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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 o f 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 in THE FACULTY OF GRADUATE STUDIES (Department o f P a t h o l o g y )  We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA October 1979  ©  Yew C h o i Pang, 1979  In presenting  this thesis in partial  fulfilment of the requirements for  an advanced degree at the University of B r i t i s h 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 by his representatives.  be granted by the Head of my  Department or  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 B r i t i s h Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1WS  D a t e  October 10, 1979  ABSTRACT  Halothane ( 1 , 1 , 1 - t r i f l u o r o - 2 - b r o m o - 2 - c h l o r o e t h a n e ) general  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 p r o c e d u r e  anaesthetic.  was  developed to q u a n t i t a t i v e l y e s t i m a t e  and  o t h e r aqueous f l u i d s .  p o r t which o b v i a t e d aqueous phase.  i s a commonly employed  the h a l o t h a n e c o n c e n t r a t i o n  T h i s used a s p e c i a l l y designed e x t e r n a l  a p r e l i m i n a r y s e p a r a t i o n of the a n a e s t h e t i c  The method was  of  blood  injection  from the  extended to i n c l u d e q u a n t i t a t i v e e s t i m a t i o n  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 ethanol was  over the approximate range 1-100  mg/100 ml.  of and  T h i s a n a l y t i c a l method  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 h a l o t h a n e w i t h major  human b l o o d  components and  the d i s t r i b u t i o n of h a l o t h a n e between c e l l s  and  plasma. The  r e s u l t s o b t a i n e d w i t h an e q u i l i b r i u m d i a l y s i s t e c h n i q u e developed f o r  t h i s study showed t h a t haemoglobin, a l b u m i n , red c e l l membranes 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 halothane i n blood. the aqueous phase.  thus the t r a n s p o r t ,  of  A s i g n i f i c a n t amount of h a l o t h a n e i s a l s o d i s s o l v e d i n The  r e s u l t s suggest t h a t h a l o t h a n e i n t e r a c t s w i t h a  number o f s u r f a c e s i t e s on haemoglobin and was  and  albumin.  finite  When the aqueous phase  s a t u r a t e d w i t h h a l o t h a n e , the average number o f h a l o t h a n e m o l e c u l e s bound  per haemoglobin and  albumin m o l e c u l e was  a p p r o x i m a t e l y 5 and  20 r e s p e c t i v e l y .  I n 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 h a l o t h a n e m o l e c u l e s appeared to be l o c a t e d w i t h i n the h y d r o p h o b i c c o r e , the amount of h a l o t h a n e s o l u b i l i z e d by the m i c e l l e s and membrane  since  increased  - iii  -  w i t h i n c r e a s i n g f r e e h a l o t h a n e c o n c e n t r a t i o n w i t h o u t showing s a t u r a t i o n of hydrophobic s i t e s .  evidence of  The r e s u l t s o b t a i n e d from the e q u i l i b r i u m  d i a l y s i s s t u d i e s 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 o f h a l o t h a n e between the c e l l s and plasma.  T h i s d i s t r i b u t i o n was  a l s o e x p e r i m e n t a l l y determined by  a n a l y s i s o f the 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 plasma a f t e r c e n t r i f u g a t i o n of whole b l o o d samples e q u i l i b r a t e d w i t h h a l o t h a n e .  There was  reasonable  agreement between the r e s u l t s o b t a i n e d by the two methods. The uptake and d i s t r i b u t i o n o f h a l o t h a n e i n dog b l o o d at d i f f e r e n t l e v e l s o f h a l o t h a n e was  inspired  s t u d i e d by a n a l y s i n g the c o n c e n t r a t i o n o f h a l o t h a n e i n  whole b l o o d and plasma o f a r t e r i a l and mixed venous b l o o d at d i f f e r e n t a f t e r the i n d u c t i o n o f a n a e s t h e s i a .  G e n e r a l l y , a steady s t a t e was  a p p r o x i m a t e l y 2 hours a f t e r i n d u c t i o n .  times  reached  The time r e q u i r e d f o r the d i s t r i b u t i o n  o f h a l o t h a n e between the plasma and c e l l s appeared time r e q u i r e d to a t t a i n the steady s t a t e .  to be much s h o r t e r than the  T h i s suggested t h a t the d i s t r i b u t i o n  o f h a l o t h a n e between b l o o d 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 studies 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 b l o o d h a l o t h a n e c o n c e n t r a t i o n c a l c u l a t e d by combining e x p e r i m e n t a l l y determined e n d - t i d a l h a l o t h a n e p a r t i a l p r e s s u r e and  the  literature  v a l u e s f o r the b l o o d 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 e x p e r i m e n t a l l y . T h i s suggested t h a t h a l o t h a n e i n the a l v e o l i h a l o t h a n e i n the a r t e r i a l b l o o d were not i n thermodynamic e q u i l i b r i u m , as commonly a c c e p t e d .  and  - iv TABLE OF CONTENTS ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF TABLES  v i  LIST OF FIGURES  viii  ACKNOWLEDGEMENTS  ix  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 p o r t  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. 2.  A n a l y t i c a l methods S t u d i e s o f the b i n d i n g o f h a l o t h a n e t o b l o o d components a. b.  S a t u r a t i o n concentration o f halothane i n s a l i n e Hemoglobin  41 41 41 42  - v -  3.  c. d. e. f.  Albumin y-globulin Triglyceride r i c h micelles Red c e l l ghosts  42 43 43 44  g.  D i a l y s i s o f b l o o d components a g a i n s t h a l o t h a n e  44  D i s t r i b u t i o n o f h a l o t h a n e between c e l l s and plasma  RESULTS  47 51  1.  Saturation c o n c e n t r a t i o n of halothane i n s a l i n e  51  2.  A d s o r p t i o n o f h a l o t h a n e to haemoglobin  51  3.  A d s o r p t i o n o f h a l o t h a n e to albumin  55  4.  y-globulin  59  5.  A b s o r p t i o n o f h a l o t h a n e to r e d c e l l ghosts  62  6.  A b s o r p t i o n o f h a l o t h a n e 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 o f h a l o t h a n e between the components o f b l o o d  67  8.  D i s t r i b u t i o n o f h a l o t h a n e 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  91  DISCUSSION  CONCLUSIONS  106  BIBLIOGRAPHY  107  - viLIST OF TABLES I II  I n h a l a t i o n a l general anaesthetic  molecules  3  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 o f i n h a l a t i o n general anaesthetics  5  D i s t i l l a 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 o f i n h a l a t i o n general anaesthetics  6  S o l v e n t e x t r a 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 i n h a l a t i o n general anaesthetics  7  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 o f i n h a l a t i o n general anaesthetics  8  VI  Performance c h a r a c t e r i s t i c s o f the flame i o n i z a t i o n d e t e c t o r , e l e c t r o m e t e r and e l e c t r o n i c i n t e g r a t o r  23  VII  G a s - l i q u i d chromatographic a n a l y s i s o f whole b l o o d samples c o n t a i n i n g known c o n c e n t r a t i o n s o f h a l o t h a n e , m e t h o x y f l u r a n e , d i e t h y l e t h e r and e t h a n o l  26  Reported dependence o f the s o l u b i l i t y of h a l o t h a n e on b l o o d components  31  Saturation concentration  52  III IV V  VIII IX X XI XII XIII XIV XV XVI  o f h a l o t h a n e i n s a l i n e a t 4 and 37°C  Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r t h e d i a l y s i s o f haemoglobin. against halothane  53  Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r the d i a l y s i s o f albumin against halothane  57  D i a l y s i s of y - g l o b u l i n against halothane  61  Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r t h e d i a l y s i s o f r e d c e l l ghosts a g a i n s t h a l o t h a n e  62  Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r the d i a l y s i s o f 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  65  D i s t r i b u t i o n o f h a l o t h a n e i n b l o o d 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  69  Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r the d i s t r i b u t i o n o f h a l o t h a n e between c e l l s and plasma  70  XVII XVIII  vii-  D i s t r i b u t i o n o f h a l o t h a n e between c e l l s and plasma  72  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  87  at 37°C  XIX  Comparison of a r t e r i a l b l o o d h a l o t h a n e c o n c e n t r a t i o n determined e x p e r i m e n t a l l y and t h a t 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 p r e s s u r e 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  96  XX  Comparison of a r t e r i a l b l o o d h a l o t h a n e c o n c e n t r a t i o n determined e x p e r i m e n t a l l y and t h a t 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 p r e s s u r e 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  97  XXI  Comparison o f a r t e r i a l b l o o d h a l o t h a n e c o n c e n t r a t i o n determined e x p e r i m e n t a l l y and t h a t 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 p r e s s u r e 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  98  XXII  Comparison of a r t e r i a l b l o o d h a l o t h a n e c o n c e n t r a t i o n determined e x p e r i m e n t a l l y and t h a t 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 p r e s s u r e 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  99  XXIII  E f f e c t o f 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 h a l o t h a n e between plasma and c e l l s  101  - viii  -  LIST OF FIGURES 1.  Diagrammatic r e p r e s e n t a t i o n o f the f r o n t view o f the e x t e r n a l i n j e c t i o n port  12  2.  E n g i n e e r i n g drawings  13  3.  Schematic  4.  Sample chromatograms from the a n a l y s i s o f h a l o t h a n e , m e t h o x y f l u r a n e , d i e t h y l e t h e r and e t h a n o l  24  5.  Diagrammatic r e p r e s e n t a t i o n o f the e q u i l i b r i u m d i a l y s i s assembly  45  6.  A d s o r p t i o n o f h a l o t h a n e to haemoglobin  54  7.  S c a t c h a r d P l o t o f h a l o t h a n e b i n d i n g to haemoglobin  56  8.  A d s o r p t i o n o f h a l o t h a n e to albumin  58  9.  S c a t c h a r d P l o t o f h a l o t h a n e b i n d i n g to albumin  60  10.  A b s o r p t i o n o f h a l o t h a n e to red c e l l ghosts  64  11.  A b s o r p t i o n o f h a l o t h a n e 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 c o n s t a n t t r i g l y c e r i d e concentration  66  12.  A b s o r p t i o n o f h a l o t h a n e 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 c o n s t a n t free halothane c o n c e n t r a t i o n  68  13.  Blood and plasma h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d at c o n s t a n t i n s p i r e d l e v e l o f 1.0%  92  14.  B l o o d and plasma h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d at c o n s t a n t i n s p i r e d l e v e l o f 1.5%  93  15.  Blood and plasma h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d at c o n s t a n t i n s p i r e d l e v e l o f 2.0%  94  16.  Blood and plasma h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d at c o n s t a n t i n s p i r e d l e v e l o f 2.5%  95  of the e x t e r n a l i n j e c t i o n p o r t  c a r r i e r gas f l o w diagram o f the e x t e r n a l i n j e c t i o n p o r t  15  - ixACKNOWLEDGEMENTS Dr. P.E. R e i d and Dr. D.E. Brooks, my r e s e a r c h 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 a d v i c e , s u p p o r t , t o l e r a n c e , and above a l l ,  their  good sense o f humour. I would l i k e t o thank Jan R e i d f o r h e r h o s p i t a l i t y , P r o f e s s o r C.F.A. C u l l i n g f o r h i s h e l p , i n p a r t i c u l a r f o r showing me t h a t ghosts a r e v i s i b l e , Dr. K.M. L e i g h t o n f o r h i s a c t i v e support o f t h i s r e s e a r c h 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 e x p e r i m e n t s ,  Dr. G. Gray f o r the haemoglobin  a n a l y s e s , Dr. W. Ramey f o r the arrangement o f the "hot room" f a c i l i t i e s , Janzen and M i c h a e l 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  e x p e r t p r e p a r a t i o n o f the m a n u s c r i p t , Dr. D. Vance f o r a sample o f o l e i c and the f o l l o w i n g b l o o d donors:  acid,  Dr. P.E. R e i d , C h a r l e s Ramey, Dr. R.H.  P e a r c e , Amir A l - S u h a i l and Dorothy E m s l i e . many u n i t s o f outdated  Johan  The Red Cross a l s o k i n d l y s u p p l i e d  blood.  T h i s r e s e a r c h 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 t o Dr. D. A p p l e g a r t h f o r a Graduate  A s s i s t a n t s h i p and t o the U n i v e r s i t y o f B.C. f o r a Summer Research  Scholarship  and U n i v e r s i t y Graduate F e l l 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 o n l y d i d he  d e s i g n and c o n s t r u c t t h e 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 r e s c u e d t h i s p r o j e c t more than once, i . e . t r o u b l e - s h o t e s s e n t i a l p i e c e s o f equipment which t h r e a t e n e d to withdraw t h e i r s e r v i c e s from time t o time. L a s t , b u t by no means l e a s t , I would l i k e t o mention C h a r l e s 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 , p r o v i d e d t e c h n i c a l h e l p , and m a i n t a i n e d an optimum management o f the l a b o r a t o r y , a l l o f which c o n t r i b u t e d to making r e s e a r c h an e n j o y a b l e  experience.  - x -  ABBREVIATIONS ESR  — Electron spin  G  - Gibbs f r e e energy  G"  - Chemical p o t e n t i a l  GLC  - Gas-liquid  MAC  -  MAP  - Minimum a l v e o l a r p a r t i a l p r e s s u r e  mg%  - Milligrams  MW  - M o l e c u l a r weight  n  - Number o f moles  N  - Avogradro's number  NMR  - N u c l e a r magnetic  P  - Pressure  P  H  resonance  chromatography  Minimum a l v e o l a r c o n c e n t r a t i o n  per 100 grams  resonance  - P a r t i a l pressure of halothane  PBS  - Phosphate b u f f e r  R  - U n i v e r s a l gas c o n s t a n t  T  - Absolute  TG  - Triglyceride  Ul  - Microlitre  V  - Volume  V  - P a r t i a l s p e c i f i c volume  saline  temperature  VLDL - Very Low D e n s i t y  Lipoprotein  - xl -  «&*  % M fk &  *fo  AS* #3  *\\  infart 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 g e n e r a l a n a e s t h e t i c i n N o r t h America,  and has a v e r y h i g h potency  a n a e s t h e t i c s (Saidman et a l . 1967;  compared to o t h e r  S t o e l t i n g et a l . 1970;  inhalation  Eger et a l . 1965b).  I t s s o l u b i l i t y i n b l o o d i s much h i g h e r than t h a t i n water or s a l i n e et a l . 1973).  T h i s c l e a r l y suggests  (Steward  t h a t h a l o t h a n e , i n a d d i t i o n to b e i n g  d i s s o l v e d i n the aqueous phase o f b l o o d , i s a l s o a s s o c i a t e d w i t h , or c a r r i e d by, one or more b l o o d components. evidence p r e s e n t e d  As y e t t h e r e has been no c o n c l u s i v e  to show t h a t the i n c r e a s e d s o l u b i l i t y o f h a l o t h a n e  as compared to s a l i n e i s due to any major b l o o d component. was  i n blood  The p r e s e n t  study  aimed at e l u c i d a t i n g which are the important b l o o d components c o n t r i b u t i n g  to the t r a n s p o r t o f h a l o t h a n e  and how  significant i s this contribution in  human whole b l o o d . The approaches commonly taken f o r 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 o f i n h a l a t i o n a n a e s t h e t i c s are based on the concept e q u i l i b r i u m thermodynamics.  S i n c e a l i v i n g organism  of  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 o f an e q u i l i b r i u m thermodynamical concept may  not be t e n a b l e .  i n the treatment  of the k i n e t i c s of a n a e s t h e t i c uptake  T h e r e f o r e i n v i v o experiments  t e s t the v a l i d i t y o f the e q u i l i b r i u m assumption. experiments  were c a r r i e d out i n dogs to In a d d i t i o n ,  these  p r o v i d e d d e t a i l e d i n f o r m a t i o n on the b l o o d l e v e l o f a n a e s t h e t i c  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 g e n e r a l a n a e s t h e t i c s are e i t h e r gaseous or v o l a t i l e compounds commonly employed i n s u r g e r y to produce a n a e s t h e t i c e f f e c t s . the c h e m i c a l  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 a n a e s t h e t i c s .  s i m p l e , e i t h e r s h o r t c h a i n hydrocarbons (eg. c y c l o p r o p a n e ) diethylether). by h a l o g e n s .  T a b l e I shows These are v e r y  or e t h e r s  (eg.  The more r e c e n t l y d i s c o v e r e d a n a e s t h e t i c s are a l l s u b s t i t u t e d Halothane (1956) i s a h y d r o c a r b o n s u b s t i t u t e d by  fluorine,  c h l o r i n e and bromine w h i l e f l u r o x e n e ( 1 9 5 3 ) , m e t h y o x y f l u r a n e  and  (1967) are e t h e r s 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  physical,  c h e m i c a l and a n a e s t h e t i c p r o p e r t i e s o f these h a l o g e n a t e d  agents have been  e x t e n s i v e l y reviewed  by Rudo and K r a n t z  (1974).  C l i n i c a l l y the amount o f a n a e s t h e t i c s a d m i n i s t e r e d  i s monitored  r e d a b s o r p t i o n w i t h a c c u r a t e l y c a l i b r a t e d f l o w meters (Wolfson method i s u n s u i t a b l e f o r e x p e r i m e n t a l gas phase can be a n a l y s e d .  by  1968).  Much e f f o r t has been d i r e c t e d to develop  methods, i n c l u d i n g a u t o r a d i o g r a p h y  (Cohen et a l . 1972), mass  et a l . 1953), and e l e c t r o e n c e p h a l o g r a p h y  been i n v e s t i g a t e d f o r t h i s purpose.  infraThis  r e s e a r c h because o n l y a n a e s t h e t i c i n the  f o r a n a l y s i n g v a r i o u s a n a e s t h e t i c s i n b l o o d and t i s s u e samples.  (Jones  enflurane  (Wolfson  Various  spectroscopy  et a l . 1967)  G a s - l i q u i d chromatography (GLC)  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 i n e x p e n s i v e and t h e o r y u n d e r l y i n g i t s o p e r a t i o n i s w e l l understood.  GLC  a method  have i s by f a r the  p r o c e d u r e s f o r the  q u a n t i t a t i v e a n a l y s i s o f i n h a l a t i o n a n a e s t h e t i c s i n b l o o d and o t h e r f l u i d s  are  o f two  on  types:  e i t h e r d i r e c t , i n which the gas chromatography i s performed  - 3Table I - I n h a l a t i o n general a n a e s t h e t i c molecules  H  H H  I  I  Cl-C-Cl  1  H H 11  H-C-C-O-C-C-H  I  H H  Cl Chloroform  F Cl  I I  F-C-C-H  I I  H H  Ether  Cl F  H  I I  I  I I  I  H-C-C-O-C-H  I I  F Br Halothane  Cl F H Methoxyflurane  H Cl F H-C H  >C-H H  Cyclopropane  I I  I  F-C-C-O-C-CH-5  III  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 t h e a n a e s t h e t i c i s s e p a r a t e d from t h e  sample p r i o r t o 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  further i n t o three general classes:  i n T a b l e I I . They can be d i v i d e d  ( a ) those i n which t h e sample i s i n j e c t e d  i n t o t h e i n j e c t i o n p o r t o f t h e chromatograph, made i n t o a removable g l a s s l i n e r  (b) those i n which i n j e c t i o n i s  i n s e r t e d i n t o t h e 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 d e v i c e i s o l a t e d the  columns  from  and t h e n , a f t e r a p e r i o d o f t i m e , t h e v o l a t i l e s a r e swept onto t h e  column by a stream o f c a r r i e r gas v i a a s w i t c h i n g 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 , a p p a r e n t l y s i m p l e and can be used w i t h s m a l l samples o f b l o o d (Yamamura e t 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 c o n t a m i n a t i o n o f t h e columns w i t h n o n - v o l a t i l e b l o o d components ( B u t l e r e t a l . 1967; Kolmer e t a l . 1975a; Cousins and Mazze 1972) and subsequent b a s e l i n e d r i f t  due t o t h e slow e l u t i o n o f such components  ( B u t l e r e t a l . 1967; Cousins and Mazze 1972), c l o g g i n g o f the s y r i n g e s used for  i n j e c t i o n (Kolmer e t a l . 1975a; Cousins and Mazze 1972), d i s t o r t i o n and  b r o a d e n i n g o f the peaks ( B u t l e r e t a l . 1967; Kolmer e t a l . 1975; Jones e t a l . 1972; C o u s i n s and Mazze 1972), ghost peaks (Kolmer e t a l . 1975; C o u s i n s and Mazze 1972) and poor r e p r o d u c i b i l i t y (Yamamura e t a l . 1966; B u t l e r e t a l . 1967).  2.  I n d i r e c t Methods The i n d i r e c t GLC methods a r e l i s t e d  (a)  distillation,  i n T a b l e s I I I , IV and V.  i n which t h e a n a e s t h e t i c s a r e d i s t i l l e d  They a r e :  or v a p o r i z e d from  T a b l e 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 f o r t h e q u a n t i t a t i v e e s t i m a t i o n o f i n h a l a t i o n general anaesthetics i n blood  Method  Precolumn Device  Lowe 1964 None Lowe and Beckham 1964  Anaesthetics Analysed Cyclopropane Ether Methoxy f l u r a n e Halothane Chloroform Trifluoroethylv i n y l ether  Detector  Calibration Method  Stationary Phase  Flame Ionization  Peak Height Calibration  Chromosorb P  Approximate Range of s e n s i t i v i t y (mg/100 m l )  L a s s b e r g and E t s t e n 1965  Glass liner  Cyclopropane  Flame Ionization  Peak Area Calibration  DC550 Silicone O i l  C o u s i n s and Mazze 1972  Glass liner  Halothane Methoxyfluorane  Flame Ionization  Peak Area Calibration  OV101  Douglas et a l . 