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Crystal growth of monosodium urate monohydrate Dutt, Yougesh Chander 1985

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CRYSTAL GROWTH OF MONOSODIUM URATE MONOHYDRATE BY YOUGESH CHANDER DUTT Diploma i n Pharmacy, Panjab U n i v e r s i t y , 1969 B. Pharm., Panjab U n i v e r s i t y , 1973 . Pharm.(Pharmaceutics), Panjab U n i v e r s i t y , 197 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( F a c u l t y of Pharmaceutical Sciences) D i v i s i o n o f Pharmaceutics We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA © Yougesh Chander Dutt, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) A B S T R A C T H y p e r u r i c e m i a and l o c a l t e m p e r a t u r e c h a n g e s i n t h e j o i n t s o f t h e e x t r e m i t i e s a r e known t o be r e s p o n s i b l e , i n p a r t , f o r t h e d e v e l o p m e n t o f g o u t y a r t h r i t i s . No s a t i s f a c t o r y e x p l a n a t i o n i s y e t a v a i l a b l e f o r (1) t h e s e l e c t i v e d e p o s i t i o n o f monosodium u r a t e m o n o h y d r a t e (MSUM) c r y s t a l s i n c o n n e c t i v e t i s s u e s (2) t h e i n c r e a s e d i n c i d e n c e o f g o u t i n t h e l a t e r y e a r s o f l i f e and (3) t h e i n c r e a s e d i n c i d e n c e o f MSUM c r y s t a l d e p o s i t i o n i n c o n n e c t i v e t i s s u e s a f t e r t r a u m a and i n j o i n t s w i t h p r e e x i s t i n g d i s e a s e . I t i s p o s s i b l e t h a t t h e a l t e r a t i o n s i n c o m p o s i t i o n o f t h e non-f i b r i l l a r m a t r i x o f c a r t i l a g e and s y n o v i a l f l u i d w h i c h a r e t h o u g h t t o o c c u r w i t h a g e i n g , trauma o r p r e e x i s t i n g d i s e a s e , may p r e d i s p o s e t h e s e t i s s u e s t o c r y s t a l d e p o s i t i o n . The o b j e c t i v e s o f t h i s s t u d y were t o d e t e r m i n e t h e e f f e c t o f t h e c a r t i l a g e and s y n o v i a l f l u i d components, c h o n d r o i t i n s u l f a t e , h y a l u r o n i c a c i d , p r o t e o g l y c a n monomer, p r o t e o g l y c a n a g g r e g a t e , p h o s p h o l i p i d s and a l b u m i n on t h e gr o w t h o f MSUM. The d e g r a d a t i o n o f MSUM s o l u t i o n s was s t u d i e d u n d e r s t e r i l e and n o n - s t e r i l e c o n d i t i o n s t o d e t e r m i n e t h e p o s s i b l e c a u s e s o f d e g r a d a t i o n and t o d e f i n e t h e t i m e span o f c r y s t a l g r o w t h e x p e r i m e n t s . The r a t e o f d e g r a d a t i o n o f MSUM s o l u t i o n s i n c r e a s e d w i t h an i n c r e a s e i n t e m p e r a t u r e . The c o n c e n t r a t i o n o f MSUM i n s o l u t i o n f e l l s h a r p l y a f t e r a u t o c l a v i n g and s o l u t i o n s s t o r e d i n c o n t a i n e r s w i t h rubber c l o s u r e s showed g r e a t e r d e g r a d a t i o n o f MSUM than a u t o c l a v e d s o l u t i o n s s t o r e d i n a l l - g l a s s c o n t a i n e r s . Rubber stoppers a p p a r e n t l y absorbed MSUM from s o l u t i o n . The degrad a t i o n o f MSUM s o l u t i o n s was thought t o be due to both b a c t e r i a l consumption and chemical decomposition i n n o n - s t e r i l e s o l u t i o n s but was due on l y t o chemical decomposition i n s t e r i l e s o l u t i o n s . The aqueous s o l u b i l i t y o f MSUM was determined a t d i f f e r e n t temperatures and i n the presence o f v a r y i n g c o n c e n t r a t i o n s o f sodium c h l o r i d e . Sodium c h l o r i d e suppressed MSUM s o l u b i l i t y . The aqueous s o l u b i l i t y o f MSUM was a l s o determined i n the presence o f s e v e r a l c o n n e c t i v e t i s s u e components a t 37°. C h o n d r o i t i n s u l f a t e (CS) decreased the s a t u r a t i o n s o l u b i l i t y o f MSUM probably due to the sodium pres e n t i n the CS samples. P r o t e o g l y c a n aggregate, p r o t e o g l y c a n monomer, h y a l u r o n i c a c i d an albumin r e s u l t e d i n very s l i g h t i n c r e a s e s i n the s o l u b i l i t y o f MSUM. The growth k i n e t i c s o f MSUM was s t u d i e d u s i n g the seeded growth technique. An equation o f the ge n e r a l form: R = K (obs) S ( C - C s ) n g o was used t o determine the o v e r a l l growth r a t e c o n s t a n t , K Q ( o b s or r a t e constant, K' (K'= K (ob s ) S ) . L i n e a r p l o t s o f the i n t e g r a t e d form o f the second order growth equation gave the be s t f i t between the p o i n t s and gave reasonably constant v a l u e s f o r K Q(obs) determined a t a gi v e n i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n and v a r y i n g seed amounts. An i n d u c t i o n p e r i o d or a p e r i o d o f slow growth was observed at both the i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n s s t u d i e d . The leng t h o f the i n d u c t i o n p e r i o d was i n v e r s e l y p r o p o r t i o n a l t o the added seed amount. D i f f e r i n g c o n c e n t r a t i o n s o f a d d i t i v e s were i n c l u d e d i n the growth medium and K' determined. C h o n d r o i t i n s u l f a t e (CS) s i g n i f i c a n t l y i n c r e a s e d the growth r a t e constant f o r MSUM growth. However, the p r o p o r t i o n o f CS decreases i n aged and o s t e o a r t h r i t i c c a r t i l a g e and thus a d e c r e a s i n g p r o p o r t i o n o f a growth a c c e l e r a t o r i s u n l i k e l y t o be a f a c t o r i n the d e p o s i t i o n o f MSUM i n c a r t i l a g e . CS has been found i n the s y n o v i a l f l u i d o f a r t h r i t i c j o i n t s and may a c t as an MSUM growth a c c e l e r a t o r i n t h i s medium. H y a l u r o n i c a c i d (HA) and albumin caused s i g n i f i c a n t i n h i b i t i o n of the growth of MSUM c r y s t a l s . T h i s e f f e c t may be due t o the a d s o r p t i o n o f these molecules onto the MSUM seed c r y s t a l s r e s u l t i n g i n the p o i s o n i n g o f the a c t i v e growth s i t e s on the c r y s t a l s u r f a c e . C a r t i l a g e HA and s y n o v i a l f l u i d albumin l e v e l s are i n c r e a s e d i n aged and/or d i s e a s e d j o i n t s . Increased p r o p o r t i o n s o f growth i n h i b i t o r s do not o f f e r l i k e l y e x p l a n a t i o n s o f c r y s t a l d e p o s i t i o n i n j o i n t t i s s u e s . - V -A t c o n c e n t r a t i o n s o f 0.1-1.0 mg mL p r o t e o g l y c a n monomer (PGM) and p r o t e o g l y c a n a g g r e g a t e (PGA) s l i g h t l y i n c r e a s e d t h e MSUM g r o w t h r a t e c o n s t a n t b u t t h i s i n c r e a s e was s t a t i s t i c a l l y i n s i g n i f i c a n t . The two p h o s p h o l i p i d s , p h o s p h a t i d y l c h o l i n e and p h o s p h a -t i d y l s e r i n e i n c r e a s e d t h e g r o w t h r a t e c o n s t a n t o f MSUM. P h o s p h a t i d y l s e r i n e , however, d i d n o t s i g n i f i c a n t l y i n c r e a s e t h e g r o w t h r a t e c o n s t a n t a t t h e c o n c e n t r a t i o n s s t u d i e d . I t i s p o s s i b l e t h a t t h e r a i s e d l e v e l s o f p h o s p h o l i p i d s i n aged o r d i s e a s e d c a r t i l a g e and s y n o v i a l f l u i d c o u l d a c c e l e r a t e t h e g r o w t h o f MSUM c r y s t a l s r e s u l t i n g i n MSUM d e p o s i t i o n i n t h e s e t i s s u e s . -vi -T A B L E O F C O N T E N T S Page ABSTRACT i i LIST OF TABLES x i v LIST OF FIGURES x i x SYMBOLS AND ABBREVIATIONS xxv ACKNOWLEDGEMENTS xxx 1 INTRODUCTION 1 2 LITERATURE SURVEY 4 2.1. C r y s t a l d e p o s i t i o n d i s e a s e s 4 2.1.1. Gout 5 2.1.2. Pseudogout 9 2.1.3. Hydroxy a p a t i t e (HAP) d e p o s i t i o n d i s e a s e 9 2.1.4. M i s c e l l a n e o u s 9 2.2. C r y s t a l - i n d u c e d inflammation 10 2.3. Hyperuricemia and epidemiology 13 - v i i -Page 2.4. Causes o f hype r u r i c e m i a 16 2.4.1. Inborn e r r o r s o f metabolism 16 2.4.2. Impaired e x c r e t i o n 17 2.4.3. Drugs and a l c o h o l 18 2.5. D e p o s i t i o n o f c r y s t a l s 19 2.5.1. O r i g i n o f c r y s t a l s and t h e i r r e l a t i o n s h i p t o j o i n t d i s e a s e 19 2.6. C a r t i l a g e 20 2.6.1. Composition of c a r t i l a g e 24 (A) Water 24 (B) C o l l a g e n 24 (C) P r o t e o g l y c a n aggregate (PGA) 25 (I) P r o t e o g l y c a n subunit (Monomer)(PGM) 25 (Ia) C h o n d r o i t i n s u l f a t e (CS) 30 (l b ) Keratan s u l f a t e (KS) 30 (II) H y a l u r o n i c a c i d (HA) 32 (D) L i p i d s o f c a r t i l a g e 33 2.6.2. F a c t o r s a f f e c t i n g the composition of c a r t i l a g e 33 (A) E f f e c t o f age 33 (B) E f f e c t o f i n j u r y 36 (C) E f f e c t o f j o i n t d i s e a s e 36 2.7. S y n o v i a l f l u i d 38 2.7.1. Composition o f s y n o v i a l f l u i d 39 -viii-Page (A) S o l u b l e c o n s t i t u e n t s d e r i v e d from the bloo d 39 (B) C o n s t i t u e n t s s e c r e t e d by the j o i n t t i s s u e s 39 (I) H y a l u r o n i c a c i d (HA) 39 (II) L u b r i c a t i n g g l y c o p r o t e i n s 41 (C) Products d e r i v e d from the c a t a b o l i s m o f j o i n t t i s s u e s 41 2.7.2. L i p i d s o f s y n o v i a l f l u i d 41 2.7.3. F a c t o r s a f f e c t i n g the composition o f s y n o v i a l f l u i d 41 (A) E f f e c t o f j o i n t d i s e a s e 41 2.8. Theory of c r y s t a l growth 42 2.8.1. S u p e r s a t u r a t i o n 43 2.8.2. N u c l e a t i o n 46 (A) Homogeneous n u c l e a t i o n 46 (B) Heterogeneous and secondary n u c l e a t i o n 51 2.8.3. C r y s t a l growth 54 (A) Surface energy t h e o r i e s 54 (B) A d s o r p t i o n l a y e r t h e o r i e s 55 (I) D i s l o c a t i o n s 57 (C) D i f f u s i o n t h e o r i e s 62 2.8.4. F a c t o r s a f f e c t i n g c r y s t a l growth r a t e s 65 (A) E f f e c t o f seed c r y s t a l s i z e and - i x -Page s u r f a c e area 65 (B) E f f e c t o f degree o f s u p e r s a t u r a t i o n 67 (C) E f f e c t o f temperature 68 (D) E f f e c t o f degree o f a g i t a t i o n 69 (E) E f f e c t o f i m p u r i t i e s 69 2.8.5. Determination o f c r y s t a l growth r a t e 70 (A) Face growth r a t e s 70 (B) O v e r a l l growth r a t e s 71 2.9. MSUM s o l u t i o n s 72 2.9.1. Degradation o f MSUM s o l u t i o n s 73 2.9.2. S o l u b i l i t y o f MSUM 75 2.10. N u c l e a t i o n and c r y s t a l growth o f MSUM 77 3 EXPERIMENTAL 81 3.1. Instruments 81 3.2. M a t e r i a l s 82 3.3. Methods 84 3.3.1. P r e p a r a t i o n o f MSUM 84 3.3.2. C h a r a c t e r i z a t i o n of MSUM c r y s t a l s 85 (A) U l t r a - v i o l e t spectroscopy 85 (B) I n f r a - r e d spectroscopy 85 (C) X-ray d i f f r a c t i o n 85 (D) D i f f e r e n t i a l scanning c a l o r i m e t e r y 86 (E) Scanning e l e c t r o n microscopy 86 (F) Determination o f s u r f a c e area o f MSUM seed c r y s t a l s 86 -X-Page 3.3.3. A n a l y s i s o f monosodium ura t e monohydrate 88 3.3.4. Degradation o f monosodium ura t e monohydrate s o l u t i o n s 88 (A) N o n - s t e r i l e s o l u t i o n s 88 (B) S t e r i l e s o l u t i o n s 89 3.3.5. Determination o f s a t u r a t i o n s o l u b i l i t y o f monosodium ura t e monohydrate 90 (A) E f f e c t o f temperature 90 (B) E f f e c t o f e l e c t r o l y t e s 91 (C) E f f e c t o f c h o n d r o i t i n s u l f a t e , h y a l u r o n i c a c i d , p r o t e o g l y c a n monomer, p r o t e o g l y c a n aggregate and albumin 91 3.3.6. C r y s t a l growth o f monosodium ura t e monohydrate 92 (A) E f f e c t o f s u p e r s a t u r a t i o n c o n c e n t r a t i o n , seed c r y s t a l b a t c h and seed amount on the c r y s t a l growth k i n e t i c s o f mono-sodium u r a t e monohydrate 94 (B) E f f e c t o f a d d i t i v e s on the c r y s t a l growth o f monosodium urate monohydrate 95 (I) C h o n d r o i t i n s u l f a t e and albumin 95 -xi -(II) H y a l u r o n i c a c i d and p r o t e o g l y c a n monomer ( I I I ) P r o t e o g l y c a n aggregate (IV) P h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l s e r i n e 3.3.7. Determination o f sodium and/or potassium content of h y a l u r o n i c a c i d , c h o n d r o i t i n s u l f a t e and pr o t e o g l y c a n samples 3.3.8. E f f e c t o f sodium and potassium ions on growth o f MSUM 3.3.9. C h a r a c t e r i z a t i o n o f MSUM c r y s t a l s a f t e r c r y s t a l growth RESULTS AND DISCUSSIONS 1. C h a r a c t e r i z a t i o n o f monosodium urate monohydrate 2. Assay o f monosodium ura t e monohydrate i n s o l u t i o n 3. Degradation o f monosodium urate monohydrate i n s o l u t i o n 4. S a t u r a t i o n s o l u b i l i t y o f MSUM 4.4.1. E f f e c t o f temperature 4.4.2. E f f e c t o f sodium c h l o r i d e 4.4.3. E f f e c t o f c h o n d r o i t i n s u l f a t e , h y a l u r o n i c a c i d , proteoglycans -xi i -Page and albumin 121 4.5. C r y s t a l growth o f MSUM 124 4.5.1. Determination o f s u p e r s a t u r a t i o n c o n c e n t r a t i o n and seed amount f o r c r y s t a l growth 124 4.5.2. S e l e c t i o n o f the method to determine the r a t e constant o f c r y s t a l growth 128 4.5.3. E f f e c t o f seed c r y s t a l s on the c r y s t a l growth 147 4.5.4. E f f e c t o f s u p e r s a t u r a t i o n on the c r y s t a l growth r a t e constant 152 4.5.5. E f f e c t o f sodium and potassium ions on the growth r a t e constants 152 4.5.6. E f f e c t o f a d d i t i v e s on the c r y s t a l growth r a t e constant 156 (A) E f f e c t o f h y a l u r o n i c a c i d on the growth r a t e constant 156 (B) E f f e c t o f c h o n d r o i t i n s u l f a t e on the growth r a t e constant 158 (C) E f f e c t o f p r o t e o g l y c a n monomer on the c r y s t a l growth r a t e constant 160 (D) E f f e c t o f p r o t e o g l y c a n aggregate on the c r y s t a l growth r a t e constant 160 (E) E f f e c t o f p h o p h a t i d y l c h o l i n e and p h o s p h a t i d y l s e r i n e on the c r y s t a l -xiii-Page growth r a t e constant o f MSUM 163 (F) E f f e c t o f albumin on MSUM c r y s t a l growth 166 4.4.7. C h a r a c t e r i z a t i o n o f MSUM c r y s t a l s a f t e r growth experiments 174 5 SUMMARY AND CONCLUSIONS 180 i 6 REFERENCES 188 APPENDIX 214 -xiv-LIST OF TABLES Table Page 1 Suggested mechanisms r e l a t e d t o i n - v i v o MSUM c r y s t a l formation. 21 2 The estimated i n c r e a s e s i n the l i p i d c ontent o f c a r t i l a g e per year o f age. 37 3 The r e l a t i o n s h i p between molecular weight and c o n c e n t r a t i o n o f serum p r o t e i n s i n s y n o v i a l f l u i d . 40 4 Powder X-ray d i f f r a c t i o n p a t t e r n o f monosodium ur a t e monohydrate. 105 5 S a t u r a t i o n s o l u b i l i t y (Cs) of mono-sodium urate monohydrate at d i f f e r e n t temperatures. 116 6 R e l a t i o n s h i p between temperature and c o n c e n t r a t i o n o f sodium c h l o r i d e o r p h y s i o l o g i c i o n c o n c e n t r a t i o n on the s a t u r a t i o n s o l u b i l i t y o f monosodium ura t e monohydrate. 120 7 S a t u r a t i o n s o l u b i l i t y o f monosodium - X V -u r a t e monohydrate i n the presence o f a d d i t i v e s a t 37°. MSUM growth r a t e c o n s t a n t s , K*, obtained from the l i n e a r r e g r e s s i o n of l o g R versus l o g (C-Cs) ( c r y s t a l growth i n 1 L c a p a c i t y apparatus; C = 5 g L - 1 ) . MSUM growth r a t e c o n s t a n t s , K', obtained from the l i n e a r r e g r e s s i o n o f l o g R versus l o g (C-Cs) ( c r y s t a l growth i n 1 L c a p a c i t y apparatus, C = 6 g L - 1 ) . MSUM growth r a t e c o n s t a n t s , K', obtained from the l i n e a r r e g r e s s i o n of l o g R versus l o g (C-Cs) ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus; C = 5 g L - 1 ) . MSUM growth r a t e c o n s t a n t s , K', obtained from the l i n e a r r e g r e s s i o n o f l o g R versus l o g (C-Cs) ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus; C = 6 g L - 1 ) . MSUM growth r a t e c o n s t a n t s , K', -xvi -Table Page obtained from the n o n - l i n e a r computer program ( c r y s t a l growth i n 1 L c a p a c i t y apparatus; C = 5 g L ^ ) . 136 13 MSUM growth r a t e constants, K', obtained from the n o n - l i n e a r computer program ( c r y s t a l growth i n 1 L c a p a c i t y apparatus, C = 6 g L ) . 137 14 MSUM growth r a t e constants, K', obtained from the n o n - l i n e a r computer program ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus, C = 5 g L -"''). 138 15 MSUM growth r a t e constants, K 1, obtained from the n o n - l i n e a r computer program ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus, C = 6 g L-"*"). 139 16 MSUM growth r a t e constants, K', obtained from p l o t s o f the i n t e g r a t e d form o f the second order growth equation ( c r y s t a l growth i n 1 L c a p a c i t y apparatus, C = 5 g L - 1 ) . 143 17 MSUM growth r a t e constants, K', obtained from p l o t s o f the i n t e g r a t e d form of the second order growth equation -xvii-Table Page ( c r y s t a l growth i n 1 L c a p a c i t y apparatus, C = 6 g L - 1 ) . 144 18 MSUM growth r a t e constants, K', obtained from p l o t s o f the i n t e g r a t e d form of the second order growth equation ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus, C = 5 g L - 1 ) . 145 19 MSUM growth r a t e constant, K', obtained from p l o t s o f the i n t e g r a t e d form of the second order growth equation ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus, C = 6 g L ) . 146 20 O v e r a l l growth r a t e c o n s t a n t s , K (obs), c a l c u l a t e d from r a t e o constants, K', ( c r y s t a l growth i n 1 L c a p a c i t y apparatus, C = 5 g L ) . 148 21 O v e r a l l growth r a t e c o n s t a n t s , K (obs), c a l c u l a t e d from r a t e o constants, K', ( c r y s t a l growth i n 1 L c a p a c i t y apparatus, C = 6 g L ^ ) . 149 22 O v e r a l l growth r a t e c o n s t a n t s , K (obs), c a l c u l a t e d from r a t e o constants, K", ( c r y s t a l growth i n 50 mL -xvi i i -c a p a c i t y apparatus, C = 5 g L - 1 ) . O v e r a l l growth r a t e constants, K Q ( o b s ) , c a l c u l a t e d from r a t e c o n s t a n t s , K' , ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus, C = 6 g L ) . Sodium and potassium content o f a d d i t i v e s E f f e c t o f sodium and potassium ions on MSUM growth r a t e constant. E f f e c t o f h y a l u r o n i c a c i d on the growth k i n e t i c s o f MSUM. E f f e c t o f c h o n d r o i t i n s u l f a t e on the growth k i n e t i c s o f MSUM. E f f e c t o f p r o t e o g l y c a n monomer on the growth k i n e t i c s o f MSUM. E f f e c t o f p r o t e o g l y c a n aggregate on the growth k i n e t i c s o f MSUM. E f f e c t o f p h o s p h a t i d y l c h o l i n e on the growth k i n e t i c s o f MSUM. E f f e c t of p h o s p h a t i d y l s e r i n e on the growth k i n e t i c s o f MSUM. -xix-LIST OF FIGURES F i g u r e Page 1 Chemical formulae of (A) Monosodium urat e monohydrate, (B) Calcium pyro-phosphate d i h y d r a t e , and (C) Hydroxy a p a t i t e . 7 2 A view of the c r y s t a l s t r u c t u r e o f MSUM viewed down the needle a x i s . 8 3 P o s s i b l e steps i n v o l v e d i n c r y s t a l -induced inflammation. 12 4 Pathways o f p u r i n e metabolism i n man. 15 5 P o s s i b l e pathways i n v o l v e d i n c r y s t a l r e l a t e d j o i n t d i s e a s e s . 22 6 P o s s i b l e r e l a t i o n s h i p s between c r y s t a l d e p o s i t i o n and j o i n t d i s e a s e s . 23 7 S t r u c t u r e o f a r t i c u l a r c a r t i l a g e : (a) Domains o f p r o t e o g l y c a n aggregate and c o l l a g e n , (b) Regions of i n t e r a c t i o n between p r o t e o g l y c a n aggregate and c o l l a g e n . 26 - X X -Figure Page 8 S t r u c t u r e o f p r o t e o g l y c a n aggregate. 27 9 S t r u c t u r e s o f : (a) p r o t e o g l y c a n monomer, (b) h y a l u r o n i c a c i d . 29 10 S t r u c t u r e s o f : (a) C h o n d r o i t i n ( i : - 4 - ; i i : - 6 - ) s u l f a t e , (b) Keratan s u l f a t e . 31 11 The s o l u b i l i t y - s u p e r s o l u b i l i t y diagram. 44 12 Free energy diagram f o r n u c l e a t i o n e x p l a i n i n g the e x i s t e n c e of a c r i t i c a l n u c leus. 50 13 E f f e c t o f s u p e r s a t u r a t i o n on the n u c l e a t i o n r a t e . 52 14 C r y s t a l growth without d i s l o c a t i o n s : (a) m i g r a t i o n towards d e s i r e d l o c a t i o n ; (b) completed l a y e r ; (c) s u r f a c e n u c l e a t i o n . 56 15 K o s s e l ' s model of a growing c r y s t a l s u r f a c e : (A) f l a t s u r f a c e ; (B) s t e p s ; (C) k i n k s ; (D) surface-adsorbed growth u n i t s ; (E) edge v a c e n c i e s ; and (F) s u r f a c e v a c e n c i e s . 58 F i g u r e 16 17 18 19 20 21 22 23 24 25 -xx "U Page D i s l o c a t i o n s i n c r y s t a l : (a) an edge d i s l o c a t i o n , and (b) screw d i s l o c a t i o n . 59 Development o f a growth s p i r a l s t a r t i n g from a screw d i s l o c a t i o n . 61 S a t u r a t i o n s o l u b i l i t y of MSUM i n normal s a l i n e . 76 C r y s t a l growth apparatus. 93 U l t r a - v i o l e t spectrum o f monosodium urat e monohydrate s o l u t i o n . 101 I n f r a - r e d spectrum o f monosodium urat e monohydrate. 102 X-ray d i f f r a c t i o n p a t t e r n o f monosodium ura t e monohydrate. 104 Scanning e l e c t r o n micrograph of monosodium ura t e monohydrate. 106 DSC scan o f monosodium urate monohydrate. 107 A standard curve f o r MSUM s o l u t i o n . 108 F i g u r e 26 27 28 29 30 31 32 - xx i i -Page Degradation o f n o n - s t e r i l e MSUM s o l u t i o n s . 110 Degradation o f s t e r i l e (0.22 pm f i l t e r ) s o l u t i o n s i n V a c u t a i n e r s . I l l Degradation o f s t e r i l e ( a u t o c l a v i n g ) MSUM s o l u t i o n s i n a l l - g l a s s c o n t a i n e r s . 112 Degradation o f s t e r i l e ( a u t o c l a v i n g ) MSUM s o l u t i o n s i n V a c u t a i n e r s . 113 E f f e c t o f temperature on the s a t u r a t i o n s o l u b i l i t y o f : (a) MSUM; (b) MSUM i n the presence o f p h y s i o l o g i c a l i o n c o n c e n t r a t i o n . 117 A van't Hoff p l o t f o r MSUM s o l u b i l i t y i n water. 118 Seeded growth curves f o r MSUM a t 37° i n 1 L c a p a c i t y apparatus and an i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n of 5 g L - 1 . 125 Seeded growth curves f o r MSUM a t 37° i n 1 L c a p a c i t y apparatus and an i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n -xxi i i -Figure Page of 6 g L - 1 . 126 34 Second-order k i n e t i c p l o t s o f the i n t e g r a t e d form o f growth equation f o r seeded growth o f MSUM a t 3 7° i n the 50 mL c a p a c i t y apparatus and an i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 5 g L - 1 . 141 35 Second-order k i n e t i c p l o t s o f the i n t e g r a t e d form o f growth equation f o r seeded growth o f MSUM a t 37° i n the 50 mL c a p a c i t y apparatus and an i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n of 6 g L - 1 . 142 36 Seeded growth k i n e t i c s o f MSUM a t 37° i n the presence o f albumin i n the 50 mL c a p a c i t y apparatus and an i n i t i a l super-s a t u r a t i o n c o n c e n t r a t i o n o f 5 g L . 167 3 7 Scanning e l e c t r o n micrograph o f MSUM c r y s t a l s a f t e r growth. I n i t i a l super-s a t u r a t i o n c o n c e n t r a t i o n , 5 g L . 175 38 Scanning e l e c t r o n micrograph o f MSUM c r y s t a l s a f t e r growth. I n i t i a l super-s a t u r a t i o n c o n c e n t r a t i o n , 5 g L ^. 176 -xxfv-Figure Page 39 Scanning e l e c t r o n micrograph o f MSUM c r y s t a l s a f t e r growth i n the presence o f albumin (50 mg). I n i t i a l super-s a t u r a t i o n c o n c e n t r a t i o n , 5 g L ^. 177 40 Scanning e l e c t r o n micrograph o f MSUM c r y s t a l s a f t e r growth i n the presence of c h o n d r o i t i n s u l f a t e (20 mg). I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n , 5 g L ^. 178 41 Scanning e l e c t r o n micrograph o f MSUM c r y s t a l s a f t e r growth i n the presence of p r o t e o g l y c a n monomer (10 mg). I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n , 5 g L ^. 179 - X X V -SYMBOLS AND ABBREVIATIONS s' Degree o f s u p e r s a t u r a t i o n C C o n c e n t r a t i o n of s o l u t e i n the s o l v e n t a t some g i v e n temperature Cs E q u i l i b r i u m s a t u r a t i o n c o n c e n t r a t i o n o f s o l u t e i n the s o l v e n t a t a g i v e n temperature C i S o l u t e c o n c e n t r a t i o n a t the c r y s t a l - s o l u t i o n i n t e r f a c e W T o t a l q u a n t i t y o f the work r e q u i r e d t o form a s t a b l e c r y s t a l nucleus Wg The work r e q u i r e d to form the s u r f a c e of the s u r f a c e of the c r y s t a l The work r e q u i r e d t o form the bulk of the c r y s t a l £ The s u r f a c e energy of the s p h e r i c a l p a r t i c l e per u n i t area a Surface area o f the p a r t i c l e A p The p r e s s u r e d i f f e r e n c e between the vapour phase and the i n t e r i o r of the l i q u i d d r o p l e t v Volume o f the p a r t i c l e r Radius o f the d r o p l e t pr Vapour p r e s s u r e over a l i q u i d d r o p l e t o f r a d i u s r -xxvi-p* Vapour p r e s s u r e over a f l a t l i q u i d s u r f a c e M Molecular weight of the substance f D e n s i t y o f the d r o p l e t T Absolute temperature R Gas constant G O v e r a l l excess f r e e energy G g Surface excess f r e e energy G v Volume excess f r e e energy G O v e r a l l excess f r e e energy, G, a t maximum c r i t ^ J where r = r c r , r a d i u s o f a c r i t i c a l s i z e nucleus c N Rate o f n u c l e a t i o n or the number o f n u c l e i formed per u n i t time A Constant of n u c l e a t i o n r a t e A G O v e r a l l f r e e energy change f o r the formation of a p a r t i c a l A G c r ^ t O v e r a l l f r e e energy change a s s o c i a t e d with homogeneous n u c l e a t i o n AG' .. O v e r a l l f r e e energy change a s s o c i a t e d with c r i t ^ J ^ heterogeneous n u c l e a t i o n A constant r e l a t e d t o the i n t e r f a c i a l energy Ssi T ^ e i n t e r f a c i a l energy between the s u r f a c e o f the seed and the l i q u i d £fcs The i n t e r f a c i a l energy between the s u r f a c e s of the c r y s t a l l i z i n g phase and the seed s u r f a c e £JC^ The i n t e r f a c i a l energy between the -xxvii-c r y s t a l l i z i n g phase and the l i q u i d a^ Area of the i t h face of the c r y s t a l g^ Surface f r e e energy per u n i t area o f the i t h face b Q Burgers v e c t o r Rg Rate o f c r y s t a l growth A Temperature dependent constant o f the growth r a t e B Temperature dependent constant of the growth r a t e S Surface area o f the s o l i d D C o e f f i c i e n t o f d i f f u s i o n o f s o l u t e h Thickness o f the l i q u i d f i l m R t Rate o f a r r i v a l o f s o l u t e a t the s u r f a c e by d i f f u s i o n from the bulk s o l u t i o n R g Rate of i n t e g r a t i o n i n t o the s o l i d s u r f a c e K T r a n s p o r t r a t e constant K S Surface i n t e g r a t i o n r a t e constant K Q ( o b s ) Observed o v e r a l l growth r a t e constant K ' Observed growth r a t e constant, ( K ' = K Q S ) K'(add) Observed growth r a t e constant f o r MSUM growth i n the presence of a d d i t i v e K ' ( c o n t r o l ) Observed growth r a t e constant i n the absence of a d d i t i v e n Order o f growth r e a c t i o n e . S^ E f f e c t i v e s u r f a c e area a t time t - x x v i i i -K T Growth r a t e constant dependent on temperature f ( o t ) Term dependent on the s u p e r s a t u r a t i o n c^ T o t a l mass of s o l i d a t time t S^/S Geometric t o t a l s u r f a c e area a t time t C ,C + C o n c e n t r a t i o n s o f anions and c a t i o n s at the s u p e r s a t u r a t i o n c o n c e n t r a t i o n Cs , C s + C o n c e n t r a t i o n s of anions and c a t i o n s at the s a t u r a t i o n c o n c e n t r a t i o n P P a r t i a l p r e s s u r e of adsorbate Po Saturated p r e s s u r e of adsorbate 2 3 Nu Avogadro's number (6.023 x 10 ) Vc Volume o f c a l i b r a t i o n gas Pa Ambient p r e s s u r e i n atmosphere A S i g n a l area or d e s o r p t i o n count o f c a l i b r a t i o n gas Acs Cross s e c t i o n a l area of adsorbate molecules i n square meters "\ Wavelength o f X-rays d Spacing between the c r y s t a l l o g r a p h i c planes of a c r y s t a l n* Is an i n t e g e r i n equation 27 0 Angle o f the i n c i d e n t X-rays A H Heat of s o l u t i o n PGA Proteoglycan aggregate PGM P r o t e o g l y c a n monomer PGs Proteoglycans HA H y a l u r o n i c a c i d -xxix-CS C h o n d r o i t i n s u l f a t e PC P h o s p h a t i d y l c h o l i n e PS P h o s p h a t i d y l s e r i n e MSUM Monosodium ura t e monohydrate CPPD Calcium pyrophosphate d i h y d r a t e HAP Hydroxy a p a t i t e , A p a t i t e , Calcium hydroxy a p a t i t e - X X X -ACKNOWLEDGEMENTS I wish to thank Dr. H.M. Burt f o r her s u p e r v i s i o n and encouragement duri n g the course of t h i s study. I am g r a t e f u l t o Dr. A.G. M i t c h e l l f o r h i s v a l u a b l e q u e s t i o n s and suggestions and to Dr. J . M c N e i l l f o r encourage-ment throughout t h i s p r o j e c t . S i n c e r e thanks t o : Dr. Mark Adams, Department of Medicine, f o r h i s suggestions and f o r p r o v i d i n g the p r o t e o g l y c a n s . Mr. R. B u t t e r s , Department of M e t a l l u r g y , f o r the X-ray d i f f r a c t i o n a n a l y s i s . Ms. M. Mager, Department o f M e t a l l u r g y , f o r the scanning e l e c t r o n microscopy. Mr. R. Burton, F a c u l t y of Pharmaceutical s c i e n c e s , f o r computer a n a l y s i s . S e v e r a l f r i e n d s (who must remain anonymous) f o r t h e i r f r i e n d s h i p . F i n a n c i a l support from the U n i v e r s i t y of B r i t i s h Columbia, Medical Research C o u n c i l of Canada and S t a n l e y Drug Products i s g r a t e f u l l y acknowledged. - 1 -1 INTRODUCTION The n u c l e a t i o n and growth o f c r y s t a l s from s u p e r s a t u r a t e d s o l u t i o n s i s an important process i n b i o l o g i c a l systems, f o r example, i n the d e p o s i t i o n o f c a l c i u m h y d r o x y a p a t i t e c r y s t a l s i n bones and t e e t h . Other compounds such as c a l c i u m o x a l a t e s , c a l c i u m pyrophosphates, u r i c a c i d , sodium u r a t e e t c . may be found as p a t h o l o g i c a l d e p o s i t s i n the body. A c r y s t a l d e p o s i t i o n d i s e a s e may be d e f i n e d as a p a t h o l o g i c a l c o n d i t i o n a s s o c i a t e d w i t h the presence o f c r y s t a l s which then c o n t r i b u t e t o the t i s s u e damage. Gout i s a s s o c i a t e d w i t h the appearance of monosodium ura t e monohydrate (MSUM) c r y s t a l s i n the s y n o v i a l f l u i d and/or i n oth e r c o n n e c t i v e t i s s u e s o f the j o i n t . Hyperuricemia i s r e q u i r e d f o r the development o f gouty a r t h r i t i s . Increased urate l e v e l s i n blood may r e s u l t from: m e t a b o l i c d i s o r d e r s , impaired r e n a l e x c r e t i o n and i n g e s t i o n o f drugs and a l c o h o l . Since the mean plasma u r a t e l e v e l s i n h e a l t h y males are n e a r l y the same as the s a t u r a t i o n s o l u b i l i t y o f ura t e i n plasma, l o c a l temperature changes may be a f a c t o r i n the formation o f t o p h i i n p e r i p h e r a l s i t e s and acute c r y s t a l l i z a t i o n i n j o i n t s . - 2-Hyperuriceraia and temperature v a r i a t i o n s alone, however, are i n s u f f i c i e n t t o e x p l a i n the observed p a t t e r n s o f MSUM c r y s t a l l i z a t i o n , such as, (1) the s e l e c t i v e d e p o s i t i o n o f MSUM c r y s t a l s i n connective t i s s u e s , (2) the i n c r e a s e d i n c i d e n c e o f gouty a r t h r i t i s i n the l a t e r years o f l i f e and (3) the involvement of connective t i s s u e s exposed t o trauma i n t h i s d i s e a s e . There are a number of bi o c h e m i c a l and metabolic changes which occur i n the a r t i c u l a r c a r t i l a g e and s y n o v i a l f l u i d o f j o i n t s as a r e s u l t o f f a c t o r s such as ageing, trauma and p r e e x i s t i n g d i s e a s e . A l t e r a t i o n s i n the composition o f the c a r t i l a g e matrix and s y n o v i a l f l u i d caused by ageing and d i s e a s e may p r e d i s p o s e these t i s s u e s t o c r y s t a l d e p o s i t i o n . Although there have been some s t u d i e s o f the f a c t o r s a f f e c t i n g the s o l u b i l i t y and n u c l e a t i o n r a t e s o f MSUM, the f a c t o r s which may i n f l u e n c e the c r y s t a l growth o f MSUM are p o o r l y understood. The o b j e c t i v e s o f the present study were: 1. To i n v e s t i g a t e the c r y s t a l growth k i n e t i c s o f MSUM. 2. To determine the e f f e c t o f s u p e r s a t u r a t i o n and seed amount on the MSUM c r y s t a l growth k i n e t i c s . 