 15 sec. These plasma samples w i l l not form v i s i b l e f i b r i n c l o t s f o r at l e a s t 48 hrs at 4°C. Presumably most of the f i b r i n c l o t s i n i t i a l l y formed were sedimented along w i t h the c e l l s . Red c e l l s and red c e l l ghosts were counted i n a Neubauer counting chamber (Dacie and Lewis 1968), i n the l a t t e r case using a phase c o n t r a s t microscope. Disodium EDTA (0.2% w/v) was used as the anticoagulant whenever necessary. Halothane concentrations were determined by g a s - l i q u i d chromatography as described i n Part I of t h i s work. 2. Studies of the Binding of Halothane to Blood Components a. S a t u r a t i o n c o n c e n t r a t i o n of halothane i n s a l i n e S a l i n e (0.85% w/v NaCl) was c a r e f u l l y layered onto a bulk phase of h a l o -thane contained i n R e a c t i - v i a l s such that the two phases occupied an approxi-mately equal volume. The v i a l s were sealed by d i s p l a c i n g excess s a l i n e i n such a manner that no a i r bubbles were trapped i n the v i a l and they were then - 42 -e q u i l i b r a t e d at e i t h e r 4 or 37°C without mixing f o r up to 98 and 168 hours, r e s p e c t i v e l y . Since the two phases were not mixed, the p o s s i b i l i t y of h a l o -thane forming m i c r o - d r o p l e t s , which would lead to erroneous high value for the s a t u r a t i o n c o n c e n t r a t i o n , was e l i m i n a t e d . E q u i l i b r i u m concentrations were determined by g a s - l i q u i d chromatography. b. Haemoglobin Red blood c e l l s were obtained from outdated Red Cross blood (not more than 1 month o l d ) by c e n t r i f u g a t i o n at 20,000 rpm f o r 20 min i n a S o r v a l l SS-34 r o t o r (max. 48,000 g). They were washed f i v e times i n four times t h e i r own volume of 150 mM sodium c h l o r i d e and were then haemolysed by the a d d i t i o n of an approximately equal volume of d i s t i l l e d water. The s o l u t i o n was c e n t r i -fuged as above and the supernatant recovered. A f t e r f i v e r e p e t i t i o n s of t h i s process the ghost free s o l u t i o n was d i a l y s e d twice for 4 and 16 hr respec-t i v e l y , against ten volumes of PBS (phosphate b u f f e r s a l i n e : pH 7.4, 0.01 M sodium phosphate, 150 mM sodium c h l o r i d e and 0.02% (w/v) sodium a z i d e ) . The r e s u l t i n g haemoglobin s o l u t i o n was c e n t r i f u g e d as above and the supernatant used f o r e q u i l i b r i u m d i a l y s i s . More than 95% of the p r o t e i n i n t h i s s o l u t i o n was haemoglobin (Wintrobe et a l . 1974) at a concentration of 9.8 g/100 ml. The concentration of 2,3 diphosphoglycerate would not be more than 0.4 mM. c. Albumin F a t t y a c i d - f r e e human albumin was d i s s o l v e d i n PBS, e i t h e r alone or together with o l e i c a c i d . The f i n a l concentration of albumin was a p p r o x i -mately 2.3 g/100 ml. The o l e i c a c i d concentration used was approximately 14 - 43 -mg/100 ml, which i s w i t h i n the normal range of u n e s t e r i f i e d f a t t y a c i d i n the plasma (Henry et a l . 1974). d. y - g l o b u l i n Human Y-gl°bulin d i s s o l v e d i n PBS at a f i n a l c o ncentration of ap p r o x i -mately 0.7 g/100 ml was used f o r d i a l y s i s against halothane. e. T r i g l y c e r i d e - r i c h m i c e l l e s These were prepared by a t e c h n i c a l m o d i f i c a t i o n of the method of Hatch and Lees (1968). Blood (50 m l ) , taken from n o n - f a s t i n g h e a l t h y donors and a n t i -coagulated w i t h EDTA, was c e n t r i f u g e d at approximately 1,000 g f o r 5 min. A l i q u o t s (approximately 4 ml) of the plasma were loaded i n t o 6 polyallomer tubes. Seven ml of a s o l u t i o n with a density of 1.006 g/ml (prepared by d i s s o l v i n g 11.40 g of NaCl, 0.1 g disodium EDTA and 1 ml of 1 N NaOH i n a t o t a l volume of 1.003 1 of water) was layered on the plasma samples w i t h a glass p i p e t t e , by a l l o w i n g i t to run continuously and slowly down the sides of the s l i g h t l y i n c l i n e d tubes. The p i p e t t e t i p was maintained at about 1 cm above the l i q u i d l e v e l i n the tubes to avoid d r i p p i n g and turbulence. The loaded tubes were c e n t r i f u g e d i n a Beckman SW 41 r o t o r at 40,000 rpm (min. 120,000 g, max. 280,000 g) at 16 to 18°C for 16 hours. One to three mis of the white suspension of t r i g l y c e r i d e - r i c h m i c e l l e s (chylomicrons and very low dens i t y l i p o p r o t e i n ) was taken from the top of each tube, pooled, and d i a l y s e d at 4°C against 1.0 1 po r t i o n s of d i s t i l l e d water for 4 and 16 hr r e s p e c t i v e l y . A sample of the suspension was taken f o r t r i g l y c e r i d e assay, and to the remainder was added e x a c t l y 1/9 i t s volume of a 10 x PBS b u f f e r c ontaining 1% (w/v) disodium EDTA. - 44 -f. Red c e l l ghosts suspension Ghosts were prepared from out dated Red Cross blood (not more than 1 month old) and resealed by warming as described by Theodore and Kant (1974). The resealed ghosts were washed twice and resuspended i n an approximately equal volume of PBS. g. D i a l y s i s of Blood Components against Halothane The apparatus employed i s shown i n F i g . 5. A d i s c of c e l l u l o s e d i a l y s i s membrane was c a r e f u l l y placed on top of a 0.3 ml R e a c t i - v i a l completely f i l l e d w i t h a s o l u t i o n or suspension of the blood component to be d i a l y s e d . The d i s c was sealed, f i n g e r t i g h t , by means of an 'O'-ring (cut from a s i l i c o n e rubber septum commercially a v a i l a b l e f o r these v i a l s ) f i t t e d i n t o the cap of the R e a c t i - v i a l . A strong rubber band was fastened l o n g i t u d i n a l l y outside the 25 ml v i a l and tw i s t e d so that i t tended to t i g h t e n the screw cap i n a clockwise d i r e c t i o n (see F i g . 5). This prevented the caps from loosening during d i a l y s i s . The v i a l s were incubated for v a r i o u s time i n t e r v a l s at 37°C w i t h s o l u t i o n s of halothane i n PBS contained i n completely f i l l e d , sealed, 25 ml screw cap v i a l s . When red c e l l ghosts, y g l o b u l i n and t r i g l y c e r i d e - r i c h m i c e l l e s were being i n v e s t i g a t e d , the outer 25 ml v i a l was r o t a t e d end to end at 10-30 r.p.m. On completion of the d i a l y s i s samples were taken from each v i a l f o r the e s t i m a t i o n of the concentration of halothane. In those experiments where the free halothane concentration was h e l d constant and the t r i g l y c e r i d e concentration v a r i e d , a l l the 0.3 ml R e a c t i -v i a l s c o n t a i n i n g d i f f e r e n t concentrations of t r i g l y c e r i d e were placed i n t o one 500 ml reagent b o t t l e . A ga s - t i g h t s e a l f o r t h i s b o t t l e was made by c u t t i n g a - 45 -Fig. 5. Diagrammatic representation of the equilibrium dialysis assembly, (a) Cross section, (b) Side view. - 46 -c i r c u l a r piece of T e f l o n (PTFE, Dupont) to f i t the i n s i d e of the p l a s t i c screw cap. Otherwise the procedures were i d e n t i c a l to the other d i a l y s i s experiments. The number of halothane molecules bound per p r o t e i n molecule was c a l c u l a t e d using the f o l l o w i n g formula: [Halothane]! _ [Halothane]o Number of Halothane Molecules = 1-VjProtein] X MW of p r o t e i n Number of P r o t e i n Molecules [ P r o t e i n ] MW of Halothane 1-VTProtein] .....[4] where [Halothane]! = Halothane concentration (g/ml) i n s i d e the 0.3 ml R e a c t i -v i a l [Halothane]o = Halothane concentration (g/ml) outside the 0.3 ml R e a c t i -v i a l [ P r o t e i n ] = P r o t e i n concentration (g/ml) V = P a r t i a l s p e c i f i c volume of the p r o t e i n For haemoglobin V" = 0.749 ml/g MW = 64,500 (Altman and Dittmer 1972) For albumin V" = 0.733 ml/g MW = 66,200 (Peters J r . 1975) The number of halothane molecules per red c e l l ghost was c a l c u l a t e d using the f o l l o w i n g formula: iMlothSBiJi - [Halothane ]o Number of Halothane Molecules = 1-KC x N Number of Red C e l l Ghosts r. C_ MW of Halothane 1-KC where C = number of ghosts per u n i t volume ^ K - volume of one ghost, approximated by the product of the thickness and surface area of the ghost membrane, taken to be 2.4 nm ( F e t t i p l a c e - 47 -2 et a l . 1971) and 136.9 um (Jay 1975) r e s p e c t i v e l y . N = Avogadro's number The number of grams of halothane per gram of t r i g l y c e r i d e was c a l c u l a t e d using the f o l l o w i n g formula: Wt. of Halothane = [Halothane]i - [Halothane]o [6] Wt. of T r i g l y c e r i d e [ T r i g l y c e r i d e ] 3. D i s t r i b u t i o n of Halothane between C e l l s and Plasma A 10 ml g a s - t i g h t syringe f i t t e d with a cap (made by breaking o f f the needle p o r t i o n of a Luer Lok syringe needle and s e a l i n g the opening with solder) was weighed without the plunger and then f i l l e d w i t h approximately 10 ml blood (anti-coagulated with EDTA) from a non- f a s t i n g healthy donor and reweighed. An a c c u r a t e l y known volume ( l e s s than 1 ml) of a normal s a l i n e s o l u t i o n saturated w i t h halothane at room temperature was added, to give a f i n a l halothane concentration of approximately 40, 20 or 5 ml/100 ml, and the plunger was immediately i n s e r t e d with the needle end of the syringe downward. The syringe was then i n v e r t e d , the cap removed, and the plunger was pressed to expel most of the a i r without l o s i n g any s i g n i f i c a n t amount of blood. The syringe was then resealed with the cap and r o t a t e d end to end at 10-30 r.p.m. for 30 min. S i x 1 ml R e a c t i - v i a l s were f i l l e d w i t h blood from t h i s syringe as described i n Part I , and were e q u i l i b r a t e d by r o t a t i n g very s l o w l y at 37°C. At the end of the e q u i l i b r a t i o n , four of the v i a l s were c e n t r i f u g e d i n a S o r v a l l HB-4 r o t o r at 4,500 rpm (max. 2,870 g) f o r 15 min, which was s u f f i c i e n t to sediment almost a l l the p l a t e l e t s (Schauberge et a l . 