1970  Glass liner  Halothane  Flame Ionization  Peak H e i g h t Calibration  Chromosorb P  Y o k o t a et a l . 1967  "Vaporizing apparatus"  Cyclopropane Halothane Methoxyflurane D i e t h y l ether N i t r o u s oxide  Flame Peak Height ' E t h y l e n e g l y c o l Ionization Calibration succinate and thermal conductivity  Cole et a l . 1975  "Precolumn Device"  Halothane Methoxyflurane  Flame Ionization  NG:  not given  Anesthetic to I n t e r n a l Standard Peak H e i g h t R a t i o  3-40  10-70 2-40  FFAP  20-50  NG  5-40  T a b l e I I I - D i s t i l l a t i o n Methods f o r t h e q u a n t i t a t i v e a n a e s t h e t i c s i n b l o o d by g a s - l i q u i d chromatography  estimation of inhalation  general  Method  Anaesthetic Analysed  Detector  Calibration Method  Stationary Phase  Gadsden e t a l . 1965  Halothane  Thermal Conductivity  Peak Height Calibration  di-2-ethylhexyl sebacate  9-55  Rackow e t a l . 1966  Diethylether  Flame Ionization  Peak Area Calibration  NG  NG  Approximate Range of s e n s i t i v i t y (mg/100 ml)  ON  NG:  not g i v e n  i  - 7 -  Table IV - Solvent extraction methods for the q u . n t i t . t i * . estimation of inhalation general anaesthetics i n blood bv gas-liquid chromatography '  Anaesthetics Anelyead  Calibration Method  Stationary Phase  Approximate range ef s e n s i t i v i t y (Kg/100 a l )  Butler and H i l l 1961  Flai Ion  Peak Beight Calibration  n-fieptane  Silicone P l a i d MS 350  1-20  Rut ledge ct a l . 1963  Thermal Conductivity  Peak Height Calibration  n—Heptane  Tide  5-20  Plane lonisation  Anaesthetic to Internal Standard (toluene) Peak Height katio  Carbon disulphide  SB 50  4-15  Anaesthetic to Internal Standard (chloroform) Peak Height t a t i o  Carbon tetrachloride  8X30  Wolfeon at a l . 1966a  Methoxyflurane  Wolfson et a l . 1966b  Halothane Ether  Hartley et a l . 1968  Plaae Ionization  Peak Height Calibration  n-Heptane  Silicone Pluid KS 550  Cervenko 1968  Plane Ion ication  Anaesthetic to Internal Standard (diethylether) Peak Height Ratio  Carbon tetrachloride  Silicone O i l MS 550  Douglas et a l . 1970  Electros Capture  Peak Area Calibration  n-Heptane  Silieooe Pluid KS 550  Allotc et a l . 1971  Plane Ionization  Anaesthetic to Internal Standard (chloroform) Peak Height t a t i o  Carbon tetrachloride  Jones et a l . 1972  Methoxyflurane  PIa  Peak Area Calibration  Silicone Pluid MS 200/20 C.S.  SZ30  Attallah and Creddes 1972  Halothane  Electron Capture  Peak Beight Calibration  n-Heptane  Silicone Pluid MS 550  Electron Capture  Peak Height Calibration  Oavii et a l . 1975 E l l i a and Sto*1 ting 1975  Pluroxene Halothane Methoxyflurane  Poobalaaingaa 1976  Halliday et a l . 1977  Toner et a l . 1977  •C:  not given  Peek Height Calibration Plane Ion ication  Cyclopropane Tr i ch1oroethene  Rnflurene  Electron Capture  Diiaodecyl phthalate Tetraehloro•thylene  Anaesthetic to Carbon Internal Standard Disulphide ( cr i ch 1 or oe t hy 1 en e ) Peak Height Ratio Anaesthetic to Internal Standard (chloroform, toluene) Peak Height Ratio  Carbon tetrachloride Carbon Disulphide  Anaesthetic to Internal Standard (methoxyflarene) Peek Beight Ratio  B-Beptaj  S i l i c o n e Pluid MS 550  0.5-8 i ole/1 0.5-4 i ole/I  Poropack Q  - 8-  Table V - Head space methods for the q u a n t i t a t i v e e s t i m a t i o n o f i n h a l a t i o n general a n a e s t h e t i c s i n blood by gas-1iquid chromatography  Method  Anaesthetic Analysed  Detector  Calibration Method  Stationary Phase  Noehren and Cudmore 1961  Diethylether  NG  Peak Height Calibration  Tetraethylene g l y c o l dimethylether  Bowes 1964  N i t r o u s oxide  Thermistor  Peak Height Calibration  Dimethylsulphoxide  Yamamura et a l . 1966  Halothane Methoxyflurane Cyclopropane Ether N i t r o u s oxide  Flame Ionization Thermal Conductivity  Peak Height Calibration  PEG  Approximate Range of S e n s i t i v i t y  25-168 mg/100 ml  NG  10-20 mg/100 ml  DOP A c t i v a t e d charcoal  100-200 mg/100 ml 10-100 mg/100 ml  B u t l e r et a l . 1967  Halothane  Flame Ionization  Peak Height Calibration  S i l i c o n e gum rubber  Theye 1968  Halothane  Thermal Conductivity  Peak Height Calibration  Amine 220 and Carbowax 400  Fink and Morikawa 1970  Halothane  Flame Ion i z a t i o n  Peak Height Calibration  SE30  Wagner et a l . 1974  Halothane Ether Cyclopropane  Flame I on iz at ion  Peak Height Calibration  Poropak T  10" -0.1X (v/v)  Kolmer et a l . 1975a  Halothane  Flame Ion i z a t i o n  Peak Height Calibration  Porasil S Carbowax 400  lppm-52 (v/v)  Heavner et a l . 1976  Halothane  Flame Ionization  Peak Height Calibration  Poropak Q  Renzi and Waud 1977  Enflurane Ether Fluroxene Halothane Isothurane Methoxyflurane  NC  Peak Height Calibration  NG  NG:  not given  NG  0.3-3.82 (v/v)  NG  4  0.02-2.5Z (v/v)  NG  - 9 the sample and s u b s e q u e n t l y t r a n s f e r r e d on t o the column,  (b) s o l v e n t  e x t r a c t i o n , i n which the a n a e s t h e t i c s are e x t r a c t e d from the sample by  direct  m i x i n g w i t h an o r g a n i c phase, an a l i q u o t o f which i s s u b s e q u e n t l y 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 a l l o w e d to  e q u i l i b r a t e w i t h a f i x e d amount o f a i r and a f t e r e q u i l i b r i u m i s r e a c h e d , 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 p r o c e d u r e s , d e s i g n e d t o s e p a r a t e the a n a e s t h e t i c s from the sample, are used t o c i r c u m v e n t the above mentioned encountered i n the d i r e c t i n j e c t i o n methods. a l s o can produce d i f f i c u l t i e s .  difficulties  However, t h e s e p r e t r e a t m e n t s  They are time consuming  (Yamamura et a l . 1966;  B u t l e r et a l . 1967; Kolmer 1975a; Y a k o t a et a l . 1967; C o u s i n s and Mazze  1972;  C o l e et a l . 1975), i n v o l v e a p o t e n t i a l l o s s of the agent ( C o u s i n s and Mazze 1972) and may be d i f f i c u l t t o a p p l y to s m a l l samples ( C o u s i n s and Mazze 1972).  F u r t h e r m o r e , s o l v e n t e x t r a c t i o n o f the a n a e s t h e t i c r e s u l t s i n the  c o n c u r r e n t e x t r a c t i o n o f l i p i d m a t e r i a l s from the b l o o d or t i s s u e  samples.  These w i l l be d e p o s i t e d onto the column upon i n j e c t i o n , p a r t i a l l y  offsetting  the i n t e n d e d advantage o f the method ( B u t l e r 1967). l e a d s to l a r g e s o l v e n t peaks which may  Solvent e x t r a c t i o n also  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  a n a e s t h e t i c peak and/or r e s u l t i n p r o l o n g e d e l u t i o n times ( W o l f s o n et a l . 1966a; Douglas et a l . 1970; Jones et a l . 1972). The head space method, a l t h o u g h f r e e o f the t e c h n i c a l d i s a d v a n t a g e s o f s o l v e n t 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 c o n c e n t r a t i o n of the anaest h e t i c i n the sample, but o n l y the p a r t i a l p r e s s u r e o f the a n a e s t h e t i c 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, u n l e s s the a n a e s t h e t i c  p a r t i a l p r e s s u r e o f the sample i s the d e s i r e d r e s u l t , another method s h o u l d be used i f p o s s i b l e .  - 10 For the reasons d i s c u s s e d above i t was  c o n s i d e r e d d e s i r a b l e to develop  a  s i m p l e and r e l i a b l e a n a l y t i c a l method f o r e x p e r i m e n t a l a n a e s t h e t i c s r e s e a r c h . The method was  to be a p p l i c a b l e to s m a l l samples, g i v e r e s u l t s d i r e c t l y i n  c o n c e n t r a t i o n u n i t s , and sample.  To t h i s end,  i n v o l v e a minimum amount of p r e t r e a t m e n t  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  p l a c e d i n the c a r r i e r gas stream b e f o r e the column, was d i r e c t i n j e c t i o n o f a b l o o d sample i n t o the preheated the gas chromatograph.  o f the  used which a l l o w e d  c a r r i e r gas  stream o f  E v a p o r a t i o n at a h i g h 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 o n l y the v o l a t i l e components e n t e r the column.  T h i s e l i m i n a t e s the c o n t a m i n a t i o n  problem  encountered i n o t h e r d i r e c t i n j e c t i o n methods and p e r m i t s the r a p i d a n l y s i s o f halothane, methoxyflurane, approximate range 1-100  d i e t h y l e t h e r and e t h a n o l i n whole b l o o d over  the  mg/100 ml.  *The e x t e r n a l i n j e c t i o n p o r t was  designed  and c o n s t r u c t e d by Mr.  R o l f Muelchen.  MATERIALS AND METHODS  H a l o t h a n e (Hoechst  P h a r m a c e u t i c a l s , M o n t r e a l , 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 ( s p e c t r o a n a l y s e d ; F i s h e r S c i e n t i f i c , M o n t r e a l , Canada) were used w i t h o u t f u r t h e r p u r i f i c a t i o n . I s o b u t a n o l ( r e a g e n t grade; M a l l i n c k r o d t , S t . L o u i s , Mo., U.S.A.) was d r i e d over anhydrous p o t a s s i u m carbonate  and p u r i f i e d by f r a c t i o n a l  distillation  (Vogel 1954).  E t h a n o l was p u r i f i e d by t h e use o f a G r i g n a r d reagent  ( V o g e l 1954).  Vacuum grease  procedure  (Dow, M i d l a n d , M i c h i g a n ) was o b t a i n e d from  F i s c h e r S c i e n t i f i c , M o n t r e a l , Canada.  Swagelok f i t t i n g s ( C r a w f o r d  Fitting  Company, S o l o n , Ohio) were o b t a i n e d from Columbia V a l v e & F i t t i n g Co., Vancouver.  A l l o t h e r c h e m i c a l s employed were o f reagent grade o r b e t t e r .  S i l i c o n e rubber 0 - r i n g s 3/16" I.D. and 5/16" O.D. HT8 low b l e e d s e p t a were o b t a i n e d from A p p l i e d S c i e n c e , S t a t e C o l l e g e , P a . , U.S.A. 103,  105, and 107 were purchased  Chromosorb 101,  from Johns M a n v i l l e , Denver, C o l o . , 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 v a l v e s from P i e r c e , R o c k f o r d , 111., U.S.A.  These v i a l s were s u p p l i e d w i t h g a s - t i g h t t e f l o n l i n e d s e a l s and  served as l e a k - p r o o f c o n t a i n e r s f o r h a l o t h a n e . equipped was  w i t h a septum t o prevent  opened f o r sampling.  Each M i n i n e r t v a l v e was  l o s s o f v o l a t i l e compounds when the v a l v e  Gas chromatographic  s y r i n g e s were o b t a i n e d from  H a m i l t o n Co., Reno, Nevada, U.S.A.  The e x t e r n a l i n j e c t i o n p o r t Design The  d e s i g n o f the e x t e r n a l i n j e c t i o n p o r t i s shown d i a g r a m m a t i c a l l y i n  F i g . 1; F i g . 2 shows d e t a i l e d e n g i n e e r i n g drawings.  The e x t e r n a l i n j e c t i o n  - 12 -  TO GLC  F i g . 1. Diagrammatic r e p r e s e n t a t i o n o f the f r o n t v i e w o f the e x t e r n a l i n j e c t i o n port.  - 13 -  F i g . 2.  E n g i n e e r i n g drawings o f the e x t e r n a l i n j e c t i o n p o r t .  - 14 p o r t c o n s i s t s e s s e n t i a l l y o f a heat r e s e r v o i r and a r o t a r y v a l v e b o t h o f which c o n t a i n channels f o r d i r e c t i n g the f l o w o f 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 v a l v e a r e c o n s t r u c t e d o f b r a s s , t h e i r r e s p e c t i v e p l a n e f a c e p l a t e s being separated  by a 1/32" T e f l o n gasket a g a i n s t which the v a l v e r o t a t e s .  Mounted on the f r o n t o f the r o t a r y v a l v e a r e :  ( a ) a s p r i n g which can be used  to t i g h t e n the v a l v e a g a i n s t the T e f l o n gasket t o a c h i e v e a gas t i g h t  seal,  (b) a gas chromatographic i n j e c t i o n p o r t o f c o n v e n t i o n a l d e s i g n s e a l e d w i t h a septum and ( c ) a p a i r o f h a n d l e s which a l l o w r o t a t i o n o f the v a l v e . r e s e r v o i r i s maintained  a t 180°C w i t h a h e a t e r  The  set into a well d r i l l e d  the body o f the r e s e r v o i r ; a s i m i l a r w e l l c o n t a i n s a thermometer.  into  The base o f  the p o r t i s a t t a c h e d , by a gas t i g h t s e a l , t o the i n j e c t i o n p o r t system o f the gas chromatograph and can be r e a d i l y m o d i f i e d f o r use w i t h a v a r i e t y o f instruments.  The c a r r i e r gas e n t e r s the p o r t through a Swagelok  fitting  s e a l e d i n t o the s i d e o f 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 o f t h e c a r r i e r gas stream p r i o r t o the p o i n t where i t e n t e r s the o r i g i n a l  injection  p o r t o f the gas chromatograph. F i g . 3 i l l u s t r a t e s the f l o w paths o f 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 p o r t when the r o t a r y v a l v e i s i n the "on" o p e r a t i o n a l and t h e " o f f " non-operational  positions.  I n 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, o f the heat r e s e r v o i r and the r o t a r y v a l v e . f i x e d channel connecting The  I t then proceeds v i a the  c l o c a t e d i n the r e s e r v o i r , i n t o a g l a s s U-tube (3/16"  c w i t h the f i x e d channel  O.D.)  d l o c a t e d i n the body o f the r e s e r v o i r .  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 v a l v e and the r e s e r v o i r and e n t e r s the gas chromatography column through the base o f the i n j e c t i o n p o r t .  The U-tube i s  - 15 -  Fig.  3.  Schematic c a r r i e r gas flow diagram o f 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 t h e l e f t s i d e ( s e e F i g . 2) and i s s e a l e d 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 - r i n g s l i g h t l y l u b r i c a t e d w i t h a h i g h temperature vacuum grease.  B e f o r e i n s e r t i o n t h e ends o f t h e U-tube were a l s o l u b r i c a t e d w i t h t h e  same vacuum grease.  A lever attached  to t h e r e s e r v o i r ( s e e F i g . 2) p r e v e n t s  the e j e c t i o n o f t h e U-tube when t h e v a l v e i s i n t h e "on" p o s i t i o n (approximate pressure  20-25 p . s . i . ) and a l s o serves t o m a i n t a i n a gas t i g h t s e a l .  Holes  s i t u a t e d i n t h e a p p r o p r i a t e p o s i t i o n s i n t h e T e f l o n gasket a l l o w f r e e 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 t h e v a l v e i s i n t h e "on" p o s i t i o n t h e needle o f a H a m i l t o n gas c h r o m a t o g r a p h i c s y r i n g e w i l l pass through b o t h t h e septum o f t h e i n j e c t i o n p o r t mounted on the f r o n t o f t h e v a l v e and t h e T e f l o n gasket and e n t e r t h e 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 t h e pre-heated c a r r i e r gas stream. When t h e v a l v e i s i n t h e " o f f " p o s i t i o n t h e U-tube i s by-passed and t h e c a r r i e r gas passes d i r e c t l y i n t o t h e gas chromatograph through channels a, b and  f . T h i s enables t h e U-tube t o be changed simply and r a p i d l y w i t h o u t  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 t o t h e f i r s t o p e r a t i o n o f the e x t e r n a l i n j e c t i o n p o r t t h e apparatus o i s maintained for  a t 180 C a t a p r e s s u r e  on the T e f l o n gasket o f 15-20 pounds  24-36 h r . Under these c o n d i t i o n s t h e T e f l o n gasket s o f t e n s and i s moulded  i n t o the shape o f the i n s i d e s u r f a c e s o f the r o t a r y v a l v e and heat r e s e r v o i r . T h i s ensures a good s e a l and p r o v i d e d remoulding i s o n l y n e c e s s a r y  the r e s e r v o i r i s m a i n t a i n e d  a t 180°C  when a new gasket i s f i t t e d .  W i t h the r o t a r y v a l v e i n the "on" p o s i t i o n a sample o f b l o o d  (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 l o o s e g l a s s wool f i l t e r p l u g i n s e r t e d i n t o t h e  - 17 U-tube.  To a v o i d c l o g g i n g o f t h e n e e d l e o f t h e s y r i n g e by c o a g u l a t e d  components i n j e c t i o n was accomplished w i t h a chaser b l o o d sample i n the s y r i n g e b a r r e l was s e p a r a t e d  technique  blood  i n which the  from a p l u g o f water by a  b u b b l e o f a i r . When the a n a l y s i s i s completed the r o t a r y v a l v e i s s w i t c h e d t o the " o f f " p o s i t i o n and t h e U-tube i s r e p l a c e d .  On s w i t c h i n g t o t h e "on"  p o s i t i o n the equipment i s ready f o r another i n j e c t i o n .  Changing the U-tube  o c c u p i e s between 15 and 60 s e c .  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  steel  column was packed w i t h Chromosorb 101 (80-100 mesh) and c o n d i t i o n e d f o r 16 h r at 200°C w i t h c a r r i e r gas f l o w .  The c o n d i t i o n e d Chromosorb 101 was then  unpacked from t h e 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.  g l a s s columns.  Gas chromatography was performed w i t h these g l a s s columns  temperature programmed (temperature  programming i s a t e c h n i q u e  i n gas  chromatography i n which the temperature o f the columns i s i n c r e a s e d from an i n i t i a l t o a f i n a l temperature a t a p r e d e t e r m i n a t e d 6°C/min i n a H e w l e t t - P a c k a r d ionization detectors.  r a t e ) from 110-180°C a t  7610A gas chromatograph f i t t e d w i t h d u a l flame  The i n j e c t i o n p o r t o f t h e chromatograph was  maintained  at 200°C, the e x t e r n a l i n j e c t i o n p o r t at 180°C and the flame i o n i z a t i o n d e t e c t o r a t 250°C.  Gas f l o w - r a t e s 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 o f the e l e c t r o m e t e r was s e t a t 8 10  ohms. Peak areas were measured w i t h a H e w l e t t - P a c k a r d  operated  3370B e l e c t r o n i c i n t e g r a t o r  a t "Up" and "Down" s l o p e s e n s i t i v i t i e s o f 0.1 mV/min.  Injection  volumes were chosen t o y i e l d a minimum a n a e s t h e t i c peak area o f 5 x 10 sec whenever p o s s i b l e , o t h e r w i s e prepared  40 u l samples were i n j e c t e d .  uV  A l l samples  f o r a n a l y s i s o f a n a e s t h e t i c c o n c e n t r a t i o n s were k e p t a t 0-4°C  d u r i n g sampling.  The s y r i n g e was r i n s e d once w i t h 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  s y r i n g e by n o n - v o l a t i l e b l o o d components a f t e r repeated  c l o g g i n g o f the  injections.  To o b v i a t e d a i l y c a l i b r a t i o n o f 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 o f t h e d e t e c t o r response f o r t h e  a n a e s t h e t i c and the s t a n d a r d , d i v i d e d by the r a t i o o f t h e i r w e i g h t s i n a sample i s a c o n s t a n t  f o r the type o f d e t e c t o r used i n t h i s study,  t h a t the d e t e c t o r i s n o t s a t u r a t e d (Novak et a l . 1970).  provided  Hence t h i s r a t i o , o r  response f a c t o r , was employed t o determine t h e a n a e s t h e t i c  concentrations.  Response f a c t o r i s d e f i n e d by the e q u a t i o n : Response f a c t o r = peak area agent peak area i s o b u t a n o l x  wt. 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 . fill  [1]  I s o b u t a n o l was added t o h a l f  a preweighed 3 ml R e a c t i - v i a l equipped w i t h a magnetic s t i r r e r and a  Mininert valve. pletely f i l l  T h i s was weighed.  The a n a e s t h e t i c was then added t o com-  t h e v i a l , which was a g a i n weighed.  A f t e r thorough m i x i n g , 2 u l  o f t h e m i x t u r e was t r a n s f e r r e d , u s i n g a H a m i l t o n 10 u l s y r i n g e , i n t o a n o t h e r 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 b l o o d w i t h EDTA.  T h i s was t h o r o u g h l y mixed f o r 2 h r a t 0-4°C.  anticoagulated  Samples from t h i s  b l o o d m i x t u r e were a n a l y s e d by gas chromatography. Analyses  o f b l o o d samples c o n t a i n i n g a n a e s t h e t i c s were performed on EDTA  a n t i c o a g u l a t e d b l o o d u s i n g 0.3 ml R e a c t i - v i a l s ( a c t u a l volume  approximately  0.5  ml)  19  each equipped w i t h 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 t h a t the r e m a i n i n g volume, when the v i a l  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 o f i s o b u t a n o l i n water ( a p p r o x i m a t e l y g) was  added and the v i a l was  analysed  (450 u l ) .  The  completely  260 mg  i n t e r n a l standard  were e s t a b l i s h e d by w e i g h i n g the v i a l at the a p p r o p r i a t e t i m e s .  c o n c e n t r a t i o n o f the a n a e s t h e t i c was  per  100  f i l l e d w i t h the b l o o d sample to be  q u a n t i t i e s o f b l o o d and  were mixed and gas chromatography was  was  employed The  performed on a l i q u o t s o f 4-40 c a l c u l a t e d from the  samples ul.  The  formula:  A n a e s t h e t i c c o n c e n t r a t i o n = peak area a n a e s t h e t i c 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. b l o o d sample x response f a c t o r [2] where I.S. = i n t e r n a l s t a n d a r d  solution.  