3. To determine the e f f e c t o f c a r t i l a g e and s y n o v i a l f l u i d « components on the MSUM growth k i n e t i c s . -3-There are a number o f methods f o r s t u d y i n g the k i n e t i c s o f c r y s t a l growth, but the seeded growth technique has been shown to be h i g h l y r e p r o d u c i b l e and was employed i n these s t u d i e s . Supersaturated sodium urate s o l u t i o n s were seeded with w e l l -c h a r a c t e r i s e d seed c r y s t a l s o f MSUM under c o n d i t i o n s o f constant temperature and a g i t a t i o n . -4-2. LITERATURE REVIEW 2.1 CRYSTAL DEPOSITION DISEASES Gout as a d i s e a s e has been known f o r s e v e r a l c e n t u r i e s . The a n c i e n t accounts of gout were summarized by F r a n c i s Adams i n 1844 i n h i s commentaries i n the t r a n s l a t i o n o f "The seven books of Paulus A e g i n e t a " (Hartung, 1957). Hippocrates (400 BC) d e s c r i b e d t h i s d i s e a s e with remarkable accuracy ( P r i t c h a r d , 1981). In h i s aphorisms on gout, Hippocr-ates r e f e r r e d s p e c i f i c a l l y t o the h e r e d i t a r y p r e d i s p o s i t i o n , the p e r i o d i c i t y o f acute episodes, the worsening of the d i s e a s e i n the s p r i n g and f a l l , the r a r i t y b e f o r e puberty, the appearance i n women a f t e r the menopause, the immunity of eunuchs t o gout and the b e n e f i t o f c o l d a p p l i c a t i o n s (Hartung, 1957). Gout has been t r a c e d t o the f i r s t c e n tury of the c h r i s t i a n e r a . In a cemetery i n Nubia, Egypt, S i m i t h and Jones (1910) found a s k e l e t o n of an e l d e r l y male whose g r e a t toe and other j o i n t s showed l a r g e t o p h i of u r a t e c r y s t a l s (Hartung, 1957). The f i r s t m i c r o s c o p i c d e s c r i p t i o n of the c r y s t a l s d e r i v e d from a gouty tophus was g i v e n by Antoni von Leewenhoek i n e a r l y 1700 (McCarty, 1970), but i t was o n l y i n 1797, when Wollaston -5-demonstrated t h a t the e x t r u s i o n s from gouty t o p h i were of " l i t h i a t e d soda" (Hartung, 1957). " L i t h i c a c i d " i s now known as u r i c a c i d . C r y s t a l s , as the cause of the inflammatory response were advocated by S i r A l f r e d B a r i n g Garrod i n 1859 ( P r i t c h a r d , 1981). He s t a t e d t h a t acute gouty a r t h r i t i s i s an inflammatory r e a c t i o n t o c r y s t a l s o f sodium u r a t e . F reudweiler (1901) and Hi s s (1900) r e p o r t e d experiments on c r y s t a l - i n d u c e d inflammation. Evidence of c r y s t a l l i n e d e p o s i t s i n and around j o i n t s was noted i n r a d i o l o g i c a l examinations as e a r l y as 1907 by P a i n t e r . T h i s was fo l l o w e d by r e p o r t s o f inflammation r e l a t e d t o these d e p o s i t s ( S c h m i t t , 1921; Sandstorm, 1938). The n a t u r e o f the p r e c i p i t a t e s i n tophaceous gout was d e s c r i b e d by Brandenberger et a l . ( 1 9 4 7 ) . I t was not u n t i l the e a r l y 1960's t h a t a c t i v e r e s e a r c h was i n i t i a t e d i n t h i s f i e l d by H o l l a n d e r and McCarty. The terms " c r y s t a l d e p o s i t i o n d i s e a s e s " and " c r y s t a l - i n d u c e d inflammation" were coined a f t e r c r y s t a l s o f sodium urate i n the s y n o v i a l f l u i d were observed u s i n g p o l a r i z e d l i g h t microscopy (McCarty and Ho l l a n d e r , 1961). T h i s method o f s y n o v i a l f l u i d examination l e d to the d i s c o v e r y o f c a l c i u m pyrophosphate d i h y d r a t e (CPPD) c r y s t a l s i n j o i n t s (McCarty e t a l . , 1962; Kohn e t a l . , 1962). T h i s a r t h r o p a t h y was c a l l e d "pseudogout" because o f i t s s i m i l a r i t i e s t o gout. 2.1.1 GOUT T h i s d i s e a s e i s caused by the d e p o s i t i o n o f monosodium ura t e - 6 -monohydrate (MSUM) c r y s t a l s i n c a r t i l a g e or i n the j o i n t f l u i d . The chemical s t r u c t u r e o f MSUM i s shown i n F i g u r e l a . C r y s t a l s o f MSUM have been e x t r a c t e d from gouty t o p h i and s y n o v i a l f l u i d and examined by p o l a r i z e d l i g h t microscopy (McCarty and H o l l a n d e r , 1961). These c r y s t a l s b e l o n g t o the t r i c l i n i c c r y s t a l system (Howell et a l . , 1963) and have an a c i c u l a r or needle shaped c r y s t a l h a b i t . The c r y s t a l s a re between 2-20 p i n l e n g t h and 0.5-2 um i n width (Dieppe and C a l v e r t , 1983). The c r y s t a l s t r u c t u r e o f MSUM has been determined by Mandel and Mandel (1976) and Rinaudo and B o i s t e l l e (1982). They have shown t h a t the MSUM c r y s t a l c o n s i s t s of urate anions stacked i n p a r a l l e l sheets i n t e r s p e r s e d w i t h sodium ions which bond t o four neighbouring ur a t e anions v i a the oxygen atoms. The water molecules form hydrogen bonds wi t h the p u r i n e r i n g s ( F i g u r e 2 ) . Recent s t u d i e s have shown t h a t the c r y s t a l s o f MSUM possess a net ne g a t i v e charge (Dieppe e t a l . , 1981a; Burt e t a l . , 1983). The e l e c t r o p h o r e t i c m o b i l i t y o f th e s e c r y s t a l s i n p h y s i o -l o g i c a l s a l i n e s o l u t i o n has been found t o be 1 um s ^ V ^ cm (Dieppe and C a l v e r t , 1983). MSUM c r y s t a l l i z e s o n l y i n con n e c t i v e t i s s u e s . The major s i t e s are a r t i c u l a r c a r t i l a g e , p e r i a r t i c u l a r s o f t t i s s u e s , bursae, on the ear and i n tendon sheaths (Dieppe and C a l v e r t , 1983). In acute gout, the s y n o v i a l f l u i d c o n t a i n s numerous MSUM c r y s t a l s o f t e n w i t h i n the l e u c o c y t e s (McCarty and Holla n d e r , 1961; Weinberger et a l • , 1979; Agudelo e t a l . , 1979; Gordon et a l . , 1982). In c h r o n i c gout a l a r g e number of MSUM -7-l C 5 H 3 ° 3 N 4 N a * H 2 0 3 (A) C a 2 P 4 ° 7 * 2 H 2 ° (B) C a 1 0 ( P O 4 ) 6 ( O H ) 2 (C) Figure 1 . Chemical formulae of (A) Monosodium urate monohydrate, IB) Calcium pyrophosphate dihydrate, and (C) Hydroxy a p a t i t e . - 8 -Figure 2. A view of the c r y s t a l structure of MSUM viewed down the needle axis - 9-c r y s t a l s are l o c a t e d i n a tophus (Dieppe and C a l v e r t , 1983). 2.1.2 PSEUDOGOUT Th i s d i s e a s e i s caused by the d e p o s i t i o n o f c a l c i u m pyro-phosphate d i h y d r a t e (CPPD) c r y s t a l s . The chemical formula f o r CPPD i s shown i n F i g u r e l b . There are two polymorphic forms of CPPD, one b e l o n g i n g to the t r i c l i n i c c r y s t a l system and the other t o the m o n o c l i n i c c r y s t a l system. Both t h e s e forms are d e p o s i t e d i n the j o i n t s (McCarty et a l . , 1962; McCarty et a l . , 1966; J a c o b e l l i et a l . , 1973; Ellman et a l . , 1981a,b). CPPD c r y s t a l s have dimensions of between 0.1-10 um. 2.1.3 HYDROXY APATITE (HAP) DEPOSITION DISEASE The chemical formula o f HAP i s shown i n F i g u r e l c . Calcium hydroxy a p a t i t e (HAP) has been i d e n t i f i e d i n degenerated a r t i c u l a r c a r t i l a g e by e l e c t r o n probe and scanning e l e c t r o n microscopy ( A l i and Wisby, 1975), i n tendons, c a r t i l a g e and other c o n n e c t i v e t i s s u e s (McCarty and G a t t e r , 1966; McCarty e t a l . , 1966; P r i t z k e r and Luc, 1976) and s y n o v i a l f l u i d (Dieppe et a l . , 1976). The common s i t e s are the shoulder j o i n t , h i p j o i n t and spine (Dieppe and Doherty, 1982; Andres and T r a i n e r , 1980). The c r y s t a l s of HAP are hexagonal and are u s u a l l y between 0.1-2 um i n s i z e . The c r y s t a l s g e n e r a l l y form s m a l l s p h e r i c a l c l u s t e r s composed of many HAP c r y s t a l s (Dieppe and C a l v e r t , 1983). 2.1.4 MISCELLANEOUS D i c a l c i u m phosphate d i h y d r a t e ( U t s i n g e r , 1977; Faure -10-et a l . , 1977), c o r t i c o s t e r o i d s (Kahn et a l . , 1970) and c h o l e s t e r o l c r y s t a l s (Zuckner et a l • , 1963) have a l s o been i d e n t i f i e d i n j o i n t t i s s u e s . U r i c a c i d and c a l c i u m o x a l a t e c r y s t a l s are r e s p o n s i b l e f o r r e n a l stones. 2.2 CRYSTAL-INDUCED INFLAMMATION The p h y s i c a l presence o f c r y s t a l s i n the j o i n t f l u i d produces an acute arthropathy, seen i n gout, pseudogout and i n some cases o f inflammatory o s t e o a r t h r i t i s . S y n t h e t i c c r y s t a l s o f MSUM and CPPD, when i n j e c t e d i n t o j o i n t s provoke an inflammatory response s i m i l a r t o the n a t u r a l m a t e r i a l (Boyle and S e e g m i l l e r , 1971). C r y s t a l s o f HAP produce an inflammatory response when i n j e c t e d i n t r a d e r m a l l y i n t o human v o l u n t e e r s (Dieppe, 1977). The s i z e o f the i n d i v i d u a l c r y s t a l s i s known to be an important f a c t o r i n acute inflammation. For example, d i f f e r e n t s i z e d u r a t e c r y s t a l s cause a v a r y i n g degree o f edema a f t e r b e i n g i n j e c t e d i n t o a r a t ' s paw (Dieppe and C a l v e r t , 1983). These workers r e p o r t e d t h a t very s m a l l c r y s t a l s (<0.1 pm) and l a r g e c r y s t a l s (>20 urn) are r e l a t i v e l y i n e f f e c t i v e i n producing inflammation, whereas c r y s t a l s o f about 5 pm i n l e n g t h are most r e a c t i v e . The na t u r e o f the c r y s t a l s u r f a c e i s thought t o be the most important f a c t o r i n determining the inflammatory e f f e c t o f the c r y s t a l s . The s t r u c t u r a l b a s i s o f the inflammatory p o t e n t i a l has been i n v e s t i g a t e d by Mandel (1976). He concluded t h a t the key f a c t o r i s the s u r f a c e roughness on an atomic s c a l e . Thus 'smooth' c r y s t a l s l i k e c y s t e i n e are i n a c t i v e , whereas u r a t e , with many charged groups p r o t r u d i n g from the s u r f a c e i s very p h l o g i s t i c . The n e g a t i v e s u r f a c e charge on the c r y s t a l faces i s thought to be r e s p o n s i b l e f o r a p a r t o f the inflammatory e f f e c t o f the c r y s t a l s . Kellermeyer (1965) proposed t h a t the negative s u r f a c e charge o f urat e c r y s t a l s c o u l d i n i t i a t e the Hageman f a c t o r and induce inflammation. Dieppe et a l . (1981a,b) r e p o r t e d t h a t a l l the c r y s t a l s capable o f causing inflammation were n e g a t i v e l y charged. They a l s o r e p o r t e d t h a t sodium u r a t e , which has the h i g h e s t inflammatory p o t e n t i a l , a l s o has the h i g h e s t negative s u r f a c e charge. Heating and g r i n d i n g s i g n i f i c a n t l y reduces the su r f a c e charge and consequently the inflammatory p o t e n t i a l (Dieppe e t a l . , 1981a,b). Burt et a l . (1983) s t u d i e d the membranolytic e f f e c t o f MSUM c r y s t a l s and r e p o r t e d a decrease i n both the z e t a p o t e n t i a l and membranolytic e f f e c t o f MSUM c r y s t a l s a f t e r h e a t i n g and r e h y d r a t i o n . Various pathways i n v o l v e d i n the mediation o f an acute inflammatory a t t a c k are shown i n F i g u r e 3 (Dieppe and C a l v e r t , 1983). F o l l o w i n g the r e l e a s e o f c r y s t a l s i n t o the s y n o v i a l f l u i d , p r o t e i n becomes bound t o the c r y s t a l s u r f a c e . I n - v i t r o s t u d i e s show t h a t MSUM c r y s t a l s adsorb a v a r i e t y o f p r o t e i n s (Kozin and McCarty, 1976, 1977; Hasselbacher, 1978, 1982; Hasselbacher and Schumacher, 1978), p a r t i c u l a r l y immunoglobulin, IgG, (Kozin and McCarty, 1976, 1977). P r o t e i n b i n d i n g i s l i k e l y t o change the s u r f a c e c h a r a c t e r i s t i c s o f the c r y s t a l s and may -12-Release of crystal into tissue space Cell-membrane interaction Release of lysosomal enzymes, activation of chemical mediators of inflammation Activation of protein/enzyme systems Activation of mediators of Increased permeability, chemotaxis etc. Coating of crystals with protein Phagocytosis Release of lysosomal ond cytoplasmic enzymes, and mediators of Inflammation, sometimes causes cell rupture Inflammation Figure 3. Possible steps involved i n crystal-induced inflammation enhance the i n t e r a c t i o n o f the c r y s t a l s with c e l l membranes, phagocytosis and a c t i v a t i o n o f c e l l f r e e inflammatory mechanisms (Doherty et a l . , 1983; G i c l a s et a l . , 1979). S e v e r a l workers have r e p o r t e d the c e n t r a l r o l e p l a y e d by the p h a g o c y t i c c e l l s i n c r y s t a l - i n d u c e d inflammation. A mechanism was proposed by McCarty (1979). The evidence f o r the r o l e o f p h a g o c y t i c c e l l s i n c l u d e s , (a) d e p l e t i n g the p h a g o c y t i c c e l l s reduces the inflammatory response i n e x p e r i m e n t a l animals and (b) the predominance of polymorphonuclear c e l l s w i t h a c t i v e p h a g o c y t o s i s i n c r y s t a l - i n d u c e d inflammation (Phelps and McCarty, 1966). S e v e r a l authors have documented the events t h a t occur on i n c u b a t i o n o f c r y s t a l s w i t h phagocytic c e l l s i n - v i t r o • Rajan (1975) observed the r e a c t i o n between polymorphs and sodium' urate c r y s t a l s u s i n g cine-microphotography. He demonstrated t h a t polymorphs engulfed or attempted t o engulf the u r a t e c r y s t a l s . Once i n s i d e the c e l l , the c r y s t a l was surrounded by a membrane (phagosome). Lysosomes then fused with the phagosome membrane. Lysosomal enzymes were t r a n s f e r e d i n t o the phagosomes but w i t h no apparent e f f e c t . Polymorphs r a p i d l y d i e d a f t e r t h a t r e s u l t i n g i n both the r e l e a s e o f c r y s t a l s f o r f u r t h e r inflammatory r e a c t i o n and l y s o s o m a l r e l e a s e . R e l e a s e o f l y s o s o m a l enzyme i s thought t o be the major cause o f inflammation. 2.3 HYPERURICEMIA AND EPIDEMIOLOGY U r i c a c i d i s the f i n a l p roduct o f c a t a b o l i s m of the p u r i n e s , -14-adenine and guanine. The p u r i n e metabolic pathways are known i n d e t a i l (Boyle and S e e g m i l l e r , 1971; Wyngaarden and K e l l e y , 1978; Nuki, 1979). The steps i n v o l v e d i n p u r i n e metabolism are shown i n F i g u r e 4. At p h y s i o l o g i c a l pH more than 90% o f the u r i c a c i d i s i o n i z e d and i t i s common to r e p r e s e n t the plasma or serum u r i c a c i d c o n c e n t r a t i o n as plasma or serum ur a t e c o n c e n t r a t i o n . Numerous e p i d e m i o l o g i c s t u d i e s have appeared i n the l i t e r a t u r e over the l a s t twenty y e a r s . More re c e n t r e p o r t s i n c l u d e , Munan e t a l . (1976); Munan e t a l . (1977); F e s s e l and B a r r (1977); S t u r g e e t a l . (1977); Yano e t a l . (1977); Simons and Jones (1978); S c o t t (1980); A k i z u k i (1982); Glynn et a l . (1983) and H a l l e t a l . (1983). These s t u d i e s show t h a t the serum urate l e v e l s vary w i t h f a c t o r s such as g e o g r a p h i c a l area, race, s o c i a l s t a t u s , age and sex. Mean serum u r a t e l e v e l s a r e g e n e r a l l y h i g h e r i n normal a d u l t males than normal a d u l t females. A sex d i f f e r e n c e i n the serum urate l e v e l s i s u s u a l l y not observed i n c h i l d h o o d but appears d u r i n g adolescence, when serum ur a t e l e v e l s i n c r e a s e more i n men than i n women (N i s h i o k a et a l . , 1974; Mikelson et a l . , 1965; Munan e t a l . , 1977). Lower mean serum ur a t e l e v e l s g e n e r a l l y makes females l e s s prone to gouty a t t a c k s than males u n t i l they reach the menopause age where, due to unknown f a c t o r s , the mean serum ur a t e l e v e l i n females r i s e s making them s u s c e p t i b l e t o gouty a r t h r i t i s . Normal serum urate l e v e l s i n h e a l t h y males i s between 5.5 mg R i b o s e - 5 - p h o s p h a t e • A T P PRPPs j 5 - P h o s p h o r i b o s y l - l - p y r o p h o s p h a t e ( P R P P ) • G l u t a m i n e PAT d G T P r dGDP ;DP •I N u c l e i c D e o x y n u c l e i c a c i d s a c i d s 5 - P h o s p h o r i b o s y l - l - a m i n e \ \ / A T P d A T P D e o x y - / G l u c o s e >NL ^ J l G T P n u c l e i c F o r m a t e J V D P d A D P a c i d s r^-~—Glutamine G D P N \ B i c a r b o n a t e N u c l e i c a c i d s - A s p a r t a t e t£^~- F o r m a t e A P R T G u a n i l i c I n o s i n i c a c i d -• A d e n y l i c - ^ A d e n i n i n e d A M P - - - - - - J a c i d ( P R P P ) 5 * N T a c i d 5 ' N T A D A G u a n o s i n e H G P R T I n o s i n e - < A d e n o s i n e k N P A G u a n i n e G u a n a s e ( P R P P ) N P X O 8 - O H a d e n i n e X O N P 2 , 8 - D i h y d r x y a d e n i n e NP H y p o x a n t h i n e r £ = 5 = ; d e o x y i n o s i n e X O X a n t h i n e X O D r i c a c i d I n t I A D A 1 D e o x y a d e n o s i n e 5 ' N T A D A = A d e n o s i n e d e a m i n a s e . A P R T - A d e n i n e p h o s p h o r i b o s y l t r a n s f e r a s e . H G P R T - H y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e . N P = N u c l e o s i d e p h o s p h o r y l a s e . 5 * N T « 5 ' n u c l e o t i d a s e . P A T = P h o s p h o r i b o s y l a m i d o t r a n s f e r a s e . P R P P S = P h o s p h o r i b o s y l p y r o p h o s p h a t e . X O = X a n t h i n e o x i d a s e . F i g u r e 4 . P a t h w a y s o f p u r i n e m e t a b o l i s m i n n a n ( N u k i , 1 9 7 9 ) . - 1 6 -dL t o 7.0 mg dL . In h y p e r u r i c e m i a the u r a t e l e v e l s are h i g h e r than normal. Hyperuricemia and gout are c l o s e l y r e l a t e d ( H a l l et a l . , 1967). The presence of h y p e r u r i c e m i a d u r i n g an a t t a c k o f acute gout i s an important d i a g n o s t i c i n d i c a t o r . However, many i n d i v i d u a l s have hyperuricemia f o r years without ever having an a t t a c k o f gouty a r t h r i t i s . 2 . 4 CAUSES OF HYPERURICEMIA 2 . 4 . 1 INBORN ERRORS OF METABOLISM Enzyme d e f i c i e n c i e s are known i n p a t i e n t s who are primary over producers o f u r a t e . However, o n l y about 5 p e r c e n t o f a l l cases o f gout are due to these d e f i c i e n c i e s . A s t r o n g f a m i l y h i s t o r y e x i s t s i n a l l types of primary gout (Graham and S c o t t , 1970). The b e s t known of these d e f i c i e n c i e s i s the t o t a l or p a r t i a l d e f i c i e n c y o f the enzyme, hypoxanthine guanine phospho-r i b o s y l t r a n s f e r a s e (HGPRTase), a f a m i l i a l n e u r o l o g i c a l d i s o r d e r and a s s o c i a t e d with h y p e r u r i c e m i a . T h i s d i s o r d e r was f i r s t d e s c r i b e d by Lesch and Nyhan i n 1964 and i s i n h e r i t e d as an x - l i n k e d r e c e s s i v e t r a i t . The enzyme d e f e c t was i d e n t i f i e d by S e e g m i l l e r et a l . (1967). Mothers of the a f f e c t e d c h i l d r e n are h e t e r o z y g o t e s and have two p o p u l a t i o n s o f f i b r o b l a s t s , one w i t h a normal p h o s p h o r i b o s y l t r a n s f e r a s e (PRTase) enzyme and others without enzyme a c t i v i t y (Rosenbloom e t a l . , 1967). Another c o n d i t i o n which may l e a d t o gout by the age of 10 years i s the Type-I glycogen storage d i s e a s e . T h i s d i s e a s e i s due to the d e f i c i e n c y o f the enzyme glucose-6-phosphatase and -17-occurs i n i n f a n c y . P a t i e n t s w i t h t h i s d i s e a s e are unable t o produce f r e e glucose r e s u l t i n g i n h y p e r l a c t i c acidemia and k e t o s i s . T h i s c o n d i t i o n has an i n h i b i t o r y e f f e c t on the r e n a l t u b u l a r e x c r e t i o n o f u r i c a c i d (Yu et a l . , 1957). D e f i c i e n c y o f an enzyme, e r y t h r o c y t e g l u t a t h i o n e reductase (EGRase) has been r e p o r t e d i n c o n j u n c t i o n with h y p e r u r i c e m i a i n negro males (Long, 1967). An autosomal mode of i n h e r i t a n c e i s i n d i c a t e d b u t the c o n n e c t i o n between the two a b n o r m a l i t i e s i s not understood. 2.4 .2 I M P A I R E D E X C R E T I O N Only t en perc e n t o f the t o t a l urate p a s s i n g through the kidneys i s f i n a l l y e x c r e t e d . There i s a g e n e r a l disagreement as t o the r e l a t i o n s h i p between gout and kidney f u n c t i o n . In a review, S t e e l e (1979) has i m p l i e d t h a t h y p e r u r i c e m i a alone has a d e l e t e r i o u s e f f e c t on kidney f u n c t i o n . Berger and Yu (1975) f a i l e d t o d e t e c t any har m f u l e f f e c t o f u n t r e a t e d h y p e r u r i c e m i a on r e n a l e f f i c i e n c y . However, K l i n e n b e r g et a l . (1975) claimed t h a t h y p e r u r i c e m i a may induce r e n a l impairment even i n the absence o f symptoms. Moderate h y p e r u r i c e m i a f r e q u e n t l y occurs i n c h r o n i c r e n a l d i s e a s e (Gresham et a l . , 1971). However, c h r o n i c r e n a l i n s u f f i c i e n c i e s are found o n l y i n a small p r o p o r t i o n o f gouty s u b j e c t s ( S a r r e , 1964; R i c h e t e t a l . , 1965). A number o f r e n a l d i s e a s e s appear t o be a s s o c i a t e d with gout, f o r example, c h r o n i c l e a d nephropathy (Emmerson, 1963; Morgan et a l . , 1966), -18-p o l y c y s t i c k i dney d i s e a s e (Newcombe, 1973; Martinez-Maldonado, 1974) and r e n a l a m i l o i d o s i s ( R i c h e t e t a l . , 1965). Although acute gouty a r t h r i t i s has been r e p o r t e d i n f r e q u e n t l y and even questioned as a c o m p l i c a t i o n o f c h r o n i c r e n a l d i s e a s e , i t i s b e l i e v e d t h a t impaired r e n a l e x c r e t i o n i s the major cause o f hy p e r u r i c e m i a ( C u r r i e , 1979; Sorensen, 1980; Gibson et a l . , 1980). 2 . 4 . 3 DRUGS AND ALCOHOL A number of drugs may i n t e r f e r e with the r e n a l t u b u l a r h a n d l i n g o f u r a t e . The hyp e r u r i c e m i a a s s o c i a t e d w i t h d i u r e t i c therapy has been found t o be due to s a l t and water l o s s ( S t e e l e and Oppenheimer, 1969). S a l i c y l a t e s , phenylbutazone, sulphinpyrazone and probenecid reduce ur a t e e x c r e t i o n a t low doses, whereas, a t h i g h e r doses these drugs i n c r e a s e the e x c r e t i o n o f urate (Wyngaarden and K e l l e y , 1978). The i n g e s t i o n o f eth a n o l may p r e c i p i t a t e an a t t a c k o f gout (Wyngaarden and K e l l e y , 1976). T h i s e f f e c t o f a l c o h o l has been a t t r i b u t e d t o decreased r e n a l c l e a r a n c e o f u r i c a c i d secondary t o the h y p e r l a c t i c acidemia a s s o c i a t e d with e t h a n o l metabolism ( L i e b e r and Davidson, 1962; L i e b e r et a l . , 1962; Beck, 1981; Yu et a l . , 1957; Maclachlan and Rodnan, 1967). A l c o h o l i n t a k e has a l s o been i m p l i c a t e d i n the i n c r e a s e d p r o d u c t i o n o f u r i c a c i d (Grunst e t a l . , 1977; Delba r r e e t a l . , 1967a,b; F a l l e r and -19-Fox, 1982). There i s a h i g h e r p r e v a l e n c e o f gout amongst i n d i v i d u a l s who consume et h a n o l r e g u l a r l y (Saker et a l . , 1967; P e l l and D'Alonzo, 1968) and p o p u l a t i o n s w i t h a h i g h e r i n c i d e n c e o f gout have a h i g h p r o p o r t i o n o f people who consume et h a n o l i n excess (Gibson and Grahame, 1974). 2.5 DEPOSITION OF CRYSTALS 2.5.1 ORIGIN OF CRYSTALS AND THEIR RELATIONSHIP TO JOINT  DISEASE The r e l a t i o n s h i p between j o i n t d i s e a s e s and c r y s t a l d e p o s i t i o n i s not w e l l understood. A number o f complex systems are p r e s e n t i n the body t o a c t i v a t e c r y s t a l f o r m a t i o n where i t i s necessary, f o r example i n the bones and t e e t h and i n h i b i t i t where i t i s u n d e s i r a b l e , f o r example i n the e x c r e t o r y organs, s a l i v a and the u r i n a r y t r a c t , a l l o f which c o n t a i n compounds which i n h i b i t c r y s t a l growth. There are s e v e r a l i n s t a n c e s where abnormal f u n c t i o n i n g o f a system may cause a s o l u t e excess l e a d i n g t o c r y s t a l l i z a t i o n . P a t i e n t s with h y p e r u r i c o s u r i a may develop r e n a l stones. In j o i n t d i s e a s e t h e r e may be e i t h e r a l o c a l or a g e n e r a l i z e d s o l u t e excess. For example, i n CPPD d e p o s i t i o n d i s e a s e there i s a l o c a l i z e d excess o f i n o r g a n i c pyrophosphate i n the j o i n t , and i n gout, t h e r e i s an i n c r e a s e d l e v e l o f serum u r a t e . However, t r u e s o l u t e c o n c e n t r a t i o n s a t v a r i o u s s i t e s cannot be e s t a b l i s h e d because o f the complex nature o f the b i o l o g i c a l system. -20-Some o f the suggested mechanisms r e l a t e d t o MSUM c r y s t a l formation are l i s t e d i n Table.1. ( S c o t t , 1983). L i t t l e i s known about these f a c t o r s except those r e l a t e d t o s u p e r s a t u r a t i o n , such as temperature. The r e l a t i o n s h i p s between c r y s t a l d e p o s i t i o n and j o i n t d i s e a s e have been s t u d i e d by Dieppe (1982) and Dieppe and C a l v e r t (1983). They have proposed v a r i o u s pathways i n v o l v e d i n c r y s t a l r e l a t e d j o i n t d i s e a s e s ( F i g u r e 5). They b e l i e v e t h a t j o i n t damage may a l s o r e s u l t i n n u c l e a t i o n and c r y s t a l growth because of the removal of the i n h i b i t o r s or i n t r o d u c t i o n of some a c t i v a t o r s . Dieppe et a l . (1983) have proposed some p o s s i b l e r e l a t i o n s h i p s between c r y s t a l d e p o s i t i o n and j o i n t d i s e a s e ( F i g u r e 6 ) . These i n c l u d e , (1) j o i n t d i s e a s e causes c r y s t a l l i z a t i o n , (2) c r y s t a l s cause j o i n t d i s e a s e , (3) c r y s t a l d e p o s i t s are a by-product o f a process causing a r t h r i t i s , (4) c r y s t a l d e p o s i t i o n and j o i n t d i s e a s e are independent and a chance r e l a t i o n s h i p e x i s t s , and (5) j o i n t damage and c r y s t a l d e p o s i t i o n i n t e r a c t t o g e t h e r . 2.6 CARTILAGE J o i n t s c o n t a i n a v a r i e t y o f co n n e c t i v e t i s s u e s such as ligaments, tendons, s y n o v i a l membrane and c a r t i l a g e . With the ex c e p t i o n o f c a r t i l a g e , l i t t l e i s known about the composition o f these t i s s u e s . Almost, a l l v e r t e b r a t e c a r t i l a g e s c o n s i s t s o f a combination of c o n n e c t i v e (or s k e l e t a l ) t i s s u e c e l l s and e x t r a c e l l u l a r - 2 1 -Table 1. Suggested mechanisms r e l a t e d t o MSUM i n - v i v o c r y s t a l formation ( S c o t t , 198 1. S u p e r s a t u r a t i o n of serum or s y n o v i a l f l u i d w i t h MSUM. 2. P r o t e i n b i n d i n g of u r a t e . 3. Turnover of p r o t e o g l y c a n s . 4. Temperature. 5. Trauma and e x e r c i s e . 6. A l t e r e d hydrogen i o n c o n c e n t r a t i o n . 7. R e s o r p t i o n of e x t r a v a s c u l a r f l u i d . 8. Ageing and a v a s c u l a r i t y . - 2 2 -SOLUTE EXCESS LOCAL TISSUE CONDITIONS ACTIVATORS/INHIBITORS DISSOLUTION^ CRYSTAL FORMATION INFLAMMATION .ASYMPTOMATIC DEPOSIT OTHER MECHANISMS ? TISSUE DAMAGE Fi g u r e 5. P o s s i b l e pathways i n v o l v e d i n c r y s t a l r e l a t e d j o i n t d i s e a s e s (Dieppe and C a l v e r t , 1983). - 2 3 -(1) X > JOINT DISEASE > CRYSTAL (2) X > CRYSTALS > JOINT DISEASE ^^JOINT DISEASE (3) X ~ CRYSTALS (4) X > JOINT DISEASE Y > CRYSTAL GROWTH (5) JOINT DISEASE <===> CRYSTAL GROWTH Fig u r e 6. P o s s i b l e r e l a t i o n s h i p s between c r y s t a l d e p o s i t i o n and j o i n t d i s e a s e s (Dieppe e t a l . , 1983). -24-substance ( f i b r o u s matrix and ground substance) (Moss and Moss-S a l e n t i j n , 1983). A r t i c u l a r c a r t i l a g e i s an a v a s c u l a r t i s s u e i n which the c e l l s (chondrocytes) are s p a r s e l y d i s t r i b u t e d i n a s t i f f and abundant matrix (Edwards and Chrisman, 1979). In the j o i n t s , t h i s t i s s u e can withstand very h i g h compressive l o a d s . 2 . 6 . 