1976). Plasma samples were then taken for a n a l y s i s of halothane concentration. Blood samples from the same set were a l s o analysed f o r halothane concentration before and a f t e r the e q u i l i b r a t i o n . - 48 -Blood samples with a r t i f i c i a l l y low and elevated triglyceride concen-trations were prepared (see flow chart) by separating cells and plasma and then centrifuging the latter in a Beckman SW 41 rotor at 41,000 rpm (min. 130,000 g, max. 290,000 g) for 4 hr. The f i r s t and the last of the 11 ml in each centrifuge tube was discarded, and the remaining plasma was reconstituted with the cells to give a haematocrit of 50. This triglyceride-poor blood sample was divided into two 10 ml portions. To one of these was added approx-imately 0.4 ml of a highly concentrated triglyceride preparation.(obtained by concentrating the triglyceride-rich micelle fraction from 25 ml of plasma to 0.5 ml using Amicon CF 50 membrane ul t r a f i l t e r s ) to yield a triglyceride-rich blood sample. To the other, triglyceride-poor sample, was added an equal amount of saline. One ml aliquots were taken from each of the triglyceride-rich and triglyceride-poor samples for the determination of triglyceride concentration. Two plasma samples obtained from these two blood aliquots were further divided into two sets of triglyceride-rich and triglyceride-poor plasma samples. They were washed with saline on Amicon CF 25 membrane u l t r a f i l t e r s to dilute the concentration of EDTA to negligible quantities. One set was washed 5 times and the other 8 times with 5 ml aliquots. A l l the f i l t r a t e s from the 1st wash and the four final retentates were then assayed for triglyceride. The remaining triglyceride-rich and triglyceride-poor blood samples were used for the study of the distribution of halothane between cells and plasma as described above. The distribution of halothane between cells and plasma was calculated from the following equations: - 49 -Flow chart of the procedure f o r the p r e p a r a t i o n of t r i g l y c e r i d e - r i c h and  t r i g l y c e r i d e ^ p o o r blood samples Blood (50 ml) a n t i c o a g u l a t e d w i t h EDTA c e n t r i f u g e 1,000 g 5 min Plasma C e l l s C e n t r i f u g e 4 hr 130,000-290,000 g D i s c a r d top 1 ml 'Discard bottom 1 ml Remaining Plasma-Blood (10 ml) r e c o n s t i t u t e Blood (10 ml) Add 0.4 ml concentrated t r i g l y c e r i d e - r i c h m i c e l l e preparation-Tr i g l y c e r i d e - r i c h blood sample blood (1 ml) c e n t r i f u g e 1,000 g 5 min d i s c a r d c e l l s wash 5 x with s a l i n e on Amicon CF25 f i l t e r Retentate T r i g l y c e r i d e - p o o r blood sample blood (1 ml) same treatment as f o r the cor-responding t r i -g l y c e r i d e - r i c h sample f o r the study of the d i s t r i b u t i o n of halothane between c e l l s and plasma plasma F i l t r a t e ^ j f 1st wash wash 8 x with s a l i n e on Amicon CF 25 f i l t e r F i l t r a t e of 1st wash Retentate 1 Assay of t r i g l y c e r i d e c o n c e n t r a t i o n [Halothanejb = _Hc [Halothane]c + (1 - He)[Halothane]p [7] 100 100 Percent of halothane i n plasma = (100-Hc)[Halothane]p [8] [Halothanejb Percent o f halothane i n ce l i s = He [Halothane ]c [9] [Halothane]b where [Halothanejb = experimentally determined halothane c o n c e n t r a t i o n (mg/100 m l ) i n blood [Halothane]p = experimentally determined halothane concentration (mg/100 [Halothane]c = halothane concentration i n c e l l s He = 100 x volume f r a c t i o n occupied by c e l l s RESULTS 1. S a t u r a t i o n c o n c e n t r a t i o n of halothane i n s a l i n e As shown i n Table IX, the concentration of halothane i n s a l i n e (0.85% w/v NaCl) e q u i l i b r a t e d w i t h a bulk phase of halothane at 37°C increased w i t h time up to 43 hr, a f t e r which there was no f u r t h e r detectable in c r e a s e . S i m i l a r l y , the concentration of halothane i n s a l i n e e q u i l i b r a t e d w i t h a bulk phase of halothane at 4°C showed no detectable increase a f t e r 72 h r. Using the average of the l a s t 5 values for 37°C and the 4 values for 4°C i n Table IX, the s a t u r a t i o n concentration of halothane i n s a l i n e was found to be 297 + 8 mg/100 ml and 481 + 12 mg/100 ml r e s p e c t i v e l y . 2. Adsorption of halothane to haemoglobin Table X shows the r e s u l t s of p r e l i m i n a r y experiments performed to estab-l i s h the time required to achieve e q u i l i b r i u m . I t can be seen that when the free halothane concentration was approximately 15 mg/100 ml, the average number of halothane molecules per haemoglobin molecule increased w i t h time up to 68 hr. When the free halothane concentration was approximately 300 mg/100 ml, there was no s i g n i f i c a n t change i n the average number of halothane molecules per haemoglobin molecule a f t e r 73 hr. Therefore 92 hr was chosen as the e q u i l i b r a t i o n time to ensure that e q u i l i b r i u m was reached. F i g . 6 shows the e q u i l i b r i u m d i a l y s i s r e s u l t s . Each data point represents one independent d i a l y s i s experiment. The haemoglobin concentration was 9.8 g/100 ml. The average number of halothane molecules bound to each haemoglobin molecule increases from 0 to approximately 5 with i n c r e a s i n g free halothane Table IX - Concentration of halothane i n s a l i n e e q u i l i b r a t e d with a bulk phase of halothane at d i f f e r e n t e q u i l i b r a t i o n time Samples of s a l i n e e q u i l i b r a t e d with a bulk phase of halothane f o r d i f -f erent time i n t e r v a l s were analyzed f o r halothane concentrations to e s t a b l i s h the time taken to reach e q u i l i b r i u m and the s a t u r a t i o n concentration of halothane i n s a l i n e 37°C 4°C E q u i l i b r a t i o n Time Concentration E q u i l i b r a t i o n Time Concentration (hr) (mg/100 ml) (hr) (mg/100 ml) 19.0 180 21.5 218 43.0 299 45.5 288 72 501 67.0 291 74 478 69.5 306 96 477 168.0 303 98 468 Table X - Time r e q u i r e d to reach e q u i l i b r i u m f or the d i a l y s i s of haemoglobin against halothane Haemoglobin s o l u t i o n s were d i a l y s e d against halothane f o r d i f f e r e n t time i n t e r v a l s . Samples of the b u f f e r containing f r e e halothane and samples of the haemoglobin s o l u t i o n c o n t a i n i n g free and bound halothane were then analyzed f o r halothane c o n c e n t r a t i o n to e s t a b l i s h the time required to reach e q u i l i b r i u m E q u i l i b r a t i o n Time (hr) Free [Halothane] i n b u f f e r mg/100 ml Average number of halothane molecules per hemoglobin molecule 22 14.2 0.06 44 14.3 0.1 48 15.8 0.1 68 12.2 0.2 92 13.6 0.2 117 16.5 0.2 73 301 5.1 144 304 4.7 - 54 -6r Fig. 6. Adsorption of halothane to haemoglobin. - 55 -concentration from 0 to s a t u r a t i o n . The slope of the curve g r a d u a l l y decreases with i n c r e a s i n g free halothane c o n c e n t r a t i o n , but i t does not l e v e l o f f to a plateau when the s a t u r a t i o n c oncentration of halothane i s reached. L i n e a r r e g r e s s i o n on those data p o i n t s obtained with a free halothane concentration of l e s s than 150 mg/100 ml gives the f o l l o w i n g equation: H h = 0.0223[Halothane]f - 0.119 [10] where H^ = number of halothane molecules bound per haemoglobin molecule [Halothane]f = f r e e halothane concentration i n mg/100 ml The r e s u l t s were al s o analysed by the Scatchard method (M a r s h a l l 1978), as shown i n F i g . 7. L i n e a r r e g r e s s i o n of the r e s u l t s obtained from e q u i l i b r i u m d i a l y s i s w i t h H^ l a r g e r than 1 using the Scatchard equation gives a t o t a l number of 20 HH 6 s i t e s (95% confidence l i m i t ) (Snedecor and Cochran 1967) f o r halothane on each haemoglobin molecule as the X - i n t e r c e p t of the f o l l o w i n g equation: , H h . = -1.08 x 10-3 H h + 2.20 x 10"2 [Halothane]f 3. Adsorption of halothane to albumin Table XI shows the r e s u l t s of p r e l i m i n a r y experiments performed to e s t a b l i s h the time r e q u i r e d to achieve e q u i l i b r i u m . When the free halothane concentration was approximately 190 mg/100 ml, there was no s i g n i f i c a n t change i n the average number of halothane molecules per albumin molecule a f t e r 96 hr. This was th e r e f o r e chosen as the e q u i l i b r a t i o n time. F i g . 8 shows the e q u i l i b r i u m d i a l y s i s r e s u l t s . The curve obtained w i t h albumin was q u a l i t a t i v e l y s i m i l a r to that f o r haemoglobin ( F i g . 6) except that - 56 -= number of halothane molecules bound per haemoglobin molecule [ H a l o t h a n e ] f = f r e e halothane c o n c e n t r a t i o n i n mg/100 ml Fig. 7. Scatchard plot of halothane binding to haemoglobin. Table XI - Time r e q u i r e d to reach e q u i l i b r i u m f or the d i a l y s i s of albumin against halothane Albumin s o l u t i o n s were d i a l y s e d against halothane f o r d i f f e r e n t time i n t e r v a l s . Samples of the b u f f e r containing free halothane and samples of the albumin s o l u t i o n c o n t a i n i n g f r e e and bound halothane were analysed f o r halothane concentration to e s t a b l i s h the time required to reach e q u i l i b r i u m E q u i l i b r a t i o n Time Free [Halothane] i n b u f f e r Average number of halothane (hr) mg/100 ml molecule per albumin molecule 68 190 9.7 96 174 14.9 114 176 16.6 U l 118 192 14.9 - 58 -20r HALOTHANE (MG/100 ML) F i g . 8. Adsorption of halothane to albumin. - 59 -the average number of halothane molecules bound to each albumin molecule i s much hig h e r , reaching approximately 20 when the b u f f e r was saturated w i t h halothane. The presence of p h y s i o l o g i c a l concentrations of o l e i c a c i d had no detectable e f f e c t on the i n t e r a c t i o n of halothane w i t h albumin. L i n e a r r e g r e s s i o n on those data p o i n t s obtained w i t h a free halothane c o n c e n t r a t i o n l e s s than 150 mg/100 ml gave the f o l l o w i n g equation: H = 0.0830[Halothane]f - 0.0294 [11] SL where H = number of halothane molecules bound per albumin molecule a r [Halothane]f = free halothane concentration i n mg/100 ml Line a r r e g r e s s i o n of the r e s u l t s w i t h H l a r g e r than 4 usi n g Scatchard cl equation ( F i g . 