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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 D i p l o m a i n Pharmacy, P a n j a b U n i v e r s i t y , 1969 B. Pharm., P a n j a b U n i v e r s i t y , 1973 . P h a r m . ( P h a r m a c e u t i c s ) , P a n j a b 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 in THE FACULTY OF GRADUATE (Faculty  of Pharmaceutical Sciences)  Division  We a c c e p t t h i s to  STUDIES  o f Pharmaceutics  thesis  the required  as c o n f o r m i n g standard  THE UNIVERSITY OF B R I T I S H COLUMBIA  ©  Y o u g e s h C h a n d e r D u t t , 1985  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by the head o f representatives.  my  It is  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department o f The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T  DE-6  (3/81)  1Y3  written  ABSTRACT  Hyperuricemia the  extremities  development yet  is  fibrillar thought  of  this  synovial  that  t o occur these  study  albumin  The and  with  tissues  monomer,  to crystal  tissues of life  deposition  i n  preexisting  fluid  the  and (3) connective  disease.  I t  o f t h e nonwhich a r e  or preexisting deposition.  (2)  disease,  may  The o b j e c t i v e s  t h e e f f e c t o f t h e c a r t i l a g e and  chondroitin  proteoglycan  sulfate, hyaluronic  aggregate,  acid,  phospholipids  and  o f MSUM.  o f MSUM  conditions  and t o d e f i n e The r a t e  an i n c r e a s e  with  trauma  t o determine  degradation  experiments.  o f monosodium  years  o f c a r t i l a g e and s y n o v i a l  on t h e growth  degradation  o f MSUM c r y s t a l  components,  non-sterile  i n the later  ageing,  for the  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  and i n j o i n t s  with  i n the joints of  i npart,  the alterations i n composition  were  fluid  proteoglycan  o f gout  trauma  matrix  predispose  t o be r e s p o n s i b l e ,  arthritis.  incidence  after  possible  changes  (MSUM) c r y s t a l s i n c o n n e c t i v e  incidence  increased  tissues  temperature  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  monohydrate  increased the  a r e known  o f gouty  available  urate  and l o c a l  solutions  was s t u d i e d  t o determine t h e time  span  of degradation  i n temperature.  under  thepossible of crystal  o f MSUM  causes o f  growth  solutions  The c o n c e n t r a t i o n  sterile  increased  o f MSUM i n  solution  fell  sharply after  containers with MSUM t h a n  rubber  autoclaved  Rubber s t o p p e r s degradation bacterial  a u t o c l a v i n g and s o l u t i o n s  stored i n  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  solutions  stored i n all-glass  a p p a r e n t l y absorbed  MSUM f r o m  o f MSUM s o l u t i o n s was t h o u g h t  consumption and c h e m i c a l  solution.  The  t o b e due t o b o t h  decomposition  s o l u t i o n s b u t was due o n l y t o c h e m i c a l  containers.  i n non-sterile  decomposition  in sterile  solutions.  The  aqueous s o l u b i l i t y  temperatures  and i n t h e p r e s e n c e  sodium c h l o r i d e . The presence  suppressed  MSUM p r o b a b l y Proteoglycan  (CS)  resulted  solubility.  decreased  i n the  components a t 3 7 ° .  the saturation s o l u b i l i t y o f  due t o t h e s o d i u m p r e s e n t aggregate,  MSUM  o f MSUM was a l s o d e t e r m i n e d  o f several connective tissue sulfate  at different  o f varying concentrations o f  Sodium c h l o r i d e  aqueous s o l u b i l i t y  Chondroitin  albumin  o f MSUM was d e t e r m i n e d  i n t h e CS s a m p l e s .  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  i n very  slight  increases i n the s o l u b i l i t y of  MSUM.  The  growth k i n e t i c s  growth t e c h n i q u e .  