1 COMPOSITION OF CARTILAGE A r t i c u l a r c a r t i l a g e c o n s i s t s mainly o f t h r e e components, water, c o l l a g e n and p r o t e o g l y c a n aggregates. The composition o f a r t i c u l a r c a r t i l a g e v a r i e s markedly with depth and l o c a l i z a t i o n (Muir, 1979). (A) WATER The water content o f a r t i c u l a r c a r t i l a g e i s about 65-75% (Venn and Maroudas, 1977; Maroudas, 1979). The p r e s e n c e of water i n c o n j u n c t i o n with p r o t e o g l y c a n s and c o l l a g e n makes c a r t i l a g e tough and e l a s t i c . (B) COLLAGEN C o l l a g e n i s the major component o f the e x t r a c e l l u l a r matrix of a l l c a r t i l a g e s and comprises approximately 10% o f the wet weight and 40-50% of the dry weight of the t i s s u e (Anderson et a l . , 1964 ; Mayne and Vonder-Mark, 1983). C o l l a g e n appears as a f i n e mesh work o f f i b r i l s . The nomenclature f o r the c o l l a g e n c h a i n s and t h e i r m o l e c u l a r o r g a n i z a t i o n was proposed by B o r n s t e i n and Sage (1980). Various types of c o l l a g e n s found i n d i f f e r e n t - 2 5 -t i s s u e s have d i f f e r e n t chains and molecular o r g a n i z a t i o n . Proteoglycans can e i t h e r i n t e r a c t w i t h c o l l a g e n d i r e c t l y (Smith et a l . , 1967) or p o s s i b l y through a p r o t e i n such as chondronectin (Hewitt et a l . , 1980) and a c a r t i l a g e matrix p r o t e i n (Paulsson and Heinegard, 1979, 1981). (C) PROTEOGLYCAN AGGREGATE (PGA) In normal a r t i c u l a r c a r t i l a g e , c o l l a g e n forms a f i b r o u s net-work, w i t h i n which a random macromolecular mesh i s p r e s e n t . T h i s macromolecular mesh i s an aggregate of some 30-40 p r o t e o g l y c a n s u b u n i t s (monomers) each w i t h a m o l e c u l a r weight of approximately one m i l l i o n and h y a l u r o n i c a c i d . F i g u r e 7 shows a schematic r e p r e s e n t a t i o n of the domains of c o l l a g e n and i t s r e g i o n s o f i n t e r a c t i o n with p r o t e o g l y c a n aggregate (Hascal and Heinegard, 1974; Hardingham and Muir, 1974). The u l t r a s t r u c t u r e of the p r o t e o g l y c a n a g g r e g a t e was r e p o r t e d by Rosenberg e t a l • (1975). F i g u r e 8 shows the most accepted model o f the p r o t e o g l y c a n -h y a l u r o n i c a c i d aggregate (Hardingham et a l . , 1976). The b i o l o g i c a l f u n c t i o n of p r o t e o g l y c a n aggregation i s not known. However, the s i z e of the aggregate immobilizes them i n the c o l l a g e n network and presumably has a r o l e p e c u l i a r to the f u n c t i o n o f c a r t i l a g e . ( I ) PROTEOGLYCAN SUBUNIT (MONOMER)(PGM) These are macromolecules which c o n t a i n one or more glycosaminoglycan (GAG) c h a i n s , normally a t t a c h e d to p r o t e i n . -26-gure 7 . S t r u c t u r e of a r t i c u l a r c a r t i l a g e : (a) Domains of p r o t e o g l y c a n aggregate and c o l l a g e n . (b) Regions of i n t e r a c t i o n between p r o t e o g l y c a n aggregate and c o l l a g e n . -27-H Y A L U R O N I C A C I D F i g u r e 8 . S t r u c t u r e of p r o t e o g l y c a n aggregate. - 28-D i f f e r e n t c l a s s e s of proteoglycans (PGs) vary i n the nature and s i z e of the core p r o t e i n , i n the type, number, average s i z e and degree of s u l f a t i o n of the GAG c h a i n s , and the types of other o l i g o s a c c h a r i d e s p r e s e n t on the core p r o t e i n . S e v e r a l reviews of the s t r u c t u r e and f u n c t i o n s of p r o t e o g l y c a n s have been p u b l i s h e d ( H a s c a l l , 1981; Muir, 1980; Roden, 1980). The c a r t i l a g e PGs are composed of a p r o t e i n core g e n e r a l l y c o m p r i s i n g about 10-20% of the weight of the molecule, to which a l a r g e number of GAG chains are attached l a t e r a l l y ( F i g u r e 9a). The s t r u c t u r e of PG was f i r s t proposed by Mathews and L o z a i t y t e (1958). In the c a r t i l a g e PGs, c h o n d r o i t i n s u l f a t e (CS) i s the major GAG w i t h s m a l l e r but v a r i a b l e amounts of k e r a t a n s u l f a t e (KS) . Most of the PG-monomers can form an aggregate with h y a l u r o n i c a c i d (HA). However, there are minor PGs t h a t cannot i n t e r a c t with HA to form an aggregate and these PGs can be e x t r a c t e d from c a r t i l a g e by non d i s s o c i a t i n g s a l t s o l u t i o n s o f p h y s i o l o g i c a l i o n i c c o n c e n t r a t i o n (Hardingham and Muir, 1974; Hardingham et a l . , 1976). These PGs are heterogeneous i n molecular s i z e and chemical composition (Brandt and Muir, 1971) and are of r e l a t i v e l y low molecular weight (Tsiganos and Muir, 1969). They c o n t a i n l e s s p r o t e i n and l e s s KS than the m a j o r i t y o f PGs (Hardingham and Muir, 1974; Brandt and Muir, 1969; Simunek and Muir, 1972a,b; Mayes, Mason and G r i f f i n , 1973). -29-Figure 9. Structures of: (a) Proteoglycan monomer, (b) hyaluronic a c i d . -30-( I a ) CHONDROITIN S U L F A T E (CS) C h o n d r o i t i n s u l f a t e c o n s i s t s of r e p e a t i n g u n i t s o f g l u c u r o n i c a c i d and N - a c e t y l galactosamine. An average c h a i n of CS c o n t a i n s about 25-30 d i s a c c h a r i d e u n i t s and about one s u l f a t e group per d i s a c c h a r i d e u n i t i n one of the two i s o m e r i c p o s i t i o n s , forming e i t h e r c h o n d r o i t i n - 6 - s u l f a t e or c h o n d r o i t i n - 4 - s u l f a t e ( F i g u r e 10a). The d i s t r i b u t i o n of the s u l f a t e r e s i d u e s i s not uniform along the c h a i n , being fewer i n the r e g i o n near the l i n k a g e o f carbohydrate to p r o t e i n (Wasteson and L i n d a h l , 1971). C h o n d r o i t i n - 6 - s u l f a t e appears to predominate i n human and bovine a r t i c u l a r c a r t i l a g e (Mourao et a l . , 1976; Murata and B j e l l e , 1977), whereas i n growth c a r t i l a g e there are approximately equal p r o p o r t i o n s o f the two isomers (Mourao et a l . , 1976). The b i o l o g i c a l s i g n i f i c a n c e o f the p o s i t i o n o f the s u l f a t e group i s not c l e a r . Both CS isomers show h i g h l y ordered h e l i c a l c o n f o r m a t i o n s . The s u l f a t e groups p r o j e c t f u r t h e r from the c h a i n i n c h o n d r o i t i n - 6 - s u l f a t e than i n c h o n d r o i t i n - 4 - s u l f a t e (Isaac and A t k i n s , 1973; A t k i n s , 1977). I t has been suggested t h a t the presence o f c h o n d r o i t i n - 6 - s u l f a t e allows PGs t o i n t e r a c t more s t r o n g l y with the b a s i c groups o f c o l l a g e n and other p r o t e i n s . ( I b ) KERATAN S U L F A T E (KS) Keratan s u l f a t e i s composed of d i s a c c h a r i d e r e p e a t i n g u n i t s of g a l a c t o s e and N-acetylglucosamine ( F i g u r e 10b). H i s t o -chemical ( S t o c k w e l l , 1970) and X-ray microprobe a n a l y s i s (Maroudas, 1972) methods i n d i c a t e t h a t i n the middle and deep -31-Figure 10. Structures of: (a) Chondroitin ( i : - 6 - ; i i : - 4 - ) s u l f a t e , (b) Keratan s u l f a t e . -32-l a y e r s o f a r t i c u l a r c a r t i l a g e the p r o p o r t i o n o f KS r e l a t i v e t o CS v a r i e s . KS i s more v a r i a b l e than CS i n both c h a i n l e n g t h and i n degree o f s u l f a t i o n (Muir and Hardingham, 1975). Two p o p u l a t i o n s o f KS a r e thought t o e x i s t . One p o p u l a t i o n has one s u l f a t e group per d i s a c c h a r i d e r e p e a t i n g u n i t , while the o t h e r p o p u l a t i o n c o n t a i n s c o n s i d e r a b l y more s u l f a t e ( H j e r t q u i s t and Lempberg, 1972). The s k e l e t a l KS has an average molecular weight o f 5000-10,000 corresponding to about 13 d i s a c c h a r i d e u n i t s (Robinson and Hopwood, 1973; H a s c a l l and R i o l o , 1972). Other s u l f a t e d c a r t i l a g e GAGs i n c l u d e dermatan s u l f a t e , h e p a r i n s u l f a t e and h e p a r i n . The composition o f v a r i o u s s u l f a t e d GAGs and t h e i r sources have been reviewed r e c e n t l y by Lash and Vasan (1983). ( I I ) HYALURONIC A C I D (HA) HA from c a r t i l a g e has been i s o l a t e d and f u l l y c h a r a c t e r i z e d ( H a s c a l l and Heinegard, 1974; Hardingham and Muir, 1974; H j e r t q u i s t and Lempberg, 1972). HA i s a n o n - s u l f a t e d GAG composed o f equimolar q u a n t i t i e s o f g l u c u r o n i c a c i d and N-acetylglucosamine. E l e c t r o n m i c r o s c o p i c s t u d i e s ( F e s s l e r and F e s s l e r , 1966) show HA t o c o n s i s t of a s i n g l e c h a i n . F i g u r e 9b shows the s t r u c t u r e o f HA. In c a r t i l a g e , HA p l a y s an e s s e n t i a l r o l e i n the aggregation o f p r o t e o g l y c a n s . I t i s p r e s e n t mainly as a component of PGAs and not i n the f r e e form. -33-(D) L I P I D S OF C A R T I L A G E L i p i d i s found i n the c e l l s and i n the matrix of human a r t i c u l a r c a r t i l a g e . I n t r a c e l l u l a r l i p i d i s c o n s i d e r e d t o be a normal c o n s t i t u e n t o f c a r t i l a g e because i t i s found i n the absence o f degenerative changes i n the c e l l s ( C o l l i n s et a l . , 1965). E x t r a c e l l u l a r l i p i d s have been i d e n t i f i e d i n h e a l t h y a r t i c u l a r c a r t i l a g e from i n d i v i d u a l s i n the second decade o f l i f e upwards ( G h a d i a l l y et a l . , 1965; S t o c k w e l l , 1965; Zbinden, 1952; Schott, 1963; M a r o t t i , 1963). L i t t l e i s known about the nature o f the l i p i d s o f a r t i c u l a r c a r t i l a g e . S t a i n i n g c h a r a c t e r i s t i c s and chemical a n a l y s i s i n d i c a t e t h a t they are p a r t l y comprised of n a t u r a l f a t s : t r i g l y c e r i d e s , c h o l e s t e r o l , c h o l e s t e r o l e s t e r s and p h o s p h o l i p i d s (Bonner e t a l . , 1975; Pearse, 1968). 2 . 6 . 2 FACTORS A F F E C T I N G THE COMPOSITION OF C A R T I L A G E (A) E F F E C T OF A G E A d u l t a r t i c u l a r c a r t i l a g e shows i n c r e a s e d degenerative changes w i t h age ( C o l l i n s and Meachim, 1961). Other changes i n c l u d e , a decrease i n water content o f c a r t i l a g e w i t h age (Ruttner et a l . , 1974) and a decrease i n the c o l l a g e n content w i t h age on a dry weight b a s i s (Werner et a l . , 1976). The e l a s t i c i t y o f c a r t i l a g e i s r e s p o n s i b l e f o r the r e s i s t a n c e t o compression and i s r e l a t e d t o the content and the s t r u c t u r e o f the p r o t e o g l y c a n s i n the c a r t i l a g e matrix ( H a r r i s et a l . , 1972; S c o t t , 1973, 1975). The r e s i s t a n c e t o -34-compression o f the c a r t i l a g e decreases with age, probably due to the a l t e r a t i o n s i n the p r o t e o g l y c a n s t r u c t u r e o f the a r t i c u l a r c a r t i l a g e ( S c h o f i e l d and Weightman, 1978; Armstrong et a l . , 1 9 7 9 ) . Evidence i n d i c a t e s t h a t the composition of the matrix o f human a r t i c u l a r c a r t i l a g e changes with age ( E l l i o t and Gardener, 1978, 1979; Venn, 1978; I n e r o t e t a l . , 1 9 7 8 ) . The f o l l o w i n g i s a summary o f the e f f e c t s of ageing on the composition o f c a r t i l a g e . ( i ) B i ochemical s t u d i e s o f changes i n the nature and q u a n t i t y of c a r t i l a g e matrix glycosaminoglycans d u r i n g ageing have shown d i f f e r e n t r e s u l t s . L i t t l e or no changes i n t o t a l GAG c o n t e n t o f a r t i c u l a r c a r t i l a g e were r e p o r t e d by Maroudas et a l . ( 1 9 7 3 ) . E l l i o t t and Gardner (1979) r e p o r t e d a decrease i n the GAG content of the s u r f a c e zone c a r t i l a g e from 15% (dry weight) at about 4 years of age to about 7% by 70 years o f age. T h i s o b s e r v a t i o n o f a decrease i n the t o t a l GAG content was a l s o noted by I n e r o t et a l . ( 1 9 7 8 ) , and Roughley and White ( 1 9 8 0 ) . ( i i ) The h y a l u r o n i c a c i d content of the c a r t i l a g e i n c r e a s e s g r a d u a l l y with ageing. During the f i r s t decade o f l i f e , v e r y l i t t l e HA i s d e t e c t e d . A f t e r ten y e a r s the HA c o n t e n t i n c r e a s e s t o 2% and reaches 6% by 60 years ( E l l i o t t and Gardner, 1 9 7 9 ) . ( i i i ) The p r o p o r t i o n of proteoglycans t h a t can be e x t r a c t e d w i t h 4 molar guanidinium c h l o r i d e decreases w i t h -35-i n c r e a s i n g age ( I n e r o t et a l . , 1978; Roughley and White, 1980). However, B a y l i s s and A l i (1978a,b); Simunek and Muir (1972b) and Sweet et a l . (1977) r e p o r t e d an i n c r e a s e i n the e x t r a c t a b l e p r o t e o g l y c a n f r a c t i o n with age. ( i v ) The average molecular weight o f c h o n d r o i t i n s u l f a t e decreases from about 20,000 i n young i n d i v i d u a l s t o 16,000 i n a d u l t s and the e l d e r l y ( H j e r t q u i s t and Wasteson, 1972; Garg and Swan, 1981; Sweet et a l . , 1979). Ageing a l s o r e s u l t s i n a decrease i n the p r o p o r t i o n of c h o n d r o i t i n s u l f a t e i n the e x t r a c t e d proteoglycans ( S t r i d e r et a l . , 1976; B a y l i s s , 1976; Venn, 1978). However, the r e l a t i v e p r o p o r t i o n of c h o n d r o i t i n - 6 -s u l f a t e t o c h o n d r o i t i n - 4 - s u l f a t e i n c r e a s e s w i t h age i n a d u l t human and bovine c a r t i l a g e ( H j e r t q u i s t and Wasteson, 1972; H j e r t q u i s t and Lempberg, 1972; Mankin and L i p p i e l l o , 1971; Lust and Pronosky, 1972; Lempberg et a l . , 1974; Murata and B j e l l e , 1979; Rougley and White, 1980; Garg and Swan, 1981). (v) The ke r a t a n s u l f a t e content of the e x t r a c t a b l e p r o t e o g l y c a n s i n c r e a s e s wih age ( B a y l i s s and A l i , 1978a,b; I n e r o t e t a l . , 1978; Sweet e t a l . , 1977; E l l i o t t and Gardner, 1979). During ageing the s u p e r f i c i a l zone of a d u l t c a r t i l a g e seems t o accumulate a h i g h e r percentage of KS ( E l l i o t t and Gardner, 1979). T h i s i n c r e a s e has been thought to be due to a f a s t e r turnover o f the GAG i n the c a r t i l a g e s u r f a c e zone caused by wear du r i n g the mechanical f u n c t i o n i n g of the s y n o v i a l j o i n t s ( E l l i o t t and Gardner, 1979). ( v i ) Both i n t r a c e l l u l a r and e x t r a c e l l u l a r l i p i d -36-c o n c e n t r a t i o n s show a d i s t i n c t i n c r e a s e w i t h advancing age ( G h a d i a l l y e t a l . , 1965; M a r o t t i , 1963; Bonner e t a l . , 1975). The estimated i n c r e a s e per year i s shown i n Table 2 (Bonner et a l . , 1975; H i r s c h and P e i f f e r , 1957; Ravetto, 1964). (B) E F F E C T OF INJURY S t u d i e s u s i n g the a r t i c u l a r c a r t i l a g e o f the r a b b i t showed th a t a f t e r a s u p e r f i c i a l i n j u r y , the f i r s t change occured i n the matrix w i t h a l o s s of p r o t e o g l y c a n s along the margin of i n j u r y f o l l o w e d by enhanced GAG s y n t h e s i s (Meachim, 1963). A f t e r i n j u r y , the chondrocytes produce enzymes capable o f degrading the c a r t i l a g e matrix (Chrisman and F e s s e l , 1962). A c a t h e p s i n - l i k e p rotease seems to be the enzyme r e s p o n s i b l e f o r c a r t i l a g e d e g r a d a t i o n (Dingle and D i n g l e , 1980; Z i f f et a l . , 1960). ( C ) E F F E C T OF J O I N T D I S E A S E Primary o s t e o a r t h r i t i s i s u s u a l l y but not e x c l u s i v e l y a d i s e a s e o f o l d age. O s t e o a r t h r i t i s secondary to d i s e a s e or trauma o f t e n occurs i n young and middle aged i n d i v i d u a l s . The changes i n the j o i n t c a r t i l a g e are s i m i l a r i n both primary and s e c o n d a r y o s t e o a r t h r i t i s ( I n e r o t et a l . , 1978; Chrisman, 1969). Kempson et a l . (1971) showed t h a t the e l a s t i c i t y o f the c a r t i l a g e decreases i n o s t e o a r t h r i t i c i n d i v i d u a l s . Since the e l a s t i c i t y of c a r t i l a g e i s l a r g e l y r e l a t e d t o the content and s t r u c t u r e of p r o t e o g l y c a n s , i t i s t h e r e f o r e l i k e l y t h a t the -37-Table 2. The estimated increase i n the l i p i d content of c a r t i l a g e per year of age (Bonner et a l . f 1975; Hirsh and P e i f f e r , 1957; Ravetto, 1964). Estimated increase per year L i p i d s (% dry weight) S u p e r f i c i a l Deep layer layer Total l i p i d 0.046 0.03 Tr i g l y c e r i d e s 0.007 0.006 Cholesterol 0.008 0.003 Phospholipids 0.001 0.004 -38-composition o f the p r o t e o g l y c a n s i s changed i n o s t e o a r t h r i t i s . I t has been suggested t h a t d e g r a d a t i o n o f the p r o t e o g l y c a n s i s an important step i n the development of o s t e o a r t h r i t i s (Mankin et a l . , 1971. S e v e r a l s t u d i e s have r e p o r t e d the f o l l o w i n g changes i n the composition o f c a r t i l a g e i n o s t e o a r t h r i t i s . ( i ) The d e g e n e r a t e d o s t e o a r t h r i t i c c a r t i l a g e has a h i g h e r water content (McDevitt and Muir, 1976; Mankin and Z a r i n s -Thrasher, 1975; L i p s h i t z e t a l . , 1976). ( i i ) P r o t e o g l y c a n monomers from d e g e n e r a t e d c a r t i l a g e are r e p o r t e d t o be s m a l l e r than those from normal c a r t i l a g e of the same age ( I n e r o t et a l . , 1978). The d e g r a d a t i o n o f p r o t e o g l y c a n s may be due to the i n c r e a s e d enzyme a c t i v i t y o f c a t h e p s i n s ( A l i and Evans, 1973) or n e u t r a l proteases ( M a r t e l -P e l l e t i e r e t a l • , 1984). ( i i i ) The t o t a l GAG content i s s l i g h t l y lower (not s i g n i f i c a n t ) i n degenerated than i n normal c a r t i l a g e o f the same age ( I n e r o t et a l . , 1978). S i m i l a r o b s e r v a t i o n s were a l s o made by B o l l e t and Nance (1966) and Mankin and L i p p e l l o (1970). ( i v ) In o s t e o a r t h r i t i s the c o n c e n t r a t i o n of c h o n d r o i t i n -4 - s u l f a t e i n c r e a s e s and the c o n c e n t r a t i o n o f k e r a t a n s u l f a t e decreases (Mankin and L i p p i e l l o , 1971). 2 . 7 S Y N O V I A L F L U I D The s y n o v i a l f l u i d i s r e s p o n s i b l e f o r the n u t r i t i o n and l u b r i c a t i o n o f the j o i n t t i s s u e s . The s y n o v i a l membrane -39-r e g u l a t e s both the volume and the macromolecular composition of the s y n o v i a l f l u i d (Horst and W a l i t z a , 1980). 2 . 7 . 1 COMPOSITION OF SYNOVIAL F L U I D The c o n s t i t u e n t s o f s y n o v i a l f l u i d are d e r i v e d from t h r e e p o s s i b l e sources (Swan, 1978). (A) S O L U B L E CONSTITUENTS DERIVED FROM T H E BLOOD The e l e c t r o l y t e components, glucose and a l l but a few p r o t e i n c o n s t i t u e n t s are d e r i v e d d i r e c t l y from the plasma. The r e l a t i v e p r o p o r t i o n s o f the c o n s t i t u e n t s depends p r i m a r i l y on t h e i r m olecular weights. The r e l a t i o n s h i p between molecular weight and c o n c e n t r a t i o n o f serum p r o t e i n i n s y n o v i a l f l u i d i s • give n i n Table 3 (Kushner and S o m e r v i l l e , 1971). Albumin, the low molecular weight p r o t e i n o f plasma, c o n s t i t u t e s about 75% of the t o t a l p r o t e i n content o f s y n o v i a l f l u i d (Sandson and Hamerman, 1958). (B) CONSTITUENTS S E C R E T E D BY T H E J O I N T T I S S U E S ( I ) HYALURONIC A C I D (HA) The HA o f the s y n o v i a l f l u i d i s s i m i l a r t o the HA of c a r t i l a g e . Only a smal l amount of HA i s pr e s e n t i n the s y n o v i a l f l u i d and i t i s thought t o be manufactured by the c e l l l i n i n g o f the s y n o v i a l membrane (Baxter et a l . , 1973). The f u n c t i o n o f HA i n the s y n o v i a l f l u i d i s not c l e a r but i t i s thought t o mai n t a i n the v i s c o s i t y o f the s y n o v i a l f l u i d . -40-Table 3. The r e l a t i o n s h i p between molecular weight and concentration of serum proteins i n synovial f l u i d . Component Molecular weight (x 10 J) Normal plasma concentration (mg. mL~ ) Normal SF/Serum r a t i o a 1 ~ a c i d glyco-protein 44 0.75- 1.0 0.23 +0.09 Transferin 74 2.0 - 3.2 0.24 +0.08 Ceruloplasmin .160 0.27- 0.39 0.16 +0.04 a2 _n>acroglobulin 820 2.2 - 3.8 0.033+0.028 IgG 158 12.0 -18.0 0.13 +0.07 IgM 1000 0.045+0.024 - 4 1 -( I I ) L U B R I C A T I N G GLYCOPROTEINS The p u r i f i e d l u b r i c a t i n g p r o t e i n s p r e s e n t i n the s y n o v i a l f l u i d r e p r e s e n t about 0.5% o f the t o t a l p r o t e i n i n the s y n o v i a l f l u i d (Swan and Radin, 1972). (C ) PRODUCTS DERIVED FROM T H E CATABOLISM OF J O I N T T I S S U E S Disease s t a t e s such as o s t e o a r t h r i t i s and rheumatoid a r t h r i t i s r e s u l t i n p e r s i s t a n t PG l o s s from the a r t i c u l a r c a r t i l a g e . S y n o v i a l f l u i d removes the d e g r a d a t i o n products by lymphatic uptake or p h a g o c y t i c uptake. C h o n d r o i t i n s u l f a t e i s p r e s e n t i n s y n o v i a l f l u i d as a normal c o n s t i t u e n t ( S i l p a n a n t a et a l . , 1967). 1 2 . 7 . 2 L I P I D S OF SYNOVIAL F L U I D Normal s y n o v i a l f l u i d c o n t a i n s some p h o s p h o l i p i d s , c h o l e -s t e r o l and some n e u t r a l l i p i d s . Among the p h o s p h o l i p i d s , p h o s p h a p h a t i d y l c h o l i n e , sphingomyelin, l y s o l e c i t h i n , p h o s p h a t i d y l i n o s i t o l and c e p h a l i n s have been i d e n t i f i e d (Bole, 1962; Chung et a l . , 1962). 2 . 7 . 3 FACTORS A F F E C T I N G THE COMPOSITION OF SYNOVIAL F L U I D (A) E F F E C T OF J O I N T D I S E A S E The t o t a l s y n o v i a l f l u i d p r o t e i n content i s u s u a l l y i n c r e a s -ed i n d e g e n e r a t i v e j o i n t d i s e a s e and rheumatoid a r t h r i t i s (Currey and Vernon-Roberts, 1976). The t o t a l p r o t e i n content i n c r e a s e d - 4 2 -from 1.8-2.1 g dL i n normal j o i n t s t o 2.9-3.9 g dL-^" i n o s t e o a r t h r i t i s and to 4.2-4.9 g d L - 1 i n rheumatoid a r t h r i t i s (Markowitz, 1983). However, the percentage of albumin decreased from about 56-63% i n normal j o i n t s to 42-52% i n rheumatoid a r t h r i t i s and the percentage of h i g h molecular weight p r o t e i n s ( g l o b u l i n s ) i n c r e a s e d (Markowitz, 1983). C h o n d r o i t i n s u l f a t e i s p r e s e n t i n the normal s y n o v i a l f l u i d and i s pro b a b l y r e l e a s e d as a r e s u l t o f the c a t a b o l i c process i n a r t i c u l a r c a r t i l a g e ( S i l p a n a n t a et a l . , 1967). The l e v e l s o f c h o n d r o i t i n s u l f a t e i n the s y n o v i a l f l u i d are e l e v a t e d i n dege n e r a t i v e j o i n t d i s e a s e and rheumatoid a r t h r i t i s (Seppala et a l . , 1972; Barker et a l . , 1966). Diseased j o i n t s i n o s t e o a r t h r i t i s and rheumatoid a r t h r i t i s show g r e a t l y i n c r e a s e d l i p i d l e v e l s (Bole, 1962; Chung et a l . , 1962; Small et a l . , 1964; Newcombe and Cohen, 1965). The i n c r e a s e i n t r i g l y c e r i d e content i s l e s s than the i n c r e a s e i n p h o s p h o l i p i d and c h o l e s t e r o l content (Bole, 1962). The c o n c e n t r a t i o n o f p h o s p h o l i p i d s i n the s y n o v i a l f l u i d a l s o shows d i s t i n c t change i n d i s e a s e s t a t e s . Chung et al.(1962) repor-ted a decrease i n the l e v e l s o f p h o s p h a t i d y l c h o l i n e and an i n c r e a s e i n c e p h a l i n and sphingomyelin l e v e l s i n the s y n o v i a l f l u i d i n o s t e o a r t h r i t i s or rheumatoid a r t h r i t i s . 2.8 THEORY OF CRYSTAL GROWTH The d e p o s i t i o n o f a s o l i d c r y s t a l l i n e phase from s o l u t i o n can o n l y occur i f some degree of s u p e r s a t u r a t i o n or s u p e r c o o l i n g -43-has f i r s t been achieved i n the system. Any c r y s t a l l i z a t i o n process can be co n s i d e r e d t o be comprised o f t h r e e b a s i c s t e p s : (A) Achievement o f s u p e r s a t u r a t i o n or s u p e r c o o l i n g . (B) Formation o f c r y s t a l n u c l e i . (C) Growth o f the c r y s t a l s . 2 . 8 . 1 SUPERSATURATION A s o l u t i o n which i s i n e q u i l i b r i u m with the s o l i d phase i s s a i d t o be s a t u r a t e d with r e s p e c t t o t h a t s o l i d . However, a s o l u t i o n c o n t a i n i n g more d i s s o l v e d s o l i d than t h a t r e p r e s e n t e d by the s a t u r a t i o n s o l u b i l i t y i s s a i d t o be s u p e r s a t u r a t e d . Uncontaminated s o l u t i o n s i n c l e a n c o n t a i n e r s , c o o l e d slowly, without d i s t u r b a n c e , i n a dust f r e e atmosphere, can r e a d i l y be made to show a p p r e c i a b l e degrees o f s u p e r s a t u r a t i o n . The s t a t e o f s u p e r s a t u r a t i o n i s an e s s e n t i a l f e a t u r e o f c r y s t a l l i z a t i o n . Below the s a t u r a t i o n s o l u b i l i t y , a l l s o l u t i o n s are s t a b l e . Ostwald i n 1897 in t r o d u c e d the terms ' l a b i l e ' (unstable) and 'metastable' s u p e r s a t u r a t i o n . These terms r e f e r t o super-s a t u r a t e d s o l u t i o n s i n which spontaneous d e p o s i t i o n o f the s o l i d phase i n the absence o f s o l i d n u c l e i , w i l l and w i l l not occur, r e s p e c t i v e l y . A s o l u b i l i t y - s u p e r s o l u b i l i t y diagram i s shown i n Fi g u r e 11. The lower continuous l i n e i s the normal s o l u b i l i t y curve f o r the p a r t i c u l a r s a l t . Temperatures and c o n c e n t r a t i o n s at which spontaneous c r y s t a l l i z a t i o n occurs are represented by the upper broken curve, r e f e r r e d t o as the s u p e r s o l u b i l i t y curve. T h i s curve i s not as w e l l d e f i n e d as the s o l u b i l i t y curve and -44-TEMPERATURE Figure 1 1 . The s o l u b i l i t y - s u p e r s o l u b i l i t y diagram. -45-i t s p o s i t i o n on the diagram depends on the degree o f a g i t a t i o n of the s o l u t i o n . Despite the f a c t t h a t the s u p e r s o l u b i l i t y curve i s i l l - d e f i n e d , t h e r e e x i s t s a r e g i o n o f m e t a s t a b i l i t y i n the s u p e r s a t u r a t e d r e g i o n above the s o l u b i l i t y curve. In the metastable r e g i o n , spontaneous c r y s t a l l i z a t i o n i s improbable. However, i f f o r e i g n p a r t i c l e s (seed c r y s t a l s ) are i n t r o d u c e d i n t o such a s o l u t i o n , c r y s t a l growth oc c u r s u n t i l the c o n c e n t r a t i o n o f s o l u t e i n s o l u t i o n reaches the s a t u r a t i o n s o l u b i l i t y . When the s u p e r s a t u r a t i o n i s g r e a t e r than the l i m i t s of the metastable r e g i o n , a r e g i o n o f unstable s o l u t i o n i s a t t a i n e d , where spontaneous c r y s t a l l i z a t i o n i s probable. I f a s o l u t i o n r e presented by p o i n t A i n F i g u r e 11 i s c o o l e d without l o s s o f s o l v e n t ( l i n e ABC), spontaneous c r y s t a l l i z a t i o n cannot o c c u r u n t i l c o n d i t i o n s r e p r e s e n t e d by p o i n t C are reached. At t h i s p o i n t , c r y s t a l l i z a t i o n may be spontaneous or i t may be induced by seeding, a g i t a t i o n or by mechanical shock. F u r t h e r c o o l i n g t o p o i n t D may be n e c e s s a r y b e f o r e c r y s t a l l i z a t i o n can be induced, p a r t i c u l a r l y with h i g h l y s o l u b l e substances. S u p e r s a t u r a t i o n can a l s o be a c h i e v e d by removing the s o l v e n t from the s o l u t i o n by e v a p o r a t i o n c a r r i e d out a t constant temper-atu r e ( l i n e A B ' C ) i n F i g u r e 11. A c o e f f i c i e n t or degree of s u p e r s a t u r a t i o n , s', can be d e f i n e d by: -46-where C i s the c o n c e n t r a t i o n o f the substance i n s o l u t i o n a t some gi v e n temperature, and Cs i s the e q u i l i b r i u m s a t u r a t i o n c o n c e n t r a t i o n o f s o l u t e i n the s o l v e n t a t the same temperature. deCoppet i n 1872 produced evidence to show t h a t the l e n g t h o f time o f s t a b i l i t y o f a s u p e r s a t u r a t e d s o l u t i o n was i n v e r s e l y p r o p o r t i o n a l t o the degree o f s u p e r s a t u r a t i o n . 2 . 8 . 2 NUCLEATION S u p e r s a t u r a t i o n alone i s not s u f f i c i e n t f o r a system to begin to c r y s t a l l i z e . Before c r y s t a l s can grow t h e r e must e x i s t i n s o l u t i o n a number o f minute s o l i d bodies known as c e n t e r s o f c r y s t a l l i z a t i o n , seeds, embryos or n u c l e i . N u c l e a t i o n may occur spontaneously or i t may be induced a r t i f i c i a l l y , r e f e r r e d to as homogeneous and heterogeneous n u c l e a t i o n r e s p e c t i v e l y . (A) HOMOGENEOUS NUCLEATION E x a c t l y how a c r y s t a l nucleus i s formed w i t h i n a homo-geneous f l u i d i s not known with any degree o f c e r t a i n t y . The condensation o f a s u p e r s a t u r a t e d vapour t o the l i q u i d phase i s o n l y p o s s i b l e a f t e r the appearance o f m i c r o s c o p i c d r o p l e t s . However, as the vapour p r e s s u r e a t the s u r f a c e o f these minute d r o p l e t s i s very h i g h , they evaporate r a p i d l y even though the surrounding vapour i s s u p e r s a t u r a t e d . New n u c l e i form while o l d ones evaporate, u n t i l e v e n t u a l l y s t a b l e d r o p l e t s are formed e i t h e r by c o a g u l a t i o n or under c o n d i t i o n s o f very h i g h s u p e r s a t u r a t i o n . The mechanism o f homogeneous n u c l e a t i o n i s -47-thought t o be as f o l l o w s : Minute s t r u c t u r e s are formed, f i r s t from the c o l l i s i o n o f two ions or molecules, then from a t h i r d with the p a i r , and so on, to form c l u s t e r s . For c r y s t a l l i z a t i o n t o occur, the energy b a r r i e r f o r n u c l e a t i o n must be exceeded. The f o r m a t i o n o f a l i q u i d d r o p l e t o r s o l i d p a r t i c l e w i t h i n a homogeneous f l u i d r e q u i r e s the e x p e n d i t u r e o f a c e r t a i n amount o f energy i n the c r e a t i o n o f the l i q u i d or s o l i d s u r f a c e . The t o t a l q u a n t i t y o f work, W, r e q u i r e d to form a s t a b l e c r y s t a l nucleus i s equal to the sum o f the work r e q u i r e d to form the s u r f a c e , Wg (a p o s i t i v e q u a n t i t y ) , and the work r e q u i r e d t o form the bulk o f the p a r t i c l e , Wv (a n e g a t i v e q u a n t i t y ) , so t h a t W = W - W (2) s v For the formation o f a s p h e r i c a l l i q u i d d r o p l e t i n a s u p e r s a t u r a t e d vapour, f o r example, equation 2 can be w r i t t e n as W = a £ - v A p (3) where & i s the s u r f a c e energy o f the s p h e r i c a l d r o p l e t per u n i t area,Ap the p r e s s u r e d i f f e r e n c e between the vapour phase and the i n t e r i o r o f the l i q u i d d r o p l e t , and a and v are the s u r f a c e area and volume, r e s p e c t i v e l y , o f the d r o p l e t . I f r i s the r a d i u s o f the d r o p l e t , then a = 4 ^ t r 2 -48-v = - 1Tr3 26 and A.P = r t h e r e f o r e equation 3 becomes 4 7 ^ W = -7{r ( 4 ) 3 The i n c r e a s e i n the vapour pressure o f a l i q u i d d r o p l e t as i t s s i z e decreases can be estimated from the Gibbs-Thomson equation pr 2M^ In = (5) p* RT/r where, p r and p* are the vapour p r e s s u r e over a l i q u i d d r o p l e t r a d i u s r and a f l a t l i q u i d s u r f a c e , r e s p e c t i v e l y , M i s the molecular weight, f , the d e n s i t y of the d r o p l e t , T, the a b s o l u t e temperature and R, the gas c o n s t a n t . The term, pr/p* i s a measure of the s u p e r s a t u r a t i o n , s', o f the system, so equation 5 becomes 2M^ In s' = -RT/r ( 6 ) -49-2M^ or r = 3(RT/l (7) n s' ) S u b s t i t u t i n g f o r r i n e quation 4 1 67fe 3M 2 W = 3(RT/ In s' ) 2 (8) Equation 8 g i v e s a measure of the work of n u c l e a t i o n i n terms o f the degree of s u p e r s a t u r a t i o n o f the system. When s'=l, (a s a t u r a t e d s o l u t i o n ) , In s'= 0 and the amount o f energy r e q u i r e d f o r n u c l e a t i o n i s i n f i n i t e . The f r e e energy changes a s s o c i a t e d with the process o f homogeneous n u c l e a t i o n are as f o l l o w s . The o v e r a l l excess f r e e e nergyAG, between a small s o l i d p a r t i c l e o f s o l u t e and the s o l u t e i n s o l u t i o n i s equal to the sum of the s u r f a c e excess f r e e energy,AG g/ t h a t i s t h e e x c e s s f r e e energy between the s u r f a c e o f the p a r t i c l e and the bulk o f the p a r t i c l e , and the volume excess f r e e energy ^ G v » t h a t i s the excess f r e e energy between a very l a r g e p a r t i c l e (r = «0 ) and the s o l u t e i n s o l u t i o n . ^ G g 2 i s a p o s i t i v e q u a n t i t y and i s p r o p o r t i o n a l t o r . In a s u p e r s a t u r a t e d s o l u t i o n A G v i s a n e g a t i v e q u a n t i t y p r o p o r t i o n a l 3 t o r . These r e l a t i o n s h i p s are shown i n F i g u r e 12. As r i n c r e a s e s , the o v e r a l l excess f r e e energy, AG, reaches a maximum value when the nucleus achieves a c r i t i c a l s i z e (r ), t h a t i s , c A G 4 f l ^ ( r c ) 2 (9) c r i t 3 -50-Size of nucleus, r Figure 12. Free energy diagram for nucleation explaining the existence of a c r i t i c a l nucleus. -51-The c r i t i c a l s i z e r c» r e p r e s e n t s the minimum s i z e o f a s t a b l e nucleus. P a r t i c l e s s m a l l e r than r w i l l d i s s o l v e . c The r a t e o f n u c l e a t i o n , N (the number o f n u c l e i formed per u n i t time per u n i t volume), i s giv e n by, N = A exp (-AG/RT) (10) where A i s a constant a ndAG i s the o v e r a l l excess f r e e energy o f the p a r t i c l e , t h a t i s , the work o f n u c l e a t i o n W, from equations 8 and 1 0 , 1 6 f l i ? M 2 N = A exp ( — ) (11) 3 R V / Z ( l n s " ) T h i s e q u a t i o n shows the dependence o f the r a t e o f n u c l e a t i o n on the temperature, T, the degree o f s u p e r s a t u r a t i o n , s', and the i n t e r f a c i a l t e n s i o n , & . A p l o t o f the n u c l e a t i o n r a t e , N versus the degree o f s u p e r s a t u r a t i o n , s', i s shown i n F i g u r e 1 3 . A r a p i d i n c r e a s e i n the r a t e o f n u c l e a t i o n i s e v i d e n t once a c r i t i c a l l e v e l of s u p e r s a t u r a t i o n i s exceeded. (B) HETEROGENEOUS AND SECONDARY NUCLEATION C r y s t a l l i z a t i o n may be induced by i n o c u l a t i n g or seeding a su p e r s a t u r a t e d s o l u t i o n with s m a l l p a r t i c l e s o f the m a t e r i a l t o be c r y s t a l l i z e d . E f f e c t i v e seed c r y s t a l s do not have t o be the -52-Q> c o a Of o Z Theoretical Experimental Superscturation, s" Figure 1 3 * Effect of supersaturation on the nucleation rate. - 5 3 -m a t e r i a l being c r y s t a l l i z e d . For example isomorphous compounds w i l l f r e q u e n t l y induce c r y s t a l l i z a t i o n o f the s u p e r s a t u r a t e d s o l u t i o n . There i s some evidence t o show t h a t n u c l e a t i o n by seeding i s dependent on the s u r f a c e charge of the n u c l e a t i n g s u b s t r a t e (Edwards and Evans, 1962). The presence o f a s u i t a b l e seeding m a t e r i a l induces n u c l e a t i o n a t degrees o f s u p e r s a t u r a t i o n lower than those r e q u i r e d f o r homogeneous n u c l e a t i o n . The o v e r a l l f r e e energy change a s s o c i a t e d with the formation o f a c r i t i c a l nucleus f o r heterogeneous n u c l e a t i o n , A G ' c r £ t » i s l e s s than the co r r e s p o n -di n g f r e e energy change, ^GcrjL^> a s s o c i a t e d with homogeneous n u c l e a t i o n i s g i v e n by, (12) where i s l e s s than u n i t y , and i s g i v e n by equation 13 (Volmer,1939). (2 + Cos 9)(1 - Cos 0 ) 2 (13) 4 ^ s l -3 where Cos e s i o f the seed and the l i q u i d , cs the i n t e r f a c i a l energy between the - 5 4 -s u r f a c e s o f the c r y s t a l l i z i n g phase and the seed s u r f a c e . the i n t e r f a c i a l energy between the c r y s t a l l i z i n g phase and the l i q u i d , and = ( S + €> , Cos 6) cs c l 2 . 