9) (Ma r s h a l l 1978) gives a t o t a l number of 130 + 29 s i t e s (95% confidence l i m i t ) (Snedecor and Cochran 1967) for halothane on each albumin molecule as the X - i n t e r c e p t of the f o l l o w i n g equation: H a = -6.95 x I t ) - * H a + 9.07 x 1 0 - 2 [Halothane]f 4. y - g l o b u l i n Table X I I shows the r e s u l t s of the d i a l y s i s of y - g l o b u l i n against h a l o -thane. There was no detectable d i f f e r e n c e between the fr e e halothane concen-t r a t i o n and the halothane concentration i n the presence of y - g l o b u l i n . This i n d i c a t e s that there was no s i g n i f i c a n t adsorption of halothane to y - g l o b u l i n at the p h y s i o l o g i c a l y - g l o b u l i n concentration employed. 5. Absorption of halothane to red c e l l ghosts Table X I I I shows the r e s u l t s of p r e l i m i n a r y experiments performed to e s t a b l i s h the time r e q u i r e d to achieve e q u i l i b r i u m . When the fr e e halothane - 60 -H [Halothane] 0 - 1 0 r 0 0 0 5 • • 1 0 1 5 H H & = number of halothane molecules bound per albumin molecule [Halothane]f = free halothane concentration in mg/100 ml F i g . 9. Scatchard p l o t of halothane b i n d i n g to albumin. Table X I I - Results of the d i a l y s i s of y - g l o b u l i n against halothane at d i f f e r e n t e q u i l i b r a t i o n time y - g l o b u l i n s o l u t i o n s were d i a l y s e d against halothane f o r d i f f e r e n t time i n t e r v a l s . Samples of the b u f f e r containing free halothane and samples of the y - g l o b u l i n s o l u t i o n were then analysed f o r halothane concentrations to e s t a b l i s h whether or not detectable b i n d i n g of halothane to y - g l o b u l i n occurred E q u i l i b r a t i o n Time (hr) Free [Halothane] i n b u f f e r mg/100 ml [Halothane] i n y - g l o b u l i n s o l u t i o n (mg/100 ml) 72 19.7 20.0 96 24.4 24.7 120 27.6 27.5 Table X I I I - Time r e q u i r e d to reach e q u i l i b r i u m for the d i a l y s i s of red c e l l ghosts against halothane Red c e l l ghosts suspensions were d i a l y s e d against halothane f o r d i f f e r e n t time i n t e r v a l s . Samples of the b u f f e r containing free halothane and samples of the red c e l l ghosts suspension containing f r e e and bound halothane were then analysed f o r halothane concentration to e s t a b l i s h the time required to reach e q u i l i b r i u m E q u i l i b r a t i o n Time Free [Halothane] i n b u f f e r [Halothane] i n ghosts suspension (hr) mg/100 ml (mg/100 ml) 20 135 142 44 127 168 68 128 166 c o n c e n t r a t i o n was approximately 130 mg/100 ml, there was no s i g n i f i c a n t change i n the halothane concentration i n the ghost suspension a f t e r 44 hr. This was chosen as the e q u i l i b r a t i o n time. F i g . 10 shows the e q u i l i b r i u m d i a l y s i s r e s u l t s . In con t r a s t to the r e s u l t s obtained w i t h haemoglobin and albumin the slopes of the curve f o r red c e l l ghosts increased w i t h i n c r e a s i n g halothane c o n c e n t r a t i o n . L i n e a r r e g r e s s i o n on the data p o i n t s obtained w i t h a f r e e halothane c o n c e n t r a t i o n of l e s s than 150 mg/100 ml gave the f o l l o w i n g equation: H = 6.28 x 10 6[Halothane]f + 3.15 x 10 7 [12] g where H^ = number of halothane molecules s o l u b i l i z e d per red c e l l ghost [Halothane]f = free halothane concentration i n mg/100 ml 6. Absorption of halothane to t r i g l y c e r i d e - r i c h m i c e l l e s (chylomicrons and  very low den s i t y l i p o p r o t e i n s ) Table XIV shows the r e s u l t s of p r e l i m i n a r y experiments performed to e s t a b l i s h the time required to achieve e q u i l i b r i u m . When the f r e e halothane concentration was approximately 30 mg/100 ml, there was no s i g n i f i c a n t change i n the halothane concentration i n the t r i g l y c e r i d e - r i c h m i c e l l e s suspension a f t e r 47 hr. This was chosen as the e q u i l i b r a t i o n time. F i g . 11 shows the e q u i l i b r i u m d i a l y s i s r e s u l t s when the t r i g l y c e r i d e c o ncentration was kept constant at 83 mg/100 ml. The slope of the curve increased with i n c r e a s i n g free halothane c o n c e n t r a t i o n , i n a s i m i l a r manner to that found f o r red c e l l ghosts ( F i g . 10), but the change i n slope was even more pronounced with the t r i g l y c e r i d e m i c e l l e s . L i n e a r r e g r e s s i o n of those data p o i n t s obtained w i t h free halothane concentration of l e s s than 150 mg/100 ml gave the f o l l o w i n g equation: - 64 -Fig. 10. Absorption of halothane to red cell ghosts. Table XIV - Time re q u i r e d to reach e q u i l i b r i u m for the d i a l y s i s of t r i g l y c e r i d e - r i c h m i c e l l e s against halothane T r i g l y c e r i d e - r i c h m i c e l l e suspensions were d i a l y s e d against halothane f o r d i f f e r e n t time i n t e r v a l s . Samples of the b u f f e r c o n t a i n i n g f r e e halothane and samples of the t r i g l y c e r i d e - r i c h m i c e l l e suspensions containing f r e e and bound halothane were than analysed f o r halothane concentration to e s t a b l i s h the time r e q u i r e d to reach e q u i l i b r i u m E q u i l i b r a t i o n Time Free [Halothane] i n b u f f e r [Halothane] i n t r i g l y c e r i d e - r i c h (hr) mg/100 ml m i c e l l e suspension (mg/100 ml) 24 17.1 21.9 47 29.2 39.1 77 30.0 41.5 95 30.0 39.7 - 66 -HALOTHANE (MG/100 ML) Fig. 11. Absorption of halothane to triglyceride-rich micelles at a constant triglyceride concentration of 83 mg/100 ml. - 67 -H = 0.00537[Halothane]f - 0.0170 [13] where H = grams of halothane s o l u b i l i z e d per grams of t r i g l y c e r i d e [Halothane]f = free halothane concentration i n mg/100 ml As shown i n F i g . 12 when the free halothane concentration was kept constant at 9.2 mg/100 ml, there was a l i n e a r r e l a t i o n s h i p between the q u a n t i t y of halothane s o l u b i l i z e d by the t r i g l y c e r i d e - r i c h m i c e l l e s and the t r i g l y c e r i d e c o ncentration ( i e . the number of t r i g l y c e r i d e - r i c h m i c e l l e s ) . L i n e a r r e g r e s s i o n of a l l the data points gives the f o l l o w i n g equation: [Halothane] t = 0.0346[TG] + 0.227 [14] where [Halothane]t = halothane (mg/100 ml) s o l u b i l i z e d i n the presence of t r i g l y c e r i d e r i c h m i c e l l e s [TG] = t r i g l y c e r i d e concentration (mg/100 ml) 7. D i s t r i b u t i o n of halothane between the components of blood Equations [10]-[13] were used to c a l c u l a t e the d i s t r i b u t i o n of halothane between water, albumin, t r i g l y c e r i d e , haemoglobin and red c e l l membrane i n blood using normal values f o r the blood components (Henry et a l . 1974) and assuming that these are the only important components c o n t r i b u t i n g to the s o l u b i l i t y of halothane i n blood. The r e s u l t s of t h i s c a l c u l a t i o n are shown i n Table XV. 8. D i s t r i b u t i o n of halothane between c e l l s and plasma Table XVI shows the r e s u l t s of p r e l i m i n a r y experiments performed to estab-l i s h the time required to achieve e q u i l i b r i u m . As expected, the c o n c e n t r a t i o n of halothane i n the plasma was higher than that i n the whole blood at the - 68 -4r o l i J 0 50 100 TRIGLYCERIDE (MG/100 ML) Fig. 12. Absorption of halothane to triglyceride-rich micelles at a constant halothane concentration of 9.2 mg/100 ml. Table XV - D i s t r i b u t i o n of halothane i n blood c a l c u l a t e d from the r e s u l t s of e q u i l i b r i u m d i a l y s i s This d i s t r i b u t i o n , c a l c u l a t e d from the l i n e a r equations [10]-[13] (which were obtained when the free halothane concentration was l e s s than 150 mg/100 ml) i s a p p l i c a b l e to the range of blood halothane concentration found during anaesthesia. Blood Component Co n t r i b u t i o n to halothane s o l u b i l i t y % of t o t a l albumin (4.3 g/100 ml) Plasma t r i g l y c e r i d e (100 mg/100 ml) water 16 37 6 15 ( f r e e halothane) red c e l l membrane (4.5 x 10 9RBC/ml) Blood hemoglobin (14.5 g/100 ml) water 40 63 13 10 ( f r e e halothane) Table XVI - Time r e q u i r e d to reach e q u i l i b r i u m f or the d i s t r i b u t i o n of halothane between c e l l s and plasma Whole blood samples c o n t a i n i n g halothane were e q u i l i b r a t e d f or d i f f e r e n t time i n t e r v a l s . Blood and plasma samples were then analysed f o r halothane concentration to e s t a b l i s h the time required to reach e q u i l i b r i u m Time (hr) [Halothane] mg/100 ml Plasma Blood 1.0 47.0 1.0 6.0 47.6 38.6 24.5 i --J o i 19.5 34.5 21.0 34.6 42.3 24.0 32.7 24.0 35.6 beginning of the e q u i l i b r a t i o n . A f t e r 19.5 hr however, the plasma halothane concentration was lower than that i n blood and appeared to have s t a b i l i z e d . This was chosen as the e q u i l i b r a t i o n time. Table XVII compares the experimentally determined d i s t r i b u t i o n of h a l o -thane between the c e l l s and plasma to the t h e o r e t i c a l d i s t r i b u t i o n c a l c u l a t e d from equation [10]-[13]. Comparison was made f o r three normal blood samples at d i f f e r e n t halothane concentrations and two samples from the same donor with d i f f e r e n t t r i g l y c e r i d e concentrations. The c e l l counts and the concentrations of albumin, haemoglobin and t r i g l y c e r i d e of these blood samples were e x p e r i -mentally determined. Since, as shown i n Table XVII, there was no detectable d i f f e r e n c e i n the halothane concentration of the whole blood before and a f t e r the e q u i l i b r a t i o n , the amount of halothane l o s t during the e q u i l i b r a t i o n was n e g l i g i b l e . The plasma t r i g l y c e r i d e c o n c e n t r a t i o n ( c o r r e c t e d f o r d i l u t i o n ) for the two sets of plasma samples, washed 5 times and 8 times with s a l i n e (to d i l u t e the concentration of EDTA) r e s p e c t i v e l y , are a l s o shown i n Table XVII. There was no detectable change i n the t r i g l y c e r i d e concentration of the more e x t e n s i v e l y washed samples, i n d i c a t i n g that there was no i n t e r f e r e n c e w i t h the t r i g l y c e r i d e assay from any remaining anticoagulant. Table XVII - D i s t r i b u t i o n of halothane between c e l l s and plasma Whole blood samples were e q u i l i b r a t e d with halothane. Blood and plasma samples were then analysed f o r halothane concentration [Halothane] mg/100 ml D i a l y s i s * * C e n t r i f u g a t i o n Plasma T r i g l y c e r i d e (mg/100 ml) i n whole : blood 7o of Halothane % of Halothane before a f t e r i n i n 5 x washed* 8 x washed* e q u i l i b r a t i o n e q u i l i b r a t i o n Plasma C e l l Plasma C e l l 42.8 41.7 38 62 48 52 23.9 23.4 37 63 49 51 3.8 4.2 38 62 49 51 30 + 35 25.1 28 72 39 61 1 8 6 + + 179 25.7 34 66 48 52 *Plasma t r i g l y c e r i d e concentrations were assayed a f t e r washing with s a l i n e to d i l u t e the disodium EDTA co n c e n t r a t i o n to n e g l i g i b l e amount * * C a l c u l a t e d from equation [10]-[13] + T r i g l y c e r i d e - p o o r plasma + + T r i g l y c e r i d e - r i c h plasma - 73 -DISCUSSION The s o l u b i l i t y of halothane i n s a l i n e (297 mg/100 ml at 37°C and 481 mg/100 ml at 4°C) i s high compared to that of n-pentane (3.1 mg/100 ml at 20°C i n Ringer s o l u t i o n ) (Haydon et a l . 