T h i s g i v e s the a n a e s t h e t i c c o n c e n t r a t i o n i n weight o f a n a e s t h e t i c per u n i t weight o f b l o o d .  I t can be converted  to a volume c o n c e n t r a t i o n by m u l t i p l y i n g  by the d e n s i t y o f b l o o d , which i s e s s e n t i a l l y a c o n s t a n t 1970).  (Diem and  Lentner,  S i m i l a r l y the volume c o n c e n t r a t i o n of an a n a e s t h e t i c i n any medium can  be o b t a i n e d by m u l t i p l y i n g the weight c o n c e n t r a t i o n c a l c u l a t e d from [2] w i t h the d e n s i t y o f the medium. a b l e from the l i t e r a t u r e , i t was d e n s i t y found was  When the r e q u i r e d d e n s i t y was  e x p e r i m e n t a l l y determined.  s u f f i c i e n t l y c l o s e t o u n i t y and was  equation not  avail-  I n most cases the  not a s i g n i f i c a n t  cor-  rection factor. The  e f f e c t o f s t o r a g e on the h a l o t h a n e  t e s t e d as f o l l o w s .  Standard s o l u t i o n s o f h a l o t h a n e  d i r e c t weighing of halothane for  2 hrs at 4°C.  c o n c e n t r a t i o n s o f b l o o d samples i n b l o o d were prepared  i n EDTA a n t i c o a g u l a t e d b l o o d .  was by  These were mixed  R e a c t i - v i a l s (1.0 m l ) , c o n t a i n i n g a g l a s s bead, were  - 20 f i l l e d w i t h these b l o o d samples i n such a manner t h a t no a i r bubble was t r a p p e d i n the v i a l .  To a c h i e v e t h i s , the v i a l was o v e r f i l l e d w i t h b l o o d , the  g l a s s bead was dropped i n t o the v i a l , horizontally  into place, displacing  screwed on f i n g e r - t i g h t .  then the t e f l o n l i n e d s e a l was  the excess b l o o d , and the cap was  slid then  The samples were s t o r e d a t 4° f o r 7 and 14 days,  t h o r o u g h l y mixed and a n a l y s e d by gas chromatography.  - 21  -  RESULTS  An e x t e r n a l i n j e c t i o n p o r t temperature of 180"C s a r y to m a i n t a i n  a gas  area for evaporation components. pre-heating  The  found to be  t i g h t s e a l between the T e f l o n gasket and  p l a t e s of the r o t a r y v a l v e and g l a s s wool p l u g served  was  the heat r e s e r v o i r .  rate of evaporation  of the c a r r i e r gas  surface  was  presumably c o n t r o l l e d by b o t h  the  stream i n the body of the heat r e s e r v o i r sample.  When l i q u i d was  deposited  and on a  evaporated  chromatography.  i n j e c t i o n s of volumes of b l o o d up to a t o t a l of 10 u l c o u l d be made  i t was  n e c e s s a r y t o i n s e r t a f r e s h U-tube but use o f samples o f 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  0 - r i n g s used to s e a l the U-tube i n t o the p o r t gave some b l e e d i n g  when new.  the  of the v o l a t i l e s and of a t r a p f o r the n o n - v o l a t i l e  s l o w l y , a phenomenon accompanied by peak b r o a d e n i n g on gas  The  face  Under these c o n d i t i o n s  p o r t i o n of the U-tube f r e e of g l a s s wool the f l u i d beaded and  before  the  the dual f u n c t i o n o f p r o v i d i n g a l a r g e , heated  the d i s p e r s i o n o f the i n j e c t e d b l o o d  Successive  neces-  These c o u l d be e l i m i n a t e d , however, by h e a t i n g  problems  the r i n g s f o r 16 h  a t 160°C b e f o r e  i n s e r t i o n i n t o the p o r t .  was  l i g h t l u b r i c a t i o n w i t h a h i g h - t e m p e r a t u r e vacuum g r e a s e (Dow).  improved by  Routinely  The  injection.  e f f e c t i v e l i f e of the  rings  the septum of the i n j e c t i o n assembly, mounted to the f r o n t of  r o t a r y v a l v e , was  changed once a week; i t c o u l d , i n any  the  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 e v a l u a t e e t h a n o l , n- and  i s o p r o p a n o l , n-,  s e c - , t e r t - and  the use  of m e t h a n o l ,  i s o b u t a n o l and  a l c o h o l as i n t e r n a l s t a n d a r d s f o r the e s t i m a t i o n of h a l o t h a n e and  isoamyl methoxy-  - 22 -  f l u r a n e on one or more o f the column p a c k i n g s Chromosorb 101, 103, 105, or 107 under e i t h e r i s o t h e r m a l or temperature-programmed c o n d i t i o n s . found t o be a cheap and convenient  I s o b u t a n o l was  i n t e r n a l s t a n d a r d s i n c e i t c o u l d 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 b l o o d a t the c o n c e n t r a t i o n s employed and had a r e t e n t i o n time i n t e r m e d i a t e between t h a t o f h a l o t h a n e a l s o proved t o be a good i n t e r n a l standard ether.  f o r both e t h a n o l and d i e t h y l  The use o f a temperature programme r e s u l t e d i n the s e p a r a t i o n o f the  water peak from the h a l o t h a n e area.  and m e t h o x y f l u r a n e ; i t  peak p e r m i t t i n g a c c u r a t e measurements o f peak  Chromosorb 101 was the p r e f e r r e d p a c k i n g m a t e r i a l s i n c e i t gave  or no b l e e d and was v e r y s t a b l e ; a column has been used w i t h 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 y e a r s . c h r o m a t o g r a p h i c peaks i s a s i g n o f d e g r a d a t i o n columns were used when t h i s o c c u r r e d .  little  satisfactory  Severe " t a i l i n g " o f  o f the column p a c k i n g .  New  Furthermore Chromosorb 101 can be  employed, under the same o p e r a t i n g c o n d i t i o n s , f o r the r a p i d a n a l y s i s o f halothane, methoxyflurane,  e t h a n o l and d i e t h y l e t h e r . U s i n g 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 o c c u r r e d c o u l d be c o r r e c t e d by a s i n g l e b l a n k program r u n or by a s h o r t p e r i o d o f c o n d i t i o n i n g a t 180°C.  Table V I l i s t s the s e n s i t i v i t y o f  the flame i o n i s a t i o n d e t e c t o r and the performance c h a r a c t e r i s t i c s o f the e l e c t r o m e t e r and i n t e g r a t o r when h a l o t h a n e , m e t h o x y f l u r a n e , e t h e r and i s o b u t a n o l were a n a l y s e d as d e s c r i b e d above. chromatograms o b t a i n e d from these  ethanol, diethyl  F i g . 4 shows sample  analyses.  Both h e p a r i n and d i s o d i u m EDTA can be used as the a n t i c o a g u l a n t . l a t t e r was used because i t was l e s s expensive  and more s t a b l e .  The  Whole b l o o d  o b t a i n e d from the Red C r o s s , 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 V I  Performance c h a r a c t e r i s t i c s o f the flame i o n i z a t i o n d e t e c t o r , e l e c t r o m e t e r and e l e c t r o n i c  integrator  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  C o n c e n t r a t i o n (mg%)  11  98  52  60  56  Volume o f i n j e c t i o n ( u l )  15  15  40  10  10  Detector output at maximum (A)  2.56  I n t e g r a t o r i n p u t at maximum (mV) (=electrometer output)  2.56  Peak a r e a  7.422 x 1 0  (uV.sec)  x 10-1°  4  3..44 x 10-10  4..18 x 10-10  6,.50 x 10-10  9,.61 x 10-10  3..44  4.,18  6,.50  9,.61  1 .363 x 1 0  4  9..543 x 1 0  4  1..222 x 1 0  5  These performance c h a r a c t e r i s t i c d a t a c o r r e s p o n d t o peaks A-E o f the sample chromatograms i n F i g . 4  2.,354 x 105  - 24 -  B  1  1I  i—. c  F  V  W  -A—A  H  D  G  w 1 1 1 1 1 0 2 4 6  1 •  Ii i i 8  10  1 1 1 1 l 0 2 4  l 6  l  l 8  l 1  i iiii i 12  14  Time in Minutes  F i g . 4. Sample chromatograms from the a n a l y s i s o f h a l o t h a n e ( A ) , methoxyf l u r a n e ( B ) , d i e t h y l ether ( c ) and e t h a n o l (D) u s i n g , i n each c a s e , i s o b u t a n o l (E, F, G, H) as i n t e r n a l s t a n d a r d . 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 b a s e l i n e s a r e event markers a r i s i n g from the i n t e g r a t o r .  - 25 -  graph, gave r i s e 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 d i r e c t l y 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 e f f i c i e n t for storing blood samples containing halothane for periods as long as two weeks at 4°C. of analyses when the experimental  Such storage permits e f f i c i e n t staging  design requires multiple  samplings.  -  26 -  Table VII G a s - l i q u i d chromatographic a n a l y s i s o f whole blood samples c o n t a i n i n g known concentrations o f halothane, methoxyflurane, d i e t h y l ether and ethanol  Compound added Halothane  Response f a c t o r (isobutanol • 1)  0.207 + 0.008  Retention time (min)*  6.21  R e l a t i v e r e t e n t i o n time (isobutanol • 1)  0.697  +0.20  Methoxyflurane  D i e t h y l ether  Ethanol  0.242 + 0.009  0.924 + 0.001  0.797 + 0.024 ~~  5.36  3.21  12.31  +0.13  1.397  +0.15  0.608  +0.08  0.382  Added (mgX)  Found (agZ)  Added (mg*)  Found (mgl)  Added (ngZ)  Found (mgX)  Added (mgX)  Found (mgZ)  102.7 102.3 94.1 93.5 92.8 54.7 15.3 14.9 14.2 13.2 11.4 4.7 1.2  105.2 103.6 90.9 97.3 90.4 53.1 15.0 13.8 14.3 12.2 12.1 4.3 1.0  110.4  111.3  111.6  115.3  119.7  121.7  54.7  54.2  67.4  66.6  62.2 24.4  63.6 23.5  10.3 5.0 0.7  10.7 4.9 0.8  12.7 5.2 1.0  11.9 5.5 0.7  11.5  11.7  d.f.***  0.26 12 N.S.  A f t e r 1 week storage A f t e r 2 weeks storage  18.8 18.4  0.69 4 N.S.  0.5 4 N.S.  0.9 3 N.S.  17.8 18.6  *In these studies the r e t e n t i o n times f o r isobutanol were 8.91 + 0.56, 8.81 + 0.56, 8.81 + 0.56 and 8.40 + 0.48 f o r a n a l y s i s o f halothane, methoxyflurane, d i e t h y l ether and ethanol r e s p e c t i v e l y . * * P a i r e d t test (Henry et a l . 1974). D i f f e r e n c e s between q u a n t i t i e s added and found were not s i g n i f i c a n t l y d i f f e r e n t from r e r o . * * * d . f . * degrees of freedom. N.S. • not s i g n i f i c a n t  DISCUSSION  Comparison of the a n a l y t i c a l system d e s c r i b e d here w i t h o t h e r d i r e c t i n j e c t i o n methods (see T a b l e I I ) , i n d i c a t e s t h a t i t appears t o combine a l l advantages o f these systems w i t h o u t any o f t h e i r d i s a d v a n t a g e s .  the  Thus i t  p e r m i t t e d 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 o f the b l o o d c o n c e n t r a t i o n s of f o u r 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 p a c k i n g . None o f the d i f f i c u l t i e s a s s o c i a t e d 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; C o u s i n s 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), c o n t a m i n a t i o n o f 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 e v a p o r a t i o n which n e c e s s i t a t e d a p r e h e a t i n g p e r i o d o f the sample w i t h i n the pre-column d e v i c e ( Y o k o t a e t a l . 1967; C o l e et a l . 1975) were encountered.  The use o f an i n t e r n a l s t a n d a r d  coupled w i t h the measurement o f peak a r e a s , r a t h e r than peak h e i g h t s , o b v i a t e d 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 o f broadened or d i s t o r t e d peaks.  The a c c u r a c y o f the i n t e r n a l  s t a n d a r d 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 c o n c e n t r a t i o n s ( E t t r e , 1967).  R e f e r e n c e to T a b l e V I I demonstrates t h a t , w i t h  the p r o c e d u r e d e s c r i b e d h e r e a c c u r a t e a n a l y s e s can be performed over a wide range o f c o n c e n t r a t i o n s . The e x t e r n a l i n j e c t i o n p o r t can be e a s i l y c o n s t r u c t e d from i n e x p e n s i v e m a t e r i a l s and has proved to be r e l i a b l e and s i m p l e t o o p e r a t e 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 o f m o d i f i c a t i o n o f e x i s t i n g  gas chromatographs and t h e p o r t 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 t o o f f e r  advantages over p r e v i o u s systems (Yokota  e t a l . 1967; C o l e e t a l . 1975) s i n c e  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 w i t h o u t c a r r i e r gas f l o w .  distinct  i n t e r r u p t i n g the  T h i s i s a v a l u a b l e f e a t u r e when l a r g e numbers o f samples  have t o be a n a l y s e d , as e x e m p l i f i e d by the experiments d e s c r i b e d i n P a r t s I I and I I I o f t h i s work. The  range o f s e n s i t i v i t y o f t h i s method i s e q u a l t o o r b e t t e r than o t h e r  r e p o r t e d methods ( l i s t e d i n Tables  I I , I I I and I V ) i n which a flame i o n i z a t i o n  d e t e c t o r was used f o r a n a l y s i n g a n a e s t h e t i c c o n c e n t r a t i o n s recovery  at approximately  97-104% r e s p e c t i v e l y .  i n blood.  The  10 mg/100 ml and 100 mg/100 ml was 92-106% and  - 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 o f H a l o t h a n e i n Human B l o o d :  A Model  Study INTRODUCTION  There i s l i t t l e known about t h e mechanism o f t h e t r a n s p o r t o f i n h a l a t i o n g e n e r a l a n a e s t h e t i c agents i n b l o o d .  Although  t h e r e have been model s t u d i e s  o f t h e i n t e r a c t i o n o f a n a e s t h e t i c agents w i t h b o t h 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 H u b b e l l  1976; K o e h l e r  e t a l . 1977)  and haemoglobin and o t h e r p r o t e i n s ( M i l l a r e t a l . 1971; L a a s b e r g and H e d l e y Whyte 1971; Schulman e t a l . 1970; H a n i s c h e t a l . 1969; B a r k e r  e t a l . 1975),  they do n o t p r o v i d e q u a n t i t a t i v e i n f o r m a t i o n on t h e c o n t r i b u t i o n o f t h e v a r i o u s components o f b l o o d t o the t r a n s p o r t o f a n a e s t h e t i c s . The mechanism o f a c t i o n o f g e n e r a l a n a e s t h e t i c s i s b e i n g investigated.  E x a m i n a t i o n o f the c h e m i c a l  intensively  structures of i n h a l a t i o n general  a n a e s t h e t i c s ( T a b l e I ) showed t h a t they l a c k a common s t r u c t u r a l  specificity.  T h i s suggests t h a t t h e r e i s no s p e c i f i c r e c e p t o r a t the s i t e o f a c t i o n . M i l l e r e t a l . (1972) found t h a t t h e potency o f i n h a l a t i o n a n a e s t h e t i c s 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 t h a t t h e s i t e o f a c t i o n i s a t t h e l i p i d r e g i o n o f t h e membrane o f the neuron.  U s i n g a l c o h o l s and e r y t h r o c y t e membrane as models, i t was shown  t h a t a n a e s t h e t i c s caused membrane e x p a n s i o n (Seeman, 1972, 1974; Seeman e t a l . 1969, phobic  1971).  A l s o , n - a l k a n e s and n - a l k a n o l s were shown t o i n c r e a s e t h e h y d r o -  t h i c k n e s s o f an a r t i f i c i a l membrane (Haydon e t a l . 1977).  Expansion o f  the membrane may cause an i n c r e a s e i n t h e d i s o r d e r o f t h e 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 e s p o n s i b l e f o r the t r a n s m i s s i o n of nerve impulse.  I n the l a t e r a l phase s e p a r a t i o n h y p o t h e s i s  a n a e s t h e t i c s a c t by f l u i d i z i n g nerve membrane so t h a t c r i t i c a l  ( T r u d e l l , 1977), lipid  regions  no l o n g e r c o n t a i n the phase s e p a r a t i o n s r e q u i r e d t o f a c i l i t a t e the conforma t i o n a l changes o f p r o t e i n s or l i p o p r o t e i n s n e c e s s a r y or the r e l e a s e o f t r a n s m i t t e r s t o o c c u r .  f o r membrane e x c i t a t i o n  F l u i d i z a t i o n o f membrane i n the  presence o f a n a e s t h e t i c m o l e c u l e s has been observed w i t h d i f f e r e n t p r o b e s , i n c l u d i n g f i r e f l y luminescence (Ueda and Kamaya, 1973; Ueda e t a l . 1974), NMR ( S h i e h et a l . 1975) and ESR ( T r u d e l l and Cohen, 1975). cept o f membrane e x p a n s i o n as a p r i m a r y observations  cause f o r a n a e s t h e s i a  t h a t a h i g h ambient p r e s s u r e was capable  (Seeman, 1977).  I n a d d i t i o n , t h e coni s supported  of reversing  by  anaesthesia  Presumably compression o f the membrane reduces the i n c r e a s e  i n f l u i d i t y due t o the presence o f a n a e s t h e t i c  molecules.  S i n c e c u r r e n t t h e o r i e s o f the a c t i o n o f a n a e s t h e t i c s f a v o r an a n a e s t h e t i c membrane i n t e r a c t i o n ( S c h n e i d e r Halsey al.  1968; M i l l e r et a l . 1973; R i c h a r d s 1976;  1974; T r u d e l l 1974; Eger et a l . 1965; Saidman e t a l . 1967; Hansch e t  1975; Haydon et a l . 1977), one might expect t h a t a s i g n i f i c a n t p r o p o r t i o n  of the a n a e s t h e t i c agent would be t r a n s p o r t e d by the r e d c e l l membrane. However, t h e r e has been no c o n c l u s i v e evidence f o r or a g a i n s t t h i s  hypothesis,  and as shown i n T a b l e V I I I i t has been v a r i o u s l y r e p o r t e d t h a t the s o l u b i l i t y of halothane  i n b l o o d has e i t h e r a p o s i t i v e or n e g a t i v e dependence on, or i s  independent o f , h a e m a t o c r i t . These a p p a r e n t l y c o n t r a d i c t o r y r e s u l t s a r e p r o b a b l y  due t o the l a c k o f  b o t h a s y s t e m a t i c approach and o f a p r e c i s e knowledge o f t h e c o m p o s i t i o n o f the system examined.  F o r example, i n most i n v e s t i g a t i o n s the c o n c e n t r a t i o n s  -  31  -  T a b l e V I I I - R e p o r t e d dependence o f t h e s o l u b i l i t y o f h a l o t h a n e components  B l o o d Components Tested  on b l o o d  Results  Authors  Species  Mapleson e t a l . 1972  Rabbit  Cowles e t a l . 1971a  Dog  Hemoglobin Hematocrit  n e g a t i v e dependence* n e g a t i v e dependence*  Steward e t a l . 1975  Dog  Hematocrit  n e g a t i v e dependence*  Larson et a l . 1962  Human  Hemoglobin  positive  dependence  Hans and H e l r i c h 1966  Human  Hemoglobin Hematocrit  positive negative  dependence dependence  Lowe and H a g l e r 1969  Human  Hematocrit  negative  dependence  L a a s b e r g and H e d l e y Whyte 1970  Human  Albumin y-globulin Hemoglobin  p o s i t i v e dependence n e g a t i v e dependence p o s i t i v e and n e g a t i v e dependence+  Cowles e t a l . 1971a  Human  Hemoglobin Hematocrit  negative negative  dependence dependence  E l l i s and S t o e l t i n g 1975  Human  Hematocrit  positive  dependence  Saraiva et a l . 1977a  Human  negative negative negative positive positive positive negative negative  dependence* dependence* dependence* dependence dependence* dependence* dependence* dependence*  Hematocrit  Hemoglobin Albumin T o t a l serum p r o t e i n Triglyceride Cholesterol Globulin Albumin/globulin r a t i o Albumin/total protein ratio Hematocrit  positive  dependence  n e g a t i v e dependence*  no dependence Human Triglyceride Munson e t a l , 1978 *Statistically insignificant +Maximum s o l u b i l i t y a t normal hemoglobin c o n c e n t r a t i o n and lower s o l u b i l i t y when hemoglobin c o n c e n t r a t i o n i s h i g h e r or lower than normal  o f the plasma p r o t e i n s were unknown, a l t h o u g h 1974)  to i n f l u e n c e the s o l u b i l i t y of h a l o t h a n e  they are known ( C h i o u and i n plasma.  Hsiao  F u r t h e r , none o f  the s t u d i e s d i s t i n g u i s h e d between a b s o r p t i o n to the red c e l l membrane and a d s o r p t i o n to haemoglobin, and many were made on e i t h e r whole b l o o d or plasma and  do not p r o v i d e i n f o r m a t i o n on the l o c a t i o n o f a n a e s t h e t i c s w i t h i n the  blood.  Most o f these s t u d i e s are d i f f i c u l t to e v a l u a t e because they d i d not  g i v e 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 e x c e p t i o n o f L a a s b e r g and Hedley-Whyte (1970).  The  study o f S a r a i v a et a l .  (1977a) i s the most complete i n terms o f the number o f b l o o d i n v e s t i g a t e d and p r o b a b l y more r e l i a b l e due  the  components  to the l a r g e r number o f b l o o d  samples used. A l l o f these s t u d i e s (Table V I I I ) made use o f 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 o f the s o l u b i l i t y halothane  i n the aqueous phase b e i n g examined.  f i c i e n t of halothane  The  blood/gas p a r t i t i o n  i s d e f i n e d as the r a t i o o f the c o n c e n t r a t i o n o f  of  coef-  halothane  i n b l o o d to t h a t i n a gas phase o f 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 [ H a l o t h a n e ] i n gas phase  Thus a h i g h blood/gas p a r t i t i o n c o e f f i c i e n t means a h i g h s o l u b i l i t y o f anaesthetic i n blood.  ^2] the  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  but e x t e n d to any 2 phases.  blood  B l o o d / g a s , 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 o f t e n used i n the t h e o r y 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 i n the body, and i n t h e o r i e s o f the m o l e c u l a r mechanism o f anaesthesia.  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 d e s c r i b e the r e l a t i v e c a p a c i t y per u n i t volume of  - 33 each phase f o r the a n a e s t h e t i c .  Thus a p o s i t i v e ( o r n e g a t i v e ) dependence o f  the s o l u b i l i t y o f h a l o t h a n e on a b l o o d component means t h a t the p a r t i t i o n c o e f f i c i e n t o f h a l o t h a n e i n c r e a s e s ( o r d e c r e a s e s ) when t h e c o n c e n t r a t i o n o f t h a t b l o o d component i s i n c r e a s e d ( o r d e c r e a s e d ) . Other terms d e s c r i b e the same or n e a r l y the same e q u i l i b r i u m o f a n a e s t h e t i c between gas and l i q u i d phases.  