8 . 3 C R Y S T A L GROWTH As soon as s t a b l e n u c l e i , t h a t i s p a r t i c l e s l a r g e r than the c r i t i c a l s i z e are formed i n a s u p e r s a t u r a t e d system, they b e g i n t o grow i n t o c r y s t a l s o f a v i s i b l e s i z e . Three t h e o r i e s o f c r y s t a l growth have been proposed. (A) S U R F A C E ENERGY T H E O R I E S A d r o p l e t o f a l i q u i d i s most s t a b l e when i t s s u r f a c e f r e e energy and thus, i t s s u r f a c e area i s a minimum. Gibbs (1928) suggested t h a t the shape o f a growing c r y s t a l would be such t h a t the s u r f a c e energy was a minimum. The t o t a l f r e e energy o f a c r y s t a l i n e q u i l i b r i u m w i t h i t s surroundings a t constant temperature and p r e s s u r e i s a minimum f o r a g i v e n volume. I f the volume f r e e energy per u n i t volume i s assumed to be constant throughout the c r y s t a l , then where a^ i s the area o f the i t h face o f the c r y s t a l bounded by n f a c e s , and g^ i s the s u r f a c e f r e e energy per u n i t area o f the i t h f a c e . I f a c r y s t a l i s allowed to grow, i t n (14) -55-should develop i n t o an e q u i l i b r i u m shape t o ensure minimum t o t a l s u r f a c e f r e e energy f o r a g i v e n volume o f the c r y s t a l . Wulff (1901) showed t h a t the e q u i l i b r i u m shape o f a c r y s t a l i s r e l a t e d t o the f r e e e n e r g i e s o f the f a c e s . He suggested t h a t the c r y s t a l faces would grow a t r a t e s p r o p o r t i o n a l t o t h e i r r e s p e c t i v e s u r f a c e e n e r g i e s . Laue (1943) m o d i f i e d W u l f f s theory p o i n t i n g out t h a t a l l p o s s i b l e combinations o f faces must be c o n s i d e r e d t o determine which o f the o v e r a l l s u r f a c e f r e e e n e r g i e s r e p r e s e n t a minimum. There i s l i t l e q u a n t i t a t i v e evidence t o support the su r f a c e energy t h e o r i e s and they have not been g e n e r a l l y accepted. (B) ADSORPTION L A Y E R T H E O R I E S A theory o f c r y s t a l growth based on the e x i s t e n c e o f an adsorbed l a y e r o f s o l u t e atoms or molecules was proposed by Volmer (1939). C o n t r i b u t i o n s and m o d i f i c a t i o n s t o Volmer's theory have been made by Brandes (1927); S t r a n s k i (1928) and Kos s e l (1934). Atoms, i o n s or molecules w i l l a t t a c h themselves onto the c r y s t a l s u r f a c e where the a t t r a c t i v e f o r c e s are g r e a t e s t , t h a t i s they migrate towards p o s i t i o n s where a maximum number o f l i k e elements are l o c a t e d ( F i g u r e 14a). T h i s stepwise b u i l d up w i l l continue u n t i l a whole plane s u r f a c e i s completed ( F i g u r e 14b). Before the c r y s t a l s u r f a c e can continue t o grow, another "center o f c r y s t a l l i z a t i o n " must be formed on the plane s u r f a c e . I t was suggested t h a t a monolayer i s l a n d nucleus (two dimensional nucleus) was c r e a t e d on the c r y s t a l s u r f a c e ( F i g u r e 14c). -56-Figure 14. C r y s t a l growth without d i s l o c a t i o n s : (a) migration towards desired l o c a t i o n ; (tO completed l a y e r ; (c) surface nucleation. -57-However, a h i g h degree o f s u p e r s a t u r a t i o n i s necessary f o r t h i s type o f two dimensional n u c l e a t i o n to occur ( M u l l i n , 1961). K o s s e l (1934) proposed a model of a growing c r y s t a l face ( F i g u r e 15). The f l a t s u r f a c e i s d i v i d e d i n t o two r e g i o n s by a monatomic step and the step i t s e l f may be incomplete, showing one or more k i n k s . In a d d i t i o n t h e r e are l o o s e l y adsorbed growth u n i t s on the c r y s t a l s u r f a c e and vacancies i n the s u r f a c e and s t e p s . Growth u n i t s are more e a s i l y i n c o r p o r a t e d a t a k i n k s i t e . The face i s completed by the movement of the k i n k along the step and a f r e s h step i s c r e a t e d by s u r f a c e n u c l e a t i o n . A c c o r d i n g t o t h i s theory, a c r y s t a l should grow f a s t e s t when i t s faces are e n t i r e l y covered w i t h k i n k s . (I) DISLOCATIONS Most c r y s t a l s c o n t a i n d i s l o c a t i o n s . L i n e d e f e c t s or d i s l o c a t i o n s are one dimensional d e f e c t s . A d i s l o c a t i o n i s a r e g i o n where the atoms are not p r o p e r l y surrounded by neighbours. There are two types o f d i s l o c a t i o n , the edge d i s l o c a t i o n and the screw d i s l o c a t i o n . A d i s l o c a t i o n can be d e f i n e d with the a i d o f a Burgers c i r c u i t . T h i s i s any atom-to-atom path made i n a c r y s t a l which forms a c l o s e d loop. I f the c i r c u i t i s made i n an i d e a l d i s l o c a t i o n - f r e e c r y s t a l , the v e c t o r t o complete the c i r c u i t i s zero. I f the Burgers c i r c u i t e n c l o s e s a d i s l o c a t i o n , the c l o s u r e f a i l u r e i s the Burgers v e c t o r ( b Q ) . In edge d i s l o c a t i o n s the Burgers v e c t o r i s p e r p e n d i c u l a r t o the d i s l o c a t i o n and i n screw d i s l o c a t i o n s i t i s p a r a l l e l ( F i g u r e 16) -53-Figure 15- Kossel's model of a growing c r y s t a l surface: (A) f l a t surfaces; (B) steps; (C) kinks; (D) surface-adsorbed growth u n i t s ; (E) edge vacancies; and (F) surface vacancies. - 5 9 -tL JJ Dislocation line Dislocation line Figure 16. D i s l o c a t i o n s i n c r y s t a l : (a) an edge d i s l o c a t i o n , and (b) screw d i s l o c a t i o n . -60-Frank (1949) proposed s e v e r a l ways i n which d i s l o c a t i o n s c o u l d a r i s e d u r i n g the growth of a c r y s t a l and these have been reviewed by Nabarro (1967) and Albon (1963). The d i s l o c a t i o n s are formed d u r i n g the growth process under the i n f l u e n c e o f s u r f a c e and i n t e r n a l s t r e s s e s . Screw d i s l o c a t i o n s are c o n s i d e r e d important f o r c r y s t a l growth, s i n c e they e l i m i n a t e the need f o r s u r f a c e n u c l e a t i o n f o r c r y s t a l growth to c o n t i n u e . Screw d i s l o c a t i o n s g i v e r i s e t o a p a r t i c u l a r mode o f growth, f i r s t p o s t u l a t e d by Frank (1949). Once a screw d i s l o c a t i o n has been formed, the c r y s t a l face can continue t o grow. F i g u r e 17 (a-c) shows the s u c c e s s i v e stages i n the development of a growth s p i r a l from a screw d i s l o c a t i o n . F r e q u e n t l y , complex s p i r a l s develop when s e v e r a l d i s l o c a t i o n s grow tog e t h e r (Verma, 1953; Read, 1953). Burton, Cabrera and Frank (1951) developed a k i n e t i c theory based on the screw d i s l o c a t i o n mechanism of c r y s t a l growth and were able to c a l c u l a t e the growth r a t e a t any s u p e r s a t u r a t i o n . The Burton, Cabrera and Frank (B.C.F) r e l a t i o n s h i p i s g i v e n by R a = h(T2 tanh (B/<y) (15) where R^ i s the growth r a t e , fT = s ' - l and s'= C/Cs. A and B are complex temperature dependent c o n s t a n t s . At low s u p e r s a t u r a t i o n c o n c e n t r a t i o n s , R^ i s p r o p o r t i o n a l 2 . . . to 9" , but a t h i g h s u p e r s a t u r a t i o n c o n c e n t r a t i o n s R i s -61-Figure 1 7 . Development of a growth s p i r a l s t a r t i n g from a screw d i s l o c a t i o n . -62-p r o p o r t i o n a l t o or the r e l a t i o n s h i p changes from a p a r a b o l i c (square) growth law t o a l i n e a r growth law as the s u p e r s a t u r a t i o n i n c r e a s e s . (C) DIFFUSION THEORIES Noyes and Whitney (1897) proposed t h a t the d e p o s i t i o n o f s o l i d on the face o f a growing c r y s t a l was the re v e r s e o f the d i s s o l u t i o n process and was e s s e n t i a l l y a d i f f u s i o n p r o c e s s . A c c o r d i n g t o them the r a t e s o f c r y s t a l growth or d i s s o l u t i o n were governed by the d i f f e r e n c e between the c o n c e n t r a t i o n s a t the s o l i d s u r f a c e and i n the bulk o f the s o l u t i o n , g i v e n i n the form where i s the growth r a t e , S i s the s u r f a c e area o f the s o l i d , C i s the s o l u t e c o n c e n t r a t i o n i n the su p e r s a t u r a t e d s o l u t i o n , Cs i s the s a t u r a t i o n s o l u b i l i t y and K t i s the t r a n s p o r t r a t e c o n s t a n t . Nernst (1904) m o d i f i e d the Noyes-Whitney equation w i t h the assumption t h a t t h e r e was a t h i n stagnant f i l m o f l i q u i d a d j a c e n t t o the growing face, through which s o l u t e molecules d i f f u s e d . The m o d i f i e d r e l a t i o n s h i p was: R = K. S (C-Cs) g t (16) R g D S (C - Cs) h (17) He concluded t h a t K. = (D/h), where D = c o e f f i c i e n t o f d i f f u s i o n o f s o l u t e and h = t h i c k n e s s o f the l i q u i d f i l m . -63-The t h i c k n e s s o f the l i q u i d f i l m depends upon the r e l a t i v e s o l i d - l i q u i d v e l o c i t y , t h a t i s on the degree o f a g i t a t i o n . Marc (1908, 1909a,b, 1910) observed t h a t K d i d not i n c r e a s e i n d e f i n a t e l y w i t h i n c r e a s i n g s o l u t i o n v e l o c i t y but reached some l i m i t i n g v a l u e . He suggested t h a t c r y s t a l l i z a t i o n i s not n e c e s s a r i l y the r e v e r s e o f d i s s o l u t i o n and t h a t f i l m d i f f u s i o n alone i s not s u f f i c i e n t t o e x p l a i n the mechanism of c r y s t a l l i z a t i o n . Berthoud (1912) and V a l e t o n (1923a,b; 1924) consequently m o d i f i e d the d i f f u s i o n theory of c r y s t a l growth and suggested t h a t c r y s t a l growth i n v o l v e d two s t e p s . F i r s t l y , a d i f f u s i o n p r o c e s s , whereby s o l u t e molecules are t r a n s p o r t e d from the b u l k of the f l u i d phase to the s o l i d s u r f a c e , f o l l o w e d by a f i r s t order r e a c t i o n where s o l u t e molecules are i n c o r p o r a t e d i n t o the c r y s t a l l a t t i c e . These two steps, o c c u r i n g under the i n f l u e n c e o f d i f f e r e n t c o n c e n t r a t i o n d r i v i n g f o r c e s can be represented by the equations: R t = K S (C-Ci) (18) and R = K S (Ci-Cs) (19) s s where i s the t r a n s p o r t r a t e constant ( c o e f f i c i e n t o f mass t r a n s f e r by d i f f u s i o n ) , K g i s the s u r f a c e r e a c t i o n r a t e i constant, C i i s the s o l u t e c o n c e n t r a t i o n a t the c r y s t a l - s o l u t i o n i n t e r f a c e , R^ i s the r a t e of a r r i v a l of s o l u t e a t the s u r f a c e by d i f f u s i o n from the bulk s o l u t i o n and R i s the r a t e o f 1 s i n t e g r a t i o n i n t o ' t h e s o l i d l a t t i c e ( s u r f a c e r e a c t i o n ) . Equations 18 and 19 are o f l i t t l e use i n p r a c t i c e because -64-t h e c o n c e n t r a t i o n C i i s d i f f i c u l t to measure. I t i s p o s s i b l e to e l i m i n a t e the term C i s i n c e at steady r a t e , = R = R . 1 t s g Then the growth r a t e can be w r i t t e n as: R = K (obs) S (C-Cs) (20) g o where K Q(obs) = observed o v e r a l l growth r a t e constant, 1 1 1 and - = - + -K (obs) K o t s Equation 20 d e s c r i b e s c r y s t a l growth when the two co n s e c u t i v e r e a c t i o n s , t r a n s p o r t o f s o l u t e by d i f f u s i o n and a s u r f a c e r e a c t i o n step, are of comparable v e l o c i t y . For the c r y s t a l l i z a t i o n o f an i o n i s i n g s o l u t e from an aqueous s o l u t i o n , the two con s e c u t i v e step mechanism of c r y s t a l growth may not e x p l a i n the growth because of the involvement o f s e v e r a l p rocesses s i m u l t a n e o u s l y ( M u l l i n , 1972). Some of these processes i n c l u d e : - Bulk d i f f u s i o n o f s o l v a t e d ions through the d i f f u s i o n l a y e r . - Bulk d i f f u s i o n o f s o l v a t e d ions through the a d s o r p t i o n l a y e r . Surface d i f f u s i o n o f s o l v a t e d or unsolva t e d i o n s . P a r t i a l or t o t a l d e s o l v a t i o n o f i o n s . I n t e g r a t i o n o f ions i n t o the l a t t i c e . - Counter d i f f u s i o n o f r e l e a s e d water through the a d s o r p t i o n l a y e r . -65-Counter d i f f u s i o n of water through the boundary l a y e r . Any o f these processes may become r a t e - c o n t r o l l i n g and render the c r y s t a l growth phenomenon very complex. Marc (1908) i n v e s t i g a t e d the r a t e o f c r y s t a l l i z a t i o n o f a number of s a l t s and found t h a t i n many cases the dependence o f growth r a t e on s u p e r s a t u r a t i o n was g r e a t e r than f i r s t o r d e r . Thus equation 20 may be w r i t t e n as: R = K (obs) S ( C - C s ) n (21) g o where n = the order o f the o v e r a l l growth p r o c e s s . 2 . 8 . 4 FACTORS A F F E C T I N G C R Y S T A L GROWTH RATES (A) E F F E C T OF S E E D C R Y S T A L S I Z E AND S E E D S U R F A C E A R E A The r a t e c onstant K g f o r the s u r f a c e i n t e g r a t i o n process i s expected t o be independent o f mass t r a n s f e r i n the s o l u t i o n , and t h e r e f o r e i s independent o f c r y s t a l s i z e . However, K t, behaves as a normal mass t r a n s f e r c o e f f i c i e n t and thus should show dependence on c r y s t a l s i z e and s o l u t i o n v e l o c i t y ( P h i l l i p s , 1973). In the seeded growth technique, r e d u c t i o n i n the s i z e o f seed c r y s t a l s would r e s u l t i n an i n c r e a s e o f the growth r a t e . During c r y s t a l growth i n the presence of seeds the s u r f a c e area o f the seeds i n c r e a s e s p r o g r e s s i v e l y . L i t t l e i s known about the e f f e c t o f t h a t i n c r e a s e on the c r y s t a l growth k i n e t i c s . In a -66-r e c e n t r e p o r t , Barone et a l . (1983) d i s c u s s e d the r e l a t i o n s h i p between the i n c r e a s e i n s u r f a c e area and mass d e p o s i t e d on the growing c r y s t a l . The f o l l o w i n g i s a b r i e f summary of t h e i r d i s c u s s i o n . The r a t e o f c r y s t a l growth f o r a seeded system can be i n t e r p r e t e d i n terms of e m p i r i c a l (Nancollas, 1978) or t h e o r e t i c a l e x p r e s s i o n s (O'Hara and Reed, 1973; C h r i s t o f f e r s e n and C h r i s t o f f e r s e n , 1976; C h r i s t o f f e r s e n , 1980). The f u n c t i o n a l form of the r a t e equation (equation 20) f o r many i o n i c compounds growing i n an aqueous s o l u t i o n has been d e t a i l e d by Barone et 8^.(1983) and can be w r i t t e n as, R g dc dt t = K ( T ) X f<°t> (22) where c ^ i s t h e t o t a l mass o f s o l i d a t t i m e t , K ( T j a r a t e constant dependent on the temperature T, S^ . the t o t a l e f f e c t i v e s u r f a c e a r e a a t time t , and f ( o t ) a term dependent on the s u p e r s a t u r a t i o n , o^ = (C - C s ) / Cs, where C and Cs are the c o n c e n t r a t i o n s o f p r e c i p i t a t i n g ions or molecules i n s o l u t i o n a t time t and a t s a t u r a t i o n r e s p e c t i v e l y . The e f f e c t i v e s u r f a c e area, S., i s d i f f e r e n t from t geometric t o t a l s u r f a c e area (S^) which depends o n l y on the geometry of the c r y s t a l and i s equal t o the q u a n t i t y measured, f o r example by BET gas a d s o r p t i o n . eS. depends on the -67-dimensional type of the growth, and i s the q u a n t i t y which determines the r a t e o f r e a c t i o n . For 1-dimensional growth o f c u b i c shaped c r y s t a l s , growth occurs o n l y along two p a r a l l e l faces r e s u l t i n g i n no i n c r e a s e i n e f f e c t i v e s u r f a c e a r e a . • . e S i m i l a r l y , f o r 3-dimensional growth S^ = Sfc and f o r two dimensional growth St>eS^. As the seed c r y s t a l s grow, the e f f e c t o f an i n c r e a s e i n s u r f a c e area on the r a t e o f c r y s t a l growth i s a p p r e c i a b l e f o r some s a l t s (Barone and N a n c o l l a s , 1978) and n e g l i g i b l e f o r o t h e r s ( L i u and N a n c o l l a s , 1970). In cases where the r a t e o f growth changes as the s u r f a c e area i n c r e a s e s the r e l a t i v e i n c r e a s e i n s u r f a c e area (S./S ) or e f f e c t i v e t o t a l s u r f a c e area t o ( S./ S ) h a s a d i r e c t r e l a t i o n s h i p w h i c h i s d e p e n d e n t on t o c the type o f growth dimension o f the c r y s t a l . For uniform t h r e e dimensional growth S t / S o - % / % = ( V mo ) 2 / 3 ( 2 3 > where m and m. are the t o t a l seed masses a t zero time o t and a t time, t . (B) E F F E C T OF DEGREE OF SUPERSATURATION From the growth equation, c r y s t a l growth r a t e i s d i r e c t l y p r o p o r t i o n a l t o s u p e r s a t u r a t i o n . Schott (1980) s t u d i e d and confirmed the d i r e c t r e l a t i o n s h i p between s u p e r s a t u r a t i o n and growth r a t e . T h e o r e t i c a l c a l c u l a t i o n s i n d i c a t e t h a t f o r -68-d i s . l o c a t i o n c o n t r o l l e d growth, the growth r a t e i s p r o p o r t i o n a l to the square of the d r i v i n g f o r c e while s u r f a c e - n u c l e a t i o n c o n t r o l l e d growth w i l l r e s u l t i n an e x p o n e n t i a l dependence ( M u l l i n , 1961). The degree o f s u p e r s a t u r a t i o n can a l s o a f f e c t the h a b i t of a c r y s t a l . A h i g h degree o f s u p e r s a t u r a t i o n can cause the p r e f e r e n t i a l growth o f a c r y s t a l i n one p a r t i c u l a r d i r e c t i o n , l e a d i n g to the formation o f needle shaped c r y s t a l s . T h i s h a b i t allows f o r a f a s t r a t e o f heat d i s s i p a t i o n from the s o l i d phase when a h i g h degree o f s u p e r s a t u r a t i o n i s p r e s e n t ( M u l l i n , 1961). (C) E F F E C T OF TEMPERATURE The r e l a t i o n s h i p between the growth r a t e constant, K Q ( o b s ) , and the a b s o l u t e temperature i s g i v e n by the A r r h e n i u s equation (Arrhenius, 1880). The e f f e c t o f temperature on c r y s t a l growth r a t e has been e x t e n s i v e l y s t u d i e d and the energy of a c t i v a t i o n f o r the growth r e a c t i o n determined. Some o f the s t u d i e s have been r e f e r e n c e d by P h i l l i p s and E p s t e i n (1974). The growth r a t e o f c r y s t a l s i n c r e a s e s w i t h an i n c r e a s e i n temperature and i s g e n e r a l l y c o n t r o l l e d by the s u r f a c e r e a c t i o n mechanism a t low temperatures, whereas at h i g h temperatures the c r y s t a l l i z a t i o n i s g e n e r a l l y c o n t r o l l e d by the d i f f u s i o n process ( M u l l i n , 1961). -69-(D) E F F E C T OF DEGREE OF A G I T A T I O N The r a t e a t which a c r y s t a l grows a t a g i v e n temperature under constant c o n d i t i o n s o f s u p e r s a t u r a t i o n and s u r f a c e area o f seed c r y s t a l s can be a l t e r e d by a g i t a t i n g the s o l u t i o n . The r a t e o f growth i n c r e a s e s c o n s i d e r a b l y i n the i n i t i a l stages as the r e l a t i v e v e l o c i t y between c r y s t a l and l i q u i d i n c r e a s e s , but c o n d i t i o n s are soon reached when f u r t h e r a g i t a t i o n has no e f f e c t . An i n c r e a s e i n the a g i t a t i o n r a t e o f the s o l u t i o n decreases the t h i c k n e s s o f the f i l m on the c r y s t a l s u r f a c e f o r d i f f u s i o n c o n t r o l e d growth. Ther e f o r e a t h i g h r a t e s o f a g i t a t i o n the r a t e o f c r y s t a l growth depends s o l e l y on the r a t e o f the s u r f a c e r e a c t i o n p r o c e s s . (E ) E F F E C T OF I M P U R I T I E S The presence of i m p u r i t i e s can have a profound e f f e c t on the growth o f a c r y s t a l . Some i m p u r i t i e s suppress growth, some enhance growth and others may modify the c r y s t a l h a b i t . I m p u r i t i e s can change the p r o p e r t i e s o f the s o l u t i o n or the e q u i l i b r i u m s a t u r a t i o n c o n c e n t r a t i o n . They can change the c h a r a c t e r i s t i c s o f the a d s o r p t i o n l a y e r a t the c r y s t a l - s o l u t i o n i n t e r f a c e . They may be adsorbed onto the c r y s t a l faces and ex e r t a b l o c k i n g e f f e c t and thus d i s r u p t the flow o f growth l a y e r s a c r o s s the f a c e s . They may be b u i l t i n t o the c r y s t a l s or may i n t e r a c t c h e m i c a l l y w i t h the c r y s t a l s . I m p u r i t i e s t h a t have a common i o n a c c e l e r a t e the growth -70-process by r e d u c i n g the s o l u b i l i t y a c c o r d i n g to the law of mass a c t i o n . I m p u r i t i e s w i t h o u t a common i o n can a c c e l e r a t e growth at low c o n c e n t r a t i o n s but reduce i t a t h i g h c o n c e n t r a t i o n s . I t has been suggested t h a t these i m p u r i t i e s a c t as d i f f u s i o n r e t a r d e r s . I m p u r i t i e s o f o r g a n i c o r i g i n r e t a r d the growth by being adsorbed onto the c r y s t a l f a c e s . I t has been suggested t h a t the a c t i o n o f i m p u r i t i e s i s due t o : (1) The e l e c t r i c f i e l d o f the ions (governed by i o n i c charge and r a d i u s ) , (2) chemical i n t e r a c t i o n between the i m p u r i t y and the c r y s t a l , or (3) formation o f complex aquo i o n s , r e s u l t i n g i n a decrease i n the growth r a t e of c r y s t a l s ( M u l l i n , 1972). 2 . 8 . 5 DETERMINATION OF C R Y S T A L GROWTH R A T E (A) F A C E GROWTH RATES The d i f f e r e n t faces of a n o n - i s o t r o p i c c r y s t a l may grow at d i f f e r e n t r a t e s under i d e n t i c a l environmental c o n d i t i o n s . A s m a l l c r y s t a l i s mounted on a tungsten wire i n a p a r t i c u l a r o r i e n t a t i o n . A s o l u t i o n o f known s u p e r s a t u r a t i o n i s c i r c u l a t e d around the mounted c r y s t a l and the r a t e o f advance o f the c r y s t a l face i s observed through a t r a v e l l i n g microscope ( M u l l i n , 1972). (B) OVERALL GROWTH RATES The measurement of c r y s t a l growth r a t e s as a f u n c t i o n o f the mass d e p o s i t e d per u n i t time per u n i t area o f c r y s t a l s u r f a c e i s convenient. T h i s can be done by determining the mass de p o s i t e d on a known mass of seed c r y s t a l s under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . Two methods have been used t o o b t a i n r e l i a b l e i n f o r m a t i o n on c r y s t a l growth k i n e t i c s . In the f l u i d i s e d bed method the seed c r y s t a l s are kept suspended i n a s u p e r s a t u r a t e d s o l u t i o n by c o n t r o l l i n g the s o l u t i o n v e l o c i t y i n a v e r t i c a l g l a s s column. S o l u t i o n c o n c e n t r a t i o n s can be determined a t i n t e r v a l s or c o n t i n u o u s l y by means o f a r e c o r d i n g d e n s i t y meter. The d e t a i l s o f the apparatus and method are g i v e n by M u l l i n (1972). A second method i n v o l v e s the use of a s t i r r e r t o suspend the seed c r y s t a l s i n a s u p e r s a t u r a t e d s o l u t i o n . T h i s method was f i r s t used by Davies and Jones (1949) to study the p r e c i p i t a t i o n k i n e t i c s o f s i l v e r c h l o r i d e from aqueous s o l u t i o n s . Many r e p o r t s have appeared i n the l i t e r a t u r e over the l a s t s e v e r a l years u s i n g t h i s technique to study the k i n e t i c s of c r y s t a l growth o f many e l e c t r o l y t e s . Davies and Jones (1949) proposed a model f o r c r y s t a l growth and suggested t h a t the r a t e s o f a d s o r p t i o n f o r the c a t i o n s and anions were d i r e c t l y p r o p o r t i o n a l t o the s u r f a c e area o f seed c r y s t a l s . They proposed t h a t i n the event o f simultaneous a r r i v a l of p o s i t i v e and n e g a t i v e ions a t the growth s i t e , the -72-r a t e of growth i s g i v e n by: R = K (obs) S ( C + -g o Cs )(C - Cs ) (24) + where C , Cs , C and Cs are the c o n c e n t r a t i o n s o f c a t i o n s and anions a t the s u p e r s a t u r a t i o n and the s a t u r a t i o n c o n c e n t r a t i o n s o f the e l e c t r o l y t e . For e q u a l i o n c o n c e n t r a t i o n s , t h i s equation reduces t o : The r a t e c o n s t a n t f o r the growth of i o n i c compounds has been g e n e r a l l y found to be independent of the s t i r r i n g r a t e and the f l u i d dynamics under experimental c o n d i t i o n s ( L i u and N a n c o l l a s , 1970). Thus the t r a n s p o r t r a t e o f s o l u t e by d i f f u s i o n i s not the r a t e c o n t r o l l i n g mechanism. During these experiments, the mass of s o l i d d e p o s i t e d may be as much as two to three times the weight of seed c r y s t a l s added i n i t i a l l y . T h e r e f o r e there i s a c o n s i d e r a b l e i n c r e a s e i n the s u r f a c e area d u r i n g the experiments. However, the k i n e t i c equation appears t o adequately d e s c r i b e the growth data without i n t r o d u c i n g a term to account f o r the i n c r e a s e i n s u r f a c e area i n t o the r a t e equation. There i s s u f f i c i e n t evidence to show t h a t i n many systems the e f f e c t i v e growth area becomes constant at some stage i n the (25) -73-growth process even though the c r y s t a l s i n c r e a s e i n s i z e (Nancollas and Purdie, 1961). The r a t e constant (K 1) i s , however, d i r e c t l y p r o p o r t i o n a l t o the amount o f seed c r y s t a l s (Doremus, 1958). Two models o f c r y s t a l growth f o r e l e c t r o l y t e s have been proposed (Doremus, 1958). A c c o r d i n g t o the f i r s t model, adsorbed ions combine t o form a n e u t r a l s a l t molecule which then d i f f u s e s to the growth step. The r a t e o f formation o f the s u r f a c e 3 molecules was p r o p o r t i n a l t o (C-Cs) f o r 1:1 and 2:2 e l e c t r o l y t e s and ( C - C s ) 4 f o r 2:1 e l e c t r o l y t e s . In the second model, the o p p o s i t e l y charged ions are i n c o r p o r a t e d i n t o the c r y s t a l s u r f a c e a l t e r n a t e l y , d i r e c t l y from the adsorbed l a y e r a t a k i n k i n a growth step. The r a t e o f c r y s t a l growth i s then 2 p r o p o r t i o n a l t o (C-Cs) f o r 1:1 and 2:2 e l e c t r o l y t e s and 3 (C-Cs) f o r 2:1 e l e c t r o l y t e s . The r a t e constant i n equation 25 i n c l u d e s a l l f a c t o r s such as p r o b a b i l i t y o f i n c o r p o r a t i o n o f molecules i n t o the c r y s t a l l a t t i c e and frequency o f c o l l i s i o n w ith growth s i t e s . 2.9 MSUM SOLUTIONS U r i c a c i d i s a d i b a s i c a c i d (pKa 5.75 and 10.3) (Bergmann and D i k s t e i n , 1955). A l k a l i n e s o l u t i o n s o f u r i c a c i d are r e a d i l y o b t a i n e d forming the s a l t o f u r i c a c i d and a l k a l i . MSUM i s formed when s o l u t i o n s o f sodium hydroxide and u r i c a c i d (pH 8.9, 50°) a r e mixed and l e f t t o s t a n d . Monosodium u r a t e d i s s o c i a t e s p a r t i a l l y i n aqueous s o l u t i o n t o sodium and urat e ions and shows -74-t y p i c a l r e a c t i o n s o f u r i c a c i d i n a l k a l i n e c o n d i t i o n s . 2 . 9 . 1 DEGRADATION OF MSUM SOLUTIONS Urates are decomposed by a number of b a c t e r i a , molds, some p l a n t s and a number of animal t i s s u e s . An enzyme u r i c a s e i s r e s p o n s i b l e f o r t h i s decomposition. Organisms b e l o n g i n g t o the C l o s t r i d i u m genus, f o r example, C l . a c i d i u r i c i convert u r i c a c i d a n a e r o b i c a l l y i n t o ammonia, carbon d i o x i d e and a c e t i c a c i d (Barker, 1938). U r i c a s e has been i d e n t i f i e d i n the t i s s u e s o f a number of animals (Truszkowski and Goldmanowna, 1933; M i k h l i n and Ruizova, 1934; F a n e l l i e t a l . , 1970). E x t r a c t s from b r e a s t and kidney t i s s u e s cause anaerobic u r i c o l y s i s and form urea ( M i k h l i n and Ruizova, 1934). Soybean seeds c o n t a i n u r i c a s e which converts u r i c a c i d i n t o a l l a n t o i n (Echevin and Brunnel, 1937). I n - v i t r o experiments have shown t h a t the end products o f u r i c a s e o x i d a t i o n o f u r i c a c i d depends on the c o n d i t i o n s o f the r e a c t i o n . In phosphate b u f f e r (pH 7.2-8.5) u r i c a s e o x i d i z e s u r i c a c i d t o a l l a n t o i n , whereas i n bor a t e b u f f e r a t pH 7.2 urea and a l l o x a n i c a c i d are formed ( C a n a n l l a k i s and Cohen, 1955). A l l a l k a l i s cause decomposition o f u r i c a c i d ( A u s t i n , 1903; May, 1911; Stevens and May, 1911). T h i s decomposition was re p o r t e d t o be s i m i l a r t o the decomposition caused by u r i c o l y t i c enzymes p r e s e n t i n organ e x t r a c t s . Water at 200°, converts u r i c a c i d , i n p a r t t o a mixture of p t e r i d i n e s and pyrimido(5,4g) p t e r i d i n e s . -75-U r i c a c i d i n a l k a l i n e s o l u t i o n o x i d i z e s r a p i d l y t o a l l a n t o i n , oxonic a c i d , a l l a n t o x a d i n or o x a l u r i c a c i d depending on the c o n d i t i o n s ( D a l g l i e s h and Neuberger, 1954). 