1977), probably due to the formation of hydrogen bonds between the f l u o r i n e s of halothane and water molecules. That halothane i s more s o l u b l e at a lower temperature i s expected for a v o l a t i l e l i q u i d and agrees w i t h the previous f i n d i n g s that the water/gas p a r t i t i o n c o e f f i c i e n t increased with decreasing temperature ( A l l o t t et a l . 1973). However, the s o l u b i l i t y of halothane i n aqueous s o l u t i o n does not seem to be s t r o n g l y dependent on s a l t c oncentrations, as the water/gas, saline/gas and Kreb's solution/gas p a r t i t i o n c o e f f i c i e n t s (0.74-0.89, 0.70-0.77 (Steward et a l . 1973) and 0.75 (Renzi and Waud 1972)) are a l l n e a r l y the same. The time taken f o r the d i a l y s i s to reach e q u i l i b r i u m i s unusually long. This was most probably due to the small surface area of the d i a l y s i s membrane. The d i a l y s i s surface ( r e l a t i v e to the volume c o n t a i n i n g the blood component, F i g . 5) was much l e s s than a ro u t i n e d i a l y s i s using a d i a l y s i s tubing. The time taken to reach e q u i l i b r i u m f o r red c e l l ghosts and t r i g l y c e r i d e r i c h m i c e l l e s (42 and 47 hours r e s p e c t i v e l y ) , were quite d i f f e r e n t from that for haemoglobin and albumin (73 and 96 hours r e s p e c t i v e l y ) , probably because the d i a l y s i s assembly was r o t a t e d i n the former case but not i n the l a t t e r . The shape of the adsorption isotherms of halothane to haemoglobin and albumin suggest that halothane i s attached to a f i n i t e number of s i t e s on the p r o t e i n surface. Since halothane i s very soluble i n o i l and q u i t e i n s o l u b l e i n water (Steward et a l . 1973), these are presumably hydrophobic s i t e s ; the - 74 -bound halothane molecules are s t a b i l i z e d by hydrophobic i n t e r a c t i o n s . I t i s a l s o p o s s i b l e that halothane i s s t a b i l i z e d by hydrogen bonding between the f l u o r i n e and a p o l a r i z e d region (e.g. a c i d i c group) of the p r o t e i n . As the isotherms do not l e v e l o f f to a p l a t e a u , the b i n d i n g s i t e s were not yet s a t u r -ated when the aqueous phase was saturated w i t h halothane. In a t h e o r e t i c a l study, Tanford (1973) i n t e r p r e t e d such phenomena as i n d i c a t i n g that the de-crease i n f r e e energy associated w i t h the b i n d i n g of hydrophobic molecules with a p r o t e i n i s l e s s favourable than that i n the s e l f - a s s o c i a t i o n between the hydrophobic molecules when the s o l u t i o n i s saturated. A l t e r n a t i v e l y , Hildebrand (1979) maintained that the increase i n free energy due to the d i s r u p t i o n of hydrogen bonds between water molecules i s too unfavourable f o r more halothane to enter i n t o the aqueous s o l u t i o n , i n which case the chemical p o t e n t i a l of halothane i n the aqueous s o l u t i o n i s not high enough f o r b i n d i n g w i t h more p r o t e i n surface s i t e s to occur. Although the adsorption isotherms f o r haemoglobin and albumin are q u a l i -t a t i v e l y s i m i l a r , many more halothane molecules were bound to albumin at any given f r e e halothane concentration. This was to be expected since albumin i s g e n e r a l l y considered to be a c a r r i e r p r o t e i n for small hydrophobic molecules (Peters 1975) such as the plasma f a t t y a c i d s . I t has been estimated that l e s s than 0.01% of the t o t a l u n e s t e r i f i e d f a t t y a c i d are free i n the plasma (Goodman 1958) and that 33% of the f a t t y a c i d bound to albumin i s o l e i c a c i d (cis-9-octadecenoic a c i d ) ( S a i f e r and Goldman 1961). Thus i t was d e s i r a b l e to i n v e s t i g a t e the e f f e c t of o l e i c a c i d upon the b i n d i n g of halothane to albumin. F i g . 6 shows that o l e i c a c i d at p h y s i o l o g i c a l concentrations had no d e t e c t a b l e e f f e c t i n the b i n d i n g of halothane to albumin. This r e s u l t i s open to s e v e r a l - 75 -i n t e r p r e t a t i o n s : ( i ) Halothane and o l e i c a c i d do not compete f o r the same s i t e s . This i s u n l i k e l y because both are expected to bind n o n - s p e c i f i c a l l y to hydrophobic s i t e s , although halothane and o l e i c a c i d , being q u i t e d i f f e r e n t i n molecular dimensions, may bind p r e f e r e n t i a l l y to d i f f e r e n t s i t e s . ( i i ) The number of s i t e s i s l a r g e enough so that the b i n d i n g does not s i g n i f i c a n t l y reduce the number of s i t e s a v a i l a b l e for halothane. ( i i i ) The b i n d i n g of o l e i c a c i d to albumin creates new hydrophobic s i t e s f o r halothane. I t should be noted that the l a s t two i n t e r p r e t a t i o n s are not mutually e x c l u s i v e . F i g . 7 and 9 show the e q u i l i b r i u m d i a l y s i s r e s u l t s p l o t t e d according to the Scatchard equation, which assumes that there i s a f i n i t e number of s i t e s on a p r o t e i n molecule f o r the i n t e r a c t i o n with halothane which are i d e n t i c a l and independent of one another. I f t h i s assumption i s v a l i d , the r e s u l t i n g graph should show a s t r a i g h t l i n e w i t h a negative slope (Marshall 1978). This i s the advantage of the Scatchard method because the v a l i d i t y of the assumption can be judged by how cl o s e the data points f i t a s t r a i g h t l i n e . The assumption i s obviously not true i n the case of b i n d i n g of halothane to albumin and haemoglobin. F i g . 7 and 9 both appear to have an i n i t i a l p o s i t i v e slope followed by a l e s s pronounced negative slope. Thus the nature of b i n d i n g does not f i t a simple assumption of i d e n t i c a l and independent s i t e s . The i n i t i a l p o s i t i v e slope suggests a p o s s i b l e p o s i t i v e cooperative e f f e c t (Jones 1975; M a r s h a l l 1978). The dimension of the surface s i t e s has been suggested as a p o s s i b l e explanation of t h i s cooperative e f f e c t (Tanford 1973). I f a s i t e i s large enough to accommodate more than one small molecule, then i t could be e n e r g e t i c a l l y more favourable for a second small molecule to bind to the same s i t e a f t e r the f i r s t small molecule i s already bound. - 76 -When the i n i t i a l parts of the two graphs with the p o s i t i v e slopes are ignored, l i n e a r r e g r e s s i o n of a l l the data p o i n t s on the second p o r t i o n s w i t h negative slope give a t o t a l number of 20 and 130 surface s i t e s on haemoglobin and albumin r e s p e c t i v e l y f o r the binding of halothane. The slope of the absorption isotherm of halothane to red c e l l ghosts sug-gests that the nature of the i n t e r a c t i o n between halothane and the red c e l l membrane, and that between halothane and haemoglobin or albumin are very d i f f e r e n t . Since halothane i s hydrophobic and not surface a c t i v e , i t would presumably be " s o l u b i l i z e d " w i t h i n the hydrophobic region of the membrane. Thus the "bound" halothane would not be l o c a l i z e d to a f i x e d s i t e w i t h i n the f l u i d membrane. The data suggest that i t i s more favourable f o r halothane molecules to move i n t o a membrane which has already incorporated some halothane molecules. P o s s i b l y the halothane molecules i n i t i a l l y accommodated w i t h i n the membrane d i s t o r t i t and lead to a l e s s organized s t r u c t u r e , capable of accommodating more halothane molecules. In a study of the e f f e c t of n-alkanes on black l i p i d b i l a y e r membrane (Haydon et a l . 1977), i t was found that an increase of alkane concentration caused an increase i n the hydrophobic t h i c k -ness of the black l i p i d b i l a y e r as c a l c u l a t e d from the e l e c t r i c a l c a p a c i t y of the b i l a y e r . The slope of the curve increased w i t h i n c r e a s i n g alkane concen-t r a t i o n i n a way s i m i l a r to the r e l a t i o n s h i p found here between the number of halothane molecules per ghost and the free halothane c o n c e n t r a t i o n . Our r e s u l t s are i n q u a l i t a t i v e agreement with those of Haydon et a l . (1977). In the plasma, t r i g l y c e r i d e i s transported as p r o t e i n - l i p i d m i c e l l e s , predominantly i n chylomicrons and very low den s i t y l i p o p r o t e i n (VLDL), c o n s i s t i n g of 88% and 56% of t r i g l y c e r i d e r e s p e c t i v e l y (Osborne and Brewer 1977). The absorption isotherm of halothane i n these t r i g l y c e r i d e - r i c h - 77 -m i c e l l e s resembles q u a l i t a t i v e l y that f o r the red c e l l membrane, c o n s i s t e n t wit h the f a c t that membranes and m i c e l l e s share a s i m i l a r a r c h i t e c t u r e , i . e . a hydrophobic core surrounded by a h y d r o p h i l i c surface. I t i s apparent (Table XV) that a l l the major components of human blood t e s t e d , except y - g l o b u l i n , ( i . e . albumin, t r i g l y c e r i d e , haemoglobin and red c e l l membrane) c o n t r i b u t e s i g n i f i c a n t l y to the s o l u b i l i t y , and hence the transport of halothane i n blood. According to the r e s u l t s obtained from e q u i l i b r i u m d i a l y s i s , red c e l l membranes, which c a r r y 40% of the t o t a l amount of halothane i n whole blood, are q u a n t i t a t i v e l y most important, followed by the water i n blood, i n which 25% of the halothane i s d i s s o l v e d . Albumin car-r i e s 16%, haemoglobin 13% and t r i g l y c e r i d e m i c e l l e s approximately 6%. Using these r e s u l t s i n combination with the saline/gas p a r t i t i o n c o e f f i c i e n t ( t a k i n g an average value of 0.74 (Steward et a l . 