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UBC Theses and Dissertations

Cell partition in aqueous polymer two phase systems Van Alstine, James Melvin 1984

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CELL PARTITION IN AQUEOUS POLYMER TWO PHASE SYSTEMS by JAMES MELVIN VAN ALSTINE B . S c , Biochemistry, The U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS PRESENTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF FOR THE DEGREE OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA - A p r i l 1984 (C) James M. Van A l s t i n e , 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 i i ABSTRACT Experimental s t u d i e s of the p a r t i t i o n of c e l l s and d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e (DMPC) liposomes i n aqueous polymer two phase systems co n t a i n i n g dextran T500 and p o ly-(ethylene g l y c o l ) (PEG) 8000 have been c a r r i e d out. Work has been completed i n f i v e areas: (1) PEG 8 0 00-l- 1 Z lC-palmitate f a t t y a c i d e s t e r , synthesized v i a a procedure i n v o l v i n g o x a l y l c h l o r i d e a c t i v a t i o n of f r e e 14 1- C - p a l m i t i c a c i d , was used i n the development of a method to c h e m i c a l l y assay PEG e s t e r i f i c a t i o n and a column chromatographic procedure f o r the p u r i f i c a t i o n of PEG-alkyl d e r i v a t i v e s . (2) A s e r i e s of dextran/PEG two phase systems compounded with v a r i o u s amounts of sodium phosphate, pH 7.2 b u f f e r and made i s o t o n i c with sodium c h l o r i d e were developed. These systems were shown to e x h i b i t e l e c t r o s t a t i c bulk phase p o t e n t i a l s whose magnitude decreased with i n c r e a s i n g sodium c h l o r i d e c o n c e n t r a t i o n . (3) The PEG-fatty a c i d e s t e r induced p a r t i t i o n of DMPC sm a l l u n i l a m e l l a r liposomes was shown to i n c r e a s e i n a s s o c i a t i o n w i t h l i p i d g e l to l i q u i d - c r y s t a l l i n e phase t r a n s i t i o n s induced by an i n c r e a s e i n ambient experimental temperature or exposure of the liposomes to free l i n o l e i c a c i d . i i i (4) The e f f e c t s of manipulation o f a wide range of experimental v a r i a b l e s on the p a r t i t i o n of normal human er t h r o c y t e s have been noted. The v a r i a b l e s chosen i n c l u d e d ; system polmer l o t , c o n c e n t r a t i o n and p u r i t y , system s a l t composition, PEG-fatty a c i d e s t e r type, c o n c e n t r a t i o n and p u r i t y , c e l l type and c o n c e n t r a t i o n , c e l l f i x a t i o n with various aldehyde f i x a t i v e s and c e l l exposure to V i b r i o c h o l e r a neuraminidase. Under appropriate c o n d i t i o n s the lo g a r i t h m of the p a r t i t i o n c o e f f i c i e n t K (equal to the r a t i o of the number of c e l l s p a r t i t i o n i n g i n t o the upper phase to the number of c e l l s not p a r t i t i o n i n g i n t o the upper phase) has been shown to be d i r e c t l y p r o p o r t i o n a l to system e l e c t r o s t a t i c i n t e r f a c i a l p o t e n t i a l , interfacial bulk phase t e n s i o n , and system PEG-fatty a c i d e s t e r c o n c e n t r a t i o n . Furthermore, i t has been shown that PEG-fatty a c i d e s t e r induced c e l l p a r t i t i o n i s hydrophobic i n nature and appears to be dependant on e s t e r - c e l l r a t h e r than ester-system i n t e r a c t i o n s . (5) E r y t h r o c y t e s from p a t i e n t s w i t h m u l t i p l e s c l e r o s i s have been demonstrated to e x h i b i t l e s s upper phase a f f i n i t y than normal s u b j e c t s ' e r y t h r o c y t e s i n a v a r i e t y of two phase systems. iv TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES i x LIST OF FIGURES x i ACKNOWLEDGEMENTS AND DEDICATION x v i i i ABBREVIATIONS xix CHAPTER 1. INTRODUCTION 1 1.1 - CHAPTER INTRODUCTION 1 1.2 - MULTIPLE SCLEROSIS 2 1.2.1 - General Background 2 1.2.2 - Variation In Serum And Blood C e l l L i p i d Metabolism In Multiple Sclerosis 17 1.3 - AQUEOUS POLYMER TWO PHASE PARTITION 37 1.3.1 - C e l l Separation 37 1.3.2 - Aqueous Polymer Two Phase P a r t i t i o n Al CHAPTER 2. MATERIALS AND METHODS 52 2.1 - CHAPTER INTRODUCTION 52 2.2 - MATERIALS 52 2.2.1 - Supplies of Chemicals And Equipment 52 2.2.2 - Synthesis of PEG-8000-l- 1 4C-Palmitate 55 2.3 - METHODS 56 2.3.1 - Two Phase System Preparation And Physical Characterization 56 2.3.1.1 - Two Phase System Preparation 56 V 2.3.1.2 - Two Phase System P h y s i c a l C h a r a c t e r i z a t i o n 58 2.3.2 - P u r i f i c a t i o n And An a l y s i s of PEG-Fatty A c i d E s t e r s and Related Compounds 60 2.3.2.1 - Se c t i o n I n t r o d u c t i o n 60 2.3.2.2 - Solvent E x t r a c t i o n of PEG And PEG-Alkyl D e r i v a t i v e s 61 2.3.2.3 - Sephadex LH-20 Gel E x c l u s i o n Column Chromatography 63 2.3.2.A - Recovery o f PEG And PEG D e r i v a t i v e s From Methanol/Water (5V/IV) Solvent 64 2.3.2.5 - Octyl-Sepharose CL-4B Hydrophobic Column Chromatography 64 2.3.2.6 - Assay of Chromatographed R a d i o a c t i v e Compounds 65 2.3.2.7 - Nessler's Reagent Assay For PEG Containing Compounds 65 2.3.2.8 - PEG-Fatty Acid Ester UV Absorption 67 2.3.2.9 - Hydroxamic Acid Assay For P E G - E s t e r i f i c a t i o n 67 2.3.2.10- Thin Layer Chromatography Of PEG Containing Compounds 69 2.3.3 - P r e p a r a t i o n , D i f f e r e n t i a l Scanning C a l o r i m e t r y and P a r t i t i o n of D i m y r i s t o y l P h o s p h a t i d y l c h o l i n e (DMPC) Small U n i l a m e l l a r Liposomes 70 2.3.3.1 - P r e p a r a t i o n of DMPC Liposomes 70 2.3.3.2 - D i f f e r e n t i a l Scanning Calorimetry 72 2.3.3.3 - P a r t i t i o n of DMPC Liposomes 73 2.3.4 - Pr e p a r a t i o n And P a r t i t i o n of Human Er y t h r o c y t e s 74 2.3.4.1 - I s o l a t i o n Of Erythrocytes 74 2.3.4.2 - S i n g l e Tube P a r t i t i o n Experiments 75 2.3.4.3 - Counter Current D i s t r i b u t i o n Experiments 79 2.3.4.4 - I n d i v i d u a l C e l l P a r t i t i o n Experiments 80 vi 2.3.4.5 - Comparative P a r t i t i o n of M u l t i p l e S c l e r o s i s And Normal Subjects' E r y t h r o c y t e s 84 2.4 - PROCEDURAL RESULTS AND DISCUSSION 87 2.4.1 - Synthesis of PEG 8 0 0 0 - l - 1 4 C - P a l m i t a t e 87 2.4.2 - Two Phase System P r e p a r a t i o n And C h a r a c t e r i z a t i o n 90 2.4.3 - P u r i f i c a t i o n And A n a l y s i s of PEG-Fatty A c i d E s t e r s And Related Compounds 92 2.4.3.1 - I n t r o d u c t i o n 92 2.4.3.2 - Solvent E x t r a c t i o n of PEG And PEG-Alkyl D e r i v a t i v e s 94 2.4.3.3 - Sephadex LH-20 Gel E x c l u s i o n Column Chromatography 95 2.4.3.4 - Octyl-Sepharose CL-4B Hydrophobic Column Chromatography 98 2.4.3.5 - Nessler's Reagent Assay For PEG Containing Compounds 101 2.4.3.6 - Assay of Radioactive Compounds 103 2.4.3.7 - PEG-Fatty Acid E s t e r UV Absorption 104 2.4.3.8 - Hydroxamic Acid Ester-Assay 106 2.4.3.9 - Thin Layer Chromatography 111 2.4.4 - P r e p a r a t i o n And P a r t i t i o n of D i m y r i s t o y l P h o s p h a t i d y l c h o l i n e Small U n i l a m e l l a r Liposomes 113 CHAPTER 3. TWO PHASE SYSTEM PARTICLE PARTITION 117 3.1 - INTRODUCTION 117 3.2 - TWO PHASE SYSTEM CHARACTERIZATION - RESULTS AND DISCUSSION 117 3.2.1 - E l e c t r o s t a t i c Bulk Phase P o t e n t i a l s 118 3.2.2 - P a r t i t i o n Of System Components Between The Phases 122 3.3 - PARTITION OF DMPC SMALL UNILAMELLAR LIPOSOMES 123 3.3.1 - S e c t i o n I n t r o d u c t i o n 1123 V l l 3.3.2 - Results 124 3.3.3 - D i s c u s s i o n And Conclusions 141 - ERYTHROCYTE PARTITION 155 3.4.1 - S e c t i o n I n t r o d u c t i o n 155 3.4.2 - E f f e c t of S e t t l i n g Time on C e l l P a r t i t i o n 156 3.4.3 - E f f e c t s Of Various System And C e l l A l t e r a t i o n s On Erythrocyte P a r t i t i o n - Results And D i s c u s s i o n 165 3.4.3.1 - E f f e c t Of Increasing Na phosphate Concentra-t i o n And Decreasing NaCl Concentration On Ery t h r o c y t e P a r t i t i o n In I s o t o n i c Ester-Free Two Phase systems 165 3.4.3.2 - E f f e c t s o f Various PEG-Fatty Acid E s t e r s On C e l l P a r t i t i o n 167 3.4.3.3 - E f f e c t Of Na phosphate/NaCl Concentration Ratio On Ester Induced C e l l P a r t i t i o n In Is o t o n i c Two Phase Systems 186 3.4.3.4 - E f f e c t Of Polymer Concentration, I n t e r f a c i a l Tension And V e r t i c a l Versus H o r i z o n t a l Tube S e t t l i n g On C e l l P a r t i t i o n 188 3.4.3.5 - E f f e c t Of Polymer Lot On C e l l P a r t i t i o n 189 3.4.3.6 - E f f e c t Of C e l l Concentration On Er y t h r o c y t e P a r t i t i o n 195 3.4.3.7 - E f f e c t s Of Temperature V a r i a t i o n , E s t e r Degradation, Load Mix Phase and Blood Sample P r e p a r a t i o n V a r i a t i o n On C e l l P a r t i t i o n 199 3.4.3.8 - E f f e c t Of Surface S i a l i c A c i d Removal By Neuraminidase Treatment On E r y t h r o c y t e P a r t i t i o n 208 3.4.3.9 - E f f e c t Of Aldehyde F i x a t i o n On C e l l P a r t i t i o n 214 3.4.3.10 - E f f e c t Of Phase System T o n i c i t y And C e l l Shape On Erythrocyte P a r t i t i o n 227 3.4.3.11 - E f f e c t Of Exposure To Ethanol, Dimethyl S u l f o x i d e And Other Solvents On E r y t h r o c y t e P a r t i t i o n 234 3.4.3.12 - E f f e c t Of Added Free Fatty Acid On C e l l P a r t i t i o n 240 v i i i 3.4.3.13 - Countercurrent D i s t r i b u t i o n Of Ery t h r o c y t e s 259 3.4.3.14 - E f f e c t Of Polymer And A f f i n i t y P a r t i t i o n D e r i v a t i v e P u r i f i c a t i o n On C e l l P a r t i t i o n 261 3.4.4 - General D i s c u s s i o n 265 CHAPTER 4. DIFFERENTIAL PARTITION OF MULTIPLE SCLEROSIS AND NORMAL SUBJECTS' ERYTHROCYTES 273 4.1 - INTRODUCTION 273 4.2 - RESULTS 279 4.2.1 - S i n g l e Tube P a r t i t i o n Of MS And Normal Subj e c t s ' E r y t h r o c y t e s 279 4.2.2 - Counter Current D i s t r i b u t i o n Of MS And Normal Subjects' E r y t h r o c y t e s 307 4.2.3 - C o n t r o l P a r t i t i o n Studies On E r y t h r o c y t e s From Normal Subjects And P a t i e n t s With Diseases Other Than MS 310 4.2.4 - Summary Of P a r i t i t i o n R e s ults 312 4.2.5 - Summary Of Re s u l t s From Related Experiments 314 4.3 - DISCUSSION AND CONCLUSIONS 315 APPENDIX 1. BIOPHYSICAL STUDIES RELATED TO AQUEOUS POLYMER TWO PHASE PARTITION 331 A l . l - INTRODUCTION 331 A1.2 - Ph.D. THESIS WORK OF K. A. SHARP 331 A1.3 - ELECTROPHORETIC MOBILITY STUDIES 338 Al.3.1 - E f f e c t s Of PEG-Fatty Acid E s t e r s On C e l l And Free Fatty Acid Ethanolasome E l e c t r o p h o r e t i c M o b i l i t y 338 Al.3.2 - E f f e c t s Of PEG-Fatty Acid E s t e r s On The E l e c t r o p h o r e t i c M o b i l i t y Of Phase Dro p l e t s Suspended In The Complimentary Phase 343 A1.4 - EFFECT OF INTERFACIAL TENSION ON PARTICLE PARTITION 346 THESIS BIBLIOGRAPHY 356 i x LIST OF TABLES 2-1. B u f f e r Systems Used In Compounding Two Phase Systems. 91 2- 2. Percent S u b s t i t u t i o n And Average Molecular Weight Estimates Of The PEG-Fatty Acid E s t e r s Used In Our Work. 112 3- 1. E l e c t r o s t a t i c Bulk Phase P o t e n t i a l s Of Various Two Phase Systems. 120 3-2. P a r t i t i o n Of DMPC Liposomes In Ester-Free Two Phase Systems 129 3-3. E f f e c t Of Temperature On The P a r t i t i o n Of DMPC Small U n i l a m e l l a r Liposomes In A (5,4) V PEG Ester Containing Phase System. 129 3-4. E f f e c t Of Polymer Lot On Fixed And Fresh E r y t h r o c y t e P a r t i t i o n . 193 3-5. E f f e c t Of Polymer Lot On Fixed And Fresh E r y t h r o c y t e P a r t i t i o n . 194 3-6. E f f e c t Of C e l l Concentration On (5,4) I System Er y t h r o c y t e P a r t i t i o n . 197 3-7. E f f e c t Of S i a l i c Acid Removal By V i b r i o c h o l e r a Neuraminidase On Human Erythrocyte P a r t i t i o n . 211 3-8. Published E f f e c t s Of Aldehyde F i x a t i o n And L i p i d E x t r a c t i o n On The P a r t i t i o n And E l e c t r o p h o r e t i c M o b i l i t y Of Human Er y t h r o c y t e s . 216 3-9. E f f e c t s Of Aldehyde F i x a t i o n On The P a r t i t i o n And E l e c t r o p h o r e t i c M o b i l i t y Of Human Er y t h r o c y t e s . 219 3-10. P a r t i c l e P a r t i t i o n Factors D i r e c t l y Related To Log K. 266 4-1. M u l t i p l e S c l e r o s i s P a r t i t i o n Test V a r i a b l e s . 275 4-2. P a r t i t i o n Of Fresh And F i x e d , MS And Normal S u b j e c t s ' E r y t h r o c y t e s In Ester Free Two Phase Systems. 281 X LIST OF TABLES CONTINUED 4-3. Summary Of Fixed MS And Normal Subjects' E r y t h r o c y t e P a r t i t i o n In (5,4) V Systems Containing PEG 6000-Linoleate (18:2) Est e r And L i n o l e i c (18:2) Acid Added In Ethanol. 289 4-4. Fresh MS And Normal Subjects' Erythrocyte P a r t i t i o n In (5,4) V Systems Containing Various PEG 6000-Fatty Acid E s t e r s . 296 4-5. Fresh MS And Normal Subjects' Erythrocyte P a r t i t i o n In (5,4) V Systems Containing E i t h e r PEG 6000-Stearate (18:0) or PEG 6000-Linoleate (18:2) E s t e r . 303 4-6. P a r t i t i o n Of Fresh Normal, Other N e u r o l o g i c a l D i s o r d e r , I n f e c t i o u s Acute, and Non-Infectious Acute S u b j e c t s ' E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate 311 (18:2) E s t e r . 4-7. P o s s i b l e Blood C e l l A l t e r a t i o n s Associated With MS And Their E f f e c t s On C e l l P a r t i t i o n In Various Two Phase Systems. 318 A l - 1 . C r i t i c a l M i c e l l e Concentrations Of PEG-Fatty Acid E s t e r s . 335 Al- 2 . E l e c t r o p h o r e t i c M o b i l i t i e s Of (5,4) I System Phase Droplets. 344 Al - 3 . P h y s i c a l Analyses Of Two Phase Systems Used In This Thesis. 347 x i LIST OF FIGURES 2-1. Apparatus For Measuring Two Phase System E l e c t r o s t a t i c Bulk Phase P o t e n t i a l s . 59 2-2. O u t l i n e Of P u r i f i c a t i o n And A n a l y s i s P r o t o c o l For PEG-Fatty A c i d E s t e r s And Related PEG D e r i v a t i v e s . 62 2-3. T y p i c a l S i n g l e Tube P a r t i c l e P a r t i t i o n Procedure. 77 2-4. P r o t o c o l For Preparing M u l t i p l e S c l e r o s i s , C o n t r o l Disease Subjects' And Normal Subjects' Blood Samples For D i f f e r e n t i a l E r y t h r o c y t e Two Phase P a r t i t i o n . 85 2-5. Separation Of PEG 6000-Linoleate (18:2) Este r And Free Fatty Acid By Sephadex LH-20 Gel F i l t r a t i o n Column Chromatography. 97 2-6. Separation Of PEG 6000 And PEG 6 0 0 0 - P a l m i t a t e - l - 1 A C E s t e r By Octyl-Sepharose CL-4B Hydrophobic A f f i n i t y Column Chromatography. 100 2-7. Standard Curve For The Assay Of PEG 6000 In Ne s s l e r ' s Reagent. 102 2-8. U l t r a v i o l e t Absorption Of PEG 6000-Fatty Acid E s t e r s And Related Compounds. 105 2-9. U l t r a v i o l e t Absorption Of PEG 6000-Fatty Acid E s t e r s And Related Compounds. 105 2-10. Standard Curve For Hydroxamic Acid Ester Assay. 108 2- 11. Separation of Large M u l t i l a m e l l a r And Small U n i l a m e l l a r DMPC Liposomes On A Sepharose 4-B Gel F i l t r a t i o n Column. 115 3- 1. E l e c t r o s t a t i c Bulk Phase P o t e n t i a l Versus NaCl Concentration In Various I s o t o n i c NaCl-Na Phosphate Containing (5,4) Systems. 121 3-2. D i f f e r e n t i a l Scanning Calorimetry Of DMPC Small U n i l a m e l l a r Liposomes Under Various Conditions. 126 3-3. P a r t i t i o n Of DMPC Small U n i l a m e l l a r Liposomes In PEG-Fatty A c i d Ester Free (5,4) V, (5,3.7) V and (5,3.5) V Phase Systems. 130 3-4. Percent P a r t i t i o n Versus Log Ester Concentration For DMPC SUV's, DMPC LMVs And Fresh Human Ery t h r o c y t e s In (5,4) V Systems Containing Ethanol Extracted And LH-20 Column P u r i f i e d PEG 6000-Linoleate (18:2) E s t e r . 133 x i i LIST OF FIGURES CONTINUED 3-5. Percent P a r t i t i o n Versus Log Ester Concentration For DMPC SUV's At 18° c A n d 26° C, And Human Ery t h r o c y t e s At 22° C In A (5,4) V System Containing PEG 6000-Stearate (18:0) E s t e r . 134 3-6. Percent P a r t i t i o n Versus Log Ester Concentration For DMPC SUV's At 18° C And 26° C, And Human E r y t h r o c y t e s At 22° c In A (5,4) V System Containing PEG 6000-Linoleate (18:0) E s t e r . 135 3-7. Percent P a r t i t i o n Versus Temperature For DMPC SUV's In A (5,4) V System Containing 0.5 uM PEG 6000-Linoleate (18:2) E s t e r . 136 3-8. Percent P a r t i t i o n Versus Temperature For DMPC SUV's In (5,4) V Systems Containing 0.5 uM PEG 6000-Linoleate (18:2) E s t e r Or 0.5 uM PEG 6000-Stearate (18:0) E s t e r . 138 3-9. Log P a r t i t i o n C o e f f i c i e n t (K) Versus Temperature For The Data Expressed In Figure 3-11. 139 3-10. Percent P a r t i t i o n Versus Log Ester Concentration For DMPC SUV's, EggPC LUV's and Human Eryth r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate (18:2) E s t e r . 154 3-11. T y p i c a l S i n g l e Tube Er y t h r o c y t e P a r t i t i o n Experiment 160 3-12. Percent P a r t i t i o n Versus Log S e t t l i n g Time For F i x e d Human Ery t h r o c y t e s In (5,4) V Systems Containing Various Amounts Of PEG 6000-Linoleate (18:2) Ester. 162 3-13. Log P a r t i t i o n C o e f f i c i e n t (K) Versus E l e c t r o s t a t i c Bulk Phase P o t e n t i a l s For Human Erythrocytes In I s o t o n i c (5,4) NaCl-Na Phosphate Containing Phase Systems. 166 3-14. P a r t i t i o n Of Erythrocytes In A (5,4) V Phase System Containing PEG 6000-Stearate (18:0) Expressed As Percent Upper Phase P a r t i t i o n , P a r t i t i o n C o e f f i c i e n t K and Log K. 169 3-15. Percent P a r t i t i o n Of Human Erythrocytes In A (5,4) V System Containing Various PEG 6000-Fatty Acid E s t e r s . 170 3-16. Percent P a r t i t i o n Versus Log Ester Concentration For Human Eryth r o c y t e s In A (5,4) System Containing Various PEG 6000-Fatty Acid E s t e r s . 171 3-17. Percent P a r t i t i o n Versus Log Ester Concentration For Fi x e d And Fresh Human Eryth r o c y t e s In A (5,4) System Containing Various PEG 6000-Fatty Acid E s t e r s . 173 x i i i LIST OF FIGURES CONTINUED 3-18. Log P a r t i t i o n C o e f f i c i e n t (K) Versus Log Es t e r Concentration For Human Erythrocytes In A (5,4) V System Containing Various PEG 6000-Fatty A c i d E s t e r s . 174 3-19. Pecent P a r t i t i o n Versus Log Ester Concentration For Human Erythrocytes In A (5,4) V System Containing PEG-Oleate (18:1) Or PEG-Ricinoleate (18:1-C 1 2-0H) F a t t y Acid E s t e r s With Various S i z e PEG Head Groups. 175 3-20. E f f e c t of PEG-Fatty Acid E s t e r A c y l T a i l Hydrophobicity On Ester Induced C e l l P a r t i t i o n 178 3-21. E f f e c t of PEG-Fatty Acid E s t e r T a i l Unsaturation On E s t e r Induced C e l l P a r t i t i o n 179 3-22. P a r t i t i o n Of Human Ery t h r o c y t e s In A (5,4) V System Cont a i n i n g PEG 6000-Palmitate (16:0) Or - P a l m i t a t e - l - 1 4 C (16:0) E s t e r . 180 3-23. P a r t i t i o n Of Human Ery t h r o c y t e s In A (5,4) V System Containing PEG 6000-Oleate (18:1) Mono- Or D i - S u b s t i t u t e d E s t e r . 182 3-24. P a r t i t i o n Of Human Ery t h r o c y t e s In A (5,4) V System Containing PEG 6000-Stearate (18:0) Ester Or PEG 6000-Octadecyl (18:0) Ether. 185 3-25. P a r t i t i o n Of Human Eryth r o c y t e s In (5,4) I , (5,4) I I and (5,4) V Systems Containing PEG 6000-Linoleate (18:2) E s t e r . 187 3-26. P a r t i t i o n ( l o g K) Of Human Erythrocytes In (5,4) V, (5,3.7) V and (5,3.5) V Systems Containing PEG 6000-Linoleate (18:2) Ester. 190 3-27. E f f e c t Of Polymer Lot Number On The P a r t i t i o n Of Ery t h r o c y t e s In (5,4) V Systems Containing PEG 6000-Linoleate (18:2) E s t e r . 192 3-28. E f f e c t Of C e l l Concentration On Hydrophobic A f f i n i t y C e l l P a r t i t i o n . 198 3-29. E f f e c t Of PEG-Fatty Acid E s t e r B i o l o g i c a l Degradation On Hydrophobic A f f i n i t y C e l l P a r t i t i o n . 202 3-30. E f f e c t Of P r i o r E q u i l i b r a t i o n Of C e l l s With Lower Compared To Upper Phase On Hydrophobic A f f i n i t y C e l l P a r t i t i o n . 203 x i v LIST OF FIGURES CONTINUED 3-31. E f f e c t Of Erythr o c y t e Washing On Hydrophobic A f f i n i t y C e l l P a r t i t i o n . 206 3-32. E f f e c t Of Surface S i a l i c A cid Removal By V i b r i o c h o l e r a Neuraminidase On Hydrophobic A f f i n i t y C e l l P a r t i t i o n . 210 3-33. E f f e c t Of C e l l F i x a t i o n By Various Lower Aldehydes On PEG 6000-Linoleate (18:2) A f f i n i t y C e l l P a r t i t i o n . 224 3-34. E f f e c t Of S a l t Concentration On Erythrocyte Percent P a r t i t i o n In pH 7.2 And pH 6.8 Na phosphate C o n t a i n i n g (5,4) Systems. 230 3-35. E f f e c t Of Glutaraldehyde F i x a t i o n - B u f f e r - T o n i c i t y On Fixed E r y t h r o c y t e Hydrophobic A f f i n i t y P a r t i t i o n . 232 3-36. E f f e c t Of Ethanol On Fixed Erythrocyte P a r t i t i o n In A (5,4) V System Containing PEG 6000-Linoleate (18:2) E s t e r . 236 3-37. E f f e c t Of L i n o l e i c (18:2) A c i d , S t e a r i c (18:0) A c i d And Ethanol On The P a r t i t i o n Of Fixed Erythrocytes In A (5,4) V System Containing PEG 6000-Stearate (18:0) E s t e r . 243 3-38. E f f e c t Of S t e a r i c (18:0) A c i d And Ethanol On The P a r t i t i o n Of Fixed E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate (18:2) Ester. 244 3-39. E f f e c t Of S t e a r i c (18:0) Acid And Ethanol On The P a r t i t i o n Of Fixed E r y t h r o c y t e s In A (5,4) V System C o n t a i n i n g PEG 6000-Linoleate (18:2) E s t e r . 245 3-40. E f f e c t Of L i n o l e i c (18:2) Acid And Ethanol On The P a r t i t i o n Of Fixed E r y t h r o c y t e s In A (5,4) V System Con t a i n i n g PEG 6000-Linoleate (18:2) E s t e r . 247 3-41. E f f e c t Of L i n o l e i c (18:2) Acid And Ethanol On The P a r t i t i o n Of Fixed E r y t h r o c y t e s In A (5,4) V System C o n t a i n i n g PEG 6000-Linoleate (18:2) E s t e r . 248 3-42. E f f e c t Of L i n o l e i c (18:2) A c i d And Ethanol On The P a r t i t i o n Of Fixed E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Arachidonate (20:4) Or - L i n o l e n a t e (18:3) E s t e r . 249 3-43. M o d i f i c a t i o n Of The E f f e c t Of L i n o l e i c (18:2) Acid And Ethanol On The P a r t i t i o n Of Fixed E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate (18:2) Est e r By A l t e r i n g Aldehyde F i x a t i v e Type And Concentration. 251 XV LIST OF FIGURES CONTINUED 3-44. Counter Current D i s t r i b u t i o n Of Human Er y t h r o c y t e s In A (5,4) V Or A (5,4) I System At 4° C. 260 3-45. E f f e c t Of Ester P u r i f i c a t i o n On The P a r t i t i o n Of Fresh And Fixed Human Er y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate Ester. 264 4-1. Summary Of P r e l i m i n a r y Experiments I n v o l v i n g The Comparative P a r t i t i o n Of MS And Normal Subjects' F i x e d E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-L i n o l e a t e (18:2) E s t e r , L i n o l e i c (18:2) Acid And Ethanol. 283 4-2. Summary Of P r e l i m i n a r y Experiments. Decrease In The Percent P a r t i t i o n Of Fixed E r y t h r o c y t e s In A (5,4) V System Conta i n i n g 10 uM PEG 6000-Linoleate (18:2) E s t e r Due To The A d d i t i o n Of L i n o l e i c A c i d P r i o r To E s t e r . 284 4-3. Comparative P a r t i t i o n Of Fixed MS And Normal S u b j e c t s ' E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-L i n o l e a t e (18:2) E s t e r , L i n o l e i c (18:2) A c i d , And Ethanol. 286 4-4. Comparative P a r t i t i o n Of Fixed MS And Normal Subj e c t s ' E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-L i n o l e a t e (18:2) E s t e r , L i n o l e i c (18:2) A c i d , And Ethanol. 287 4-5. Comparative P a r t i t i o n Of Fresh MS And Normal Subj e c t s ' E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-St e a r a t e (18:0) Ester. 292 4-6. Comparative P a r t i t i o n Of Fresh MS And Normal Subj e c t s ' E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-L i n o l e a t e (18:2) E s t e r . 293 4-7. Comparative P a r t i t i o n Of Fresh MS And Normal S u b j e c t s ' E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-L i n o l e n a t e (18:3) E s t e r . 294 4-8. Comparative P a r t i t i o n Of Fresh MS And Normal Subj e c t s ' E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Arachidonate (20:4) E s t e r . 295 4-9. Comparative Average P a r t i t i o n (%) Of Th i r t y - N i n e MS And Twenty-Two Normal Subjects' Fresh Erythrocytes In A (5,4) V System Containing PEG 6000-Stearate (18:0) Es t e r . 298 x v i LIST OF FIGURES CONTINUED 4-10. Comparative Average P a r t i t i o n C o e f f i c i e n t s (K) Of T h i r t y - N i n e MS And Twenty-Two Normal Subjects' Fresh E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Stearate (18:0) E s t e r . 299 4-11. Comparative Average P a r t i t i o n (Log K) Of T h i r t y - N i n e MS And Twenty-Two Normal Subjects' Fresh E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Stearate (18:0) E s t e r . 300 4-12. Comparative Average P a r t i t i o n (%) Of T h i r t y - N i n e MS And Twenty-Two Normal Subjects' Fresh Er y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate (18:2) E s t e r . 301 4-13. Comparative Average P a r t i t i o n C o e f f i c i e n t s (K) Of T h i r t y - N i n e MS And Twenty-Two Normal Subjects' Fresh E r y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate (18:2) E s t e r . 302 4-14. Comparative Average P a r t i t i o n (Log K) Of T h i r t y - N i n e MS And Twenty-Two Normal Subjects' Fresh Er y t h r o c y t e s In A (5,4) V System Containing PEG 6000-Linoleate (18:2) E s t e r . 302 4-15. MS Subjects' Fresh Eryt h r o c y t e Maximal P a r t i t i o n , Expressed As A Percentage Of Matching Normal Subjects' Fresh E r y t h r o c y t e Maxiaml P a r t i t i o n In (5,4) V Systems Containing E i t h e r PEG 6000-Stearate (18:0) Or - L i n o l e a t e (18:2) E s t e r . 305 4-16. MS Subjects' Fixed E r y t h r o c y t e Maximal P a r t i t i o n , Expressed As A Percentage Of Matching Normal Subjects' Fixed E r y t h r o c y t e Maxiaml P a r t i t i o n In A (5,4) V System Containing PEG 6000-L i n o l e a t e (18:2) E s t e r . 306 4-17. Counter Current D i s t r i b u t i o n s Of MS And Normal Sub j e c t s ' Fresh E r y t h r o c y t e s In A (5,3.5) V System At 4° C. 309 A l - 1 . E r y t h r o c y t e E l e c t r o p h o r e t i c M o b i l i t y Versus Log PEG-Fatty Acid E s t e r Suspending B u f f e r Concentration. 340 Al - 2 . Log P a r t i t i o n C o e f f i c i e n t (K) Versus I n t e r f a c i a l Tension For Er y t h r o c y t e s And P a l m i t i c (16:0) Acid Enriched Acholeplasma  l a i d l a w i i B C e l l s In Various Two Phase Systems Containing 0.15 M NaCl and 0.01 M NaPhosphate, pH 7.2. 351 x v i i ACKNOWLEDGEMENTS I would l i k e to take t h i s opportunity to f o r m a l l y thank c e r t a i n i n d i v i d u a l s and groups because t h i s t h e s i s would have been much more d i f f i c u l t to complete without t h e i r f a i t h , help and example. They are: Don Brooks - whose c h a l l e n g i n g example of what a s c i e n t i s t should be would have been t e r r i b l y f r u s t r a t i n g without the i n s p i r a t i o n of h i s f r i e n d s h i p and the g i f t of h i s wife's f r i e n d s h i p . Everyone i n Don Brooks l a b o r a t o r y i n c l u d i n g Kim Sharp, Tim Webber, Mansur Y a l p a n i , R u s s e l l G r i e g , Johan Janzen, B a s i l Chiu, Rosemarie Rupps, John Cavanaugh, Poul Sorrensen (who deserves a great deal of the c r e d i t f o r the s y n t h e s i s of P E G - l - l ^ c - p a l m i t a t e and the success of the liposome p a r t i t i o n s t u d i e s ) , Mandy Hoskins, Richard A. Van Wagenen, E r i c Brown, Wai Pan Chan, Y. C. Pang, The Snoek-Sturgeons, and Raymond Norris-Jones (who provided meticulous t e c h n i c a l help with the e r y t h r o c y t e p a r t i t i o n s t u d i e s ) . The U.B.C. Department of Pathology e s p e c i a l l y , P h i l Reid, Richard Pearce, Evan Evans, J u r i F r o h l i c h , Derek Applegarth, W.L. Dunn, Charles Ramey, and the very supportive support s t a f f . Harry Walter, M i l t o n and Mrs. H a r r i s , G.V.F. Seaman, Roy Swank, Cherry Tamblyn, Chip Zukoski IV, Stephan Bamberger, Gote Johansson, Eva E r i k s s o n , K a r l - E r i c Magnusson and Per-Ake A l b e r t s s o n . The M u l t i p l e S c l e r o s i s S o c i e t y of Canada f o r patience and f i n a n c i a l support above and beyond what I deserved. Dr. D. W. Paty and h i s a s s o c i a t e s at the U.B.C. Acute Care H o s p i t a l M u l t i p l e S c l e r o s i s C l i n i c . Dr. M. Jones of Shaughnessy H o s p i t a l . Dr. Peter C u l l i s and co-workers. L y l e Brown, whose f r i e n d s h i p i n the face of misunderstanding withstood too many years of shared accomodation, mutual postgraduate education, l o s t love and the Saturn Return. My f a m i l y , e s p e c i a l l y my mother. My brother P e t e r , my s i s t e r B a r b l , Brenda, J u l i a , Genevieve, Dianne, C e c i l i a , and every other s p e c i a l f r i e n d and s o u l who has helped me l e a r n to love myself as a human being. x v i i i DEDICATION I dedicate t h i s t h e s i s to U r a n t i a , my wi f e , Wilhelm Reich, the Comet Creature, the Peur Aeternus, one day at the beach, and "The l i g h t which climbs and s u r f a c i n g d i v i d e s , becoming two; one f o r the eart h to hold, one free a l i v e held never, one f o r my l i f e to mold, one without form f o r e v e r . " x i x ABBREVIATIONS ACTH Ad r e n o c o r t i c o t r o p i n hormone ADCC Antibody dependent c e l l u l a r c y t o t o x i c i t y ADP Adenosine-diphosphate BHA Beta-hydroxy-anisole BHT Beta-hydroxy-toluene CCD Countercurrent d i s t r i b u t i o n CNS Ce n t r a l nervous system CPM Counts per minute CSF C e r e b r a l s p i n a l f l u i d D (Dextran), Poly -(alpha-1, 6-glucose) DMPC D i m y r i s t o y l p h o s p h a t i d y l c h o l i n e DNA Deoxyribonucleic a c i d DPM D i s i n t e g r a t i o n s per minute EAE Experimental a l l e r g i c e n c e p h a l o m y e l itis EDTA Et h y l e n e d i a m i n e - t e t r a c e t i c a c i d EUFA Ery t h r o c y t e unsaturated f a t t y a c i d GLA Gama-linolenic a c i d GSH Glutathione (reduced) GSSG Glutathione ( o x i d i z e d ) HLA Human h i s t o c o m p a t i b i l i t y antigen HPLC High pressure l i q u i d chromatography igc Immunoglobulin -l-ype G IgM Immunoglobulin Type M XX LA L i n o l e i c a c i d LAD L i n o l e i c a c i d ( e l e c t r o p h o r e t i c m o b i l i t y ) depression LCAT L e c i t h i n - c h o l e s t e r o l a c y l t r a n s f e r a s e LMV Large m u l t i l a m e l l a r v e s i c l e (liposome) LUV Large u n i l a m e l l a r v e s i c l e (liposome) MBP Myelin b a s i c p r o t e i n MEM Macrophage e l e c t r o p h o r e t i c m o b i l i t y MIF M i g r a t i o n i n h i b i t i o n f a c t o r MS M u l t i p l e s c l e r o s i s N Normal NANA N-acetyl neuraminic a c i d OND Other n e u r o l o g i c a l d i s o r d e r (than MS) PBS Phosphate buf f e r e d s a l i n e PEG Po l y - ( e t h y l e n e g l y c o l ) PG P r o s t a g l a n d i n PGEj P r o s t a g l a n d i n type E]_ PNS P e r i p h e r a l nervous system PPD P u r i f i e d p r o t e i n d e r i v a t i v e (of M. t u b e r c u l o s i s ) PUFA Polyunsaturated f a t t y a c i d RNA Ri b o n u c l e i c a c i d RRI R e l a t i v e r e f r a c t i v e index SRRI S p e c i f i c r e l a t i v e r e f r a c t i v e index SSPE Subacute s c l e r o s i n g panencephalomyelitis SUV Small u n i l a m e l l a r v e s i c l e Tris-HCI T r i s (hydroxy methyl) aminomethane-HCI UFA Unsaturated f a t t y a c i d VCN V i b r i o c h o l e r a Neuraminidase - 1 -CHAPTER ONE - INTRODUCTION 1.1 GENERAL INTRODUCTION This t h e s i s i s concerned with the p a r t i t i o n of c e l l s and phos p h a t i d y l c h o l i n e liposomes i n two aqueous polymer phase systems. In p a r t i c u l a r i t discusses the p a r t i t i o n of human e r y t h r o c y t e s i n such systems and a s u c c e s s f u l attempt to d i f f e r e n t i a l l y p a r t i t i o n normal and m u l t i p l e s c l e r o s i s (MS) su b j e c t s ' e r y t h r o c y t e s . The importance of the l a t t e r l i e s i n the demonstration of the p o s s i b l e d i a g n o s t i c usefulness of polymer two phase p a r t i t i o n and i n the demonstration t h a t MS i s as s o c i a t e d with blood c e l l surface a b n o r m a l i t i e s . A l l of the f o l l o w i n g work was accomplished using two phase systems compounded with the polymers dextran T500 and po l y - ( e t h y l e n e g l y c o l ) 6000 ( r e c e n t l y redesignated PEG 8000 by Union Carbide). Countercurrent d i s t r i b u t i o n (CCD) was used minimally since the p a r t i t i o n d i f f e r e n c e s found were u s u a l l y l a r g e enough to be seen v i a s i n g l e tube p a r t i t i o n . In s p i t e of t h i s l i m i t a t i o n , p r a c t i c a l experience convinces me that the f o l l o w i n g m a t e r i a l should be of i n t e r e s t to anyone working i n the f i e l d of two phase p a r t i t i o n , independent of the p a r t i c u l a r techniques, polymer phase systems, or substances being used. - 2 -1.2 MULTIPLE SCLEROSIS  1.2.1 - General Background M u l t i p l e s c l e r o s i s i s the most common n e u r o l o g i c a l disease a f f e c t i n g young adults i n temperate cl i m a t e s (1-10). I t s incidence can be higher than 1 case per 1,000 population (11). This incidence plus the d e b i l i t a t i n g sometimes deadly nature of the disease has p r e c i p i t a t e d a tremendous e f f o r t i n recent years aimed at a more fundamental understanding of MS. To date a great deal of v a l u b l e i n f o r m a t i o n has been accumulated but a good understanding of MS, e s p e c i a l l y i n regard to i t s fundamental cause, i s s t i l l l a c k i n g (2,3,A,12,13). The b a s i c l e s i o n i n m u l t i p l e s c l e r o s i s was described as e a r l y as 1868 by J . M. Charcot (IA, see a l s o 15). I t i s autoimmune demyelination of the nerve f i b r e s or axons i n the c e n t r a l nervous system (CNS) (3,15,16,17). Myelin, which i s a c t u a l l y part of the o l i g o d e n d r o c y t i c plasma membrane, coats nerve c e l l axonal c y l i n d e r s i n an i n s u l a t i n g manner i n c r e a s i n g the speed and c l a r i t y of s i g n a l s conducted v i a the axons. The demyelination occurs i n d i s c r e t e l o c i or "plaques" d i f f u s e i n both time of appearance and l o c a t i o n which range i n diameter from 0.1 to s e v e r a l centimeters. Plaque formation occurs during attacks of the disease. These a t t a c k s vary considerably i n frequency and s e v e r i t y of r e s u l t i n g n e u r o p a t h o l o g i c a l symptoms. Symptoms may incl u d e b l i n d n e s s , l a c k of limb c o o r d i n a t i o n , l o s s of bladder c o n t r o l , tremor and i n v o l u n t a r y spasms (A,7) though, " i t i s now c l e a r l y understood that the dysfunction experienced by the p a t i e n t i s not - 3 -d i r e c t l y p r o p o r t i o n a l to the amount of h i s t o l o g i c a l l y demonstratable demyelination" (18). The myelin membranes of o l i g o d e n d r o g l i a c e l l s are p r i m a r i l y a f f e c t e d with secondary o l i g o d e n d r o c y t i c l o s s , a s t r o c y t i c and phagocytic m i c r o g l i a l c e l l p r o l i f e r a t i o n and l o c a l i z e d i n f l a m a t o r y response. Neuronal c e l l s and t h e i r axons are not i n i t i a l l y a f f e c t e d . Plaques are u s u a l l y i n the white matter and have a tendency to occur, f o r unknown reasons, at c e r t a i n anatomical s i t e s such as c l o s e to t e r m i n a l veins i n the o p t i c nerves, p e r i v e n t r i c u l a r regions and s p i n a l cord. "Old l e s i o n s appear grey i n c o l o r and fi r m i n consistency (hence Charcot's d e s c r i p t i o n 'sclerose en plaques'): more recent l e s i o n s have an oedematous edge, are yellow or pink and of s o f t c o n s i s t e n c y . " (15). Some i n v e s t i g a t o r s now suspect that some p e r i p h e r a l nerve demyelination may als o occur i n MS (19,20). This demyelination may not be as s e r i o u s as CNS aemyelination due to the a b i l i t y of p e r i p h e r a l nervous system (PNS) Schwann c e l l s to regenerate myelin much f a s t e r than t h e i r CNS oligodendrocyte analogues (12). This l i m i t e d a b i l i t y of the CNS to res t o r e myelin i n plaque areas e x p l a i n s the need to develop treatments capable of h a l t i n g demyelination during attacks of the disease and to develop a d i a g n o s t i c t e s t capable of d e t e c t i n g MS at an e a r l y stage. Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e concerning the i n i t i a l stages of plaque formation. In 1981 A l l e n (15) o u t l i n e d three c o n t r a s t i n g views which suggest that the i n i t i a l l e s i o n may i n v o l v e m i c r o g l i a l c e l l h y p e r p l a s i a , p e r i v a s c u l a r lymphocytic and monocytic/macrophagic r e a c t i o n or n o n - c e l l mediated myelin d i s i n t e g r a t i o n with each of the two other phenomena f o l l o w i n g ( f o r a d e s c r i p t i o n of the pathology accompanying each - 4 -type of demyelination see reference 21). The edema i n e a r l y plaques i s probably due to v a s c u l a r damage as w e l l as normal inflammatory response mechanisms, while the f i r m consistency of older l e s i o n s i s p r i m a r i l y due to a s t r o c y t e s p r o l i f e r a t i n g and l a y i n g down g l i a l f i b r e s ( 2 ) . In a c t i v e l y demyelinating plaque areas secondary (chronic) inflammatory responses are evident with macrophage c e l l s i n abundance. P e r i v a s c u l a r lymphocyte " c u f f i n g " i s a l s o evident and i s i n f a c t somewhat c h a r a c t e r i s t i c of MS. In a d d i t i o n antibody-producing plasma c e l l s can be demonstrated both p e r i v a s c u l a r l y and w i t h i n the l e s i o n . These may be r e s p o n s i b l e i n l a r g e part f o r the immunoglobulins found i n the c e r e b r o s p i n a l f l u i d (CSF) i n MS (15,17 and 22). Approximately 15% of MS p a t i e n t s have abnormally increased CSF IgG p r o t e i n ( p o s s i b l y due to a l t e r a t i o n of the blood b r a i n b a r r i e r , 22) and more than 90% have o l i g o c l o n a l IgG suggestive of an immunoproliferative antibody c e l l response to s p e c i f i c antigens (15,17 and vide i n f r a ) IgG appears to be present both free and i n the form of complement-fixing (Clq) immune complexes (15). In 1979 Prineas (23) reported the presence of lymphoid-like t i s s u e i n the CNS of MS p a t i e n t s r e i n f o r c i n g the b e l i e f , based on the presence of CSF-IgG, that a s p e c i f i c antigen was being processed i n a r e c c u r i n g mannner. P r i c e and Cuzner reported decreased CSF l e v e l s of complement components C^ and C^ and i n the proteinase i n h i b i t o r a l p h a - l - a n t i t r y p s i n (24, see a l s o 25). In a d d i t i o n to these a l t e r a t i o n s the a c t i v e plaque areas c o n t a i n many biochemical a l t e r a t i o n s (17 and see below) i n c l u d i n g decreased l e v e l s of t o t a l l i p i d , increased percentages of u n e s t e r i f i e d , e s p e c i a l l y unsaturated, f a t t y a c i d s (15), lysosomal enzyme a c t i v i t y (26), and - 5 -decreased myelin-associated, myelin b a s i c p r o t e i n and a myelin a s s o c i a t e d g l y c o p r o t e i n (27) and g l y c o l i p i d (17, 28). Some of these a l t e r a t i o n s can be detected i n the otherwise normal appearing white matter at the edge of the plaques, suggesting that myelin l o s s i s a secondary symptom of the disease (15). The best s t u d i e d animal model of MS i s experimental a l l e r g i c encephalomyelitis (EAE), i n which a CNS autoimmune a t t a c k i s induced by i n j e c t i n g animals with whole white matter or myelin b a s i c p r o t e i n mixed with an adjuvant such as Freund's complete adjuvant (2,21,29-33). The r e s u l t i n g demyelination i s c e l l mediated and, as i n MS, a n t i m y e l i n serum antibodies are a l s o detectable although passive t r a n s f e r of EAE can only be made by t a n s f e r i n g T c e l l s and not serum from s e n s i t i z e d donors (2,21,32). O r i g i n a l l y EAE was unable to model the r e c u r r e n t p r o g r e s s i v e patte r n of MS however such a course has now be induced i n many animals which e x h i b i t abnormal immune r e g u l a t i o n such as SJL mice (2,32). Recent EAE s t u d i e s suggest that animals s e n s i t i z e d to MBP alone do not show as extensive demyelination as those s e n s i t i z e d to myelin. This and other experiments suggest that the t i s s u e d e s t r u c t i o n i n MS probably i n v o l v e s two or more d i s t i n c t antigens (with one antigen p o s s i b l y r e o c c u r r i n g at random) and i s not mediated by antibody alone ( 2 ) . This antigen could be a v i r u s or a non-MBP myelin component such as a p r o t e o l i p i d p r o t e i n , g l y c o p r o t e i n or g l y c o l i p i d . (34) In f a c t MBP may not be s p e c i f i c a l l y i n volved at a l l (35). V i r a l induced primary demyelinating diseases are a l s o being s t u d i e d as MS model systems, as are other many other models such as t o x i n and chemical induced demyelination (2,15). Both the EAE and - 6 -v i r a l models show some MS l i k e dependence on genetic f a c t o r s , but are d i s i m i l a r to MS i n c e r t a i n other regards (21,32,33,36). None of the above animal models are i d e a l . For example they a l l e x h i b i t c onsiderable post attack r e m y e l i n a t i o n ; which does not occur a p p r e c i a b l y i n MS (2,15,21). Wisniewski (21) has pointed out that demyelinating diseases can g e n e r a l l y be d i v i d e d i n t o those i n v o l v i n g demyelination i n the pesence of a cell-mediated immune response and those where demyelination occurs without inflammation. Both MS and v i r u s induced distemper as w e l l as EAE f i t i n t o the former c l a s s while t o x i n and inborn e r r o r ( i . e n e u r o l i p i d o s e s ) induced demyelinations are examples of the l a t t e r . Diagnosis of MS (discussed i n more d e t a i l below) can only be made by an experienced p h y s i c i a n on the basis of a p a t i e n t ' s h i s t o r y and c l i n i c a l c o n d i t i o n (37). Diagnosis i s only completely p o s i t i v e at death when CNS white matter can be inspected g r o s s l y and/or h i s t o l o g i c a l l y f o r m u l t i p l e plaques of varying age. This i s not to s t a t e that the disease i s n e c e s s a r i l y f a t a l . In f a c t i t s course i s extremely v a r i a b l e , the s e v e r i t y of symptoms being r e l a t e d to the l o c a t i o n and extensiveness of the plaques. The most common course f o r MS i n v o l v e s remission and r e l a p s e , though p a t i e n t s may a l s o experience a long l a s t i n g or permanent benign form of the disease, a chronic progressive d e t e r i o r a t i o n or even a malignant form which can lead to death i n a few years (7,16,18 a l s o 37,38). Kurtzke et a l . (39) c a l c u l a t e d that 75% of a l l MS p a t i e n t s were a l i v e 20 years a f t e r diagnosis and 50% were a l i v e 30 years l a t e r . Often diagnosis w i l l be made at death i n a person who possesses numerous plaques yet i n l i f e showed l i t t l e s i g n of n e u r o l o g i c a l impairment (37,40). - 7 -A l a r g e amount of e p i d e m i o l o g i c a l data on MS has been assessed. This data has l e d to considerable i n s i g h t regarding the geographical and population d i s t r i b u t i o n of MS, as w e l l as a v a r i e t y of r i s k f a c t o r s (see above) and p o s s i b l e causes of the disease. I t i s noteworthy that g e n e r a l l y a l l e p i d e m i o l o g i c a l s t u d i e s on MS have s u f f e r e d from, "...the l a c k of an e a s i l y c a r r i e d out s p e c i f i c and s e n s i t i v e d i a g n o s t i c t e s t . . . " (11, see a l s o 10,41,42). Geographically, MS world occurence can be d i v i d e d i n t o three zones of high, medium and low frequency corresponding approximately to prevalence rates of over 30, 5 to 25, and below 5 cases per 100,000 p o p u l a t i o n . These areas correspond roughly to Europe and North America and southern A u s t r a l i a and New Zealand between 45° and 65° l a t i t u d e , southern Europe and most of the United States and A u s t r a l i a , and A s i a and A f r i c a with the exception of medium occurence i n the white p o p u l a t i o n i n South A f r i c a , r e s p e c t i v e l y (1,10,41). MS appears to occur predominantly i n Caucasians e s p e c i a l l y those of European o r i g i n . In America, Negros and O r i e n t a l s have much lower r a t e s of incidence though they show the same geographical s u s e p t a b i l i t y (10,11,41,43). People m i g r a t i n g a f t e r the age of 15 carry the r i s k of t h e i r b i r t h p l a c e . M i g r a t i o n below t h i s average age r e s u l t s i n the migrant assuming much of the r i s k of the area migrated to (11). The onset of MS i s u s u a l l y between the ages of 15 and 50 (2) with the d i a g n o s i s , but not n e c e s s a r i l y the i n i t i a l a t t a c k , being made i n the e a r l y part of the f o u r t h decade of l i f e ( 3 ) . The above data suggests that MS i s an exogenous environmental disease, aquired p o s s i b l y i n e a r l y adolescence, which possesses a long l a t e n t p e r i o d (2,3,10,11,41,43,44). A - 8 -v i r u s has been proposed as the most l i k e l y candidate (2,10). Though no d i r e c t support of t h i s hypothesis has been forthcomming (vi d e i n f r a ) i t i s i n t e r e s t i n g to note that a stepwise increased incidence of MS i n the Faroe Islands and i n Iceland f o l l o w i n g the second world war suggests epidemics f o l l o w i n g exposure to occupying troops and equipment (41,44). As w e l l as a geographic p r e d i s p o s i t i o n race and f a m i l i a l r i s k f a c t o r s a l s o e x i s t . Female p a t i e n t s outnumber male p a t i e n t s by a r a t i o of 1.5 to 1 (2,11). There i s some tendency f o r the disease to occur i n f a m i l i e s (42) although the genetic l i n k i s not w e l l defined (7,10) and could be due to s i m i l a r d i e t , geographic and emotional (45) environmental f a c t o r s . There i s now good evidence that c e r t a i n h i s t o c o m p a t a b i l i t y antigens, [ g l y c o p r o t e i n c e l l surface antigens i n v o l v e d i n both immune c e l l mutual r e c o g n i t i o n and immune response to f o r e i g n c e l l s (46,47)], occur to a greater extent i n p a t i e n t s with MS than i n unaffected persons l i v i n g i n the same general area. The antigens i n question i n c l u d e HLA-A3, HLA-B7 and HLA-Dw2 which has been reported to occur i n up to 50% of a l l p a t i e n t s (48). Because of l i n k a g e d i s e q u i l i b r i u m , [the tendency of a p a i r of a l l e l e s at two d i f f e r e n t l o c i w i t h i n the same chromosomal area or complex to occur i n a s s o c i a t i o n more frequently than would be c a l c u l a t e d on the basis of t h e i r gene frequencies ( 4 8 ) ] , these antigens might not be d i r e c t l y l i n k e d to the disease but they do i m p l i c a t e the h i s t o c o m p a t a b i l i t y complex and p o s s i b l y a l t e r e d immune responsiveness i n MS patho g e n i c i t y and/or the course of the disease. I n t e r e s t i n g l y , the above antigens have a much higher frequency of occurence i n high r i s k areas and among Caucasians (11,42,48,49). Black Americans with MS show a - 9 -s i g n i f i c a n t percentage of Dw2 even though t h i s antigen i s uncommon i n the t o t a l Black American population (42,50). In a d d i t i o n to the above there are current r e p o r t s of a G-m antibody gene l i n k a g e to MS which i s i n t e r e s t i n g due to recent reports of G-m a l l o t y p e - and HLA-linked c o n t r o l i n autoimmune disease (32, see a l s o 47 and 48). Autoimmunity a s s o c i a t e d with a n t i g e n i c b r a i n g l y c o l i p i d s (51) or c e r t a i n c e l l surface HLA antigens (48) has been suggested as a p o s s i b l e cause of MS and a number of s t u d i e s have r e i n f o r c e d t h i s b e l i e f on the basis of the a s s o c i a t i o n of the disease with a number of HLA a n t i g e n i c types (2,48). I t has been s t a t e d that a major d i f f i c u l t y i n s t u d i e s i n v o l v i n g HLA t y p i n g and MS has been a l a c k of c e r t a i n t y , at times, of the diagnosis of MS, mild cases being overlooked (13). The above tend to support the idea of a genetic l i n k to MS however i t should be noted that t h i s l i n k and i t s true pathogenic s i g n i f i c a n c e i s not w e l l understood. Another theory concerning the s i g n i f i c a n c e of the a s s o c i a t i o n of c e r t a i n HLA antigens with MS i s t h a t these g l y c o p r o t e i n s increase the bi n d i n g of a s p e c i f i c v i r u s to c e l l s i n the CNS (47) which share many such antigens with other c e l l s i n the body (2,52). Theories advanced to e x p l a i n the e t i o l o g y of MS cover the e n t i r e spectrum of known disease-producing mechanisms. A v i r a l theory has been widely discussed (4,13,53,54, and see below) and although no unequivocal abnormality has been found i n the response of MS lymphocytes to the v i r a l antigens t e s t e d (56), an immune-viral theory of MS i s i n t e l l e c t u a l l y s a t i s f y i n g . The strongest evidence f o r a v i r a l e t i o l o g y i s the ep i d e m i o l o g i c a l c o n s i d e r a t i o n s o u t l i n e d above, the l a t e n t r e m i t t i n g nature - 10 -of the disease, the known demyelinating a b i l i t y of c e r t a i n v i r u s e s (myxoviruses) i n animal models and n a t u r a l l y o ccuring diseases such as subacute s c l e r o s i n g p a n e n c e p h a l i t i s (SSPE) and distemper, unusual MS lymphocyte r e a c t i v i t y to c e r t a i n c e r t a i n v i r a l antigens ( i . e . measles), reports of abnormally high antimeasles v i r u s a n t i b o d i e s i n MS serum, and reports of i d e n t i f i c a t i o n of v i r a l p a r t i c l e s i n MS t i s s u e i n c l u d i n g the CNS ( f o r reviews see 15,55,56). In s p i t e of the above no v i r u s or v i r a l agent has been c o n v i n c i n g l y l i n k e d d i r e c t l y to m u l t i p l e s c l e r o s i s and a l l attempts to i s o l a t e a s p e c i f i c v i r u s from MS t i s s u e s have f a i l e d ( 2 ) . Recent r e p o r t s have brought i n t o question the s p e c i f i c i t y of v i r u s - s p e c i f i c serum a n t i b o d i e s which may be r e l a t e d to HLA type not MS (42,57,58), the transmissable MS-associated agent of Carp et a l . (59,60,61) and other groups (62) and the paramyxovirus-like i n c l u s i o n s (63) reported to occur i n c e l l s of the nervous system of MS p a t i e n t s (15,64). Modern b e l i e f i s that any v i r a l involvement i n MS i s probably as one of many f a c t o r s . For instance i t i s b e l i e v e d that SSPE develops because of hindered c e l l mediated immunity to measles v i r u s r e s u l t i n g i n a p e r s i s t e n t i n f e c t i o n . Such immunodeficiencey might i t s e l f a r i s e as a r e s u l t of a v i r a l i n f e c t i o n (65, a l s o 66). As i n d i c a t e d above there i s considerable evidence f o r an immunopathogenesis of MS. At t h i s time however i t i s not c l e a r what type(s) of immune r e a c t i o n s are involved nor what f o r e i g n or auto-antigens. Evidence f o r a s p e c i f i c antigen i s suggested from the h i s t o l o g y of plaque formation and i t s s i m i l a r i t y to EAE as w e l l as the presence of CSF o l i g o c l o n a l IgG and CNS lymphoid t i s s u e . Autoimmunity i s - 11 -suggested by the r e l a t i o n s h i p of the disease to v a r i o u s HLA antigens and the i n a b i l i t y to i s o l a t e a s p e c i f i c e x t e r n a l cause of the disease, although i n a c o n t r o l l e d study of 120 d e f i n i t e cases of MS there was no evidence of an i n c r e a s e i n other autoimmune d i s o r d e r s or of malignancy (67). Modern b e l i e f i s that MS may be a disease of m u l t i p l e e t i o l o g y i n v o l v i n g one or more immunopathogenic mechanisms t r i g g e r e d by various p e r s i s t e n t antigens (32). There appears to be no s o l i d evidence i n d i c a t i n g t h a t a type I immunopathogenic mechanism ( i n v o l v i n g the i n t e r a c t i o n of IgE antibody with a s p e c i f i c antigen followed by the r e l e a s e of c e l l born vasoactive substances) i s important i n MS (33). The increase i n CSF- and CNS-IgG i n MS, and the o l i g o c l o n a l nature of these immunoglobulins s t r o n g l y suggests a r o l e f o r antibody-mediated immunopathogenic mechanisms a c t i n g i n MS. However, the nature of the antigen(s) i n v o l v e d i s unknown. The antibody could be d i r e c t e d against a v i r a l antigen i n a normal immune response or a s e l f - a n t i g e n i n an autoimmune response (15,33). The s i g n i f i c a n c e of the reported MS p a t i e n t antibody s p e c i f i c i t y f o r myelin, b a s i c myelin p r o t e i n o l i g o d e n d r o g l i a , and s e v e r a l v i r a l antigens are questionable (2,17,33,35) as i s the s i g n i f i c a n c e and s p e c i f i c i t y of the o l i g o c l o n a l IgG i t s e l f (2,68). I t should be r e s t a t e d that a n t i m y e l i n a n t i b o d i e s alone are not capable of inducing EAE. A t a b l e of a b n o r m a l i t i e s r e l a t e d to immunoglobulins i n MS can be found i n reference 2. C i r c u l a t i n g immune complexes have been found i n the serum i n about 50% of p a t i e n t s with MS and other n e u r o l o g i c a l diseases. (2,25,69). The CSF a l s o contains such complexes ( 2 ) . Immune complex a s s o c i a t e d d e s t r u c t i o n i s probably of secondary importance i n MS since i t would not e x p l a i n the - 12 -CNS s p e c i f i c i t y of the i n i t i a l t i s s u e d e s t r u c t i o n which i s u s u a l l y l i m i t e d to white matter r a t h e r than the more h e a v i l y v a s c u l a r i z e d grey matter. The immune complexes probably form as a r e s u l t of CNS t i s s u e d e s t r u c t i o n r a t h e r than v i c e versa (33). Modern immunology i s r e v e a l i n g ever more complex i n t e r a c t i o n s between the c e l l u l a r components of the immune system. S p e c i f i c i t y i n these i n t e r a c t i o n s comes from HLA and r e l a t e d c e l l surface antigens (47,48). An example of t h i s i s the a b i l i t y that monoclonal a n t i b o d i e s have given us to recognize s p e c i f i c T lymphocyte c e l l subsets on the b a s i s of t h e i r s p e c i f i c c e l l surface g l y c o p r o t e i n antigens. B r i e f l y , T c e l l s can be d i v i d e d i n t o inducer c e l l s which, upon a n t i g e n i c s t i m u l a t i o n , c o l l a b o r a t e with B lymphocytes to produce antibody, c y t o t o x i c e f f e c t o r c e l l s capable of l y s i n g c e l l s b i n d i n g s p e c i f i c a n t i b o d i e s and suppressor c e l l s which modulate immune responses by i n f l u e n c i n g the behaviour o f the other T c e l l s . (70,71) Normal humoral and cell-mediated immune responses are balanced as a r e s u l t of such i n t e r a c t i o n s whose abnormalcy can be pathogenic. In r e m i t t i n g experimental a l l e r g i c e n c e p h a l o m y e l i t i s , f o r instance, i t i s now p o s s i b l e to separate the i n d u c t i o n o f both e f f e c t o r and suppressor T c e l l subsets by manipulation of the antigen immunization schedule (32). The hypothesis that most immunopathologic r e a c t i o n s and c l i n i c a l autoimmunity may r e s u l t from a b n o r m a l i t i e s i n the i n t e r a c t i o n s of lymphocyte subtypes c u r r e n t l y enjoys more favor than t h e o r i e s Of c l o n a l e l i m i n a t i o n or s e n s i t i z a t i o n to hidden antigens (33). The s i m i l a r i t y of the demyelinating l e s i o n s i n acute MS and EAE st r o n g l y suggest that c e l l mediated immune mechanisms are f u n c t i o n i n g i n m u l t i p l e s c l e r o s i s . At a June 1982 meeting sponsored by the Kroc - 13 -Foundation i t was g e n e r a l l y agreed that the demyelination i s "...T c e l l mediated and seems to be caused p r i n c i p a l l y by the Ly 1+ c l a s s of e f f e c t o r c e l l s . Conventional immunological wisdom would have us b e l i e v e t h a t Ly 1+, l a - r e s t r i c t e d T c e l l s are the i n s t i g a t o r s of demyelinating damage, but there i s no evidence that Ia antigens are present on myelin or on the oligodendrocyte. Also morphological evidence would suggest t h a t c e l l u l a r damage to the myelin sheath i s a f u n c t i o n of monocytes/macrophages r a t h e r than T c e l l s . " (32). The T c e l l s might thus be f u n c t i o n i n g v i a lymphokine or p r o s t a g l a n d i n a c t i v a t i o n of the monocytes/macrophages. The disease may be due to a b n o r m a l i t i e s i n cell-mediated immunity (2,13,72). For inst a n c e , i t has been reported that lymphocytes from MS p a t i e n t s e x h i b i t low responsiveness to T c e l l mitogens (13,72) although hyporesponsive transformation r e a c t i v i t y to myxovirus antigens i n MS p a t i e n t s may be a n o n - s p e c i f i c e f f e c t of n e u r o l o g i c a l disease (73). L i s a k et a l . (74) reported that CSF lymphocytes from MS p a t i e n t s i n the a c t i v e stage of the disease were more r e a c t i v e to MBP than blood lymphocytes from the same subject and that t h i s d i f f e r e n c e could not be demonstrated i n MS p a t i e n t s i n s t a b l e c o n d i t i o n or i n other c o n t r o l s . S i m i l a r r e s u l t s have been reported by other groups. I t i s not known i f they represent increased r e a c t i v i t y of MBP-sensitive lymphocytes or a decrease i n suppressor c e l l f u n c t i o n s i n c e both have been reported i n MS (33). Of these two hypotheses the l a t t e r i s c u r r e n t l y r e c e i v i n g more a t t e n t i o n . Many s t u d i e s have demonstrated a decrease i n suppressor T c e l l numbers and/or f u n c t i o n i n MS (2,71,75,76,77). This decrease c o i n c i d e s with the acute phase of the disease; suppressor c e l l numbers/activity n o r m a l i z i n g or even becoming - 14 -abnormally high during subsequent recovery periods. In a few cases the abnormality has been reported to have occurred s h o r t l y before the onset of an attack . Abnormalities i n MS p a t i e n t s ' monocyte subpopulations have a l s o been reported ( 2 ) . Recently i t has been shown that the r e s i s t a n c e to EAE of an inbred s t r a i n of Lewis r a t s may be based on r e g u l a t i o n of the f u n c t i o n a l behavior of a n t i - s e l f , e f f e c t o r T lymphocytes (36). A l t e r a t i o n s such as those above have been detected using monoclonal a n t i b o d i e s and fluorescence a c t i v a t e d c e l l s o r t i n g as w e l l as other techniques such as r o s e t t i n g (2,78). I t i s a l s o of i n t e r e s t that a z a t h i o p r i n e and s t e r o i d treatment of p a t i e n t s (79) as w e l l as other f a c t o r s (78) may a l t e r apparent lymphocyte responsiveness and subpopulation r a t i o s . The presence of CSF/CNS o l i g o c l o n a l IgG and the extreme and v a r i e d c e l l u l a r i t y ( i . e . macrophages and plasma c e l l s as w e l l as lymphocytes) of the plaques i n MS suggests that antibody dependant c e l l u l a r c y t o t o x i c i t y (ADCC) may c e r t a i n l y be responsible f o r the i n i t i a l t i s s u e d e s t r u c t i o n i n MS. The f a c t that anti-MBP antibody does not cause demyelination on i t s own i n d i c a t e s that ADCC "may be superimposed on the inflammation r e s u l t i n g from T-cell-mediated immunity when whole myelin serves as the immunizing agent." ( 2 ) . Some i n v e s t i g a t o r s have reported increases i n a c t i v a t e d T c e l l s bearing Fc surface receptors i n MS p a t i e n t s (34,80). Other groups have reported decreases i n Fc-receptor bearing c e l l s of the Ty c l a s s i n acute a t t a c k s of MS (2,69). N a t u r a l k i l l e r c e l l s appear to be d e f i c i e n t i n a t h i r d or more of MS p a t i e n t s . They are e s p e c i a l l y low i n the CSF. This could be l i n k e d to a diminished a b i l i t y to produce i n t e r f e r o n f o l l o w i n g a n t i g e n i c ( i . e . v i r a l ) - 15 -s t i m u l a t i o n (2,34,81). A recent t r i a l of i n t e r f e r o n treatment showed promising r e s u l t s i n terms of decreased exacerbation ra t e and improved c l i n i a l c o n d i t i o n over a period of 1.5 years (82). I t i s a l s o i n t e r e s t i n g to note that even i n periods of c l i n i c a l remission s t i m u l a t e d b l a s t - l i k e lymphocytes may account f o r up to 20% of the white c e l l s i n the c i r c u l a t i o n of MS p a t i e n t s and an even higher p r o p o r t i o n of the CSF lymphocytes (2). A t a b l e of abnormalities of lymphoid c e l l s i n MS , i n c l u d i n g the few noted above, can be found i n reference ( 2 ) . Whether these a l t e r a t i o n s are r e l a t e d symptomatically or p a t h o g e n i c a l l y to MS i s not known. The case f o r a c i r c u l a t i n g t o x i n being r e s p o n s i b l e f o r MS has been overshadowed i n recent years by general s c i e n t i f i c i n t e r e s t and awareness i n biochemical, immunological and v i r a l e t i o l o g i c a l t h e o r i e s . Nevertheless the case f o r a p o s s i b l e t o x i n i s q u i t e sound (19). Of p a r t i c u l a r i n t e r e s t are reports that a heat s t a b l e f a c t o r can be i s o l a t e d from MS serum or plasma which i s capable of i n h i b i t i n g the phytohemagglutinin s t i m u l a t i o n of human T lymphocytes and i s c y t o t o x i c to human T lymphoblasts both i n vivo and i n v i t r o (83). The f a c t o r has been hypothesized as re s p o n s i b l e f o r both decreased T c e l l (subset) a c t i v i t y and number i n MS p a t i e n t s (83, see a l s o 84). With a l l of the above a l t e r a t i o n s i n lymphocyte subsets and hence average lymphocyte surface s t r u c t u r e i t i s perhaps not s u r p r i s i n g that a number of unusual MS lymphocyte surface p r o p e r t i e s have been reported (34). For in s t a n c e , Meyer-Rienecker has reported t h a t MS p a t i e n t s ' lymphocytes possess unusually low membrane surface p o t e n t i a l s as measured by c e l l e l e c t r o p h o r e s i s (34). Levy et a l . (85) and others (86-88) have - 16 -reported that lymphocytes from MS p a t i e n t s form stronger r o s e t t e s w i t h measles-infected e p i t h e l i a l c e l l s than do c o n t r o l s , as estimated by shear r e s i s t a n c e during p i p e t t i n g . This has been suggested as the b a s i s f o r an MS d i a g n o s t i c t e s t . Barna et a l . (89) reported t h a t i n some cases they had observed a degree of leukocyte adherence to noninfected c e l l s and that i n t h e i r experiments MS and c o n t r o l group data s e t s overlapped, although they were s i g n i f i c a n t l y (p ^ 0.050) d i f f e r e n t . Salmi and Frey (86) reported t h a t s t a t i s t i c a l l y s i g n i f i c a n t r e s u l t s were not obtained, t h a t MS blood c e l l types have abnormal surface p r o p e r t i e s , and that changes i n the surface of MS blood c e l l s , r a t h e r than r e a c t i v i t y with a s p e c i f i c v i r a l antigen, may account f o r t h e i r f i n d i n g s . A s i m i l a r c o n c l u s i o n was reached by Angers et a l . (88) who were able to e x t r a c t an antigen from pooled whole MS blood which was a c t i v e i n i n h i b i t i n g leukocyte adherence of white c e l l s from MS p a t i e n t s but not normals. I t remains to be seen i f other groups w i l l confirm these r e s u l t s which a l s o suggest the p o s s i b i l i t y of an abnormal plasma f a c t o r ( s ) present i n MS p a t i e n t s r e s p o n s i b l e f o r the above d i f f e r e n c e s (2,19,83,90). More complex d i a g n o s t i c procedures have a l s o been proposed. V i s u a l l y evoked p o t e n t i a l and r e l a t e d e l e c t r o p h y s i o l o g i c a l measurements (18,91,92), c r a n i a l computer tomography ((93,94), c e r e b r o s p i n a l f l u i d (CSF) immunoglobulin a n a l y s i s (70,95,96), CSF myelin b a s i c p r o t e i n radioimmunoassay (97) and other approaches (91) have been a p p l i e d but a l l are e i t h e r o p e r a t i o n a l l y complex or have been shown to possess s e r i o u s drawbacks (91-93). For i n s t a n c e , although increased CSF-IgM i s known to occur i n MS p a t i e n t s (95,98,99) and i s absent i n healthy c o n t r o l s , p o s i t i v e r e s u l t s can a l s o be produced by b a c t e r i a l or v i r a l i n f e c t i o n s , - 17 -abnormal blood - CSF exchange or as a consequence of n e u r o l o g i c a l d i s o r d e r s other than MS (95,99). Also the o l i g o c l o n a l IgG bands seen f o l l o w i n g e l e c t r o p h o r e s i s of up to 90% of MS p a t i e n t ' s CSF can r e s u l t from n o n s p e c i f i c p o l y c l o n a l a c t i v a t i o n (70). The number of f a l s e p o s i t i v e s due to c o n d i t i o n s other than MS such as n e u r o s y p h i l i s may be as high as 40% (18). There i s e l e c t r o p h y s i o l o g i c a l evidence of past o p t i c n e u r i t i s i n 90% of c l i n i c a l l y d e f i n i t e cases of MS (18,37) however t h i s and other such t e s t s are based on s u b s t a n t i a l demyelination having already occured, l i m i t i n g t h e i r e a r l y d i a g n o s t i c usefulness. The remaining d i a g n o s t i c t e s t s to be discussed are a l l a s s o c i a t e d with reported v a r i a t i o n s i n l i p i d metabolism i n i n d i v i d u a l s s u f f e r i n g from MS, as evidenced by a v a r i e t y of reported c h a r a c t e r i s t i c s or responses. These c h a r a c t e r i s t i c s can be accommodated by a l l of the major e t i o l o g i c a l t h e o r i e s of MS (100,101) so they do not provide d e f i n i t i v e i n f o r m a t i o n i n t h i s regard. 