distribution  The Ostwald S o l u b i l i t y  Coef-  f i c i e n t i s d e f i n e d as the volume o f gas absorbed p e r u n i t volume o f s o l v e n t a t the temperature o f the measurement when the p a r t i a l p r e s s u r e o f the d i s s o l v e d gas e q u a l s 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  t h a t 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 o n c e n t r a t i o n o f 1. S i n c e the gas phase  i s always the denominator o f the r a t i o and a s s i g n e d a  v a l u e o f 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 e q u a l s the partition  coefficient.  The t h e o r e t i c a l b a s i s f o r 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 s t a t e s t h a t , at e q u i l i b r i u m , the p a r t i a l p r e s s u r e o f 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 t o i t s c o n c e n t r a t i o n i n the s o l v e n t 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 t o a n a e s t h e t i c s , t h e p a r t i t i o n  c o e f f i c i e n t would be a c o n s t a n t .  O r i g i n a l l y Henry's Law was developed f o r the  treatment o f the b e h a v i o u r o f gas m o l e c u l e s d i s s o l v e d i n a homogeneous l i q u i d phase a t i n f i n i t e d i l u t i o n .  S i n c e a l l the gas m o l e c u l e s d i s s o l v e d i n the  homogeneous l i q u i d phase e x p e r i e n c e the same p h y s i c o c h e m i c a l environment a t i n f i n i t e d i l u t i o n , the p a r t i a l p r e s s u r e i s a measure o f the a c t i v i t y o f a l l gas m o l e c u l e s d i s s o l v e d i n the s o l v e n t (Moore, 1962).  The concept o f p a r t i a l  p r e s s u r e has g a i n e d widespread acceptance i n a n a e s t h e s i o l o g y .  - 34 The use o f p a r t i a l p r e s s u r e arose from t h e thermodynamic treatment o f 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. e q u i l i b r i u m a t c o n s t a n t temperature f u n c t i o n G (Gibb's f r e e e n e r g y ) .  Chemical  thermodynamic  and p r e s s u r e i s d e f i n e d i n terms o f t h e  F o r a s i m p l e two-phase system, t h e  d i f f e r e n c e i n t h e v a l u e o f G f o r each phase i s a measurement o f how f a r a p a r t the two phases a r e from e q u i l i b r i u m .  When the two phases a r e a t e q u i l i b r i u m ,  the d i f f e r e n c e o f t h e v a l u e o f G f o r t h e two phases, AG, i s z e r o . G a p p l i e s t o a phase or a system as a whole.  Another  function derived  from G, c a l l e d t h e c h e m i c a l p o t e n t i a l , a p p l i e s t o a s i n g l e c h e m i c a l s p e c i e s . The  c h e m i c a l p o t e n t i a l G^ f o r t h e i t h c h e m i c a l s p e c i e s , i s d e f i n e d as  follows:  where n. = number o f moles o f t h e i t h c h e m i c a l s p e c i e s 1  When c h e m i c a l e q u i l i b r i u m between two phases A and B i s reached a t c o n s t a n t temperature 1962)  and p r e s s u r e , i . e . when AG = 0, i t can be proven  (Moore,  t h a t the c h e m i c a l p o t e n t i a l o f any g i v e n c h e m i c a l s p e c i e s i n t h e two  phases a r e e q u a l , or  where G.  = c h e m i c a l p o t e n t i a l o f the i t h c h e m i c a l s p e c i e s i n phase A = c h e m i c a l p o t e n t i a l o f the i t h c h e m i c a l s p e c i e s i n phase B  Thus, w h i l e G i s used t o d e s c r i b e t h e e q u i l i b r i u m between two m a c r o s c o p i c phases, G. i s t h e c o r r e s p o n d i n g parameter f o r e q u i l i b r i u m between the m o l e c u l e s o f a p a r t i c u l a r c h e m i c a l s p e c i e s 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 v a l u e o f the c h e m i c a l p o t e n t i a l G^ between the two phases i s a measure o f how f a r a p a r t t h e c h e m i c a l s p e c i e s i n t h e two phases a r e from  - 35 e q u i l i b r i u m w i t h each o t h e r .  -  I f the c h e m i c a l p o t e n t i a l o f the i t h s p e c i e s i n  ~A phase A, i . e . G^ i s l a r g e r than the c o r r e s p o n d i n g c h e m i c a l p o t e n t i a l -B . . . G^ i n phase B, t h e r e w i l l be a net f l o w o f the i t h c h e m i c a l s p e c i e s phase A to phase B u n t i l the two c h e m i c a l p o t e n t i a l s p o t e n t i a l as such cannot be e a s i l y measured.  A  Such a r e l a t i o n s h i p i s  I t can be shown (Moore 1962)  t h a t , f o r a vapor  such as h a l o t h a n e , i t s c h e m i c a l 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 as i n the f o l l o w i n g where G„ H  =  equation: %  " H ?  +  R T  l n  P  chemical  T h e r e f o r e i t i s n e c e s s a r y 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 . a v a i l a b l e f o r a gas phase.  are e q u a l .  from  pressure  H  c h e m i c a l p o t e n t i a l o f h a l o t h a n e i n the gas phase  G° = s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l of h a l o t h a n e H  i n the gas phase.  T h i s i s a c o n s t a n t , d e f i n e d as the v a l u e o f the c h e m i c a l p o t e n t i a l when the h a l o t h a n e p a r t i a l p r e s s u r e i s 1 atmosphere. P  H  = P a r t i a l p r e s s u r e of h a l o t h a n e . halothane molecules which may  I t i s the p r e s s u r e e x e r t e d by  i n the gas phase, independent  o f other  a l s o be p r e s e n t .  The above e q u a t i o n assumes the v a l i d i t y o f the i d e a l gas PV =  nRT  where P = p r e s s u r e V = volume n = number o f moles o f the gas or vapor R = universal  gas  constant  T = absolute  temperature  law:  molecules  T h i s i d e a l gas law i s a c c u r a t e under common ambient c o n d i t i o n s . F o r example, the d e v i a t i o n o f carbon d i o x i d e from i d e a l b e h a v i o u r at 40°C and 1 atmosphere i s 0.1%, and a t 10 atmosphere i s 1% (Moore,  1962).  I d e a l l y , t h e r e s h o u l d a l s o be a c o r r e s p o n d i n g e q u a t i o n r e l a t i n g the c h e m i c a l p o t e n t i a l o f h a l o t h a n e d i s s o l v e d i n b l o o d or any o t h e r f l u i d s t o a q u 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 , t h e r e has y e t 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 t o such an e q u a t i o n . the c h e m i c a l p o t e n t i a l o f h a l o t h a n e  i n s o l u t i o n has t o be i n d i r e c t l y  Thus,  related  to the c h e m i c a l p o t e n t i a l o f h a l o t h a n e vapor i n a gas phase i n e q u i l i b r i u m with that i n s o l u t i o n .  S i n c e , as d i s c u s s e d above, the c h e m i c a l p o t e n t i a l i s  the same f o r a c h e m i c a l s p e c i e s ( i n t h i s case h a l o t h a n e ) 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 c h e m i c a l 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 p r e s s u r e o f h a l o t h a n e i n t h e gas phase i s n u m e r i c a l l y e q u a l t o the c h e m i c a l p o t e n t i a l o f h a l o t h a n e  i n the  aqueous phase. T h i s i s the b a s i s f o r the use o f p a r t i a l p r e s s u r e t o d e s c r i b e the b e h a v i o u r o f i n h a l a t i o n a n a e s t h e t i c s because t h e r e i s a one t o one correspondence  between  the c h e m i c a l p o t e n t i a l and the p a r t i a l p r e s s u r e i n the gas phase. Chemical p o t e n t i a l i s a fundamental  quantity a p p l i c a b l e to a chemical  s p e c i e s whether or not the system i s i n thermodynamic e q u i l i b r i u m .  However,  the use o f p a r t i a l p r e s s u r e as a p r a c t i c a l a l t e r n a t i v e t o d e s c r i b e the b e h a v i o u r o f i n h a l a t i o n a n a e s t h e t i c s i n aqueous s o l u t i o n ( o r i n any medium f o r t h a t m a t t e r ) n e c e s s a r i l y i m p l i e s 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 p r e s s u r e i s measured.  to use p a r t i a l p r e s s u r e under n o n - e q u i l i b r i u m c o n d i t i o n s .  I t i s meaningless  - 37 A n a e s t h e t i c c o n c e n t r a t i o n s i n b l o o d and o t h e r b i o l o g i c a l media a r e t r a d i t i o n a l l y expressed i n terms o f p a r t i a l p r e s s u r e s o f the a n a e s t h e t i c 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 c o n c e n t r a t i o n u n i t s and t h e p a r t i t i o n c o e f f i c i e n t s a r e c a l c u l a t e d from these p a r t i a l p r e s s u r e measurements.  Attempts  to c o r r e l a t e t h e p a r t i t i o n c o e f f i c i e n t w i t h t h e c o n c e n t r a t i o n o f d i f f e r e n t b l o o d components g i v e r i s e t o the r e s u l t s summarized i n Table V I I I .  However,  when t h e s o l v e n t i s a m u l t i p h a s e , heterogeneous m i x t u r e , e.g. whole b l o o d , t h e i n t e r p r e t a t i o n o f the p a r t i a l p r e s s u r e becomes more d i f f i c u l t .  The same  p a r t i a l p r e s s u r e o f an a n a e s t h e t i c 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 t h e c o n c e n t r a t i o n s o f t h e a n a e s t h e t i c i n t h e d i f f e r e n t phases a r e n o t t h e same.  Thus t h e p a r t i a l p r e s s u r e p e r se i s i n s u f f i c i e n t t o  d e s c r i b e t h e q u a n t i t a t i v e d i s t r i b u t i o n o f an a n a e s t h e t i c i n t h e d i f f e r e n t phases o f b l o o d . T h e r e f o r e , t o study t h e 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 o f t h e d i f f e r e n t b l o o d components t o t h e s o l u b i l i t y o f h a l o t h a n e i n b l o o d and t o c o n s t r u c t a model f o r t h e mode o f 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 n e c e s s a r y . i b r i u m d i a l y s i s was chosen f o r t h i s purpose.  Equil-  I n t h e t e c h n i q u e t o be d e s c r i b e d ,  an aqueous s o l u t i o n o f t h e b l o o d component was p l a c e d i n one compartment and an aqueous s o l u t i o n o f h a l o t h a n e was p l a c e d i n another compartment.  The two  compartments were s e p a r a t e d by a semi-permeable membrane which a l l o w e d a s m a l l molecule  (such as h a l o t h a n e ) b u t n o t a macromolecule (such as t h e b l o o d  components used i n t h i s study) t o pass through. a net d i f f u s i o n o f halothane molecules  During the d i a l y s i s , there i s  i n t o t h e compartment w i t h t h e b l o o d  component u n t i l t h e c h e m i c a l p o t e n t i a l s o f h a l o t h a n e i n t h e two compartments are e q u a l ( i . e . a t 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, t h e number o f  - 38 h a l o t h a n e m o l e c u l e s bound p e r m o l e c u l e o f t h e b l o o d component i s g i v e n by the following  equation (Marshall,  1978, Sophianopoulos  e t a l . 1978):  Number o f h a l o t h a n e m o l e c u l e s = Number o f m o l e c u l e s o f b l o o d component [Halothane].; - [ H a l o t h a n e ] [ B l o o d component] where [Halothane]  o  0 x  MW o f b l o o d component MW o f h a l o t h a n e  = h a l o t h a n e c o n c e n t r a t i o n (g/ml) i n the compartment w i t h o u t the b l o o d component  [ H a l o t h a n e ] ^ = h a l o t h a n e c o n c e n t r a t i o n (g/ml) i n the compartment w i t h the b l o o d component [Blood Component] = c o n c e n t r a t i o n o f the b l o o d component (g/ml) I f the c o n c e n t r a t i o n o f the b l o o d component and c o n c e n t r a t i o n o f h a l o t h a n e i n the two compartments a r e known, the number o f h a l o t h a n e m o l e c u l e s bound p e r m o l e c u l e o f the b l o o d component can be c a l c u l a t e d . t a k e i n t o account  T h i s e q u a t i o n does n o t  the volume o c c u p i e d by the b l o o d 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 o f the volume o c c u p i e d by the b l o o d component i n i t s compartment. volume a v a i l a b l e  Then 1-Q i s the e f f e c t i v e  f r a c t i o n o f the  f o r h a l o t h a n e i n the compartment w i t h the b l o o d component.  The e x p e r i m e n t a l l y determined  c o n c e n t r a t i o n s i n the compartment w i t h the b l o o d  component a r e c o r r e c t e d by d i v i d i n g w i t h 1-Q, and the above e q u a t i o n becomes: Number o f h a l o t h a n e m o l e c u l e s Number o f m o l e c u l e s o f b l o o d component [Halothane] - - [ H a l o t h a n e ] 1-Q [ B l o o d component] n  =  Q  x MW o f b l o o d component MW o f h a l o t h a n e  1-Q The a n a l y t i c a l method developed quantitative  i n P a r t I o f t h i s work was used f o r the  d e t e r m i n a t i o n o f h a l o t h a n e c o n c e n t r a t i o n s 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 o b t a i n e d 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 o f h a l o t h a n e w i t h haemoglobin, r e d c e l l  ghosts,  albumin ( b o t h f a t t y a c i d f r e e and i n the presence o f 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 ( c h y l o m i c r o n s and v e r y low d e n s i t y lipoproteins).  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 o f  h a l o t h a n e between c e l l s and plasma and were compared to those o b t a i n e d by independent  an  method, i n which whole b l o o d samples c o n t a i n i n g d i s s o l v e d  h a l o t h a n e , and the c o r r e s p o n d i n g plasma samples o b t a i n e d by were a n a l y s e d f o r h a l o t h a n e c o n c e n t r a t i o n .  centrifugation,  Comparison o f the r e s u l t s o b t a i n e d  by these two d i f f e r e n t methods p r o v i d e s a t e s t f o r 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 s e p t a and 25 ml screw cap v i a l s were o b t a i n e d from P i e r c e Chemical Co., R o c k f o r d , 111., U.S.A.  These v i a l s were s u p p l i e d w i t h g a s - t i g h t  t e f l o n l i n e d s e a l s and served as l e a k p r o o f c o n t a i n e r s f o r h a l o t h a n e .  Gas-  t i g h t s y r i n g e s were o b t a i n e d from H a m i l t o n Co., Reno, Nevada, U.S.A.  Amicon  CF50 and CF25 membrane u l t r a f i l t e r s were o b t a i n e d from Amicon Corp., L e x i n g t o n , Mass.  A l l t h e reagents f o r t r i g l y c e r i d e assay were o b t a i n e d from H y c e l I n c . ,  Houston, Texas, U.S.A.  3 , 3 ' , 5 , 5 - t e t r a b r o m o - m - c r e s o l s u l f o n p h t h a l e i n were 1  o b t a i n e d from American M o n i t o r Corp., I n d i a n a p o l i s , I n d i a n a , U.S.A.  Human  Y - g l o b u l i n , f a t t y a c i d f r e e human albumin ( l e s s than 0.005% f a t ) and d i a l y s i s t u b i n g s were o b t a i n e d from Sigma Chemical Co., S t . L o u i s , Mo., U.S.A. A l l o t h e r o r g a n i c and i n o r g a n i c c h e m i c a l s used were o f reagent grade from F i s c h e r S c i e n t i f i c Co., M o n t r e a l , 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 t h e method o f Miyada et a l . (1972) and t r i g l y c e r i d e by t h e t e c h n i q u e o f Schmidt and Von Dahl (1968).  S i n c e a n t i c o a g u l a n t s may  i n t e r f e r e w i t h t h e t r i g l y c e r i d e a s s a y , e s t i m a t i o n o f albumin and t r i g l y c e r i d e was performed on plasma samples p r e p a r e d from b l o o d samples t a k e n w i t h a p l a s t i c s y r i n g e and w i t h o u t added a n t i c o a g u l a n t .  The b l o o d sample so o b t a i n e d  was i m m e d i a t e l y 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 a t 10,000 g f o r 15 s e c . These plasma samples w i l l n o t form v i s i b l e f i b r i n c l o t s f o r a t l e a s t 48 h r s a t 4°C.  Presumably most o f the f i b r i n c l o t s i n i t i a l l y  formed  were sedimented a l o n g w i t h t h e c e l l s . Red c e l l s and r e d c e l l g h o s t s were counted i n a Neubauer c o u n t i n g chamber ( D a c i e and Lewis 1968), i n the l a t t e r case u s i n g a phase c o n t r a s t m i c r o s c o p e . D i s o d i u m EDTA (0.2% w/v) was used as the a n t i c o a g u l a n t whenever n e c e s s a r y . H a l o t h a n e c o n c e n t r a t i o n s were determined by g a s - l i q u i d  chromatography as  d e s c r i b e d i n P a r t I o f t h i s work.  2.  S t u d i e s o f the B i n d i n g o f Halothane t o B l o o d Components  a.  Saturation concentration 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 l a y e r e d onto a b u l k phase o f h a l o -  thane c o n t a i n e d i n R e a c t i - v i a l s m a t e l y e q u a l volume.  such t h a t the two phases o c c u p i e d an a p p r o x i -  The v i a l s were s e a l e d by d i s p l a c i n g  excess s a l i n e i n  such a manner t h a t 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 a t e i t h e r 4 or 37°C w i t h o u t m i x i n g f o r up to 98 and 168 respectively.  hours,  S i n c e the two phases were not mixed, the p o s s i b i l i t y o f h a l o -  thane forming m i c r o - d r o p l e t s , which would l e a d to erroneous h i g h v a l u e f o r 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 c o n c e n t r a t i o n s were determined by g a s - l i q u i d chromatography.  b.  Haemoglobin Red b l o o d c e l l s were o b t a i n e d from outdated Red Cross b l o o d (not more than  1 month o l d ) by c e n t r i f u g a t i o n a t 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 ) .  volume o f 150 mM  They were washed f i v e times i n f o u r times t h e i r  sodium c h l o r i d e and were then haemolysed by the a d d i t i o n o f  an a p p r o x i m a t e l y e q u a l volume of d i s t i l l e d water. fuged as above and the s u p e r n a t a n t r e c o v e r e d . p r o c e s s the ghost f r e e s o l u t i o n was t i v e l y , a g a i n s t t e n volumes o f PBS sodium phosphate,  150 mM  used f o r e q u i l i b r i u m d i a l y s i s .  s o l u t i o n was  centri-  d i a l y s e d t w i c e f o r 4 and 16 h r r e s p e c (phosphate b u f f e r s a l i n e : (w/v)  pH 7.4,  0.01  sodium a z i d e ) .  M The  c e n t r i f u g e d as above and the s u p e r n a t a n t  More than 95% o f the p r o t e i n i n t h i s  was haemoglobin (Wintrobe et a l . 1974) The c o n c e n t r a t i o n o f 2,3  The  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  sodium c h l o r i d e and 0.02%  r e s u l t i n g haemoglobin s o l u t i o n was  c.  own  at a c o n c e n t r a t i o n o f 9.8  solution  g/100  ml.  d i p h o s p h o g l y c e r a t e would not be more than 0.4  mM.  Albumin F a t t y a c i d - f r e e human albumin was  together with o l e i c a c i d . m a t e l y 2.3 g/100  ml.  The  d i s s o l v e d i n PBS, e i t h e r a l o n e or  f i n a l c o n c e n t r a t i o n o f albumin was a p p r o x i -  The o l e i c a c i d c o n c e n t r a t i o n used was  approximately  14  - 43 mg/100 m l , which i s w i t h i n the normal range o f 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-globulin Human Y gl°bulin d i s s o l v e d i n PBS a t a f i n a l c o n c e n t r a t i o n o f a p p r o x i -  m a t e l y 0.7 g/100 ml was used f o r d i a l y s i s a g a i n s t h a l o t h a n e .  e.  Triglyceride-rich micelles These were p r e p a r e d by a t e c h n i c a l m o d i f i c a t i o n o f t h e method o f Hatch and  Lees (1968).  B l o o d (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 -  c o a g u l a t e d w i t h EDTA, was c e n t r i f u g e d a t a p p r o x i m a t e l y 1,000 g f o r 5 min. A l i q u o t s ( a p p r o x i m a t e l y 4 m l ) o f the plasma were loaded i n t o 6 p o l y a l l o m e r tubes.  Seven ml o f a s o l u t i o n w i t h a d e n s i t y of 1.006 g/ml ( p r e p a r e d by  d i s s o l v i n g 11.40 g o f N a C l , 0.1 g d i s o d i u m EDTA and 1 ml o f 1 N NaOH i n a t o t a l volume o f 1.003 1 o f water) was l a y e r e d on the plasma samples w i t h a g l a s s p i p e t t e , by a l l o w i n g i t t o r u n c o n t i n u o u s l y and s l o w l y down t h e s i d e s o f 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 m a i n t a i n e d a t about 1 cm  above t h e l i q u i d l e v e l i n the tubes t o a v o i d d r i p p i n g and t u r b u l e n c e .  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) a t 16 t o 18°C f o r 16 h o u r s .  One t o t h r e e mis o f  the w h i t e s u s p e n s i o n o f t r i g l y c e r i d e - r i c h m i c e l l e s ( c h y l o m i c r o n s and v e r y low d e n s i t y l i p o p r o t e i n ) was taken from the t o p o f each tube, p o o l e d , and d i a l y s e d at 4°C a g a i n s t 1.0 1 p o r t i o n s o f d i s t i l l e d water f o r 4 and 16 h r respectively.  A sample o f the s u s p e n s i o n was taken f o r t r i g l y c e r i d e  assay,  and t o the remainder was added e x a c t l y 1/9 i t s volume o f a 10 x PBS b u f f e r c o n t a i n i n g 1% (w/v) d i s o d i u m EDTA.  - 44 f.  Red c e l l ghosts s u s p e n s i o n Ghosts were p r e p a r e d from out dated Red Cross b l o o d ( n o t more than 1 month  old)  and r e s e a l e d by warming as d e s c r i b e d by Theodore and Kant (1974).  r e s e a l e d ghosts were washed t w i c e and resuspended  The  i n an a p p r o x i m a t e l y e q u a l  volume o f PBS.  g.  D i a l y s i s o f B l o o d Components a g a i n s t Halothane The apparatus employed i s shown i n F i g . 5.  