2.9.2 SOLUBILITY OF MSUM The s o l u b i l i t y o f MSUM i n water has been the s u b j e c t o f s e v e r a l s t u d i e s . The s o l u b i l i t y of MSUM was determined by the e q u i l i b r i u m s o l u b i l i t y method (Loeb, 1972), hot stage microscopy (Wilcox and Khalaf, 1975) and by seeding a su p e r s a t u r a t e d s o l u t i o n o f MSUM fol l o w e d by repeated a n a l y s i s o f the s o l u t i o n u n t i l a c o n s t a n t r e s u l t was o b t a i n e d ( F i d d i s e t a l . , 1983). The s o l u b i l i t y o f MSUM i s a f u n c t i o n o f temperature i n the presence o f a p h y s i o l o g i c a l sodium i o n c o n c e n t r a t i o n . F i g u r e 18 shows the r e l a t i o n s h i p between the temperature and the s o l u b i l i t y determined by v a r i o u s groups o f workers. Amorphous MSUM has an ap p a r e n t s o l u b i l i t y o f 2.03 g L - 1 a t 18°. On storage o f t h i s s o l u t i o n the apparent s o l u b i l i t y decreases and becomes t h a t o f the c r y s t a l l i n e form, which i s about 0.85 g L - 1 a t 18° (Barkan, 1922). Supersaturated s o l u t i o n s o f MSUM prepared by h e a t i n g s o l u t i o n s c o n t a i n i n g excess s o l i d t o 70° fo l l o w e d by c o o l i n g were found t o be t r u e s o l u t i o n s (Kohler 1913). However, when MSUM was aut o c l a v e d i n water a t 121° f o r 30 minutes the formation o f a su p e r s a t u r a t e d c o l l o i d a l d i s p e r s i o n r e s u l t e d (Schade, 1922). The c o l l o i d a l form o f sodium u r a t e i n water i s converted i n t o a s t a b l e g r a n u l a r form a f t e r storage (Barkan, -76-1924). MSUM i n a sup e r s a t u r a t e d s o l u t i o n may e x i s t i n a metastable c o n d i t i o n , i n which spontaneous p r e c i p i t a t i o n does not take p l a c e . The upper l i m i t o f t h i s metastable s t a t e f o r a pure s o l u t i o n o f MSUM was found t o be about 2.5 times the s a t u r a t i o n c o n c e n t r a t i o n (Kohler, 1913). Sodium urate i s ten times more s o l u b l e i n pure water than i t i s i n a 1% s o l u t i o n o f sodium c h l o r i d e , but i n a 1% sodium c h l o r i d e s o l u t i o n the upper l i m i t o f the metastable s t a t e f o r sodium ur a t e i s 5 times the s a t u r a t i o n c o n c e n t r a t i o n f o r t h i s s o l u t i o n . The s a t u r a t i o n s o l u b i l i t y o f MSUM i n plasma and s y n o v i a l f l u i d from normal and a r t h r i t i c p a t i e n t s has been determined. There i s a small d i f f e r e n c e i n the s o l u b i l i t y o f MSUM i n plasma and s y n o v i a l f l u i d i n h e a l t h y i n d i v i d u a l s . However, t h i s d i f f e r e n c e i s g r e a t e r i n p a t i e n t s with v a r i o u s a r t h r i t i d e s (Dorner et a l . , 1981). Bovine n a s a l c a r t i l a g e has been shown t o have an a p p r e c i a b l e s o l u b i l i z i n g e f f e c t on MSUM i n b u f f e r (Katz, 1978). Katz and Schubert (1970) s t u d i e d the e f f e c t o f pr o t e o g l y c a n s , c h o n d r o i t i n s u l f a t e and albumin on the s o l u b i l i t y o f MSUM and rep o r t e d t h a t the MSUM s o l u b i l i t y was g r e a t l y enhanced by small amounts o f prot e o g l y c a n , whereas c h o n d r o i t i n s u l f a t e and albumin produced a s l i g h t i n c r e a s e i n s o l u b i l i t y even when p r e s e n t i n l a r g e amounts. The e f f e c t o f c h o n d r o i t i n s u l f a t e on the s o l u b i l i t y o f MSUM was i n disagreement w i t h Laurent (1964) who r e p o r t e d a decrease i n the s o l u b i l i t y o f MSUM i n the presence o f c h o n d r o i t i n s u l f a t e . -77-P e r r i c o n e and Brandt (1978, 1979) s t u d i e d the enhancement o f urate s o l u b i l i t y by connec t i v e t i s s u e components and found t h a t p r o t e o g l y c a n aggregate f a c i l i t a t e d the d i s s o l u t i o n o f about 2.5 times more sodium urate than non-aggregated p r o t e o g l y c a n s . T h i s e f f e c t o f p r o t e o g l y c a n on urat e s o l u b i l i t y was a b o l i s h e d by d i g e s t i o n o f the aggregate with an enzyme h y a l u r o n i c a c i d B l-> 3 hy d r o l a s e . T h i s i n d i c a t e d t h a t t o i n c r e a s e u r a t e s o l u b i l i t y the pr o t e o g l y c a n must e x i s t as a l a r g e macromolecular aggregate ( P e r r i c o n e and Brandt, 1978). They a l s o r e p o r t e d t h a t the aggregate d i d not s u s t a i n sodium urate c o n c e n t r a t i o n i n sup e r s a t u r a t e d s o l u t i o n s a f t e r 24 hours. P e r r i c o n e and Brandt (1979) r e p o r t e d t h a t the enhancement o f sodium ur a t e s o l u b i l i t y by p r o t e o g l y c a n aggregate (prepared i n the presence o f potassium ions) was due to the c a t i o n i c exchange o f potassium with sodium i o n s . Potassium entered the s o l u t i o n and potassium u r a t e b e i n g more s o l u b l e than sodium ur a t e r e s u l t e d i n the observed i n c r e a s e i n sodium ur a t e s o l u b i l i t y . 2 . 1 0 NUCLEATION AND CRYSTAL GROWTH OF MSUM Kha l a f and Wilox (1973) and Wilcox and Khal a f (1975) developed the hot stage microscope technique t o observe the n u c l e a t i o n o f MSUM and found t h a t c a l c i u m and hydrogen ions i n c r e a s e d the n u c l e a t i o n r a t e o f MSUM as determined by the time f o r the appearance o f observable n u c l e i . C u p r i c ions were found to have a mixed e f f e c t on the number o f n u c l e i formed. S y n o v i a l f l u i d from gouty p a t i e n t s i n c r e a s e d the number o f n u c l e i formed. However, s y n o v i a l f l u i d from rheumatoid p a t i e n t s was found t o -78-i n h i b i t n u c l e a t i o n . Tak et a l . (1980) s t u d i e d the n u c l e a t i o n o f MSUM under p h y s i o l o g i c a l c o n d i t i o n s o f temperature, pH and i o n i c c o n c e n t r a t i o n and found t h a t the n u c l e a t i o n o f MSUM was g r e a t l y enhanced by s y n o v i a l f l u i d from gouty p a t i e n t s , whereas s y n o v i a l f l u i d from degenerative j o i n t d i s e a s e p a t i e n t s moderately enhanced n u c l e a t i o n and f l u i d from rheumatoid a r t h r i t i s p a t i e n t s i n h i b i t e d t he n u c l e a t i o n . Tak e t a l . (1980) a l s o r e p o r t e d t h a t h y a l u r o n i c a c i d and p u r i n e s had a minimal e f f e c t on u r a t e n u c l e a t i o n and ot h e r c o n n e c t i v e t i s s u e components had no e f f e c t . N u c l e a t i o n o f sodium urate has been shown t o be more s e n s i t i v e to urat e i o n c o n c e n t r a t i o n (Tak and Wilcox, 1980) and a d d i t i o n o f p h y s i o l o g i c q u a n t i t i e s o f potassium and magnesium ions s l i g h t l y i n h i b i t e d the n u c l e a t i o n o f MSUM. The growth o f MSUM c r y s t a l s from s u p e r s a t u r a t e d s o l u t i o n s has been s t u d i e d . A l l e n et al.(1965a,b) r e p o r t e d t h a t the growth o f the needle-shaped c r y s t a l s was i n h i b i t e d by s u r f a c e a c t i v e agents. The l i n e a r growth r a t e o f MSUM c r y s t a l s i n the presence o f 0.1% benzalkonium c h l o r i d e was reduced ( A l l e n et a l . , 1965a). Dyes such as bismarck brown and methylene b l u e were shown t o i n h i b i t the c r y s t a l growth o f MSUM ( A l l e n e t a l . , 1965b). The seeded growth technique was used by Erwin and Na n c o l l a s (1981) t o determine the r a t e o f growth o f MSUM and the e f f e c t o f a d d i t i v e s on the growth o f MSUM. They showed t h a t the growth o f MSUM fo l l o w e d the square law which i n d i c a t e d a s u r f a c e r e a c t i o n c o n t r o l l e d growth p r o c e s s . Methylene blu e , a c a t i o n i c dye, -79-s i g n i f i c a n t l y reduced the r a t e o f c r y s t a l growth o f MSUM a t 13 ppm, whereas, o r g a n i c phosphonate and sodium h e p a r i n had a n e g l i g i b l e e f f e c t . F i d d i s et a l . (1983) s t u d i e d the p o i s o n i n g e f f e c t o f n e u t r a l red dye and serum albumin on the c r y s t a l growth o f MSUM. They d e t e r m i n e d the c r y s t a l l i z a t i o n time a t 37° i n the p r e s e n c e o f the a d d i t i v e s . A d d i t i o n o f 33 mmol L - 1 n e u t r a l red to a 70 mmol L ^ sodium urate s o l u t i o n i n c r e a s e d the c r y s t a l l i z a t i o n time by 50 minutes. Albumin a t a c o n c e n t r a t i o n o f 10 g L _ 1 i n c r e a s e d the c r y s t a l l i z a t i o n time by a f a c t o r o f 4. Heparin (0.01%) was shown t o be an e f f e c t i v e i n h i b i t o r o f urate c y s t a l l i z a t i o n . F i d d i s et a l . (1983) r e p o r t e d the dependence of the l i n e a r growth r a t e o f MSUM on the s u p e r s a t u r a t i o n . Expressed as a power law, the l i n e a r growth r a t e was found t o vary with s u p e r s a t u r a t i o n d e f i n e d as: ((Na+)(HU-)) 1/2 4.5 •( (Na+) (HU-) ) 1/2 which they proposed f i t t e d the screw d i s l o c a t i o n model o f c r y s t a l growth. -80-• • <9 • O O i 0.03 0.06 0.09 SODIUM URATE CONCENTRATION, mmol L Figure 18. Solubility of sodium urate in normal saline. (•) Wilcox et a l . , 1975 by hot stage; (o) Wilcox et a l . . 1975; (•) Allen et a l . . 1965; (o) Erwin and Nancollas, 1981; and (*) Fiddis et a l . . .1983-- 8 1 -3 EXPERIMENTAL 3 . 1 . INSTRUMENTS A u t o c l a v e , AMSCO General purpose, American S t e r i l i z e r . Atomic A b s o r p t i o n Flame Emission Spectrometer, J e r a l d Ash. Cahn E l e c t r o b a l a n c e , Gram Ventron C o r p o r a t i o n . Constant Temperature Water C i r c u l a t o r , Haake FT. D i f f e r e n t i a l Scanning C a l o r i m e t e r , DSC-1B, P e r k i n Elmer. E l e c t r o n i c Stroboscope, F l a s h t a c E l e c t r o n i c C o r p o r a t i o n . I n f r a red Spectrophotometer, IR 10, Beckman. Oven, Isotemp, F i s h e r S c i e n t i f i c . pH Meter, Accumet, F i s h e r S c i e n t i f i c . Rotary Vacuum Evaporator, Rotavapor, Buchi L a b o r a t o r i e s . Scanning E l e c t r o n Microscope, ETEC Autoscan. S t i r r e r , F i s h e r S t e d i Speed S t i r r e r , F i s h e r S c i e n t i f i c . S u r f a c e Area A n a l y z e r , Quantasorb , Quantachrome C o r p o r a t i o n U.V Spectrophotometer, Becman Model 24. U l t r a s o n i c Cleaner, M e t t l e r E l e c t r o n i c s . Water Bath Shaker, Aquatherm, New Brunswick S c i e n t i f i c Co. Inc. Water Bath C o o l i n g U n i t , F r i d g e d f l o w Bath C i r c u l a t o r , New Brunswick S c i e n t i f i c Co. Inc. X-Ray D i f f r a c t o m e t e r , Wide Angle, P h i l i p s . -82-3 . 2 . M A T E R I A L S Albumin, f r a c t i o n - V , from bovine serum, Sigma Chemicals. A s e p t i c f i l t r a t i o n u n i t , M i l l i p o r e C o r p o r a t i o n . Calcium c h l o r i d e , B.D.H. C h o n d r o i t i n s u l f a t e , sodium s a l t , from Whale or Shark c a r t i l a g e (99%), Sigma Chemicals. D i s p o s a b l e f i l t r a t i o n u n i t s , (Millex-GS,0.22um, Millex-HA,0.8 urn), M i l l i p o r e C o r p o r a t i o n . H y a l u r o n i c a c i d , s a l t (0.1% sodium, 9.7% potassium), from Human U m b i l i c a l Cord, Sigma Chemicals. Membrane f i l t e r s , M i l l i p o r e C o r p o r a t i o n . P h o s p h a t i d y l c h o l i n e (40%), from Soybean, Sigma Chemicals. P h o s p h a t i d y l s e r i n e (80%), from Bovine B r a i n , Sigma Chemicals. Potassium c h l o r i d e , Analar, B.D.H. * P r o t e o g l y c a n aggregate, ( A l f r a c t i o n ) . * P r o t e o g l y c a n monomer, (A1D1 f r a c t i o n ) . (*) obtained from Dr. Mark Adams, F a c u l t y o f Medicine, U n i v e r s i t y o f B.C. Method o f p r e p a r a t i o n g i v e n i n Appendix. - 8 3 -Sodium c h l o r i d e , ACS, F i s h e r S c i e n t i f i c . Sodium hydroxide, ACS, F i s h e r S c i e n t i f i c . S t e r i l e evacuated g l a s s tubes, red top V a c c u t a i n e r , Becton D i c k i n s o n . U r i c a c i d (99%), Sigma Chemicals. -84-3 . 3 METHODS 3 . 3 . 1 PREPARATION OF MSUM C r y s t a l s o f MSUM were grown a c c o r d i n g t o the method d e s c r i b e d by Denko and Whitehouse (1976). A s o l u t i o n o f u r i c a c i d (6.0 g L 1 ) and 1 M sodium hydroxide a t 55° and pH 8.9 was l e f t t o stand o v e r n i g h t a t room temperature. The c r y s t a l s formed were separated by s u c t i o n f i l t r a t i o n and were r i n s e d s e v e r a l times w i t h c o l d d i s t i l l e d water and d r i e d a t 60° f o r 12 hours i n a c i r c u l a t i n g hot a i r oven. These c r y s t a l s were used f o r the p r e p a r a t i o n o f s o l u t i o n s i n v a r i o u s experiments and as seed c r y s t a l s i n the p r e l i m i n a r y c r y s t a l growth experiments (batch A seed c r y s t a l s ) . Batch B seed c r y s t a l s , used t o study the e f f e c t o f s u p e r s a t u r a t i o n and presence of a d d i t i v e s on c r y s t a l growth, was prepared by a m o d i f i c a t i o n o f the method d e s c r i b e d above. A s o l u t i o n o f u r i c a c i d (6.0 g L ) and 1 M sodium hydroxide a t 55° and pH 8.9 was f i l t e r e d through a 0.22 um M i l l i p o r e membrane f i l t e r w h i l e h o t . The f i l t r a t e was l e f t t o stand o v e r n i g h t . The c r y s t a l s were separated, r i n s e d s e v e r a l times with c o l d , f i l t e r e d , d i s t i l l e d water and d r i e d a t 60° f o r 12 hours i n a c i r c u l a t i n g hot a i r oven. Drying o f MSUM samples r e s u l t e d i n the formation o f a hard cake. Samples were t h e r e f o r e ground i n a g l a s s mortar and - 8 5 -p e s t l e p r i o r t o t h e i r use i n v a r i o u s experiments. The ground batch B seed c r y s t a l s were passed through a s e t o f s i e v e s (# 50/80, US standard) o f mesh s i z e 180 jim t o 300 jam i n an attempt to o b t a i n a g r e a t e r degree o f s i z e u n i f o r m i t y o f seed c r y s t a l s . 3 . 3 . 2 C H A R A C T E R I Z A T I O N OF MSUM C R Y S T A L S (A) U L T R A - V I O L E T SPECTROSCOPY Approximately 10 mg of ground MSUM c r y s t a l s were d i s s o l v e d i n 100 mL o f d i s t i l l e d water. A s u i t a b l e d i l u t i o n o f t h i s s o l u t i o n was scanned from 320 nm to 230 nm on an u l t r a - v i o l e t spectrophotometer. (B) I N F R A - R E D SPECTROSCOPY Approximately 2 mg of an MSUM sample was mixed with approximately 200 mg o f anhydrous potassium bromide. A p o r t i o n o f t h i s mixture was compressed i n t o a d i s c at 10 ton pre s s u r e u s i n g a d i e and punch i n a h y d r a u l i c p r e s s . The compressed d i s c was scanned i n the 625-3800 cm ^ range u s i n g an i n f r a r e d spectrophotometer. (C) X - R A Y D I F F R A C T I O N Approximately 100 mg of a ground MSUM sample was t i g h t l y packed i n a g l a s s sample h o l d e r ( t h i c k n e s s = 0.1cm) and exposed t o CuKCC r a d i a t i o n a wide angle X-ray diffTactometer a t a scanning r a t e o f 2 degrees o f 20 per minute. The l o c a t i o n and i n t e n s i t y o f the peaks a t d i f f e r e n t values -86-of 29 were s t u d i e d . (D) D I F F E R E N T I A L SCANNING CALORIMETERY Ground samples o f MSUM were analysed u s i n g a d i f f e r e n t i a l scanning c a l o r i m e t e r . Samples o f 1-5 mg were weighed on a Cahn-Gram e l e c t r o b a l a n c e and a n a l y s e d a t a s c a n n i n g r a t e o f 20° per minute i n open aluminum sample pans. V a p o r i z a t i o n o f the water o f h y d r a t i o n from the open pans was estimated q u a n t i t a t i v e l y by weighing the pan a f t e r the appearance o f the endothermic peak, and the perc e n t water l o s s c a l c u l a t e d . (E ) SCANNING E L E C T R O N MICROSCOPY A s m a l l q u a n t i t y o f an MSUM seed c r y s t a l sample was d i s p e r s e d on an SEM sample h o l d e r . The sample was coated with s p e c t r o g r a p h i c g r a p h i t e under vacuum. Scanning e l e c t r o n micrographs were taken a t 4000 x or 8000 x m a g n i f i c a t i o n u s i n g a Scanning E l e c t r o n Microscope. ( F ) DETERMINATION OF SURFACE A R E A OF MSUM S E E D C R Y S T A L S The s u r f a c e areas o f the two batches o f MSUM seed c r y s t a l s were determined u s i n g the Quantasorb s u r f a c e area a n a l y s e r . Three p o i n t BET s u r f a c e area determinations were made us i n g 0.072%, 0.104% and 0.184% mole f r a c t i o n k r y p t o n i n helium gas mixtures. An a c c u r a t e l y weighed sample (approximately O.lg) was p l a c e d in a g l a s s c e l l and degassed a t the out g a s s i n g p o r t f o r one hour -87-a t 60° under a slow stream of n i t r o g e n gas. The c e l l was t r a n s f e r e d to the adsorbing gas p o r t and c o o l e d with l i q u i d n i t r o g e n . Krypton from one of the three krypton-helium mixtures was allowed to adsorb onto the MSUM sample f o r 30 min. Krypton was desorbed by removing the l i q u i d n i t r o g e n f l a s k and q u i c k l y b r i n g i n g the c e l l t o room temperature by d i p p i n g the c e l l i n water at room temperature. The d e s o r p t i o n count was recorded. The procedure was repeated t h r e e times f o r each o f the three krypton-helium mixtures. The s u r f a c e area was c a l c u l a t e d from the mean d e s o r p t i o n count (at each krypton c o n c e n t r a t i o n ) c a l i b r a t e d with the d e s o r p t i o n counts accumulated from measured volumes of n i t r o g e n gas u s i n g the f o l l o w i n g equation P A Nu Acs Pa S = (1 ) (—) Vc( ) metre square (26) Po Ac RT where P = p a r t i a l p r e s s u r e o f adsorbate, Po =saturated p r e s s u r e of adsorbate, 23 Nu = Avogadro s number = 6.023 x 10 , R = gas constant = 82.1 cc atm./mole.degree, Vc = volume o f c a l i b r a t i o n gas, Pa = ambient p r e s s u r e i n atmosphere, A = s i g n a l area ( d e s o r p t i o n count o f sample ), Ac = area of c a l i b r a t i o n ( d e s o r p t i o n count of c a l i b r a t i o n gas, Acs= c r o s s s e c t i o n a l area of adsorbate molecules i n _5 square meters ( f o r krypton, 19.5 xlO meter - 8 8 -square per atm.), T = temperature o f c a l i b r a t i o n volume f o r ambient temperature i n degree K e l v i n . 3.3.3 A N A L Y S I S OF MONOSODIUM URATE MONOHYDRATE Urate c o n c e n t r a t i o n s i n s o l u t i o n were analyzed by measuring the absorbances a t 292 nm u s i n g an u l t r a - v i o l e t r e c o r d i n g spectrophotometer. A u r a t e standard curve was prepared by d i s s o l v i n g an a c c u r a t e l y weighed, 200 mg sample o f MSUM i n water i n a vo l u m e t r i c f l a s k and making up t o 100 mL wit h water. A f t e r f u r t h e r d i l u t i o n , the absorbances were immediately read a t 292 nm. 3.3.4 DEGRADATION OF MONOSODIUM URATE MONOHYDRATE SOLUTIONS (A) N O N - S T E R I L E SOLUTIONS A 0.2 g L 1 s o l u t i o n o f MSUM i n water was p r e p a r e d and 125 mL of the s o l u t i o n added t o each o f f i v e , 150 mL g l a s s Erlenmeyer f l a s k s f i t t e d w ith ground g l a s s s toppers. One f l a s k was s t o r e d i n the r e f r i g e r a t o r (4°), one f l a s k was kept a t room temperature (22°), one f l a s k was s t o r e d i n a water bath e q u i l i b r a t e d a t 45° and remaining f l a s k s were s t o r e d i n a c i r c u l a t i n g h ot a i r oven a t 35° and 65°. A l i q u o t s o f 3 mL of s o l u t i o n were withdrawn from each f l a s k a f t e r 6, 24, 48, 72, and 96 hours, and a f t e r f u r t h e r d i l u t i o n , the absorbance o f the s o l u t i o n s was read a t 292 nm. -89-(B) STERILE SOLUTIONS A l l s o l u t i o n s were prepared with f r e s h l y d i s t i l l e d , d e i o n i z e d water which had been s t e r i l i s e d by a u t o c l a v i n g a t 121° f o r 30 minutes. A 0.2 g L s o l u t i o n o f MSUM i n water was prepared and approximately 10 mL o f s o l u t i o n f i l t e r e d through a g l a s s s y r i n g e M i l l i p o r e f i l t r a t i o n u n i t f i t t e d w ith a 0.22 jam f i l t e r d i r e c t l y i n t o each o f s i x s t e r i l e , red top Vacutainer tubes (Becton D i c k i n s o n ) . The tubes were s t o r e d a t 4°. A f t e r 6 hours, a 3 mL sample was removed from one tube using a s t e r i l e d i s p o s a b l e s y r i n g e and needle and the remaining s o l u t i o n i n the tube d i s c a r d e d . Samples were a l s o taken a t time i n t e r v a l s o f 24, 48, 72, and 96 h o u r s . The samples were d i l u t e d and the absorbance o f the s o l u t i o n s was read a t 292 nm. Th i s procedure was repeated f o r tubes s t o r e d a t 22°, 35°, 45° and 65°. An MSUM s o l u t i o n (0.2 g L ^) was f i l t e r e d through a l a r g e s c a l e M i l l i p o r e f i l t r a t i o n u n i t f i t t e d w i t h a 0.22 urn f i l t e r and 125 mL o f the s o l u t i o n p l a c e d i n t o each o f f i v e , s t e r i l e Erlenmeyer f l a s k s with g l a s s s t o p p e r s . The MSUM s o l u t i o n was a l s o f i l t e r e d d i r e c t l y i n t o s t e r i l e , red top Va c u t a i n e r tubes as d e s c r i b e d above. The f l a s k s and tubes c o n t a i n i n g MSUM s o l u t i o n were then a u t o c l a v e d a t 121° f o r 35 minutes. The f l a s k s and tubes were allowed t o c o o l t o room temperature and the s o l u t i o n s i n one o f each o f the f l a s k s and Vacutainer tubes were -90-immediately analyzed f o r the urat e c o n c e n t r a t i o n . The remaining tubes and f l a s k s were s t o r e d a t 22°, 35°, 45°, and 65° and samples removed as d e s c r i b e d above u s i n g s t e r i l e , d i s p o s a b l e s y r i n g e s and n e e d l e s , a f t e r 12, 24, 48, 72 and 96 hours. Samples were f u r t h e r d i l u t e d and the absorbance o f the s o l u t i o n s was read a t 292 nm. 3 . 3 . 5 DETERMINATION OF SATURATION S O L U B I L I T Y OF MONOSODIUM  U R A T E MONOHYDRATE (A) E F F E C T OF TEMPERATURE Into each o f thr e e 125 mL Erlenmeyer f l a s k s c o n t a i n i n g f r e s h l y washed and d r i e d g l a s s marbles, was p l a c e d 100 mL o f d i s t i l l e d water and the f l a s k s f i t t e d with ground g l a s s s t o p p e r s . The f l a s k s were e q u i l i b r a t e d a t a giv e n temperature i n a water bath shaker f i t t e d w i t h h e a t i n g and c o o l i n g u n i t s . An excess (200-500mg sample) o f MSUM was added t o each o f the f l a s k s and shaken v i g o r o u s l y . A l i q u o t s o f a p p r o x i m a t e l y 2 mL were withdrawn from each f l a s k a f t e r v a r i o u s time i n t e r v a l s , immediately f i l t e r e d through a 0.22 um Millex-GS d i s p o s a b l e f i l t e r u n i t and the f i l t r a t e assayed f o r the MSUM c o n c e n t r a t i o n a f t e r s u i t a b l e d i l u t i o n . Sampling was terminated when three c o n s e c u t i v e assays gave i d e n t i c a l r e s u l t s . S o l u b i l i t i e s were determined a t 4.4°, 9.0°, 15.1°, 24.7°, 29.9°, 35.0°, 37.0°, 40.0°, 41.8°, 51.0° and 55.0°. -91-(B) E F F E C T OF E L E C T R O L Y T E S ON T H E SATURATION S O L U B I L I T Y OF  MONOSODIUM URATE MONOHYDRATE IN WATER To determine the e f f e c t o f sodium c h l o r i d e and other e l e c t r o l y t e s p r e s e n t i n plasma and s y n o v i a l f l u i d on the s o l u b i l i t y o f MSUM, v a r i o u s c o n c e n t r a t i o n s o f sodium c h l o r i d e (0.2, 0.4, 0.6 and 1.0 % w/v, f i n a l c o n c e n t r a t i o n ), and a mixture o f sodium c h l o r i d e (0.78% w/v), potassium c h l o r i d e (0.03 % w/v) and c a l c i u m c h l o r i d e (0.035% w/v) ( a l l are f i n a l c o n c e n t r a t i o n s ) were added t o 125 mL Erlenmeyer f l a s k s c o n t a i n i n g p r e v i o u s l y washed and d r i e d g l a s s marbles and 100 mL o f d i s t i l l e d water which had been e q u i l i b r a t e d a t a gi v e n temperature as de s c r i b e d p r e v i o u s l y . MSUM samples (200mg) were added t o these f l a s k s and shaken v i g o r o u s l y . Sample withdrawal and analyses were c a r r i e d out as d e s c r i b e d i n s e c t i o n 3.3.5.(A). S o l u b i l i t i e s were determined a t 4.4°, 10.4°, 17.6°, 24.7°, 29.9°, 41.8° and 51.1°. (C ) E F F E C T OF CHONDROITIN S U L F A T E , HYALURONIC A C I D , A L B U M I N ,  PROTEOGLYCAN MONOMER AND PROTEOGLYCAN AGGREGATE ON THE  SATURATION S O L U B I L I T Y OF MSUM The s a t u r a t i o n s o l u b i l i t y o f MSUM i n water a t 37° was determined i n the presence o f these a d d i t i v e s a t s e v e r a l c o n c e n t r a t i o n s . The a d d i t i v e was added t o 125 mL Erlenmeyer f l a s k s c o n t a i n i n g p r e v i o u s l y washed and d r i e d g l a s s marbles and 100 mL o f d i s t i l l e d water. The f l a s k s were e q u i l i b r a t e d a t -92 -37°, MSUM (200 mg) added and the s o l u b i l i t y determined as d e s c r i b e d i n s e c t i o n 3.3.5(A). E i t h e r 0.45 urn o r 0.8 pm M i l l e x d i s p o s a b l e f i l t e r s were used t o f i l t e r s o l u t i o n s c o n t a i n i n g HA and PGs. The f o l l o w i n g a d d i t i v e s were used: c h o n d r o i t i n s u l f a t e (20, 40, and 60 mg dL ^ ) h y a l u r o n i c a c i d (10, 20 and 40 mg dL ^ ) albumin (10, 20, 40, 60 and 100 mg d L - 1 ) pr o t e o g l y c a n monomer (10, 20, 40 and 100 mg dL ) pr o t e o g l y c a n aggregate (20, 40 and 100 mg dL 1 ) 3 . 3 . 6 CRYSTAL GROWTH OF MONOSODIUM URATE MONOHYDRATE A diagram o f the c r y s t a l growth apparatus i s shown i n F i g u r e 19. The s u p e r s a t u r a t e d MSUM s o l u t i o n (1000 mL or 50 mL) was p l a c e d i n t h e 1000 mL or 50 mL c a p a c i t y g l a s s r e a c t i o n v e s s e l and e q u i l i b r a t e d i n a water bath a t 37° u s i n g a constant temperature water c i r c u l a t o r . The r e a c t i o n v e s s e l was f i t t e d w ith a cover t o minimize s o l v e n t e v a p o r a t i o n . An a l l g l a s s s t i r r e r w i t h two paddles was lowered i n t o the s o l u t i o n through a c e n t r a l p o r t t o a constant depth (2.5 cm from the bottom f o r the 1 L v e s s e l and 1 cm from the bottom f o r the 50 mL r e a c t i o n v e s s e l ) and r o t a t e d a t a constant speed (200 rpm) by means o f a motor. Constant checks on the r o t a t i o n speed were made through-out a l l the experiments u s i n g an e l e c t r o n i c stroboscope. The temperature o f the su p e r s a t u r a t e d s o l u t i o n was monitored Figure 19. Crystal growth apparatus -94-c o n t i n u o u s l y u s i n g a thermometer r e a d i n g t o 0.1 p l a c e d through the second p o r t . The t h i r d p o r t was f i t t e d w i t h a ground g l a s s stopper and was used f o r sample withdrawal. S o l u t i o n s of d i f f e r i n g degrees of s u p e r s a t u r a t i o n were prepared by d i s s o l v i n g a c c u r a t e l y weighed samples o f MSUM (4.0 g, 5.0 g, 6.0 g) i n d i s t i l l e d water (950 mL) w i t h the a i d of heat (85 t o 90°) and constant s t i r r i n g . The s o l u t i o n was c o o l e d s l o w l y to about 45°, the pH was a d j u s t e d t o 7.4 and the volume made up to 1 L w i t h pH 7.4-adjusted d i s t i l l e d water. The s o l u t i o n was f i l t e r e d through a 0.22 pm membrane f i l t e r and t r a n s f e r e d t o the r e a c t i o n v e s s e l . (A) EFFECT OF SUPERSATURATION CONCENTRATION, SEED CRYSTAL BATCH AND SEED AMOUNT ON THE CRYSTAL GROWTH KINETICS OF  MONOSODIUM URATE MONOHYDRATE I n i t i a l c r y s t a l growth s t u d i e s were c a r r i e d out u s i n g 1 L of a s u p e r s a t u r a t e d s o l u t i o n i n a 1 L r e a c t i o n v e s s e l . To determine the e f f e c t o f c o n c e n t r a t i o n of seed c r y s t a l s , experiments were c a r r i e d out by adding e i t h e r 100 mg, 200 mg, 300 mg, 500 mg or 1 g of seed c r y s t a l s t o the s u p e r s a t u r a t e d s o l u t i o n i n the r e a c t i o n v e s s e l . A l i q u o t s o f 1 mL of growth medium were withdrawn a t predetermined time i n t e r v a l s up to 360 minutes, f i l t e r e d immediately through a 0.22 urn Millex-GS d i s p o s a b l e f i l t e r u n i t , d i l u t e d and analyzed f o r MSUM content as d e s c r i b e d i n s e c t i o n 3.3.5.(A). These experiments were repeated using a s m a l l e r s c a l e 6 - 9 5 -apparatus. Supersaturated s o l u t i o n s (50 mL) c o n t a i n i n g 5 or 6 g L MSUM were prepared as d e s c r i b e d above and p l a c e d i n a 50 mL g l a s s r e a c t i o n v e s s e l . To the su p e r s a t u r a t e d s o l u t i o n s were added e i t h e r 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg o f seed c r y s t a l s . A l i q u o t s o f 0.3 mL were withdrawn, f i l t e r e d through a 0.22 um Mi l l e x - G S d i s p o s a b l e f i l t e r u n i t , d i l u t e d and analysed f o r MSUM content (see s e c t i o n 3.3.5 ( A ) ) . (B) E F F E C T OF A D D I T I V E S ON T H E C R Y S T A L GROWTH OF  MONOSODIUM URATE MONOHYDRATE The s m a l l s c a l e (50 mL) growth apparatus was used f o r a l l the experiments. ( I ) CHONDROITIN S U L F A T E AND ALBUMIN A c c u r a t e l y weighed q u a n t i t i e s o f 10 mg, 20 mg, or 30 mg o f c h o n d r o i t i n s u l f a t e or 10 mg, 50 mg, 100 mg, or 200 mg, of albumin were d i s s o l v e d i n 5 mL of d i s t i l l e d water and f i l t e r e d through a 0.22 um Millex-GS f i l t e r u n i t d i r e c t l y i n t o the r e a c t i o n v e s s e l . To the r e a c t i o n v e s s e l were added 45 mL o f a sup e r s a t u r a t e d s o l u t i o n such t h a t a f i n a l MSUM c o n c e n t r a t i o n o f 5.0 g L was o b t a i n e d . The s o l u t i o n was e q u i l i b r a t e d a t 37° f o r 30 minutes, s t i r r i n g c o n s t a n t l y a t a s t i r r e r speed o f 200 rpm. Seed c r y s t a l s (40mg) were then added and 0.3 mL a l i q u o t s o f growth medium withdrawn a t predetermined times up t o 360 minutes. Samples were f i l t e r e d immediately and assayed f o r MSUM content as d e s c r i b e d i n s e c t i o n 3.3.5 (A). -96-( I I ) HYALURONIC A C I D AND PROTEOGLYCAN MONOMER A 5 mg, 10 mg or 20 mg sample o f h y a l u r o n i c a c i d or 5mg or 10 mg of p r o t e o g l y c a n monomer (Al DI f r a c t i o n from bovine c a r t i l a g e ) was added t o the r e a c t i o n v e s s e l c o n t a i n i n g 5 mL f i l t e r e d (0.22 urn Mi l l e x - G S f i l t e r ) d i s t i l l e d water and d i s p e r s e d i n the water. To the r e a c t i o n v e s s e l were added 45 mL o f a sup e r s a t u r a t e d s o l u t i o n t o o b t a i n a f i n a l MSUM c o n c e n t r a t i o n o f ""1 — 1 o 5 . 0 g L o r 6 . 