1.2.2 - V a r i a t i o n In Serum and Blood C e l l L i p i d Metabolism In  M u l t i p l e S c l e r o s i s L i p i d s comprise approximately 70% of the dry weight of myelin (21% c h o l e s t e r o l , 16% g l y c o l i p i d and 32% phospholipid) (17) and a s u b s t a n t i a l p o r t i o n of the damage i n MS plaques i n v o l v e s l i p i d . I t i s t h e r e f o r e n a t u r a l that l i p i d would be i m p l i c a t e d i n t h e o r i e s concerning the e t i o l o g y of MS. L i p i d involvement i n MS might be r e l a t e d to an MS s p e c i f i c ( g l y c o l i p i d ) antigen, or to abnormal myelin c o n s t r u c t i o n causing increased s u s c e p t i b i l i t y to unusual atrophy, v i r a l i n f e c t i o n or peroxide at t a c k . - 18 -Abnormal myelin formation could be due to abnormal serum l i p i d l e v e l s and/or to abnormal l i p i d metabolism. Increased awareness of the r o l e of l i p i d s i n the behaviour and f u n c t i o n of immune and other c e l l s suggests other mechanisms whereby serum and membrane l i p i d a l t e r a t i o n s could c o n t r i b u t e to the development of MS (100). Increased awareness of the a n t i g e n i c i t y of g l y c o l i p i d s (51,102-104) has prompted s p e c u l a t i o n of an MS-specific g l y c o l i p i d antigen (51). I t i s i n t e r e s t i n g to note that i n MS plaque and plaque border areas myelin cerebroside i s reduced q u i t e s i g n i f i c a n t l y (17) and decreased plasma l e v e l s of g l y c o l i p i d s ( p o s s i b l y r e l a t e d to a n t i - g l y c o l i p i d a n t i b o d i e s (51,105) have been reported i n MS (106). In the e a r l y 1950's, e p i d e m i o l o g i c a l data was u t i l i z e d by Swank and c o l l a b o r a t o r s to b r i n g out c o r r e l a t i o n s between the incidence of MS and consumption of d i e t s r i c h i n saturated animal and d a i r y f a t s (107,108), r e s u l t s l a t e r v e r i f i e d (101). A d i e t low i n sa t u r a t e d f a t s was t h e r e f o r e proposed by Swank as a treatment f o r MS, a regimen which was subsequently reported to be ass o c i a t e d with l e s s d i s a b i l i t y and a lower death r a t e (109). These claims were considerably weakened, however, by the l a c k of an appropriate c o n t r o l group (100). The r a t i o n a l e behind t h i s approach was the suggestion that saturated f a t i n g e s t i o n leads to increased s u s c e p t i b i l i t y to thrombosis (84,101,110-114) as evidenced by enhanced p l a t e l e t adhesiveness (110,115,116). In 1956 S i n c l a i r extended Swank's theory to one of MS r e s u l t i n g from a d e f i c i e n c y i n polyunsaturated f a t t y a c i d s (PUFA) brought about by a d i e t r i c h i n animal f a t (117). The b r a i n i s a major s i t e of l i p i d metabolism e s p e c i a l l y p o s t - n a t a l l y when a l o t of b r a i n growth and myelin s y n t h e s i s occurs (100, 118-122). - 19 -A l t e r a t i o n s i n l i p i d i ntake i n animal infancy have been shown to produce permanent myelin l i p i d compositional a l t e r a t i o n s (100,118,119,121). In 1961 G e r s t e l et a l . reported that the l e v e l of s a t u r a t e d f a t t y a c i d s was increased r e l a t i v e to the l e v e l of unsaturated f a t t y a c i d s i n the b r a i n s of m u l t i p l e s c l e r o s i s p a t i e n t s (123). S i m i l a r r e s u l t s were found i n 1963 by Baker et a l . who noted that the f a t t y a c i d composition of p h o s p h a t i d y l c h o l i n e s from MS p a t i e n t s ' b r a i n s e x h i b i t e d s i m i l a r a b n o r m a l i t i e s even i n non-plaque areas (124). Though s t i l l somewhat c o n t r o v e r s i a l (15,100) the above a l t e r a t i o n has been v e r i f i e d by a number of independent i n v e s t i g a t o r s (125) and a l s o appears to occur i n the free f a t t y a c i d (120) and c h o l e s t e r o l e s t e r i f i e d f a t t y a c i d l e v e l s i n the b r a i n (126) . In 1974 Agranoff and Goldberg (118) suggested that MS might be caused by the use of PUFA-poor cow's milk i n i n f a n t nursing programs and such use might be r e f l e c t e d i n the geographical d i s t r i b u t i o n of the disease. Baker, Thompson and Z i l k h a reported i n 1964 t h a t the l i n o l e i c a c i d ( l i n o l e i c a c i d and l i n o l e a t e are used interchangably i n the f o l l o w i n g s e c t i o n ) content of serum l i p i d s was decreased i n m u l t i p l e s c l e r o s i s (127) . Serum l i p i d a l t e r a t i o n , s p e c i f i c a l l y decreased free and e s t e r i f i e d l i n o l e i c a c i d has s i n c e been reported by a number of authors (2,115,125,127-138) though some researchers have not been able to confirm t h i s abnormality (129,131,132,139,140) and/or have questioned i t s s i g n i f i c a n c e (125,130,138). I t does appear to be r e l a t e d to myelin damage (2). I t does not n e c e s s a r i l y appear to be due to a b n o r m a l i t i e s of d i e t and/or malabsorbtion (100,134,141, although see a l s o 142-144). M i c k e l has suggested t h a t the f a l l i n serum l i n o l e a t e i s due to abnormally increased - 20 -p e r o x i d a t i o n , caused p o s s i b l y by increased e n t e r i c absorption of e n t e r o b a c t e r i a l peroxides, and that MS i s the r e s u l t of t h i s increased systemic p e r o x i d a t i o n which might r e s u l t i n increased CNS t i s s u e damage or increased p l a t e l e t a g g r e g a b i l i t y (84,145). I f t h i s hypothesis i s c o r r e c t the value of d i e t s r i c h i n PUFA and poor i n s a t u r a t e d f a t t y a c i d s might l i e i n t h e i r increased concentrations of n a t u r a l a n t i o x i d a n t s (144). In 1966 Thompson reported that not only were f r e e and e s t e r i f i e d l i n o l e a t e l e v e l s reduced i n the serum of MS p a t i e n t s but t h a t the serum l e v e l of l i n o l e a t e i n v e r s e l y c o r r e l a t e d with the degree of p a t i e n t d i s a b l i l i t y (146). This report was subsequently v e r i f i e d (128). Thompson suggested that m u l t i p l e s c l e r o s i s may be a biochemically-based d i s o r d e r , p o s s i b l y accentuated by d i e t and which predisposed the subject to MS by an unknown mechanism (146). Further suggestive evidence l i n k i n g MS and l i p i d metabolism was provided i n 1967 by Clausen and M o l l e r (29) who showed that animals fed a d i e t d e f i c i e n t i n e s s e n t i a l f a t t y acids ( i n c l u d i n g l i n o l e a t e , arachidonate and hence pr o s t a g l a n d i n precursor) were more s u s c e p t i b l e to experimental a l l e r g i c encephalomyelitis than those on normal d i e t s and that a d d i t i o n of d i e t a r y l i n o l e a t e afforded some p r o t e c t i o n against EAE, r e s u l t s s i n c e confirmed by others (30,31). An extension of t h i s type of work to human n e u r o l o g i c a l d i s o r d e r s was reported by M i l l a r and colleagues (116) who published the r e s u l t s of a two year double b l i n d study on the e f f e c t s of supplementing the d i e t s of MS p a t i e n t s with l i n o l e i c and o l e i c a c i d s . The subpopulation r e c e i v i n g l i n o l e a t e supplementation had l e s s frequent and l e s s severe a t t a c k s but c l e a r evidence that treatment decreased the c l i n i c a l d e t e r i o r a t i o n rate was not found. Negative t h e r a p e u t i c r e s u l t s - 21 -have been reported by other groups (147-150), i n s p i t e of the f a c t that the l i p i d a l t e r a t i o n s seen i n MS resemble those seen i n e s s e n t i a l f a t t y a c i d ( i . e . l i n o l e a t e ) d e f i c i e n c y (2,116,125,126,150,151) and l i n o l e i c a c i d d i e t supplementation i n the form of sunflower seed o i l or N a u d i c e l l e capsules has been shown to increase serum and blood c e l l membrane l i n o l e a t e l e v e l s (104,133). I t i s re l e v a n t to note that the patter n of l i p i d a l t e r a t i o n seen i n m u l t i p l e s c l e r o s i s , (decreased l i n o l e a t e with decreased arachidonate and s l i g h t l y increased o l e a t e , palmitate and p a l m i t o l e a t e ) , a l s o occurs i n many other neuropathies (125,131,132,152) and i n f a c t may be a general phenomenon as s o c i a t e d with acute i l l n e s s (125) and/or p a t i e n t drug therapy (153). Numerous s t u d i e s concerning the i n v i t r o and i n vivo i n c o r p o r a t i o n of f a t t y a c i d s and phospholipids i n t o erythrocytes have been reported (100, 104,115,133, 154-156). They i n d i c a t e that a l t e r a t i o n s i n serum l i n o l e a t e and other f a t t y a c i d s a l s o a f f e c t blood c e l l membranes (115,154,156). Lymphocytes and other blood c e l l s such as monocytes are probably s i m i l a r l y a f f e c t e d (133,157) as are t i s s u e c e l l s such as o l i g o d e n d r o g l i a and t h e i r myelin membranes. C e r t a i n l y i n t i s s u e c u l t u r e systems i t has been shown that the membrane l i p i d composition i s responsive to changes i n free f a t t y a c i d content of the growth medium (17) sin c e i t i s known that under these co n d i t i o n s membrane f a t t y acids are obtained p r i m a r i l y from t h i s source (158). Membrane l i p i d v a r i a t i o n s can i n turn r e s u l t i n a l t e r e d c e l l morphology, growth c o n t r o l , membrane enzyme a c t i v i t y , s u b s t r a t e adhesion, d i f f e r e n t i a t i o n and l e c t i n a g g l u t i n a t i o n (157,158,159,160,161). - 22 -Thompson and co-workers have al s o published r e s u l t s i n d i c a t i n g that the f a t t y a c i d changes seen i n the serum of MS p a t i e n t s were a l s o found i n blood c e l l membranes. In 1970 Gul et a l . (115) reported s i g n i f i c a n t l y (p ^ 0.001) decreased t o t a l serum l i n o l e a t e as w e l l as e r y t h r o c y t e and p l a t e l e t p h o s p holipid l i n o l e a t e i n MS. In both c e l l types p h o s p h o l i p i d p a l m i t a t e , s t e a r a t e , oleate and arachidonate were normal. Further s t u d i e s i n d i c a t e d normal e r y t h r o c y t e phospholipid ( l e c i t h i n , p - s e r i n e , p-ethanolamine and sphingomyelin) composition except p o s s i b l y f o r a 50% increase i n l y s o l e c i t h i n . I t was a l s o found that an inverse c o r r e l a t i o n between e r y t h r o c y t e l i n o l e a t e and arachidonate which e x i s t e d i n normal erythrocytes (162,170) d i d not e x i s t i n the MS c e l l s suggesting a p o s s i b l e defect i n unsaturated l i p i d metabolism (162). Further s t u d i e s a l s o detected a 30% increase (p ^ 0.001) i n er y t h r o c y t e sphingomyelin e s t e r i f i e d l i g n o c e r a t e , (a saturated 24 carbon f a t t y a c i d ) (163). This l a s t d i f f e r e n c e was apparently e l i m i n a t e d by d i e t a r y sunflower seed o i l supplementation (163) however the defect i n the membrane l i n o l e a t e and arachidonate c o r r e l a t i o n was not (162). A l t e r a t i o n s i n erythrocyte l i p i d composition are o f t e n r e l a t e d to a l t e r e d morphology (104) and osmotic f r a g i l i t y (104) as w e l l as membrane enzyme a c t i v i t y (80,81). In m u l t i p l e s c l e r o s i s increased e r y t h r o c y t e s i z e (volume) (34,115,164,165) and osmotic f r a g i l i t y (166-169) have been reported. S i m i l a r a l t e r a t i o n s have been noted i n a number of diseases. In such instances the c o r r e l a t i o n between decreased l i n o l e a t e and increased f r a g i l i t y may have to do with the average age of the c e l l s (104,170). - 23 -Increased p l a t e l e t a g g r e g a b i l i t y , s e n s i t i v i t y to ADP and g l a s s adhesiveness have been reported by many researchers i n MS (115,171) and even d i r e c t l y i m p l i c a t e d i n the disease e t i o l o g y (84). A s i g n i f i c a n t c o r r e l a t i o n has been shown between serum c h o l e s t e r o l l i n o l e a t e and p l a t e l e t adhesiveness (172) although the authors b e l i e v e d t h a t both observations were due to other a l t e r a t i o n s i n the plasma. Boulton et a l . (171) b e l i e v e d t h a t increased plasma/platelet membrane l y s o l e c i t h i n i n MS was resp o n s i b l e f o r increased p l a t e l e t ADP s e n s i t i v i t y and adhesiveness to g l a s s , as w e l l as t h e i r observed p l a t e l e t e l e c t r o p h o r e t i c m o b i l i t y a b n o r m a l i t i e s . Increased plasma (173) and red c e l l (162) but not p l a t e l e t membrane l y s o l e c i t h i n (104) was subsequently reported i n MS. A l t e r e d p l a t e l e t responsiveness might a l s o be due to the abnormal plasma unsaturated l i p i d l e v e l s mentioned above (115,156, 174-178) and/or to p l a t e l e t p r o s t a g l a n d i n synthesis abnormalities (114,157,179-182). Diet induced increases i n serum PUFA have been shown to a l t e r p l a t e l e t membrane PUFA acc o r d i n g l y and to r e s u l t i n decreased a g g r e g a b i l i t y (107,116,156,183). Many r e p o r t s suggest that a s p e c i f i c plasma f a c t o r may be as s o c i a t e d with m u l t i p l e s c l e r o s i s . Boulton et a l . (171) t h e o r i z e d that the increased l y s o l e c i t h i n they noted i n the plasma and p l a t e l e t membranes of MS p a t i e n t s was due to increases i n plasma l e c i t h i n - c h o l e s t e r o l a c y l t a n s f e r a s e (LCAT, EC 2.3.1.43) a c t i v i t y (184,185). Homozygous LCAT d e f i c i e n t p a t i e n t s e x h i b i t low plasma c h o l e s t e r o l - f a t t y a c i d e s t e r and l y s o l e c i t h i n l e v e l s and increased erythrocyte c h o l e s t e r o l , l e c i t h i n and l i n o l e a t e . Their e r y t h r o c y t e s a l s o e x h i b i t increased r e s i s t a n c e to hypotonic l y s i s (186,104). Heterozygous LCAT d e f i c i e n t p a t i e n t s possess - 24 -approximately normal plasma LCAT a c t i v i t y and plasma l i p i d l e v e l s but s t i l l e x h i b i t a l t e r e d e r y t h rocyte membrane phospholipid and osmotic f r a g i l i t y (185,186). Reports (100, and see above) of decreased plasma and blood c e l l membrane l i n o l e a t e and increased plasma and blood c e l l l y s o l e c i t h i n , (both l i p i d s which are known to be l y t i c and demyelinating, 104,187), i n MS prompted an e a r l y study which found that plasma LCAT a c t i v i t y was s i g n i f i c a n t l y increased i n t h i s disease and th a t t h i s increase appeared to be r e l a t e d to the phase of the disease (188). In another study researchers concluded that MS s u b j e c t s ' plasma LCAT a c t i v i t y was normal when incubated with normal s u b j e c t s ' l i p o p r o t e i n s u b s t r a t e and that normal s u b j e c t s ' plasma LCAT a c t i v i t y was increased i n the presence of MS su b j e c t s l i p o p r o t e i n substrate (189). In s p i t e of t h i s i n t e r e s t i n g r e s u l t Waksmann r e c e n t l y noted (2) that l i p o p r o t e i n a b n o r m a l i t i e s , which can a f f e c t c e l l membranes and a number of r e l a t e d phenomena (190), have not been looked f o r i n MS. Decreased serum l i n o l e a t e and LCAT a c t i v i t y have been found i n p a t i e n t s with h e r e d i t a r y motor and sensory neuropathies (152). During the past year we were able to detect an abnormal (9%) increase i n MS p a t i e n t s ' plasma LCAT a c t i v i t y (191). L i p o p r o t e i n l i p a s e has a l s o been implemented i n t h e o r i e s of MS e t i o l o g y (192). Reports that serum a 2-macroglobulin, which p o s s i b l y has a suppressor r o l e i n immunological response, i s decreased i n MS (144,193) appear to be questionable as i t s protease binding a c t i v i t y i s normal (194). The t r i p e p t i d e g l u t a t h i o n e ( L - Y - g l u t a m y l - L - c y s t e i n y l - g l y c i n e ) (GSH) fun c t i o n s i n an impressive number and v a r i e t y of c e l l u l a r phenomena i n c l u d i n g p r o s t a g l a n d i n , l e u k o t r i e n e , estrogen and melanin biochemistry, as a c a r r i e r i n the -glutamyl amino a c i d c y c l e , as a coenzyme, as an - 25 -i n t r a c e l l u l a r and membrane reductant to maintain p r o t e i n t h i o l groups i n a reduced a c t i v e non-crosslinked s t a t e , and to p r o t e c t against free r a d i c a l s and peroxides (195). The l a t t e r f u n c t i o n i n v o l v e s selenium dependent and independent GSH peroxidase enzyme a c t i v i t i e s and the conversion of GSH to the t h i o l b r i d g e - l i n k e d d i g l u t a t h i o n e (GSSG) which i n turn i s reduced by g l u t a t h i o n e reductase (195,196). As noted e a r l i e r , a major f u n c t i o n a l r o l e of g l u t a t h i o n e peroxidase i s thought to be the p r o t e c t i o n of erythrocyte membranes and hemoglobin as w e l l as other t i s s u e s from organic peroxide and f r e e r a d i c a l damage, such as caused by exposure to unsaturated free f a t t y acids (100,195,196). In t h i s r o l e i t f u n c t i o n s c l o s e l y with vitamin E (197,198) which i s a l s o i n t i m i t e l y l i n k e d to prostaglandin metabolism (198). White matter samples from brains of MS p a t i e n t s have been reported to possess s i g n i f i c a n t l y greater concentrations of calcium, i r o n , manganese and z i n c , and s i g n i f i c a n t l y l e s s phosphorous (199). Decreased plasma magnesium (200) and z i n c (199,201, though see 202) as w e l l as copper (199) have al s o been reported i n MS. Normal plasma selenium l e v e l s but decreased whole blood selenium l e v e l s were reported by Wikstrom et a l . (197) i n F i n n i s h MS p a t i e n t s . Zinc and copper d e f i c i e n c y can lead to abnormal myelin formation (199). In 1977 decreased erythrocyte g l u t a t h i o n e p e r i o x i d a s e a c t i v i t y was reported i n MS and l i n k e d to reports of decreased selenium (203). This report has since been confirmed by other s t u d i e s (204-206) however a report of a s i m i l a r decrease i n enzyme a c t i v i t y i n MS lymphocytes and granulocytes (205) was r e c e n t l y not v e r i f i e d f o r lymphocyte or p l a t e l e t selenium dependent or independent g l u t a t h i o n e peroxidase a c t i v i t y (206). - 26 -The cause of decreased a c t i v i t y i n erythrocytes and p o s s i b l y i n other (blood) c e l l types remains u n c e r t a i n . P o s s i b l y connected serum amino a c i d a b n o r m a l i t i e s have a l s o been reported i n MS, serum glutamate being elevated p r i o r to and during the onset of c l i n i c a l r e l a p s e s (202). Also decreased a b i l i t y of blood to reduce methylene blue dye (200), and increased s e n s i t i v i t y to i o n i z i n g r a d i a t i o n (207) and perhaps p e r o x i d a t i o n (100) have been reported i n the disease. These l a s t observations and those mentioned above i n regard to plasma and blood c e l l membrane l i p i d a l t e r a t i o n i n MS may a l l be l i n k e d to the known r e l a t i o n s h i p between gl u t a t h i o n e peroxidase, vitamin E and pr o s t a g l a n d i n metabolism (196,197). In keeping with the above both plasma exchange and plasmapheresis therapy have been reported to be b e n e f i c i a l i n t r e a t i n g MS, e s p e c i a l l y i n p a t i e n t s undergoing acute exacerbations (208-210). The reason(s) f o r such b e n e f i t are l a r g e l y unknown (211) and have been hypothesized as immunosuppressive. As with p l a t e l e t s and erythrocytes (115), MS p a t i e n t s ' p e r i p h e r a l blood lymphocytes have been reported to possess s i g n i f i c a n t l y low l e v e l s of membrane l i n o l e a t e (133). Lymphocyte a c t i v a t i o n by antigen r e s u l t s i n increased membrane PUFA l e v e l s and f l u i d i t y (34,100,157). In turn t h i s a l t e r a t i o n might r e s u l t i n a l t e r e d surface HLA antigen expression and r e c o g n i t i o n (157,212). Decreases i n membrane l i n o l e a t e are known to a f f e c t lymphocyte f u n c t i o n e s p e c i a l l y i n regard to mitogenic s t i m u l a t i o n (100,157,213). Lymphocyte f u n c t i o n appears to be a f f e c t e d by membrane c h o l e s t e r o l (214) and phospholipid (215) as w e l l as PUFA l e v e l s . These i n turn are a f f e c t e d by l i p o p r o t e i n s (2,190) and enzymes such as lymphocyte c e l l surface associated LCAT (33,157). As noted e a r l i e r many lymphocyte - 27 -functions appear to be reduced or otherwise abnormal i n MS. For instance p e r i p h e r a l blood lymphocytes from p a t i e n t s with MS appear to be more adherent to measles i n f e c t e d human e p i t h e l i a l c e l l s than normal lymphocytes. They a l s o tend to form E-rosettes l e s s a v i d l y w i t h sheep red c e l l s . Dore-Duffy and Z u r i e r have reported that p r o s t a g l a n d i n PGE^ reduces a v i d E-rosette formation i n normal lymphocytes and a l s o i n c r e a s e s t h e i r adherence to measles-infected c e l l s (17,216,217). Furthermore i n v i t r o a d d i t i o n of a s p i r i n or indomethacin, (both i n h i b i t o r s of pros t a g l a n d i n formation), to p e r i p h e r a l blood c e l l s normalized lymphocyte adherence to measles i n f e c t e d c e l l s . A s p i r i n had s i m i l a r e f f e c t s i n v i v o (17,216,217). Furthermore MS p a t i e n t s ' leukocytes e x h i b i t decreased migration i n h i b i t i o n to measles v i r u s antigen. E type p r o s t a g l a n d i n s from monocytes supposedly i n h i b i t the formation of mi g r a t i o n i n h i b i t i o n f a c t o r (MIF) lymphokine (87,217,218). These r e s u l t s may be due to the f a c t that MS p a t i e n t s have a quicker turnover r a t e of lymphocytes and therefore on the average have more immature p r o s t a g l a n d i n s e n s i t i v e lymphocytes i n t h e i r p e r i p h e r a l c i r c u l a t i o n (2,219). They may a l s o be due to abnormal p r o s t a g l a n d i n formation i n MS monocytes (87). Pr o s t a g l a n d i n s are known to have a number of immunoregulatory p r o p e r t i e s i n c l u d i n g i n h i b i t i o n of lymphokine release from a c t i v a t e d T lymphocytes and a c t i v a t i o n of suppressor c e l l s (17,220). Kirby reported t h a t lymphocytes from MS p a t i e n t s d i s p l a y e d d e f e c t i v e PGE-mediated i n h i b i t i o n of leukocyte MIF r e l e a s e (221). Other researchers were not able to v e r i f y t h i s o bservation, but i t does appear that the immuno-regulation a b n o r m a l i t i e s known to occur i n MS could have a basis i n abnormal p r o s t a g l a n d i n metabolism, which i n turn could be due to abnormal serum and/or blood c e l l - 28 -membrane PUFA l e v e l s (222). In a 1979 review Horrobin o u t l i n e d the r a t i o n a l e behind the theory that the fundamental ( l i p i d ) defect i n MS was one of p r o s t a g l a n d i n metabolism and that t h i s defect could be t r e a t e d w i t h evening primrose o i l which i s r i c h i n l i n o l e i c and T f - l i n o l e n i c a c i d (GLA) and c o l c h i c i n e (223). He noted t h a t i n the body l i n o l e i c a c i d (C18:2) i s converted i n t o GLA (C18:3) which becomes dihomo-GLA (C20:3), a precursor of PG^ ^ pros t a g l a n d i n s , and arac h i d o n i c a c i d (C20:4) which i t s e l f i s a precursor of the PG 2 s e r i e s of prostaglandins i n c l u d i n g thromboxane A 2 (154,175,180,212,223,224). The e s s e n t i a l f a t t y a c i d nature of l i n o l e i c a c i a l i e s i n i t s a b i l i t y to be converted to GLA (223,225). Zinc i s important i n the above r e a c t i o n s and the symptoms of z i n c d e f i c i e n c y , which has been reported i n MS, are the same as e s s e n t i a l f a t t y a c i d , dihomo-GLA or PGE^ d e f i c i e n c y (223). Prostaglandins of the E s e r i e s appear to have i n h i b i t o r y / r e g u l a t o r y e f f e c t s on the immune system i n c l u d i n g T suppressor c e l l s (157,181,212,224). These e f f e c t s are v a r i e d and may depend on the r e l a t i v e concentrations o f the various prostaglandins present. Horrobin b e l i e v e s that decreased plasma l i n o l e a t e i n MS comes from increased production of PGE 2 and decreased plasma thromboxane l e v e l s . The r e s u l t i n g reduction i n the PGE^/PGE2 plasma r a t i o could lead to abnormal immune system f u n c t i o n i n g . Evening primrose o i l was advocated as i n c r e a s i n g serum l i n o l e i c a c i d l e v e l s while p r o v i d i n g GLA f o r production of PGE^. Chol c h i c i n e apparently reduces the production of PGE 2 enhancing thromboxane s y n t h e s i s . He saw the f a i l u r e of t r i a l s of l i n o l e a t e d i e t supplementation to show a c l e a r t h e r a p e u t i c e f f e c t as p o s s i b l y due to t h e i r i n a b i l i t y to c o r r e c t the e r r o r - 29 -i n p r o s t a g l a n d i n metabolism. He al s o noted that two negative t r i a l s of evening primrose o i l d i e t supplementation may have been compromised by the low dose of the o i l which was administered and/or the f a c t that i t was packaged i n N a u d i c e l l e capsules colored with dyes known to i n h i b i t p r o s t a g l a n d i n s y n t h e s i s (223,226). In l i n e with t h i s l a s t observation i t has been shown that the p r o t e c t i v e e f f e c t of polyunsaturated f a t t y a c i d s i n suppressing EAE i n r a t s (227) i s abolished by treatment with indomethacin which i n h i b i t s prostaglandin s y n t h e s i s (223,228,229). The above suggests that f u r t h e r d i e t a r y t r i a l s of PUFA and e s p e c i a l l y GLA should probably be undertaken i n MS (18). Dihomo-GLA i s now a v a i l a b l e f o r such s t u d i e s and may prove to be of more obvious t h e r a p e u t i c b e n e f i t to MS p a t i e n t s (154). At t h i s time the s i g n i f i c a n c e of f a t t y a c i d s i n m u l t i p l e s c l e r o s i s appears to be undecided. The p o s s i b l e r e l a t i o n s h i p s between low serum and membrane l e v e l s of l i n o l e a t e and other l i p i d a b n o r m a l i t i t e s and MS e t i o l o g y have been mentioned b r i e f l y above and have been discussed i n d e t a i l by a number of other authors (2,17,100,101,105,125,126,128,129, 131,133). Waksman has noted (2) that the a b n o r m a l i t i e s discussed above can c e r t a i n l y account f o r some of the unusual lymphocytic, p l a t e l e t and erythrocyte p r o p e r t i e s a s s o c i a t e d with MS. The e a r l y f a t t y a c i d composition s t u d i e s mentioned above, while i n c o n c l u s i v e , prompted a 1973 study by Mert i n , Shenton and F i e l d on the e f f e c t s of o l e a t e , l i n o l e a t e and arachidonate on lymphocyte lymphokine generation measured v i a the macrophage e l e c t r o p h o r e t i c m o b i l i t y (MEM) t e s t (230-232). In t h i s t e s t lymphokine production by lymphocytes stimulated by l e c t i n or an antigen such as MBP, t h y r o g l o b u l i n or a p u r i f i e d p r o t e i n - 30 -d e r i v a t i v e of Mycobacterium t u b e r c u l o s i s (PPD), i s measured v i a the i n h i b i t o r y e f f e c t such production has on the e l e c t r o p h o r e t i c m o b i l i t y of guinea p i g p e r i t o n e a l macrophages exposed to con d i t i o n e d medium from the lymphocyte/antigen i n c u b a t i o n . Less reduction occurs i n the absence of antigen. Normal, s e n s i t i z e d lymphocytes produced a 10-20% MEM decrease when exposed to PPD (230-232). A d d i t i o n of 80 ug/ml of l i n o l e a t e or arachidonate (approximately double the normal serum l e v e l of l i n o l e a t e ) i n h i b i t e d lymphokine production from normal and MS lymphocytes by 60% and 80% r e s p e c t i v e l y , as estimated by MEM. The r e s u l t s depended on the order i n which the f a t t y a c i d s and antigen were presented to the lymphocytes, a response only o c c u r r i n g i f the c e l l s were p r e t r e a t e d with f a t t y a c i d before s t i m u l a t i o n . A d d i t i o n of 80 ug/ml of o l e a t e (approximately the normal serum l e v e l ) had a n e g l i g i b l e e f f e c t . Support f o r some of the above conclusions was subsequently reported (233) based on lymphocyte transformation experiments. F i e l d , Shenton and Joyce (234) subsequently suggested t h a t the degree of i n h i b i t i o n of MEM slowing was s u f f i c i e n t l y greater with lymphocytes from MS p a t i e n t s compared to normals or p a t i e n t s w i t h other n e u r o l o g i c a l d i s o r d e r s (ONDs) that the MEM-linoleic a c i d depression (MEM-LAD) t e s t could be used as a s p e c i f i c l a b o r a t o r y t e s t f o r MS d i a g n o s i s . Very r a p i d l y , however, a d i s c l a i m e r was published by M e r t i n , Hughes, Caspary et a l (235), colleagues of F i e l d ' s i n Newcastle, based on the r e s u l t s of three f u r t h e r double b l i n d t r i a l s of the MEM-LAD t e s t . This paper p o i n t s out the d i f f i c u l t y of making o b j e c t i v e e l e c t r o p h o r e t i c m o b i l i t y measurements on macrophages and s t a t e s , " I r r e s p e c t i v e o f the t e c h n i c a l hazards of the method, the extremely small standard d e v i a t i o n s and narrow - 31 -ranges reported by F i e l d et a l f o r each of t h e i r c l i n i c a l c a t e g o r i e s i m p l i e s an unexpectedly high l e v e l of b i o l o g i c a l homogeneity w i t h i n c a t e g o r i e s . I t t h e r e f o r e seems reasonable to express s k e p t i c i s m , e s p e c i a l l y since some d u p l i c a t e measurements made under b l i n d c o n d i t i o n s on specimens from the same p a t i e n t s disagreed as widely as the o v e r a l l range of the reported d i a g n o s t i c s c a l e . " . The v a l i d i t y of the MEM-LAD i s s t i l l i n dispute (236). F i e l d (237) and a r e l a t e d group i n East Germany (238) s t i l l c l a i m i t can d i s t i n g u i s h among not only normals, ONDs and MS p a t i e n t s but r e l a t i v e s of MS s u f f e r e r s , i n c l u d i n g c h i l d r e n of MS p a t i e n t s i n whom they c l a i m to be able to detect the disease (239). Most other workers i n the f i e l d remain to be convinced, however, and the t e s t has not been widely accepted. In 1976 another, s i m p l i f i e d t e s t f o r MS was published by F i e l d and c o l l a b o r a t o r s based on the e l e c t r o p h o r e t i c behaviour of er y t h r o c y t e s (236). They found t h a t i n the presence of 80 ug/ml of l i n o l e i c or arac h i d o n i c , but not o l e i c , a c i d the e l e c t r o p h o r e t i c m o b i l i t i e s of red c e l l s from three MS p a t i e n t s were decreased approximately 7% by the unsaturated f a t t y a c i d s (UFA) while m o b i l i t i e s of e r y t h r o c y t e s from normals or ONDs were increased approximately 4%. This erythrocyte-UFA t e s t f o r m u l t i p l e s c l e r o s i s (239) has a l s o been claimed by F i e l d and co-workers to be s u c c e s s f u l at d i f f e r e n t i a t i n g among MS, OND and normal p a t i e n t s and at d e t e c t i n g MS p o s i t i v e readings i n the o f f s p r i n g and near r e l a t i v e s of MS s u f f e r e r s (239,240,241). The t e s t procedure has a l s o been used to f o l l o w the response of MS p a t i e n t s to d i e t supplementation with - l i n o l e n a t e and l i n o l e i c a c i d i n the form of N a u d i c e l l e capsules. Test r e s u l t s are reported to approach normal values a f t e r 4-6 months of treatment (239,240,242). - 32 -While four groups have been unable to confirm F i e l d ' s r e s u l t s (243-246), r e c e n t l y two l a b o r a t o r i e s have published r e p o r t s supporting the a p p l i c a b i l i t y of the erythrocyte m o b i l i t y t e s t to MS diagnosis (247,248 and 90,249-251). The most experienced of these groups, Seaman et a l . found a decrease i n red c e l l m o b i l i t y i n the presence of free l i n o l e i c a c i d of 7.0 _+ 2.5% f o r 65 MS p a t i e n t s compared to a 0.8 +_ 1.2% decrease i n 27 normals (p < 0.001) and a 1.3 + 0.8% decrease i n 5 ONDs (249). They als o pointed out that because of the small d i f f e r e n c e s observed that an experienced operator must make the measurements, a c r i t e r i o n amply f u l f i l l e d by members of t h i s group (250-253). L i k e F i e l d et a l . they found that glutaraldehyde treatment of washed MS c e l l s f i x e d the abnormality i n the MS t e s t i n d e f i n i t e l y (249,251,252). Rather than d e f i b r i n a t i n g blood samples with glass beads (239) they were able to achieve equal (251) and i n some cases b e t t e r (90) r e s u l t s using t r i s o d i u m c i t r a t e as an a n t i c o a g u l a n t . Blood samples could be s t o r e d up to 36 hours at 4° C (251). The optimal l i n o l e i c a c i d t e s t c o n c e n t r a t i o n f o r f r e s h c e l l s was found to be 150juM ( f i n a l ) added i n 28 mM ethanol ( f i n a l ) to a tube c o n t a i n i n g 1.5 x 10 7 c e l l s / m l i n B i o - C u l t medium 199 from Gibco (251). Medium 199 appeared to slow down the hemolytic a c t i o n of the f r e e f a t t y a c i d (251,252). No obvious c o r r e l a t i o n s were found between the the t e s t r e s u l t s and the s t a t u s , duration or current a c t i v i t y of the disease nor the d i e t of the p a t i e n t as regards intake of s a t u r a t e d f a t t y a c i d s (251). Seaman et a l . (90,250) have reported that the MS e r y t h r o c y t e surface abnormalities detected v i a the LAD e l e c t r o p h o r e t i c m o b i l i t y t e s t were acquired from the plasma and that cross i n c u b a t i o n of MS and normal - 3 3 -erythrocytes i n f r e s h compatible c i t r a t e d plasma, but not d e f i b r i n a t e d sera ( 9 0 , 2 5 4 ) f o r 1 . 