A disc of cellulose  dialysis  membrane was c a r e f u l l y p l a c e d on top o f a 0.3 ml R e a c t i - v i a l c o m p l e t e l y f i l l e d w i t h a s o l u t i o n or s u s p e n s i o n o f the b l o o d component t o be d i a l y s e d .  The d i s c  was s e a l e d , f i n g e r t i g h t , by means o f an 'O'-ring ( c u t from a s i l i c o n e rubber septum c o m m e r c i a l l y 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 o f the Reacti-vial.  A s t r o n g rubber band was f a s t e n e d l o n g i t u d i n a l l y o u t s i d e the 25  ml v i a l and t w i s t e d so t h a t i t tended to t i g h t e n the screw cap i n a c l o c k w i s e d i r e c t i o n (see F i g . 5 ) . dialysis.  T h i s p r e v e n t e d the caps from l o o s e n i n g d u r i n g  The v i a l s were i n c u b a t e d f o r v a r i o u s time i n t e r v a l s a t 37°C w i t h  s o l u t i o n s o f h a l o t h a n e i n PBS c o n t a i n e d i n c o m p l e t e l y f i l l e d , screw cap v i a l s .  s e a l e d , 25 ml  When r e d c e l l g h o s t s , 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 b e i n g i n v e s t i g a t e d , the o u t e r 25 ml v i a l was r o t a t e d end t o end at  10-30 r.p.m.  On c o m p l e t i o n o f 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 c o n c e n t r a t i o n o f halothane. In  those experiments where the f r e e h a l o t h a n e c o n c e n t r a t i o n was h e l d  c o n s t a n t and the 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 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 c o n c e n t r a t i o n s o f t r i g l y c e r i d e were p l a c e d i n t o one 500 ml reagent b o t t l e .  A g a 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 p i e c e o f T e f l o n (PTFE, Dupont) t o f i t the i n s i d e o f t h e p l a s t i c cap.  O t h e r w i s e the p r o c e d u r e s were i d e n t i c a l  screw  t o the o t h e r d i a l y s i s  experiments. The number o f h a l o t h a n e m o l e c u l e s bound p e r p r o t e i n m o l e c u l e was c a l c u l a t e d u s i n g the f o l l o w i n g f o r m u l a : [Halothane]! _ [Halothane]o Number o f H a l o t h a n e M o l e c u l e s = 1 - V j P r o t e i n ] Number o f P r o t e i n M o l e c u l e s [Protein] 1-VTProtein]  X MW o f p r o t e i n MW o f Halothane .....[4]  where [ H a l o t h a n e ] ! = Halothane c o n c e n t r a t i o n (g/ml) i n s i d e t h e 0.3 ml R e a c t i vial [ H a l o t h a n e ] o = Halothane c o n c e n t r a t i o n (g/ml) o u t s i d e the 0.3 ml R e a c t i vial [Protein]  = P r o t e i n c o n c e n t r a t i o n (g/ml) V = P a r t i a l s p e c i f i c volume o f t h e p r o t e i n  For  haemoglobin  V" = 0.749 ml/g MW = 64,500 (Altman and D i t t m e r 1972)  For  albumin  V" = 0.733 ml/g MW = 66,200 ( P e t e r s J r . 1975)  The number o f h a l o t h a n e m o l e c u l e s per r e d c e l l ghost was c a l c u l a t e d u s i n g the  f o l l o w i n g formula:  Number o f Halothane M o l e c u l e s = Number o f Red C e l l Ghosts  iMlothSBiJi - [Halothane ]o 1-KC r. C_ 1-KC  x  N MW o f Halothane  where C = number o f ghosts per u n i t volume  ^  K - volume o f one g h o s t , approximated by the p r o d u c t o f the t h i c k n e s s and s u r f a c e area o f the ghost membrane, taken t o be 2.4 nm  (Fettiplace  - 47 et a l . 1971)  and  136.9  um  2  -  (Jay 1975) r e s p e c t i v e l y .  N = Avogadro's number The number of grams of h a l o t h a n e  per gram of t r i g l y c e r i d e was  calculated  u s i n g the f o l l o w i n g f o r m u l a :  3.  Wt.  of H a l o t h a n e  = [ H a l o t h a n e ] i - [Halothane]o  Wt.  of T r i g l y c e r i d e  [6]  [Triglyceride]  D i s t r i b u t i o n o f H a l o t h a n e between C e l l s and  Plasma  A 10 ml g a s - t i g h t s y r i n g e f i t t e d w i t h a cap (made by b r e a k i n g o f f the n e e d l e p o r t i o n of a Luer Lok s y r i n g e n e e d l e and s o l d e r ) was ml b l o o d  weighed w i t h o u t  the p l u n g e r  f i n a l halothane plunger was  and  added, to g i v e a  40, 20 or 5 ml/100 m l , and  the  i n s e r t e d w i t h the n e e d l e end o f the s y r i n g e downward.  then i n v e r t e d , the cap removed, and the p l u n g e r was  e x p e l most of the a i r w i t h o u t  30 min.  at room temperature was  c o n c e n t r a t i o n of approximately  immediately  s y r i n g e was  s y r i n g e was  10  An a c c u r a t e l y known volume ( l e s s than 1 ml) o f a normal s a l i n e  s o l u t i o n saturated w i t h halothane  for  and then f i l l e d w i t h a p p r o x i m a t e l y  ( a n t i - c o a g u l a t e d w i t h EDTA) from a n o n - f a s t i n g h e a l t h y donor  reweighed.  The  s e a l i n g the opening w i t h  pressed  l o s i n g any s i g n i f i c a n t amount of b l o o d .  then r e s e a l e d w i t h the cap and r o t a t e d end  to  The  to end at 10-30  r.p.m.  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 b l o o d from t h i s s y r i n g e as  d e s c r i b e d i n P a r t I , and were e q u i l i b r a t e d by r o t a t i n g v e r y s l o w l y at 37°C. At the end o f the e q u i l i b r a t i o n , f o u r o f 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 to  r o t o r at 4,500 rpm  2,870 g) f o r 15 min, which was  sediment almost a l l the p l a t e l e t s (Schauberge et a l . 1976).  were then taken  f o r a n a l y s i s of h a l o t h a n e  the same s e t were a l s o a n a l y s e d the  (max.  equilibration.  concentration.  f o r halothane  Blood  sufficient  Plasma samples samples from  c o n c e n t r a t i o n b e f o r e and  after  - 48 Blood samples with a r t i f i c i a l l y low and elevated t r i g l y c e r i d e concentrations were prepared (see flow chart) by separating c e l l s and plasma and then centrifuging the l a t t e r i n a Beckman SW 41 rotor at 41,000 rpm 130,000 g, max.  290,000 g) for 4 hr.  (min.  The f i r s t and the last of the 11 ml i n  each centrifuge tube was discarded, and the remaining plasma was  reconstituted  with the c e l l s 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 t r i g l y c e r i d e preparation.(obtained by concentrating the t r i g l y c e r i d e - r i c h micelle f r a c t i o n from 25 ml of plasma to 0.5 ml using Amicon CF 50 membrane u l t r a f i l t e r s ) to y i e l d a t r i g l y c e r i d e - r i c h blood sample.  To the other, triglyceride-poor sample, was  added an equal  amount of saline. One ml aliquots were taken from each of the t r i g l y c e r i d e - r i c h and triglyceride-poor samples for the determination of t r i g l y c e r i d e concentration. Two plasma samples obtained from these two blood aliquots were further divided into two sets of t r i g l y c e r i d e - r i c h 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 n e g l i g i b l e quantities. and the other 8 times with 5 ml aliquots.  One set was washed 5 times  A l l the f i l t r a t e s from the 1st wash  and the four f i n a l retentates were then assayed for t r i g l y c e r i d e . The remaining t r i g l y c e r i d e - r i c h and triglyceride-poor blood samples were used for 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 as described above. The d i s t r i b u t i o n of halothane between c e l l s and plasma was calculated from the following equations:  - 49 Flow c h a r t o f the p r o c e d u r e f o r t h e p r e p a r a t i o n o f 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 b l o o d samples B l o o d (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  Cells  Centrifuge 4 hr 130,000-290,000 g D i s c a r d top 1 ml  ' D i s c a r d bottom 1 ml Remaining Plasmareconstitute B l o o d (10 ml)  B l o o d (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 preparationTr i g l y c e r i d e - r i c h b l o o d sample  Triglyceride-poor b l o o d sample b l o o d (1 ml) same t r e a t m e n t as f o r the corresponding t r i glyceride-rich sample  b l o o d (1 ml)  c e n t r i f u g e 1,000 g 5 min discard  cells  f o r t h e study o f t h e d i s t r i b u t i o n o f h a l o t h a n e between c e l l s and plasma plasma  wash 5 x with saline on Amicon CF25 f i l t e r  Filtrate^jf 1st wash  Retentate  Assay o f t r i g l y c e r i d e  wash 8 x with saline on Amicon CF 25  F i l t r a t e of 1st wash  1  concentration  filter  Retentate  [ H a l o t h a n e j b = _Hc [ H a l o t h a n e ] c + (1 - H e ) [ H a l o t h a n e ] p 100 100  [7]  Percent of halothane  i n plasma = (100-Hc)[Halothane]p [Halothanejb  [8]  Percent o f halothane  i n ce l i s  [9]  = He [Halothane ]c [Halothane]b  where [ H a l o t h a n e j b = e x p e r i m e n t a l l y determined m l ) i n blood  halothane  c o n c e n t r a t i o n (mg/100  [Halothane]p = e x p e r i m e n t a l l y determined  halothane  c o n c e n t r a t i o n (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 o c c u p i e d 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 T a b l e I X , the c o n c e n t r a t i o n o f h a l o t h a n e i n s a l i n e (0.85% w/v  N a C l ) e q u i l i b r a t e d w i t h a b u l k phase o f h a l o t h a n e at 37°C i n c r e a s e d w i t h time up to 43 h r , a f t e r which t h e r e was no f u r t h e r d e t e c t a b l e i n c r e a s e . S i m i l a r l y , the c o n c e n t r a t i o n o f h a l o t h a n e 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 b u l k phase o f h a l o t h a n e a t 4°C showed no d e t e c t a b l e i n c r e a s e a f t e r 72 h r .  Using  the average o f the l a s t 5 v a l u e s f o r 37°C and the 4 v a l u e s f o r 4°C i n T a b l e I X , the s a t u r a t i o n c o n c e n t r a t i o n o f h a l o t h a n e i n s a l i n e was found t o 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.  A d s o r p t i o n o f h a l o t h a n e t o haemoglobin T a b l e X shows the r e s u l t s o f p r e l i m i n a r y experiments performed  l i s h the time r e q u i r e d t o a c h i e v e e q u i l i b r i u m .  to estab-  I t can be seen t h a t when t h e  f r e e h a l o t h a n e c o n c e n t r a t i o n was a p p r o x i m a t e l y 15 mg/100 m l , the average number o f h a l o t h a n e m o l e c u l e s per haemoglobin hr.  m o l e c u l e i n c r e a s e d w i t h time up t o 68  When the f r e e h a l o t h a n e c o n c e n t r a t i o n was a p p r o x i m a t e l y 300 mg/100 m l ,  t h e r e was no s i g n i f i c a n t change i n the average number o f h a l o t h a n e m o l e c u l e s per haemoglobin m o l e c u l e a f t e r 73 h r .  T h e r e f o r e 92 h r was chosen as the  e q u i l i b r a t i o n time t o ensure t h a t 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 . one independent d i a l y s i s experiment. g/100 m l .  Each data p o i n t r e p r e s e n t s  The haemoglobin  c o n c e n t r a t i o n was 9.8  The average number o f h a l o t h a n e m o l e c u l e s bound t o each  haemoglobin  m o l e c u l e i n c r e a s e s from 0 t o a p p r o x i m a t e l y 5 w i t h i n c r e a s i n g f r e e h a l o t h a n e  Table IX - C o n c e n t r a t i o n o f h a l o t h a n e 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 b u l k phase o f h a l o t h a n e 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 o f s a l i n e e q u i l i b r a t e d w i t h a b u l k phase of h a l o t h a n e f o r d i f f e r e n t time i n t e r v a l s were a n a l y z e d f o r h a l o t h a n e c o n c e n t r a t i o n s t o e s t a b l i s h the time taken t o r e a c h e q u i l i b r i u m and t h e 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  37°C  E q u i l i b r a t i o n Time (hr)  4°C  Concentration (mg/100 ml)  E q u i l i b r a t i o n Time (hr)  Concentration (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 t o r e a c h e q u i l i b r i u m f o r the d i a l y s i s o f haemoglobin against halothane Haemoglobin s o l u t i o n s were d i a l y s e d a g a i n s t h a l o t h a n e f o r d i f f e r e n t time intervals. Samples o f the b u f f e r c o n t a i n i n g f r e e h a l o t h a n e and samples of t h e haemoglobin s o l u t i o n c o n t a i n i n g f r e e and bound h a l o t h a n e were then a n a l y z e d f o r h a l o t h a n e c o n c e n t r a t i o n t o e s t a b l i s h the time r e q u i r e d t o 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)  F r e e [Halothane] i n b u f f e r mg/100 ml  Average number o f h a l o t h a n e m o l e c u l e s p e r hemoglobin m o l e c u l e  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  F i g . 6.  Adsorption of halothane to haemoglobin.  - 55 c o n c e n t r a t i o n from 0 t o s a t u r a t i o n .  The s l o p e o f t h e curve g r a d u a l l y d e c r e a s e s  w i t h i n c r e a s i n g f r e e h a l o t h a n e c o n c e n t r a t i o n , b u t i t does n o t l e v e l o f f t o a p l a t e a u when t h e s a t u r a t i o n c o n c e n t r a t i o n o f h a l o t h a n e i s reached.  Linear  r e g r e s s i o n on those d a t a p o i n t s o b t a i n e d w i t h a f r e e h a l o t h a n e c o n c e n t r a t i o n o f l e s s than 150 mg/100 ml g i v e s t h e f o l l o w i n g e q u a t i o n : H where  h  = 0.0223[Halothane]f - 0.119  [10]  H^ = number o f h a l o t h a n e m o l e c u l e s bound p e r haemoglobin m o l e c u l e  [ H a l o t h a n e ] f = f r e e h a l o t h a n e c o n c e n t r a t i o n i n mg/100 ml The r e s u l t s were a l s o a n a l y s e d by the S c a t c h a r d 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 o f t h e r e s u l t s o b t a i n e d 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 u s i n g t h e S c a t c h a r d e q u a t i o n g i v e s a t o t a l number o f 20 HH 6 s i t e s ( 9 5 % c o n f i d e n c e l i m i t ) (Snedecor and Cochran 1967) f o r h a l o t h a n e on each haemoglobin m o l e c u l e as the X - i n t e r c e p t o f t h e f o l l o w i n g equation: , h . [Halothane]f H  3.  = -1.08 x 10-3 H  h  + 2.20 x 10"2  A d s o r p t i o n o f h a l o t h a n e t o albumin T a b l e X I shows t h e r e s u l t s o f p r e l i m i n a r y experiments performed t o  e s t a b l i s h the time r e q u i r e d to a c h i e v e e q u i l i b r i u m .  When t h e f r e e h a l o t h a n e  c o n c e n t r a t i o n was a p p r o x i m a t e l y 190 mg/100 m l , t h e r e was no s i g n i f i c a n t change i n t h e average number o f h a l o t h a n e m o l e c u l e s p e r albumin m o l e c u l e a f t e r 96 hr.  T h i s was t h e r e f o r e chosen as t h e 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 o b t a i n e d 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 t o t h a t f o r haemoglobin ( F i g . 6) except  that  - 56 -  = number o f h a l o t h a n e m o l e c u l e s bound p e r haemoglobin [Halothane]  Fig. 7.  f  = f r e e h a l o t h a n e c o n c e n t r a t i o n i n mg/100 m l  Scatchard plot of halothane binding to haemoglobin.  molecule  T a b l e X I - Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r t h e 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 a g a i n s t h a l o t h a n e f o r d i f f e r e n t time i n t e r v a l s . Samples o f t h e b u f f e r c o n t a i n i n g f r e e h a l o t h a n e and samples o f t h e 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 h a l o t h a n e were a n a l y s e d f o r h a l o t h a n e c o n c e n t r a t i o n t o e s t a b l i s h the time r e q u i r e d t o 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 [ H a l o t h a n e ] i n b u f f e r mg/100 ml  Average number o f h a l o t h a n e m o l e c u l e per albumin molecule  68  190  9.7  96  174  14.9  114  176  16.6  118  192  14.9  U l  - 58 -  20r  HALOTHANE (MG/100 ML)  F i g . 8.  Adsorption of halothane to albumin.  - 59 the average number o f h a l o t h a n e m o l e c u l e s bound t o each a l b u m i n m o l e c u l e i s much h i g h e r , r e a c h i n g a p p r o x i m a t e l y 20 when t h e b u f f e r was s a t u r a t e d w i t h halothane.  The presence o f p h y s i o l o g i c a l c o n c e n t r a t i o n s o f o l e i c a c i d had no  d e t e c t a b l e e f f e c t on the i n t e r a c t i o n o f h a l o t h a n e w i t h albumin.  Linear  r e g r e s s i o n on those d a t a p o i n t s o b t a i n e d w i t h a f r e e h a l o t h a n e c o n c e n t r a t i o n l e s s than 150 mg/100 ml gave t h e f o l l o w i n g e q u a t i o n : H  = 0.0830[Halothane]f - 0.0294  [11]  SL  where H  a  = number o f h a l o t h a n e m o l e c u l e s bound p e r albumin m o l e c u l e r  [ H a l o t h a n e ] f = f r e e h a l o t h a n e c o n c e n t r a t i o n i n mg/100 ml L i n e a r r e g r e s s i o n o f the r e s u l t s w i t h H  l a r g e r than 4 u s i n g S c a t c h a r d cl  e q u a t i o n ( F i g . 9) ( M a r s h a l l 1978) g i v e s a t o t a l number o f 130 + 29 s i t e s ( 9 5 % c o n f i d e n c e l i m i t ) (Snedecor and Cochran 1967) f o r h a l o t h a n e on each albumin m o l e c u l e as t h e X - i n t e r c e p t o f t h e f o l l o w i n g e q u a t i o n : H = -6.95 x I t ) * H [Halothane]f -  a  4.  a  + 9.07 x 1 0  - 2  y-globulin T a b l e X I I shows the r e s u l t s o f the d i a l y s i s o f y - g l o b u l i n a g a i n s t h a l o -  thane.  There was no d e t e c t a b l e d i f f e r e n c e between t h e f r e e h a l o t h a n e concen-  t r a t i o n and t h e 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 presence o f y - g l o b u l i n .  This  i n d i c a t e s t h a t t h e r e was no s i g n i f i c a n t a d s o r p t i o n o f h a l o t h a n e t o y - g l o b u l i n at t h e p h y s i o l o g i c a l y - g l o b u l i n c o n c e n t r a t i o n employed.  5.  Absorption of halothane to r e d c e l l  ghosts  T a b l e X I I I shows the r e s u l t s o f p r e l i m i n a r y experiments performed t o e s t a b l i s h t h e time r e q u i r e d t o a c h i e v e e q u i l i b r i u m .  When t h e f r e e h a l o t h a n e  - 60 H [Halothane]  r  0 -10  •  •  0  0  05  10  15  H H  &  = number of halothane molecules bound per albumin molecule  [Halothane] = free halothane concentration i n mg/100 ml f  F i g . 9.  Scatchard p l o t o f halothane b i n d i n g t o 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 a g a i n s t h a l o t h a n e f o r d i f f e r e n t time intervals. Samples o f t h e b u f f e r c o n t a i n i n g f r e e h a l o t h a n e and samples of t h e y - g l o b u l i n s o l u t i o n were then a n a l y s e d f o r h a l o t h a n e c o n c e n t r a t i o n s t o e s t a b l i s h whether or not d e t e c t a b l e b i n d i n g o f h a l o t h a n e t o y - g l o b u l i n o c c u r r e d  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 t o reach e q u i l i b r i u m f o r t h e d i a l y s i s o f r e d c e l l ghosts a g a i n s t h a l o t h a n e Red c e l l g h o s t s s u s p e n s i o n s were d i a l y s e d a g a i n s t h a l o t h a n e f o r d i f f e r e n t time i n t e r v a l s . Samples o f t h e b u f f e r c o n t a i n i n g f r e e h a l o t h a n e and samples o f the r e d c e l l g h o s t s s u s p e n s i o n c o n t a i n i n g f r e e and bound h a l o t h a n e were then a n a l y s e d f o r h a l o t h a n e c o n c e n t r a t i o n to e s t a b l i s h t h e time r e q u i r e d to reach equilibrium  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 ghosts s u s p e n s i o n (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 a p p r o x i m a t e l y i n the halothane  c o n c e n t r a t i o n i n t h e ghost suspension  chosen as t h e e q u i l i b r a t i o n Fig.  130 mg/100 m l , t h e r e was no s i g n i f i c a n t change a f t e r 44 h r .  T h i s was  time.  10 shows t h e 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 .  I n c o n t r a s t to the r e s u l t s  o b t a i n e d w i t h haemoglobin and albumin t h e s l o p e s o f t h e curve f o r r e d c e l l ghosts i n c r e a s e d w i t h i n c r e a s i n g h a l o t h a n e  concentration.  on t h e data p o i n t s o b t a i n e d w i t h a f r e e h a l o t h a n e  Linear regression  c o n c e n t r a t i o n o f l e s s than  150 mg/100 ml gave the f o l l o w i n g e q u a t i o n : H  = 6.28 x 1 0 [ H a l o t h a n e ] f 6  + 3.15 x 1 0  [12]  7  g where H^ = number o f h a l o t h a n e [Halothane]f = f r e e halothane 6.  Absorption o f halothane  molecules s o l u b i l i z e d per r e d c e l l  ghost  c o n c e n t r a t i o n i n mg/100 ml  t o t r i g l y c e r i d e - r i c h m i c e l l e s ( c h y l o m i c r o n s and  v e r y low d e n s i t y l i p o p r o t e i n s ) T a b l e XIV shows t h e r e s u l t s o f p r e l i m i n a r y experiments performed t o e s t a b l i s h t h e time r e q u i r e d t o a c h i e v e e q u i l i b r i u m . c o n c e n t r a t i o n was a p p r o x i m a t e l y i n the halothane a f t e r 47 h r . Fig.  When t h e f r e e  halothane  30 mg/100 m l , t h e r e was no s i g n i f i c a n t change  c o n c e n t r a t i o n 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  T h i s was chosen as the e q u i l i b r a t i o n  suspension  time.  11 shows t h e 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 t h e 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 was k e p t c o n s t a n t  a t 83 mg/100 m l . The s l o p e o f t h e curve  increased with i n c r e a s i n g f r e e 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 t o  t h a t found f o r r e d c e l l ghosts ( F i g . 