1973)), the blood/gas p a r t i t i o n c o e f f i c i e n t was c a l c u l a t e d to be 2.6. This i s i n reasonable agreement with the experimentally determined value of 2.3 (Steward et a l . 1973). The data i n d i c a t e s therefore that the s o l u b i l i t y of halothane i n blood w i l l increase with increases i n albumin, t r i g l y c e r i d e , haemoglobin and haematocrit, e i t h e r s i n g l y or i n combinations. Experimental observations on these r e l a t i o n s h i p s , however, are i n c o n f l i c t . Several i n v e s t i g a t o r s (see Table V I I I ) have reported a decrease i n s o l u b i l i t y w i t h an increase i n haematocrit (Cowles et a l . 1971a; Han and H e l r i c h 1966; Lowe and Hagler 1969) while other studies have shown that the s o l u b i l i t y of halothane i s independent of haematocrit (Saraiva et a l . 1977a), that halothane i s l e s s s oluble i n blood of low haematocrit or more s o l u b l e i n blood of high haematocrit (Laasberg and Hedley-Whyte 1970) and that halothane i s l e s s s oluble i n anemic blood ( E l l i s and S t o e l t i n g 1975). I t has also been reported that l i p i d c o n centration either increased (Saraiva et a l . 1977a; Larson et a l . 1962; Wagner et a l . 1974) or was not correlated (Munson et a l . 1978) with the solubility of halothane, and that the solubility was dependent upon the albumin to globulin ratio (Laasberg and Hedley-Whyte 1970). Saraiva et a l . (1977a) studied the effect of the concentrations of haemoglobin, serum albumin, globulin, total protein, triglyceride, cholesterol, the haematocrit, the albumin to globulin ratio and the albumin to total protein ratio upon the solubility of halothane in blood and were able to demonstrate a s t a t i s t i c a l l y significant correlation only with serum triglyceride. Munson et a l . (1978) were, however, unable to demonstrate this correlation, possibly because they used a narrower range of triglyceride concentrations and a smaller number of samples. In the studies referred to above the effect of various blood components upon the solubility of halothane was inferred indirectly from changes in the blood/gas partition coefficient (see Introduction, Part II). Such changes are slight even when the concentration of a contributing blood component changes drastically, as exemplified by the apparently conflicting reports of Saraiva et a l . (1977a) and Munson et a l . (1978) referred to above. Thus i f the normal range of the concentration of a particular blood component is small, as is the case with most blood components, any change in the partition coefficient may be too small to be detected. Furthermore, when whole blood samples are used, the effect of random changes in the concentration of a l l the blood components tends to mask the effect of the change in concentration of a particular com-ponent, thereby decreasing the chance of finding a s t a t i s t i c a l l y significant correlation. In other words, the lack of a s t a t i s t i c a l l y significant correlation between the partition coefficient and the concentration of a - 79 -c e r t a i n blood component does not n e c e s s a r i l y imply that the blood component does not c o n t r i b u t e to the s o l u b i l i t y of halothane, i t may simply mean that the a n a l y t i c a l method being used i s not s e n s i t i v e enough to detect the c o n t r i b u t i o n of the v a r i o u s blood components as found i n t h i s study. I t i s not s u p r i s i n g , t h e r e f o r e , that the only s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n found by Saraiva et a l . (1977) was that for t r i g l y c e r i d e . In that study t r i g l y c e r i d e has the widest normal range i n the blood samples used. As can be seen i n Table XVII a large increase i n the plasma t r i g l y c e r i d e c o n c e n t r a t i o n r e s u l t e d i n an increase i n the plasma halothane concentration. The d i s t r i b u t i o n of halothane between c e l l s and plasma c a l c u l a t e d from the e q u i l i b r i u m a n a l y s i s studies agrees reasonably w e l l w i t h that from the study of whole blood (Table XVII). There seems to be a systematic d i f f e r e n c e , however, between the d i s t r i b u t i o n s found by the two d i f f e r e n t methods, the p r o p o r t i o n of halothane found by the d i r e c t assay of the plasma being c o n s i s -t e n t l y higher than that c a l c u l a t e d from the r e s u l t s of the d i a l y s i s method. This discrepancy can be p a r t l y accounted for by those blood components which were not i n v e s t i g a t e d by e q u i l i b r i u m d i a l y s i s , namely, plasma p r o t e i n s other than albumin and y g l o b u l i n , and c e l l s other than red blood c e l l s . Since the t o t a l number of white blood c e l l s i n a normal blood sample i s only approxi-mately 2 per 1000 red c e l l s (Ganong 1975), t h e i r c o n t r i b u t i o n can s a f e l y be ignored. F u r t h e r , although the r a t i o of p l a t e l e t s to red c e l l s i s l e s s than 1/10 (Ganong 1975), the t o t a l membrane surface area of p l a t e l e t s i s l e s s than 1% of the red c e l l (assuming diameters of 3 um and 7 um f o r p l a t e l e t s and red c e l l s r e s p e c t i v e l y (Leeson and Leeson 1976) and that the r a t i o of surface area - 80 -i s the r a t i o of the square of diameters). Therefore the c o n t r i b u t i o n from p l a t e l e t s can al s o be ignored. Plasma p r o t e i n s other than albumin and y - g l o b u l i n c o n s t i t u t e approximately 30% of the t o t a l plasma p r o t e i n (Henry et a l . 1974). I f i t i s assumed that equal weights of albumin and of plasma p r o t e i n s other than albumin and y - g l o b u l i n bind the same amount of halothane then the discrepancies between the two estimates of d i s t r i b u t i o n disappear. However t h i s assumption i s u n l i k e l y to be v a l i d , since the unaccounted f o r plasma p r o t e i n s have probably l e s s b i n d i n g c a p a c i t y f o r halothane than albumin and give only a p a r t i a l e x p l a n ation f o r these d i s c r e p a n c i e s . The concentration of haemoglobin may al s o account f o r these d i s c r e p a n c i e s . The haemoglobin co n c e n t r a t i o n used i n the d i a l y s i s experiments was a p p r o x i -mately 2/3 that of the normal haemoglobin concentration i n the whole blood, or l e s s than 1/3 of the a c t u a l haemoglobin concentrations w i t h i n the red c e l l s , since haemoglobin molecules are concentrated w i t h i n t h i s volume. Aggregation of haemoglobin molecules i n i n t a c t red c e l l s may s h i e l d some of the surface s i t e s f o r halothane, i n which case the e q u i l i b r i u m d i a l y s i s r e s u l t s f o r the adsorption of halothane to haemoglobin would be an overestimate of the a c t u a l adsorption o c c u r r i n g i n whole blood. Consequently an overestimate would be made of the d i s t r i b u t i o n of halothane between c e l l s and plasma i n favour of the c e l l f r a c t i o n . F u r t h e r , since the same r e g r e s s i o n equations were used i n the c a l c u l a t i o n s f o r a l l the blood samples, any random e r r o r i n the e s t i m a t i o n of the halothane bound to a p a r t i c u l a r blood component w i l l be converted to a systematic e r r o r . The claims that the s o l u b i l i t y of halothane i n blood has a negative dependence on haematocrit (Cowles et a l . 1971a; Han and H e l r i c h 1966; Lowe and - 81 -Hagler 1969) are d i f f i c u l t to evaluate because there were no data regarding the time required to reach e q u i l i b r i u m . In these studies halothane was i n t r o -duced i n t o the aqueous phase p r i o r to the e q u i l i b r a t i o n w i t h a head space (gas phase) for the determination of the blood/gas p a r t i t i o n c o e f f i c i e n t . I t i s conceivable that the rat e of v a p o r i z a t i o n of halothane from the aqueous phase to the head space (the gas phase i n which the p a r t i a l pressure was measured) i s much f a s t e r than the rat e of halothane b i n d i n g to one or more of the major blood components. I f t h i s were t r u e , a r a p i d i n i t i a l r i s e of the p a r t i a l pressure of halothane i n the head space would be observed while the halothane was d i s s o l v i n g i n the aqueous phase, followed by a period when the p a r t i a l pressure would f a l l very slowly. I f the p a r t i a l pressure were measured during t h i s slowly f a l l i n g p e r i o d , the p a r t i t i o n c o e f f i c i e n t c a l c u l a t e d from the p a r t i a l pressure would be lower than the true e q u i l i b r i u m value. This e f f e c t i s more pronounced when the haematocrit i s higher, i . e . the measured p a r t i t i o n c o e f f i c i e n t would be f u r t h e r below the true e q u i l i b r i u m p a r t i t i o n c o e f f i c i e n t at a higher haematocrit, l e a d i n g to the conclusion that the s o l u b i l i t y of halothane i n blood has a negative dependence on haematocrit. - 82 -PART I I I - Uptake of halothane i n dog blood i n v i v o INTRODUCTION There have been very few measurements of the a c t u a l c o n c e n t r a t i o n of i n h a l a t i o n anaesthetics i n blood during anaesthesia and those reported (Chenoweth et a l . 1962; Cervenko 1968; Lowe 1964a) are not s u f f i c i e n t l y comprehensive to be used f o r c o r r e l a t i o n w i t h theories of the uptake and d i s t r i b u t i o n of a n a e s t h e t i c s . This i s probably due i n part to the l a c k of a r e l i a b l e method f o r the d i r e c t determination of i n h a l a t i o n anaesthetics i n blood, and i n part to the g e n e r a l l y accepted formulation of the pharmaco-k i n e t i c s of i n h a l a t i o n a n a e s t h e t i c s . I t i s a widely h e l d assumption t h a t , when a s u f f i c i e n t l y long p e r i o d of time has elapsed a f t e r the i n d u c t i o n of anaesthesia, thermodynamic e q u i l i b r i u m i s almost achieved and t h e r e f o r e the chemical p o t e n t i a l s of the anaesthetic i n the a l v e o l i , blood and nervous t i s s u e are almost the same (Halsey 1974; Cowles et a l . 