5 hours r e s u l t e d i n the two c e l l types e x h i b i t i n g opposite m o b i l i t y c h a r a c t e r i s t i c s . This r e s u l t was i n t e r p t e t e d to support the hypothesis that e r y t h r o c y t e s from MS p a t i e n t s do not possess an i n t r i n s i c membrane defect but rather acquire membrane d i f f e r e n c e s r e v e r s i b l y from the plasma. S i m i l a r r e s u l t s have a l s o been found f o r p l a t e l e t s ( 1 7 1 ) where the requirement f o r f r e s h blood was a l s o noted. Seaman's group found v a r i a b i l i t y i n the absolute m o b i l i t y values of glutaraldehyde f i x e d c e l l s , p o s s i b l y due to v a r i a t i o n s i n f i x a t i v e p u r i t y ( 2 5 5 ) and are now p r i m a r i l y working with f r e s h c e l l samples. Their e a r l y f i x e d c e l l s t u d i e s were important however as regards understanding the basis of the PUFA m o b i l i t y t e s t f o r MS. These s t u d i e s , which were based on a very small number of MS samples ( 2 5 2 ) , suggested that the e l e c t r o p h o r e t i c m o b i l i t y of f i x e d erythrocytes i n bicarbonate buffered s a l i n e increased w i t h l i n o l e a t e concentration up to approximately 2 0 0 Ajg/ml ( 7 0 0 AJM) and that i t s t a r t e d to l e v e l o f f at 3 5 0 yuM. This increase appeared to be due to the adsorption of l i n o l e i c f a t t y a c i d d r o p l e t s ranging i n diameter from 0 . 5 to 5 . 0 (um) onto the the c e l l s u r f a c e . In the s a l i n e s o l u t i o n these d r o p l e t s had a m o b i l i t y of approximately 4 . 0 Oum/sec/volts/cm) or roughly four times the normal e r y t h r o c y t e m o b i l i t y of 1 . 1 ( 2 5 3 ) . The d r o p l e t s p r e f e r e n t i a l l y adsorbed onto normal (N) compared to MS eryt h r o c y t e s r e s u l t i n g i n the d i f f e r e n t i a l MS-N in c r e a s e i n m o b i l i t y which was found to be optimal at pH 7 . 2 and a c e l l c o n c e n t r a t i o n of 6 x 1 0 6 c e l l s / m l and a f a t t y a c i d concentration of 5 0 0 /JM ( f i n a l ) added i n 2 8 mM ethanol ( f i n a l ) though even 1 7 0 /uM l i n o l e i c a c i d r e s u l t e d i n a l a r g e MS-N d i f f e r e n c e . F i e l d had p r e v i o u s l y s t a t e d that the speed of - 34 -the l i n o l e i c a c i d e f f e c t on c e l l m o b i l i t y suggested i t was a surface e f f e c t (239). The c o n t r a d i c t i o n i n the behaviour of c e l l s exposed to l i n o l e i c a c i d i n s a l i n e compared to medium 199, where t h e i r m o b i l i t y decreased, was found to be due to the s u r f a c t a n t Tween 80 contained as a s o l u b i l i z i n g agent i n the medium. Tween 80 (polyoxyethylene-20-sorbitan mono-oleate e s t e r ) (256) i s a PEG based f a t t y a c i d e s t e r s u r f a c t a n t s i m i l a r to the PEG-fatty a c i d e s t e r s used i n hydrophobic a f f i n i t y p a r t i t i o n , although i t possesses l e s s a f f i n i t y f o r c e l l s (257). The Tween 80 i n Medium 199 adsorbed onto the d r o p l e t surfaces decreasing t h e i r e l e c t r o p h o r e t i c m o b i l i t y to 0.6 such that d r o p l e t adsorbtion onto f i x e d c e l l s lowered c e l l m o b i l i t y . PEG-fatty a c i d e s t e r adsorption onto c e l l s decreases t h e i r m o b i l i t y i n a s i m i l a r fashion (Appendix 1). Whether droplet adsorption increased or decreased c e l l m o b i l i t y was dependant on the concentration of Tween 80 i n the medium. V a r i a t i o n s i n t h i s c oncentration from source to source was c i t e d as p o s s i b l y e x p l a i n i n g d i f f e r e n c e s between the absolute m o b i l i t y r e s u l t s of the various groups using the t e s t . The f a c t that f r e s h and f i x e d erythrocytes behave e s s e n t i a l l y i d e n t i c a l l y i n the above MS m o b i l i t y t e s t suggests t h a t d i f f e r e n t i a l d r o p l e t adsorption i s res p o n s i b l e f or the MS-N d i f f e r e n c e s seen i n both c e l l types and that the various e f f e c t s l i n o l e i c a c i d has on f r e s h but not on f i x e d e r y t h r o c y t e s [ i . e . discocyte-echinocyte-spherocyte t r a n s f o r m a t i o n (258) and c l u s t e r i n g of surface groups (259)] are of secondary importance. The b a s i s f o r the d i f f e r e n t i a l a dsorption remains mysterious. I t appears to depend on the d i f f e r e n t i a l e f f e c t o f various f a t t y a c i d s on the c e l l s (260,261,262) si n c e o l e a t e does not induce MS-N - 35 -m o b i l i t y d i f f e r e n c e s (236,239) and i t i s d i f f i c u l t to e n v i s i o n a d i f f e r e n c e between oleate and l i n o l e i c a c i d d r o p l e t s which could e x p l a i n t h i s e f f e c t . One i s l e f t assuming that the d i f f e r e n t i a l a dsorption r e f l e c t s unknown membrane surface d i f f e r e n c e s between the two c e l l types which are p o s s i b l y more evident, as regards d r o p l e t a d s o r p t i o n , a f t e r exposure of the c e l l s to the approximately 30 yuM l i n o l e a t e which would be free i n s o l u t i o n (246). Treatment and management of m u l t i p l e s c l e r o s i s i s beyond the scope of t h i s i n t r o d u c t i o n and has been reviewed by other authors (12,18,263,264 and the general reviews mentioned above i n c l u d i n g 2 ) . I t should be appreciated that u n t i l more knowledge of MS e t i o l o g y i s amassed and an accurate method to diagnose MS at an e a r l y stage i s a v a i l a b l e much of our e f f o r t s can only be concerned with the symptoms of MS and t h e i r emotional e f f e c t on the p a t i e n t . This i s not to take away from the importance of such treatment, which i n the l a s t f i f t y years has apparently almost doubled the l i f e expectancy of the average p a t i e n t (18,263). Generally management i n v o l v e s a d m i n i s t r a t i o n of anti-inflammatory drugs such as the s t e r o i d s ACTH and prednisolone during and a f t e r acute a t t a c k s . On a d a i l y b a s i s a n t i - s p a s t i c drugs such as baclofen are p r e s c r i b e d as i s vi t a m i n B22 a i d i n myelin regeneration and to decrease demyelination ( 1 2 ) ) , and general t r a n q u i l i z e r s . Regular c l i n i c a l e v a l u a t i o n and treatment m o d i f i c a t i o n i s suggested. T r i a l treatments such as the "low f a t d i e t " mentioned above u s u a l l y r e f l e c t a p a r t i c u l a r theory concerning the e t i o l o g y of MS. As such they are many and v a r i e d , w i t h as yet no r e a l l y noteable successes. The f a c t that i n a d d i t i o n to immuno-suppressive agents, immuno-potentiating drugs such as levamisole, which improves c e l l - 36 -mediated immune f u n c t i o n and decreases serum antibody l e v e l s , have been used on a t r i a l b a s i s to t r e a t MS demonstrates how c o n f l i c t i n g the p h i l o s o p h i c a l bases of many of these treatments are, as w e l l as how l i m i t e d i s our b a s i c knowledge of MS and our a b i l i t y to diagnose and monitor t h i s very complicated disease. I t should be c l e a r from the above d i s c u s s i o n t h a t at present there e x i s t s no simple, widely accepted l a b o r a t o r y t e s t procedure which c o r r e l a t e s c l o s e l y with m u l t i p l e s c l e r o s i s (91). The l i p i d - a s s o c i a t e d e r y t h r o c y t e e l e c t r o p h o r e t i c m o b i l i t y t e s t appears promising but the f a c t that three l a b o r a t o r i e s were unable to obtain p o s i t i v e r e s u l t s suggests that considerably more developmental work w i l l be r e q u i r e d before i t can be considered a r e l i a b l e procedure, even i n the hands of experts. The lack of good l a b o r a t o r y t e s t s f o r MS i s s e r i o u s , h i n d e r i n g e p i d e m i o l o g i c a l , c l i n i c a l and bas i c research s t u d i e s of the disease (10,265). A confident diagnosis can only be made at t h i s time by an experienced n e u r o l o g i s t a f t e r c o n s i d e r a t i o n of a l a r g e number of f a c t o r s (37,265), requirements which are not e a s i l y met i n many cases of the disease. Those cases which are e a s i l y diagnosed may only represent the " t i p of the i c e b e r g " of subjects s u f f e r i n g without any c l i n i c a l treatment from t h i s disease/syndrome. The development of a simple and r e l i a b l e l a b o r a t o r y c o r r e l a t e f o r MS, e s p e c i a l l y f o r e a r l y or s u b - c l i n i c a l cases of MS, would t h e r e f o r e be valuable from many po i n t s of view. I t was towards t h i s goal that experimental work on the d i f f e r e n t i a l p a r t i t i o n o f blood c e l l s from MS p a t i e n t s and normal s u b j e c t s , which i s discussed i n the f o l l o w i n g chapters, was aimed . - 37 -1.3 AQUEOUS POLYMER TWO PHASE PARTITION 1.3.1 - C e l l Separation C e l l biology i s as dependant on methods of c e l l s e p a r a t i o n as biochemistry i s on methods of molecular and macromolecular f r a c t i o n a t i o n . An important p r e r e q u i s i t e f o r studying any c e l l u l a r or molecular system i s the a b i l i t y to i s o l a t e i t s components from one another so they can be c h a r a c t e r i z e d , q u a n t i t a t e d , manipulated and recombined under c o n t r o l l e d c o n d i t i o n s . A number of elegant and powerful procedures are a v a i l a b l e f o r the i d e n t i f i c a t i o n and i s o l a t i o n of pure molecular species from complex s o l u t i o n s . These i n c l u d e u l t r a c e n t r i f u g a t i o n , u l t r a f i l t r a t i o n , a f f i n i t y chromatography, i o n exchange chromatography and various e l e c t r o k i n e t i c methods such as i s o e l e c t r i c focussing. Molecules are separated by these techniques on the b a s i s of a v a r i e t y of p h y s i c a l p r o p e r i t e s i n c l u d i n g s i z e , shape, d e n s i t y , hydrophobicity, and charge, as w e l l as on the b a s i s of b i o l o g i c a l or f u n c t i o n a l p r o p e r t i e s such as a n t i g e n i c i t y or a f f i n i t y f o r a s p e c i f i c molecule. Often the a p p l i c a t i o n of one technique w i l l b r i n g about the d e s i r e d s e p a r a t i o n . Complex mixtures or d i f f i c u l t s e paration problems may r e q u i r e a procedure i n v o l v i n g a combination of methods such as g e l f i l t r a t i o n column chromatography followed by hydrophobic a f f i n i t y column chromatography. Some techniques may allow the a p p l i c a t i o n of two methods at the same time such as two-dimensional s l a b gel e l e c t r o p h o r e s i s where a macromolecular mixture can be separated on the basis of i s o e l e c t r i c pH p o i n t and molecular weight (266). E q u a l l y v a r i e d and elegant techniques e x i s t f o r the separation of c e l l s . (For reviews - 38 -see references 267 to 271). In many cases the techniques used have been adapted from molecular biochemistry, however the s e p r a t i o n s achieved are often of poor q u a l i t y . The reasons f o r t h i s are due i n par t to b a s i c d i f f e r e n c e s between c e l l s and molecules. Molecules are small enough to d i s t r i b u t e evenly i n s o l u t i o n v i a d i f f u s i o n and are not e a s i l y denatured by being sieved i n passage through f i l t e r s or the g e l matrices used i n column and tube or s l a b g e l techniques. They are reasonably immune to v a r i a t i o n s i n s o l u t i o n temperature, pH and s a l t c o n c e n t r a t i o n . Molecular homogeneity i s f i x e d by a chemical s t r u c t u r e which i s o f t e n unique, c o n f e r i n g s i m i l a r l y unique p r o p e r t i e s and separation behaviour on each molecular species or group. C e l l s , on the other hand, are f r a g i l e , complex, v a r i e d c o l l e c t i o n s of molecules. In suspension they tend to sediment r a p i d l y and to aggregate. They are l y s e d f a i r l y e a s i l y by s i e v i n g a c t i o n ; a property which coupled with t h e i r s i z e hinders column chromatography, g e l e l e c t r o p h r o r e s i s and many other s e p a r a t i o n procedures. C e l l s are g e n e r a l l y very s e n s i t i v e to a l t e r a t i o n s i n pH, temperature and s o l u t i o n s a l t c o ncentration. They cannot withstand long term exposure to l e s s than i d e a l c o n d i t i o n s . The o v e r a l l s i m i l a r i t i e s between types of c e l l s can o f t e n f a r outweigh t h e i r d i f f e r e n c e s . This i s e s p e c i a l l y true f o r subpopulations of c e l l s such as lymphocytes. Even members of a w e l l defined subpopulation of c e l l s may vary according to age, developmental stage and other f u n c t i o n a l or b i o p h y s i c a l f a c t o r s . Each c e l l and i t s clones are p h e n o t y p i c a l l y unique as to t h e i r environmental response. C e l l p opulations are th e r e f o r e defined to a greater extent than molecules by the se p a r a t i o n methods used i n t h e i r i s o l a t i o n and i d e n t i f i c a t i o n . S i m i l a r problems and - 39 -con s i d e r a t i o n s apply to the separation of c e l l o r g a n e l l e s , c e l l fragments, b a c t e r i a , b a c t e r i a l fragments, v i r u s e s , v i r a l fragments, e t c . and complicated, e a s i l y denatured macromolecules ( i . e . DNA) and macromolecular systems ( i . e . l i p o p r o t e i n s ) . In l i g h t of the problems mentioned above much work i n recent years has gone i n t o f u r t h e r i n g the development of c e l l s e p a r a t i o n technology because of the p o t e n t i a l bio-medical research and t e c h n o l o g i c a l progress which i s c u r r e n t l y being held back by the lack of s u i t a b l y powerful, cheap and e f f i c i e n t techniques (272). C u r r e n t l y c e l l s eparation techniques can be d i v i d e d up i n t o methods separating on the ba s i s of b i o l o g i c a l growth and s u r v i v a l , morphology and density ( i . e . sedimentation), and c e l l surface p r o p e r t i e s (267). At present there are four broad c l a s s e s of c e l l c h a r a c t e r i z a t i o n or separation procedures which s e l e c t on the basis of surface p r o p e r t i e s ; procedures which depend on an adherence r e a c t i o n between c e l l s and a surface or matrix (267,271-273), c e l l e l e c t r o p h o r e s i s (274,275) or i s o e l e c t r i c focussing (276), i n d i v i d u a l c e l l flow s o r t i n g a c t i v a t e d by fl u o r e s c e n t c e l l surface l a b e l s or l i g h t s c a t t e r i n g s i g n a t u r e (75,267,277), and p a r t i t i o n of c e l l s i n phase separated aqueous polymer s o l u t i o n s (278,279 and see below). Some methods, such as c l o n i n g , are only s u i t a b l e f o r r a p i d l y i s o l a t i n g s m a ll q u a n t i t i e s of c e l l s . Some methods such as c e n t r i f u g a t i o n are s u i t a b l e f o r bulk separations and many methods ( i . e . flow s o r t i n g , e l e c t r o p h o r e s i s ) are s u i t a b l e f o r intermediate s i z e s e p a r a t i o n s . The most s u c c e s s f u l techniques are n a t u r a l l y those which operate e f f i c i e n t l y on w e l l understood p r i n c i p l e s such that they provide i n f o r m a t i o n on the s t r u c t u r e of the separated m a t e r i a l and can be - 40 -se l e c t e d and, i f necessary, modified to f i t s p e c i f i c s e p a r a t i o n requirements. Given current a c t i v i t y i n biotechnology, a p a r t i c u l a r technique's speed, s i m p l i c i t y , r e l i a b i l i t y , v e r s a t i l i t y , cost and a b i l i t y to be scaled up f o r large s c a l e a p p l i c a t i o n are a l l becoming i n c r e a s i n g l y important (280). C e l l s often tend to e x h i b i t more overlap and range i n t h e i r p h y s i c a l p r o p e r t i e s than i n t h e i r f u n c t i o n a l p r o p e r t i e s . There seems l i t t l e reason to b e l i e v e , f o r instance that a subpopulation of lymphocytes engaged i n a p a r t i c u l a r immune f u n c t i o n should e x h i b i t a unique and uniform c e l l geometry or d e n s i t y . In approaching the problem of how to i d e n t i f y or separate c e l l s which c a r r y out p a r t i c u l a r normal or abnormal f u n c t i o n s one would t h e r e f o r e l i k e to apply procedures which operate on the ba s i s of c h a r a c t e r i s t i c s which are c l o s e l y associated with the f u n c t i o n of i n t e r e s t . Where those f u n c t i o n s i n v o l v e a c t i v i t i e s a s s o c i a t e d with c e l l s u r f a c e s , such as c e l l u l a r r e c o g n i t i o n phenomena, i t i s to be expected that separation techniques based on c e l l membrane p r o p e r t i e s would have the highest l i k e l i h o o d of success (347). Of the four types of c e l l separation procedures based on membrane surface p r o p e r t i e s mentioned above the l e a s t popular technique i s c e l l p a r t i t i o n i n phase separated aqueous polymer s o l u t i o n s . I t seems f a i r to say, i n s p i t e of extensivce work by c e r t a i n groups, p a r t i c u l a r l y i n Sweden where the technique o r i g i n a t e d (278), that the other techniques have a l l been s t u d i e d and a p p l i e d s c i e n t i f i c a l l y and commericially more widely than has c e l l p a r t i t i o n . E l e c t r o p h o r e s i s i s a complicated procedure r e q u i r i n g f a i r l y elaborate equipment. I t i s subject t e c h n i c a l l y to many problems e s p e c i a l l y when s c a l e d up f o r prep a r a t i v e work (281) and i t s most - 41 -noteworthy separations have been a n a l y t i c a l i n nature. Adherence based techniques ( i n c l u d i n g some types of flow s o r t i n g ) o f t e n i n v o l v e p u r i f i e d and/or modified l e c t i n s or antibodies and can be expensive. They are a l s o t e c h n i c a l l y d i f f i c u l t to s c a l e up. A problem of u n i v e r s a l concern with these techniques i s the a l t e r a t i o n or l y s i s of c e l l s t h a t may r e s u l t from i n t e r a c t i o n with a supporting matrix, the adherence r e a c t i o n and the c e l l surface l o c a l i z a t i o n of f o r e i g n m a t e r i a l or the procedures necessary to remove such m a t e r i a l . In many cases a s p e c i f i c antibody i s r e q u i r e d , implying t h a t s u f f i c i e n t knowledge about the a n t i g e n i c p r o p e r t i e s of the c e l l surface of i n t e r e s t to allow the d e t e c t i o n and i s o l a t i o n of conventional or monoclonal a n t i b o d i e s , i s already possessed. In perhaps the m a j o r i t y of cases wherein c e l l s eparation i s a l i m i t i n g problem, l i t t l e i f any knowledge e x i s t s about the p a r t i c u l a r c e l l subpopulation p r i o r to i t s i s o l a t i o n and study. In the case of flow s o r t i n g , the above co n s i d e r a t i o n s plus the cost of the apparatus, the t e c h n i c a l d i f f i c u l t y of the technique and the low sample s i z e s associated w i t h a procedure i n which p a r t i c l e s are s o r t e d one at a time are a l l l i m i t i n g . Despite these problems flow s o r t i n g , e l e c t r o p h o r e s i s and adherence methods are c u r r e n t l y the most w e l l known and a p p l i e d techniques. 1.3.2 - Aqueous Polymer Two Phase P a r t i t i o n P a r t i t i o n i n g of m a t e r i a l s between two immiscible phases i s one of the o l d e s t separatory methods. C l a s s i c a l l y i t i s used i n organic chemistry to separate p o l a r and apolar m a t e r i a l s between a hydrophobic phase such as chloroform and a h y d r o p h i l l i c solvent phase such as water i n a two phase - 42 -system (282,283). I f two substances show dramatic preferences f o r opposite phases a good separation of a mixture of these substances can be obtained i n one step by simply d i s s o l v i n g the mixture i n the two phase system, a l l o w i n g the phases to separate and p h y s i c a l l y i s o l a t i n g the phases and hence the substances from each other. The p a r t i t i o n of each substance between the phases i s c h a r a c t e r i z e d by a p a r t i t i o n c o e f f i c i e n t , K, which i s equal to the amount of that p a r t i c u l a r substance i n the upper phase d i v i d e d by the amount i n the lower phase. I d e a l l y K i s a f u n c t i o n of the substance, the phase system and the temperature and i s independent of the concentration of the substance, the volume r a t i o of the two phases, the geometry of the apparatus used i n the separation and the time the phases are e q u i l i b r a t e d beyond the minimum required f o r phase s e p a r a t i o n . At the microscopic l e v e l the substances are p a r t i t i o n e d during mixing and s e t t l i n g between the d r o p l e t s of one phase suspended i n the other, but e q u i l i b r i u m w i l l be a t t a i n e d even i n the absence of mixing. M a c r o s c o p i c a l l y they are p a r t i t i o n e d as s i m i l a r phase d r o p l e t s coalesce to form two bulk phases dri v e n by the force of g r a v i t y ( i . e . d e n s i t y d i f f e r e n c e s between the phases) and i n t e r f a c i a l t e n s i o n ( i . e . m i n i m i z a t i o n of i n t e r f a c i a l area and hence i n t e r f a c i a l f r e e energy). Comparing the p a r t i t i o n c o e f f i c i e n t s of two d i f f e r e n t substances i n a known two phase system allows one to a c c u r a t e l y p r e d i c t the nature of c e r t a i n b a s i c surface d i f f e r e n c e s between the substances and t h e i r a b i l i t y to be separated by p a r t i t i o n i n that system. When p a r t i t i o n c o e f f i c i e n t s are appreciably but not d r a s t i c a l l y d i f f e r e n t a s a t i s f a c t o r y s e p a r a t i o n of substances can o f t e n be obtained by c a r r y i n g out m u l t i p l e p a r t i t i o n s v i a a procedure termed countercurrent d i s t r i b u t i o n (CCD) i n which the s e t t l e d - 43 -phases are repeatedly i s o l a t e d and recombined with f r e s h complimentary phases (278,282,283). T h i r t y years ago two phase p a r t i t i o n i n simple s o l v e n t systems was a popular technique f o r the separation of low molecular weight biochemicals. Macromolecules such as p r o t e i n s were subject to denaturation as w e l l as to degradation caused by the s t r e s s of repeated mechanical a g i t a t i o n i n the simple solvent systems which possessed appreciable i n t e r f a c i a l tensions ( i . e . 10^ /jN/m) between the organic and aqueous phases (278,284). C e l l s and other b i o l o g i c a l p a r t i c l e s were s i m i l a r l y damaged by p a r t i t i o n i n g i n these systems. In a d d i t i o n they g e n e r a l l y p a r t i t i o n e d i n t o the aqueous phase and l i t t l e i f any separation occurred. The advent of chromatographic techniques such as gel and i o n exchange column chromatography i n the 1960's e l i m i n a t e d the usefulness of the technique f o r the separation of low molecular weight biochemicals. When d i l u t e aqueous s o l u t i o n s of two d i f f e r e n t polymers are mixed above c e r t a i n concentrations l i q u i d immiscible two phase systems o f t e n form even though the s o l u t i o n s may be 95% or more water by weight. This phenomena i s discussed i n references 278, 285, 286 and 287. Each phase i s enriched i n one of the polymers. Phase diagrams can be constructed i n which the % (w/w) c o n c e n t r a t i o n of each polymer i s s c a l e d on perpendicular separate axes (278). P o i n t s i n d i c a t i n g polymer concentrations i n the t o t a l system and i n each phase define s t r a i g h t l i n e s ( t i e - l i n e s ) whose r e l a t i v e length i s i n d i c a t i v e of the magnitude of the d i f f e r e n c e s i n phase compositions as w e l l as system i n t e r f a c i a l tensions (278,284). In the work described i n t h i s t h e s i s we have used the n e u t r a l polymers dextran [poly-(0C -1,6-glucose)] (D) and poly-(ethylene g l y c o l ) (PEG) which form a - 44 -two phase system with a dextran-enriched lower phase and a PEG-enriched upper phase. These two polymer aqueous phase systems can be buffered to p h y s i o l o g i c a l pH and made i s o t o n i c by the a d d i t i o n o f various s a l t s or other low molecular weight compounds. I n t e r f a c i a l t e n s i o n s i n the system are extremely low ( i . e . 