1 0 ) , b u t t h e change i n s l o p e was even more pronounced w i t h t h e t r i g l y c e r i d e m i c e l l e s . data p o i n t s o b t a i n e d w i t h f r e e h a l o t h a n e ml gave t h e f o l l o w i n g e q u a t i o n :  L i n e a r r e g r e s s i o n o f those  c o n c e n t r a t i o n o f l e s s t h a n 150 mg/100  - 64 -  Fig. 10.  Absorption of halothane to red c e l l ghosts.  T a b l e XIV - Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r t h e d i a l y s i s o f 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 a g a i n s t halothane f o r d i f f e r e n t time i n t e r v a l s . Samples o f the b u f f e r c o n t a i n i n g f r e e h a l o t h a n e and samples o f 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 c o n t a i n i n g f r e e and bound h a l o t h a n e were than a n a l y s e d f o r h a l o t h a n e 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)  F r e e [Halothane] i n b u f f e r mg/100 ml  [Halothane] i n t r i g l y c e r i d e - r i c h m i c e l l e s u s p e n s i o n (mg/100 m l )  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 t r i g l y c e r i d e - r i c h micelles at a constant t r i g l y c e r i d e concentration of 83 mg/100 ml.  - 67 -  H where H  = 0.00537[Halothane]f  - 0.0170  [13]  = grams o f h a l o t h a n e s o l u b i l i z e d per grams o f  triglyceride  [ H a l o t h a n e ] f = f r e e h a l o t h a n e c o n c e n t r a t i o n i n mg/100 ml As shown i n F i g . 12 when the f r e e h a l o t h a n e c o n c e n t r a t i o n was c o n s t a n t at 9.2 mg/100 m l , t h e r e was a l i n e a r r e l a t i o n s h i p  kept  between the  q u a n t i t y of h a l o t h a n e 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 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 ( i e . the number o f t r i g l y c e r i d e - r i c h  the  micelles).  L i n e a r r e g r e s s i o n o f a l l the data p o i n t s g i v e s the f o l l o w i n g e q u a t i o n : [Halothane]  t  = 0.0346[TG] + 0.227  [14]  where [ H a l o t h a n e ] t = h a l o t h a n e (mg/100 ml) s o l u b i l i z e d i n the presence o f triglyceride rich micelles [TG] = 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 (mg/100 ml)  7.  Distribution  o f h a l o t h a n e between the components of b l o o d  E q u a t i o n s [ 1 0 ] - [ 1 3 ] 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 o f h a l o t h a n e between water, a l b u m i n , t r i g l y c e r i d e , haemoglobin and r e d c e l l membrane i n b l o o d u s i n g normal v a l u e s f o r the b l o o d components (Henry et a l . 1974)  and  assuming t h a t these are the o n l y i m p o r t a n t 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. i n Table  8.  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  XV.  Distribution  o f h a l o t h a n e between c e l l s and plasma  T a b l e XVI shows the r e s u l t s  o f p r e l i m i n a r y experiments performed  l i s h the time r e q u i r e d to a c h i e v e e q u i l i b r i u m .  to e s t a b -  As e x p e c t e d , the c o n c e n t r a t i o n  of h a l o t h a n e i n the plasma was h i g h e r than t h a t i n the whole b l o o d a t the  - 68 -  4r  ol 0  i 50  J 100  TRIGLYCERIDE (MG/100 ML)  F i g . 12. Absorption of halothane to t r i g l y c e r i d e - r i c h micelles at a constant halothane concentration of 9.2 mg/100 ml.  T a b l e XV - D i s t r i b u t i o n o f h a l o t h a n e i n b l o o d c a l c u l a t e d from the r e s u l t s o f equilibrium dialysis T h i s 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 e q u a t i o n s [10]-[13] (which were o b t a i n e d when the f r e e h a l o t h a n e c o n c e n t r a t i o n was l e s s than 150 mg/100 ml) i s a p p l i c a b l e t o the range o f b l o o d h a l o t h a n e c o n c e n t r a t i o n found d u r i n g anaesthesia.  C o n t r i b u t i o n to halothane  B l o o d Component  solubility  % of t o t a l  Plasma  albumin (4.3 g/100 m l ) t r i g l y c e r i d e (100 mg/100 ml) water  37  16 6 15 ( f r e e h a l o t h a n e )  Blood  r e d c e l l membrane (4.5 x 10 RBC/ml) hemoglobin (14.5 g/100 ml) water  63  40 13 10 ( f r e e h a l o t h a n e )  9  T a b l e XVI - Time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r t h e d i s t r i b u t i o n of h a l o t h a n e between c e l l s and plasma Whole b l o o d samples c o n t a i n i n g h a l o t h a n e were e q u i l i b r a t e d f o r d i f f e r e n t time i n t e r v a l s . B l o o d and plasma samples were then a n a l y s e d f o r h a l o t h a n e c o n c e n t r a t i o n t o e s t a b l i s h t h e time r e q u i r e d t o reach e q u i l i b r i u m  Time ( h r )  [Halothane] mg/100 ml Plasma  1.0  47.0  1.0  47.6  6.0  38.6  19.5  34.5  21.0  34.6  24.0  32.7  24.0  35.6  Blood  24.5  i --J  o i  42.3  b e g i n n i n g o f the e q u i l i b r a t i o n . c o n c e n t r a t i o n was T h i s was  lower  A f t e r 19.5  h r however, the plasma  halothane  than t h a t i n b l o o d and appeared to have s t a b i l i z e d .  chosen as the e q u i l i b r a t i o n  time.  T a b l e X V I I compares the e x p e r i m e n t a l l y 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 from e q u a t i o n  [10]-[13].  at d i f f e r e n t h a l o t h a n e different  Comparison was  o f a l b u m i n , haemoglobin and m e n t a l l y determined.  negligible. f o r the two  The  samples  two samples from the same donor w i t h  The  c e l l counts and the  concentrations  t r i g l y c e r i d e o f these b l o o d samples were e x p e r i -  S i n c e , as shown i n T a b l e X V I I , t h e r e was  d i f f e r e n c e i n the h a l o t h a n e the e q u i l i b r a t i o n ,  made f o r t h r e e normal b l o o d  c o n c e n t r a t i o n s and  t r i g l y c e r i d e concentrations.  distribution calculated  no  detectable  c o n c e n t r a t i o n o f the whole b l o o d b e f o r e and  the amount o f h a l o t h a n e  after  l o s t d u r i n g the e q u i l i b r a t i o n  was  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 )  s e t s of plasma samples, washed 5 times and 8 times w i t h s a l i n e  (to  d i l u t e the c o n c e n t r a t i o n o f EDTA) r e s p e c t i v e l y , are a l s o shown i n T a b l e X V I I . There was  no d e t e c t a b l e change i n the 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 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 t r i g l y c e r i d e assay from any r e m a i n i n g  t h a t t h e r e was anticoagulant.  no i n t e r f e r e n c e w i t h  the  T a b l e X V I I - D i s t r i b u t i o n o f h a l o t h a n e between c e l l s and plasma Whole b l o o d samples were e q u i l i b r a t e d w i t h h a l o t h a n e . B l o o d and plasma samples were t h e n analysed f o r halothane concentration  Plasma T r i g l y c e r i d e 5 x washed*  30  +  186  + +  (mg/100 m l ) 8 x washed*  [ H a l o t h a n e ] mg/100 ml i n whole: b l o o d after before equilibration equilibration  Dialys i s * * 7o o f H a l o t h a n e in Plasma Cell  Centrifugation % o f Halothane in Plasma Cell  42.8  41.7  38  62  48  52  23.9  23.4  37  63  49  51  3.8  4.2  38  62  49  51  35  25.1  28  72  39  61  179  25.7  34  66  48  52  *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 s were assayed a f t e r washing w i t h s a l i n e t o d i l u t e t h e d i s o d i u m EDTA c o n c e n t r a t i o n t o n e g l i g i b l e amount * * C a l c u l a t e d from e q u a t i o n [ 1 0 ] - [ 1 3 ] 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 h a l o t h a n e i n s a l i n e (297 mg/100 ml a t 37°C and  mg/100 ml at 4°C)  481  i s h i g h compared to t h a t o f n-pentane (3.1 mg/100 ml at  20°C i n R i n g e r s o l u t i o n ) (Haydon e t a l . 1977), p r o b a b l y due to the f o r m a t i o n o f hydrogen bonds between the f l u o r i n e s o f h a l o t h a n e and water m o l e c u l e s . That h a l o t h a n e i s more s o l u b l e at a lower temperature i s expected f o r a v o l a t i l e l i q u i d and agrees w i t h the p r e v i o u s f i n d i n g s t h a t the  water/gas  p a r t i t i o n c o e f f i c i e n t i n c r e a s e d w i t h d e c r e a s i n g temperature ( A l l o t t et a l . 1973).  However, the s o l u b i l i t y o f h a l o t h a n e 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 o n c e n t r a t i o n s , as the water/gas, s a l i n e / g a s and Kreb's s o l u t i o n / g a s 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, et a l . 1973) and 0.75 The  0.70-0.77 (Steward  ( R e n z i and Waud 1972)) are a l l n e a r l y the same.  time taken f o r the d i a l y s i s to r e a c h e q u i l i b r i u m i s u n u s u a l l y l o n g .  T h i s was most p r o b a b l y due to the s m a l l s u r f a c e a r e a o f the d i a l y s i s membrane. The d i a l y s i s s u r f a c e ( r e l a t i v e to the volume c o n t a i n i n g the b l o o d component, F i g . 5) was much l e s s than a r o u t i n e d i a l y s i s u s i n g a d i a l y s i s t u b i n g . time taken to r e a c h e q u i l i b r i u m f o r r e d c e l l ghosts and t r i g l y c e r i d e  The  rich  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 q u i t e d i f f e r e n t from t h a t f o r haemoglobin  and albumin (73 and 96 hours r e s p e c t i v e l y ) , p r o b a b l y 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 o f the a d s o r p t i o n isotherms o f h a l o t h a n e to haemoglobin  and  albumin suggest t h a t h a l o t h a n e i s a t t a c h e d to a f i n i t e number of s i t e s on the protein surface.  S i n c e h a l o t h a n e i s v e r y s o l u b l e i n o i l and q u i t e  insoluble  i n water (Steward et a l . 1973), these are presumably h y d r o p h o b i c s i t e s ;  the  - 74 bound h a l o t h a n e m o l e c u l e s are s t a b i l i z e d by h y d r o p h o b i c i n t e r a c t i o n s .  It is  a l s o p o s s i b l e t h a t h a l o t h a n e 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 r e g i o n (e.g. a c i d i c group) of the p r o t e i n . isotherms  As  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 y e t  ated when the aqueous phase was  saturated with halothane.  satur-  In a t h e o r e t i c a l  s t u d y , T a n f o r d (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 t h a t the crease  the  de-  i n f r e e energy a s s o c i a t e d w i t h the b i n d i n g of h y d r o p h o b i c m o l e c u l e s  with a p r o t e i n i s less favourable  than t h a t i n the s e l f - a s s o c i a t i o n between  the hydrophobic m o l e c u l e s when the s o l u t i o n i s s a t u r a t e d . Hildebrand  (1979) m a i n t a i n e d  Alternatively,  t h a t the i n c r e a s e i n f r e e energy due  to  the  d i s r u p t i o n of hydrogen bonds between water m o l e c u l e s i s too u n f a v o u r a b l e more h a l o t h a n e to e n t e r i n t o the aqueous s o l u t i o n , i n which case the  for  chemical  p o t e n t i a l of h a l o t h a n e i n the aqueous s o l u t i o n i s not h i g h enough f o r b i n d i n g w i t h more p r o t e i n s u r f a c e s i t e s to A l t h o u g h the a d s o r p t i o n  occur.  isotherms  f o r haemoglobin and albumin are  quali-  t a t i v e l y s i m i l a r , many more h a l o t h a n e m o l e c u l e s were bound to albumin at given f r e e halothane concentration. g e n e r a l l y considered ( P e t e r s 1975) than 0.01%  T h i s was  any  to be expected s i n c e albumin i s  to be a c a r r i e r p r o t e i n f o r s m a l l h y d r o p h o b i c m o l e c u l e s  such as the plasma f a t t y a c i d s .  I t has been e s t i m a t e d  that l e s s  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 f r e e i n the plasma  (Goodman 1958)  and  (cis-9-octadecenoic  t h a t 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 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 o f h a l o t h a n e to a l b u m i n . F i g . 6 shows t h a t o l e i c a c i d at p h y s i o l o g i c a l c o n c e n t r a t i o n s had no e f f e c t i n the b i n d i n g o f h a l o t h a n e to a l b u m i n .  detectable  T h i s r e s u l t i s open to s e v e r a l  - 75 interpretations: sites.  ( i ) Halothane and o l e i c a c i d do not compete f o r the same  T h i s i s u n l i k e l y because both are expected to b i n d n o n - s p e c i f i c a l l y to  hydrophobic s i t e s , a l t h o u g h h a l o t h a n e and o l e i c a c i d , b e i n g q u i t e d i f f e r e n t i n m o l e c u l a r d i m e n s i o n s , may  b i n d 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 .  number o f s i t e s i s l a r g e enough so t h a t the b i n d i n g does not reduce the number o f s i t e s a v a i l a b l e f o r h a l o t h a n e .  (iii)  ( i i ) The  significantly The b i n d i n g of  o l e i c a c i d t o albumin c r e a t e s new h y d r o p h o b i c s i t e s f o r h a l o t h a n e .  I t should  be noted t h a t the l a s t two i n t e r p r e t a t i o n s are not m u t u a l l y e x c l u s i v e . Fig.  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 a c c o r d i n g t o  the S c a t c h a r d e q u a t i o n , which assumes t h a t t h e r e 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 m o l e c u l e f o r the i n t e r a c t i o n w i t h h a l o t h a n e which are i d e n t i c a l and independent  o f one a n o t h e r .  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 s h o u l d show a s t r a i g h t l i n e w i t h a n e g a t i v e s l o p e ( M a r s h a l l 1978).  This  i s the advantage of the S c a t c h a r d method because the v a l i d i t y of the assumption can be judged by how The assumption  c l o s e the data p o i n t s f i t a s t r a i g h t  line.  i s o b v i o u s l y not t r u e i n the case of b i n d i n g of h a l o t h a n e to  albumin and haemoglobin.  F i g . 7 and 9 b o t h appear to have an i n i t i a l  s l o p e f o l l o w e d by a l e s s pronounced n e g a t i v e s l o p e .  Thus the n a t u r e of  b i n d i n g does not f i t a s i m p l e assumption o f i d e n t i c a l and independent The  positive  sites.  i n i t i a l p o s i t i v e s l o p e suggests a p o s s i b l e p o s i t i v e c o o p e r a t i v e e f f e c t  (Jones 1975; M a r s h a l l 1978).  The dimension o f the s u r f a c e s i t e s has been  suggested as a p o s s i b l e e x p l a n a t i o n of t h i s c o o p e r a t i v e e f f e c t ( T a n f o r d 1973).  I f a s i t e i s l a r g e enough to accommodate more than one s m a l l m o l e c u l e ,  then i t c o u l d be e n e r g e t i c a l l y more f a v o u r a b l e f o r a second s m a l l m o l e c u l e t o b i n d to the same s i t e a f t e r the f i r s t s m a l l m o l e c u l e i s a l r e a d y bound.  - 76 When t h e i n i t i a l p a r t s o f t h e two graphs w i t h the p o s i t i v e s l o p e s a r e i g n o r e d , l i n e a r r e g r e s s i o n o f a l l t h e data p o i n t s on the second p o r t i o n s w i t h n e g a t i v e s l o p e g i v e a t o t a l number o f 20 and 130 s u r f a c e s i t e s on haemoglobin and albumin r e s p e c t i v e l y f o r t h e b i n d i n g o f h a l o t h a n e . The  slope of the absorption isotherm of halothane  t o r e d c e l l ghosts sug-  g e s t s t h a t the n a t u r e o f the i n t e r a c t i o n between h a l o t h a n e membrane, and t h a t between h a l o t h a n e different.  Since halothane  and t h e r e d c e l l  and haemoglobin or albumin a r e v e r y  i s h y d r o p h o b i c and n o t s u r f a c e 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 t h e h y d r o p h o b i c r e g i o n o f t h e membrane. Thus the "bound" h a l o t h a n e f l u i d membrane.  would n o t be l o c a l i z e d t o a f i x e d s i t e w i t h i n t h e  The data suggest t h a t i t i s more f a v o u r a b l e f o r  halothane  m o l e c u l e s t o move i n t o a membrane which has a l r e a d y i n c o r p o r a t e d some molecules.  Possibly  the halothane  m o l e c u l e s 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 l e a d t o a l e s s o r g a n i z e d accommodating more h a l o t h a n e  halothane  molecules.  s t r u c t u r e , c a p a b l e of  I n a study o f t h e e f f e c t o f n - a l k a n e s  on b l a c k l i p i d b i l a y e r membrane (Haydon e t a l . 1977), i t was found t h a t an i n c r e a s e o f a l k a n e c o n c e n t r a t i o n caused an i n c r e a s e i n t h e h y d r o p h o b i c t h i c k ness o f the b l a c k 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 s l o p e o f t h e curve i n c r e a s e d w i t h i n c r e a s i n g a l k a n e  concen-  t r a t i o n i n a way s i m i l a r t o the r e l a t i o n s h i p found here between t h e number o f halothane  m o l e c u l e s p e r ghost and the f r e e h a l o t h a n e  concentration.  Our  r e s u l t s a r e i n q u a l i t a t i v e agreement w i t h those of Haydon e t a l . (1977). I n the plasma, t r i g l y c e r i d e i s t r a n s p o r t e d 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 v e r y low d e n 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 o f 88% and 56% o f 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 a b s o r p t i o n i s o t h e r m o f h a l o t h a n e  i n these  triglyceride-rich  - 77 m i c e l l e s resembles  q u a l i t a t i v e l y t h a t f o r the r e d c e l l membrane, c o n s i s t e n t  w i t h the f a c t t h a t 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  I t i s apparent  by a h y d r o p h i l i c s u r f a c e .  ( T a b l e XV) t h a t a l l the major components o f human b l o o d  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 r e d 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 t o the s o l u b i l i t y , and hence the transport o f halothane i n blood.  A c c o r d i n g t o the r e s u l t s o b t a i n e d  from  e q u i l i b r i u m d i a l y s i s , r e d c e l l membranes, which c a r r y 40% o f the t o t a l amount of  h a l o t h a n e i n whole b l o o d , a r e q u a n t i t a t i v e l y most i m p o r t a n t , f o l l o w e d by  the water  i n b l o o d , i n which 25% o f the h a l o t h a n e i s d i s s o l v e d .  Albumin c a r -  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 a p p r o x i m a t e l y 6%.  Using  these r e s u l t s i n c o m b i n a t i o n w i t h the s a l i n e / g a s p a r t i t i o n c o e f f i c i e n t  (taking  an average v a l u e o f 0.74 (Steward et a l . 1973)), the blood/gas c o e f f i c i e n t was c a l c u l a t e d t o be 2.6. the e x p e r i m e n t a l l y determined  partition  T h i s i s i n r e a s o n a b l e agreement w i t h  v a l u e o f 2.3 (Steward e t a l . 1973).  The d a t a  i n d i c a t e s t h e r e f o r e t h a t the s o l u b i l i t y o f h a l o t h a n e i n b l o o d w i l l i n c r e a s e w i t h i n c r e a s e s i n a l b u m i n , t r i g l y c e r i d e , haemoglobin and h a e m a t o c r i t , e i t h e r s i n g l y or i n c o m b i n a t i o n s . however, a r e i n c o n f l i c t . r e p o r t e d a decrease al.  E x p e r i m e n t a l o b s e r v a t i o n s on these  relationships,  S e v e r a l i n v e s t i g a t o r s (see Table V I I I ) have  i n s o l u b i l i t y w i t h an i n c r e a s e i n h a e m a t o c r i t  (Cowles e t  1971a; Han and H e l r i c h 1966; Lowe and H a g l e r 1969) w h i l e o t h e r s t u d i e s  have shown t h a t the s o l u b i l i t y o f h a l o t h a n e i s independent ( S a r a i v a et a l . 1977a),  t h a t h a l o t h a n e i s l e s s s o l u b l e i n b l o o d o f low  h a e m a t o c r i t or more s o l u b l e i n b l o o d o f h i g h h a e m a t o c r i t Hedley-Whyte 1970) and t h a t h a l o t h a n e and S t o e l t i n g 1975).  of haematocrit  (Laasberg and  i s l e s s s o l u b l e i n anemic b l o o d  (Ellis  I t has a l s o been r e p o r t e d t h a t l i p i d c o n c e n t r a t i o n  either 1974)  increased (Saraiva et a l . 1977a; Larson et a l . 1962;  Wagner et a l .  or was not correlated (Munson et a l . 1978) with the s o l u b i l i t y of  halothane,  and that the s o l u b i l i t y was  dependent upon the albumin to globulin  r a t i o (Laasberg and Hedley-Whyte 1970).  Saraiva et a l . (1977a) studied the  e f f e c t of the concentrations of haemoglobin, serum albumin, globulin, protein, t r i g l y c e r i d e , cholesterol,  the haematocrit,  total  the albumin to globulin  r a t i o and the albumin to t o t a l protein r a t i o upon the s o l u b i l i t y of halothane in blood and were able to demonstrate a s t a t i s t i c a l l y s i g n i f i c a n t  correlation  only with serum t r i g l y c e r i d e .  Munson et a l . (1978) were, however, unable to  demonstrate this correlation,  possibly because they used a narrower range of  t r i g l y c e r i d e concentrations and a smaller number of samples. In the studies referred to above the effect of various blood components upon the s o l u b i l i t y of halothane was blood/gas p a r t i t i o n c o e f f i c i e n t  inferred  i n d i r e c t l y from changes i n the  (see Introduction, Part I I ) .  Such changes are  s l i g h t even when the concentration of a contributing blood component changes d r a s t i c a l l y , as exemplified by the apparently c o n f l i c t i n g 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 i s small, as i s the case with most blood components, any change i n the p a r t i t i o n c o e f f i c i e n t be too small to be detected.  