1968b). Thus a measurement of the a l v e o l a r e n d - t i d a l anaesthetic p a r t i a l pressure which i s d i r e c t l y r e l a t e d to chemical p o t e n t i a l , s u f f i c e s to i n d i c a t e the anaesthetic chemical p o t e n t i a l i n nervous t i s s u e . Based upon t h i s reasoning, the concept of Minimum A l v e o l a r Concentration (MAC) was developed as a standard of anaesthetic potency (Eger et a l . 1965). MAC i s defined as the minimum a l v e o l a r concentration of anaesthetic necessary to prevent movement i n response to a p a i n f u l stimulus i n 50% of the experimental subjects. S t r i c t l y speaking, i t i s the minimum anaesthetic p a r t i a l pressure (MAP) which i s being used (Fink 1971; Eger 1971), but at the common ambient - 83 -atmospheric pressure, where d e v i a t i o n from the i d e a l gas law i s s l i g h t , MAC i s d i r e c t l y p r o p o r t i o n a l to MAP. There i s a reasonable c o r r e l a t i o n between MAC and the o i l / g a s p a r t i t i o n c o e f f i c i e n t (the r a t i o of the c o n c e n t r a t i o n of anaes-t h e t i c i n an o l i v e o i l phase to that i n a gas phase when the two phases are i n e q u i l i b r i u m ) (Saidman et a l . 1967). Since current t h e o r i e s of the mechanism of anaesthesia favour an a n a e s t h e t i c - l i p i d i n t e r a c t i o n (Kaufman 1977; M i l l e r 1977) there i s a general acceptance of the use of p a r t i a l pressure (of a gas phase i n e q u i l i b r i u m w i t h the t i s s u e or blood being s t u d i e d ) , r a t h e r than con c e n t r a t i o n , as a q u a n t i t a t i v e measurement of anaesthetics (Cowles et a l . 1971a, 1972a; Kolmer et a l . 1975a; A l l o t et a l . 1976; Saraiva et a l . 1977a,b; Munson et a l . 1978). Mathematical models of the uptake and d i s t r i b u t i o n of anaesthetic are a l s o forumulated i n terms of p a r t i a l pressure rather than concentration (Eger 1963; Bourne 1964; Eger and Severinghaus 1964; Munson and Bowes 1967; Ashman et a l . 1970; Cowles et a l . 1968a,b, 1971b, 1972b; Mapleson 1963, 1964a,b, 1973; Kolmer et a l . 1975b; Zwart et a l . 1972). Models of the uptake and d i s t r i b u t i o n of i n h a l a t i o n anaesthetics can be d i v i d e d i n t o two types. 1. E m p i r i c a l Models These include mathematical equations and p h y s i c a l devices which produce an uptake and d i s t r i b u t i o n curve w i t h any given set of input data. Kolmer et a l . (1975b) used a f i r s t order r a t e equation as a model f o r the uptake of halothane i n dogs. D i f f e r e n t compartments i n the body were repre-sented by d i f f e r e n t terms with d i f f e r e n t parameters (time constants and f r a c -t i o n a l c o e f f i c i e n t s ) i n the l i n e a r r a t e equation. The number of terms i n the equation and the values of the parameters were adjusted to give the best f i t - 84 -to experimentally measured end-tidal partial pressure and the halothane partial pressures in a head space in equilibrium with blood samples. An equation with two terms (4 parameters) yielded the highest s t a t i s t i c a l significance with an F-test, and i t was claimed that this suggested the presence of two body compartments. The physical device most widely used as a model for studying the uptake and distribution of anaesthetic is the analog circuit (Cowles et a l . 1968a,b; Mapleson 1963, 1964b). These are DC circuits with a power source (battery), resistors and capacitors representing the anaesthetic vaporizer, blood vessels and organs respectively. Thus the inspired anaesthetic level, the rate of transport of anaesthetic in the blood, and the rate of uptake of anaesthetic by the organs can be regulated by changing the EMF, resistances and capaci-tances of the circuit respectively to mimic the conditions during anaesthesia. Another physical model consists of cylinders of different diameters inter-connected at the base, to represent different organs with different capacity for the anaesthetic (Eger 1974). Water, representing the anaesthetic, is added to the cylinder representing the lung and the resulting water level and its rate of rise in the different cylinders represent the anaesthetic level and its rate of rise in the different organs. As with the electric analog, the inspired anaesthetic level, transport of anaesthetic in blood, and the uptake of the anaesthetic can be regulated by changing the rate of water input into the "lung" cylinder, the diameters of the interconnecting pipes and that of the "organ" cylinders, respectively. The usefulness of these empirical models is judged entirely by the closeness of the f i t to experimentally derived data. Since the f i t can be a r b i t r a r i l y improved by the appropriate s e l e c t i o n of the number and values of parameters (constants i n mathematical models, r e s i s t a n c e and capacitance i n analog c i r c u i t s , pipe and c y l i n d e r diameters i n hydrodynamic models), an e m p i r i c a l model which a c c u r a t e l y describes the q u a n t i t a t i v e aspects of the uptake and d i s t r i b u t i o n of a n a e s t h e t i c s , although u s e f u l as a pedagogical device, does not n e c e s s a r i l y c o n t r i b u t e to the understanding of the r e a l process i n v o l v e d . 2. Computation-simulation Models These models p r e d i c t the l e v e l of anaesthetic and i t s r a t e of r i s e i n d i f f e r e n t organs by computing f o r a given i n s p i r e d anaesthetic l e v e l the q u a n t i t y of anaesthetic t r a n s f e r r e d from the a l v e o l i to a r t e r i a l blood and that t r a n s f e r r e d from the a r t e r i a l blood to the t i s s u e s . The v e n t i l a t i o n was e i t h e r assumed to be continuous (Cowles et a l . 1972a,b), or i n more elaborate models, stepwise computation corresponded to the discontinuous v e n t i l a t i o n (Munson and Bowes 1967; Munson et a l . 1973; Mapleson 1973; A l l o t et a l . 1976). The computation can become very in v o l v e d and r e s u l t s can only be obtained with the a i d of a d i g i t a l computer. To s i m p l i f y the c a l c u l a t i o n s , many assumptions were made. For example, none of the models took i n t o account the metabolism of anaesthetics as part of the computation, although i t has been used as a c o r r e c t i o n f a c t o r ( A l l o t et a l . 1976). One b a s i c assumption i s inherent to a l l these models. The anaesthetic contained i n the a l v e o l a r gas, t i s s u e and pulmonary c a p i l l a r y blood i s e i t h e r i n continuous e q u i l i b r i u m (Cowles et a l . 1972a,b) or achieve e q u i l i b r i u m w i t h i n one i n h a l a t i o n - e x h a l a t i o n i n t e r v a l (Mapleson 1973). This assumption, i n combination with the blood/gas p a r t i t i o n c o e f f i c i e n t , allows the exact q u a n t i t y of anaesthetic t r a n s f e r r e d from the - 86 -lung to the a r t e r i a l blood to be e a s i l y c a l c u l a t e d f o r any given time i n t e r v a l , provided that the blood/gas p a r t i t i o n c o e f f i c i e n t i s a constant. Although the blood/gas p a r t i t i o n c o e f f i c i e n t i s not a r e a l constant, values for dog blood are, as shown i n Table X V I I I , s u f f i c i e n t l y c l o s e for i t to be t r e a t e d as such. Morris (1974), however, has pointed out that e q u i l i b r i u m thermodynamics i s not n e c e s s a r i l y a p p l i c a b l e to an open system (eg. a l i v i n g organism), where m a t e r i a l t r a n s f e r between the system and i t s environment occurs. Furthermore, the v a l i d i t y of using the e n d - t i d a l p a r t i a l pressure as an absolute index of the a r t e r i a l anaesthetic p a r t i a l pressure has been c r i t i c i z e d on p h y s i o l o g i c a l grounds (Eger and Bahlman 1971). Although the f i n d i n g that the MAC i s unaltered by periods of anaesthesia of up to 8 hours (Eger et a l . 1965a) appears to i n d i c a t e that thermodynamic e q u i l i b r i u m i s achieved (Halsey 1974) there has been as yet no d e f i n i t i v e study which v e r i f i e s t h i s g e n e r a l l y accepted assumption (Mapleson 1963; Cowles 1972). In the experiments to be described, the e n d - t i d a l p a r t i a l pressures and the concentrations of halothane i n blood and plasma during anaesthesia were measured under c o n t r o l l e d c o n d i t i o n s . The e q u i l i b r i u m assumption w i l l be discussed i n the l i g h t of these r e s u l t s . Table XVIII - Blood/gas p a r t i t i o n c o e f f i c i e n t f o r dog blood at 37°C Authors Mean P a r t i t i o n C o e f f i c i e n t Measure of Uncertainty No. of Animals No. of Determinations Haematocrit Steward et a l . 1975 3.51 + 0.31 95% confidence l i m i t 7 14 37.3 Cowles et a l . 1971 3.16 + 0.18 95% confidence l i m i t 7 88 45 Ikeda 1972 2.18 + 0.16 Standard D e v i a t i o n NG 13 42 NG: not given - 88 -MATERIALS AND METHODS Male mongrel dogs (17-25 kg) were starved overnight but allowed f r e e access to water and then anaesthesia was induced w i t h sodium thiopentone (20 mg/kg i v ) . The dogs were intubated with an endotracheal tube and halothane was administered w i t h a Drager v a p o r i z e r i n a c i r c u i t w i t h a carbon d i o x i d e absorber. Halothane i n s p i r e d concentrations were kept constant at 1.0, 1.5, 2.0 or 2.5% (of 1 atmosphere) r e s p e c t i v e l y ; when 1% halothane was used neuro-muscular blockade was maintained w i t h pancuronium. V e n t i l a t i o n was maintained wit h a B i r d mark 8 r e s p i r a t o r . During the course of the experiment, the blood gases were maintained i n the f o l l o w i n g ranges PCO^^ 30-40 mm Hg, pH: 7.35-7.45; VO^'- greater than 100 mm Hg, and the body temperature was maintained at 38°C (see Tables XIX-XXII) with a heating pad. A r t e r i a l blood pressure and e n d - t i d a l halothane p a r t i a l pressure were monitored by a Statham P23AC transducer and by a Beckman LB 2 i n f r a r e d analyzer as described by Leighton et a l . (1978). Blood was withdrawn from the femoral a r t e r y and from the r i g h t atrium ( v i a a catheter i n s e r t e d through the j u g u l a r vein) at approximately (exact time known) 15, 30, 60, 120, 240, 300 minutes a f t e r commencing anaesthesia. (These parts of the experiments were c a r r i e d out by Ms. C a r o l i n e Bruce i n the Depart-ment of Pharmacology, U n i v e r s i t y of B.C.). The