5 juN/m) (284,288) so that when the phases are mixed b i o l o g i c a l p a r t i c l e s and macromolecules are subjected t o minimal mechanical s t r e s s . Such p a r t i c l e s or macromolecules f r e q u e n t l y possess an asymmetric preference f o r one of the phases, or the i n t e r f a c e and t h i s preference can be used as the basis of t h e i r p r e p a r a t i v e and/or a n a l y t i c a l p a r t i t i o n as was f i r s t shown and i n v e s t i g a t e d e x t e n s i v e l y by Alb e r t s s o n (278). Soluble substances p a r t i t i o n mainly between the two phases j u s t as i n organic-aqueous phase systems except that the gentleness o f the phase systems warrant t h e i r use with d e l i c a t e b i o l o g i c a l macromolecules such as pro t e i n s and n u c l e i c a c i d s . Many types of p r o t e i n s i n c l u d i n g iso-enyzmes have been d i f f e r e n t i a l l y p a r t i t i o n e d (278,280,289-291). The sep a r a t i o n of DNA molecules of d i f f e r e n t base p a i r composition, molecular weight, species o r i g i n and c o i l s t r u c t u r e has been accomplished. DNA and contaminating RNA have a l s o been separated (278,292,293). Generally macromolecules can be e a s i l y t r a n s f e r e d from one phase to the other by s e l e c t i v e l y a l t e r i n g the phase system polymer or s a l t composition ( i . e . pH) (278). Soluble substances tend to p a r t i t i o n i n a manner such that the p a r t i t i o n c o e f f i c i e n t K i s r e l a t e d to the d i f f e r e n c e i n surface free energy of the substance i n the two phases by: - 45 -( A G / k T ) [1-1] K = C e where C = the constant of p r o p o r t i o n a l i t y , G = the d i f f e r e n c e i n surface free energy and kT = Boltzmann's constant times absolute temperature. This equation derived by Bronsted (278) a p p l i e s s t r i c t l y to sm a l l p a r t i c l e s which d i s t r i b u t e evenly i n s o l u t i o n by Brownian motion. The surface free energy of the macromolecule-phase i n t e r f a c e ( G ) i s given by: [1-2] G = AY where A i s the t o t a l surface area of the macromolecule which i s p r o p o r t i o n a l to the molecular weight, and V i s the s p e c i f i c surface f r e e energy o f the i n t e r f a c e between the substance and the phase i n which i t i s l o c a t e d . Therefore: [1-3] In K = In (X x/X 2) = C AAYVkT where and X 2 are the s o l u t e molar concentrations i n the upper and lower phases r e s p e c t i v e l y and A Y = ( Yi - Y 2 ) (278, see a l s o 288). Equation 1-3 serves to i l l u s t r a t e why low molecular weight substances tend to d i s t r i b u t e f a i r l y evenly between the phases and why as molecular weight and molecular surface area increase small d i f f e r e n c e s i n the i n t e r a c t i o n between the s o l u t e and the phases r e s u l t i n large p a r t i t i o n c o e f f i c i e n t s . Thus small d i f f e r e n c e s i n phase system composition or substance molecular s t r u c t u r e can r e s u l t i n l a r g e p a r t i t i o n d i f f e r e n c e s . I t should be noted t h a t the f a c t o r A Y i s r e a l l y the summation of a la r g e number of f a c t o r s comprising the i n t e r a c t i o n s of the p a r t i t i o n e d substance with the phases i n c l u d i n g hydrogen, hyrophobic and i o n i c bond a s s o c i a t i o n between the s o l u t e and the polymers or the s o l u t e and the water molecules i n each phase. The f a c t t h a t we have l i m i t e d understanding of the f a c t o r s a f f e c t i n g Y means th a t i t i s d i f f i c u l t to a c c u r a t e l y a s c e r t a i n the type and magnitude of the d i f f e r e n c e s responsible - 46 -f o r a p a r t i t i o n d i f f e r e n c e between two macromolecules or p a r t i c l e s i n a two polymer aqueous phase system. Albertsson has pointed out that d i f f e r e n c e s of even a few k i l o c a l o r i e s per mole i n a substances t o t a l f r e e energy i n one phase versus the other, are t h e o r e t i c a l l y l a r g e enough to cause more or less-complete p a r t i t i o n i n t o the favored phase (278). Hence two phase p a r t i t i o n i s amply s u i t e d to the separation of substances d i f f e r i n g even s l i g h t l y i n t h e i r surfaces as "seen" by the phases. The a r t l i e s i n being able to s e l e c t the proper system. B i o l o g i c a l p a r t i c l e s such as c e l l s , b a c t e r i a , v i r u s e s and o r g a n e l l e s have a strong tendency to c o l l e c t at the i n t e r f a c e i n these two phase systems, decreasing the area of the l i q u i d - l i q u i d i n t e r f a c e and lowering the net free energy of the system. As noted p r e v i o u s l y low system i n t e r f a c i a l t e n sion p r o t e c t s these p a r t i c l e s from mechanical d i s r u p t i o n when the phases are mixed and allowed to separate. The f a c t that such p a r t i c l e s p a r t i t i o n s e l e c t i v e l y between the upper phase, the i n t e r f a c e and the lower phase increases the separation c a p a b i l i t y of the technique but a l s o i n c r e a s e s i t s complexity (278,289). This complexity i s enhanced by the adsorption of p a r t i c l e s to the surfaces of phase system d r o p l e t s formed upon mixing which a f f e c t s d r o p l e t f u s i o n and bulk phase s e p a r a t i o n . P a r t i c l e s a l s o tend to s e t t l e to the bottom of the p a r t i t i o n v e s s e l and s e l e c t i v e p a r t i t i o n i s only p o s s i b l e because phase separation occurs at a much f a s t e r rate than p a r t i c l e sedimentation. As a r e s u l t of the above, p a r t i c l e p a r t i t i o n i s much more complicated and l e s s w e l l understood than macromolecule p a r t i t i o n or c l a s s i c a l two phase p a r t i t i o n . P a r t i c l e s i z e , shape, density and concentration as w e l l as phase system v i s c o s i t y , phase volume r a t i o , s e t t l i n g time and s e t t l i n g chamber geometry are a l l important c o n s i d e r a t i o n s , due to the f a c t that p a r t i c l e s e p a r a t i o n i s - 47 -dependent on the mechanics as w e l l as the thermodynamics of the p a r t i t i o n (278,288,294, see a l s o chapter 3). Consideration of the above f a c t o r s leads to the n e c e s s i t y of extending equation [1-3] f o r the case of p a r t i t i o n o f b i o l o g i c a l p a r t i c u l a t e s (11,278,288,294). From t h i s e l a b o r a t i o n i t i s s t i l l apparent that p a r t i c l e p a r t i t i o n i n t o the upper phase should increase e x p o n e n t i a l l y with the f a c t o r s f a v o r i n g t h i s p a r t i t i o n . Thus i n a phase system p a r t i t i o n i n g on the b a s i s of c e l l surface charge we might expect p a r t i t i o n to be more s e n s i t i v e to s u b t l e surface charge d i f f e r e n c e s than a technique such as e l e c t r o p h o r e s i s i n which p a r t i c l e m o b i l i t y i s l i n e a r l y r e l a t e d to surface charge (278,288). This i n f a c t appears to be the case as has been shown by Walter et a l . f o r d i f f e r e n t s p e c i e s ' e r y t h r o c y t e s , although i t should be noted that p a r t i t i o n and e l e c t r o p h o r e s i s appear to r e f l e c t d i f f e r e n t p a r t i c l e surface charge c h a r a c t e r i s t i c s (278,279,295,296). Many i n t e r e s t i n g and u s e f u l p a r t i t i o n s i n v o l v i n g c e l l s , b a c t e r i a , v i r u s e s , c e l l fragments, liposomes and other b i o l o g i c a l p a r t i c u l a t e s have been reported (278,279,294,295,297, and see below). Those which p e r t a i n d i r e c t l y to the present work are discussed i n the relevant s e c t i o n s of chapters 3 and 4. In a d d i t i o n to systems p a r t i t i o n i n g b i o l o g i c a l p a r t i c l e s i n a manner r e l a t e d to t h e i r surface charge, some systems appear to p a r t i t i o n c e l l s on the b a s i s of non-charge r e l a t e d c h a r a c t e r i s t i c s (295,298,299). The charge and non-charge r e l a t e d membrane c h a r a c t e r i s t i c s r e s p o n s i b l e f o r the separations achieved with these two phase systems are as yet not w e l l understood. Nevertheless the separations are impressive and i n many cases cannot be d u p l i c a t e d by any other s i n g l e technique. C e l l s can be separated according to type (278,279,294,295,298-302), age (295,302-305) and a l s o developmental stage - 48 -(302,306-310). Lymphocytes have been separated i n t o subpopulations ( i . e . B,T,N, and NK) d i f f e r i n g i n fu n c t i o n and surface s t r u c t u r e (295,311-315). Human p e r i p h e r a l blood monocytes have been separated by CCD i n t o f r a c t i o n s d i f f e r i n g i n t h e i r phagocytic a c t i v i t y . Monocyte c e l l surface p r o p e r t i e s were a l s o demonstrated to be a l t e r e d as a consequence of p a r t i c l e engulfment (316). Murine lymphosarcoma c e l l s were subdivided i n t o groups possessing d i f f e r e n t m etastatic p o t e n t i a l as judged by r e i n j e c t i o n i n t o r e c e p t i v e hosts (312, see a l s o 302). The e f f e c t s of various i n v i t r o treatments on c e l l s have a l s o been s t u d i e d using CCD (278,279,294,295,318-321) as have c e l l - c e l l i n t e r a c t i o n s (295,313,308,322). Liposomes have been shown to p a r t i t i o n i n a manner r e l a t e d to t h e i r membrane l i p i d composition (323-327). B a c t e r i a l subpopulations d i f f e r i n g i n t h e i r s u s c e p t i b i l i t y to the a n t i b i o t i c s erythromycin (328) and streptomycin (329) have been d i f f e r e n t i a l l y p a r t i t i o n e d . In the l a t t e r case the subpopulations d i f f e r e d by a s i n g l e S-12 ribosomal p r o t e i n amino a c i d mutation, which was somehow a l s o r e f l e c t e d at the c e l l s u r f a ce. In the l a s t few years a f f i n i t y p a r t i t i o n i n two aqueous polymer phase systems has been developed. The technqiue i n v o l v e s c o u p l i n g a l i g a n d to one of the polymers (279,294,295,300,301,323,324,330-332). S p e c i f i c i n t e r a c t i o n of the l i g a n d with a macromolecule or p a r t i c l e r e s u l t s i n the obje c t having an increased a f f i n i t y f o r the phase enriched i n the s p e c i f i c polymer. T y p i c a l l y the l i g a n d i s coupled to PEG so that the c e l l or macromolecule i n t e r a c t i n g with the PEG coupled l i g a n d w i l l e x h i b i t enhanced p a r t i t i o n i n t o the upper PEG-enriched phase i n a dextran-PEG two-phase system (295,300,301). C e l l s , b a c t e r i a , liposomes, c e l l fragments and various enzymes have been s u c c e s s f u l l y p a r t i t i o n e d by t h i s technique, one advantage of which i s that the i n t e r a c t i o n r e s p o n s i b l e f o r the p a r t i t i o n i s reasonably w e l l understood. - 49 -One might speculate that exposure to the n e u t r a l polymer-containing phases would have a d e l e t e r i o u s e f f e c t on c e l l s u r v i v a l , however the opposite i s i n f a c t t r u e . E r y t h r o c y t e s are much more r e s i s t a n t to hypotonic l y s i s i n the presence of the phase system polymers than i n low i o n i c s t r e n g t h s a l i n e (278,333). The v i a b i l i t y and responsiveness of a number of c e l l l i n e s has been shown to remain high even a f t e r the repeated p a r t i t i o n i n g of CCD (302,311,312,314,315,317,334). C e l l s can be c u l t u r e d i n a normal f a s h i o n f o l l o w i n g CCD (302,310,317,335,336) and cancer c e l l s r e t a i n t h e i r a b i l i t y to l i v e i n a host and metastasize (303,317). R e t i c u l o c y t e s i s o l a t e d v i a CCD mature normally when r e - i n j e c t e d i n t o autologous hosts (337). Enzymes and p r o t e i n s p a r t i t i o n e d i n two aqueous polymer phase systems a l s o r e t a i n t h e i r n a t i v e s t r u c t u r e and f u n c t i o n (278,290-293,338). In many cases the l i g a n d s employed i n a f f i n i t y p a r t i t i o n i n t e r a c t r e v e r s i b l y with the d e s i r e d c e l l s or macromolecules causing no a l t e r a t i o n of c e l l v i a b i l i t y and responsiveness or enzyme a c t i v i t y . From the above i n t r o d u c t i o n i t should be apparent t h a t two aqueous polymer phase p a r t i t i o n i s a g e n t l e , powerful, v e r s a t i l e technique f o r the a n a l y t i c a l and p r e p a r a t i v e p a r t i t i o n of ( b i o l o g i c a l ) macromolecules and p a r t i c u l a t e s . What may not be as apparent i s the speed, r e l i a b i l i t y , s i m p l i c i t y and inexpensiveness of the technqiue, whose l i q u i d - l i q u i d nature makes i t i d e a l f o r both l a r g e i n d u s t r i a l and small s c a l e separations (278,280,339). Because two polymer aqueous phase p a r t i t i o n appears to separate l a r g e l y on the basis of surface s t r u c t u r e i t i s complementary to p h y s i c a l techniques such as c e n t r i f u g a t i o n (278,340). The major drawbacks to p a r t i t i o n are the d e f i n i t e lack of published knowledge concerning the f a c t o r s a f f e c t i n g p a r t i t i o n , as w e l l as a cohesive p a r t i t i o n theory (278,288,294,341). Lack of the l a t t e r - 50 -l i m i t s the i n t e r p r e t a t i o n of any r e s u l t s obtained. These weaknesses have not e l i m i n a t e d growing use of the technique which has been shown, i n many cases, to provide separations unattainable by any other method. However, i t cannot be denied that a more complete knowledge of the f a c t o r s governing p a r t i t i o n would make the choice of a s u i t a b l e system much e a s i e r , the a p p l i c a b i l i t y of the technique to s p e c i f i c problems more a t t r a c t i v e and the a n a l y t i c a l i n t e r p r e t a t i o n of r e s u l t s more c e r t a i n . This t h e s i s grew out of an i n t e r e s t i n i n v e s t i g a t i n g c e r t a i n f a c t o r s governing the p a r t i t i o n of c e l l s i n two polymer aqueous phase systems. My goal was to u t i l i z e t h i s knowledge i n the a p p l i c a t i o n of two phase p a r t i t i o n to the d i f f e r e n t i a t i o n of c e l l s from normal and diseased i n d i v i d u a l s f o r a n a l y t i c a l and c l i n i c a l purposes. M u l t i p l e s c l e r o s i s (MS) was chosen as a model disease because of the urgent need of a simple r e l i a b l e l a b o r a t o r y c o r r e l a t e f o r MS and because various c e l l membrane surface charge and non-charge r e l a t e d a b n o r m a l i t i e s had been reported i n e r y t h r o c y t e s and other blood c e l l types from MS s u b j e c t s . The main hypothesis was t h a t MS p a t i e n t blood c e l l surface a l t e r a t i o n s should, under w e l l c o n t r o l l e d c o n d i t i o n s , be detectable by two phase p a r t i t i o n . The approach taken i n v o l v e d i n v e s t i g a t i n g various phase system p a r t i t i o n f a c t o r s such as polymer p u r i t y , and phase system s a l t composition as w e l l as the c o n t r i b u t i o n s of c e r t a i n c e l l surface components to the p a r t i t i o n . These l a t t e r s t u d i e s were c a r r i e d out d i r e c t l y by studying the p a r t i t i o n e f f e c t s of various i n v i t r o c e l l surface treatments and i n d i r e c t l y by studying the p a r t i t i o n of pure p h o s p h o l i p i d liposomes. Related s t u d i e s , not discussed d i r e c t l y i n t h i s t h e s i s i n v o l v e d studying the p a r t i t i o n behaviour of b a c t e r i a possessing a v a r i e t y of known c e l l surface l i p i d , p r o t e i n and carbohydrate a l t e r a t i o n s (343,344), as w e l l as the - 51 -p a r t i t i o n behaviour of numerous c e l l types (302,345,346). The systems i n v e s t i g a t e d included those p a r t i t i o n i n g on the apparent b a s i s of membrane surface charge and non-charge r e l a t e d c h a r a c t e r i s t i c s . Hydrophobic a f f i n i t y p a r t i t i o n , i n which p a r t i t i o n i n t o the PEG-enriched upper phase i s increased d i f f e r e n t i a l l y on the b a s i s of hydrophobic i n t e r a c t i o n s between the substance or p a r t i c l e of i n t e r e s t and long chain f a t t y a c y l l i g a n d s e s t e r i f i e d to PEG, was i n v e s t i g a t e d q u i t e e x t e n s i v e l y . - 52 -CHAPTER 2. TECHNICAL PROCEDURES 2.1 - INTRODUCTION This chapter d e s c r i b e s the m a t e r i a l s and methods used to obt a i n the r e s u l t s presented and discussed i n chapters 3 and 4. The t o p i c s covered i n c l u d e ; two phase system p r e p a r a t i o n and p h y s i c a l c h a r a c t i z a t i o n , s y n t h e s i s p u r i f i c a t i o n and a n a l y s i s of PEG-fatty a c i d e s t e r s and r e l a t e d hydrophobic a f f i n i t y p a r t i t i o n l i g a n d s , p r e p a r a t i o n and p a r t i t i o n of d i m y r i s t o y l p h o s p h a t i d y l - c h o l i n e small u n i l a m e l l a r liposomes as w e l l as the p r e p a r a t i o n and p a r t i t i o n of human e r y t h r o c y t e s . As chapters 3 and 4 are only concerned with two phase p a r t i t i o n r e s u l t s , t h i s chapter i n c l u d e s a procedural r e s u l t s and d i s c u s s i o n s e c t i o n . This s e c t i o n was added due to the l a c k of published i n f o r m a t i o n concerning such procedures. 2.2 - MATERIALS 2.2.1 - Supp l i e s Of Chemicals and Equipment Unless s p e c i f i e d a l l reagents used were of ACS or b e t t e r q u a l i t y from various common sources. Dextran T500, (M approx. = 500,000, M n approx = 190,000) was purchased from Pharmacia Fine Chemicals, Uppsala Sweden: l o t 7693, 7830 or 16207. I t s density was approximately 1.637 (g/cc) (1) and i t s o p t i c a l r o t a t i o n at 25° C i s [ a ] n = +199° (2,3,4). I t s p a r t i a l s p e c i f i c volume i s 0.611 (cc/g) ( 5 ) . I t s s p e c i f i c r e l a t i v e r e f r a c t i v e index, i n water, i s 1.47 x 103 per % (w/v) (5). - 53 -Po l y - ( e t h y l e n e g l y c o l ) (PEG) 6000, r e c e n t l y redesignated 8000 (6 ) , [M = w 6650 + 50, from high pressure l i q u i d chromatography ( 7 ) ] , was obtained from Union Carbide Corp., Piscataway, N.J.: l o t M-68546, B-529-9104 and B-688-0232-2. I t s d e n s i t y i s approximately 1.20 (g/cc) ( 5 ) , and i t s s p e c i f i c r e l a t i v e r e f r a c t i v e index i s 1.33 x IO 3 per % (w/v) ( 3 ) , and i t s p a r t i a l s p e c i f i c volume i s 0.833 (cc/g) (5). Indubiose A45 e l e c t r o p h o r e s i s grade agarose was from F i s h e r S c i e n t i f i c as was Nessler's reagent. Sephadex LH-20, Octyl-Sepharose CL-4B and Sepharose 4B were a l l purchased from Pharmacia Fine Chemicals. P l u r o n i c p o l y o l s were obtained from BASF Wynadotte Corp (Wyandotte, Michigan). T r i t o n X-100 from Harleco and Tween 80 from F i s h e r . Pure 99.5+% Gold Label Spectro-analyzed ethanol was obtained from A l d r i c h . A l l n o n - r a d i o l a b e l l e d p h o s p h o l i p i d s , f a t t y a c i d s and f a t t y a c i d methyl-esters were 99% pure (Sigma Chemical Co.) PEG-fatty a c i d e s t e r s were purchased custom synthesized from Chem Ser v i c e Chemicals Inc. ( P h i l a d e l p h i a , PA). They were made using 90-99% pure f r e e f a t t y a c i d s . Other PEG-alkyl d e r i v a t i v e s were s u p p l i e d by Dr. M. H a r r i s (1). They were a l l p u r i f i e d and analyzed as described below ( 7 ) . Sodium c i t r a t e , sodium ethylenediamine t e t r a c e t i c a c i d (EDTA), T r i s - b u f f e r and D i l u i d 3, a z i d e - f r e e , i s o t o n i c d i l u e n t were bought from J . T. Baker Corporation. V i b r i o c h o l e r a neuraminidase enzyme (VCN) (E.C. 3.2.1.18) was obtained i n p u r i f i e d form i n 1ml v i a l s from Behringwerke GmbH (German Democratic R e p u b l i c ) . Each v i a l contained one Unit of enzyme a c t i v i t y , defined as the amount of enzyme re q u i r e d to r e l e a s e 1 micromole of s i a l i c a c i d per minute at 37° C, pH 5.5. Paraformaldehyde was from F i s h e r Chemical Co. ACS grade acetaldehyde was from Eastman Kodak Co - 54 -(8). Pure glutaraldehyde (pentanedialdehyde) (9) was purchased as a 70% aqueous s o l u t i o n , under freon, i n 2ml ampoules from Ladd Labs ( B u r l i n g t o n , VT). 1 4C-PEG4000 (50 juCi/ml i n H 20, 87.2 mCi/millimole) was purchased from New England Nuclear (NEN). l- 1 Z tC-octadecanoic ( s t e a r i c , 18:0) a c i d (30 juCi/ml i n hexane, 56 mCi/millimole) and 1-^C-hexadecanoic ( p a l m i t i c , 16:0) a c i d (30 uCi/ml i n hexane, 59 mC i / m i l l i m o l e ) were both obtained from Amersham. 3 H - p a l m i t i c a c i d (99% pure 5 mCi/0.5ml i n benzene, 12.3 C i / m i l l i m o l e ) was obtained from NEN. Custom synthesized "^C-DMPC (95% pure, 50 uCi/ml i n benzene, 55 mCi/ m i l l i m o l e ) was obtained from A p p l i e d Science Labs. Inc (PO Box 440, Penn., 16801). Atomlight (NEN) s c i n t i l l a t i o n f l u i d was used throughout. S i l i c a g e l p l a s t i c - b a c k e d 0.25 mm t h i c k p l a t e s (Polygram S i l G/HY) were obtained from Macherey, Nagel, and Co., West Germany. They were purchased from Brinkman L t d , Toronto. A l l g e l chromatographic columns were purchased from Pharmacia. S t e r i l e 0.45 (urn d i s p o s a b l e f i l t e r u n i t s were from Sybron/Nalge (Rochester, New York). Amicon CF-25 u l t r a f i l t r a t i o n cones were purchased from Amicon Inc. Evergreen brand d i l u i t o n v i a l s (VWR s c i e n t i f i c ) were used f o r e l e c t r o n i c p a r t i c l e counting. S t e r i l e 13mm x 100mm g l a s s c u l t u r e tubes and s i z e 000 rubber stoppers were purchased from F i s h e r . Polypropylene 13mm x 100mm c u l t u r e tubes (Western S c i e n t i f i c ) and 1.5ml Eppendorf p l a s t i c c e n t r i f u g a t i o n tubes (Brinkman L t d . , Toronto) were a l s o used. The f o l l o w i n g equipment was a l s o used; a Drs. Steeg and Reuter Polarimeter (Hamburg, FDR), an Abbe serum p r o t e i n r e f r a c t o m e t e r (Bausch and Lomb, I n c . ) , an Osmette I I osmometer ( P r e c i s i o n Systems Inc., Waltham, - 55 -Mass.), a v e r t i c a l p i p e t t e p u l l e r (David Kopf Instruments, I n c . ) , a Hewlet Packard (HP) 7100B s t r i p c h a r t recorder (10 6 ohm inp u t r e s i s t a n c e ) equipped with a 17505 A input module, an HP 3465 A ( I O 1 0 ohm input r e s i s t a n c e ) d i g i t a l multimeter, micromanipulators (model 7165, Warishige Co., Tokyo), a Braun Thermomix 1440 c i r c u l a t i n g water bath heater (B. Braun, Melsungen AG, FDRO, a two channel l i q u i d s c i n t i l l a t i o n counter (model LS-233, Beckman I n s t . , Inc.) equipped with an i n t e r n a l Barium standard, a 1000 ml c a p a c i t y B u c h i i r o t a r y evaporator, a p e r i s t a l t i c pump (model P - l , Pharmacia), various Pipetteman automatic p i p e t t o r s ( G i l s o n , France), a 1 ml Cornwall automatic p i p e t t o r (Beckson-Dickinson), a dual beam re c o r d i n g UV/VIS spectrophotometer (model 25, Beckman I n s t . , I n c . ) , a model 10-100 free dryer ( V i r t i s Corp.), a -60° C f r e e z e r ( H a r r i s Corp.), a model 150 Sonic-Dismembrator s o n i c a t o r equipped with a 0.5 i n c h diameter c y l i n d r i c a l t i t a n i u m probe ( F i s h e r S c i e n t i f i c ) , a S o r v a l l RC-5 c e n t r i f u g e , a bench top c e n t r i f u g e (model HN, I n t . Equip. Co), a Perkin-Elmer model DSC-1B d i f f e r e n t i a l scanning c a l o r i m e t e r , an ElectroZone c e l l o s c o p e (model 112 CLTH, P a r t i c l e Data Inc., Elmhurst, I L ) , equipped w i t h a 76 micron diameter ruby o r i f i c e , and a CCD 1200 t h i n l a y e r counter c u r r e n t apparatus made by IRD, Bromma, Sweden, and designed by P.-A. A l b e r t s s o n (4). 14 2.2.2 - Procedure For The Synthesis Of PEG 8000-1- C- P a l m i t a t e 20g o f PEG 8000 p u r i f i e d by ether p r e c i p i t a t i o n from acetone (4,7), was d i s s o l v e d i n 100 ml toluene, and 20 ml toluene was d i s t i l l e d out of the s o l u t i o n to remove any moisture using a r o t a r y evaporator ( B u c h i i ) . To t h i s s o l u t i o n was added 0.4g (55 /ul) of t r i e t h y l a m i n e which had been - 56 -d i s t i l l e d over calcium hydride. Approximately 0.10 g p a l m i t i c a c i d - l - ^ C (Amersham) which had been d i l u t e d 1 to 9 with n o n-radioactive p a l m i t i t c a c i d to produce a s p e c i f i c a c t i v i t y of 5.9 mCi per m i l l i m o l e was d i s s o l v e d i n 2 ml of methanol c o n t a i n i n g 1.0 M NaOH. This s o l u t i o n was then l y o p h y l l i z e d to remove a l l o f the methanol. The l y o p h y l l a t e was d i s s o l v e d i n 10 ml of anhydrous d i e t h y l ether. To t h i s s o l u t i o n was added dropwise 2 ml of o x a l y l c h l o r i d e (10). The s o l u t i o n was r o t a r y evaporated to dryness and the residue was resuspended i n 10 ml of toluene. This suspension was added to the s o l u t i o n of dry PEG i n toluene. The mixture was g e n t l y r e f l u x e d f o r 15 minutes and then cooled to 4° C to p r e c i p i t a t e unreacted PEG and PEG-fatty a c i d e s t e r from s o l u t i o n . The PEG r e a c t i o n product mixture was 99 % recovered by f i l t r a t i o n through a buchner funnel equipped with a paper f i l t e r (Whatman No.1). I t was then p u r i f i e d and analyzed f o r % s u b s t i t u t i o n as described below. The % y i e l d o f the r e a c t i o n was c a l c u l a t e d by comparing the assayed % s u b s t i t u t i o n to the expected (100 % y i e l d ) s u b s t i t u t i o n . 2.3 - METHODS 2.3.1 - Two Phase System P r e p a r a t i o n And P h y s i c a l C h a r a c t e r i z a t i o n 2.3.1.1 - Two Phase System P r e p a r a t i o n Two phase systems were prepared a f t e r the method of Walter (2,11, see a l s o 4) from the f o l l o w i n g stock s o l u t i o n s which were made fr e s h weekly and s t o r e d at 4° C. - 57 -(A) Dextran (D). An approximately 20% (w/w) stock s o l u t i o n was made by adding 22 g D to an equal weight of H 20. The mixture was s t i r r e d i n t o a paste. Then 56 g H 20 was added. The mixture was s t i r r e d m a g n e t i c a l l y f o r two hours u n t i l a c l e a r , t h i c k syrup formed. A 2 ml sample was d i l u t e d 12.5 times (v/v) with water and i t s dextran c o n c e n t r a t i o n determined p o l a r i m e t r i c a l l y . The o r i g i n a l (w/v) stock s o l u t i o n c o n c e n t r a t i o n was 6.2814 times the d i l u t e d samples o p t i c a l r o t a t i o n . The % (w/w) co n c e n t r a t i o n was c a l c u l a t e d assuming a d e n s i t y of 1.06 f o r a 20% (w/v) dextran stock s o l u t i o n ( 1 ) . (B) P o l y - ( e t h y l e n e g l y c o l ) (PEG). A 30% (w/w) stock s o l u t i o n was made by up d i s s o l v i n g 30.0 grams of PEG 8000 i n 70.0 g H 20. A l t e r n a t i v e l y , one could measure the stock s o l u t i o n % (w/w) conc e n t r a t i o n by d i v i d i n g i t s r e l a t i v e r e f r a c t i v e index by the s p e c i f i c r e l a t i v e r e f r a c t i v e index of PEG 8000 [1.33 x 10 , (5)] and con v e r t i n g t h i s t o % (w/w) based on the density o f PEG [1.20 g/cc (5, 6)1. (C) NaCl. 600 mM (D) Na phosphate, pH 7.2. 218 mM Na 2HP0 4 and 69 mM NaH 2P0 4, pH7.2 at 20°C. Phase systems were prepared by mixing appropriate amounts of the above stock s o l u t i o n s , f o r example; 100 g of 5% (w/w) D, 4% (w/w) PEG, 150 mM NaCl, 10 mM Na phosphate, pH 7.2 system was made by combining 25.0 g of dextran s t o c k , 13.3 g of PEG stoc k , 25.0 g of Nacl stock, 3.4 g of Na phosphate stock and making the s o l u t i o n up t o 100.0 g w i t h water. There i s a 3% e r r o r i n s t a t e d composition as the system d e n s i t y i s 1.03 (g / c c ) . - 58 -Systems were s t i r r e d m a g n e t i c a l l y f o r two hours and immediately vacuum f i l t e r e d through a s t e r i l e 0.45 jum disposable media f i l t e r u n i t . F i l t e r e d system was t r a n s f e r r e d to a graduated, c y l i n d r i c a l separatory funnel and allowed to s i t overnight at the temperature i t would be used a t . Phase volumes were then recorded and the c l e a r phases were i s o l a t e d , care being taken to d i s c a r d the i n t e r f a c e . Phase systems or i s o l a t e d phases could be st o r e d f o r up to one week at 4° C provided t h a t they were allowed to e q u i l i b r a t e at the temperature they were to be used a t . 2.3.1.1.- Two Phase System P h y s i c a l C h a r a c t e r i z a t i o n R e l a t i v e phase volumes were measured f o l l o w i n g phase se p a r a t i o n i n graduated c y l i n d r i c a l separatory funnels or i n more accurate graduated c y l i n d e r s . Phase polymer compositions and system i n t e r f a c i a l tensions were measured by Dr. Stefan Bamberger as described i n references (3,5,12). E l e c t r o s t a t i c bulk phase p o t e n t i a l s between the top and bottom phases were estimated by measuring the change i n voltage detected between two s a l t b ridges to r e v e r s i b l e Ag/AgCl e l e c t r o d e s when one e l e c t r o d e was d i s p l a c e d to the non-reference phase. The method, described below and i n Figure 2-1, was e s s e n t i a l l y t h a t o f Rietherman et a l . (13) as modified by Walter, Krob and Brooks (11) to ensure that the input r e s i s t a n c e of the voltage r e c o r d i n g device was at l e a s t 1 megohm. Ag/AgCl e l e c t r o d e s were made from two i n c h long s i l v e r wire as described by Van Wagenen (14, see a l s o 15). Once p l a t e d they were shorted and allowed to stand overnight i n 1 M KCI s o l u t i o n . The p l a t e d wires were s t a b i l i z e d by rubber stoppers i n g l a s s e l e c t r o d e s f i l l e d w i t h 1 M KCI - 59 -To Ground Figure 2-1. Apparatus f o r measuring two phase system e l e c t r o s t a t i c i n t e r f a c i a l po ten t i a l s . - 60 -s o l u t i o n . Three types of e l e c t r o d e s were used, 2 ml g l a s s Pasteur p i p e t t e s whose t i p s contained 2% (w/w) e l e c t r o p h o r e s i s grade agarose i n 1 M KCI, 2 ml Pasteur p i p e t t e s whose t i p s had been p u l l e d t o approximately 50 AJTI i n n e r t i p diameter, or 1.5 megohm, 30 MTI inner t i p diameter e l e c t r o d e s produced with a v e r t i c a l p i p e t t e p u l l e r which were su p p l i e d by Dr. Evan Evans. Bulk phase p o t e n t i a l s were measured with a d i g i t a l multimeter, i n p a r a l l e l w i t h a s t r i p chart r e c o r d e r . A l l measurements were conducted at 22° C, unless s p e c i f i e d , using 20 ml of phase system i n a c l e a r p l a s t i c s c i n t i l l a t i o n v i a l . Two e l e c t r o d e s , held by micromanipulators, were s t a t i o n e d i n one phase. A f t e r the monitored p o t e n t i a l had s t a b i l i z e d , one e l e c t r o d e was moved i n t o the other phase, the p o t e n t i a l d i f f e r e n c e being recorded a f t e r the second p o t e n t i a l had s t a b i l i z e d . This procedure was repeated a number of times using a d i f f e r e n t reference phase each time. The p a r t i t i o n of 1 AC-PEG 4000, PEG 8 0 0 0 - l - 1 A C - p a l m i t a t e and 14 C-octadecanoic a c i d between the phases i n various systems were measured by adding to a sample of upper phase enough l a b e l l e d m a t e r i a l such t h a t , at a counting e f f i c i e n c y of 30%, a d d i t i o n of 200 ;ul of phase to 10ml s c i n t i l l a t i o n f l u i d y i e l d e d 500 to 2000 counts per minute. 2.3.2 - P u r i f i c a t i o n And A n a l y s i s of PEG-Esters And Related Compounds 2.2.2.1 - Sec t i o n I n t r o d u c t i o n The b a s i c p u r i f i c a t i o n and a n a l y s i s p r o t o c o l i s given i n Figure 2-2. PEG ( f o r use i n a f f i n i t y p a r t i t i o n d e r i v a t i v e s y n t h e s i s ) or PEG-alkyl - 61 -d e r i v a t i v e s were i n i t i a l l y p u r i f i e d by solvent e x t r a c t i o n . PEG-alkyl d e r i v a t i v e s were f u r t h e r p u r i f i e d by d i s s o l v i n g i n methanol/H 20 (5v/lv) s o l v e n t and chromatographing on a Sephadex LH-20 g e l e x c l u s i o n column, with the same solvent system (16,17). The e l u t i o n p r o f i l e of PEG c o n t a i n i n g compounds was followed by Nessler's reagent assay. The e l u t i o n p r o f i l e o f e s t e r group c o n t a i n i n g compounds was followed by hydroxamic a c i d ester-assay and/or eluent u l t r a - v i o l e t a bsorption. R a d i o l a b e l e d compounds' e l u t i o n p r o f i l e s were monitored v i a l i q u i d s c i n t i l l a t i o n counting. The above a n a l y t i c a l methods were t y p i c a l l y used i n combination. F o l l o w i n g g e l f i l t r a t i o n , PEG c o n t a i n i n g compounds could be recovered by r o t a r y evaporation of the methanol i n the methanol/H 20 so l v e n t system followed by l y o p h i l l i z a t i o n . F o l l o w i n g methanol removal PEG-alkyl d e r i v a t i v e s were of t e n separated from u n d e r i v a t i z e d PEG by hydrophobic a f f i n i t y chromatography on an Octyl-Sepharose CL-4B column. The mixture was e l u t e d onto the column i n aqueous s o l u t i o n . E l u t i o n with H 20 removed u n d e r i v a t i z e d PEG while e l u t i o n wth methanol/H 20 (5V/IV) removed PEG-alkyl compounds which were i s o l a t e d i n pure form by r o t a r y evaporation and l y o p h i l l i z a t i o n . The p u r i f i e d d e r i v a t i v e was then assayed by t h i n l a y e r chromatography, as w e l l as chemical ( ^ h y d r o x a m i c a c i d ester-assay) or s c i n t i l l a t i o n counting techniques. 