0 g L . A f t e r e q u i l i b r a t i n g a t 37 f o r 30 minutes and a s t i r r e r speed o f 200 rpm, 30 mg, 40 mg, or 50 mg of seed c r y s t a l s were added. A l i q u o t s o f 0.3 mL o f growth medium were removed a t predetermined time i n t e r v a l s , f i l t e r e d , d i l u t e d and assayed f o r MSUM content (see s e c t i o n 3.3.5 ( A ) ) . ( I I I ) PROTEOGLYCAN AGGREGATE A sample o f p r o t e o g l y c a n aggregate (10 mg to 50 mg) was d i s p e r s e d i n 5 mL of f i l t e r e d (0.22 jim f i l t e r ) d i s t i l l e d water i n the growth c e l l . To the growth c e l l were added 45 mL of a p r e v i o u s l y f i l t e r e d (0.22 jim f i l t e r ) s u p e r s a t u r a t e d s o l u t i o n o f MSUM t o o b t a i n a f i n a l c o n c e n t r a t i o n of 5.0 g L . The ^ s t i r r e r speed was 200 rpm and the mixture was e q u i l i b r a t e d a t 37° f o r 30 minutes. An accurate q u a n t i t y (40 mg) of seed c r y s t a l s were added and a l i q u o t s o f 0.3 mL of growth medium were withdrawn at predetermined time i n t e r v a l s . The samples were immediately f i l t e r e d through 0.8 um Millex-HA f i l t e r s , d i l u t e d and assayed f o r MSUM content as d e s c r i b e d p r e v i o u s l y ( s e c t i o n 3.3.5 ( A ) ) . -97-(D) PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLSERINE A 10 mg, 20 mg or 30 mg sample of p h o s p h a t i d y l c h o l i n e or 10 mg or 20 mg o f p h o s p h a t i d y l s e r i n e was d i s s o l v e d i n 10 mL of a chl o r o f o r m - methanol mixture (2:1) i n a 50 mL round bottomed f l a s k . The s o l v e n t mixture was evaporated under vacuum at 60° usin g a r o t a r y vacuum evaporator. The t h i n f i l m o f p h o s p h o l i p i d formed on the i n s i d e w a l l o f the f l a s k was d i s p e r s e d i n 5 mL f i l t e r e d (0.22 pm f i l t e r ) d i s t i l l e d water u s i n g an u l t r a -s o n i c a t o r . The p h o s p h o l i p i d suspension was t r a n s f e r r e d i n t o the r e a c t i o n v e s s e l and a su p e r s a t u r a t e d MSUM s o l u t i o n (45 mL) was added t o o b t a i n a f i n a l c o n c e n t r a t i o n o f 5.0 g L-"^. A f t e r e q u i l i b r a t i n g a t 37° f o r 30 minutes and s t i r r e r speed o f 200 rpm, 40 mg or 50 mg of seed c r y s t a l s were added. A l i q u o t s o f 0.3 mL o f growth medium were f i l t e r e d , d i l u t e d and assayed f o r MSUM content as d e s c r i b e d i n s e c t i o n 3.3.5 (A). 3.3.7 DETERMINATION OF SODIUM A N D / O R POTASSIUM CONTENT OF HYALURONIC A C I D , CHONDROITIN S U L F A T E AND PROTEOGLYCAN  MONOMER SAMPLES An a c c u r a t e l y weighed 100 mg q u a n t i t y o f sodium c h l o r i d e or potassium c h l o r i d e was d i s s o l v e d i n d i s t i l l e d water i n a v o l u m e t r i c f l a s k and the volume was made up t o 100 mL. Sodium or potassium standard s o l u t i o n s were made by f u r t h e r d i l u t i n g these s o l u t i o n s t o o b t a i n a c o n c e n t r a t i o n range o f 0.2 mg dL ^ t o 2.0 mg dL ^  o f sodium c h l o r i d e or potassium c h l o r i d e . The percent e m i s s i o n o f these s o l u t i o n s was measured on a flame photometer a t -98-t h e maximum wavelength o f 585 nm f o r sodium and 765 n>m f o r potassium. A c a l i b r a t i o n curve o f percent emission versus c o n c e n t r a t i o n o f sodium i o n or potassium i o n was p l o t t e d . A 10 mg sample o f c h o n d r o i t i n s u l f a t e , h y a l u r o n i c a c i d , p r o t e o g l y c a n monomer or p r o t e o g l y c a n aggregate was d i s s o l v e d i n d i s t i l l e d water i n a v o l u m e t r i c f l a s k and the volume made t o 100 mL. The pe r c e n t emission f o r c h o n d r o i t i n s u l f a t e and pr o t e o g l y c a n was measured a t 585 nm and f o r h y a l u r o n i c a c i d , p ercent emission was measured a t 765 nm and 585 nm. The co n c e n t r a t i o n s o f sodium i o n and potassium i o n i n the samples were determined from the standard curve. 3 . 3 . 8 E F F E C T OF SODIUM AND POTASSIUM IONS ON GROWTH OF MSUM A 0.7 mL volume o f 0.1 M sodium h y d r o x i d e ( e q u i v a l e n t t o 1.6 mg sodium ion) r e p r e s e n t i n g 5.6% w/w sodium i n a 30 mg sample o f the sodium s a l t o f c h o n d r o i t i n s u l f a t e , was added t o 45 mL of a sup e r s a t u r a t e d s o l u t i o n o f MSUM (pH 7.4). The volume o f the s o l u t i o n was a d j u s t e d t o 50 mL by the a d d i t i o n o f d i s t i l l e d water (pH 7.4) so t h a t the f i n a l MSUM c o n c e n t r a t i o n o f the sup e r s a t u r a t e d s o l u t i o n was 5.0 g L ^. The s o l u t i o n was f i l t e r e d through a 0.22 um Millex-GS d i s p o s a b l e f i l t e r u n i t i n t o the growth c e l l and e q u i l i b r a t e d a t 3 7° f o r 30 minutes. Seed c r y s t a l s (40 mg) were added and the growth experiment c a r r i e d out as d e s c r i b e d i n s e c t i o n 3.3.6. T h i s experiment was repeated using 0.34 mL of a 0.1 M potassium h y d r o x i d e s o l u t i o n ( e q u i v a l e n t t o 1.3 mg potassium ion) -99-r e p r e s e n t i n g 6.5% w/w of potassium i n a 20 mg sample o f h y a l u r o n i c a c i d . The c o n c e n t r a t i o n of the s u p e r s a t u r a t e d s o l u t i o n was 5.0 g L ^ and 50 mg of seed c r y s t a l s were used. 3 . 3 . 9 C H A R A C T E R I Z A T I O N O F MSUM C R Y S T A L S A F T E R C R Y S T A L GROWTH I m p u r i t i e s and a d d i t i v e s i n the growth medium can r e s u l t i n a change i n c r y s t a l morphology or c r y s t a l s t r u c t u r e . To determine whether the a d d i t i v e s had caused any change i n the c r y s t a l h a b i t or s t r u c t u r e , scanning e l e c t r o n microscopy and powder X-ray d i f f r a c t i o n s t u d i e s were c a r r i e d out on the MSUM c r y s t a l s o b t a i n e d a f t e r v a r i o u s c r y s t a l growth experiments. Where ever p o s s i b l e , the s u p e r s a t u r a t e d s o l u t i o n c o n t a i n i n g the c r y s t a l s was f i l t e r e d through a 0.22 um membrane f i l t e r and the c r y s t a l s on the f i l t e r were d r i e d i n a c i r c u l a t i n g hot a i r oven at 60°. The d r i e d c r y s t a l s were ground i n a g l a s s p e s t l e and mortar and s u b j e c t e d t o SEM and powder X-ray d i f f r a c t i o n a n a l y s e s as d e s c r i b e d i n s e c t i o n 3.3.2(E) and 3.3.2(C). -100-4 R E S U L T S A N D D I S C U S S I O N 4 . 1 C H A R A C T E R I Z A T I O N O F M O N O S O D I U M U R A T E M O N O H Y D R A T E U l t r a - v i o l e t and i n f r a - r e d a b s o r p t i o n spectroscopy methods are used t o determine and c o n f i r m the f u n c t i o n a l groups pres e n t i n known compounds. The u l t r a - v i o l e t spectrum f o r an aqueous s o l u t i o n o f MSUM (Figure 20) showed the ^ max a t 235 and a t 292 nm. These two value s c o i n c i d e with the ^ max value s of i o n i s e d u r i c a c i d (West, 1970) i n d i c a t i n g the formation o f the sodium s a l t o f u r i c a c i d . The i n f r a - r e d spectrum o f MSUM (Fi g u r e 21) showed peaks a t 3580 cm - 1 (-OH); 2850-3150 cm" 1 (=CH); 1750 cm - 1 (=C=0); 1750-1650 cm - 1 (=C=C=, =C=N-,-COO~); 1610 cm" 1; 1525 cm - 1; 1425 cm - 1; 1390 cm - 1; 1250 cm - 1; 1000 cm - 1 and 900 cm 1 . The IR a b s o r p t i o n s p e c t r a o f MSUM was s i m i l a r t o t h a t r e p o r t e d i n the l i t e r a t u r e (Dieppe and C a l v e r t , 1983). The r e l a t i o n s h i p between the wavelength o f the X-rays, and the spac i n g between the c r y s t a l l o g r a p h i c p l a nes, d, o f a c r y s t a l i s g i v e n by Bragg 1s law: n ^ = 2d Sine (27) where n i s an i n t e g e r and 9 i s the angle o f the i n c i d e n t X-rays. -101-Figure 20. U l t r a - v i o l e t spectrum of monosodium urate monohydrate s o l u t i o n . W<*.l«ngth(um) 5 6 7 0 9 10 12 K £ 100- 1 ' 1 » 1 1 1 1 j F i g u r e 21. I n f r a - r e d spectrum of monosodium u ra t e monohydrate. - 103 -Since s i n Q must be between 0 and 1 the r e are u s u a l l y no more than one or two d i f f r a c t i o n orders i f ^ i s comparable t o d. The powder X-ray d i f f r a c t i o n p a t t e r n of MSUM i s shown i n Fi g u r e 22; The c a l c u l a t e d d-values are shown i n Table 4. The d-values were c h a r a c t e r i s t i c o f monosodium ur a t e monohydrate ( S e l e c t e d Powder D i f f r a c t i o n Data f o r M i n e r a l s , 1974; Rinaudo and B o i s t e l l e , 1982). A scanning e l e c t r o n micrograph o f the monosodium ur a t e monohydrate c r y s t a l s i s shown i n F i g u r e 23. The c r y s t a l s had a long, well-formed needle-shaped or a c i c u l a r c r y s t a l h a b i t . A t y p i c a l DSC scan o f an MSUM sample i s shown i n F i g u r e 24. A broad endothermic peak between 180° t o 240° accounted f o r the l o s s o f 8.78 % w/w of water o f h y d r a t i o n . The percent water l o s s corresponded t o the l o s s o f one mole o f water, c o n f i r m i n g t h a t the c r y s t a l s were the monohydrate. The t h e o r e t i c a l v alue o f p e r c e n t water l o s s f o r MSUM i s 8.65% w/w. 4.2 ASSAY OF MONOSODIUM URATE MONOHYDRATE IN SOLUTION The s t a n d a r d curve f o r an MSUM s o l u t i o n i n water i s shown i n Fi g u r e 25. The p l o t was l i n e a r over a c o n c e n t r a t i o n range o f 0.002 g L ^ t o 0.034 g L ^, the c o r r e l a t i o n c o e f f i c i e n t , 2 r , was 0.999 and the slope ( a b s o r p t i v i t y , absorbance/ c o n c e n t r a t i o n / c e l l width) was 56.8 L g ^cm ^ . F i g u r e 22. X - r a y d i f f r a c t i o n p a t t e r n of monosodium u r a t e monohydrate -105-4. Powder X-ray d i f f r a c t i o n p a t t e r n o f monosodium urate monohydrate. I n t e r p l a n a r R e l a t i v e d i s t a n c e i n t e n s i t y (d-spacing, A) 10.52 W 9.4 MS 7.55 MS 5.27 W 4.91 M 4.68 S 4.56 w 3.52 w 3.46 w 3.38 w 3.18 VS 3.02 w 2.97 w 2.65 M 2.61 w 2. 53 w 2.47 w 2.42 w 2.36 w very s t r o n g s t r o n g medium s t r o n g medium weak VS = S = MS = M = W = -106-Figure 23. Scanning electron micrograph of monosodium urate monohydrate. -107-195# Figure 24. DSC scan of monosodium urate monohydrate. -108-Figure 25. A standard curve for MSUM solution (n = 5; r = 0.999) -109-4 . 3 DEGRADATION OF MONOSODIUM URATE MONOHYDRATE IN SOLUTION The r e s u l t s o f degra d a t i o n s t u d i e s are gi v e n i n F i g u r e s 26-29 and are p l o t t e d as c o n c e n t r a t i o n o f MSUM remaining i n s o l u t i o n versus time o f i n c u b a t i o n . F i g u r e 26 shows the degra d a t i o n o f n o n - s t e r i l e MSUM s o l u t i o n s . MSUM s o l u t i o n s s t o r e d a t 4° were r e l a t i v e l y s t a b l e , showing l i t t l e change i n MSUM c o n c e n t r a t i o n w i t h t i m e . At 22°, 35°, 45° and 65°, there was a gradual decrease i n the c o n c e n t r a t i o n o f MSUM as the time o f i n c u b a t i o n was i n c r e a s e d . As the temperature o f i n c u b a t i o n was i n c r e a s e d , d e g r a d a t i o n o f MSUM occ u r e d more r a p i d l y , w i t h the e x c e p t i o n o f s o l u t i o n s s t o r e d a t 45°, which were more s t a b l e than s o l u t i o n s s t o r e d a t 35°. S i m i l a r r e s u l t s were gi v e n by s o l u t i o n s s t e r i l i z e d by a s e p t i c f i l t r a t i o n through M i l l i p o r e f i l t e r s i n t o rubber stoppered V a c u t a i n e r s ( F i g u r e 27). However, whereas n o n - s t e r i l e s o l u t i o n s showed a smooth d e c l i n e i n MSUM c o n c e n t r a t i o n w i t h time, the s t e r i l e s o l u t i o n s s t o r e d i n V a c u t a i n e r s showed some f l u c t u a t i o n s i n u r a t e c o n c e n t r a t i o n , p a r t i c u l a r l y f o r s o l u t i o n s s t o r e d a t 22°, 35° and 65°. These experiments were repeated s e v e r a l times, and the s o l u t i o n s s t e r i l i z e d by f i l t r a t i o n and s t o r e d i n Vac u t a i n e r s always showed v a r i a b i l i t y i n urate c o n c e n t r a t i o n at each time i n t e r v a l . During the course o f these s t u d i e s , i t was found t h a t the presence o f rubber or p l a s t i c c o n t a i n e r s o r c l o s u r e s appeared t o cause a l o s s o f MSUM from s o l u t i o n and t h a t t h i s e f f e c t was more -110-Figure 26. Degradation of n o n - s t e r i l e MSUM s o l u t i o n s . 4°C (o ) ; ;22°C (D ) ; 35°C (*); 45°C (v) ; 65°C (•) - I l l -F i g u r e 27. D e g r a d a t i o n o f s t e r i l e (0.22 um f i l t e r ) M S U M s o l u t i o n s i n V a c u t a i n e r s . _ 4 ° C ( O ) : 2 2 ° C ( • ) ; 3 ^ ° C 4 5 ° C (V); -112-TIME, hours Figure 28. Degradation of s t e r i l e (by autoclaving) MSUM solutions in a l l glass containers. 22°C (O); 3^C (•); ^ 0 ( A ) ; 65°C (•) -113-Figure 29. Degradation of s t e r i l e (by autoclaving) MSUM solutions i n Vacutainers. 22°C (•); 3 ? C (•); ( A ) ; 65°C (•) -114-pronounced a t h i g h e r temperatures. To co n f i r m t h i s o b s e r v a t i o n , the d e g r a d a t i o n o f s t e r i l e s o l u t i o n s o f MSUM ( s t e r i l i z e d by a u t o c l a v i n g ) was s t u d i e d i n a l l - g l a s s c o n t a i n e r s and Va c u t a i n e r s with rubber c l o s u r e s . The r e s u l t s are shown i n F i g u r e s 29 and 30. A f t e r a u t o c l a v i n g a t 121° f o r 35 minutes, the c o n c e n t r a t i o n o f MSUM i n s o l u t i o n s c o n t a i n e d i n a l l - g l a s s v e s s e l s decreased from 0.198 g L _ 1 t o 0.183 g L _ 1 ( F i g u r e 28) and from 0.198 g L - 1 to 0.165 g L - 1 f o r s o l u t i o n s i n the Vac u t a i n e r s ( F i g u r e 29). S o l u t i o n s s t o r e d i n g l a s s f l a s k s a t 22° and 45° were r e l a t i v e l y s t a b l e with l i t t l e change i n the c o n c e n t r a t i o n o f MSUM i n s o l u t i o n from 0 t o 96 hours. At 35° and 65° the MSUM c o n c e n t r a t i o n g r a d u a l l y decreased to 0.168 g L 1 and 0.117 g L 1 , r e s p e c t i v e l y , a t 96 hours. S o l u t i o n s s t o r e d i n Va c u t a i n e r s with rubber stoppers a t 22° and 35° were r e l a t i v e l y s t a b l e . At 45° and 65°, MSUM c o n c e n t r a t i o n s d e c r e a s e d t o 0.147 g L - 1 and 0.092 g L - 1 , r e s p e c t i v e l y , a t 96 hours. Heating the s o l u t i o n s i n an autoclave gave a decrease i n MSUM c o n c e n t r a t i o n . T h i s decrease was g r e a t e r f o r s o l u t i o n s s t o r e d i n V a c u t a i n e r s , i n d i c a t i n g t h a t some ura t e may have been l o s ^ by a b s o r p t i o n i n t o the rubber st o p p e r s . The data i n F i g u r e s 26-29 i n d i c a t e t h a t n o n - s t e r i l e s o l u t i o n s and s t e r i l e s o l u t i o n s o f MSUM undergo decomposition w i t h t ime. In n o n - s t e r i l e s o l u t i o n s , MSUM c o n c e n t r a t i o n may have decreased i n two ways, by b a c t e r i a l consumption and chemical -115-d e g r a d a t i o n . F or bo t h n o n - s t e r i l e and s t e r i l e systems, s o l u t i o n s s t o r e d a t 4 5 ° were more s t a b l e t h a n t h o s e s t o r e d a t 3 5 ° . The reason f o r t h i s b e h a v i o r i s not understood. No s t u d i e s were done t o determine the deg r a d a t i o n products o f MSUM. The d e g r a d a t i o n s t u d i e s i n d i c a t e t h a t c r y s t a l growth s t u d i e s may be conducted under n o n - s t e r i l e c o n d i t i o n s f o r about 8 hours without any a p p r e c i a b l e decrease i n MSUM c o n c e n t r a t i o n due to decomposition. 4 . 4 SATURATION S O L U B I L I T Y OF MSUM The s o l u b i l i t y o f MSUM i n - v i t r o i s i n f l u e n c e d by f a c t o r s such as temperature, the presence o f ions and the presence o f i m p u r i t i e s . The presence o f amorphous m a t e r i a l i n commercially prepared MSUM has been r e p o r t e d t o show an i n c r e a s e d s o l u b i l i t y (about 50% more) o f MSUM (Kippen e t a l . , 1974). The MSUM prepared i n our l a b o r a t o r y was used i n a l l experiments t o determine the e f f e c t o f f a c t o r s such as temperature and a d d i t i o n o f sodium c h l o r i d e , h y a l u r o n i c a c i d , c h o n d r o i t i n s u l f a t e , p r o t e o g l y c a n s and albumin on the aqueous s o l u b i l i t y o f MSUM. 4 . 4 . 1 E F F E C T OF TEMPERATURE The s a t u r a t i o n s o l u b i l i t y (Cs) o f MSUM as a f u n c t i o n o f temperature i s g i v e n i n Ta b l e 5 and F i g u r e 30. A van't H o f f p l o t (shown i n F i g u r e 31) o f l o g a r i t h m of s a t u r a t i o n s o l u b i l i t y versus r e c i p r o c a l o f abs o l u t e temperature should y i e l d a s t r a i g h t l i n e , Table 5. S a t u r a t i o n s o l u b i l i t y (Cs) of monosodium u r a t e monohydrate at d i f f e r e n t temperatures. Temperature 4.4 9.0 15.1 24.7 29.9 35.0 37.0 40.0 41.8 51.0 55.0 (°C) S a t u r a t i o n s o l u b i l i t y 0.476 0.560 0.605 0.998 1.050 1.300 1.346 1.617 1.688 1.900 2.493 (g L" 1) -117-Figure 30. Effect of temperature on the saturation s o l u b i l i t y of: (a) MSUM; (b) MSUM in the presence of physiological ion concentration. -118-Figure 31. A van't Hoff plot for MSUM so l u b i l i t y in water. -119-t h e slope o f which i s - A H/2.303 R , where A H i s the heat o f s o l u t i o n . However, a requirement o f t h i s r e l a t i o n s h i p i s t h a t the temperature range i s not too l a r g e , so t h a t A H remains a constant. Assuming t h a t over the 50° temperature range, the A H f o r MSUM i n water i s constant, the slope o f the van't Hoff p l o t was found t o be - 1267.7 °K, g i v i n g a ^ .H of + 5.8 K c a l m o l - 1 or + 24.3 KJ mo l " 1 . A l l e n et a l . (1965b) found the slope o f the van't Hoff p l o t f o r the s o l u b i l i t y o f MSUM i n water t o be - 1132 °K. The s a t u r a t i o n s o l u b i l i t y at 37°, determined by i n t e r p o l a t i o n o f the van't Hoff p l o t was 1.379 g L " 1 . I t i s w e l l documented t h a t temperature has a s i g n i f i c a n t e f f e c t on MSUM s o l u b i l i t y (Loeb, 1972; A l l e n et a l . , 1965a,b; Wilcox et a l . , 1972; F i d d i s et a l . , 1983). There are some v a r i a t i o n s i n the r e p o r t e d v a l u e s o f the aqueous s o l u b i l i t y o f MSUM a t d i f f e r e n t t emperatures. T h i s may be due to the d i f f e r e n t methods used i n the d e t e r m i n a t i o n o f the s a t u r a t i o n s o l u b i l i t y . Our s o l u b i l i t y data i s w i t h i n the range o f value s r e p o r t e d by other workers. 4 . 4 . 2 EFFECT OF SODIUM CHLORIDE The s a t u r a t i o n s o l u b i l i t y o f MSUM i n the presence o f d i f f e r i n g c o n c e n t r a t i o n s o f sodium c h l o r i d e and the p h y s i o l o g i c c o n c e n t r a t i o n s o f c h l o r i d e , sodium, c a l c i u m and potassium p r e s e n t i n plasma o r s y n o v i a l f l u i d was s t u d i e d as a f u n c t i o n of tempera-t u r e . The r e s u l t s are g i v e n i n Table 6. The r e l a t i o n s h i p -120-TABLE 6. R e l a t i o n s h i p between temperature and v a r i o u s c o n c e n t r a t i o n s o f sodium c h l o r i d e or p h y s i o l o g i c i o n c o n c e n t r a t i o n on the s a t u r a t i o n s o l u b i l i t y o f monosodium ura t e monohydrate. S a t u r a t i o n s o l u b i l i t y o f MSUMa (g L ) i n the presence o f Temperature 0.2% 0.4% 0.6% 1.0% 0.88% NaCl ( C) sodium sodium sodium sodium 0.032%CaCl c h l o r i d e c h l o r i d e c h l o r i d e c h l o r i d e 0.03% KC1 4.4 0.047 0.023 10.4 0.664 0.035 17.8 0.106 0.053 24.7 0.143 0.064 29.9 0.240 0.116 41.8 0.471 0.229 51.1 0.599 0.336 0.018 0.012 0.015 0.031 0.013 0.021 0.032 0.022 0.030 0.042 0.027 0.035 0.072 0.042 0.063 0.155 0.096 0.139 0.241 0.142 0.216 a: Mean o f 3 d e t e r m i n a t i o n s . -121-between MSUM s a t u r a t i o n s o l u b i l i t y i n water and temperature i n the presence and absence o f e l e c t r o l y t e s i s shown i n F i g u r e 30. The s a t u r a t i o n s o l u b i l i t y o f MSUM decreased s i g n i f i c a n t l y even i n the presence o f low c o n c e n t r a t i o n s o f sodium c h l o r i d e (Kippen et a l . , 1974). The c o n c e n t r a t i o n s o f the d i f f e r e n t i o n i c s p e c i e s i n plasma and s y n o v i a l f l u i d are very s i m i l a r and these ions a l s o caused a l a r g e decrease i n the s o l u b i l i t y o f MSUM at each temperature. Khalaf and Wilcox (1973) showed t h a t c a l c i u m ions reduced the MSUM s o l u b i l i t y i n water, whereas potassium and c u p r i c ions i n c r e a s e d the s o l u b i l i t y . 4.4.3 EFFECT OF CHONDROITIN SULFATE, HYALURONIC ACID,  PROTEOGLYCANS AND ALBUMIN The s a t u r a t i o n s o l u b i l i t y o f MSUM i n water was determined i n the presence o f these a d d i t i v e s a t 37°. These values were sub s e q u e n t l y used i n the d e t e r m i n a t i o n o f r a t e c o n s t a n t s f o r MSUM c r y s t a l growth i n the presence o f a d d i t i v e s . The r e s u l t s are shown i n Table 7. The presence o f 60 mg dL 1 CS decreased the s a t u r a t i o n s o l u b i l i t y o f MSUM from 1.379 g L - 1 (no a d d i t i v e ) t o 1.329 g L 1 . Laurent (1964) a l s o showed a s i m i l a r e f f e c t . However, Katz and Schubert (1970) r e p o r t e d a smal l i n c r e a s e i n MSUM s o l u b i l i t y i n the presence o f CS. C h o n d r o i t i n s u l f a t e o b t a i n e d commercially i s the sodium s a l t o f a mixture o f c h o n d r o i t i n - 6 - s u l f a t e and c h o n d r o i t i n - 4 - s u l f a t e -122-TABLE 7. S a t u r a t i o n s o l u b i l i t y o f MSUM i n the presence o f a d d i t i v e s a t 37 . S a t u r a t i o n s o l u b i l i t y o f MSUMa (g L 1 ) A d d i t i v e A d d i t i v e c o n c e n t r a t i o n s (mg dL 1 ) 10 20 40 60 100 C h o n d r o i t i n s u l f a t e 1.358 1.347 1.329 Hy a l u r o n i c a c i d 1.392 1.387 1.401 Albumin 1.382 1.391 1.397 1.395 P r o t e o g l y c a n monomer 1.402 1.397 1.399 1.401 P r o t e o g l y c a n aggregate 1.408 1.397 1.403 a: mean o f thr e e d e t e r m i n a t i o n s . -123-c o n t a i n i n g about 5.6% w/w of sodium. The decrease i n the s a t u r a t i o n s o l u b i l i t y o f MSUM i n water i n the presence o f c h o n d r o i t i n s u l f a t e may be due to the presence o f sodium i n c h o n d r o i t i n s u l f a t e sample (common i o n e f f e c t ) . H y a l u r o n i c a c i d , albumin, p r o t e o g l y c a n monomer and p r o t e o g l -can aggregate caused very s l i g h t i n c r e a s e s i n the s o l u b i l i t y o f MSUM. Katz and Schubert (1970) r e p o r t e d a s l i g h t i n c r e a s e i n the s o l u b i l i t y o f MSUM i n the presence of p r o t e o g l y c a n s . They observed enhancement o f MSUM s o l u b i l i t y i n the presence o f small amounts o f p r o t e o g l y c a n s . C h o n d r o i t i n s u l f a t e and albumin were found to cause o n l y a s l i g h t i n c r e a s e i n MSUM s o l u b i l i t y even i n the presence o f l a r g e c o n c e n t r a t i o n s o f these a d d i t i v e s . P e r r i c o n e and Brandt (1978) r e p o r t e d an i n c r e a s e i n ura t e s o l u b i l i t y i n the presence o f PGA. However, the i n c r e a s e i n MSUM s o l u b i l i t y was not s u s t a i n e d beyond 4 hours. P e r r i c o n e and Brandt (1979) r e p o r t e d a g r e a t l y i n c r e a s e d s o l u b i l i t y o f MSUM i n the presence o f p r o t e o g l y c a n aggregate (prepared i n the presence o f potassium i o n s ) . They concluded t h a t the observed i n c r e a s e i n s o l u b i l i t y was due to the c a t i o n i c exchange o f potassium ions o f the p r o t e o g l y c a n aggregate w i t h sodium ions o f MSUM. -124-4 . 4 C R Y S T A L GROWTH OF MSUM 4 . 4 . 1 DETERMINATION OF SUPERSATURATION CONCENTRATION AND S E E D  AMOUNT FOR C R Y S T A L GROWTH P r e l i m i n a r y experiments conducted i n the IL apparatus showed no c r y s t a l growth a t a s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 4 g L ^. The r e s u l t s o f some o f the experiments c a r r i e d out a t s u p e r s a t u r a t i o n c o n c e n t r a t i o n s o f 5 g L 1 and 6 g L 1 are shown i n F i g u r e s 32 and 33. No growth was observed when the s u p e r s a t u r a t i o n c o n c e n t r a t i o n was 5 g L - 1 and the seed amount was 100 mg. In the presence o f 200 mg seeds and a su p e r s a t u r a -t i o n c o n c e n t r a t i o n o f 5 g L 1 there was slow l i n e a r growth fo l l o w e d by r a p i d n o n - l i n e a r growth showing a p a r a b o l i c c o n c e n t r a t i o n - t i m e curve. S i m i l a r l y when the s u p e r s a t u r a t i o n c o n c e n t a t i o n o f MSUM was 6 g L 1 , the a d d i t i o n o f 200 mg seeds showed slow growth f o r about 2 hours f o l l o w e d by a r a p i d n o n - l i n e a r growth phase. A d d i t i o n o f 500 mg and 1000 mg seeds to the s u p e r s a t u r a t e d s o l u t i o n s , r e s u l t e d i n t y p i c a l n o n - l i n e a r c o n c e n t r a t i o n versus time curves throughout the time course o f the experiment. S i m i l a r r e s u l t s were ob t a i n e d using the 50 mL apparatus. S u p e r s a t u r a t i o n c o n c e n t r a t i o n s necessary f o r c r y s t a l growth were 5 g L 1 and g r e a t e r . Greater than 30 mg seed c r y s t a l s were r e q u i r e d t o g i v e the n o n - l i n e a r growth curves. In the seeded growth s t u d i e s o f MSUM by Erwin and Na n c o l l a s (1981) no p e r i o d o f slow growth o r i n d u c t i o n p e r i o d was observed. -125-< CL l±J O z o F i g u r e 32. TIME, min. S e e d e d g r o w t h c u r v e s f o r M S U M a t 37°» i n 1 L c a p a c i t y a p p a r a t u s a n d a n i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 5 g L - l . A d d e d s e e d a m o u n t : 100 m g ( v ) ; 200 m g ( • ) ; 300 m g ( A ) ; 500 m g ( • ) ; 1000 m g (o) . -126-< LU O o o 120 240 TIME, min. 360 F i g u r e 33. S e e d e d g r o w t h c u r v e s f o r M S U M a t 37? i n 1 1 c a p a c i t y a p p a r a t u s a n d a n i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 6 g L"1. A d d e d s e e d a m o u n t : 200 m g ( • ) ; 500 m g ( • ) ; 1000 m g ( O ) . -127-At an i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f approximately 2 g -1 -1 L urate and seed c o n c e n t r a t i o n o f 0.18 g L and 0.54 g L 1 t h e y o b s e r v e d the t y p i c a l second o r d e r , n o n - l i n e a r r e l a t i o -n s h i p between u r a t e c o n c e n t r a t i o n and time. Our r e s u l t s , however show t h a t a very h i g h i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 5 g L 1 and l a r g e amount o f seed c r y s t a l s ( g r e a t e r than 200 mg) are p r e r e q u i s i t e s f o r n o n - l i n e a r growth curves. Other s t u d i e s have demonstrated i n d u c t i o n e f f e c t s i n seeded growth systems. van Hook (1940) observed i n d u c t i o n p e r i o d s i n the c r y s t a l l i z a t i o n of s i l v e r chromate when s u p e r s a t u r a t e d s o l u t i o n s were i n o c u l a t e d w i t h seed c r y s t a l s . He showed t h a t e i t h e r i n c r e a s i n g the i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n f o r a g i v e n seed amount or i n c r e a s i n g the seed amount at a d e f i n i t e i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n decreased the l e n g t h o f the i n d u c t i o n p e r i o d . He a t t r i b u t e d h i s o b s e r v a t i o n s t o a n u c l e a t i o n p r o c e s s . However, Howard and N a n c o l l a s (1957) subsequently showed t h a t the i n d u c t i o n p e r i o d s i n the growth o f s i l v e r chromate were probably due to s u r f a c e contamination by a h y d r o l y s i s product. Davies et a l . (1955) s t u d i e d the seeded growth of s i l v e r c h l o r i d e and found t h a t delayed c r y s t a l l i z a t i o n occured when surface-contaminated seed c r y s t a l s were added t o s u p e r s a t u r a t e d s o l u t i o n s . The i n d u c t i o n p e r i o d was i n v e r s e l y p r o p o r t i o n a l to the q u a n t i t y of seed added. Even when i m p u r i t i e s were absent, i n d u c t i o n p e r i o d s have s t i l l been observed. In the seeded growth o f magnesium o x a l a t e , N a n c o l l a s and Purdie (1961) showed t h a t the r a t e constant f o r -128-growth depended on the number of c r y s t a l l i z a t i o n s i t e s p r e s e n t i n the i n o c u l a t i n g seed c r y s t a l s . They found t h a t the d u r a t i o n o f the i n d u c t i o n p e r i o d was i n v e r s e l y p r o p o r t i o n a l t o the s u p e r s a t u r a t i o n c o n c e n t r a t i o n and bulk n u c l e a t i o n was observed d u r i n g the time l a g . I t was thought t h a t the h i g h c o n c e n t r a t i o n g r a d i e n t s tended t o b u i l d up s o l u t e by a d i f f u s i o n p rocess f a s t e r than i t c o u l d be accommodated on the a v a i l a b l e growth s i t e s . At lower s u p e r s a t u r a t i o n c o n c e n t r a t i o n s , or when the amount o f i n o c u l a t i n g seed was i n c r e a s e d , the i n d u c t i o n p e r i o d disappeared, e n a b l i n g second order growth t o be g i n immediately. Our o b s e r v a t i o n t h a t an i n c r e a s e i n the amount o f i n o c u l a t i n g seed m a t e r i a l a t a g i v e n degree of s u p e r s a t u r a t i o n , reduced and f i n a l l y a b o l i s h e d the i n d u c t i o n p e r i o d may be due to the achievement o f a c r i t i c a l number of a c t i v e growth s i t e s on the seed c r y s t a l s . As the amount o f seed c r y s t a l s added t o the s u p e r s a t u r a t e d s o l u t i o n i s i n c r e a s e d , achievement o f the c r i t i c a l number o f a c t i v e growth s i t e s would then l e a d t o immediate second order growth of the seed c r y s t a l s with the t y p i c a l n o n - l i n e a r c u r v e s . 4 . 4 . 2 SELECTION OF THE METHOD TO DETERMINE THE  RATE CONSTANT OF CRYSTAL GROWTH The r a t e constants f o r c r y s t a l growth were determined u s i n g s e v e r a l d i f f e r e n t methods. The data from experiments without a d d i t i v e s were used f o r t h i s purpose. An equation o f the e m p i r i c a l form: •129-= K'(C-Cs) n (28) where Rg = the r a t e o f growth (decrease i n c o n c e n t r a t i o n per u n i t t i m e ) , K'(the r a t e constant o f the r e a c t i o n ) = K (obs) S o K Q ( o b s ) = o b s e r v e d o v e r a l l r a t e c o n s t a n t o f c r y s t a l growth, S = s u r f a c e area o f the seed c r y s t a l s , C = s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f MSUM s o l u t i o n , Cs= s a t u r a t i o n s o l u b i l i t y o f MSUM a t 37°, and n = order o f r e a c t i o n , was used t o c a l c u l a t e the growth r a t e constant as f o l l o w s . Logarithms o f equation 28 were taken t o g i v e : l o g R g = l o g K' + n l o g ( C - C s ) (29) L i n e a r l e a s t square f i t s were performed on l o g R g versus l o g (C - Cs) on the UBC computer. The r a t e constant o f the r e a c t i o n , K', was obtained from the i n t e r c e p t o f the f i t . The slope o f the f i t gave n, the order o f the r e a c t i o n . The value s of the r a t e c o n s t a n t s , K', and the order o f the r e a c t i o n , n, along w i t h the c o r r e l a t i o n c o e f f i c i e n t s o f the f i t s are giv e n i n Tables 8 t o 11 In most o f the experiments, the l i n e a r l e a s t - s q u a r e f i t s gave poor c o r r e l a t i o n c o e f f i c i e n t s r e s u l t i n g i n r a t e constant v a l u e s which were i n poor agreement f o r a g i v e n seed amount. The values o f n, the order o f the r e a c t i o n , v a r i e d between 0.4 t o -130-Table 8. MSUM growth r a t e c o n s t a n t s , K', obtained from the l i n e a r r e g r e s s i o n o f l o g R versus l o g (C-Cs). ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 5 g L ) Growth r a t e constant, Experiment Seed Seed C o r r e l a t i o n Reaction b a t c h amount K'x 10 c o e f f i c i e n t order (mg) (L g min ) 4 A 100 ——— 5 A 200 592.9 0.128 1.0 6 A 300 65.5 0.574 1.7 7 A 500 63.7 0.893 2.8 14 B 500 40.0 0.414 1.9 15 B 500 34.7 0.465 2.7 16 B 1000 45.9 0.877 3.4 8 A 1000 36.9 0.934 3.4 -131-Table 9. MSUM growth r a t e c o n s t a n t s , K', ob t a i n e d from the l i n e a r r e g r e s s i o n o f l o g R a v e r s u s l o g (C-Cs). ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 6 g L ) Growth r a t e constant, c C o r r e l a t i o n R e action Experiment Seed Seed K'x 10 c o e f f i c i e n t order # batch amount (mg) IT "I • "I \ (L g mm ) (n) 9 A 200 355.6 0.364 0.6 3 A 500 15.1 0.215 0.4 10 B 500 80.4 0.903 2.3 13 B 500 97.5 0.966 2.1 11 B 1000 97.3 0.964 2.7 12 B 1000 89.5 0.958 2.8 -132-T a b l e 10. MSUM growth r a t e c o n s t a n t s , K', o b t a i n e d from the l i n e a r r e g r e s s i o n o f l o g R-versus l o g (C-Cs). ( C r y s t a l growth i n 50 mL c a p a c i t y appratus; C = 5 g L seed c r y s t a l s from batch B) Growth r a t e c o n s t a n t Seed c C o r r e l a t i o n Reaction Experiment amount K'x 10 C o e f f i c i e n t order # (mg) (L g mxn ) (n) 41 5 40 10 39 10 7.2 0.47 7.2 38 20 3.6 0.51 3.5 37 20 0.5 0.69 5.5 36 30 6.4 0.58 3.5 35 30 1.5 0.79 4.9 34 40 6.6 0.76 3.9 33 40 4.7 0.88 4.1 32 50 10.2 0.88 4.1 31 50 2.4 0.77 5.0 30 50 40.5 0.97 2. -133-Table 11. MSUM growth r a t e c o n s t a n t s , K', obtained from the l i n e a r r e g r e s s i o n s o f l o g Raversus l o g (C-Cs). ( C r y s t a l growth i n 50 mL c a p a c i t y apparatus; C = 6 g L ; seed c r y s t a l s from batch B) Growth r a t e c o n s t a n t , Experiment Seed C o r r e l a t i o n Reaction # amount K'x 10 c o e f f i c i e n t order (mg) _ 1 _ 1 (n) (L g min ) 29 5 385.3 0.03 — 22 10 289.2 o.45 0.8 21 10 202.3 0.21 0.9 20 10 372.4 0.05 1.0 26 20 80.0 0.85 1.7 24 20 61.5 0.74 2.0 23 20 49.9 0.71 2.0 28 30 89.3 0.94 2.0 27 30 60.1 0.91 2.3 19 50 47.0 0.89 3.0 17 50 62.2 0.96 2.8 -134-7.2. However, when the c o r r e l a t i o n c o e f f i c i e n t s were good (>0.95), n was between 2 t o 3. Due to the poor c o r r e l a t i o n of the data p o i n t s t h i s method was not used to determine the e f f e c t o f a d d i t i v e s on the growth r a t e constant f o r MSUM. Marc (1908) observed t h a t the r a t e of c r y s t a l growth o f h i g h l y and moderately s o l u b l e substances was u s u a l l y p r o p o r t i o n a l 2 t o (C - Cs) . A s i m i l a r o b s e r v a t i o n was made by Davies and N a n c o l l a s (1955). I t has been suggested t h a t t h i s r e l a t i o n s h i p i s due to s p i r a l step c o n t r o l l e d growth c l o s e to e q u i l i b r i u m ( N i e l s e n , 1964). The r a t e o f c r y s t a l growth, R, then i s g i v e n by R = K' (C - C s ) 2 (30) g A number o f 1:1 and 2:2 e l e c t r o l y t e s have been shown t o f o l l o w the same growth law. These i n c l u d e , s i l v e r c h l o r i d e (Davies and N a n c o l l a s , 1955), magnesium o x a l a t e (Nancollas and Purdie, 1961), c a l c i u m s u l f a t e d i h y d r a t e ( L i u and N a n c o l l a s , 1970). A number of other e l e c t r o l y t e s which f o l l o w the same growth law have been r e f e r e n c e d by N a n c o l l a s (1979). More r e c e n t l y , Erwin and N a n c o l l a s (19.81) s t u d i e d the d i s s o l u t i o n and c r y s t a l growth o f sodium ura t e and r e p o r t e d the a p p l i c a b i l i t y of the same e q u a t i o n over a range of s u p e r s a t u r a t i o n , s',(s'= C/Cs), o f 0.9 t o 3.0. F u r t h e r evidence f o r the a p p l i c a b i l i t y o f the p a r a b o l i c r a t e law was shown by the independence of the r a t e o f - 1 3 5 -c r y s t a l l i z a t i o n on the s t i r r i n g dynamics, i n d i c a t i n g a s u r f a c e c o n t r o l l e d mechanism f o r the c r y s t a l l i z a t i o n o f sodium u r a t e . The i n t e g r a t e d form of equation 30 has been used over the years t o determine the r e a c t i o n r a t e constant f o r v a r i o u s compounds. I n t e g r a t i o n of equation 30 g i v e s the f o l l o w i n g equation: (C-Cs) (Ci-Cs) = K' t (31) where C i s the c o n c e n t r a t i o n a t v a r i o u s time i n t e r v a l s and C i i s the i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n . Equation 31 can be rearranged to equation 32: 1 C = + Cs (32) 1 K't + (Ci-Cs) T h i s equation shows the n o n - l i n e a r r e l a t i o n s h i p between C and t . A computer program ( n o n - l i n , M e t z l e r et a l . , 1974) was used to estimate the r e a c t i o n r a t e constant, K*. The v a l u e s o f K' and the c o r r e l a t i o n c o e f f i c i e n t s o b tained by t h i s n o n - l i n e a r f i t are g i v e n i n Tables 12 t o 15. Good c o r r e l a t i o n c o e f f i c i e n t s (>0.98) were obtained by t h i s method. I f the o r d e r of the growth r e a c t i o n , n, i s 2 then a p l o t o f - 1 3 6 -Table 12. MSUM growth r a t e c o n s t a n t s , K', obtained from the n o n - l i n e a r computer program. ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 5 g L ) Experiment Seed # batch 4 A 100 5 A 200 30.0 0.981 6 A 300 83.9 0.997 7 A 500 222.1 0.998 14 B 500 62.7 0.997 15 B 500 92.7 0.994 16 B 1000 306.1 0.999 8 A 1000 533.5 0.997 Growth Rate constant, Seed C o r r e l a t i o n amount K'xlO c o e f f i c i e n t ( m g ) fr - 1 • " I i (L g min ) -137-Table 13. MSUM growth r a t e constants obtained from the n o n - l i n e a r computer program. ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 6 g L ) Experiment # Seed batch Seed amount (mg) Growth r a t e constant, K'x 10 5 IT "I • "I \ (L g min ) C o r r e l a t i o n c o e f f i c i e n t 9 A 200 34.3 0.986 3 A 500 49.4 0.998 10 B 500 138.6 0.998 13 B 500 134.4 0.998 11 B 1000 388.2 1.000 12 B 1000 402.0 0.999 - 1 3 8 -Table 14. MSUM growth r a t e constants obtained from the n o n - l i n e a r computer program. ( c r y s t a l g r o w t h i n 50 mL c a p a c i t y apparatus; C = 5 g L ; seed c r y s t a l s from batch B) Growth r a t e constant, Experiment Seed C o r r e a l a t i o n # amount K'xlO c o e f f i c i e n t ( m g ) ij - 1 • (L g min ) 41 5 109.1 0.939 39 10 218.1 0.987 40 10 150.9 0.984 38 20 61.1 0.995 37 20 305.8 0.984 36 30 126.0 0.996 35 30 118.6 0.997 34 40 152.9 0.998 33 40 156.4 0.998 32 50 405.1 0.996 31 50 391.2 0.997 30 50 402.4 0.997 -139-Table 15. MSUM growth r a t e constants o b t a i n e d from the n o n - l i n e a r computer program. ( c r y s t a l growth i n 50 mL c a p a c i t y apparatus; C = 6 g L ; seeds from batch B) Growth r a t e constant, Experiment Seed C o r r e l a t i o n # amount K'xlO c o e f f i c i e n t (mg) , , (Lg 1min 1 ) 29 5 21.3 0.977 22 10 23.9 0.983 21 10 32.7 0.998 20 10 22.9 0.993 26 20 32.8 0.998 24 20 35.4 0.998 23 20 34.4 0.998 28 30 80.0 0.999 27 30 112.8 0.999 19 50 431.4 0.996 17 50 343.6 0.998 -140-(C-Cs) (Ci-Cs) versus t should g i v e a s t r a i g h t l i n e with a slope o f K'. T y p i c a l p l o t s f o r some o f the experiments are shown i n F i g u r e s 34 and 35. L i n e a r r e g r e s s i o n analyses on the values of ( l / ( C - C s ) ) -( l / ( C i - C s ) ) and t were performed on a pocket c a l c u l a t o r . E x c e l l e n t c o r r e l a t i o n c o e f f i c i e n t s were o b t a i n e d . The v a l u e s of K' and the r e s p e c t i v e c o r r e l a t i o n c o e f f i c i e n t s f o r d i f f e r e n t experiments are g i v e n i n Tables 16-19. Both the methods, n o n - l i n program and l i n e a r r e g r e s s i o n of (1/(C-Cs)) - ( l / ( C i - C s ) ) versus t (Tables 12-15 and 16-19 r e s p e c t i v e l y ) gave good c o r r e l a t i o n c o e f f i c i e n t s i n most o f the experiments (> 0.99). S i n c e the added seed amount v a r i e d between experiments, then the s u r f a c e area o f seeds a v a i l a b l e f o r growth was a l s o d i f f e r e n t . T h e r e f o r e the growth r a t e c o n s t a n t , K', was c o r r e c t e d f o r the s u r f a c e area of added seeds i n each experiment. The observed o v e r a l l c r y s t a l growth r a t e constant, K Q ( o b s ) , was determined from K'/S, where S i s the s u r f a c e area of added seed c r y s t a l s . The s u r f a c e areas of seed c r y s t a l s from batches A and B were c o r r e c t e d f o r the weight o f seeds taken i n each experiment and these v a l u e s were used as v a l u e s o f S to determine the observed o v e r a l l growth r a t e constant, K Q ( o b s ) . I t i s e v i d e n t t h a t f o r a s e t o f experiments where the same s u p e r s a t u r a t i o n c o n c e n t r a t i o n and batch of seed c r y s t a l s were -141-120 240 360 TIME, min. F i g u r e 3 4 . S e c o n d - o r d e r k i n e t i c p l o t s o f t h e i n t e g r a t e d t f o r m o f g r o w t h e q u a t i o n f o r t h e s e e d e d g r o w t h o f M S U M a t 3 7 ° i n t h e 5 0 m L c a p a c i t y a p p a r a t u s I a n d a n i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 5 S 'JJ« [ S e e d a m o u n t : 5 m g , e x p 4 1 (o); 1 0 m g , e x p 3 9 ( A ) ; j 2 0 m g , e x p 3 8 ( o ) ; 3 0 m g , e x p 3 6 (•;; 4 0 m g , e x p 3 4 ( O ; 5 0 m g , e x p 3 2 ( • ) . 142-0.4 1 TIME, min Figure 35. Second-order k i n e t i c p l o t s of the integrated form of growth equation f o r the seeded growth of MSUM at 3 7 ° i n the 5 0 mL capacity apparatus and an . i n i t i a l supersaturation concentration of 6 g L " Seed amount: 5 mg, exp 29 ( ° ) ; 10 mg, exp 20 (A); 20 mg, exp 26 (o); 40 mg, exp 2 7 (•); 50 mg, exp 30 -143-Table 16. MSUM growth r a t e constants, K', obtained from p l o t s o f the i n t e g r a t e d form o f the second order growth equation. ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 5 g L~ ) Growth r a t e constant, Experiment seed Seed ^ C o r r e l a t i o n # batch amount K'xlO c o e f f i c i e n t ( m g ) tr - 1 • (L g min ) 4 A 100 5 A 200 69.9 0.998 6 A 300 62.3 0.997 7 A 500 135.2 0.996 14 B 500 69.9 0.996 15 B 500 72.0 0.990 16 B 1000 153.2 0.986 8 A 1000 121.9 0.991 -144-Table 17. MSUM growth r a t e c o n s t a n t s , K', obtained from p l o t s o f the i n t e g r a t e d form o f the second order growth equation. ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 6 g L ) Experiment # Seed batch Seed amount (mg) Growth r a t e constant, j. C o r r e l a t i o n K'x 10 c o e f f i c i e n t IT -1 • "1\ (L g min ) 9 A 200 44.8 0.998 (upto 180 min) 3 A 500 68.6 0.991 10 B 500 121.4 0.990 13 B 500 127.6 0.999 11 B 1000 170.7 0.986 12 B 1000 182.1 0.993 -145-Table 18. MSUM growth r a t e c o n s t a n t s , K', obtained from p l o t s o f the i n t e g r a t e d form o f the second order growth equation. ( c r y s t a l growth i - n 50 mL c a p a c i t y apparatus; C = 5 g L seed c r y s t a l s from batch B) Growth r a t e constant, Experiment Seed c C o r r e l a t i o n # amount (mg) K'x 10 IT "I • ~1\ (L g min ) c o e f f i c i e n t 41 5 7.7 0.946 39 10 10.3 0.986 40 10 12.1 0.977 38 20 22.6 0.995 37 20 18.6 0.988 36 30 38.8 0.990 35 30 34.6 0.986 34 40 48.4 0.988 33 40 42.6 0.993 32 50 62.7 0.975 31 50 51.4 0.978 30 50 72.3 0.995 -146-Table 19. MSUM growth r a t e constants, K', obtained from p l o t s o f the i n t e g r a t e d form o f the second order growth equation. ( c r y s t a l g j o w t n i - n 50 mL c a p a c i t y apparatus; C = 6 g L ; seed c r y s t a l s from batch B) Experiment # Seed amount (mg) Growth r a t e constant, K'xlO 5 (L g min ) C o r r e l a t i o n c o e f f i c i e n t 29 5 32.2 0.957 22 10 41.2 0.974 21 10 39.5 0.949 20 10 39.8 0.996 26 20 63.1 0.997 24 20 72.0 0.994 23 20 61.1 0.995 28 30 108.2 0.997 27 30 97.0 0.998 19 50 121.3 0.978 17 50 167.1 0.996 -147-used, K Q(obs) values should be s i m i l a r . The v a l u e s of K (obs) shown i n Tables 20 t o 23 were o c a l c u l a t e d from the values of K' generated by the two d i f f e r e n t methods. The observed o v e r a l l c r y s t a l growth r a t e c o n s t a n t s , K Q ( o b s ) e s t i m a t e d from the n o n - l i n computer program show a wide v a r i a t i o n i n v a l u e s f o r a g i v e n s e t experiments. However, K Q(obs) determined from the l i n e a r , r e c i p r o c a l p l o t gave s i m i l a r v a l u e s w i t h i n a g i v e n s e t o f experiments. Therefore c r y s t a l growth r a t e c o n s t a n t s , K Q(obs) were determined by the l i n e a r r e g r e s s i o n o f ( l / ( C - C s ) ) - ( l / ( C i - C s ) ) versus t f o r a l l f u r t h e r experiments on the e f f e c t o f a d d i t i v e s on the growth r a t e of MSUM. 4.4.3 E F F E C T OF S E E D C R Y S T A L S ON THE C R Y S T A L GROWTH Two batches o f seed c r y s t a l s A and B were used i n the c r y s t a l growth experiments. The mean s u r f a c e area o f Batch A 2 —1 2 seed c r y s t a l s was 1.0447 m g (n = 3; range 0.908 m -1 2 -1 g t o 1.179 m g ) and the mean s u r f a c e area of Batch B 2 — 1 2 seed c r y s t a l s was 1.4521 m g (n = 3; range: 1.417 m — 1 2 —1 g t o 1.548 m g ). The r e s u l t s of the e f f e c t on the r a t e c onstants f o r growth o f these two batches o f seed c r y s t a l s are shown i n Table 20. The seeds from the two d i f f e r e n t batches showed d i f f e r e n t growth r a t e c o n s t a n t s . The methods used i n the p r e p a r a t i o n o f the two batches -148-Table 20. O v e r a l l growth r a t e c o n s t a n t s , K (obs) c a l c u l a t e d from r a t e c o n s t a n t s , . ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 5 g L 1 ) O v e r a l l growth r a t e constant, 3 Experiment Seed Seed K (obs) x 10 # batch amount _^ _^ ^ (mg) (L g min m ) (a) (b) 4 A 100 5 A 200 1.4 3.3 6 A 300 2.7 2.0 7 A 500 4.2 2.6 14 B 500 0.9 1.0 15 B 500 1.3 1.0 16 B 1000 2.1 1.1 8 A 1000 5.1 1.2 (a) C a l c u l a t e d from r a t e from non-1in program constant • o b t a i n e d (b) C a l c u l a t e d from l i n e a r (1/(C-Cs) -from r a t e constant r e g r e s s i o n of l / ( C i - C s ) ) versus obtained t . -149-Table 21. O v e r a l l growth r a t e c o n s t a n t s , K (obs), c a l c u l a t e d from r a t e c o n s t a n t s , R'. ( c r y s t a l growth i n IL c a p a c i t y apparatus; C = 6 gL ) O v e r a l l growth r a t e constant 3 Experiment Seed Seed K (obs) x 10 # batch amount _ 1 _^ _ 2 (mg) (L g min m~ ) (a) (b) 9 A 200 1.6 (upto 180 min) 3 A 500 1.0 1.3 10 B 500 1.9 1.7 13 B 500 1.8 1.8 11 B 1000 2.7 1.2 12 B 1000 2.8 1.2 (a) c a l c u l a t e d from r a t e constants obtained from non-1in program (b) c a l c u l a t e d from r a t e constants o b t a i n e d from l i n e a r r e g r e s s i o n o f ( l / ( C - C s ) - l / ( C i - C s ) ) versus t -150-Table 22. O v e r a l l growth r a t e c o n s t a n t s , K (obs), c a l c u l a t e d from r a t e c o n s t a n t s , 8'. (crystal_c|rowth i n 50 mL c a p a c i t y apparatus; C = 5 gL ; seed c r y s t a l s from batch B) O v e r a l l growth r a t e c o n s t a n t Experiment Seed K (obs) x ft 1 0 3 # amount (mg) (L g min m-2) (a) (b) 41 5 150.3 10.7 39 10 150.2 7.1 40 10 103.9 8.3 38 20 21.0 7.8 37 20 105.3 6.4 36 30 28.9 8.9 35 30 27.2 7.9 34 40 26.3 8.3 33 40 26.9 7.3 32 50 55.9 8.6 31 50 53.8 7.1 30 50 69.2 9.9 (a) C a l c u l a t e d from r a t e constants o b t a i n e d from non-1in program. (b) C a l c u l a t e d from r a t e constants obtained from l i n e a r r e g r e s s i o n o f ( l / ( C - C s ) - l / ( C i - C s ) ) versus t . -151-TABLE 23. O v e r a l l growth r a t e c o n s t a n t s , K , c a l c u l a t e d from r a t e c o n s t a n t s , 8 ' . (crystal_tjjrowth i n 50 mL c a p a c i t y apparatus? C = 6 gL ; seed c r y s t a l s from batch B) O v e r a l l growth r a t e constant, Experiment Seed K x 1 0 3 # amount (mg) (L g" -1 . -1 -2 \ min m ) (a) (b) 29 5 29.3 44.3 22 10 16.4 28.4 21 10 22.5 27.2 20 10 15.8 27.4 26 20 11.3 21.7 24 20 12.2 24.8 23 20 11.8 21.0 28 30 18.4 24.8 27 30 25.9 22.3 19 50 59.4 16.7 17 50 47.3 23.0 (a) c a l c u l a t e d from r a t e c onstants o b t a i n e d from non-1in program. (b) c a l c u l a t e d from r a t e constants obtained from l i n e a r r e g r e s s i o n o f ( l / ( C - C s ) - l / ( C i - C s ) ) versus t . -152-v a r i e d i n s e v e r a l aspects (see s e c t i o n 3.3.1). Ther e f o r e i t i s l i k e l y t h a t these v a r i a t i o n s i n p r e p a r a t i o n l e d t o s i g n i f i c a n t d i f f e r e n c e s i n the type and number of d e f e c t s p r e s e n t i n the two batches o f MSUM c r y s t a l s . I t i s w e l l documented t h a t the d e f e c t content o f c r y s t a l s and t h e i r c r y s t a l growth r a t e s are c l o s e l y r e l a t e d (Van B a t c h e l d e r and Vaughan, 1967; Brooks e t a l . , 1968; P a t e l and Rao, 1979). The d i f f e r e n c e s i n growth r a t e constants shown by seeds from Batches A and B may be due to the d i f f e r e n c e s i n d e f e c t content o f the seed c r y s t a l s . S i m i l a r o b s e r v a t i o n s were r e p o r t e d by Erwin and Na n c o l l a s (1981) who showed t h a t seed c r y s t a l s o f MSUM from d i f f e r e n t batches had d i f f e r e n t growth r a t e c o n s t a n t s . 4 . 4 . 4 EFFECT OF SUPERSATURATION ON THE CRYSTAL GROWTH RATE  CONSTANT A change i n the s u p e r s a t u r a t i o n c o n c e n t r a t i o n from 5 g L to 6 g L 1 i n the 50 mL apparatus, i n the presence o f seed c r y s t a l s from the same batch i n c r e a s e d the c r y s t a l growth r a t e constant v a l u e s by about 3 t o 4 f o l d . The growth r a t e constant _3 v a l u e , K Q ( o b s ) , i n c r e a s e d from approximately 7 x 10 L -1 -1 -2 -3 -1 -1 g min m to approximately 24 x 10 L g min _ 2 m when the s u p e r s a t u r a t i o n c o n c e n t r a t i o n was changed from 5 L - 1 to 6 g L _ 1 (Tables 22 and 23). 4 . 4 . 5 EFFECT OF SODIUM AND POTASSIUM IONS ON THE GROWTH RATE  CONSTANTS C h o n d r o i t i n s u l f a t e , h y a l u r o n i c a c i d , p r o t e o g l y c a n monomer -153-and p r o t e o g l y c a n aggregate c o n t a i n a c o n c e n t r a t i o n o f ions which are i n t r o d u c e d d u r i n g t h e i r p r e p a r a t i o n . The sodium and potassium contents o f these c a r t i l a g e components were determined by flame photometry and are shown i n Table 24. The e f f e c t on the growth r a t e constants o f the concentra-t i o n s o f sodium o r potassium p r e s e n t i n the maximum q u a n t i t i e s o f a d d i t i v e s used i n c r y s t a l growth experiments was determined. Sodium or potassium ions e q u i v a l e n t t o the h i g h e s t c o n c e n t r a t i o n p r e s e n t i n any o f the a d d i t i v e s t u d i e s were added t o a sup e r s a t -urated s o l u t i o n (5.0 g L 1 ) . Rate constants, K', were determined and are shown i n Table 25. In the presence o f 1.6 mg ( i n 50 mL) o f sodium the r a t e constant ranged from 42.5 x 10 ^ L g 1 min 1 t o 48.5 x -5 . -5 -1 -1 10 with a mean of 44.1 x 10 L g min . T h i s value i s not s i g n i f i c a n t l y d i f f e r e n t from t h a t obtained i n the absence o f sodium i o n s . S i m i l a r l y i n the presence o f 1.3 mg ( i n 50 mL) -5 -1 of potassium, a mean r a t e constant o f 44.3 x 10 L g min 1 was obta i n e d . I t has been shown p r e v i o u s l y ( s e c t i o n 4.4.2) t h a t the a d d i t i o n o f sodium ions suppresses the s a t u r a t i o n s o l u b i l i t y o f MSUM, by the common i o n e f f e c t . However, i t i s apparent t h a t the small a d d i t i o n a l c o n t r i b u t i o n made by the sodium or potassium i o n s p r e s e n t i n the a d d i t i v e s t o the o v e r a l l h i g h c o n c e n t r a t i o n o f sodium ions i n the s u p e r s a t u r a t e d s o l u t i o n s , was i n s u f f i c i e n t t o cause any s i g n i f i c a n t change i n the r a t e constant f o r growth. - 1 5 4 -Table 24. Sodium and potassium content o f a d d i t i v e s . ADDITIVE SODIUM POTASSIUM (% w/w) (% w/w) CHONDROITIN SULFATE 5.6 HYALURONIC ACID 0.11 6.3 PROTEOGLYCAN MONOMER 5.8 PROTEOGLYCAN AGGREGATE 5.2 -155-Table 2&. E f f e c t o f sodium and potassium ions on MSUM c r y s t a l growth r a t e c o n s t a n t . ( c r y s t a l growth i n 50 mL apparatus; C = 5 gL Seed Sodium/ Growth r a t e Experiment amount potassium constant,K', xlO # (mg) added _ 1 _^ (mg) (L g min ) 33 40 42.6 34 " 48.4 101 " 1.6(Na +) 42.5 102 " " 48.5 103 " " 43.3 104 " 1.3(K +) 44.9 105 " " 45.7 -156-4.4.6 EFFECT OF ADDITIVES ON CRYSTAL GROWTH RATE CONSTANT The f o l l o w i n g method was used f o r the a n a l y s i s o f the data generated by c r y s t a l growth experiments i n the presence o f the a d d i t i v e s , h y a l u r o n i c a c i d , c h o n d r o i t i n s u l f a t e , p r o t e o g l y c a n monomer, p r o t e o g l y c a n aggregate, p h o s p h a t i d y l c h o l i n e and phosph-a t i d y l s e r i n e . The observed r a t e constant, K'(add), f o r the growth o f MSUM i n the presence o f a d d i t i v e s , was c a l c u l a t e d from e q u a t i o n 31 as d i s c u s s e d p r e v i o u s l y ( s e c t i o n 4.4.2), where Cs was taken t o be the s a t u r a t i o n s o l u b i l i t y o f MSUM i n the presence o f the same c o n c e n t r a t i o n o f a d d i t i v e (see Table 7 ) . The r a t i o o f observed r a t e constant i n the presence o f a d d i t i v e , K'(add) t o K ' ( c o n t r o l ) was c a l c u l a t e d , where K ' ( c o n t r o l ) i s the c o n t r o l growth r a t e constant f o r MSUM i n the absence o f any a d d i t i v e s , determined under i d e n t i c a l c o n d i t i o n s o f s u p e r s a t u r a t i o n c o n c e n t r a t i o n , seed amount and sodium or potassium i o n c o n c e n t r a t i o n . I f the r a t i o K'(add)/ K " ( c o n t r o l ) was g r e a t e r than or l e s s than u n i t y , the a d d i t i v e under study e i t h e r i n c r e a s e d or decreased the growth r a t e constant and hence a l t e r e d the growth r a t e . S t a t i s t i c a l a n a l y s e s were performed u s i n g a one way a n a l y s i s of v a r i a n c e f o l l o w e d by a Neuman-Keul's t e s t . The l e v e l o f s i g n i f i c a n c e was p<0.05. (A) EFFECT OF HYALURONIC ACID ON THE GROWTH RATE CONSTANT The e f f e c t o f HA on the growth r a t e constant o f MSUM i s shown i n Table 26. In the presence o f HA (5 mg to 20 mg) i n the -157-Table 26. E f f e c t o f h y a l u r o n i c a c i d on the growth k i n e t i c s o f MSUM i n 50 mL apparatus. Experiment # H y a l u r o n i c a c i d (mg) K ' ( a d d ) / K ' ( c o n t r o l ) a (a) I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n = 5 g L 1 . 82 5 1.148 83 5 0.987 81 10 1.060 84 10 0.876 80 20 0.867 85 20 1.010 (b) I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n = 6 g L 1 . 87 5 0.783 88 5 0.778 86 10 0.742 89 10 0.762 a: R a t i o o f the observed r a t e constant i n the presence o f HA to the observed r a t e constant i n the absence o f HA. -158-50 mL apparatus with an i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 5 g L 1 and seed amount o f 50 mg the r a t i o s between the K'(add) and K ' ( c o n t r o l ) showed onl y s m a l l v a r i a t i o n s from u n i t y . How-ever, when the i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n was 6 g L 1 and the seed amount was 30 mg the K'(add)/ K ' ( c o n t r o l ) decreased t o 0.78 (±0.003) f o r 5 mg of HA and t o 0.752 (± 0.010) f o r 10 mg of HA. The a n a l y s i s o f v a r i a n c e and Neuman-Keul's t e s t showed no s i g n i f i c a n t d i f f e r e n c e i n growth r a t e constants i n the presence o f HA from those o f the c o n t r o l s (absence o f HA) at the l e v e l o f s i g n i f i c a n c e , p< 0.05 when the s u p e r s a t u r a t i o n c o n c e n t r a t i o n was 5 g L 1 . However, the same s t a t i s t i c a l a n a l y s i s showed a s i g n i f i c a n t decrease i n the c r y s t a l growth r a t e constant i n the presence o f 5 and 10 mg of HA when the s u p e r s a t u r a t i o n c o n c e n t r a -t i o n was 6 g L 1 . Hence, although HA decreased the growth r a t e constant f o r MSUM growth a t b o t h 5 g L 1 and 6 g L 1 s u p e r s a t u r a t i o n c o n c -e n t r a t i o n s , the decrease from c o n t r o l was s i g n i f i c a n t o n l y a t the hi g h e r degree o f s u p e r s a t u r a t i o n . (B) E F F E C T OF CHONDROITIN S U L F A T E ON THE GROWTH RATE  CONSTANT The r a t i o s o f K'(add) ( r a t e constant i n the presence o f c h o n d r o i t i n s u l f a t e ) t o K ' ( c o n t r o l ) f o r d i f f e r e n t amounts o f a d d i t i v e are gi v e n i n Table 27. The r a t i o i n c r e a s e d from 1.455 (± 0.004) f o r 10 mg c h o n d r o i t i n s u l f a t e t o 1.66 (± 0.040) f o r 30 -159-Table 27. E f f e c t o f c h o n d r o i t i n s u l f a t e on the growth k i n e t i c s of MSUM i n 50 mL apparatus. I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n , 5 g L Experiment C h o n d r o i t i n K ' ( a d d ) / K ' ( c o n t r o l ) 3 # s u l f a t e (mg) 91 10 1.734 92 10 1.496 98 10 1.414 93 20 1.094 96 20 1.520 97 20 1.624 94 30 1.624 95 30 1.697 a: R a t i o o f the observed r a t e constant i n the presence of CS to the observed r a t e constant i n the absence o f CS. -160-mg c h o n d r o i t i n s u l f a t e . The a n a l y s i s o f v a r i a n c e and Newman-Keul's t e s t showed a s i g n i f i c a n t i n c r e a s e i n the r a t e c onstant f o r MSUM c r y s t a l growth i n the presence o f c h o n d r o i t i n s u l f a t e over t h a t o f c o n t r o l s a t a l e v e l o f s i g n i f i c a n c e , p, o f <0.05. (C) E F F E C T OF PROTEOGLYCAN MONOMER ON THE C R Y S T A L GROWTH  RATE CONSTANT The r a t i o s o f K'(add) ( r a t e constant i n the presence of p r o t e o g l y c a n monomer) t o K ' ( c o n t r o l ) a t 5 mg and 10 mg added amounts o f p r o t e o g l y c a n monomer are given i n Table 28. Sin c e , the r a t i o i s s l i g h t l y g r e a t e r than u n i t y a s m a l l i n c r e a s e i n the growth r a t e constant i n the presence of 5 mg and 10 mg of PGM i s e v i d e n t . However, the a n a l y s i s o f v a r i a n c e and the Newman-Keul 1s t e s t showed no s i g n i f i c a n t d i f f e r e n c e between the c o n t r o l experiments and the growth experiments i n the presence o f PGM a t the 5% l e v e l o f s i g n i f i c a n c e . (D) E F F E C T OF PROTEOGLYCAN AGGREGATE ON T H E C R Y S T A L GROWTH  R A T E CONSTANT The r a t i o s o f K'(add) ( r a t e constant i n the presence of p r o t e o g l y c a n aggregate) t o K ' ( c o n t r o l ) a t d i f f e r e n t added amounts of PGA are g i v e n i n Table 29. In the presence of 10 mg, 20mg and 50 mg of PGA the r a t i o s were 0.966 (±0.050), 0.987 (± 0.140) and 1.163 (± 0.025) -161-Table 28. E f f e c t o f p r o t e o g l y c a n monomer on the growth k i n e t i c s o f MSUM i n 50 mL apparatus. I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n , 5 g L Experiment P r o t e o g l y c a n # monomer (mg) K ' ( a d d ) / K ' ( c o n t r o l ) a 121 5 0.965 122 5 1.028 123 5 1.047 124 10 1.071 125 10 1.027 a: R a t i o o f the observed r a t e constant i n the presence of PGMto the observed r a t e constant i n the absence o f PGM. -162-Table 29. E f f e c t of p r o t e o g l y c a n aggregate on the growth k i n e t i c s of MSUM i n 50 mL apparatus. I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n , 5 g L~ . Experiment P r o t e o g l y c a n K ' ( a d d ) / K ' ( c o n t r o l ) a # aggregate (mg) 201 10 0.910 202 10 1.023 203 20 0.850 204 20 1.124 205 50 1.139 206 50 1.187 a: R a t i o of the observed r a t e constant i n the presence o f PGA t o the observed r a t e constant i n the absence of PGA. -163-r e s p e c t i v e l y . The one way a n a l y s i s o f v a r i a n c e followed by the Neuman-Keul's t e s t showed no s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t the 5% l e v e l o f s i g n i f i c a n c e . (E) EFFECT OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLSERINE  ON THE CRYSTAL GROWTH RATE CONSTANT OF MSUM The r e s u l t s o f the e f f e c t s o f the p h o s p h o l i p i d s , p h - c h o l i n e and p h o s p h a t i d y l s e r i n e expressed as r a t i o s o f K'(add) ( r a t e constant i n the presence of e i t h e r of the two p h o s p h o l i p i d s ) t o K ' ( c o n t r o l ) on the MSUM c r y s t a l growth are shown i n Tables 30 and 31. P h o s p h a t i d y l c h o l i n e caused an i n c r e a s e i n the growth r a t e constant i n most o f the experiments. The r a t i o s (K'(add)/ K ' ( c o n t r o l ) ) were 1.120 (± 0.070), 1.325 (± 0.220) and 1.070 (± 0.220) i n the presence o f 10 mg, 20 mg and 30 mg o f p h o h p h a t i d y l c h o l i n e r e s p e c t i v e l y (Table 30). As i s e v i d e n t from the r e s u l t s , l a r g e v a r i a t i o n s i n the r a t e c o n s t a n t v a l u e s were observed i n the presence o f p h - c h o l i n e . The p h o s p h a t i d y l c h o l i n e used i n t h i s study was o n l y 80% p h o s p h a t i d y l -c h o l i n e ( l a b e l c l a i m ) . T h e r e f o r e the samples added t o the sup e r s a t u r a t e d s o l u t i o n contained s u b s t a n t i a l l e v e l s o f i m p u r i t i e s . These i m p u r i t i e s may have caused the h i g h degree o f v a r i a b i l i t y i n these experiments. I t i s a l s o p o s s i b l e t h a t the observed i n c r e a s e i n the growth r a t e constant i n the presence o f p h o s p h a t i d y l c h o l i n e was due to a combination o f both the phosph-a t i d y l c h o l i n e and other i m p u r i t i e s present i n the p h o s p h a t i d y l --164-Table 30. E f f e c t o f p h o s p h a t i d y l c h o l i n e on the growth k i n e t i c s of MSUM i n 50 mL apparatus. I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n , 5 g L Experiment P h o s p h a t i d y l - K ' ( a d d ) / K ' ( c o n t r o l ) a # c h o l i n e (mg) 53 10 1.050 54 10 1.190 55 20 1.293 56 20 1.357 57 30 1.299 58 30 0.862 a: R a t i o of the observed r a t e constant i n the presence o f PC to the observed r a t e constant i n the absence of PC. -165-Table 31. E f f e c t o f p h o s p h a t i d y l s e r i n e on the growth k i n e t i c s o f MSUM i n 50 mL apparatus. I n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n , 5 g L Experiment P h o s p h a t i d y l - K ' ( a d d ) / K 1 ( c o n t r o l ) a # s e r i n e (mg) 71 10 1.171 75 10 1.071 72 20 1.027 73 20 1.047 74 20 1.028 a: R a t i o of the observed r a t e constant i n the presence of PS to the observed r a t e constant i n the absence o f PS. -166-c h o l i n e sample. P h o s p h a t i d y l s e r i n e showed a very s m a l l i n c r e a s e i n the growth r a t e c o n s t a n t . T h i s i s evidenced by a s m a l l i n c r e a s e from u n i t y , i n the r a t i o s , K* (a d d ) / K ' ( c o n t r o l ) (Table 31). In the presence o f 10 mg and 20 mg of ph- s e r i n e the r a t i o s were 1.120 (± 0.050) and 1.030 (± 0.010). The a n a l y s i s o f v a r i a n c e and the Neuman-Keul's t e s t on the r e s u l t s showed a s i g n i f i c a n t i n c r e a s e (p < 0.05) i n the growth r a t e c o n s t a n t i n the presence o f p h - c h o l i n e over t h a t o f c o n t r o l . However, a t the same l e v e l o f s i g n i f i c a n c e there was no s i g n i f i c a n t d i f f e r e n c e i n the growth r a t e constant i n the presence o f ph - s e r i n e over t h a t o f the c o n t r o l . (F) EFFECT OF ALBUMIN ON MSUM CRYSTAL GROWTH The e f f e c t o f albumin on the c r y s t a l growth o f MSUM i s shown i n F i g u r e 36. The growth r a t e c o n s t a n t s f o r MSUM i n the presence of albumin are not gi v e n because very poor c o r r e l a t i o n s were obtained when the co n c e n t r a t i o n - t i m e data were s u b s t i t u t e d i n t o equation 31. At an i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 5 g L - 1 , the c o n c e n t r a t i o n - t i m e p l o t showed a l i n e a r decrease i n c o n c e n t r a t i o n with time i n the presence of 10 and 50 mg of albumin. The growth r a t e i n the presence of 10 mg and 50 mg o f albumin was l e s s than the growth r a t e o f MSUM i n the absence of albumin ( c o n t r o l ) . Complete i n h i b i t i o n o f c r y s t a l growth was seen i n the presence o f 200 mg of albumin. -167-6 i o OH 1 r - r 0 120 240 360 TIME, minutes Figure 3 6 . Seeded growth of MSUM i n the 5 0 mL capacity apparatus and an i n i t i a l supersaturation concentration of 5 6 . Quantities of albumin added; 1 0 mg (O); 5 0 mg (•); 1 0 0 mg (A); 2 0 0 mg (v); control ( •) . -168-Th e presence o f ions or molecules other than those o f the m a t e r i a l b e i n g c r y s t