f i r s t 3 ml of blood were discarded to avoid erroneous measurement of blood halothane c o n c e n t r a t i o n due to the dead volume of the catheter and then the blood samples were t r a n s f e r r e d to 1 ml R e a c t i - v i a l s ( P i e r c e Chemical Co., Rockford, 111., USA) c o n t a i n i n g s u f f i c i e n t disodium EDTA to give a f i n a l c oncentration of approximately 4.5 mg/ml. A glass bead was dropped i n t o the v i a l , which was then sealed w i t h no - 89 -t r a p p e d a i r b u b b l e . A f t e r t h o r o u g h m i x i n g p l a s m a was o b t a i n e d f r om one s e t o f samp les by c e n t r i f u g a t i o n i n a S o r v a l l HB-4 r o t o r a t 4 , 5 0 0 rpm (max. 2 , 8 7 0 g) f o r 15 m i n u t e s . P l a s m a and who le b l o o d samp les were a n a l y s e d f o r h a l o t h a n e by the gas l i q u i d c h r o m a t o g r a p h i c p r o c e d u r e d e s c r i b e d i n P a r t I o f t h i s w o r k . The p o s s i b i l i t y o f l o s s o f h a l o t h a n e d u r i n g t he t r a n s f e r o f t he b l o o d samp les was t e s t e d as f o l l o w s . H a l o t h a n e was added i n v i t r o to a human b l o o d sample i n a s y r i n g e c o n t a i n i n g a g l a s s b e a d . The s y r i n g e was capped w i t h a n e e d l e s e a l e d w i t h s o l d e r , t h e n the b l o o d was t h o r o u g h l y m i x e d and a samp le was t a k e n f o r a n a l y s i s o f h a l o t h a n e . The s y r i n g e was t h e n i n c u b a t e d a t 37°C f o r 30 m in and t he h a l o t h a n e c o n c e n t r a t i o n a g a i n d e t e r m i n e d . The d i s t r i b u t i o n o f h a l o t h a n e be tween the c e l l s and p l a s m a was d e t e r m i n e d as d e s c r i b e d i n P a r t I I o f t h i s work and c a l c u l a t e d f r om e q u a t i o n [ 8 ] , page 5 0 . I n o r d e r to d e t e r m i n e w h e t h e r or no t the d i s t r i b u t i o n changed a f t e r the b l o o d samp les had been removed f r om the dog b l o o d samp les were r o t a t e d i n R e a c t i v i a l s f o r a f u r t h e r 2 and 4 h o u r s b e f o r e d e t e r m i n i n g the h a l o t h a n e d i s t r i b u t i o n . The a r t e r i a l h a l o t h a n e c o n c e n t r a t i o n was c a l c u l a t e d f rom the end t i d a l p a r t i a l p r e s s u r e u s i n g e q u a t i o n [17] d e r i v e d as f o l l o w s . A t e q u i l i b r i u m X = [ H a l o t h a n e ] b [ H a l o t h a n e ] g where A = p a r t i t i o n c o e f f i c i e n t [ H a l o t h a n e ] g = e q u i l i b r i u m h a l o t h a n e c o n c e n t r a t i o n i n the gas phase [ H a l o t h a n e ] b = e q u i l i b r i u m h a l o t h a n e c o n c e n t r a t i o n i n t he b l o o d phase The c o n c e n t r a t i o n u n i t used f o r b o t h p h a s e s has to be the same, so t h a t t he p a r t i t i o n c o e f f i c i e n t i s a d i m e n s i o n l e s s q u a n t i t y . - 90 -Therefore i f the halothane i n the a r t e r i a l blood i s i n e q u i l i b r i u m w i t h halothane i n the a l v e o l a r gas X = [Halothane] A [15] [Halothane]a where [Halothane]A = halothane concentration i n a r t e r i a l blood [Halothane]a = a l v e o l a r halothane concentration The e n d - t i d a l p a r t i a l pressure i s expressed as the % of 1 atmosphere, thus i t i s numerically equal to the p a r t i a l volume occupied by halothane per 100 ml of gas i n the a l v e o l i . Assuming the i d e a l gas law the volume occupied by 1 mole of halothane at 37°C and 1 atmosphere pressure i s 2.54 x 10 4 ml. where ET = e n d - t i d a l p a r t i a l pressure of halothane expressed i n % of 1 atmosphere. Then [Halothane]a = ET ( i n mole/100 ml) 2.54 x 10 4 or [Halothane]a = (ET)(MW of Halothane)10 3 ( i n mg/100 ml) [16] 2.54 x 10 4 S u b s t i t u t i n g [16] i n t o [15] gives [Halothane]A = X(ET)(MW of HalothaneUO 3 ( i n mg/100 ml) [17] 2.54 x 10 4 - 91 -RESULTS AND DISCUSSION Figures 13-15 show that the appearance of the halothane i n the blood during anaesthesia w i t h constant i n s p i r e d l e v e l s of 1.0, 1.5 and 2.0 percent (of 1 atmosphere) r e s p e c t i v e l y was g e n e r a l l y c h a r a c t e r i z e d by two phases: ( i ) a r a p i d increase of the halothane concentration i n both a r t e r i a l and venous whole blood, during which time the a r t e r i a l halothane concentrations were i n general higher followed by ( i i ) a steady s t a t e , reached 2-3 hours a f t e r i n d u c t i o n of anaesthesia, where the venous halothane concentration g e n e r a l l y approached or equalled the a r t e r i a l halothane concentration. The plasma halothane concen-t r a t i o n s followed the same trends as observed w i t h whole blood, but were always l e s s than those of whole blood. S i m i l a r r e s u l t s were obtained when the i n s p i r e d l e v e l of halothane was maintained at 2.5% ( F i g . 16) except that the venous halothane concentration d i d not reach the a r t e r i a l l e v e l a f t e r 5 h r. As shown i n Tables XIX- XXII, the a r t e r i a l blood pressure throughout the 5 hr period was i n the range of 78-135 mmHg f o r i n s p i r e d halothane l e v e l s of 1.0, 1.5 and 2.0% but was depressed at 2.5%. This may be due to e i t h e r a depressed cardiac output and/or decreased p e r i p h e r a l r e s i s t a n c e . As a t e n t a t i v e explan-a t i o n a given volume of blood may have a longer c i r c u l a t i o n time, f a c i l i t a t i n g gain of halothane from the a l v e o l i and l o s s of halothane to the p e r i p h e r a l t i s s u e s ; both of which f a c t o r s would c o n t r i b u t e to a d i f f e r e n c e i n halothane concentration between the a r t e r i a l and venous blood. Hughes (1973) has shown that " . . . . i n dogs myocardial depression, i n d i c a t e d by marked reductions i n maximum a c c e l e r a t i o n , c o n t r i b u t e d g r e a t l y to the f a l l i n c a r d i a c output and the consequent hypotension w i t h i n s p i r e d concentrations of 1, 2 and 4% halothane." X END-TIDAL 2 f TIME (HR.) ,Fig. 13. Blood and plasma halothane concentration i n a r t e r i a l and mixed venous blood at a constant i n s p i r e d l e v e l of 1.0% halothane. 3| 2h TIME (HR.) F i g . 14. Blood and plasma halothane concentration i n a r t e r i a l and mixed venous blood at a constant i n s p i r e d l e v e l of 1.5% halothane. 3r TIME (HR.) F i g . 15. Blood and plasma halothane concentration i n a r t e r i a l and mixed venous blood at a constant i n s p i r e d l e v e l of 2.0% halothane. TIME (HR.) F i g . 16. Blood and plasma co n c e n t r a t i o n i n a r t e r i a l and mixed venous blood at a constant i n s p i r e d l e v e l of 2.5% halothane. IS1!*!?? " C " p * r i , O B o f a r t eF i «J * l o o d halothane concentration determined experimentally and that calculated from the end-tidal p a r t i a l pressure at 1.0% inspired l e v e l Time (hr) Mean A r t e r i a l Pressure (mo Hg) Temperature (°C) He A r t e r i a l Venous % Halothane A r t e r i a l i n Plasma Venous A r t e r i a l [Halothane](mg/100 ml) Calculated Experimentally Assuming Equilibrium Determined .25 103 39.0 23 10 .50 88 39.0 40.3 39.4 33 36 26 12 1 117 38.8 39.4 - 36 - 28 13 I 2 106 38.8 41.1 - 37 - 31 19 1 4 108 39.2 - - - - 31 22 5 39.3 41.3 - 34 - 32 20 Table XX - Comparison of a r t e r i a l blood halothane concentration determined experimentally and that calculated from the end-tidal p a r t i a l pressure at 1.5Z inspired l e v e l A r t e r i a l [Halothane](mg/100 ml) Time Mean A r t e r i a l Pressure Temperature He Z Halothane i n Plasma Calculated Experimentally (hr) (mm Hg) (°C) A r t e r i a l Venous A r t e r i a l Venous Assuming Equilibrium Determined .25 90 39.3 44.0 - 36 - 35 12 .50 78 39.3 41.2 40.7 37 35 39 17 1 83 39.0 - 40.3 - 38 . 4 2 22 2 102 38.6 - 40.8 - 43 44 25 4 120 . 38.2 45.9 44.4 40 37 46 25 5 113 38.0 46.0 45.6 33 33 47 25 Table XXI - Comparison of a r t e r i a l blood halothane concentration determined experimentally and that calculated from the end-tidal p a r t i a l pressure assuming thermodynamic equilibrium at 2.OX inspired l e v e l _ . „ . . , A r t e r i a l [Halothane](mg/100 ml) Time Mean A r t e r i a l Pressure Temperature He % Halothane i n Plasma Calculated Experimentally (hr) (ma Hg) (°C) A r t e r i a l Venous A r t e r i a l Venous Assuming Equilibrium Determined .25 135 34 16 .75 115 38.5 31.8 32.0 49 52 41 22 1 98 35.7 - 30.3 - 43 42 2 85 35.7 33.1 30.6 46 42 46 22 4 87 39.0 - 28.6 41 48 25 5 95 39.2 28.7 29.1 50 44 48 25 Table XXII - Comparison of a r t e r i a l blood halothane concentration determined experimentally and that calculated from the end-tidal p a r t i a l pressure assuming thermodynamic equilibrium at 2.531 inspired l e v e l A r t e r i a l [Halothane](mg/100 ml) lme Mean A r t e r i a l Pressure Temperature He X Halothane i n Plasma Calculated . Experimental h r ) ("» H8> ( o c> A r t e r i a l Venous A r t e r i a l Venous Assuming Equilibrium Determined .25 53 38.6 45 21 .50 73 38.2 35.4 34.4 43 43 55 23 1 55 38.0 32.9 33.1 39 38 54 28 2 40 38.0 30.8 - 39 - 59 32 4 60 37.8 - 31.1 - 36 59 30 5 63 37.8 32.2 31.0 42 42 60 33 l - 100 -As shown i n Tables XIX-XXII a r t e r i a l and venous blood d i d not d i f f e r s i g n i f i c a n t l y i n the f r a c t i o n of the halothane found i n the plasma. The halothane concentration i n the c e l l f r a c t i o n was always greater than that i n the plasma and appeared to be independent of the time at which the blood sample was taken. Table XXIII shows that t h i s d i s t r i b u t i o n d i d not change appr e c i a b l y a f t e r i n v i t r o e q u i l i b r a t i o n at 37°C for 2 or 4 hr. This suggests that the d i s t r i b u t i o n of halothane between the blood components i s s u f f i c i e n t l y r a p i d to be independent of e i t h e r the a d d i t i o n of halothane from the lung to the blood or the lo s s of halothane from the blood to the t i s s u e s . In Part I I of t h i s work i t was found t h a t , i n human blood, albumin, t r i -g l y c e r i d e , red c e l l membrane and haemoglobin c o n t r i b u t e d s i g n i f i c a n t l y to the s o l u b i l i t y of halothane i n whole blood. I t i s very l i k e l y t h a t , i n dog blood, these blood components a l s o act as c a r r i e r s f o r halothane, although the amounts of halothane c a r r i e d by each component may be d i f f e r e n t . However, the d i s t r i -b u t ion of halothane between c e l l s and plasma