2.3.2.2 - Solvent E x t r a c t i o n of PEG And PEG-Alkyl D e r i v a t i v e s This procedure was adapted from Albertsson (4). Approximately 12 grams of PEG or PEG d e r i v a t i v e was ground to a powder and d i s s o l v e d i n - 62 -PURIFICATION OF PEG-FATTY ACID ESTER DERIVATIVES ESTER SYNTHESIS REACTION PEG + FFA PEG + FA-C1 * PEG-DCCI + FFA PRODUCT MIXTURE PEG PEG-FA , FA-PEG-FA FFA , X «= DCCI , FA-C1 , ETC. H.P.L.C. ( SIZE EXCLUSION ) ( KYDROPHOBICITY ) SOLVENT EXTRACTION AND ETHER PRECIPITATION ( SOLUBILITY / POLARITY ) ACETONE OR BENZENE FOLLOWED BY DIETHYL ETHER I DISSOLVE PPT IN MEOH / H„0, 5 v / n FFA AND X SEPHADEX LH-20 COLUMN ( SIZE EXCLUSION ) ( HEAD GROUP POLARITY ) HZ REMOVE MEOH FROM PEG / PEG-ESTER FRACTIONS OCTYL-SEPHAROSE CL-4B COLUMN FFA AND X ( HYDROPHOBIC AFFINITY ) PEG / PEG-ESTERS APPLIED IN H20 I PEG-ESTER ELUTED IN MEOH / HjO, 5 V / 1 V PEG ELUTED IN H20 REMOVE MEOH FROM PEG-ESTER FRACTIONS LYOPHYLLIZE STORE UNDER NITROGEN AT — 60 °C CHARACTERIZATION OF PEG-ESTER T.L.C. ESTER ASSAY PARTITION H.P.L.C. " Figure 2-2. Outline of purification and analysis protocol for PEG-fatty acid esters and other PEG derivatives. - 63 -200 ml o f acetone i n a 600 ml beaker by heating to 60° C i n a warm water bath. The acetone s o l u t i o n was then placed i n an i c e water bath and 300 ml o f anhydrous d i e t h y l ether was added to p r e c i p i t a t e the PEG compound. This p r e c i p i t a t e was recovered by f i l t r a t i o n through a buchner funnel equipped with a paper f i l t e r (Whatman No. 1). A f t e r f i l t r a t i o n a f u r t h e r 300 ml of anhydrous d i e t h y l ether was poured over the re s i d u e which was then allowed to dry. The e n t i r e procedure was repeated four more times. Afterwards the p r e c i p i t a t e was d i s s o l v e d i n methanol/H 20 ( 5 v / l v ) and ap p l i e d to a Sephadex LH-20 column or l y o p h i l l i z e d and s t o r e d under n i t r o g e n a t -60° C. 2.3.2.3 - Sephadex LH-20 Gel E x c l u s i o n Column Chromatography The f o l l o w i n g was performed e n t i r e l y at 4° C. Approximately 350 g of Sephadex LH-20 g e l , l o t 11627, preswollen i n methanol/H 20 (5v/lv) s o l v e n t and with the f i n e s removed was poured i n t o a 5cm diameter Pharmacia K50/100 column to form a 1320 ml g e l bed with a length of 66 cm (18). T y p i c a l l y 10.5 grams of a PEG/PEG d e r i v a t i v e mixture, p r e v i o u s l y p u r i f i e d by repeated e i t h e r p r e c i p i t a t i o n from acetone, was d i s s o l v e d i n 180ml o f the above s o l v e n t . Approximately 180ml of t h i s mixture was p e r i s t a t i c a l l y pumped onto the column at a flow r a t e of 120 ml/hr (6 2 ml/cm bed cross s e c t i o n a l a r e a / h r ) . Then 2 l i t r e s o f s o l v e n t was pumped through the column at the same flow r a t e , 12 ml f r a c t i o n s being c o l l e c t e d every 6 minutes. Twenty-four hours l a t e r a f u r t h e r 2 l i t r e s of so l v e n t was pumped through the column and discarded. - 64 -A n a l y s i s of the f r a c t i o n s was determined i n p a r t by the nature of the PE G - d e r i v a t i v e chromatographed. T y p i c a l l y 25 ;ul of each f i f t h f r a c t i o n was added to 5 ml of Nessler's reagent and s p e c t r o p h o t o m e t r i c a l l y monitored at 600 nm. PEG-alkyl d e r i v a t i v e s were monitored by 280 nm UV absorbance. L i q u i d s c i n t i l l a t i o n counting of 200 /ul samples of each f r a c t i o n was performed under the appropriate circumstances. 2.3.2.4 - Recovery Of PEG And PEG D e r i v a t i v e s From Methanol/Water ( 5 v / l v ) Solvent Methanol was removed from the pooled PEG peak f r a c t i o n s by r o t a r y evaporation using a pear shaped 1 l i t r e f l a s k equipped w i t h a standard taper 24/40 neck. The PEG compounds i n H^O were then a p p l i e d to an Octyl-Sepharose column or were recovered by l y o p h i l l i z a t i o n and stored by -60° C under H^. A standard taper freeze d r y i n g chimney (Labconco) allowed the same f l a s k to be used throughout the e n t i r e procedure. 2.3.2.5 - Octyl-Sepharose CL-4B Hydrophobic Column Chromatography 200 ml of O c t y l Sepharose CL-4B (19) was preswollen i n 500 ml of methanol/H 20 (5v / l v ) s o l v e n t and l e f t f o r 24 hours at 4° C. The f i n e s were decanted three times and the f i n a l g e l s l u r r y volume was reduced to 300 ml. The s l u r r y was then poured i n t o a 2.6 cm x 40 cm (Pharmacia K 2 26/40) column. The f i n a l column dimensions were 38 cm x 5.3 cm cross s e c t i o n a l area with a 200 ml bed volume. The column was prepared and used i n a 4° C c o l d room. - 65 -In a t y p i c a l run 7 g of PEG and PEG d e r i v a t i v e mixture c o l l e c t e d o f f o f the LH-20 column was made up t o 175 ml i n H 20. This 4.0% (w/v) s o l u t i o n was then pumped i n t o the column at a flow r a t e o f 40 ml/hr using a p e r i s t a l t i c pump (Pharmacia). Following t h i s water was pumped through the column at 60 ml/hr and f r a c t i o n s were assayed by Nessler's assay (see below) f o r PEG. I t took approximately 100 ml of eluent per gram of PEG to wash a l l o f the u n d e r i v a t i z e d PEG o f f the column. A f t e r PEG could no longer be detected i n the eluent 500 ml of methanol/H 20 ( 5 v / l v ) solvent was pumped through the column and c o l l e c t e d . Twenty-four hours l a t e r another 500 mis of t h i s s o l v e n t followed by 1 l i t r e of H 20 was pumped through the column and discarded. The presence of d e r i v a t i z e d PEG i n the c o l l e c t e d f r a c t i o n s was determined by various assay methods (see below) and the compounds were i s o l a t e d from the methanol/H 20 ( 5 v / l v ) solvent by r o t a r y evaporation and freeze d r y i n g as described p r e v i o u s l y . They were then s t o r e d under n i t r o g e n at -60° C u n t i l used. 2.3.2.6 - Assay of Chromatographed Radioactive Compounds 0.5 to 1.0 ml of column eluent was added to a g l a s s s c i n t i l l a t i o n v i a l c o n t a i n i n g 10 ml of s c i n t i l l a t i o n f l u i d . The samples' counts per minute (CPM) were determined to 10% or l e s s e r r o r and c o r r e c t e d f o r v a r i a t i o n s counting e f f i c i e n c y v i a use of an e x t e r n a l standard (S r a t i o ) . 2.3.2.7 - Nessler's Reagent Assay For PEG Containing Compounds This procedure was modified from (20) a l l o w i n g f o r e f f e c t s noted i n - 66 -(21,22). N e s s l e r ' s s o l u t i o n concentrate ( F i s h e r S c i e n t i f i c Co.) was stored i n the dark, at 4° C, u n t i l use. A working s o l u t i o n was made up according to l a b e l d i r e c t i o n s , f i l t e r e d through a paper f i l t e r (Whatman #1) and allowed to come to room temperature p r i o r to use. For accurate quantative r e s u l t s a standard curve was const r u c t e d each time and three determinations averaged on each sample. Samples were a l s o allowed to e q u i l i b r a t e to room temperature. When an a l y z i n g column f r a c t i o n s standard curves and r e p l i c a t e determinations were u s u a l l y unnecessary. Absorbance readings g r e a t e r than 0.6 were remeasured on d i l u t e d s o l u t i o n s . A separate s et of cuvettes was used f o r t h i s assay because the cuvettes employed can b u i l d up r e s i d u a l mercury (21). 5 ml of Nessler's working s o l u t i o n was added to each o f a s u f f i c i e n t number of 13 x 100 mm g l a s s t e s t tubes which were then capped with rubber stoppers. 25 u l of s e l e c t e d standard or sample i n water or methanol/H 20 (5 v / l v ) s o l v e n t was added t o one of the t e s t tubes which was re-stopped, mixed by i n v e r s i o n 5x, and allowed 2 minutes to s e t t l e . The absorbance was then read at 600 nm against a Nessler's reagent blank. I f the absorbance was greater than 0.6 the sample was d i l u t e d w i t h Nessler's reagent by a known amount and reread. When assaying column f r a c t i o n s every f i f t h f r a c t i o n was sampled to determine the general peak e l u t i o n p r o f i l e then every peak f r a c t i o n ' s absorbance was determined. A t y p i c a l Sephadex LH-20 run y i e l d e d a peak whose 600 nm absorbance was 3.0 to 5.0, n e c e s s i t a t i n g d i l u t i n g the peak f r a c t i o n s f i v e to ten times before reading (Figure 2-4). The 1 ml sample cuv e t t e was r i n s e d out with Nessler's reagent between determinations. - 67 -2.3.2.8 - PEG-Fatty Acid E s t e r UV Absorption The UV Absorption of PEG-fatty a c i d e s t e r s and r e l a t e d compounds was monitored from 205 to 350nm u t i l i z i n g a dual beam spectrophotometer. 2.3.2.9 - Hydroxamic Acid Assay For P E G - E s t e r i f i c a t i o n The f o l l o w i n g was adapted from Downs and Pigman (23) using i n f o r m a t i o n s u p p l i e d by Goddu et a l (24) and by Reid and Ramey (25). Unless s t a t e d otherwise a l l assay reagents were purchased from F i s h e r S c i e n t i f i c . The reagents could be stored f o r up to one year at 4° C. Reagents used were: (1) Hydroxylamine Reagent:- 2 M hydroxylamine h y d r o c h l o r i d e i n methanol/H 20 (3v/2v) (2) A l k a l i n e Reagent:- 3.5 M NaOH i n methanol (3) A c i d Reagent:- 2M HCl i n propanol/H 20 (4v/lv) (4) F e r r i c Reagent:- 0.37 M F e C l 3 ( i r o n ( I I I ) c h l o r i d e ) i n 0.1 M HCl, f i l t e r e d a f t e r a f t e r 24 hours through a Whatman No. 1 paper f i l t e r . (5) A l k a l i n e Hydroxylamine Reagent:- Prepared f r e s h s h o r t l y before use by mixing equal volumes o f reagents (1) and (2); s t a b l e f o r only 3 hours at room temperature. ( I t i s e a s i e r to keep any Nacl p r e c i p i t a t e suspended i n s o l u t i o n during the assay r a t h e r than f i l t e r i n g i t . ) (6) Methyl-ester Standards:- Methyl-esters purchased i n 100-500 mg amounts from Sigma (99% p u r i t y ) were stored i n the dark at -60° C u n t i l used. 10 ml of 24.0 mM stock s o l u t i o n i n n-propanol was - 68 -prepared. This s o l u t i o n was then s e r i a l l y d i l u t e d by weight to cr e a t e a set of standards from 1.0 mM to 20.0 mM whose co n c e n t r a t i o n s would be d i l u t e d 12-times i n the f i n a l assay. Duplicate s e t s of standards were always made up. Pure propanol was used f o r the corresponding blank standards against which the e s t e r standards were read. (7) PEG - e s t e r Samples:- PEG-fatty a c i d e s t e r samples were c a r e f u l l y weighed out i n t o 1.5 ml c o n i c a l capped m i c r o c e n t r i f u g e p l a s t i c Eppendorf tubes; t y p i c a l l y 0.100 gram of e s t e r . Then 1 ml of methanol/H 20 (5 v / l v ) solvent was added and the weight added was noted so that the tru e volume o f solvent could be determined l a t e r on from the s o l v e n t d e n s i t y (0.848 g/ml at 20° C). Normally 3-5 i n d i v i d u a l samples were made up. The corresponding blank samples c o n s i s t e d of approximately equal weights of u n e s t e r i f i e d PEG i n the same volume of s o l v e n t . A l l procedures were c a r r i e d out i n 1.5 ml Eppendorf tubes. F i v e sample tubes, f i v e sample blank tubes, f i v e standard blank tubes and two tubes f o r each standard i n each s et were used per assay. A l l reagents were brought to room temperature, and 400 /ul of hydroxylamine reagent was added to each tube. Then 100 jul of methyl e s t e r , PEG-ester or blank sample was added to each tube. The tubes were capped and mixed by i n v e r s i o n b r i n g i n g any p r e c i p i t a t e i n t o suspension. A f t e r 20 to 25 minutes at room temperature 400 /ul of 2 M HCl i n propanol/H 20 ( 4 v / l v ) was added to the tubes which were recapped and mixed. Then 100 /ul of propanol was added to each PEG-ester sample c o n t a i n i n g tube and each PEG blank tube. S i m i l a r l y 100 /ul of methanol/H 90 ( 5 v / l v ) was added to the - 69 -remaining tubes. F i n a l l y 200 pl of 0.37 M FeCLj i n 0.1M HC1 was added to each tube which was then recapped and mixed. The r e s u l t s were read at approximately 515 nm ten minutes l a t e r . An estimate of the concentration of e s t e r i f i e d groups i n each PEG-ester sample was c a l c u l a t e d by i n t e r p o l a t i o n from a standard curve constructed from the assay r e s u l t s on the methyl-ester standards. An example c a l c u l a t i o n i s presented i n s e c t i o n 2.4.3.8. 2.3.2.10 - Thin Layer Chromatography JThe f o l l o w i n g was adapted from (26). S i l i c a g e l , p l a s t i c - b a c k e d 0.25mm t h i c k p l a t e s were pre-run with methanol/H 20 (5 v / l v ) s o l v e n t . They were scraped f r e e of g e l f o r 0.5 cm on each edge then spotted w i t h 20 pl samples of PEG or PEG-fatty a c i d e s t e r d i s s o l v e d i n the same sol v e n t at 0.2 g/ml. They were then spotted w i t h 20 u l samples of the appropriate f r e e f a t t y a c i d standards d i s s o l v e d i n the same s o l v e n t at 2, 1, 0.5 and 0.25 mg/ml. T^hese concentrations were chosen so that for ease of analysis the PEG-containing spots thus represented samples of 4 mg each and the f a t t y a c i d spots samples of 40, 20, 10 and 5 ^jg r e s p e c t i v e l y . A f t e r the samples had d r i e d , the s o l v e n t was allowed to t r a v e l 10 cm up the p l a t e . The s o l v e n t f r o n t was marked at the end of the run and the p l a t e was d r i e d and placed i n a covered j a r s a t u r a t e d with i o d i n e vapor provided by i o d i n e c r y s t a l s ( F i s h e r S c i e n t i f i c ) . A f t e r one hour the p l a t e was removed and the spots were o u t l i n e d w i t h a p e n c i l . Free f a t t y a c i d i n the PEG-ester was estimated v i s u a l l y by comparison with the standards' spots. The PEG s t a i n e d more or l e s s permanently w h i l e the f a t t y a c i d s t a i n e d f o r only a few hours. The 5 /jg spot represented the l i m i t s o f our a b i l i t y (0.13 % - 70 -w/w) to detect f r e e f a t t y a c i d i n a PEG 6000-18:2 e s t e r sample by t h i s method. TLC of PEG f a t t y a c i d e s t e r s synthesized with ^ C - l a b e l l e d free f a t t y a c i d s was done i n a s i m i l a r manner only l e s s m a t e r i a l was a p p l i e d to the p l a t e i n sm a l l e r (5 u l ) spots. A f t e r each run the p l a t e was cut between spots i n t o s e c t i o n s p e r p e n d i c u l a r to the sol v e n t f r o n t . These s e c t i o n s were then cut i n t o t h i r d s which were scraped c l e a n and placed i n 15 ml of l i q u i d s c i n t i l l a t i o n f l u i d and counted. 2.3.3 - P r e p a r a t i o n And P a r t i t i o n o f D i m y r i s t o y l P h o s p h a t i d y l c h o l i n e  Small U n i l a m e l l a r Lipsomes 2.3.3.1 - Pr e p a r a t i o n of D i m y r i s t o y l P h o s p h a t i d y l c h o l i n e Liposomes D i m y r i s t o y l p h o s p h a t i d y l c h o l i n e (DMPC) small u n i l a m e l l a r liposomes a l s o r e f e r r e d to as sm a l l u n i l a m e l l a r v e s i c l e s or SUV's, o f supposedly approximately 200 angstrom diameter and 2 x 10 6 d a l t o n molecular weight (27), were prepared by a m o d i f i c a t i o n of the method des c r i b e d by Huang (28, see a l s o 29). This m o d i f i c a t i o n , suggested by Dr. P. C u l l i s i n v o l v e d l y o p h i l l i z i n g the r a d i o l a b e l e d and no n - l a b e l l e d l i p i d mixture (30), r a t h e r than d r y i n g i t under N^ ,, i n order to more evenly d i s t r i b u t e the l a b e l . A 250-300mg sample of DMPC c o n t a i n i n g approximately 17 /uCi o f DMPC or 16 /uCi o f ^ H - p a l m i t i c a c i d was placed i n a 20 ml standard g l a s s s c i n t i l l a t i o n v i a l c o n t a i n i n g r a d i o a c t i v e DMPC or 16:0 p a l m i t i c f r e e f a t t y a c i d (see below) and was d i s s o l v e d i n three mis of benzene. Complete - 71 -l i p i d d i s s o l u t i o n was e f f e c t e d by p l a c i n g i n a 37 C water bath. This s o l u t i o n was l y o p h i l l i z e d f o r three hours or u n t i l a l l of the benzene had been removed, and 8 ml of pH 7.0 b u f f e r c o n t a i n i n g 10 mM tris(hydroxymethyl)aminomethane-HCL ( T r i s - H C l ) , 0.1 M KCI, 0.2 mM EDTA and 0.3% (w/w) NaN-j was added to the s c i n t i l l a t i o n v i a l . The l y o p h i l l i z e d l i p i d was suspended i n the b u f f e r by v o r t e x i n g . This suspension was sonicated f o r s i x t y minutes ( u n t i l c l e a r ) on a Sonic Dismembrator, model 150 ( F i s h e r S c i e n t i f i c ) , equipped with a 1/2 inch diameter c y l i n d r i c a l t i t a n i u m probe and set at 90% of maximum power output. S o n i c a t i o n was done i n three minute i n t e r v a l s at a temperature s l i g h t l y above the 21° C phase t r a n s i t i o n temperature of DMPC (vide i n f r a ) . To avoid excessive heating during s o n i c a t i o n the v i a l was f r e q u e n t l y cooled i n an i c e water bath. A f t e r s o n i c a t i o n , the liposome s o l u t i o n was concentrated to 2 ml by u l t r a f i l t r a t i o n using Amicon CF-25 membrane f i l t e r cones (Amicon I n c . ) . This procedure, which took approximately 60 minutes, a l s o separated the liposomes mixture from unincorporated, l a b e l l e d f r e e f a t t y a c i d and t i t a n i u m fragments re l e a s e d from the s o n i c a t i o n probe. The 2 ml r e t e n t a t e was then subjected to descending g e l f i l t r a t i o n chromatography. Sepharose 4B [ p u r i f i e d 4% (w/w) aqueous agarose g e l i n bead form (30)] packed using the above t r i s b u f f e r , as des c r i b e d p r e v i o u s l y f o r Sephadex LH-20, at 4° C i n t o a Pharmacia K26/76 column to form a g e l bed 2.5 cm i n diameter and 50 cm i n len g t h (approximately 245 ml) was used. F i l t r a t i o n was c a r r i e d out at 4° C, using the t r i s b u f f e r at a flow r a t e o f 0.5 ml/minute. F i v e ml f r a c t i o n s were c o l l e c t e d , and those c o n t a i n i n g liposomes were immendiately sealed under n i t r o g e n to minimize l i p i d o x i d a t i o n (28,31). - 72 -Chromatography was followed by monitoring the t u r b i d i t y o f a 0.4 ml sample of each f r a c t i o n at 300 nm and/or by monitoring the f r a c t i o n s r a d i o a c t i v i t y by adding 100 u l sample of each f r a c t i o n to 10 ml s c i n t i l l a t i o n f l u i d and counting i t f o r ten minutes or 0.2% e r r o r . 2.3.3.2 - D i f f e r e n t i a l Scanning Calorimetry D i f f e r e n t i a l scanning c a l o r i m e t r y (DSC) (32) was performed using a Perken-Elmer model DSC-1B. Liposome samples were prepared f o r DSC by co n c e n t r a t i n g the two or three most concentrated f r a c t i o n s from the second peak of the Sepharose 4B e l u t i o n p r o f i l e to approximately 400 pl using Amicon CF-25 cones. DMPC d i s p e r s i o n s were prepared at 1 mg/ml conc e n t r a t i o n s by d i s s o l v i n g DMPC i n the 0.1 M KCI t r i s b u f f e r used f o r column e l u t i o n . In some cases 2.5 /JM PEG 6 0 0 0 - l i n o l e a t e (18:2) f a t t y a c i d e s t e r or 2.86 mM 18:2 free f a t t y a c i d (FFA) i n 0.56 mM ethanol ( a l l f i n a l c o n c e n t r a t i o n s ) were added to the column eluent peak f r a c t i o n s p r i o r to t h e i r being concentrated f o r DSC. These substances were added as a l i q u o t s from the standard e s t e r and f a t t y a c i d stock s o l u i o n s discussed below. DSC was c a r r i e d out at approximately 1200 to 1800 times the conce n t r a t i o n of l i p i d found i n the two phase systems during p a r t i t i o n experiments (vide  i n f r a ) . The e s t e r and FFA i n ethanol were t h e r e f o r e added at lOx normal p a r t i t i o n c o ncentrations i n order t o make t h e i r bulk s o l u t i o n DSC concen t r a t i o n s c l o s e r to p a r t i t i o n experiment c o n d i t i o n s . - 73 -2.3.3.3 - P a r t i t i o n of DMPC Liposomes The f o l l o w i n g general procedure was used i n a l l liposome p a r t i t i o n experiments. Liposomes were added to the top phase, forming a " s o - c a l l e d " load mix. 6 ml pooled from two peak Sepharose column f r a c t i o n s were added to 140 ml of upper phase. Samples of the load mix were removed i n order to determne the counts per minute (cpm) corresponding to 100% r e p a r t i t i o n i n g of liposomes i n t o the upper phase. Then equal a l i q u o t s ( t y p i c a l l y 2.5 ml) of upper and lower phase were combined i n 13 x 75 mm g l a s s , or i n some cases p l a s t i c , t e s t tubes and mixed by capping and i n v e r t i n g 20 times. The tubes were e q u i l i b r a t e d f o r one h a l f hour, i n a water bath equipped with a Braun Thermomix 1440 c i r c u l a t i n g water bath heater, at a temperature s l i g h t l y below the lowest e x p e r i m e n t a l l y d e s i r e d temperature . The bath heater c o i l c i r c u l a t e d i n a separate i c e water bath a l l o w i n g us to vary the experimental temperature from 15° C to 30° C _+ 0.5° C. A f t e r the i n i t i a l temperature e q u i l i b r a t i o n the tubes were r e q u i l i b r a t e d f o r one hour at the lowest d e s i r e d experimental temperature. Varying amounts of PEG f a t t y a c i d e s t e r or f r e e f a t t y a c i d i n ethanol were added to the tubes i n a l i q u o t s as d e s c r i b e d above. The tubes were remixed and allowed to e q u i l i b r a t e at the experimental temperature f o r 30 minutes between the a d d i t i o n of each substance. F i n a l l y the tubes were allowed 30 minutes to s e t t l e and were sampled by t a k i n g 500 ;ul of upper phase or lower phase, (sampled by p i e r c i n g the p l a s t i c tubes at the bottom with a 19 guage needle), i n t o 15 ml of s c i n t i l l a t i o n f l u i d f o r l i q u i d s c i n t i l l a t i o n counting to an e r r o r of 2% or l e s s . The p a r t i t i o n was expressed as the percent r e p a r t i t i o n i n t o the - 7 4 -upper phase, defined as: (cpm) upper phase - background (cpm) lo a d mix - background A l t e r n a t i v e l y , the data could be expressed i n terms of the p a r t i t i o n c o e f f i c i e n t , K, defined as: (cpm) upper phase - background  [(cpm) load mix - background]-[(cpm) upper phase - background)] A schematic r e p r e s e n t a t i o n o f a s i n g l e tube p a r t i t i o n experiment i s shown ( f o r c e l l s ) i n Figure 2 - 3 . Four separate tubes were averaged f o r each experimental c o n d i t i o n s t u d i e d . Load mix cpm's were determined by averaging ten or more load mix samples. Standard d e v i a t i o n s a s s o c i a t e d with the mean cpm's of four or more tubes were u s u a l l y l e s s than 5% of the mean. PEG f a t t y a c i d e s t e r stock s o l u t i o n s were t y p i c a l l y d i l u t e d from a stock s o l u t i o n which y i e l d e d a f i n a l c o n c e n t r a t i o n o f 1 0 uM when 1 0 0 f j l was added to 5 ml of system. The e s t e r stock s o l u t i o n s , had to be kept on i c e at a l l times. A l l liposome p a r t i t i o n experiments were conducted as described below f o r c e l l p a r t i t i o n . In experiments i n v o l v i n g p a r t i t i o n i n g liposomes at two d i f f e r e n t temperatures two water baths were used, 2 5 0 JJI of the upper phase being sampled at each temperature. 2 . 3 . 4 - PREPARATION AND PARTITION OF HUMAN ERYTHROCYTES 2 . 3 . 4 . 1 - I s o l a t i o n Of Er y t h r o c y t e s Blood samples were obtained by venipuncture o f health y normal v o l u n t e e r s . 4 . 5 ml of blood was drawn i n t o a 1 0 ml p l a s t i c s y r i n g e . The - 75 -needle was immediately removed from the syringe and the sample was i n j e c t e d i n t o a c l e a n , s t e r i l e 15 ml p l a s t i c c e n t r i f u g e tube c o n t a i n i n g 0.5 ml of 3.8% (w/w) sodium c i t r a t e i n 0.15 M NaCl, 7.3 mM Na 2HP0 4, 2.3 mM NaH 2P0 A, pH 7.2 (PBS) b u f f e r . The tube was capped and mixed gently by i n v e r s i o n . In some cases the blood was drawn i n t o a s i m i l a r a n t i c o a g u l a n t s o l u t i o n c o n t a i n i n g 7.5 mg of sodium ethylenediamine-t e t r a a c e t i c a c i d (EDTA) The blood was then c e n t r i f u g e d at 500-1000 x g f o r ten minutes i n a bench top c e n t r i f u g e . The buffy coat was removed using a s y r i n g e equipped with an 18 guage needle. The plasma was removed i n a s i m i l a r manner. The e r y t h r o c y t e s were resuspended i n 20 volumes (10 ml) of PBS b u f f e r and c e n t r i f u g e d as described above. The above washing procedure was then repeated two more times. 2.3.A.2 - S i n g l e Tube P a r t i t i o n Experiments Washed e r y t h r o c y t e s were resuspended i n a sample of the appropriate phase system's upper phase at a c o n c e n t r a t i o n of approximately 2 x 10 /ml. This concentrated suspension was added to f r e s h upper phase to prepare a load mix with the d e s i r e d c e l l c o n c e n t r a t i o n . C e l l c o n c e n t r a t i o n s were determined u s i n g an e l e c t r o n i c impedance c e l l counter equipped w i t h two v i s u a l d i s p l a y channels. The f i r s t channel was set to count p a r t i c l e s up to but not i n c l u d i n g the s i z e of two aggregated red c e l l s . The second channel was s e t to count p a r t i c l e s equal t o , or l a r g e r than, two aggregated red c e l l s . The channel s e t t i n g s were determined using both e r t h r o c y t e and l a t e x p a r t i c l e suspensions ( P a r t i c l e Information S e r v i c e s , Grants Pass, OR). Second channel counts were t y p i c a l l y l e s s - 76 -than 2% o f the counts i n channel 1. The e l e c t r o n i c c e l l counter was used i n c o n j u n c t i o n with a 247:1 d i l u t o r which d i l u t e d a 40 jul sample with 9.88 ml of i s o t o n i c d i l u e n t . Thus a t y p i c a l phase system c o n c e n t r a t i o n of 1 x 1 0 7 c e l l s / m l became a counter d i s p l a y of 4000. Varying the d i l u t i o n r a t i o allowed us to a c c u r a t e l y determine phase system c e l l c oncentrations 5 8 ranging from 1 x 10 to 1 x 10 c e l l s / m l . A t y p i c a l s i n g l e tube c e l l p a r t i t i o n experiment i s o u l i n e d i n Figure 2-3. A f t e r a system had phase separated and the phases had been i s o l a t e d c e l l s were added, as described above, to the upper phase. (In a few c o n t r o l experiments the c e l l s were added i n lower phase.) The c o n c e n t r a t i o n of c e l l s i n the load mix was determined e l e c t r o n i c a l l y and 2.5 ml of load mix was added to an equivalent amount of the complementary c e l l - f r e e phase i n a c l e a n , s t e r i l e 13mm x 100mm g l a s s or p l a s t i c t e s t tube. The tubes were capped with c l e a n , s t e r i l e rubber stoppers and mixed 20 times by i n v e r s i o n . The system was then given 30 minutes to e q u i l i b r a t e . At t h i s stage any substances whose e f f e c t s on c e l l p a r t i t i o n were being s t u d i e d were added to the tubes (see below). Each substance was i n d u v i d u a l l y added, thoroughly mixed by i n v e r s i o n and the tubes were given given 30 minutes to e q u i l i b r a t e . The tubes were then mixed by i n v e r s i o n a f i n a l time and allowed t o phase separate u n t i l a d e f i n i t e i n t e r f a c e was v i s i b l e . This corresponded to 30 minutes f o r (5,4) systems s e t t l e d i n the v e r t i c a l tube p o s i t i o n , 45 minutes f o r (5,3.5) systems s e t t l e d i n the v e r t i c a l p o s i t i o n and 5 minutes f o r systems s e t t l e d i n the h o r i z o n t a l tube p o s i t i o n (the tubes being s l o w l y moved i n t o the v e r t i c a l p o s i t i o n f o r sampling). The importance of sample s e t t l i n g time i s discussed i n chapter 3. Three samples of were obtained from the center of - 77 -SINGLE TUBE CELL PARTITION TWO-PHASE SYSTEM +CELLS LOAD MIX I i SAMPLE =Xo ADD PEG-F A ,F F A Etc. MIX AND LET SETTLE ¥ SAMPLE =XI PERCENT PART I T ION z J & * i 0 ° Cu XI PARTITI ON COEFF = K= Cl ~ Xo-Xt Figure 2-3. . Typical single tube particle partition procedure illustrated for cells. - 78 -the upper phase i n the tube. Each of these three samples was then counted two or more times t o give an averaged c e l l c o n c e n t r a t i o n whose standard d e v i a t i o n was l e s s than or equal to 5% of the mean. The c e l l p a r t i t i o n was then expressed as the percent o f c e l l s which ( r e p a r t i t i o n e d i n t o the upper phase or as the p a r t i t i o n c o e f f i c i e n t K (Figure 2-3). PEG 6000-fatty a c i d e s t e r s were added to the system as 20 u l to 100 jul a l i q u o t s from aqueous stock s o l u t i o n s (20 jul maximal i n the case of (5,3.5) systems). Pipetteman p i p e t t o r s were used e x t e n s i v e l y f o r t h i s purpose. E s t e r stock s o l u t i o n s were prepared from the a n a l y t i c a l data given i n Table 2-2. The most concentrated stock s o l u t i o n needed was made up and then s e r i a l l y d i l u t e d to o b t a i n the other stock s o l u t i o n s . E s t e r molecular c o n c e n t r a t i o n can be c a l c u l a t e d i n terms of e s t e r i f i e d end groups or e s t e r i f i e d molecules. In the l a t t e r case d i e s t e r i f i e d molecules are t r e a t e d as mo n o e s t e r i f i e d . Since most o f our e s t e r mixtures were n e g l i g i b l y d i e s t e r i f i e d we decided to express our e s t e r induced p a r t i t i o n i n terms of the c o n c e n t r a t i o n of e s t e r i f i e d molecules. To prepare the stock s o l u t i o n of an e s t e r o f 6680 average molecular weight and (10.5% m o n o e s t e r i f i e d , 0.9% d i e s t e r i f i e d and) 11.4% e s t e r i f i e d , so that 50 /ul added to 5 ml of phase system r e s u l t s i n an e s t e r c o n c e n t r a t i o n of 30 /uM, the amount of e s t e r p r e p a r a t i o n used (X) can be c a l c u l a t e d as f o l l o w s : X = (6680 g/mole) x (100%/11.4%) x (30 x IO*"6 mole/1) x (5050/ul/50/ul) = 0.178 g/ml Assuming a PEG d e n s i t y of 1.20 g/ml the above stock s o l u t i o n could be made by adding 0.178 g e s t e r to 0.852 g water. PEG e s t e r stock s o l u t i o n s were always made up f r e s h and kept on i c e while being used, as they appear to be s u s e p t i b l e to b a c t e r i a l and/or chemical degradation. - 79 -2.3.4.3 - Thin Layer Counter Current D i s t r i b u t i o n P a r t i t i o n Experiments Thin l a y e r counter c u r r e n t d i s t r i b u t i o n was performed e s s e n t i a l l y as described by Walter et a l (1,2), using a t h i n l a y e r CCD 1200 apparatus designed by A l b e r t s s o n (7) which made by Incentive Research and Development (Bromma, Sweden). The machine used had one s e t of c i r c u l a r p l e x i g l a s s p l a t e s f o r 120 c a v i t y CCD and one set f o r 60 c a v i t y CCD. The 120 c a v i t y p l a t e was used i n only two experiments. The procedure given below i s f o r the 60 c a v i t y p l a t e , (of average bottom c a v i t y volume 0.55 ml), when used to run one c e l l sample f o r 28 t r a n s f e r s on one s i d e of the p l a t e and another sample f o r 28 t r a n s f e r s on the other s i d e of the p l a t e . In a l l the CCD's shown dextran T500 - l o t 16207 and PEG 8000 Sentry Grade - l o t B-688-0232-2 were used. Great care was taken to keep the p l a t e s l e v e l during the CCD procedure. The phase system used was allowed to e q u i l i b r a t e overnight i n the 4° C c o l d room c o n t a i n i n g the CCD apparatus. I f PEG-fatty a c i d e s t e r s were to be used they were added t o the e n t i r e upper phase f o l l o w i n g i t s i s o l a t i o n ( i . e before c e l l a d d i t i o n or l o a d i n g onto the CCD). A 3-5 ml upper phase load mix of approximately 1 x 10 c e l l s per ml was then made up. Using a 1.0 ml Cornwall automatic p i p e t t o r (Beckson-Dickinson) 0.5 ml of lower phase.was added to a l l 60 c a v i t i t e s and 0.6 ml upper phase was added to c a v i t i t e s 2 to 29 and 32 t o 59. Then 0.6 ml of one c e l l sample's load mix was added to c a v i t i e s 0 and 1 and 0.6 ml of the other c e l l sample's load mix was added to c a v i t i t e s 30 and 31. The c e l l s were run at shaking speed 3, 30 second shake, and 8 minute s e t t l i n g f o r a t o t a l of 28 t r a n s f e r s . CCD c a v i t y f r a c t i o n s were c o l l e c t e d i n t o 10.5 mm x 100 mm - 80 -p l a s t i c t e s t tubes as described by Albertsson (4). The f r a c t i o n s were inspected to ensure an even d i s t r i b u t i o n o f both upper and lower phase. Then 0.5 ml of PBS was added to each f r a c t i o n , which was capped and mixed by i n v e r s i o n to break the phases. F r a c t i o n s were sampled and counted as described above f o r s i n g l e tube p a r t i t i o n . The data was expressed as the percent o f t o t a l c e l l s recovered which d i s t r i b u t e d i n t o a c e r t a i n c a v i t y . The recovery of v i a b l e c e l l s was t y p i c a l l y g r e a t e r than 90%. Peak heights were t y p i c a l l y 15% i n a p o p u l a t i o n whose peak c o i n c i d e d with the center of the c a v i t y t r a i n (vide i n f r a ) . Comparative CCD's were performed with two c e l l types being p a r t i t i o n e d under i d e n t i c a l c o n d i t i o n s , o f t e n at the same time on opposite halves of the same CCD p l a t e . 