may  Furthermore, when whole blood samples are used,  the effect of random changes i n the concentration of a l l the blood components tends to mask the e f f e c t of the change i n concentration of a p a r t i c u l a r component, thereby decreasing the chance of finding a s t a t i s t i c a l l y correlation.  In other words, the lack of a s t a t i s t i c a l l y  significant  significant  correlation between the p a r t i t i o n c o e f f i c i e n t and the concentration of a  - 79 -  c e r t a i n b l o o d component does not n e c e s s a r i l y imply t h a t the b l o o d 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 h a l o t h a n e , i t may  s i m p l y mean t h a t  the a n a l y t i c a l method b e i n g used i s not s e n s i t i v e enough t o d e t e c t the c o n t r i b u t i o n o f the v a r i o u s b l o o d components as found i n t h i s s t u d y . not s u p r i s i n g , t h e r e f o r e , t h a t the o n l y s t a t i s t i c a l l y s i g n i f i c a n t found by S a r a i v a et a l . (1977) was  that for t r i g l y c e r i d e .  It is  correlation  I n t h a t study  t r i g l y c e r i d e has the w i d e s t normal range i n the b l o o d samples used.  As can  be  seen i n T a b l e XVII a l a r g e i n c r e a s e 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 i n c r e a s e i n the plasma h a l o t h a n e c o n c e n t r a t i o n . The d i s t r i b u t i o n o f h a l o t h a n e 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 s t u d i e s agrees r e a s o n a b l y w e l l w i t h t h a t from the study of whole b l o o d ( T a b l e X V I I ) .  There seems to be a s y s t e m a t i c 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 o f h a l o t h a n e found by the d i r e c t assay of the plasma b e i n g c o n s i s t e n t l y h i g h e r than t h a t c a l c u l a t e d from the r e s u l t s o f the d i a l y s i s method. T h i s d i s c r e p a n c y can be p a r t l y accounted  f o r by those b l o o d 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 o t h e r than albumin and y g l o b u l i n , and c e l l s o t h e r than r e d b l o o d c e l l s .  S i n c e the  t o t a l number o f w h i t e b l o o d c e l l s i n a normal b l o o d sample i s o n l y a p p r o x i m a t e l y 2 per 1000 ignored. 1/10  r e d 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  F u r t h e r , a l t h o u g h the r a t i o o f p l a t e l e t s to r e d c e l l s i s l e s s  (Ganong 1975), the t o t a l membrane s u r f a c e area o f p l a t e l e t s i s l e s s  1% o f the r e d c e l l (assuming  than than  d i a m e t e r s o f 3 um and 7 um f o r p l a t e l e t s and r e d  c e l l s r e s p e c t i v e l y (Leeson and Leeson 1976)  and t h a t the r a t i o of s u r f a c e a r e a  - 80 -  i s t h e r a t i o o f t h e square o f d i a m e t e r s ) . p l a t e l e t s can a l s o be i g n o r e d .  Therefore the c o n t r i b u t i o n  from  Plasma p r o t e i n s o t h e r than albumin and  y - g l o b u l i n c o n s t i t u t e a p p r o x i m a t e l y 30% o f t h e t o t a l plasma p r o t e i n (Henry e t a l . 1974).  I f i t i s assumed t h a t e q u a l w e i g h t s o f albumin and o f plasma  p r o t e i n s o t h e r than albumin and y - g l o b u l i n b i n d t h e same amount o f h a l o t h a n e then t h e d i s c r e p a n c i e s between the two e s t i m a t e s o f d i s t r i b u t i o n However t h i s assumption  disappear.  i s u n l i k e l y t o be v a l i d , s i n c e t h e unaccounted f o r  plasma p r o t e i n s have p r o b a b l y l e s s b i n d i n g c a p a c i t y f o r h a l o t h a n e than  albumin  and g i v e o n l y a p a r t i a l e x p l a n a t i o n f o r these d i s c r e p a n c i e s . The c o n c e n t r a t i o n o f haemoglobin may a l s o account  f o r these d i s c r e p a n c i e s .  The haemoglobin c o n c e n t r a t i o n used i n t h e d i a l y s i s experiments  was a p p r o x i -  m a t e l y 2/3 t h a t o f the normal haemoglobin c o n c e n t r a t i o n i n t h e whole b l o o d , o r l e s s than 1/3 o f t h e a c t u a l haemoglobin c o n c e n t r a t i o n s w i t h i n t h e r e d c e l l s , s i n c e haemoglobin m o l e c u l e s of haemoglobin m o l e c u l e s  a r e c o n c e n t r a t e d w i t h i n t h i s volume.  Aggregation  i n i n t a c t r e d c e l l s may s h i e l d some o f t h e s u r f a c e  s i t e s f o r h a l o t h a n e , 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 a d s o r p t i o n o f h a l o t h a n e t o haemoglobin would be an o v e r e s t i m a t e o f the a c t u a l a d s o r p t i o n o c c u r r i n g i n whole b l o o d .  Consequently  an o v e r e s t i m a t e would be  made o f t h e d i s t r i b u t i o n o f h a l o t h a n e between c e l l s and plasma i n f a v o u r o f the c e l l  fraction.  F u r t h e r , s i n c e the same r e g r e s s i o n e q u a t i o n s were used i n  the c a l c u l a t i o n s f o r a l l the b l o o d samples, of  any random e r r o r i n t h e e s t i m a t i o n  the h a l o t h a n e bound t o a p a r t i c u l a r b l o o d component w i l l be c o n v e r t e d t o a  systematic error. The c l a i m s t h a t the s o l u b i l i t y o f h a l o t h a n e i n b l o o d has a n e g a t i v e dependence on h a e m a t o c r i t (Cowles  et a l . 1971a; Han and H e l r i c h 1966; Lowe and  - 81 H a g l e r 1969)  are d i f f i c u l t t o e v a l u a t e because t h e r e were no d a t a r e g a r d i n g  the time r e q u i r e d t o r e a c h e q u i l i b r i u m .  I n these s t u d i e s h a l o t h a n e was i n t r o -  duced i n t o the aqueous phase p r i o r t o t h e e q u i l i b r a t i o n w i t h a head space (gas phase) f o r the d e t e r m i n a t i o n o f the blood/gas  partition coefficient.  It is  c o n c e i v a b l e t h a t t h e r a t e o f v a p o r i z a t i o n o f h a l o t h a n e from the aqueous phase to  the head space ( t h e gas phase i n which  the p a r t i a l p r e s s u r e was measured)  i s much f a s t e r than t h e r a t e o f h a l o t h a n e b i n d i n g t o one o r more o f t h e major b l o o d 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 o f the p a r t i a l  p r e s s u r e o f h a l o t h a n e i n t h e head space would be observed w h i l e t h e h a l o t h a n e was d i s s o l v i n g i n the aqueous phase, f o l l o w e d by a p e r i o d when t h e p a r t i a l p r e s s u r e would f a l l v e r y s l o w l y .  I f t h e p a r t i a l p r e s s u r e were measured d u r i n g  t h i s s l o w l y 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 p r e s s u r e would be lower than t h e t r u e e q u i l i b r i u m v a l u e .  This e f f e c t  i s more pronounced when the h a e m a t o c r i t i s h i g h e r , 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 t h e t r u e e q u i l i b r i u m p a r t i t i o n at  coefficient  a h i g h e r h a e m a t o c r i t , l e a d i n g t o the c o n c l u s i o n t h a t the s o l u b i l i t y o f  h a l o t h a n e i n b l o o d has a n e g a t i v e dependence on h a e m a t o c r i t .  - 82 -  PART I I I - Uptake o f h a l o t h a n e  i n dog b l o o d i n v i v o  INTRODUCTION  There have been v e r y few measurements of the a c t u a l c o n c e n t r a t i o n o f i n h a l a t i o n anaesthetics i n blood during anaesthesia (Chenoweth et a l . 1962;  Cervenko 1968;  and those  Lowe 1964a) are not  reported  sufficiently  comprehensive t o be used f o r c o r r e l a t i o n w i t h t h e o r i e s of the uptake d i s t r i b u t i o n of anaesthetics.  This i s probably  due  and  i n p a r t 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 d e t e r m i n a t i o n  of i n h a l a t i o n a n a e s t h e t i c s i n  b l o o d , and  f o r m u l a t i o n of the pharmaco-  i n p a r t to the g e n e r a l l y accepted  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 w i d e l y h e l d assumption t h a t , when a s u f f i c i e n t l y l o n g p e r i o d of time has e l a p s e d a f t e r the i n d u c t i o n o f a n a e s t h e s i a , thermodynamic e q u i l i b r i u m i s almost a c h i e v e d  and t h e r e f o r e the c h e m i c a l p o t e n t i a l s of the a n a e s t h e t i c i n  the a l v e o l i , b l o o d and nervous t i s s u e are almost the same ( H a l s e y 1974; et a l . 1968b).  Thus a measurement o f the a l v e o l a r e n d - t i d a l a n a e s t h e t i c  p a r t i a l p r e s s u r e w h i c h i s d i r e c t l y r e l a t e d to c h e m i c a l  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 a n a e s t h e t i c c h e m i c a l p o t e n t i a l i n nervous t i s s u e . t h i s reasoning,  the concept o f Minimum A l v e o l a r C o n c e n t r a t i o n  Based upon (MAC)  developed as a s t a n d a r d o f a n a e s t h e t i c potency (Eger et a l . 1965).  was MAC  d e f i n e d as the minimum a l v e o l a r c o n c e n t r a t i o n o f a n a e s t h e t i c n e c e s s a r y prevent movement i n response to a p a i n f u l s t i m u l u s i n 50% o f the subjects. (MAP)  Cowles  S t r i c t l y speaking,  to  experimental  i t i s the minimum a n a e s t h e t i c p a r t i a l  which i s b e i n g used ( F i n k 1971;  is  pressure  Eger 1971), but at the common ambient  - 83 atmospheric  p r e s s u r e , 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  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 r e a s o n a b l e c o r r e l a t i o n between  is  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 ( t h e r a t i o o f the c o n c e n t r a t i o n o f anaest h e t i c i n an o l i v e o i l phase to t h a t 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).  S i n c e c u r r e n t t h e o r i e s o f the mechanism  of a n a e s t h e s i a f a v o u r 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; 1977)  t h e r e i s a g e n e r a l acceptance  Miller  o f the use o f p a r t i a l p r e s s u r e ( o f 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 b l o o d b e i n g s t u d i e d ) , r a t h e r than c o n 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 o f a n a e s t h e t i c s (Cowles e t a l . 1971a, 1972a; Kolmer et a l . 1975a; A l l o t et a l . 1976; Munson et a l . 1978).  Mathematical  a n a e s t h e t i c are a l s o forumulated c o n c e n t r a t i o n (Eger 1963; Bowes 1967; 1963,  models o f the uptake and d i s t r i b u t i o n o f i n terms of p a r t i a l p r e s s u r e r a t h e r than  Bourne 1964;  Ashman et a l . 1970;  S a r a i v a et a l . 1977a,b;  Eger and Severinghaus  1964; Munson and  Cowles et a l . 1968a,b, 1971b, 1972b; Mapleson  1964a,b, 1973; Kolmer et a l . 1975b; Zwart et a l . 1972).  Models o f the  uptake and d i s t r i b u t i o n o f i n h a l a t i o n a n a e s t h e t i c s 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 i n c l u d e m a t h e m a t i c a l  e q u a t i o n s and p h y s i c a l d e v i c e s which produce an  uptake and d i s t r i b u t i o n curve w i t h any g i v e n s e t of i n p u t d a t a . Kolmer et a l . (1975b) used a f i r s t o r d e r r a t e e q u a t i o n as a model f o r the uptake o f h a l o t h a n e  i n dogs.  D i f f e r e n t compartments i n the body were r e p r e -  sented by d i f f e r e n t terms w i t h d i f f e r e n t parameters ( t i m e c o n s t a n t s and 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 e q u a t i o n .  frac-  The number o f terms i n the  e q u a t i o n and the v a l u e s o f the parameters were a d j u s t e d t o g i v e the b e s t f i t  - 84 -  to experimentally measured end-tidal p a r t i a l pressure and the halothane pressures i n a head space i n equilibrium with blood samples.  partial  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 d i s t r i b u t i o n of anaesthetic is the analog c i r c u i t (Cowles et a l . 1968a,b; Mapleson 1963,  1964b).  These are DC c i r c u i t s with a power source (battery),  r e s i s t o r s and capacitors representing the anaesthetic vaporizer, blood vessels and organs respectively. Thus the inspired anaesthetic l e v e l , the rate of transport of anaesthetic i n 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 c i r c u i t 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 d i f f e r e n t capacity for the anaesthetic (Eger 1974).  Water, representing the anaesthetic, i s  added to the cylinder representing the lung and the resulting water l e v e l and i t s rate of r i s e i n the different cylinders represent the anaesthetic l e v e l and i t s rate of r i s e i n the d i f f e r e n t organs.  As with the e l e c t r i c  analog,  the inspired anaesthetic l e v e l , transport of anaesthetic i n 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 e n t i r e l y 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 a p p r o p r i a t e s e l e c t i o n of the number and v a l u e s o f parameters ( c o n s t a n t s i n m a t h e m a t i c a l models, r e s i s t a n c e and c a p a c i t a n c e i n analog c i r c u i t s , p i p e and c y l i n d e r d i a m e t e r s i n hydrodynamic m o d e l s ) , an e m p i r i c a l model which a c c u r a t e l y d e s c r i b e s the q u a n t i t a t i v e a s p e c t s o f the uptake and d i s t r i b u t i o n o f a n a e s t h e t i c s , a l t h o u g h u s e f u l as a p e d a g o g i c a l d e v i c e , does not n e c e s s a r i l y c o n t r i b u t e t o the u n d e r s t a n d i n g of the r e a l process involved. 2.  C o m p u t a t i o n - s i m u l a t i o n Models These models p r e d i c t the l e v e l of a n a e s t h e t i c 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 g i v e n i n s p i r e d a n a e s t h e t i c l e v e l the q u a n t i t y o f a n a e s t h e t i c t r a n s f e r r e d from the a l v e o l i t o a r t e r i a l b l o o d and t h a t t r a n s f e r r e d from the a r t e r i a l b l o o d t o 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 t o be c o n t i n u o u s (Cowles e t a l . 1972a,b), or i n more e l a b o r a t e models, s t e p w i s e computation corresponded t o the d i s c o n t i n u o u s 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 v e r y i n v o l v e d and r e s u l t s can o n l y be o b t a i n e d w i t h the a i d of a d i g i t a l computer. were made.  To s i m p l i f y the c a l c u l a t i o n s , many assumptions  For example, none o f the models took i n t o account the m e t a b o l i s m  of a n a e s t h e t i c s as p a r t o f the c o m p u t a t i o n , a l t h o u g h 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). a l l t h e s e models.  One b a s i c assumption i s i n h e r e n t t o  The a n a e s t h e t i c c o n t a i n e d 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 b l o o d i s e i t h e r i n c o n t i n u o u s e q u i l i b r i u m (Cowles et a l . 1972a,b) or a c h i e v e 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 (Mapleson 1973).  interval  T h i s assumption, i n c o m b i n a t i o n 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 , a l l o w s the e x a c t q u a n t i t y of a n a e s t h e t i c t r a n s f e r r e d from the  - 86 l u n g to the a r t e r i a l b l o o d to be e a s i l y c a l c u l a t e d f o r any g i v e n  time  i n t e r v a l , p r o v i d e d t h a t 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 c o n s t a n t . 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 c o n s t a n t , v a l u e s  f o r dog b l o o d a r e , 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 f o r i t to be t r e a t e d as  such.  M o r r i s (1974), however, has p o i n t e d out t h a t 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 o r g a n i s m ) , m a t e r i a l t r a n s f e r between the system and i t s environment o c c u r s .  where  Furthermore,  the v a l i d i t y o f u s i n g the e n d - t i d a l p a r t i a l p r e s s u r e as an a b s o l u t e i n d e x of the a r t e r i a l a n a e s t h e t i c p a r t i a l p r e s s u r e has been c r i t i c i z e d on  physiological  grounds (Eger and Bahlman 1971). Although  the f i n d i n g t h a t the MAC  i s u n a l t e r e d by p e r i o d s of a n a e s t h e s i a  o f up to 8 hours (Eger et a l . 1965a) appears to i n d i c a t e t h a t thermodynamic e q u i l i b r i u m i s achieved  ( H a l s e y 1974)  t h e r e has been as y e t no  definitive  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; 1972).  I n the experiments  to be d e s c r i b e d , the e n d - t i d a l p a r t i a l  and the c o n c e n t r a t i o n s o f h a l o t h a n e  pressures  i n b l o o d and plasma d u r i n g a n a e s t h e s i a  were measured under c o n t r o l l e d c o n d i t i o n s . d i s c u s s e d i n the l i g h t o f these  Cowles  results.  The e q u i l i b r i u m assumption w i l l  be  T a b l e X V I I I - Blood/gas p a r t i t i o n c o e f f i c i e n t  Authors  Mean P a r t i t i o n Coefficient  f o r dog b l o o d a t 37°C  Measure o f Uncertainty  No. o f Animals  No. o f Determinations  Haematocrit  Steward e t a l . 1975  3.51  + 0.31 95% c o n f i d e n c e l i m i t  7  14  37.3  Cowles et a l . 1971  3.16  + 0.18 95% c o n f i d e n c e l i m i t  7  88  45  Ikeda  2.18  + 0.16 Standard D e v i a t i o n  NG  13  42  1972 NG:  not given  - 88 MATERIALS AND METHODS  Male mongrel dogs (17-25 kg) were s t a r v e d o v e r n i g h t but a l l o w e d f r e e access to water and then a n a e s t h e s i a was  induced w i t h sodium t h i o p e n t o n e  mg/kg i v ) . The dogs were i n t u b a t e d w i t h an e n d o t r a c h e a l tube and was  halothane  a d m i n i s t e r e d 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. 2.0  (20  Halothane i n s p i r e d c o n c e n t r a t i o n s were kept c o n s t a n t at 1.0,  or 2.5%  ( o f 1 atmosphere) r e s p e c t i v e l y ; when 1% h a l o t h a n e was  muscular b l o c k a d e was m a i n t a i n e d w i t h pancuronium. w i t h a B i r d mark 8 r e s p i r a t o r . gases were m a i n t a i n e d  used neuro-  V e n t i l a t i o n was  maintained  D u r i n g the course o f the experiment,  i n the f o l l o w i n g ranges PCO^^  7.35-7.45; VO^'- g r e a t e r than 100 mm  30-40 mm  Hg,  Hg, and the body temperature  m a i n t a i n e d at 38°C (see Tables X I X - X X I I ) w i t h a h e a t i n g pad.  1.5,  the b l o o d  pH: was  Arterial  p r e s s u r e and e n d - t i d a l h a l o t h a n e p a r t i a l p r e s s u r e were m o n i t o r e d  blood  by a Statham  P23AC t r a n s d u c e r and by a Beckman LB 2 i n f r a r e d a n a l y z e r as d e s c r i b e d by L e i g h t o n et a l . (1978). B l o o d was withdrawn from the f e m o r a l a r t e r y and from the r i g h t a t r i u m ( v i a a c a t h e t e r i n s e r t e d through  the j u g u l a r v e i n ) at a p p r o x i m a t e l y  (exact  time  known) 15, 30, 60, 120, 240, 300 minutes a f t e r commencing a n a e s t h e s i a . p a r t s o f the experiments  were c a r r i e d out by Ms.  ment of Pharmacology, U n i v e r s i t y of B.C.).  The  C a r o l i n e Bruce i n the  (These Depart-  f i r s t 3 ml of b l o o d were  d i s c a r d e d to a v o i d erroneous measurement o f b l o o d h a l o t h a n e  c o n c e n t r a t i o n due  to the dead volume o f the c a t h e t e r and then the b l o o d 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., R o c k f o r d , 111., USA)  containing  s u f f i c i e n t d i s o d i u m EDTA to g i v e a f i n a l c o n c e n t r a t i o n of a p p r o x i m a t e l y mg/ml.  A g l a s s bead was  dropped i n t o the v i a l , which was  4.5  then s e a l e d w i t h no  -  trapped a i r  bubble.  After  s a m p l e s by c e n t r i f u g a t i o n for  15 m i n u t e s .  the  gas l i q u i d  89  -  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 o m one s e t i n a S o r v a l l HB-4 r o t o r  chromatographic procedure described i n Part  s a m p l e s was t e s t e d  of  loss of  needle sealed with solder,  as d e s c r i b e d i n P a r t  of  the  H a l o t h a n e was a d d e d i n v i t r o  to  a human b l o o d  during  In  order  to  of  concentration  this  determine whether  or not  again  the  work. blood  The s y r i n g e was c a p p e d w i t h a m i x e d and a s a m p l e incubated at  37°C  determined.  c e l l s and p l a s m a was  w o r k and c a l c u l a t e d f r o m e q u a t i o n  b l o o d samples had b e e n removed from the Reacti v i a l s  the  The s y r i n g e was t h e n  o f h a l o t h a n e between the II  distribution  determined [8],  page 5 0 .  changed a f t e r  dog b l o o d s a m p l e s w e r e r o t a t e d  for  a further  2 and 4 h o u r s b e f o r e  determining  the  The a r t e r i a l  halothane  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 r o m t h e  the in  halothane  distribution.  partial At  pressure using equation  [17]  d e r i v e d as  end  tidal  follows.  equilibrium X  =  [Halothane]b [Halothane]g  where A = p a r t i t i o n  coefficient  [Halothane]g  = equilibrium halothane  concentration  in  the gas phase  [Halothane]b  = equilibrium  concentration  in  the b l o o d phase  The c o n c e n t r a t i o n u n i t the p a r t i t i o n  coefficient  by  transfer  a n a l y s i s of halothane.  The d i s t r i b u t i o n  halothane  this  t h e n t h e b l o o d was t h o r o u g h l y  30 m i n and t h e h a l o t h a n e  g)  of  halothane  as f o l l o w s .  2,870  I  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 .  for  4 , 5 0 0 rpm ( m a x .  