i s even more i n favour of the c e l l f r a c t i o n i n dog blood, compared to that i n human blood as described i n Part I I of t h i s study. The reason f o r t h i s d i f f e r e n c e i s not c l e a r . I t i s i n t e r e s t i n g to note that the s l i g h t p o s i t i v e dependence of Ostwald s o l u b i l i t y c o e f f i c i e n t upon haematocrit reported by Steward et a l . (1975) implies a d i s t r i b u t i o n of halothane i n blood i n favour of the c e l l f r a c t i o n . To t e s t whether thermodynamic e q u i l i b r i u m was e s t a b l i s h e d between h a l o -thane i n the a l v e o l i and halothane i n the blood, the e n d - t i d a l halothane p a r t i a l pressure was used to c a l c u l a t e the expected halothane c o n c e n t r a t i o n i n a r t e r i a l blood assuming that e q u i l i b r i u m was a t t a i n e d . This was c a l c u l a t e d from equation [17] (page 90) using the Ostwald s o l u b i l i t y c o e f f i c i e n t Table XXIII - E f f e c t of i n v i t r o e q u i l i b r a t i o n on the i n v i v o d i s t r i b u t i o n of halothane between plasma and c e l l s Blood samples from dogs anaesthetized w i t h halothane were e q u i l i b r a t e d i n v i t r o at 37°C. The d i s t r i b u t i o n of halothane between c e l l s and plasma was then determined. Blood [Halothane] E q u i l i b r a t i o n Time He % Halothane  mg/100 ml (hr) Plasma C e l l s 40 0 2 4 35.6 38 35 35 62 65 65 23 0 2 4 36 36.5 39 39 63 61 61 - 102 -(numerically equal to the blood-gas p a r t i t i o n c o e f f i c i e n t at 1 atmosphere (Eger 1974)) reported by Steward et a l . (1975) and t a k i n g i n t o account the s l i g h t p o s i t i v e dependence of t h i s Ostwald s o l u b i l i t y c o e f f i c i e n t upon haematocrit. As shown i n Tables XIX-XXII the c a l c u l a t e d concentrations were much higher than those determined experimentally. This discrepancy may be due to one or more of s e v e r a l p o s s i b i l i t i e s : ( i ) E r r o r i n the measurement of halothane concentration i n t h i s study due to l o s s of halothane during sampling, ( i i ) e r r o r i n the reported value of the Ostwald s o l u b i l i t y c o e f f i c i e n t ; ( i i i ) the e n d - t i d a l halothane p a r t i a l pressure does not represent a l v e o l a r halothane p a r t i a l pressure; ( i v ) an e r r o r i n the determination of e n d - t i d a l p a r t i a l pressure and (v) the assumption that thermodynamic e q u i l i b r i u m e x i s t s between halothane i n the a l v e o l i and halothane i n a r t e r i a l blood i s i n v a l i d . The f i r s t p o s s i b i l i t y was e l i m i n a t e d i n a c o n t r o l study i n which a human blood sample c o n t a i n i n g (by a n a l y s i s ) 74.8 mg/100 ml halothane was found to con t a i n 74.6 + 2.6 mg/100 ml (n=4) a f t e r being sampled i n the same way as dog blood. The second p o s s i b i l i t y i s u n l i k e l y because the determination of the Ostwald s o l u b i l i t y c o e f f i c i e n t s of halothane i n dog blood i s very simple. I t s value determined by d i f f e r e n t workers agree reasonably w e l l (see Table XVIII) (Cowles et a l . 1975a; Ikeda 1972; Steward et a l . 1975) and they are u n l i k e l y to be i n c o r r e c t . The use of a l i t e r a t u r e value for the Ostwald s o l u b i l i t y coef-f i c i e n t other than that obtained by Steward et a l . (1975) w i l l not s i g n i f i -c a n t l y change the r e s u l t s of the c a l c u l a t i o n . The t h i r d p o s s i b i l i t y may help to account f o r the discrepancy. Eger and Bahlman (1971) argued that the halothane concentration i n unperfused a l v e o l i , present even i n normal i n d i v i d u a l s , was equal to that of the i n s p i r e d l e v e l , - 103 -and was not lowered by l o s s of halothane to the blood. Thus the e n d - t i d a l halothane p a r t i a l pressure which i s an average of a l l the a l v e o l i (perfused and unperfused) i s an over-estimate of that i n the perfused a l v e o l i . This i s only s i g n i f i c a n t , however, when there i s a large d i f f e r e n c e between the end-t i d a l and i n s p i r e d p a r t i a l pressure. Eger and Bahlman (1971) estimated that the e n d - t i d a l p a r t i a l pressure would be 10% higher than the a r t e r i a l p a r t i a l pressure when the i n s p i r e d l e v e l was 50% higher than the e n d - t i d a l p a r t i a l pressure. This e f f e c t would be i n s u f f i c i e n t to account f o r the discrepancy e s p e c i a l l y during the steady s t a t e when the e n d - t i d a l p a r t i a l pressure approached or equalled the i n s p i r e d p a r t i a l pressure. I t i s u n l i k e l y that the e n d - t i d a l p a r t i a l pressure measurement was erroneous because, as expected (Eger 1974), i t showed an i n i t i a l r a p i d r i s e and then l e v e l l e d o f f approaching the i n s p i r e d l e v e l . At t h i s p o i n t the e r r o r i n the e n d - t i d a l measurement must be small and would not be expected to s i g n i f i c a n t l y a f f e c t the c a l c u l a t i o n made from steady s t a t e values where there was le s s than 10% d i f f e r e n c e between the e n d - t i d a l and i n s p i r e d l e v e l s . I t i s l i k e l y , t h e r e f o r e , that there i s no thermodynamic e q u i l i b r i u m between halothane i n the a l v e o l i and halothane i n a r t e r i a l blood. In f a c t , i n any pharmacokinetic theory which takes i n t o account the continuous l o s s of halothane, through metabolism ( S t i e r et a l . 1964; Sawyer et a l . 1971; A t a l l a k and Geddes 1973; Cohen et a l . 1975; Tinker et a l . 1976; Widger et a l . 1976; Cascorbi 1970; Mukai et a l . 1977) by d i f f u s i o n through the s k i n ( S t o e l t i n g and Eger 1969) and prolonged absorption by adipose t i s s u e ( S araiva et a l . 1977b), a chemical p o t e n t i a l gradient of halothane from the a l v e o l i to the a r t e r i a l blood i s necessary f o r the t r a n s f e r of halothane from the i n s p i r e d gas to the - 104 -blood f o r the replenishment of the halothane l o s t during steady s t a t e . The f i n d i n g that MAC d i d not change during prolonged anaesthesia (Eger et a l . 1965) does not mean that e q u i l i b r i u m was e s t a b l i s h e d , but rather the r a t e of h a l o -thane l o s t i s r e l a t i v e l y constant during the steady s t a t e , so that the demand f o r a d d i t i o n a l halothane ( t o compensate f o r the l o s s ) to maint a i n anaesthesia, i s a l s o r e l a t i v e l y constant. I n the two most comprehensive studies (Cowles et a l . 1972; A l l o t t et a l . 1976) i n which experimental r e s u l t s were c o r r e l a t e d w i t h a computation-s i m u l a t i o n model assuming the e q u i l i b r i u m c o n d i t i o n , the values f o r halothane p a r t i a l pressure i n e q u i l i b r i u m w i t h a r t e r i a l blood p r e d i c t e d by the models were s y s t e m a t i c a l l y higher (approximately 7% i n both cases) than the c o r r e s -ponding experimentally determined values, and the di s c r e p a n c i e s were r a t i o n -a l i z e d i n terms of metabolism. The magnitude of the d i f f e r e n c e (50-100%) between the c a l c u l a t e d and measured a r t e r i a l halothane concentrations i s much higher i n t h i s study (Tables XTX-XXII). Although i t i s not c l e a r why the systematic d i f f e r e n c e found here should be d i f f e r e n t from those of Cowles et a l . (1972) and A l l o t t et a l . (1976), they are a l l p o s i t i v e e r r o r s of p r e d i c t i o n i n the same d i r e c t i o n . The r e s u l t s presented here suggest that an experimentally measured halothane p a r t i a l pressure i n e q u i l i b r i u m w i t h a sample of a r t e r i a l blood should be much lower than the corresponding e n d - t i d a l halothane p a r t i a l pressure when the i n s p i r e d halothane concentration i s held constant. Further work w i l l be necessary to t e s t t h i s hypothesis. There i s good agreement between the blood halothane concentrations found i n t h i s study and those reported by other workers. For human p a t i e n t s , Lowe - 105 -and Beckham (1964) reported blood halothane concentrations of approximately 8mg/100 ml during " a n a l g e s i a " and 12-17 mg/100 ml during " s u r g i c a l planes"; whereas A t a l l a h and Geddes (1973) found that the venous halothane concentrations were i n the range of 7.60-10.65 mg/100 ml at the end of 20 min. of halothane anaesthesia at 1%% i n s p i r e d halothane c o n c e n t r a t i o n . For dogs, Chenoweth et a l . (1962) judged that 17.5-22.5 mg/100 g of halothane i n a r t e r i a l blood was s a t i s f a c t o r y f o r major surgery; and Cervenko (1968) found that the halothane concentration i n a o r t i c blood samples were i n the range of 17.5-23.6 mg/100 g " a f t e r exposure to 2% halothane f o r 30 min.". In t h i s study, the a r t e r i a l blood halothane concentrations a f t e r 15 and 45 min of anaesthesia at a constant 2.0% i n s p i r e d halothane concentration were found to be 16 and 22 mg/100 ml r e s p e c t i v e l y . - 106 -CONCLUSIONS Halothane, a r e p r e s e n t a t i v e i n h a l a t i o n general a n a e s t h e t i c , i s c a r r i e d i n human blood by albumin, haemoglobin, red c e l l membrane and t r i g l y c e r i d e - r i c h m i c e l l e s (chylomicrons and very low density l i p o p r o t e i n ) but not i n s i g n i f i c a n t amounts by y - g l o b u l i n at p h y s i o l o g i c concentration. A s i g n i f i c a n t f r a c t i o n of the t o t a l amount of halothane i s also present free ( i . e . d i s s o l v e d ) i n the aqueous phase of blood. I t i s conceivable that halothane i s a l s o c a r r i e d by other blood components, but since a l l the major blood components have been accounted f o r , the r e s u l t s presented here represent a reasonably complete p i c t u r e of the transport of halothane i n human blood. At 37°C, approximately h a l f or more of the halothane i s present i n the c e l l f r a c t i o n of human whole blood. In v i v o studies a l s o showed that more than h a l f of the halothane i n dog blood i s present i n the c e l l f r a c t i o n , although the percentage of halothane i n the c e l l f r a c t i o n i s higher f o r dog blood than f o r human blood. 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