a l l i z e d can have number o f e f f e c t s on the growth k i n e t i c s o r on the c r y s t a l morphology (see s e c t i o n 2.8.4). Atoms or molecules may enter the c r y s t a l l a t t i c e and can l e a d to the formation of d e f e c t s i n the c r y s t a l s . Some compounds s p e c i f i c a l l y adsorb onto a p a r t i c u l a r face o f a c r y s t a l and i n h i b i t the growth of the face, l e a d i n g to a change i n c r y s t a l morphology or h a b i t . For example, s u r f a c e a c t i v e agents and t r i v a l e n t i ons such as C r 3 + , F e 3 + and A l 3 + are commonly used to change c r y s t a l h a b i t s ( M u l l i n , 1980). Gron and Hay (1976) showed t h a t the i n h i b i t o r y e f f e c t of s a l i v a r y s e c r e t i o n s on the p r e c i p i t a t i o n o f d i c a l c i u m phosphate was a s s o c i a t e d with the macromolecular p r o t e i n f r a c t i o n of s a l i v a . The s t i m u l a t e d s a l i v a , which c o n t a i n s more p r o t e i n was found to be about two times more i n h i b i t o r y than unstimulated s a l i v a . However, no i n h i b i t o r y e f f e c t on the p r e c i p i t a t i o n o f d i c a l c i u m phosphate was observed when serum p r o t e i n s ( c o n c e n t r a t i o n range 0.6-5.5 mg mL - 1) were i n c l u d e d i n the growth medium. Moreno e t a l . (1979) a l s o o b served a r e d u c t i o n i n the r a t e o f c a l c i u m h y d r o x y a p a t i t e p r e c i p i t a t i o n i n the presence of human s a l i v a r y p r o t e i n s . They suggested t h a t the r e d u c t i o n i n the growth r a t e induced by the macromolecules was produced by b l o c k i n g of the c r y s t a l growth s i t e s through a d s o r p t i o n of the macromolecules onto the s u r f a c e of the c a l c i u m h y d r o x y a p a t i t e seed c r y s t a l s . -169-Erwin and Nan c o l l a s (1981) s t u d i e d the e f f e c t o f h e p a r i n sodium on the growth r a t e o f MSUM and r e p o r t e d no e f f e c t o f t h i s macromolecule a t l e v e l s o f 0.001-10 ppm. In our s t u d i e s both i n c r e a s e d and decreased growth r a t e s of MSUM were observed when the macromolecules, h y a l u r o n i c a c i d , c h o n d r o i t i n s u l f a t e , p r o t e o g l y c a n monomer, p r o t e o g l y c a n aggregate and albumin were i n c l u d e d i n the growth medium. H y a l u r o n i c a c i d and albumin i n h i b i t e d MSUM growth, c h o n d r o i t i n s u l f a t e and pr o t e o g l y c a n monomer a c c e l e r a t e d MSUM growth and pr o t e o g l y c a n aggregate had no e f f e c t on MSUM growth k i n e t i c s . Although c h o n d r o i t i n s u l f a t e s i g n i f i c a n t l y i n c r e a s e d the growth r a t e constant, t h i s i n c r e a s e was not due to the sodium c o n t e n t o f the CS sample as shown by the r e s u l t s i n s e c t i o n 4.4.5 (Table 25). MSUM has been shown t o a v i d l y b i n d with a number o f p r o t e i n s (Kozin and McCarty, 1976) and although MSUM c r y s t a l s have a h i g h negative s u r f a c e p o t e n t i a l , they b i n d s i g n i f i c a n t amounts o f n e g a t i v e l y charged p r o t e i n s such as the immunoglobulin IgG. C h o n d r o i t i n s u l f a t e i s a h i g h l y n e g a t i v e l y charged macromolecule, the n e g a t i v e charge a r i s i n g from the s u l f a t e (SO^) groups on a the m o l e c u l e . I t i s p o s s i b l e t h a t CS adsorbs onto the s u r f a c e o f the MSUM seed c r y s t a l s and t h a t f o l l o w i n g a d s o r p t i o n , CS a c t s as an a c c e l e r a t o r o f c r y s t a l growth. I f the SO^ groups on the CS molecule p r o j e c t e d outwards away from the c r y s t a l s u r f a c e these groups might a t t r a c t sodium ions from the s u p e r s a t u r a t e d s o l u t i o n -170-and these c e n t e r s would then a c t as " c a t a l y s t s " f o r the subsequent i n c o r p o r a t i o n o f urate ions i n t o the MSUM c r y s t a l l a t t i c e . In t h i s way, CS might f u n c t i o n as a growth a c c e l e r a t o r . T h i s proposed f u n c t i o n o f CS i s s i m i l a r t o the r o l e o f pro t e o g l y c a n s i n the c a l c i f i c a t i o n o f bone. Before a t i s s u e . c a l c i f i e s , i t must be converted from a n o n - c a l c i f i a b l e to a c a l c i f i a b l e m atrix ( U r i s t , 1976). I t i s thought t h a t the 2+ p r o t e o g l y c a n s o f c a r t i l a g e b i n d c a l c i u m ions (Ca ) which then a t t r a c t s phosphate i o n s (PO^) and forms an i n o r g a n i c n u c l e a t i o n c e n t e r . I t i s a l s o b e l i e v e d t h a t the n e g a t i v e l y charged, a c i d i c 2+ p h o s p h o l i p i d s o f matrix v e s i c l e s b i n d Ca through a two-point e l e c t r o s t a t i c i n t e r a c t i o n which then allows the ca l c i u m ions t o i n t e r a c t w i t h phosphate ions i n s o l u t i o n ( U r i s t , 1976). There i s some evidence t h a t p r e e x i s t i n g d i s e a s e such as o s t e o a r t h r i t i s may l e a d t o c r y s t a l d e p o s i t i o n and a l s o t h a t the i n c i d e n c e o f gouty a r t h r i t i s i n c r e a s e s w i t h advancing age. S i n c e the p r o p o r t i o n o f CS i n both ageing and o s t e o a r t h r i t i c c a r t i l a g e decreases, i t becomes d i f f i c u l t t o e x p l a i n how a decrease i n the amounts o f the growth a c c e l e r a t o r , CS, cou l d be a major cause o f MSUM d e p o s i t i o n i n c a r t i l a g e . However, i t should be noted t h a t i n our growth experiments, a mixture o f unknown composition o f the two isomers o f CS, c h o n d r o i t i n - 4 - s u l f a t e and c h o n d r o i t i n - 6 -s u l f a t e , was used. I t i s p o s s i b l e t h a t the two isomers c o u l d have q u i t e d i f f e r e n t e f f e c t s on MSUM c r y s t a l growth which might c o r r e l a t e w i t h the observed a l t e r a t i o n s i n the r a t i o s o f c h o n d r o i t i n - 6 - s u l f a t e t o c h o n d r o i t i n - 4 - s u l f a t e i n aged and -171-o s t e o a r t h r i t i c c a r t i l a g e . C r y s t a l s of MSUM can a l s o p r e c i p i t a t e d i r e c t l y i n the syno-v i a l f l u i d . CS i s found i n the s y n o v i a l f l u i d o f p a t i e n t s w i t h a r t h r i t i s (Barker et a l . , 1966) but the r e i s c o n t r o v e r s y as t o whether CS i s a normal c o n s t i t u e n t o f s y n o v i a l f l u i d (Barker e t a l . , 1966; S i l p a n a n t a et a l . , 1967). I t i s p o s s i b l e t h a t the low l e v e l s o f CS i n p a t h o l o g i c a l f l u i d s may be s u f f i c i e n t t o cause s i g n i f i c a n t a c c e l e r a t i o n o f MSUM c r y s t a l growth l e a d i n g t o the appearance o f c r y s t a l s i n the s y n o v i a l f l u i d . The two p h o s p h o l i p i d s s t u d i e d , p h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l s e r i n e , a l s o i n c r e a s e d the growth r a t e constant o f MSUM. However, a t the c o n c e n t r a t i o n l e v e l s o f p h o s p h a t i d y l s e r i n e used i n the growth experiments, the i n c r e a s e i n the growth r a t e constant was not s i g n i f i c a n t . P h o s p h a t i d y l s e r i n e i s an a c i d i c p h o s p h o l i p i d , b e a r i n g a net ne g a t i v e charge a t p h y s i o l o g i c pH, whereas p h o s p h a t i d y l c h o l i n e c a r r i e s both a n e g a t i v e l y charged secondary phosphate and a p o s i t i v e l y charged q u a t e r n a r y amine and i s i s o e l e c t r i c i n the pH range 3-10. Hence, these p h o s p h o l i p i d s may f u n c t i o n as MSUM growth a c c e l e r a t o r s by the same mechanisms as d e s c r i b e d above f o r CS. There i s a s i g n i f i c a n t i n c r e a s e i n the i n t r a c e l l u l a r and e x t r a c e l l u l a r l i p i d content o f a r t i c u l a r c a r t i l a g e w i t h age and e x t r a c e l l u l a r l i p i d s are prominent i n the s u r f a c e l a y e r s o f c a r t i l a g e . These l i p i d s are comprised o f t r i g l y c e r i d e s , c h o l e s t e r o l or c h o l e s t e r o l e s t e r s , p h o s p h o l i p i d s and g l y c o l i p i d s . Normal s y n o v i a l f l u i d c o n t a i n s s m a l l amounts o f p h o s p h o l i p i d s -172-and c h o l e s t e r o l and s y n o v i a l f l u i d from p a t i e n t s with rheumatoid a r t h r i t i s and o s t e o a r t h r i t i s show i n c r e a s e d amounts o f phospho-l i p i d s , c h o l e s t e r o l and n e u t r a l l i p i d s . The p h o s p h o l i p i d comp-o s i t i o n o f normal s y n o v i a l f l u i d i s s i m i l a r t o t h a t o f plasma with p h o s p h a t i d y l c h o l i n e b e i n g the major c o n s t i t u e n t . I t i s p o s s i b l e t h a t the r a i s e d l e v e l s o f p h o s p h o l i p i d s i n aged or d i s e a s e d c a r t i l a g e and s y n o v i a l f l u i d c o u l d a c c e l e r a t e the growth o f MSUM c r y s t a l s , r e s u l t i n g i n MSUM d e p o s i t i o n i n these t i s s u e s . Albumin and HA caused s i g n i f i c a n t decreases i n the MSUM growth r a t e constant, probably due to a d s o r p t i o n o f these molecules onto the MSUM c r y s t a l s u r f a c e w i t h subsequent p o i s o n i n g o f the a c t i v e growth s i t e s on the c r y s t a l s u r f a c e . Albumin l e v e l s have been found t o be r a i s e d i n the s y n o v i a l f l u i d s o f gouty p a t i e n t s . Thus i n c r e a s e d l e v e l s o f the growth i n h i b i t o r albumin c o u l d not account f o r the appearance o f MSUM c r y s t a l s i n gouty s y n o v i a l f l u i d s . S i m i l a r l y , c a r t i l a g e HA l e v e l s i n c r e a s e w i t h advancing age and although HA has been shown t o be a c r y s t a l l i z a t i o n i n h i b i t o r , i t i s u n l i k e l y t h a t HA i s a s i g n i f i c a n t f a c t o r i n the c r y s t a l l i z a t i o n o f MSUM i n a r t i c u l a r c a r t i l a g e . A t c o n c e n t r a t i o n s o f p r o t e o g l y c a n monomer between 0.1-0.2 mg mL 1 and p r o t e o g l y c a n a g g r e g a t e , 0.2-1.0 mg mL 1 , we found no s i g n i f i c a n t e f f e c t o f e i t h e r PG monomer or PG aggregate on the growth k i n e t i c s o f MSUM. Blumenthal et a l . (1979) s t u d i e d the e f f e c t o f bovine n a s a l c a r t i l a g e PG aggregate and PG monomer on the d i r e c t p r e c i p i t a t i o n o f h y d r o x y a p a t i t e from low c o n c e n t r a t i o n -173-c a l c i u m phosphate s o l u t i o n s . They found t h a t with i n c r e a s i n g c o n c e n t r a t i o n , the aggregate i n c r e a s e d the time o f onset ( i n d u c t i o n time) of h y d r o x y a p a t i t e formation compared t o the PG-free c o n t r o l . The i n d u c t i o n time was measured from mixing o f rea g e n t s t o on s e t o f h y d r o x y a p a t i t e f o r m a t i o n . The aggregate was more e f f e c t i v e on a weight b a s i s than the monomer i n d e l a y i n g the formation o f a p a t i t e . Although the onset o f a p a t i t e formation was a f f e c t e d by pr o t e o g l y c a n s , the subsequent growth k i n e t i c s were not a f f e c t e d . The l a t t e r o b s e r v a t i o n i s i n agreement wi t h our f i n d i n g s t h a t p r o t e o g l y c a n s d i d not s i g n i f i c a n t l y a l t e r the growth k i n e t i c s o f MSUM. The PG c o n c e n t r a t i o n s used by Blumenthal et a l . (1979) were i n the range o f 0.1-1.5 mg mL 1 . Since the c o n c e n t r a t i o n o f p r o t e o g l y c a n s i n c a r t i l a g e was found by Maroudas (1975) t o be i n the range o f 15-60 mg mL - 1, Blumenthal e t a l . , (1979) p r e d i c t e d t h a t such h i g h PG c o n c e n t r a t i o n s would have a potent i n h i b i t o r y e f f e c t on a p a t i t e d e p o s i t i o n . I t i s p o s s i b l e t h a t these h i g h PG c o n c e n t r a t i o n s i n c a r t i l a g e , i n a d d i t i o n t o i n h i b i t i n g the onset o f a p a t i t e formation c o u l d a l s o a f f e c t the growth k i n e t i c s o f MSUM. Stu d i e s o f the growth k i n e t i c s o f MSUM employing h i g h PG c o n c e n t r a t i o n s are needed t o con f i r m t h i s . I t i s e v i d e n t t h a t the e f f e c t s of the d i f f e r e n t c a r t i l a g e and s y n o v i a l f l u i d components on the seeded growth o f MSUM from s u p e r s a t u r a t e d s o l u t i o n s are very complex. Our r e s u l t s would suggest t h a t a l t e r a t i o n s i n the c o n c e n t r a t i o n o f some components -174-as a r e s u l t o f ageing, p r e e x i s t i n g d i s e a s e or a l t e r e d matrix metabolism c o u l d p l a y s i g n i f i c a n t r o l e s i n the growth o f MSUM c r y s t a l s i n j o i n t s . However, c r y s t a l d e p o s i t i o n may be a p r o c e s s i n v o l v i n g the i n t e r p l a y o f s e v e r a l f a c t o r s . For ins t a n c e , a l t e r a t i o n s i n more than one c a r t i l a g e or s y n o v i a l f l u i d component may be necessary b e f o r e MSUM growth proceeds. F a c t o r s such as l o c a l temperature and pH, c o n c e n t r a t i o n s o f ions such as magnesium, cal c i u m , sodium, pyrophosphate e t c . , water content o f c a r t i l a g e and o t h e r c a r t i l a g e components not s t u d i e d t o date such as g l y c o p r o t e i n s and ke r a t a n s u l f a t e may a l s o i n f l u e n c e the d e p o s i t i o n and growth o f MSUM c r y s t a l s . 4.4.7 CHARACTERIZATION OF MSUM AFTER GROWTH EXPERIMENTS MSUM c r y s t a l s a f t e r growth experiments both i n the presence and absence o f a d d i t i v e s were c h a r a c t e r i z e d u s i n g scanning e l e c t r o n microscopy and X-ray d i f f r a c t i o n . Scanning e l e c t r o n micrographs o f MSUM c r y s t a l s a f t e r growth i n the presence o f a d d i t i v e s are shown i n F i g u r e s 37 t o 41. The f i g u r e s show t h a t the needle shaped c r y s t a l h a b i t o f MSUM i s r e t a i n e d on completion o f the growth experiments. The d-values f o r a l l MSUM samples a f t e r growth e i t h e r i n the presence or absence o f a d d i t i v e s were i d e n t i c a l t o d-values obtained f o r the o r i g i n a l MSUM seed c r y s t a l s , c o n f i r m i n g t h a t t h e r e was no a l t e r a t i o n i n the c r y s t a l l i n e s t r u c t u r e o f MSUM a f t e r growth. -175-Scanning electron micrograph of MSUM crystals after growth. I n i t i a l supersaturation concentration 5 g i T i -176-Figure 38. Scanning electron micrograph of MSUM crystals after growth. Initial supersaturation concentration, 6 g L ~ l . -177-Figure 39. Scanning electron micrograph of MSUM crystals after growth i n the presence of albumin (50mg). I n i t i a l supersaturation concentration, 5 g L ~ i . -178-Figure 40. Scanning electron micrograph of MSUM crystals after growth i n the presence of chondroitin sulfate (20 mg). I n i t i a l supersaturation concentration, 5 g IT*-. -179-Figure 41. Scanning electron micrograph of MSUM crystals after growth i n the presence of proteoglycan monomer (10 mg). I n i t i a l supersaturation concentration, 5 g L--'-. -180-5 SUMMARY AND CONCLUSIONS (A) The deg r a d a t i o n o f MSUM i n aqueous s o l u t i o n s was s t u d i e d under n o n - s t e r i l e and s t e r i l e c o n d i t i o n s . S t e r i l e s o l u t i o n s were produced by ( i ) f i l t r a t i o n through 0.22 um M i l l i p o r e f i l t e r s i n t o rubber stoppered V a c u t a i n e r s ( i i ) a u t o c l a v i n g i n a l l - g l a s s c o n t a i n e r s and ( i i i ) a u t o c l a v i n g i n V a c u t a i n e r s . The s o l u t i o n s were incubated a t d i f f e r e n t temperatures. The f o l l o w i n g r e s u l t s were o b t a i n e d : 1. N o n - s t e r i l e MSUM s o l u t i o n s s t o r e d i n g l a s s f l a s k s w i t h g l a s s stoppers a t 4° were r e l a t i v e l y s t a b l e , whereas a t 22°, 35°, 45° and 65°, th e r e was a gradual decrease i n the c o n c e n t r a t i o n of MSUM. As the temperature o f i n c u b a t i o n was in c r e a s e d , d e g r a d a t i o n o f MSUM occured more r a p i d l y with the ex c e p t i o n o f s o l u t i o n s s t o r e d a t 45°, which were more s t a b l e than s o l u t i o n s s t o r e d a t 3 5°. 2. MSUM s o l u t i o n s s t e r i l i z e d by f i l t r a t i o n through 0.22 um M i l l i p o r e f i l t e r s and s t o r e d i n Vac u t a i n e r s showed s i m i l a r r e s u l t s t o the n o n - s t e r i l e s o l u t i o n s . However, repeated d e t e r m i n a t i o n s showed t h a t these s o l u t i o n s demonstrated marked v a r i a b i l i t y i n urate c o n c e n t r a t i o n s a t each time i n t e r v a l a t -181 22°, 35° and 65°. 3. MSUM s o l u t i o n s s t e r i l i z e d by a u t o c l a v i n g showed a decrease i n ur a t e c o n c e n t r a t i o n immediately a f t e r a u t o c l a v i n g . The decrease i n MSUM c o n c e n t r a t i o n was g r e a t e r f o r s o l u t i o n s s t o r e d i n Va c u t a i n e r s than f o r s o l u t i o n s s t o r e d i n a l l - g l a s s c o n t a i n e r s . T h i s i n d i c a t e d t h a t t h e r e was some u r a t e l o s s i n the presence o f rubber stoppers, probably due t o a b s o r p t i o n i n t o the rubber s t o p p e r s . 4. A u t o c l a v e d MSUM s o l u t i o n s i n a l l - g l a s s c o n t a i n e r s s t o r e d a t 22°, 35° and 45° were r e l a t i v e l y s t a b l e upto 96 hours a f t e r an i n i t i a l decrease i n urate c o n c e n t r a t i o n d u r i n g a u t o c l a v i n g , whereas s o l u t i o n s s t o r e d a t 65° showed a gr a d u a l decrease i n urat e c o n c e n t r a t i o n with time. In V a c u t a i n e r s , the auto c l a v e d MSUM s o l u t i o n s were r e l a t i v e l y s t a b l e a t 22° and 45° upto 96 hours a f t e r an i n i t i a l l o s s i n urat e c o n c e n t r a t i o n d u r i n g a u t o c l a v i n g , whereas s o l u t i o n s s t o r e d a t 35° and 65° showed a grad u a l decrease i n urat e c o n c e n t r a t i o n from 0 t o 96 hours. 5. N o n - s t e r i l e and s t e r i l e MSUM s o l u t i o n s underwent decomposition w i t h time. In n o n - s t e r i l e s o l u t i o n s , MSUM c o n c e n t r a t i o n may have decreased by both b a c t e r i a l consumption and chemical degradation, whereas i n s t e r i l e s o l u t i o n s the decrease i n MSUM c o n c e n t r a t i o n was probably due only t o chemical d e g r a d a t i o n . I t was e v i d e n t from these experiments t h a t the c r y s t a l -182-growth o f MSUM co u l d be s t u d i e d upto 8 hours under n o n - s t e r i l e c o n d i t i o n s a t 37° without a p p r e c i a b l e d e g r a d a t i o n o f MSUM. (B) The s a t u r a t i o n s o l u b i l i t y o f MSUM i n water was s t u d i e d a t d i f f e r e n t temperatures and i n the presence o f d i f f e r i n g c o n c e n t r a t i o n s o f sodium c h l o r i d e , c h o n d r o i t i n s u l f a t e , p r o t e o g l y c a n s , h y a l u r o n i c a c i d and albumin. The f o l l o w i n g i s a summary of the r e s u l t s : 1. The heat o f s o l u t i o n determined from a van't Hoff p l o t over a temperature range o f 4 . 4 ° to 55° was 5 . 8 K c a l . -1 mole 2. The s a t u r a t i o n s o l u b i l i t y o f MSUM decreased s i g n i f i c a n -t l y i n the presence o f sodium c h l o r i d e . 3 . C h o n d r o i t i n s u l f a t e decreased the s o l u b i l i t y o f MSUM, probably due t o a common i o n e f f e c t from the sodium prese n t i n the c h o n d r o i t i n s u l f a t e . 4 . H y a l u r o n i c a c i d , p r o t e o g l y c a n monomer, p r o t e o g l y c a n aggregate and albumin caused v e r y s l i g h t i n c r e a s e s i n the s a t u r a t i o n s o l u b i l i t y o f MSUM a t 3 7 ° . (C) The c r y s t a l growth k i n e t i c s o f MSUM was s t u d i e d by the seeded growth technique. The e f f e c t s o f s u p e r s a t u r a t i o n c o n c e n t r a t i o n , seed amount and the a d d i t i v e s , c h o n d r o i t i n s u l f a t e , h y a l u r o n i c a c i d , p r o t e o g l y c a n s (monomer and aggregate), albumin and p h o s p h o l i p i d s ( p h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l s e r i n e ) on the growth 183 k i n e t i c s of MSUM were s t u d i e d . A b r i e f summary o f the r e s u l t s i s g i v e n below: 1. Three methods were used to determine the growth r a t e constant, K' (K'= K Q(obs) S ; K Q(obs) = observed o v e r a l l growth r a t e constant, S = s u r f a c e area o f the added seed c r y s t a l s ) . An equation o f the e m p i r i c a l form: R = K' ( C - C s ) n was used f o r t h i s purpose. L e a s t square f i t s were performed on l o g R versus l o g (C-Cs). The r a t e constant, K', was obtained from the i n t e r c e p t of the f i t . In most o f the experiments the l i n e a r l e a s t square f i t s gave poor c o r r e l a t i o n c o e f f i c i e n t s and r e s u l t e d i n r a t e constant v a l u e s which were i n poor agreement f o r a g i v e n seed amount. The v a l u e s o f n, the order o f r e a c t i o n , obtained from the slope o f the curves v a r i e d between 0.4 to 7.2. However, when the c o r r e l a t i o n c o e f f i c i e n t was good (>0.95), n was between 2 t o 3. A number o f 1:1 and 2:2 e l e c t r o l y t e s f o l l o w the second order (n=2) c r y s t a l growth e q u a t i o n . The i n t e g r a t e d form of the growth equation w i t h n = 2 was rearranged to o b t a i n an equation which d e f i n e d the n o n - l i n e a r r e l a t i o n s h i p between the c o n c e n t r a t i o n and time (equation 32). A n o n - l i n e a r computer program (Metzler e_t a l . , 1974) was used to estimate the growth r a t e constant, K'. Good c o r r e l a t i o n c o e f f i c i e n t s were obtained by t h i s method. Since , the o r d e r of the growth process was assumed to be second, p l o t s of the i n t e g r a t e d form of the growth equation were c o n s t r u c t e d and the growth r a t e c o n s t a n t s , K", were o b t a i n e d from -184-t h e s l o p e s o f these s t r a i g h t l i n e p l o t s . E x c e l l e n t c o r r e l a t i o n c o e f f i c i e n t s were obt a i n e d . The growth r a t e constant, K Q ( o b s ) , was determined from K'/S. The growth r a t e c o n s t a n t s , K Q ( o b s ) , estimated from the n o n - l i n e a r computer program showed wide v a r i a t i o n s i n the values f o r a s e t o f experiments a t a g i v e n i n i t i a l s u p e r s a t u r a t i o n c o n c e n t r a t i o n and seed amount. However, K (obs), estimated o from the l i n e a r p l o t s o f the i n t e g r a t e d form o f the growth equation gave s i m i l a r v a l u e s w i t h i n a giv e n s e t o f experiments. The method employing the l i n e a r p l o t o f the i n t e g r a t e d form o f the growth equation t o determine K Q(obs) was used i n a l l experiments i n v o l v i n g the e f f e c t o f a d d i t i v e s on MSUM growth k i n e t i c s . 2. The growth o f MSUM seed c r y s t a l s r e q u i r e d a.high degree of s u p e r s a t u r a t i o n and a c r i t i c a l q u a n t i t y o f seed c r y s t a l s . No c r y s t a l growth was observed a t a s u p e r s a t u r a t i o n c o n c e n t r a t i o n o f 4 g L 1 . In the 1 L c a p a c i t y apparatus when the s u p e r s a t u r a -t i o n c o n c e n t r a t i o n was 5 g L 1 , no growth was observed i n the presence o f 100 mg seed c r y s t a l s . In the presence o f 200 mg o f seeds t h e r e was a slow l i n e a r growth f o r about 3 hours f o l l o w e d by r a p i d n o n - l i n e a r growth. S i m i l a r l y when the s u p e r s a t u r a t i o n c o n c e n t r a t i o n was 6 g L 1 , a slow growth p e r i o d f o r about 2 hours f o l l o w e d by r a p i d n o n - l i n e a r growth was observed when 200 mg seeds were p r e s e n t . S i m i l a r r e s u l t s were ob t a i n e d using the 50 mL c a p a c i t y -185-apparatus. A p e r i o d o f slow growth ( i n d u c t i o n p e r i o d ) was observed i n the presence of 5 mg t o 20 mg seeds when the s u p e r s a t u r a t i o n c o n c e n t r a t i o n was 5 g L - 1 and i n the presence o f 5 mg to 10 mg seeds when the s u p e r s a t u r a t i o n c o n c e n t r a t i o n was 6 g L 1 . An i n c r e a s e i n the added seed amount a t a g i v e n degree o f s u p e r s a t u r a t i o n decreased the l e n g t h o f the i n d u c t i o n p e r i o d u n t i l i t was a b o l i s h e d a t a c e r t a i n " c r i t i c a l " added seed amount. In the presence o f seed c r y s t a l s from the same batch, an i n c r e a s e i n s u p e r s a t u r a t i o n c o n c e n t r a t i o n from 5 g L 1 t o 6 g L 1 i n t h e 50 mL c a p a c i t y a p p a r a t u s i n c r e a s e d K Q ( o b s ) by 3 t o 4 f o l d . 3. The two d i f f e r e n t batches of seed c r y s t a l s used i n the c r y s t a l growth experiments showed d i f f e r e n t growth r a t e c o n s t a n t s . The growth r a t e c o n s t a n t s , K Q ( o b s ) , from batch A seeds were h i g h e r than the growth r a t e constants from batch B seed c r y s t a l s . These d i f f e r e n c e s i n K Q(obs) valu e s were thought t o be due to the d i f f e r e n c e s i n the type and number o f d e f e c t s p r e s e n t i n the two batches of seed c r y s t a l s . 4. In the presence o f sodium and potassium ions (1.6 mg i n 50 mL and 1.3 mg i n 50 mL r e s p e c t i v e l y ) there was no s i g n i f i c a n t change i n the growth r a t e c o n s t a n t v a l u e s . In the presence o f 5 t o 20 mg of h y a l u r o n i c a c i d the growth r a t e constant decreased a t both 5 g L 1 and 6 g L 1 -185-s u p e r s a t u r a t i o n c o n c e n t r a t i o n s . However, the decrease from the c o n t r o l was s i g n i f i c a n t o n l y a t 6 g L 1 s u p e r s a t u r a t i o n c o n c e n t r a t i o n . C h o n d r o i t i n s u l f a t e (10-30 mg) s i g n i f i c a n t l y i n c r e a s e d the r a t e constant o f MSUM growth. A small i n c r e a s e i n the growth r a t e constant was observed i n the presence of 5 mg to 10 mg of p r o t e o g l y c a n monomer. T h i s i n c r e a s e i n the growth r a t e constant was not s i g n i f i c a n t a t the 5% l e v e l o f s i g n i f i c a n c e . P r o t e o g l y c a n aggregate (5 mg t o 20 mg) showed no s i g n i f i c a n t change i n MSUM growth r a t e c o n s t a n t . In the presence o f the p h o s p h o l i p i d s , p h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l s e r i n e , an i n c r e a s e i n the growth r a t e constant was observed. There was a s i g n i f i c a n t i n c r e a s e i n the growth r a t e caused by p h o s p h a t i d y l c h o l i n e , whereas f o r p h o s p h a t i d y l s e r i n e the i n c r e a s e was not s i g n i f i c a n t . Albumin (10 mg t o 200 mg) caused i n h i b i t i o n o f MSUM growth. At lower c o n c e n t r a t i o n s (10 mg and 50 mg), a decrease i n the growth r a t e was observed from the c o n c e n t r a t i o n - t i m e p l o t . Complete i n h i b i t i o n o f MSUM c r y s t a l growth was seen i n the presence o f lOOmg and 200 mg of albumin. In these s t u d i e s both i n c r e a s e d and decreased growth r a t e s of MSUM were observed when the macromolecules: h y a l u r o n i c a c i d , c h o n d r o i t i n s u l f a t e , p r o t e o g l y c a n s (monomer and aggregate) and albumin were i n c l u d e d i n the growth medium. 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(1978) Gout, i n "Metabolic B a s i s o f I n h e r i t e d Diseases " 3rd.Ed., Stanbury, J.B., Wyngaarden, J.B. and F r e d r i c k s o n , D.S.,(eds.), McGraw H i l l , N.Y. pp 916-1010. Yano, K., Rhoads, G.G. and Kagan, A. (1977) Epidemiology o f serum u r i c a c i d among 8000 japanese-american men i n h a w a i i . J.Chronic D i s . , 30: 171-84. Yu, T.F., S i r o t a , J.H., Berger, L., Halpern, M. and Gutman, A.B. (1957) E f f e c t o f sodium l a c t a t e i n f u s i o n on ur a t e c l e a r a n c e i n man. Proc.Soc.Exptl.Biol.Med., 96: 809-13. Zbinden, V.G. (1952) E l e c t r o n m i c r o s c o p i c study o f the u l t r a -s t r u c t u r e and age r e l a t e d changes of h y a l i n e c a r t i l a g e and c o n t r i b u t i o n t o the understanding o f l i p i d accumulation i n the ground substance o f c a r t i l a g e . Schweiz Z. A l l e r g . P a t h o l . , 16: 165-89. Z i f f , M., G r i b e t z , H.J. and L o s p a l l u t o , J . (1960) E f f e c t o f l e u c o c y t e s and s y n o v i a l membrane e x t r a c t s on c a r t i l a g e mucoprotein. J . C l i n . I n v e s t . , 39: 400-12. Zuckner, J . , Uddin, J . , Gantner, G.E.Jr. and Dorner, R.W. (1963) C h o l e s t e r o l c r y s t a l s i n s y n o v i a l f l u i d . Ann.Intern.Med., 60: 436-46. -210-APPENDIX EXTRACTION OF PROTEOGLYCANS FROM BOVINE NASAL CARTILAGE The cesium c h l o r i d e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n method of Muir and Hardingham (1975) was used to i s o l a t e p r o t e o g l y c a n s from the c a r t i l a g e . The bovine n a s a l c a r t i l a g e was cut i n t o s m a l l and t h i n s l i c e s . Ten volumes of 4 M guanidinium h y d r o c h l o r i d e e x t r a c t i o n b u f f e r (see page 211) were added to each gram of the c a r t i l a g e s l i c e s . The mixture was r o l l e d f o r 24 hours i n the c o l d room and the e x t r a c t was separated from the r e s i d u e by f i l t r a t i o n through g l a s s wool. F i v e volumes o f the 4 M guanidinium h y d r o c h l o r i d e e x t r a c t i o n b u f f e r was then added to the r e s i d u e and the mixture was r o l l e d f o r 3 hours a t 4°. The e x t r a c t was separated from the r e s i d u e by f i l t r a t i o n through g l a s s wool. The two e x t r a c t s were pooled and were kept f r o z e n t i l l f u r t h e r treatment. The e x t r a c t was p l a c e d i n a d i a l y s i n g tube and d i a l y s e d a g a i n s t a l a r g e volume of 0.5 M guanidinium h y d r o c h l o r i d e e x t r a c t i o n b u f f e r . The mixture was d i a l y s e d o v e r n i g h t . The e x t r a c t was p l a c e d i n a weighed measuring c y l i n d e r and the d e n s i t y o f the mixture was a d j u s t e d to 1.69 g mL 1 by the a d d i t i o n o f cesium c h l o r i d e . The mixture was mixed g e n t l y t o a v o i d foaming, p l a c e d i n the u l t r a c e n t r i f u g e tubes and u l t r a c e n t r i f u g e d f o r 48 hours a t 100,000g a t 20°. - 2 1 1 -Th e c e n t r i f u g e tubes were then cut i n t o two p o r t i o n s : (1) bottom 1/4 p o r t i o n = A l (2) top 3/4 p o r t i o n = A2-4 To h a l f o f the A l f r a c t i o n was added equal volumes of 7.5 M guanidinium h y d r o c h l o r i d e b u f f e r and the d e n s i t y o f t h i s mixture was a d j u s t e d to 1.69 g mL 1 . The mixture was c e n t r i f u g e d a t 100,000 g and 20° f o r 48 hours. The tubes were cut i n t o t h r e e p o r t i o n s : (1) bottom 1/4 p o r t i o n = A1D1 (2) middle 2/4 p o r t i o n = A1D2-3 (3) top 1/4 p o r t i o n = A1D4 The remaining h a l f o f the A l f r a c t i o n and the A1D1 f r a c t i o n were d i a l y s e d a g a i n s t three changes o f 0.2 M sodium c h l o r i d e and the products l y o p h i l i z e d . The A l f r a c t i o n was p r o t e o g l y c a n aggregate and A1D1 f r a c t i o n was p r o t e o g l y c a n monomer. E x t r a c t i o n b u f f e r : 4.0 M Guanidinium h y d r o c h l o r i d e 0.05 M Sodium a c e t a t e t r i h y d r a t e 0.01 M Tetrasodium EDTA 0.10 M Ethylamino c a p r o l i c a c i d . 0.005 M Benzamidine h y d r o c h l o r i d e 0.001 M Phenylmethyl s u l f o n y l f l o u r i d e 0.004 M n - e t h y l maleimide 

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