P l a s m a and w h o l e b l o o d s a m p l e s w e r e a n a l y s e d f o r  The p o s s i b i l i t y  was t a k e n f o r  at  of  halothane used for  b o t h p h a s e s has to be the  is a dimensionless  quantity.  s a m e , so  that  - 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 h a l o t h a n e i n the a l v e o l a r gas X=  [Halothane] A [Halothane]a  [15]  where [Halothane]A = h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l b l o o d [Halothane]a = a l v e o l a r halothane c o n c e n t r a t i o n The e n d - t i d a l p a r t i a l p r e s s u r e i s e x p r e s s e d as the % o f 1 atmosphere, thus it  i s n u m e r i c a l l y e q u a l t o t h e p a r t i a l volume o c c u p i e d by h a l o t h a n e p e r 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 o c c u p i e d by 1 mole o f h a l o t h a n e a t 37°C and 1 atmosphere p r e s s u r e i s 2.54 x 1 0 m l . 4  where ET = e n d - t i d a l p a r t i a l p r e s s u r e o f h a l o t h a n e e x p r e s s e d i n % o f 1 atmosphere. Then [ H a l o t h a n e ] a =  ET ( i n mole/100 m l ) 2.54 x 1 0 [ H a l o t h a n e ] a = (ET)(MW o f H a l o t h a n e ) 1 0 ( i n mg/100 m l ) 2.54 x 1 0 4  or  3  [16]  4  Substituting  [16] i n t o  [15] g i v e s  [Halothane]A = X(ET)(MW o f H a l o t h a n e U O 2.54 x 1 0 4  3  ( i n mg/100 m l )  [17]  - 91 RESULTS AND  F i g u r e s 13-15  DISCUSSION  show t h a t the appearance  o f the h a l o t h a n e i n the b l o o d d u r i n g  a n a e s t h e s i a w i t h c o n s t a n t i n s p i r e d l e v e l s o f 1.0, atmosphere) r e s p e c t i v e l y was  1.5  and 2.0 p e r c e n t ( o f 1  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 i n c r e a s e o f the h a l o t h a n e c o n c e n t r a t i o n i n b o t h a r t e r i a l and venous whole b l o o d , d u r i n g which time 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 s were i n g e n e r a l h i g h e r f o l l o w e d 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 a n a e s t h e s i a , where the venous h a l o t h a n e c o n c e n t r a t i o n g e n e r a l l y approached e q u a l l e d 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 . The plasma h a l o t h a n e  or  concen-  t r a t i o n s f o l l o w e d the same t r e n d s as observed w i t h whole b l o o d , but were always l e s s than those o f whole b l o o d .  S i m i l a r r e s u l t s were o b t a i n e d when the  i n s p i r e d l e v e l o f h a l o t h a n e was m a i n t a i n e d at 2.5%  ( F i g . 16) e x c e p t t h a t the  venous h a l o t h a n e c o n c e n t r a t i o n d i d not r e a c h 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 T a b l e s XIX- X X I I , the a r t e r i a l b l o o d p r e s s u r e throughout the 5 h r p e r i o d was 1.5  i n the range o f 78-135 mmHg f o r i n s p i r e d h a l o t h a n e l e v e l s o f  and 2.0% but was  depressed at 2.5%.  T h i s may  1.0,  be due to e i t h e r a depressed  c a r d i a c 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 e x p l a n -  a t i o n a g i v e n volume o f b l o o d may have a l o n g e r c i r c u l a t i o n t i m e , f a c i l i t a t i n g g a i n o f h a l o t h a n e from the a l v e o l i  and l o s s o f h a l o t h a n e 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 h a l o t h a n e c o n c e n t r a t i o n between the a r t e r i a l and venous b l o o d .  Hughes (1973) has shown  t h a t " . . . . i n dogs m y o c a r d i a l d e p r e s s i o n , i n d i c a t e d by marked r e d u c t i o n s 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 t o the f a l l i n c a r d i a c output and the consequent h y p o t e n s i o n w i t h i n s p i r e d c o n c e n t r a t i o n s o f 1, 2 and 4% h a l o t h a n e . "  X  END-TIDAL f 2  TIME (HR.)  ,Fig. 13. B l o o d and plasma h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d a t a c o n s t a n t i n s p i r e d l e v e l o f 1.0% h a l o t h a n e .  3| 2h  TIME (HR.)  F i g . 14. B l o o d and plasma h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d a t a c o n s t a n t i n s p i r e d l e v e l o f 1.5% h a l o t h a n e .  3r  TIME (HR.)  F i g . 15. B l o o d and plasma h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d a t a c o n s t a n t i n s p i r e d l e v e l o f 2.0% h a l o t h a n e .  TIME (HR.)  F i g . 16. B l o o d and plasma c o n c e n t r a t i o n i n a r t e r i a l and mixed venous b l o o d a t a constant i n s p i r e d l e v e l o f 2.5% h a l o t h a n e .  IS !*!?? " " * end-tidal partial 1  Time (hr)  C  p  Fi«J * halothane c o n c e n t r a t i o n determined e x p e r i m e n t a l l y and that c a l c u l a t e d from the p r e s s u r e at 1.0% i n s p i r e d l e v e l  r i , O B  o f  arte  Mean A r t e r i a l P r e s s u r e (mo Hg)  l  o  o  d  Temperature (°C)  He Arterial  Venous  % Halothane i n Plasma Arterial Venous  .25  103  39.0  .50  88  39.0  40.3  39.4  33  1  117  38.8  39.4  -  2  106  38.8  41.1  4  108  39.2  -  -  -  39.3  41.3  -  5  A r t e r i a l [Halothane](mg/100 ml) Calculated Experimentally Assuming E q u i l i b r i u m Determined  23  10  36  26  12  36  -  28  13  37  -  31  19  31  22  32  20  34  -  I  1  T a b l e XX - Comparison o f a r t e r i a l b l o o d h a l o t h a n e c o n c e n t r a t i o n determined e x p e r i m e n t a l l y 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 at 1.5Z i n s p i r e d l e v e l  Time (hr)  Mean A r t e r i a l P r e s s u r e (mm Hg)  Temperature (°C)  He Arterial  .25  90  39.3  44.0  .50  78  39.3  41.2  1  83  39.0  2  102  38.6  -  4  120  . 38.2  5  113  38.0  Venous  40.7  Z Halothane i n Plasma Arterial Venous  A r t e r i a l [Halothane](mg/100 ml) Calculated Experimentally Assuming E q u i l i b r i u m Determined  36  -  35  12  37  35  39  17  40.8  -  43  44  25  45.9  44.4  40  37  46  25  46.0  45.6  33  33  47  25  40.3  38  . 4 2  22  T a b l e XXI - Comparison o f a r t e r i a l blood halothane c o n c e n t r a t i o n determined e x p e r i m e n t a l l y 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.OX i n s p i r e d l e v e l  _ . Time (hr)  „ . . , Mean A r t e r i a l P r e s s u r e (ma Hg)  Temperature (°C)  He Arterial  Venous  .25  135  .75  115  38.5  31.8  32.0  1  98  35.7  -  30.3  2  85  35.7  33.1  30.6  4  87  39.0  -  28.6  5  95  39.2  28.7  29.1  % Halothane i n Plasma Arterial Venous  49  46  50  A r t e r i a l [Halothane](mg/100 ml) Calculated Experimentally Assuming E q u i l i b r i u m Determined  34  16  52  41  22  43  42  42  46  22  41  48  25  44  48  25  T a b l e XXII - Comparison o f a r t e r i a l b l o o d halothane c o n c e n t r a t i o n determined e x p e r i m e n t a l l y 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 p r e s s u r e assuming thermodynamic e q u i l i b r i u m at 2.531 i n s p i r e d l e v e l  Arterial lme h  r  Mean A r t e r i a l P r e s s u r e ("» 8>  )  H  Temperature He ( > Arterial oc  Venous  .25  53  38.6  .50  73  38.2  35.4  34.4  43  1  55  38.0  32.9  33.1  2  40  38.0  30.8  4  60  37.8  -  5  63  37.8  32.2  l  [Halothane](mg/100 ml)  X Halothane i n Plasma C a l c u l a t e d . Experimental Arterial Venous Assuming E q u i l i b r i u m Determined  45  21  43  55  23  39  38  54  28  39  -  59  32  31.1  -  36  59  30  31.0  42  42  60  33  -  - 100 As shown i n T a b l e s XIX-XXII a r t e r i a l and venous b l o o d 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 o f the h a l o t h a n e  found i n the plasma.  The  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 h e c e l l f r a c t i o n was always g r e a t e r than t h a t i n the plasma and appeared t o be independent sample was taken.  o f the time a t which the b l o o d  Table X X I I I shows t h a t t h i s d i s t r i b u t i o n d i d not change  a p p r 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 f o r 2 o r 4 h r .  This  suggests t h a t t h e d i s t r i b u t i o n o f h a l o t h a n e between t h e b l o o d components i s s u f f i c i e n t l y r a p i d t o be independent  o f e i t h e r the a d d i t i o n o f h a l o t h a n e  from  the l u n g t o the b l o o d or t h e l o s s o f h a l o t h a n e from the b l o o d t o the t i s s u e s . In P a r t I I o f t h i s work i t was found t h a t , i n human b l o o d , albumin,  tri-  g l y c e r i d e , r e d 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 t o t h e s o l u b i l i t y o f h a l o t h a n e i n whole b l o o d .  I t i s v e r y l i k e l y t h a t , i n dog b l o o d ,  these b l o o d components a l s o a c t as c a r r i e r s f o r h a l o t h a n e , a l t h o u g h the amounts of h a l o t h a n e c a r r i e d by each component may be d i f f e r e n t .  However, t h e d i s t r i -  b u t i o n o f h a l o t h a n e between c e l l s and plasma i s even more i n f a v o u r o f t h e c e l l f r a c t i o n i n dog b l o o d , compared t o t h a t i n human b l o o d as d e s c r i b e d i n P a r t 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 n o t c l e a r .  to note t h a t the s l i g h t p o s i t i v e dependence o f Ostwald upon h a e m a t o c r i t r e p o r t e d by Steward  I t i s interesting  solubility  coefficient  e t a l . (1975) i m p l i e s a d i s t r i b u t i o n o f  h a l o t h a n e i n b l o o d i n f a v o u r o f the c e l l  fraction.  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 h a l o t h a n e i n the b l o o d , the e n d - t i d a l  halothane  p a r t i a l p r e s s u r e was used t o c a l c u l a t e t h e expected h a l o t h a n e c o n c e n t r a t i o n i n a r t e r i a l b l o o d assuming t h a t e q u i l i b r i u m was a t t a i n e d . from e q u a t i o n [17] (page 90) u s i n g t h e Ostwald  T h i s was c a l c u l a t e d  solubility  coefficient  Table X X I I I - E f f e c t o f 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 o f h a l o t h a n e between plasma and c e l l s B l o o d samples from dogs a n a e s t h e t i z e d w i t h h a l o t h a n e 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 h a l o t h a n e between c e l l s and plasma was then determined.  B l o o d [Halothane] mg/100 ml  E q u i l i b r a t i o n Time (hr)  He  % Halothane Plasma  Cells  40  0 2 4  35.6  38 35 35  62 65 65  23  0 2 4  36.5  36 39 39  63 61 61  - 102  -  ( n u m e r i c a l l y e q u a l 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)) r e p o r t e d by Steward et a l . (1975) and t a k i n g i n t o account s l i g h t p o s i t i v e dependence o f t h i s Ostwald haematocrit.  As shown i n Tables XIX-XXII  much h i g h e r than those determined to  halothane  s o l u b i l i t y c o e f f i c i e n t upon the c a l c u l a t e d c o n c e n t r a t i o n s were  experimentally.  one or more o f s e v e r a l p o s s i b i l i t i e s :  the  T h i s d i s c r e p a n c y may  be  due  ( i ) E r r o r i n the measurement o f  c o n c e n t r a t i o n i n t h i s study due to l o s s o f h a l o t h a n e  ( i i ) e r r o r i n the r e p o r t e d v a l u e of the Ostwald  during  sampling,  solubility coefficient; ( i i i )  the e n d - t i d a l h a l o t h a n e p a r t i a l p r e s s u r e does not r e p r e s e n t a l v e o l a r  halothane  p a r t i a l p r e s s u r e ; ( i v ) an e r r o r i n the d e t e r m i n a t i o n of e n d - t i d a l p a r t i a l p r e s s u r e and ( v ) the assumption t h a t thermodynamic e q u i l i b r i u m e x i s t s between halothane The  i n the a l v e o l i and h a l o t h a n e  f i r s t p o s s i b i l i t y was  i n a r t e r i a l blood i s i n v a l i d .  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  b l o o d 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 h a l o t h a n e was c o n t a i n 74.6  + 2.6 mg/100 ml (n=4)  found  a f t e r b e i n g sampled i n the same way  to  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 d e t e r m i n a t i o n of the  s o l u b i l i t y c o e f f i c i e n t s of halothane determined et  I t s value  by d i f f e r e n t workers agree r e a s o n a b l y w e l l (see T a b l e X V I I I ) (Cowles  a l . 1975a; Ikeda 1972;  incorrect.  i n dog b l o o d i s v e r y s i m p l e .  Steward et a l . 1975)  and they are u n l i k e l y to be  The use o f a l i t e r a t u r e v a l u e f o r the Ostwald  solubility  f i c i e n t o t h e r than t h a t o b t a i n e d by Steward et a l . (1975) w i l l not c a n t l y change the r e s u l t s o f the The  Ostwald  t h i r d p o s s i b i l i t y may  coefsignifi-  calculation.  h e l p to account  f o r the d i s c r e p a n c y .  Eger  and  Bahlman (1971) argued t h a t the h a l o t h a n e  c o n c e n t r a t i o n i n unperfused  alveoli,  p r e s e n t even i n normal i n d i v i d u a l s , was  e q u a l to t h a t o f the i n s p i r e d  level,  - 103 and was not lowered by l o s s o f h a l o t h a n e t o the b l o o d . h a l o t h a n e p a r t i a l p r e s s u r e which and unperfused)  Thus the e n d - t i d a l  i s an average o f a l l the a l v e o l i ( p e r f u s e d  i s an o v e r - e s t i m a t e o f t h a t i n the p e r f u s e d a l v e o l i .  This i s  o n l y s i g n i f i c a n t , however, when t h e r e i s a l a r g e d i f f e r e n c e between the endt i d a l and i n s p i r e d p a r t i a l p r e s s u r e .  Eger and Bahlman (1971) e s t i m a t e d t h a t  the e n d - t i d a l p a r t i a l p r e s s u r e would be 10% h i g h e r than the a r t e r i a l p r e s s u r e when the i n s p i r e d l e v e l was 50% h i g h e r than t h e e n d - t i d a l pressure.  T h i s e f f e c t would be i n s u f f i c i e n t t o account  partial  partial  f o r the d i s c r e p a n c y  e s p e c i a l l y d u r i n g t h e steady s t a t e when t h e e n d - t i d a l p a r t i a l p r e s s u r e approached or e q u a l l e d the i n s p i r e d p a r t i a l p r e s s u r e . I t i s u n l i k e l y t h a t the e n d - t i d a l p a r t i a l p r e s s u r e measurement was erroneous because, as expected  (Eger 1974),  i t showed an i n i t i a l r a p i d  and then l e v e l l e d o f f a p p r o a c h i n g t h e i n s p i r e d l e v e l .  rise  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 s m a l l and would not be expected t o s i g n i f i c a n t l y a f f e c t t h e c a l c u l a t i o n made from steady s t a t e v a l u e s where t h e r e was l e 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  levels.  I t i s l i k e l y , t h e r e f o r e , t h a t t h e r e i s no thermodynamic e q u i l i b r i u m between h a l o t h a n e i n the a l v e o l i and h a l o t h a n e pharmacokinetic  t h e o r y which  h a l o t h a n e , through metabolism  i n a r t e r i a l blood.  takes i n t o account  I n f a c t , i n any  the c o n t i n u o u s l o s s o f  ( S t i e r et a l . 1964; Sawyer e t a l . 1971; A t a l l a k  and Geddes 1973; Cohen e t a l . 1975; T i n k e r e t a l . 1976; Widger et a l . 1976; C a s c o r b i 1970; Mukai e t 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 p r o l o n g e d a b s o r p t i o n by adipose t i s s u e ( S a r a i v a e t a l . 1977b), a c h e m i c a l p o t e n t i a l g r a d i e n t o f h a l o t h a n e from the a l v e o l i t o the a r t e r i a l b l o o d i s n e c e s s a r y f o r the t r a n s f e r o f h a l o t h a n e from the i n s p i r e d gas t o t h e  -  b l o o d f o r the r e p l e n i s h m e n t  104  -  o f the halothane  l o s t d u r i n g steady s t a t e .  The  f i n d i n g t h a t MAC d i d not change d u r i n g prolonged a n a e s t h e s i a (Eger e t a l . 1965) does not mean t h a t e q u i l i b r i u m was e s t a b l i s h e d , b u t r a t h e r t h e r a t e o f h a l o thane l o s t i s r e l a t i v e l y c o n s t a n t d u r i n g the steady s t a t e , so t h a t the demand f o r a d d i t i o n a l halothane is also r e l a t i v e l y  ( t o compensate f o r t h e l o s s ) t o m a i n t a i n a n a e s t h e s i a ,  constant.  I n the two most comprehensive s t u d i e s (Cowles et a l . 1972; A l l o t t et a l . 1976)  i n which e x p e r i m e n t a l 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 , t h e v a l u e s f o r h a l o t h a n e p a r t i a l p r e s s u r e 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 b l o o d p r e d i c t e d by t h e models were s y s t e m a t i c a l l y h i g h e r ( a p p r o x i m a t e l y 7% i n both c a s e s ) than t h e c o r r e s ponding e x p e r i m e n t a l l y determined a l i z e d i n terms o f metabolism.  v a l u e s , and t h e d i s c r e p a n c i e s were r a t i o n -  The magnitude o f the d i f f e r e n c e (50-100%)  between t h e c a l c u l a t e d and measured 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 s i s much h i g h e r i n t h i s study ( T a b l e s XTX-XXII).  Although  i t i s n o t c l e a r why the  s y s t e m a t i c d i f f e r e n c e found h e r e s h o u l d be d i f f e r e n t from those o f Cowles e t al.  (1972) and A l l o t t e t a l . (1976), they a r e a l l p o s i t i v e e r r o r s o f  p r e d i c t i o n i n t h e same d i r e c t i o n . The r e s u l t s p r e s e n t e d h e r e suggest  t h a t an e x p e r i m e n t a l l y measured  h a l o t h a n e p a r t i a l p r e s s u r e i n e q u i l i b r i u m w i t h a sample o f a r t e r i a l s h o u l d be much lower than the c o r r e s p o n d i n g e n d - t i d a l h a l o t h a n e  blood  partial  p r e s s u r e when the i n s p i r e d h a l o t h a n e c o n c e n t r a t i o n i s h e l d c o n s t a n t . work w i l l be n e c e s s a r y  Further  to t e s t t h i s hypothesis.  There i s good agreement between t h e b l o o d h a l o t h a n e c o n c e n t r a t i o n s i n t h i s study and those r e p o r t e d by o t h e r workers.  found  F o r human p a t i e n t s , Lowe  - 105 -  and Beckham (1964) r e p o r t e d b l o o d h a l o t h a n e c o n c e n t r a t i o n s o f a p p r o x i m a t e l y 8mg/100 ml d u r i n g " a n a l g e s i a " and 12-17 mg/100 ml d u r i n g " s u r g i c a l p l a n e s " ; whereas A t a l l a h and Geddes (1973) found t h a t t h e venous h a l o t h a n e c o n c e n t r a t i o n s were i n t h e range o f 7.60-10.65 mg/100 ml a t t h e end o f 20 m i n . o f h a l o t h a n e a n a e s t h e s i a a t 1%% i n s p i r e d h a l o t h a n e c o n c e n t r a t i o n .  F o r dogs,  Chenoweth e t a l . (1962) judged t h a t 17.5-22.5 mg/100 g o f h a l o t h a n e i n a r t e r i a l b l o o d was s a t i s f a c t o r y f o r major s u r g e r y ; and Cervenko (1968) found t h a t t h e h a l o t h a n e c o n c e n t r a t i o n i n a o r t i c b l o o d samples were i n t h e range o f 17.5-23.6 mg/100 g " a f t e r exposure  t o 2% h a l o t h a n e f o r 30 min.".  In this  s t u d y , t h e a r t e r i a l b l o o d h a l o t h a n e c o n c e n t r a t i o n s a f t e r 15 and 45 min o f a n a e s t h e s i a a t a c o n s t a n t 2.0% i n s p i r e d h a l o t h a n e c o n c e n t r a t i o n were found t o be 16 and 22 mg/100 ml r e s p e c t i v e l y .  - 106 CONCLUSIONS  Halothane,  a representative i n h a l a t i o n general anaesthetic, i s c a r r i e d i n  human b l o o d by albumin, haemoglobin, r e d 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 ( c h y l o m i c r o n s and v e r y low d e n s i t y l i p o p r o t e i n ) b u t n o t i n s i g n i f i c a n t amounts by y - g l o b u l i n a t p h y s i o l o g i c c o n c e n t r a t i o n .  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 o f h a l o t h a n e i s a l s o p r e s e n t f r e e ( i . e . aqueous phase o f b l o o d .  d i s s o l v e d ) i n the  I t i s c o n c e i v a b l e t h a t h a l o t h a n e i s a l s o c a r r i e d by  o t h e r b l o o d components, b u t s i n c e a l l t h e major b l o o d components have been accounted  f o r , t h e r e s u l t s p r e s e n t e d here r e p r e s e n t a r e a s o n a b l y complete  p i c t u r e o f the t r a n s p o r t o f h a l o t h a n e i n human b l o o d . A t 37°C, a p p r o x i m a t e l y h a l f or more o f the h a l o t h a n e c e l l f r a c t i o n o f human whole b l o o d .  i s p r e s e n t i n the  I n v i v o s t u d i e s a l s o showed t h a t more  than h a l f o f the h a l o t h a n e i n dog b l o o d i s p r e s e n t i n the c e l l a l t h o u g h the percentage  fraction,  o f h a l o t h a n e i n the c e l l f r a c t i o n i s h i g h e r f o r dog  b l o o d than f o r human b l o o d . The uptake o f h a l o t h a n e i n dog b l o o d d u r i n g a n a e s t h e s i a went through an i n i t i a l i n d u c t i o n phase f o l l o w e d by a steady s t a t e when the b l o o d  halothane  c o n c e n t r a t i o n s t a b i l i z e d , b u t the h a l o t h a n e i n the lung and the h a l o t h a n e i n the a r t e r i a l b l o o d were not i n thermodynamic e q u i l i b r i u m a f t e r 5 hours o f a n a e s t h e s i a at c o n s t a n t i n s p i r e d h a l o t h a n e c o n c e n t r a t i o n , when the steady s t a t e was a l r e a d y e s t a b l i s h e d .  -  107  -  BIBLIOGRAPHY A l l o t t , P.R., Steward, A. and Mapleson, W.W. 1971. Determination h a l o t h a n e i n gas, b l o o d and t i s s u e s by c h e m i c a l e x t r a c t i o n and chromatography. B r . J . Anaesth. 43, 913-917.  of gas  A l l o t t , P.R., Steward, A., F l o o k , V. and Mapleson, W.W. 1973. 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