2.3.4.4 - I n d i v i d u a l C e l l P a r t i t i o n Experiments Experiments i n v o l v i n g the e f f e c t s o f c e l l c o n c e n t r a t i o n on p a r t i t i o n r e q u i r e d a l t e r a t i o n i n our c e l l counting procedure which was designed f o r loa d mix c e l l c o n c e n t r a t i o n s of from 10 6 to 1 0 7 c e l l s / m l . In experiments i n v o l v i n g l o a d mix c o n c e n t r a t i o n s of 10^ to 1 0 5 c e l l s / m l , a 1 ml sample of upper phase added to 10 ml of d i l u e n t was counted. In P experiments i n v o l v i n g load mix c o n c e n t r a t i o n s of 10 or more c e l l s / m l , a 0.5 ml sample of upper phase was added to 4.5 ml of d i l u e n t i n a t e s t tube. A 40 /ul sample from t h i s tube was then d i l u t e d 247:1 (v/v) and counted i n a normal manner. The procedure used to remove c e l l surface s i a l i c a c i d with V i b r i o  c h o l e r a neuraminidase (VCN) (E.C. 3.2.1.18) was adopted from a p r o t o c o l used r o u t i n e l y i n the l a b o r a t o r y o f Dr. D. E. Brooks at the U n i v e r s i t y o f - 81 -B r i t i s h Columbia and which has been found, v i a t h i o b a r b i t u r i c a c i d assay (33), to remove 90 to 100% of human ery t h r o c y t e surface s i a l i c a c i d (25,3A). E r y t h r o c y t e s i s o l a t e d from 5 mis of healthy f r e s h c i t r a t e d blood by c e n t r i f u g a t i o n three times i n 20 volumes of 0.15 M NaCl s o l u t i o n b u f f e r e d to pH 7 by the dropwise a d d i t i o n o f 0.5 M NaHCO^ were suspended at a f i n a l hematocrit of 20% (v/v) i n 10 ml of the same b u f f e r at 37° C. A 0.5 ml a l i q u o t of t h i s suspension was then washed twice i n the same b u f f e r at 20° C and a f i n a l time i n (5,A) V system upper phase before being p a r t i t i o n e d i n a normal manner. A 3.5 ml a l i q u o t of the 10 ml suspension was d i l u t e d 1:1 (v/v) with s a l i n e b u f f e r c o n t a i n i n g 5 mM C a C l 2 . The r e s u l t i n g 10% hematocrit (HCT), 2.5 mM C a C l 2 , pH 7.0 suspension was incubated f o r one hour at 37° C. The pH was c o n t i n u a l l y r eadjusted. These c o n t r o l i n c u b a t i o n e r y t h r o c y t e s were then washed three times i n the o r i g i n a l s a l i n e b u f f e r and a f i n a l time i n upper phase before being p a r t i t i o n e d i n a normal f a s h i o n . The remaining 5 ml a l i q o t of the o r i g i n a l 20% HCT suspension was recombined with 5 ml of 5 mM C a C l 2 c o n t a i n i n g b u f f e r and 25 pi of neraminidase s o l u t i o n (Test-Neuraminidase, Behring D i a g n o s t i c s ) . The f i n a l 10% HCT s o l u t i o n contained approximately 0.0125 U n i t s of VCN a c t i v i t y per ml, or approximately 300 o l d u n i t s of VCN a c t i v i t y per 10"^ RBC. These c e l l s , whose e l e c t r o p h o r e t i c m o b i l i t y was determined by Dr. B. Chiu to have decreased from approximately 1.16 to 0.A1 Gum x sec-"'" x V-"*" x cm) (Appendix 1 ) , were then incubated, washed and p a r t i t i o n e d as described above. The procedures used to f i x our c e l l s with glutaraldehyde and/or other aldehydes were taken from Vassar et a l (8). Instead of bicarbonate - 82 -b u f f e r e d s a l i n e we used 0.15 M NaCl, 7.3 mM Ha^PO^, 2.3 mM NahtjPO^ (PBS) b u f f e r . Formaldehyde was prepared by adding 2% (w/w) paraformaldehyde ( F i s h e r Chemical Co.) to PBS and heating the s o l u t i o n at 60° C f o r 30 minutes, i n a fume hood, before i t was cooled, f i l t e r e d and used. Pure acetaldehyde (ACS grade, Eastman Kodak Co.) was obtained i n 25 ml ampules stored under n i t r o g e n . Glutaraldehyde of high p u r i t y (9) was obtained from Ladd Research L a b o r a t o r i e s ( B u r l i n g t o n , VT) as a 70% aqueous s o l u t i o n s t o r e d i n 2 ampoules under Freon. The glutaraldehyde and acetaldehyde e x h i b i t e d a s i n g l e u l t r a - v i o l e t absorbtion peak at 280 nm, i n d i c a t i n g l i t t l e , i f any, contamination or p o l y m e r i z a t i o n (8,9). The aldehyde-buffer f i x a t i v e s o l u t i o n s were prepared j u s t before use. C e l l s were t y p i c a l l y f i x e d i n 1% (w/v) glutaraldehyde although f o r experimental reasons they were a l s o f i x e d i n 3.8% (w/v) glutaraldehyde, 2% (w/v) acetaldehyde or 2% (w/v) formaldehyde c o n t a i n i n g b u f f e r . In each case c e l l s i s o l a t e d from f r e s h c i t r a t e d blood by c e n t r i f u g a t i o n and washed three times i n 20 volumes of PBS were then suspended i n 20 volumes of the f i x a t i v e s o l u t i o n . The c e l l c o n t a i n i n g f i x a t i o n s o l u t i o n was slowly r o t a t e d f o r 24 hours on a Roto-Rack ( F i s h e r ) . The 5% HCT i n the f i x a t i o n s o l u t i o n ensured ample f i x a t i v e f o r the c e l l s and decreased the p o s s i b i l i t y of hemoglobin from l y s e d c e l l s becoming f i x e d onto the c e l l s urface ( 8 ) . This l a t t e r p o s s i b i l i t y was a l s o decreased by changing the f i x a t i o n b u f f e r every 24 hours f o r three days i n the case of glutaraldehyde f i x a t i o n and at 2, 5, 12, 24, 48, 72, 96 and 120 hours i n the case o f acetaldehyde and formaldehyde (8). Appreciable hemolysis was only noted during formaldehyde f i x a t i o n . - 83 -Ethanol was t y p i c a l l y added t o a two phase system as 16.7 jul o f 99.5+ % pure ethanol (Gold L a b e l , A l d r i c h Chemical Corp.) to 5 ml of system i n a 13 x 100 mm g l a s s t e s t tube. This corresponds approximatley to 56 mM e t h a n o l . S t e a r i c (18:0) (cis - 9-octadecanoic a c i d ) , l i n o l e i c a c i d (18:2) ( c i s - 9 , c i s - 1 2 - o c t a d e c a d i e n o i c a c i d ) and a l l other f r e e f a t t y a c i d s (FFA) were 99% pure (grade I , Sigma Chemical Co.). The f a t t y a c i d s were purchased i n 100 mg a l i q u o t s i n sealed ampoules f i l l e d w i t h n i t r o g e n . These ampoules were st o r e d at -60° C u n t i l needed. A t y p i c a l experiment i n v o l v e d adding 60 mg of l i n o l e i c a c i d (approx. MW = 281) to 1.93 grams o f ethanol ( d e n s i t y = 0.789 g/cc) to make a stock s o l u t i o n such that when 16.7 / j l was added t o 5 ml of system the f i n a l c o n c e n t r a t i o n would be 286 /JM 18:2 FFA and 56 mM e t h a n o l . Other FFA concentrations were achieved by var y i n g the stock s o l u t i o n FFA c o n c e n t r a t i o n such t h a t approximately the same c o n c e n t r a t i o n of ethanol was added each time. A d d i t i o n o f FFA to a c e l l c o n t a i n i n g two phase system before or a f t e r e s t e r i n v o l v e d adding the FFA, stoppering the t e s t tube with a rubber stopper, mixing the tube by i n v e r s i o n twenty times and w a i t i n g one h a l f hour f o r the FFA t o e q u i l i b r a t e with the system before proceding with the experiment. P a r t i t i o n was t y p i c a l l y f o l l o w e d m i c r o s c o p i c a l l y using a Ze i s s phase contrastir.esearch photomicroscope and samples of system h e l d between p l a s t i c c o v e r s l i p s . S t a t i s t i c a l analyses were not u s u a l l y performed on the p a r t i t i o n data given i n chapter 3 because the p a r t i t i o n d i f f e r e n c e s found were t y p i c a l l y so l a r g e as to render such a n a l y s i s redundant, or so small as to be i n s i g n i f i c a n t when considered i n the l i g h t o f the sm a l l number o f - 84 -completely i n d u v i d u a l experiments performed ( i . e . 3 to 5). In chapter 4 l a r g e sample populations made s t a t i s t i c a l analyses p o s s i b l e . S i n g l e tube c e l l p a r t i t i o n data i s normally d i s t r i b u t e d according to chi-square a n a l y s i s (p } 0.10) (35). Such data can be compaired using a pa i r e d or unpaired Student's t-Test (35). A l t e r n a t i v e l y , they can be compared non-parametrically using using a Wilcoxon Rank-Sum Test f o r a paired experiment or a Mann-Whitney U Test f o r unpaired data (36). Ester-induced p a r t i t i o n curves were t y p i c a l l y analyzed by comparing the mean p a r t i t i o n values f o r each e s t e r c o n c e n t r a t i o n y i e l d i n g a p preciable p a r t i t i o n , as i n a p a i r e d experiment. In our experience data which was normally d i s t r i b u t e d gave the same r e s u l t s whether analyzed by parametric or non-parametric t e s t s . In such cases the t s t a t i s t i c , degrees of freedom and corresponding p r o b a b i l i t y have been given. 2.3.4.5 - P a r t i t i o n Of M u l t i p l e S c l e r o s i s And Normal Subjects'  Erythrocytes The procedures used i n our attempts to d i f f e r e n t i a l l y p a r t i t i o n m u l t i p l e s c l e r o s i s (MS) and normal s u b j e c t s ' e r y t h r o c y t e s were described above. The r a t i o n a l e behind our phase system choices are discussed i n Chapter 4. Figure 2-4 o u t l i n e s the c e l l p r e p a r a t i o n procedure f o l l o w e d . One normal and one or more matching coded MS blood sample were studi e d per experiment. MS samples were taken from p a t i e n t s with v a r i o u s forms of the disease ( i . e . c h r o n i c p r o g r e s s i v e , r e l a p s i n g and r e m i t t i n g and s t a b l e benign) (37, see a l s o chapter 1) who at the time of sampling were i n s t a b l e or post-acute a t t a c k c o n d i t i o n and could v i s i t the UBC-MS - 85 -5 ml BLOOD INTO CITRATE •REMOVE BUFFY COAT RESUSPEND CELLS IN PBS-156 GLUTARALDEHYDE STORE ROOM TEMP. CENTRIFUGE CELLS 2x RESUSPEND IN UPPER PHASE FOR WASHED FIXED CELL PARTITION CENTRIFUGE BLOOD (lOOOg x 10 min) REMOVE.PLASMA RESUSPEND CELLS IN PBS AND CENTRIFUGE END CELLS IN PBS FREEZE 1.5 ml PLASMA -60°C FOR PLASMA ENZYME ANALYSIS FREEZE 0.5 ml PLASMA -60°C FOR PLASMA CELL INCUBATION AND CENTRIFUGE RESUSPEND CELLS IN PBS THAW 0.5 ml PLASMA STORE AT A°C OVERNIGHT CENTRI RESUSP FUGE CELLS 3END IN UPPER PHASE FOR FRESH WASHED CELL PARTITION 0.5 ml PACKED CELLS -»-IN 0.5 ml PLASMA 37°C, 90 MINUTES ADD TO UPPER PHASE FOR FRESH UNWASHED CELL PARTITION Figure 2-4... Protocol f o r preparing MS, c o n t r o l disease and normal subjects' blood samples f o r d i f f e r e n t i a l erythrocyte p a r t i t i o n studies. - 86 -C l i n i c headed by Dr. D.W. Paty. Many p a t i e n t s were t a k i n g drugs some were not. MS and normal sample blood types were not matched as there appears to be no r e l a t i o n s h i p between ABO/Rh blood type and e i t h e r p a r t i t i o n (38,39) or MS (40). Non-MS c o n t r o l disease samples are discussed i n chapter 4. These samples were s u p p l i e d by Dr. M. Jones, S t a f f N e u r o l o g i s t , Shaughnessy H o s p i t a l . Complete medical h i s t o r i e s were a v a i l a b l e f o r a l l p a t i e n t s . We a l s o had access to the r e s u l t s of other i n v e s t i g a t o r s working with these p a t i e n t s . A 4.5 ml sample of blood obtained by venipuncture was drawn i n t o a s t e r i l e c o l l e c t i o n tube c o n t a i n i n g 0.5 ml sodium c i t r a t e 3.8 % (w/v) (0.38% w/v f i n a l c o n c e n t r a t i o n ) . S i l i c o n e f r e e tubes were used due to the p o s s i b i l i t y o f the s i l i c o n e p o l y e l e c t r o l y t e adsorping onto the red c e l l s and a f f e c t i n g t h e i r p a r t i t i o n (41). The blood was t r a n s f e r e d to a 15 ml s t e r i l e p l a s t i c tube and c e n t r i f u g e d at 1000 x g f o r ten minutes at room temperature i n a t a b l e top c e n t r i f u g e . A two ml sample of plasma was removed, d i v i d e d i n t o a 1.5 ml p o r t i o n and a 0.5 ml p o r t i o n ; both of which were s t o r e d frozed i n c l e a n , s t e r i l e Eppendorf tubes at -60° C. The remaining plasma and b u f f y coat were removed. The c e l l p e l l e t was resuspended i n 10 ml of pH 7.2 PBS, r e c e n t r i f u g e d and the buffy coat once more removed. The c e l l washing procedure was repeated a t h i r d time a f t e r d i v i d i n g the c e l l suspension i n two. The washed e r y t h r o c y t e s were e i t h e r resuspended i n PBS and s t o r e d o v e r n i g h t at 4° C or resuspended i n PBS-1% (w/v) glutaraldehyde monomer f i x a t i v e s o l u t i o n and stored at room temperature. The next day the u n f i x e d c e l l s i n PBS were c e n t r i f u g e d . 0.5 ml of packed c e l l s was added to the 0.5 ml sample of matching plasma which had been thawed and warmed to room temperature. C e l l s from the - 87 -approximately 0.5 ml packed f r e s h c e l l p e l l e t were resuspended i n upper phase and a l i q u o t s of t h i s suspension were d i l u t e d with upper phase to make a load mix of 1.2 x 1 0 7 c e l l s / m l f o r our f r e s h washed c e l l p a r t i t i o n experiments. The c e l l s i n plasma were incubated f o r 1.5 hours at 37° C. Then 1.4 ml o f t h i s suspension was added t o 3 ml of upper phase to make load mix c e l l suspension which contained approximately 2.3% Q (v/v) e r y t h r o c y t e s (2 x 10 c e l l s / m l ) and 2.3% (v/v) plasma. Fi x e d e r y t h r o c y t e s were allowed to stand f o r at l e a s t one week at room temperature before being p a r t i t i o n e d . During t h i s p e r i o d the f i x a t i v e s o l u t i o n was changed every two days. 2.4 - PROCEDURAL RESULTS AND DISCUSSION 2.4.1 - Synthesis of PEG 8000-1- 1 4C-Palmitate A procedure to sy n t h e s i z e s m a l l amounts of PEG 8000-1-^C-palmitate was developed due to a need f o r r a d i o l a b e l e d e s t e r to t e s t the e f f i c i e n c y of the PEG-alkyl d e r i v a t i v e p u r i f i c a t i o n and assay techniques described l a t e r on i n t h i s chapter. Once p u r i f i e d , the r a d i o l a b e l e d e s t e r was used i n other s t u d i e s (Appendix 1 ) . We chose to s y n t h e s i z e P E G - l - ^ C - p a l m i t a t e v i a p r i o r a c t i v a t i o n o f the free f a t t y a c i d (FFA), by co n v e r t i n g i t to the a c i d c h l o r i d e form, as t h i s r e a c t i o n r e q u i r e s l i t t l e molar excess of the expensive "^C-FFA and has been shown to proceed i r r e v e r s i b l y and q u a n t i t a t i v e l y (42,43). Both 1 4C-PEG and "^C-FFA are commercially a v a i l a b l e (44). L a b e l l e d FFA was chosen as i t i s l e s s expensive, could be purchased i n a range of s p e c i f i c a c t i v i t i e s - 88 -and because the d i f f e r e n c e s i n p h y s i c a l p r o p e r t i e s on which a s e p a r a t i o n o f FFA from e s t e r could be based were greater than those between e s t e r i f i e d and u n e s t e r i f i e d PEG. PEG-palmitate was chosen due to the p o p u l a r i t y of t h i s e s t e r as a hydrophobic a f f i n i t y l i g a n d (43,45) and because of the a v a i l a b i l i t y of data concerning the i n t e r a c t i o n of 1 4 C - p a l m i t i c a c i d and e r y t h r o c y t e s (46,47). O x a l y l c h l o r i d e was used r a t h e r than p h o s p h o r o u s - t r i c h l o r i d e or t h i o n y l c h l o r i d e , due to the lack of dangerous s i d e products ( i . e . H-jPOjj, HCl) produced i n the o x a l y l r e a c t i o n and the f a c t t h a t , i n f u t u r e a p p l i c a t i o n s , i t would not a l t e r unsaturated f a t t y a c y l t a i l s (48,49). I t can r e a c t e x p l o s i v e l y with water, however (48). The s y n t h e s i s p r o t o c o l given above was adapted from F i e s e r and F i e s e r (48), f o r the production o f the a c i d c h l o r i d e , and reference (42) f o r e s t e r i f i c a t i o n of the a c i d c h l o r i d e and the e s t e r . Calcium hydride was used i n place of p t h a l l i c anhydride to dry the t r i e t h y l a m i n e . The d r y i n g steps i n the r e a c t i o n are very important s i n c e any water i n the PEG could r e a c t with the a c i d c h l o r i d e , and any methanol i n the f a t t y a c i d s a l t c o u l d react w i t h the o x a l y l c h l o r i d e . The f a t t y a c i d s a l t was b a r e l y s o l u b l e i n the ether. A d d i t i o n of o x a l y l c h l o r i d e to the r e a c t i o n mixture r e s u l t e d i n formation of the n o n - r a d i o a c t i v e p r e c i p i t a t e , b e l i e v e d to the NaCl. I t was not necessary to add p y r i d i n e to the r e a c t i o n mixture (10). The 20 g of PEG used i n the r e a c t i o n had p r e v i o u s l y been p u r i f i e d by ether p r e c i p i t a t i o n from acetone and Sephadex LH-20 column chromatography. I t s molecular weight was estimated, by high pressure chromatography to be 6650+_ 200 g (50). T y p i c a l l y a sample of 1-Wc - p a l m i t a t e with a s p e c i f i c a c t i v i t y of 59 m C i / m illimole was used - 89 -following 1:9 dilution with unlabelled palmitate. Out- protocol was designed to maximally derivitize only 6.5% of the PEG hydroxyl groups. The reaction was carried out twice with an average yield of 1.1% of the group substituted. Increasing the fatty acylchloride concentration would most probably have increased the yield considerably (42,43). Linear PEG molecules can become derivatized at either or both hydroxyl end groups. If i t i s assumed that these end groups react independently of one another and that they are equally reactive we can estimate, i f the percentage of end groups substituted i s known, the percentage of PEG molecules in a sample which are mono-, d i - and underivatized. If a fraction "1/X" of the PEG end groups in a sample are 2 derivatized the fraction of molecules di-substituted i s ( 1/X ) , the fraction unsubstituted i s (1 - 1 / X ) 2 = (1 - 2/X + 1/X2) and the fraction of molecules mono-substituted at either end i s (2/X - 2/X ). It can easily be calculated that when 10 % of the end groups are esterified (1/X=10) that 18 % of the PEG molecules are mono-substituted and only 1% of the PEG molecules are di-substituted. When 30 % of the end groups are esterified 42 % of the PEG molecules are mono-substituted and 9 % of the molecules are di-substituted. The possibility that di-substituted PEG molecules may cause c e l l partition and c e l l aggregation effects different from mono-substituted PEG'S (43, chapter 3) suggests that unless the mono- and di-substituted PEG'S can be separated from each other (which i s not presently the case) substutition reactions should be kept to a low yield to ensure most of the product i s mono-derivatized or kept to a high yield (90 % or more) to ensure most of the product i s di-derivatized. Alternately one can produce mono-substituted material in - 90 -high y i e l d by s t a r t i n g with PEG mono-methyl ether, which possesses only one r e a c t i v e hydroxyl end group (50,51). 2.4.2 - Two Phase System P r e p a r a t i o n And C h a r a c t e r i z a t i o n The most commonly used two phase systems f o r e r y t h r o c y t e p a r t i t i o n are the 5% (w/w) Dextran T500, 4% (w/w) PEG 6000, (5,4) pH 6.8 systems of Walter (2, 11). These range from a (5,4) #5 system c o n t a i n i n g 0.11 M Na phosphate to (5, 4) #5 system c o n t a i n i n g 0.15 M NaCl and 0.01 M Na phosphate. The f r e e z i n g p o i n t depression o s m o l a r i t y o f the #1 b u f f e r i s approximately 220 mOsm and of the #5 b u f f e r i s 290 mOsm. The c o n t r i b u t i o n of both polymers i s approximately 25 mOsm (1 ) . As system t o n i c i t y has been reported to i n f l u e n c e p a r t i t i o n (38) and as pH 6.8 i s c l o s e to the p i of hemoglobin, where Hb from l y s e d c e l l s could be expected to e x h i b i t maximal c e l l s u rface a d s o p t i o n / a l t e r a t i o n (52), a s e r i e s of pH 7.2, at 22° C, systems made with i s o t o n i c 290+5 mOsm b u f f e r s (53) were con s t r u c t e d . I s o t o n i c b u f f e r s were chosen s i n c e phase system h y p e r t o n i c i t y , due to the osmolar c o n t r i b u t i o n of the polymers, was expected to a f f e c t c e l l p a r t i t i o n l e s s than h y p o t o n i c i t y (38). The s e r i e s of b u f f e r s used i s given i n Table 2-1. The b u f f e r system was i n i t i l l y coded A to G. Subsequently A, C and G have become known as I, I I and V, due to s i m i l a r i t i e s with Walter's systems #1, #2, and #5. Phase system c h a r a c t e r i z a t i o n i s important i n order to i n t e r p r e t and reproduce r e s u l t s as w e l l as to compare r e s u l t s obtained with d i f f e r e n t systems. In a d d i t i o n to c h a r a c t e r i z a t i o n by measuring the r e l a t i v e p a r t i t i o n o f var i o u s exogenous substances ( i . e . c e l l s ) between the phases, - 91 -Table 2-1. Bu f f e r Systems Used In Two Phase Systems B u f f e r System Na^HPO^ NaHoPO^ NaCl (mM) (mM) (mM) A = I 109 35 0 #1 55 55 0 B 91 29 25 C = I I 73 23 50 #2 45 45 30 D 55 17 75 E 34 11 100 F 18 6 . 125 G = V 7.3 2.3 150 #5 5 5 150 The phosphate con c e n t r a t i o n s f o r the a l p h a b e t i c a l l y l a b e l l e d systems allow f o r approximately 8% (w/w) h y d r a t i o n . See t e x t f o r d i s c u s s i o n . - 92 -phase systems can be c h a r a c t e r i z e d by measuring the p a r t i t i o n of water (phase volumes), polymers, and s a l t s ( i n c l u d i n g bulk phase p o t e n t i a l s ) , as w e l l as by measuring i n t e r f a c i a l t e n s i o n s and the z e t a p o t e n t i a l s r e l a t e d to the e l e c t r o p h o r e t i c m o b i l i t y of d r o p l e t s of one phase suspended i n the other i n the presence of an e l e c t r i c f i e l d (3, 4, 5, Chapter 3, Appendix 1). As regards the measurement of bulk phase p o t e n t i a l s , a l l three types of e l e c t r o d e s used y i e l d e d s i m i l a r r e s u l t s (see 5). Although open ended e l e c t r o d e s s t a b i l i z e d f a s t e r and produced more reproducble r e s u l t s they could not be used with the s t r i p c h a r t recorder whose input r e s i s t a n c e was only 1 megaohm. C o n t r o l s t u d i e s i n d i c a t e d t h a t e l e c t r o d e s f i l l e d w i t h 0.5 M KCI y i e l d e d the same r e s u l t s as those f i l l e d with 1 M KCI suggesting that e l e c t r o s t a t i c bulk phase p o t e n t i a l s , r a t h e r than electrode-phase, l i q u i d - j u n c t i o n p o t e n t i a l s were being measured (13). The presence of PEG-fatty a c i d e s t e r s , f r e e f a t t y a c i d s , 5° C v a r i a t i o n s i n temperature or e r y t h r o c y t e s ( 1 0 7 c e l l s / m l system) d i d not a p p r e c i a b l y a f f e c t our r e s u l t s . 2.4.3 - P u r i f i c a t i o n And A n a l y s i s Of PEG-Fatty Acid E s t e r s And Related  Compounds 2.4.3.1 - I n t r o d u c t i o n The s y n t h e s i s of a wide range of PEG d e r i v a t i v e s i n c l u d i n g ether, e s t e r and amide l i n k e d hydrophobic a f f i n i t y p a r t i t i o n l i g a n d s have been discussed by ourselves and others i n the l i t e r a t u r e (7,50,51,54,55). - 93 -PEG-fatty a c i d e s t e r s were g e n e r a l l y purchased from Chem S e r v i c e Chemicals Incorporated ( P h i l a d e l p h i a ) as custom synt h e s i z e d chemicals. They were p u r i f i e d as described below. PEG-fatty a l c o h o l ethers and PEG-fatty amines a l s o appear to make e x c e l l e n t hydrophobic a f f i n i t y p a r t i t i o n l i g a n d s (7). They are harder to c h e m i c a l l y assay f o r the degree of s u b s t i t u t i o n , but e x h i b i t i n c r e a s e d s t a b i l i t y to chemical and b i o l o g i c a l degradation (50). PEG-fatty a l c o h o l ethers are commonly a v a i l a b l e detergents e.g. B r i j and Lubrol W (54,55). PEG - f a t t y a c i d e s t e r s can be purchased under the trade names Sterox CO, Myrj and Span (55). We chose to custom order our e s t e r s i n order to ensure that f a t t y a c i d s of high p u r i t y ( i . e . 90-99%) were used i n t h e i r s y n t h e s i s . Our experience i s that the PEG head group should possess more than 50 oxyethylene u n i t s i n order f o r a PEG e s t e r c e l l a f f i n i t y l i g a n d to f u n c t i o n e f f e c t i v e l y (chapter 3 ) . PEG d e r i v a t i v e product mixtures, whether purchased commercially or s y n t h e s i z e d , c o n t a i n contaminants whose nature depends on the r e a c t i o n used i n t h e i r syntheses. Contaminants may i n c l u d e s m a l l molecular weight polyethylene g l y c o l molecules, f r e e f a t t y a c i d s or a c i d c h l o r i d e s , f r e e f a t t y a l c o h o l s , amines, toluene, e t h e r , e t h a n o l , and d i c y c l o h e x y l carbo d i i m i d e (DCCI) (50). The f o l l o w i n g s e c t i o n i s concerned w i t h the p u r i f i c a t i o n and a n a l y s i s of PEG-fatty a c i d e s t e r product mixtures. The procedures given, w i t h the e x c e p t i o n of the hydroxamic a c i d e s t e r assay, work e q u a l l y w e l l with ether and amine l i n k e d PEG d e r i v a t i v e s and with s l i g h t m o d i f i c a t i o n should a l s o be a p p l i c a b l e to a wide v a r i e t y of water s o l u b l e polymer d e r i v a t i v e s . - 94 -At the time these procedures were devised the PEG-fatty a c i d e s t e r s u t i l i z e d f o r two phase a f f i n i t y p a r t i t i o n by other research groups were being p u r i f i e d by p r e c i p i t a t i o n twice from absolute ethanol (42,43,56). They were then analyzed f o r t h e i r degree of s u b s t i t u t i o n , independent of any knowledge of the t r u e MW of the PEG, on the b a s i s of NaOH s a p o n i f i c a t i o n followed by HC1 t i t r a t i o n f o r s a t u r a t e d f a t t y a c i d e s t e r s or i n the case of unsaturated f a t t y a c i d e s t e r s on the b a s i s of t h e i r c a p a c i t y to bind i o d i n e bromide (43,56). We found, however, that ethanol e x t r a c t i o n d i d not remove a l l of the f a t t y a c i d and other contaminants from the product (chapter 3 ) . The remaining f a t t y a c i d molecules would n a t u r a l l y a f f e c t the percent s u b s t i t u t i o n a n a l y s i s however t h i s was not the prime reason f o r d e v i s i n g the e x t e n s i v e PEG-ester p u r i f i c a t i o n p r o t o c o l o u t l i n e d below. This p r o t o c o l was developed i n order to e l i m i n a t e the appreciable p a r t i t i o n e f f e c t s t h a t even s m a l l amounts of f r e e f a t t y a c i d contaminants are capable of c r e a t i n g due to t h e i r a b i l i t y to aggregate and l y s e c e l l s as w e l l as to a l t e r e s t e r - c e l l i n t e r a c t i o n (chapter 3 ) . 2.4.3.2. - Solvent E x t r a c t i o n of PEG And PEG-Alkyl D e r i v a t i v e s This procedure was used to remove most of the f r e e f a t t y a c i d , a n t i o x i d a n t and other organic contaminants i n PEG and PEG-derivative samples. I t i s analogous to r e c r y s t a l l i z a t i o n of PEG compounds from absolute ehtanol s o l u t i o n s (42,43,56)) except t h a t e i t h e r and acetone are u s u a l l y purer than ethanol (49). The procedure given was u s u a l l y repeated f i v e or more times. In the case of PEG SOOO-l-^C-Palmitate t h i s was s u f f i c i e n t to leave only 7.1% (mole/mole) or 0.3% (w/w) of the - 95 -r a d i o l a b e l l e d m a t e r i a l as free f a t t y a c i d as judged by t h i n l a y e r chromatography (vide i n f r a ) . 2.4.3.3 - Sephadex LH-20 Gel E x c l u s i o n Column Chromatography Column chromatography was used to p u r i f y grams of PEG-fatty a c i d e s t e r s and r e l a t e d compounds (PEG-amines and ethers) s i n c e p r e p a r a t i v e HPLC was u n a v a i l a b l e to us. Ion exchange chromatography may have removed f r e e f a t t y acides (57,58) contaminants from our e s t e r p r e p a r a t i o n s but i t would not have removed low molecular weight PEG e s t e r s or f a t t y a l c o h o l contaminants i n e i t h e r p r e p a r a t i o n s . Even though a methanol/H 20 ( 3 v / l v ) s o l v e n t system was as e f f e c t i v e as the ( 5 v / l v ) s o l v e n t system at s o l u b i l i z i n g PEG and PEG-alkyl d e r i v a t i v e s , the l a t t e r was chosen to avoid the p o s s i b i l i t y of m i c e l l a t i o n (Appendix 1) decreasing the e f f i c i e n c y of t h i s procedure. Sephadex G-25 had p r e v i o u s l y been u t i l i z e d i n the f r a c t i o n a t i o n of f a t t y a c i d s o f v a r i o u s molecular weights (58,59). In c o n t r o l s t u d i e s PEG-6000- s t e a r a t e and ^ C - s t e a r i c a c i d were found to e l u t e together o f f of a Sephadex G-25 Fine column under c o n d i t i o n s i d e n t i c a l to those which were s u c c e s s f u l when using Sephadex LH-20. Sephacryl S-200 g e l (Pharmacia) when used with ethanol has a l s o been reported t o be s u i t a b l e f o r s i m i l a r purposes (62,63). Sephadex LH-20 was chosen as i t s 4000 to 5000 d a l t o n e x c l u s i o n l i m i t (16) s u i t e d our needs i d e a l l y . LH-20 chromatography i n chloroform had p r e v i o u s l y been employed by Downey et a l (17,60) to separate various l i p i d s . They reported that s t e a r i c a c i d (C18:0) and l i n o l e i c a c i d (C18:2) elu t e d together, j u s t past the bed volume, but that lower molecular weight - 96 -f a t t y a c i d s were e l u t e d from the column s i g n i f i c a n t l y past the bed volume. This i s probably due to secondary i o n i c i n t e r a c t i o n between the g e l and the f a t t y a c i d s (16,17,60,61). I t may a l s o be due to more p o l a r compounds p a r t i t i o n i n g to a g r e a t e r extent i n t o the g e l s o l v e n t environment s i n c e some phase s e p a r a t i o n o f so l v e n t can occur i n these columns. In F i g u r e 2-5 a sample of PEG 6000-18:2 e s t e r c o n t a i n i n g 1 AC-FFA was chromatographed on an LH-20 column. I t can be seen that the free f a t t y a c i d e l u t e d o f f of the column near the bed volume (1320 ml) while the PEG-and e s t e r group-containing compounds were e l u t e d i n the v o i d volume. PEG 17500- and 1000-fatty a c i d e s t e r s e l u t e d w i t h i n 100 mis of the PEG 6000-esters (not shown). The method was found, over a p e r i o d of two years, t o y i e l d very r e p r o d u c i b l e r e s u l t s . U n d e rivatized PEG and d e r i v a t i z e d PEG could not be separated. PEG-fatty a c i d e s t e r s behaved s i m i l a r l y to PEG-alkyl amines or a l k y l e thers. TLC of PEG 8000-1-^C-palmitate samples p u r i f i e d by t h i s method i n d i c a t e d the e s t e r to be g r e a t e r than 99.9% (w/w) p u r i f i e d of FFA contamination, with b e t t e r than 95% recovery i n the f i r s t 2 l i t r e s of s o l v e n t c o l l e c t e d . Varying the column flow r a t e or the load mix volume by 25% had l i t t l e e f f e c t on the s e p a r a t i o n achieved. Increasing the PEG load mix c o n c e n t r a t i o n from 5.5% to 7% (w/v) s i g n i f i c a n t l y i n c r e a s e d load mix v i s c o s i t y and the base l i n e width o f the PEG column e l u t i o n peak. - 97 -- 98 -2.A.3.5 - Octyl-Sepharose CL-4B Hydrophobic Column Chromatography O c t y l - and Phenyl-Sepharose C1-4B columns (19) have t y p i c a l l y been u t i l i z e d f o r the f r a c t i o n a t i o n and p u r i f i c a t i o n of p r o t e i n s . A f t e r being a p p l i e d onto the column the p r o t e i n s are d i f f e r e n t i a l l y e l u t e d by gently a l t e r i n g the st r e n g t h of t h e i r hydrophobic i n t e r a c t i o n w i t h the column v i a e l u t i o n w i t h b u f f e r s o f lower i o n i c s t r e n g t h , b u f f e r s c o n t a i n i n g c h a o t r o p i c ions or b u f f e r s c o n t a i n i n g amphipathic nonion i c PEG-hydrocarbon detergents such as Tween, B r i j or B e r o l (19,55,64). In the l a t t e r case the hydrophobic detergent molecules d i s p l a c e the p r o t e i n s from the hydrophobic matrix. Of the two g e l types, Octyl-Sepharose C1-4B i s more hydrophobic than Phenyl-Sepharose C1-4B (19). Fo l l o w i n g Sephadex LH-20 s i z e e x c l u s i o n column chromatography our product mixture c o n s i s t s of PEG and PEG-derivatives. The f a c t that amphipathic PEG-fatty a c y l detergents such as Tween possess a strong a f f i n i t y f o r O c t y l - and Phenyl-Sepharose C1-4B g e l s suggested to us th a t hydrophobic chromatography could be used to separate the f a t t y a c y l d e r i v a t i z e d and u n d e r i v a t i z e d PEG molecules (19,55,64-66). Such a se p a r a t i o n was d e s i r a b l e i n case the u n d e r i v a t i z e d PEG contaminants hindered f u r t h e r chemical m o d i f i c a t i o n of the d e r i v i t i z e d molecules (50) or c r i t i c a l m i c e l l e c o n c e n t r a t i o n determinations (67, Appendix 1). We decided t o use Octyl-Sepharose C1-4B i n order to o b t a i n as strong a hydrophobic i n t e r a c t i o n as p o s s i b l e . Due to the s t a b i l i t y of the PEG molecules i n v o l v e d we d i d not have to worry about the harshness of the eluent c o n d i t i o n s used. - 99 -The r e s u l t of a t y p i c a l Octyl-Sepharose column run i s shown i n Figure 2-6. In t h i s run, 7 g of a PEG 6000 and PEG 6000-16:0-l- 1 AC pa l m i t a t e e s t e r mixture were separated on the column. Recovery of the PEG and the r a d i o a c i t i v i t y was greater than 95%. Before chromatography the mixture contained approximately one e s t e r i f i e d PEG molecule i n 60. The methanol/K^O ( 5 v / l v ) s o l v e n t e l u t e d a mixture which contained approximately 46 e s t e r i f i e d molecules i n 60. We d i d not determine the maximal c a p a c i t y o f the column. However samples c o n t a i n i n g 200 mg of l a b e l l e d e s t e r r e t a i n e d a l l of the e s t e r on the column during aqueous e l u t i o n . Use of an Octyl-Sepharose C1-4B column and a methanol/H 20 gradient or p o l y s t y r e n e r e s i n beads (Bio-Beads SM-2 from Biorad L a b o r a t o r i e s ) (64) to separate mono- and d i - d e r i v i t i z e d PEG molecules might be p o s s i b l e . However no experiments of t h i s type were performed because the use o f mono-methyl e t h e r i f i e d PEG and low ( l e s s than 20%) y i e l d s y n t h e s i s r e a c t i o n s guaranteed a predominance of mono-derivatized PEG i n our samples. The p o s s i b l i t y of e l i m i n a t i n g the LH-20 Sephadex column chromotography step from our e s t e r p u r i f i c a t i o n procedure was examined. Experiments with l a b e l l e d f r e e f a t t y a c i d s (FFA) i n d i c a t e d that under the c o n d i t i o n s given the e s t e r and the a c i d were e l u t e d together. Eluent g r a d i e n t s might have made t h i s s e p a r a t i o n p o s s i b l e but the f a c t t h a t a complete s e p a r a t i o n of the FFA and the e s t e r was r e q u i r e d f o r our p a r t i t i o n experiments s t r o n g l y supported the use of both columns. I t should be noted that Octyl-Sepharose hydrophobic a f f i n i t y chromatography was used s u c c e s s f u l l y to p u r i f y long hydrocarbon t a i l e d PEG-fatty a c i d e s t e r s , ethers and amines. PEG d e r i v a t i v e s with short RADIOACTIVITY (CPM) PERCENT OF TOTAL RECOVERED $ - 101 -hydrocarbon t a i l s may r e q u i r e the use of acyl-agaroses of stronger hydrophobic nature than we have used such as those produced by H a l p e r i n and S h a l t i e l (68). The use of a f f i n i t y chromatography to p u r i f y a v a r i e t y o f polymer a f f i n i t y p a r t i t i o n l i g a n d s (50,69,70) should be p o s s i b l e . 2.4.5.5 - Nessler's Reagent Assay For Peg Containing Compounds The use of Nessler's reagent f o r the q u a l i t a t i v e d e t e c t i o n of PEG i n aqueous s o l u t i o n s was f i r s t r e p o r t e d by Poison et a l (71). Ingham and Ling developed the technique f o r the simple r a p i d q u a n t i t a t i v e determination of PEG (20). The s i m p l i c i t y and accuracy of the assay suggested i t over more commonly c i t e d and complex methods f o r monitoring PEG compounds i n our column eluent f r a c t i o n s (20,73,74). Nessler's reagent c o n s i s t s of a double i o d i d e of potassium and mercury (HgI 2-KI) i n a l k a l i n e s o l u t i o n . I t i s commonly used i n the c l i n i c a l determination of serum nonprotein n i t r o g e n (urea, amino a c i d s , ammonia e t c . ) by the K j e l d a h l - N e s s l e r method (21,22). When a PEG-containing s o l u t i o n i s added to Nessler's reagent a t u r b i d orange suspension forms. This r e a c t i o n i s b e l i e v e d to i n v o l v e the formation of i n s o l u b l e m u l t i d e n t a t e c o l l o i d complexes when the polyether oxygens are exposed to the mercuric c a t i o n s (21,22). The assay procedure given above was modified from reference 20. I t was very accurate over the range 0 to 50 mg/1 PEG 6000 i n N e s s l e r ' s reagent, as shown by the standard curve (Figure 2-7) and y i e l d e d s i m i l a r r e s u l t s f o r normal or d e r i v a t i z e d PEG 1000, PEG 6000 and PEG 17500 as w e l l as such PEG c o n t a i n i n g compounds such as T r i t o n X-100, Tween 80 and P l u r o n i c P-123 (74). - 103 -2.A.3.6 ,- Assay Of Radioactive Compounds 14 R a d i o l a b e l e d - C compounds were assayed as described p r e v i o u s l y . In some cases 0.5 uCi of a s u i t a b l e r a d i o l a b e l e d f a t t y a c i d was added t o an otherwise n o n - r a d i o a c t i v e PEG-fatty a c i d e s t e r product mixture p r i o r to i t s column chromatography i n order to determine the e l u t i o n p r o f i l e of any u n e s t e r i f i e d f a t t y a c i d i n the mixture (Figure 2-5). Synthesis of PEG-derivatives u t i l i z i n g r a d i o l a b e l e d compounds o f known s p e c i f i c a c t i v i t y provides a very easy method of determining PEG d e r i v a t i z a t i o n . Generally speaking the determination i s c a r r i e d out a f t e r Sephadex LH-20 column chromatography has removed most of the free compound and TLC has been used to estimate the amount of free m a t e r i a l remaining i n the PEG sample. Assuming the s p e c i f i c a c t i v i t y o f the f r e e and PEG-bound compound i s the same and knowing the molecular weight of the PEG and the e f f i c i e n c y of the counting technique employed, one can estimate the % s u b s t i t u t i o n from the r a d i o a c t i v i t y per u n i t weight of the PEG/PEG-derivative mixture. Thus cpm/g i s r e c a l c u l a t e d as dpm/g and f i n a l l y as dpm/millimole of the o r i g i n a l f r e e l i g a n d m a t e r i a l to o b t a i n a primary estimate of the % s u b s t i t u t i o n , as f o l l o w s : s p e c i f i c a c t i v i t y PEG-ester = 1.20 x 1 0 9 dpm/millimole PEG (6650 MW) s p e c i f i c a c t i v i t y f a t t y a c i d 1.23 x 10 1 0dpm/millimole (5.9mCi/mMole) = 9.8% PEG molecules s u b s t i t u t e d = 4.9% PEG end groups s u b s t i t u t e d The % s u b s t i t u t i o n i s then r e c a l c u l a t e d using a b e t t e r estimate of the average MW of the PEG/PEG-derivative mixture as o u t l i n e d p r e v i o u s l y above. S u b s t i t u t i o n estimates obtained i n t h i s manner f o r PEG-fatty a c i d e s t e r s match those d e r i v e d from the hydroxamic e s t e r assay. - 104 -2.4.3.7 - PEG-Fatty Acid E s t e r UV Absorption PEG-fatty a c i d e s t e r u l t r a v i o l e t absorption s t u d i e s can be summarized as f o l l o w s : (A) The UV absorption of p u r i f i e d PEG 6000 i s n e g l i g i b l e (Figure 2-8). (B) The UV absorption of PEG 6000-18:2 f a t t y a c i d e s t e r i s a p p r e c i a b l e with peak absorption at 280 nm and 230 nm (Figure 2-8). (C) The 230 and 280 nm UV a b s o r p t i o n bands of PEG 6000-18:2 f a t t y a c i d e s t e r appear to be due to a chromophoric group(s) s i n c e over the c o n c e n t r a t i o n range 0.05 mM to 0.50 mM i n water t h e i r absorption i s d i r e c t l y p r o p o r t i o n a l ( r = 0.999 i n both cases) to the e s t e r c o n c e n t r a t i o n and hence f o l l o w s the Lambert-Beer Law (75). The molar ab s o r b t i o n c o e f f i c i e n t s were estimated as 1400 at 280 nm and 4200 at 230 nm. (D) UV absorption i s present when the e s t e r i s d i s s o l v e d i n e i t h e r water or methanol/H 20 ( 5 v / l v ) solvent though i n water the peaks are s h i f t e d approximately 4 nm t o longer wavelengths (Figure 2-8). (E) The UV absorption spectrum of PEG 6000 18:2 e s t e r i s somewhat l i k e t hat of the 18:2 free f a t t y a c i d ' s . However, at 0.2 mM the e s t e r posesses the same 280 nm a b s o r p t i o n as 20.0 mM 18:2 f r e e f a t t y a c i d (FFA) and the same 230 nm abs o r p t i o n as 6.0 mM FFA i n methanol/H 20 ( 5 v / l v ) solvent (Figure 2-9) (F) PEG 6000 and 18:2 FFA i n combination, or 18:2 f a t t y a c i d methyl e s t e r , or PEG 6000 and 18:2 f a t t y a c i d methyl e s t e r i n combination, at comparable con c e n t r a t i o n s do not possess the same absorption spectrum Figure 2-8. Figure 2-9. WAVELENGTH (nm) WAVELENGTH (nm) Figures 2 -8 and 2-9. U l t r a v i o l e t absorption of PEG 6000-fatty a c i d esters and r e l a t e d compounds. ( Unless s t a t e d the solvent used was methanol/H 00 ( 5 v / l v ) ) . - 106 -as the e s t e r (Figure 2-9). (G) The s p e c t r a of the PEG 6000-18:0 and -18:1 e s t e r are s i m i l a r to the PEG 6000-18:2 spectrum but the molar absorbtion c o e f f i c i e n t s decrease with s a t u r a t i o n ( F i g u r e 2-8). PEG 6000-18:3 and -20:4 e s t e r s e x h i b i t much greater a b s o r p t i o n at 280 nm than PEG 6000-18:2 (not shown). I t would appear that e s t e r i f y i n g free f a t t y a c i d s to PEG d r a m a t i c a l l y increases t h e i r UV a b s o r p t i o n . The nature of the e f f e c t i s not understood. I t could be due to the formation of m i c e l l e s r e s u l t i n g i n a proximal l o c a l i z a t i o n of PEG head or f a t t y a c y l t a i l group. However, the formation of m i c e l l e s of PEG 6000-18:2 e s t e r at 50 /uM c o n c e n t r a t i o n i n methanol/H 20 so l v e n t ( 5 v / l v ) does not appear very l i k e l y (see Appendix 1). Although the molecular b a s i s f o r the a p p r e c i a b l e 280 nm absorbtion of PEG unsaturated f a t t y a c i d e s t e r s i s c u r r e n t l y unknown, t h i s absorbance does a l l o w us to e a s i l y determine t h e i r column eluent p r o f i l e s . At present we do not know i f changes i n absorption s p e c t r a occur f o l l o w i n g covalent bonding of f a t t y a c y l groups to PEG i n ether and amine l i n k e d d e r i v a t i v e s . 2.4.3.8 - Hydroxamic A c i d PEG-Ester Assay E s t e r s , anhydrides, a c i d c h l o r i d e s and other h y d r o l y z a b l e d e r i v a t i v e s o f c a r b o x y l i c a c i d s react with hydroxylamine under a l k a l i n e c o n d i t i o n s to form hydroxamic a c i d s which under a c i d c o n d i t i o n s w i l l r e a c t q u a n t i t a t i v e l y with f e r r i c i o n s t o form a orange-brown chromophoric c h e l a t e . This r e a c t i o n has been used as the b a s i s f o r a number of r a p i d , - 107 -s e n s i t i v e , q u a n t i t a t i v e assays and i s i d e a l f o r s m a l l samples c o n t a i n i n g a s i n g l e r e a c t i v e substance (23,24,76,77). The b a s i c e s t e r r e a c t i o n i s as f o l l o w s : RC00R* + NH20H 0 H ~ ^ RC0NH0H + R'OH RCONHOH + F e + 3 H + , Orange Brown Chelate A l k a l i n e h y d r o l y s i s c o n d i t i o n s are re q u i r e d so th a t the hydroxylamine w i l l r e a c t n u c l e o p h i l i c a l l y with, the e s t e r ' s c a r b o x y l i c carbon. Acid c o n d i t i o n s are req u i r e d to s t a b i l i z e the i r o n i n the f e r r i c s t a t e . Goddu et a l - (24) have st u d i e d a number o f r e a c t i o n v a r i a b l e s i n c l u d i n g the e f f e c t s o f va r i o u s s o l v e n t s . The major problem faced i n adapting t h i s assay to our needs l a y i n developing a p r o t o c o l capable of maintaining the various compounds i n r e a c t i v e s o l u t i o n . I t was found t h a t the assay tubes could be st o r e d f o r up to 48 hours at 4°C before reading and that c o l o r i n t e n s i t y improved on storage. Acid c h l o r i d e s may r e a c t i n t h i s assay (75). However, the presence o f u n e s t e r i f i e d PEG or f r e e f a t t y a c i d s do not i n t e r f e r e unless present i n the r e a c t i o n tube i n unusually high c o n c e n t r a t i o n s (10 mM). Methyl-ester standard curves were i d e n t i c a l independent of a c y l group. Methyl e s t e r standards could be s t o r e d f o r up to one year at 4°C p r o v i d i n g care was taken to prevent solvent evaporation. A t y p i c a l assay standard curve i s shown i n Figure 2-10. This "curve" represents the average o f four assays performed using d i f f e r e n t m e t h y l - f a t t y a c i d e s t e r standards over a period of one year. In s p i t e of such r e p r o d u c i b i l i t y f r e s h standards were made up f o r each assay. ESTER CONCENTRATION (mM) IN SAMPLE ASSAYED F i g u r e 2-10. S tandard curve f o r m e t h y l - f a t t y a c i d e s t e r i n hydroxamic a c i d e s t e r assay. - 109 -The hydroxamic e s t e r assay r e s u l t s i n an estimate of the molar c o n c e n t r a t i o n of e s t e r bonds i n a PEG-fatty a c i d ester/PEG mixture d i s s o l v e d i n methanol/hyD. Assuming the d e n s i t y o f the PEG/PEG e s t e r mixture i s equal to that of the corresponding PEG, knowing the weight of the s o l v e n t the mixture was added tOj and the s o l v e n t density^ the volume of each sample i t s PEG c o n c e n t r a t i o n and the t o t a l number of e s t e r bonds present can be c a l c u l a t e d . This number can be converted t o e s t e r bonds per gram of m a t e r i a l which can be averaged f o r a l l the samples. This average can then be compared to the number of PEG molecules i n a gram of PEG-ester/PEG mixture as c a l c u l a t e d from the molecular weight of the PEG determined by high pressure l i q u i d chromatography. The % s u b s t i t u t i o n so c a l c u l a t e d w i l l only be ah estimate because i t does not a l l o w f o r the e f f e c t o f mono- and d i - s u b s t i t u t i o n of some of the PEG molecules on the mixture's average molecular weight. However from t h i s f i r s t estimate of the % s u b s t i t i o n a second estimate can be c a l c u l a t e d which takes i n t o account the probable degree of mono- and d i - s u b s t i t u t i o n i n the sample (see previous s e c t i o n on PEG-derivative s y n t h e s i s ) . I f necessary a t h i r d i t e r a t i o n can be undertaken and u n t i l a c o n s i s t e n t estimate of the s u b s t i t u t i o n and average MW of the PEG-ester/PEG mixture i s obtained. 2.A.3.8 - Example C a l c u l a t i o n : PEG 6000 - 18:2 L i n o l e a t e E s t e r i f i c a t i o n The d e n s i t y of various PEG'S has been estimated g r a v i m e t r i c a l l y to be 1.20 g/ml at 20° C f o r PEG 4000 and PEG 6000 and 1.10 g/ml at 20° C f o r PEG 1000 (78,79). We have estimated the d e n s i t y of methanolAtyD ( 5 v / l v ) s o l v e n t t o be 0.848 g/ml at 20° C. Dr. M i l t o n H a r r i s has - 110 -estimated the average molecular weight of va r i o u s PEG f r a c t i o n s determined v i a s t e r i c e x c l u s i o n HPLC (50,80,38) to be as f o l l o w s : PEG 6000 = (6650 + 3%), PEG 4000 = (3350 + 3%) and PEG 1000 = (1000 + 4%) - values very c l o s e to those reported by other sources (19,64). Five samples of a PEG 6000-18:2 e s t e r mixture were assayed as possessing an average of (1.90 _+ 8%) x 10 m i l l i m o l e s o f e s t e r i f i e d groups per gram. The degree o f s u b s t i t u t i o n i s c a l c u l a t e d as f o l l o w s . 1 g PEG 6000 (6650 + 3% MW) = (15.04 + 3%) x IO" 2 m i l l i m o l e s PEG = (30.08 + 3%) x IO" 2 m i l l i m o l e s o f end groups % s u b s t i t u t i o n f i r s t estimate = (1.90 + 8%) x 100% = 6 . 3 2 + 1 1 % (30.08 + 3%) By p r o b a b i l i t y c o n s i d e r a t i o n s o u t l i n e d p r e v i o u s l y i n t h i s chapter of t h i s t h e s i s the % of PEG molecules d i - e s t e r i f i e d = (0.40 + 0.09) %, mono-esterified = (11.84 +_ 1.56)%, and u n e s t e r i f i e d = (87.76 + 1.40) %. So the average MW estimate of the mixture assayed i s (0.0040 +_ 22 %) x (7175 + 3 %) + (0.1184 + 13 %) x (6913 + 3 %) + (0.8776 + 1.6 %) x (6650 + 3 %) = (6683 + 6.1 %) g based on the MW of 18:2 FFA as 280.5 g. 1 g of PEG/PEG-ester mixture (14.96 + 6 %) x I O - 2 m i l l i m o l e s PEG = 29.92 _+ 6 %) x I O - 2 m i l l i m o l e s end groups % s u b s t i t u t i o n second estimate = (1.90 + 8 %) x 100% = 6.35 % _+ 14 % (29.92 + 6 %) I t can be seen that the f i r s t estimate was s u f f i c i e n t l y accurate f o r our purposes and no f u r t h e r e s t i m a t i o n of MW or s u b s t i t u t i o n i s r e q u i r e d . This mixture would be described as a PEG 6000-18:2/PEG 6000 mixture o f - I l l -average MW (6683 +_ 6 %) . which was (11.8 _+ 1.6)% mono-esterified, (0.4 + 0.1)% d i - e s t e r i f i e d and (87.8 + 1.4)% unesterified PEG. Thus (12.2 + 1.6)% of the PEG molecules are e s t e r i f i e d . In general the percentage of substituted molecules i n a mixture decreased d r a s t i c a l l y after solvent extracion with ethanol,or with acetone followed by ether. This presumably was due to the greater s o l u b i l i t y of contaminating lower MW e s t e r i f i e d PEG molecules. Values for various LH-20 column p u r i f i e d PEG-fatty acid esters used i n our work are given i n Table 2-2. It can be seen that d i - e s t e r i f i e d molecules were estimated to be only a small proportion of each of our PEG-ester mixture, with the exception of a d i e s t e r i f i e d sample custom produced commercially by the extensive substitution of PEG. 2.4.3.9 - Thin Layer Chromatography Thin layer chromatography (TLC) procedures were i n i t i a l l y developed to test the purity of non-radioactive PEG-fatty acid ester preparations with regard to free fatty acid contamination. Other methods considered included HPLC, infrared spectroscopy (IR) and gas l i q u i d chromatography. However, they were rejected on the grounds of lack of a suitable apparatus, overlap of ester and acid main IR absorbtion bands, and the non-volatile nature of PEG. TLC was chosen due to i t s ease a v a i l a b i l i t y , inexpensiveness and proven c a p a b i l i t y . TLC also provided a good way to is o l a t e for quantitation^ the amount of radioactive free fatty acid i n a sample of labelled PEG-fatty acid ester. I n i t i a l l y an organic solvent system was sought which would separate PEG-fatty acid esters and free - 11.2 -Table 2-2 _______ Q % S u b s t i t u t i o n of Various PEG-Fatty Acid Ester Compounds Used In Our Work  As Determined By Hydroxamic Acid Ester Assay. Ester % % % % Average End Groups Mono-ester- D i - e s t e r i - E s t e r i - moleculai e s t e r i f i e d f i e d PEG f i e d PEG f i e d PEG weight molecules molecules molecules PEG 1000-18:1 25.6 38.1 6.5 44.6 1135 PEG 1000-18:1-12-0H 24.1 36.6 5.8 42.4 1136 PEG 4000-18:1 16.8 27.9 2.8 30.7 3446 PEG 6000-12:0 4.0 7.6 0.2 7.8 6664 PEG 6000^14:0 8.2 15.0 0.7 15.7 6691 PEG 6000-16:0 9.9 17.9 1.0 18.9 6698 PEG 6000-16:1 4.0 7.7 0.2 7.9 6666 PEG 6000-18:0 15.7 26.4 2.5 28.9 6734 PEG 6000-18:1 13.6 23.5 1.8 25.3 6712 PEG 6000-18:2 6.3 11.8 0.4 12.2 6683 PEG 6000-18:3 7.5 13.9 0.6 14.5 6690 PEG 6000-20:4 12.2 12.5 1.5 23.0 6695 PEG 6000-18:1 ( D i - e s t e r i f i e d ) 23.4 - 23.4 23.4 6774 A l l PEG-esters were LH-20 column p u r i f i e d and assayed as described i n the t e x t . In a l l cases except PEG - 18:2 (see previous page) the e r r o r associated with the % s u b s t i t u t i o n or molecular weight estimate was l e s s than or equal to 0.1 of the stated value. The d i - e s t e r i f i e d ester was made commerically and mixed with u n e s t e r i f i e d PEG p r i o r to being p u r i f i e d and assayed. - 113 -f a t t y a c i d s . A number were t r i e d on a v a r i e t y of s i l i c a g e l p l a t e s . The system o f choice i s methanol/h^O (5v / l v ) which e x h i b i t e d PEG 6000 and PEG 6000-fatty a c i d e s t e r s Rf's = 0.08 and long chain f a t t y a c i d Rf's = 0.85. In a t y p i c a l TLC of Sephadex LH-20 column chromatographed PEG 6000-l-" L 4C-palmitate e s t e r , 96.4% o f the r a d i o a c t i v e molecules (CPM's) a p p l i e d onto the TLC p l a t e were recovered i n the f i r s t t h i r d of the p l a t e and 1.8% recovered i n each of the remaining s e c t i o n s . The middle t h i r d was b e l i e v e d to represent low molecular weight PEG-esters and the top t h i r d f r e e f a t t y a c i d molecules. The p u r i f i e d e s t e r p r e p a r a t i o n thus appeared t o be 98.2 % PEG-fatty a c i d e s t e r on a mole b a s i s and 99.9 % on a weight b a s i s with minor contamination by small molecular weight e s t e r s . 2.4.4 - P r e p a r a t i o n And P a r t i t i o n of D i m y r i s t o y l P h o s p h a t i d y l c h o l i n e  Liposomes The e l u t i o n p r o f i l e s o f son i c a t e d DMPC liposomes obtained using a sepharose 4B column (Figure 2-11) were s i m i l a r to those reported by Huang (81) and others (27,31,82). The sma l l e r f i r s t peak which e l u t e d i n the vo i d volume, was b e l i e v e d to c o n s i s t of a mixture of m u l t i l a m e l l a r liposomes o f high molecular weight. The l a r g e r second peak supposedly c o n s i s t s o f s m a l l , u n i l a m e l l a r v e s i c l e s (SUV's) with anhydrous weights of 1.9-2.0 x 10 6 daltons and diameters, at 20° C, of 19-20 nm (127). Other, unconfirmed, p h y s i c a l p r o p e r t i e s of such DMPC SUV's can be found i n reference 27. This data leads to estimates o f approximately 2900 DMPC molecules, (of molecular weight 661) per liposome. Approximately 4.2 x 10~4 moles of DMPC were used i n each experiment. I f we assume, from the - 114 -4B peak d i s t r i b u t i o n s of l a b e l l e d - f a t t y a c i d j that 90% of the DMPC used was inc o r p o r a t e d i n t o SUV's, then our second peak might be estimated to co n t a i n 8 x IO"*"6 liposomes. I n c o r p o r a t i o n of ^ C-DMPC i n t o sonicated liposomes was 99.9%, only 0.1% of the t o t a l CPM's passing through an Amicon CF-25 f i l t e r cone. S i m i l a r l y , ^ H - p a l m i t i c a c i d CPM's e l u t e d from the Sepharose 4B column a f t e r the SUV peak. This phenomena was not observed with "^C-DMPC. Even d i s t r i b u t i o n of l a b e l l e d l i p i d through the sm a l l u n i l a m e l l a r v e s i c l e s was, i n a l l cases, suggested by the l i n e a r i t y of the 4B column f r a c t i o n ' s t u r b i d i t y versus r a d i o a c t i v i t y graph (not shown). Assuming that l a b e l l e d l i p i d was evenly d i s t r i b u t e d one can estimate that on the average 2 _2 l a b e l l e d DMPC molecules or 3.4 x 10 l a b e l l e d p a l m i t i c a c i d molecules were i n c o r p o r a t e d i n t o each liposome. The approximate c o n c e n t r a t i o n o f liposomes i n the p a r t i t i o n system load mixes can be c a l c u l a t e d as f o l l o w s . I f i t i s c o n s e r v a t i v e l y estimated t h a t the two peak f r a c t i o n s from the SUV peak o f the Sepharose 4B column c o n t a i n 30% of the prepared liposomes, then 6ml from t h i s 10ml pool w i l l c o n t a i n approximately 18% of the t o t a l number of liposomes or 1.4 x IO"*"6 liposomes. The load mix thus contained approximately 1 x 10"^ liposomes/ml and each t e s t tube contained 2.5 times t h i s number o f liposomes. I t should be noted t h a t i n experiments i n v o l v i n g (5,3.5) V systems the amount o f column e l u e n t added to the load mix was halved i n order to not ov e r l y d i l u t e the system. The experimental procedure o u t l i n e d above y i e l d s very r e p r o d u c i b l e p a r t i t i o n r e s u l t s . One experiment i n which we added ten times the amount of l a b e l l e d l i p i d t o the liposomes and t r i e d to decrease the liposome c o n c e n t r a t i o n i n the phase system to 5 -•115 -- 116 -x 10 liposomes/ml r e s u l t e d i n v a r i a b l e r e s u l t s . C o n t r o l experiments i n v o l v i n g the p a r t i t i o n of l a r g e m u l t i l a m e l l a r v e s i c l e s (LMV's), taken from the f i r s t CL-4B column peak f r a c t i o n s , were performed i n a s i m i l a r manner to those described above. The s m a l l e r number of m u l t i l a m e l l a r liposomes o b t a i n a b l e l i m i t e d the number of p a r t i t i o n s t u d i e s which could be performed. CHAPTER 3. TWO PHASE SYSTEM PARTICLE PARTITION 3.1 - INTRODUCTION This chapter d i s c u s s e s p a r t i t i o n experiments done i n c o n j u n c t i o n with those, presented i n chapter 4, p e r t a i n i n g to the d i f f e r e n t i a l p a r t i t i o n of m u l t i p l e s c l e r o s i s and normal s u b j e c t s ' e r y t h r o c y t e s . These c o l l a b o r a t i v e experiments were performed i n order to b e t t e r understand the f a c t o r s and v a r i a b l e s a f f e c t i n g c e l l p a r t i t i o n . They are q u i t e d i v e r s e i n scope and i n c l u d e s t u d i e s concerned with the p a r t i t i o n of liposome model membranes, as w e l l as measurements of system bulk phase e l e c t r o s t a t i c p o t e n t i a l s . Due t o such d i v e r s i t y separate r e s u l t s and d i s c u s s i o n have been provided f o r each major s e c t i o n and subsection. 3.2 - TWO PHASE SYSTEM CHARACTERIZATION - RESULTS AND DISCUSSION A l t e r a t i o n o f two phase system p r o p e r t i e s with polymer and s a l t composition, s e t t l i n g temperature and other r e l e v a n t v a r i a b l e s has been discussed by a number of d i f f e r e n t authors i n c l u d i n g A l b e r t s s o n ( 1 ) , Johansson ( 2 ) , Walter (3,4), Bamberger et a l (5,6) and Sharp (7). For example, Bamberger et a l have noted a d i r e c t r e l a t i o n s h i p between phase system polymer c o n c e n t r a t i o n and i n t e r f a c i a l t e n s i o n (5) and the f a c t t h a t both t e n s i o n and phase polymer p a r t i t i o n are i n c r e a s e d i n Na phosphate-containing as compared to NaCl-containing two phase systems. A l t e r a t i o n of one system v a r i a b l e may appreciably a f f e c t a number of system p r o p e r t i e s and hence the p a r t i t i o n behaviour of a s p e c i f i c p a r t i c l e - 118 -or s o l u t e . I t i s perhaps l e s s complicated to group such a l t e r a t i o n s according t o e f f e c t ( i . e . a l t e r e d i n t e r f a c i a l t ension) r a t h e r than cause ( a l t e r a t i o n i n polymer c o n c e n t r a t i o n or average molecular weight, a l t e r a t i o n i n system s a l t c o n c e n t r a t i o n or s e t t l i n g temperature), although i n p r a c t i c e one must be aware of the myriad p o s s i b l e e f f e c t s of phase system a l t e r a t i o n s (vide i n f r a ) . One of the e f f e c t s o f t e n overlooked i s that of a c e r t a i n system a l t e r a t i o n on the i n t e r a c t i o n between the p a r t i t i o n e d m a t e r i a l and the phase polymers. This i s e s p e c i a l l y important i n the case of a f f i n i t y p a r t i t i o n i n g . 3.2.1 - E l e c t r o s t a t i c Bulk Phase P o t e n t i a l s Table 3-1 presents the e l e c t r o s t a t i c p o t e n t i a l s a s s o c i a t e d with the two phase systems described at the beginning of Chapter 2 (Table 2-1). I t should be noted t h a t : (A) S l i g h t v a r i a t i o n s i n p o t e n t i a l s occur between systems made with d i f f e r e n t polymer l o t s . (B) There was an a p preciable p o t e n t i a l e x h i b i t e d by systems c o n t a i n i n g no added s a l t s . This may be due to s a l t contamination of the polymers themselves (5,6). A d d i t i o n of as l i t t l e as 10 mM NaCl to the " s a l t f r e e " systems e l i m i n a t e d such p o t e n t i a l s while adding 10 mM Na 2HP0 4/NaH 2P0 4 (Na phosphate), pH 7.2 increased the p o t e n t i a l s . (C) No appreciable p o t e n t i a l was found i n any of the 0.15 M NaCl type V/G or #5 two phase systems at any polymer c o n c e n t r a t i o n . Decreasing the polymer c o n c e n t r a t i o n i n a (5,4) I/A system to - 119 -(5,3.5) s l i g h t l y decreased the p o t e n t i a l i n keeping w i t h the r e s u l t s o f Walter et a l ( 3 ) . (D) The e f f e c t s of 10 pM PEG 6000-fatty a c i d e s t e r or 286 pM f r e e f a t t y a c i d and 56 mM ethanol on two phase system e l e c t r o s t a t i c i n t e r f a c i a l p o t e n t i a l s appear to be n e g l i g i b l e , i n keeping w i t h t h e i r e f f e c t on phase system i n t e r f a c i a l t e n s i o n s (Appendix 1 ) . Walter et a l . (8) r e p o r t e d t h a t PEG 6000-palmitate had no e f f e c t on the n e g l i g i b l e p o t e n t i a l found i n a (5,4) #5 system (Table 3-1). (E) Figure 3-1 i n d i c a t e s the p o t e n t i a l s a s s o c i a t e d w i t h the pH 6.8 and pH 7.2 systems c o n t a i n i n g dextran l o t 7693 and PEG l o t M68546 from Table 3-1. For both s e r i e s o f two phase systems the p o t e n t i a l s appear t o decrease w i t h i n c e a s i n g NaCl c o n c e n t r a t i o n . The s l i g h t l y higher p o t e n t i a l s a s s o c i a t e d w i t h the pH 7.2 systems are probably due to t h e i r higher c o n c e n t r a t i o n o f the more asymmetrically p a r t i t i o n i n g ^ 2 ^ 0 ^ (1,2). (F) The r a t i o s o f the p o t e n t i a l s i n the (5,4) I compared t o the (5,4) I I or the (5,4) V systems do not appear to vary w i t h polymer l o t suggesting t h a t the r e l a t i o n i n d i c a t e d i n F i g u r e 3-1 holds f o r systems c o n t a i n i n g other dextran o r PEG l o t s . (G) There was no v a r i a t i o n i n the n e g l i g i b l e p o t e n t i a l o f the (5,4) v systems when the temperature was v a r i e d between 18 and 26° C. (H) A system c o n t a i n i n g (5,4) 7.3 mM Na 2HP0 4, 2.3 mM N a h y ^ e x h i b i t e d a 2 m i l l i v o l t p o t e n t i a l which was very s i m i l a r to th a t e x h i b i t e d by the (5,4) I system which c o n t a i n s f i f t e e n times the phosphate c o n c e n t r a t i o n . This may have been i n p a r t due to s a l t s i n the polymers (see B above). Table 3-1. E l e c t r o s t a t i c Bulk Phase P o t e n t i a l s Of Various Two Phase Systems System E l e c t r o s t a t i c Bulk Phase P o t e n t i a l ( m i l l i v o l t s ) Code Comment Na 2P N a i P NaCl MOPS D l o t 7830 D l o t 7830 D l o t 7830 D l o t 16027 D l o t 7693 mM mM mM mM PEG l o t PEG l o t PEG l o t PEG l o t PEG l o t B529-9104 B529-9104 B688-0232-2 B688-0232-2 M68546 P u r i f i e d (5,3.5) I/A 109 35 0 0 1.93 + .21 N. D. N. D. N. D. N. D. (5,3.5) V/G 7.3 2.3 150 0 0.23 + .04 N. D. N. D. N. D. N. D. (5,4) 0 0 0 0 1.35 + .04 1.33 + .01 N. D. N. D. N. D. (5,4) 7.3 2.3 0 0 2.26 + .03 N. D. N. D. N. D. N. D. (5,4) 0 ^ 0 10 0 0.23 + .04 N. D. N. D. N. D. N. D. (5,4) pH 7.4 7.0 1.3 3.0 0 1.83 + .11 N. D. N. D. N. D. N. D. (5,4) I/A 109 35 0 0 2.23 + .13 2.09 + .10 2.39 + .16 2.10 + .13 1.80 + .10 (5,4) I/A + ESTER 109 35 0 0 2.08 + .09 N. D. N. D. N. D. N. D. (5,4) I/A + FFA 109 35 0 0 2.14 + .04 N. D. N. D. N. D. N. D. (5,4) B 91 29 25 0 N. D. N. D. N. D. N. D. 1.48 + .07 (5,4) II/C 73 23 50 0 1.93 + .08 N. D. 1.68 + .04 1.66 + .05 1.35 + .08 (5,4) D 55 17 75 0 N. D. N. D. N. D. N. D. 1.14 + .05 (5,4) E 34 11 100 0 N. D. N. D. N. D. N. D. 0.78 + .03 (5,4) F 18 6 125 0 N. D. N. D. N. D. N. D. 0.50 + .03 (5,4) V/G 7.3 2.3 150 0 0.28 + .14 0.27 + .04 0.28 + .04 0.08 + .04 0.08 + .04 (5,4) V/G + ESTER 7.3 2.3 150 0 0.22 + .06 N. D. N. D. 0.06 + .05 N. D. (5,4) V/G MOPS 0 0 150 10 0.18 + .07 N. D. N. D. N. D. N. D. (7,4) V/G 7.3 2.3 150 0 0.20 + .04 N. D. N. D. N. D. N. D. (5,4) #1, pH 6.8 55 55 0 0 N. D. N. D. N. D. N. D. 1.57 + .16 (5,4) #2, pH 6.8 45 45 30 0 N. D. N. D. N. D. N. D. 1.44 + .09 (5,4) #3, pH 6.8 30 30 75 0 N. D. N. D. N. D. N. D. 0.65 + .06 (5,4) #4, pH 6.8 15 15 120 0 N. D. N. D. N. D. N. D. 0.26 + .04 (5,4) #5, pH 6.8 5 5 150 0 N. D. N. D. N. D. N. D. 0.05 + .05 Data i n d i c a t e s mean _+ SD of ten or more determinations at 22°C. Top phase was always p o s i t i v e r e l a t i v e to bottom phase. Unless s p e c i f i e d