An e q u a t i o n  R  was or  used  g  = K (obs) o  t o determine  rate constant,  o f MSUM was s t u d i e d u s i n g t h e s e e d e d o f thegeneral  S (C-Cs)  the overall  n  growth  K' (K'= K ( o b s ) S ) .  form:  rate constant,  K (obs Q  Linear  plots of the integrated  growth e q u a t i o n reasonably initial  constant  f i t between t h e p o i n t s  f o r K (obs) determined  or a period  supersaturation  order  a n d gave  a t a given  Q  concentration  induction period  both the i n i t i a l  length  values  supersaturation  An at  gave t h e b e s t  form o f t h e second  and v a r y i n g  seed  amounts.  o f s l o w g r o w t h was o b s e r v e d concentrations  studied.  The  o f t h e 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 t h e  added s e e d  amount.  Differing  concentrations  o f a d d i t i v e s were i n c l u d e d  i n the  g r o w t h medium a n d K' d e t e r m i n e d . Chondroitin rate  constant  decreases  found an  f o r MSUM g r o w t h .  proportion  i n the synovial  inhibition  crystals the  fluid  of arthritic  levels  surface.  are increased  proportions of crystal  o f growth deposition  a n d may a c t a s  medium.  (HA) a n d a l b u m i n c a u s e d  significant This  e f f e c t may b e  o f t h e s e m o l e c u l e s o n t o t h e MSUM s e e d  r e s u l t i n g i n thepoisoning  crystal  CS h a s b e e n  joints  o f t h e g r o w t h o f MSUM c r y s t a l s .  t o the adsorption  o f CS  o f a g r o w t h 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  MSUM g r o w t h a c c e l e r a t o r i n t h i s acid  t h e growth  c a r t i l a g e and t h u s a  i n t h e d e p o s i t i o n o f MSUM i n c a r t i l a g e .  Hyaluronic  due  increased  However, t h e p r o p o r t i o n  i n aged a n d o s t e o a r t h r i t i c  decreasing factor  s u l f a t e (CS) s i g n i f i c a n t l y  o f t h e a c t i v e g r o w t h s i t e s on  C a r t i l a g e HA and s y n o v i a l i n aged a n d / o r d i s e a s e d inhibitors i n joint  joints.  do n o t o f f e r  tissues.  fluid  likely  albumin Increased explanations  -V-  At (PGM)  concentrations  and proteoglycan  MSUM g r o w t h  rate  o f 0 . 1 - 1 . 0 mg mL aggregate  constant  proteoglycan  (PGA) s l i g h t l y  but this  increase  monomer  increased the  was  statistically  insignificant.  The  two p h o s p h o l i p i d s ,  tidylserine  increased  Phosphatidylserine, growth  rate  constant  possible  that  diseased  cartilage  of  MSUM  t h e growth  however,  rate  constant  levels  and s y n o v i a l  resulting  and phosphao f MSUM.  d i d not significantly  a t the concentrations  the raised  crystals  phosphatidylcholine  studied.  o f phospholipids fluid  could  i n MSUM d e p o s i t i o n  increase the I t  i s  i n aged o r  accelerate i n these  t h e growth tissues.  -vi -  TABLE  OF  CONTENTS  Page  ABSTRACT  i i  L I S T OF TABLES  xiv  L I S T OF FIGURES  xix  SYMBOLS AND ABBREVIATIONS  xxv  ACKNOWLEDGEMENTS  xxx  1  INTRODUCTION  1  2  LITERATURE SURVEY  4  2.1.  Crystal  deposition diseases  4  2.1.1.  Gout  5  2.1.2.  Pseudogout  9  2.1.3.  H y d r o x y a p a t i t e (HAP) deposition  2.1.4.  disease  Miscellaneous  9 9  2.2.  Crystal-induced inflammation  10  2.3.  Hyperuricemia  13  and e p i d e m i o l o g y  -vi i -  Page 2.4.  2.5.  Causes o f h y p e r u r i c e m i a 2.4.1.  Inborn  errors  2.4.2.  Impaired  2.4.3.  D r u g s and a l c o h o l  o f metabolism  16  excretion  17 18  Deposition of crystals 2.5.1.  Origin  19  of crystals  relationship 2.6.  16  and t h e i r  to joint  disease  19  Cartilage 2.6.1.  20  Composition  of cartilage  24  (A)  Water  24  (B)  Collagen  24  (C)  Proteoglycan (I)  aggregate  Proteoglycan  (PGA)  25  subunit  (Monomer)(PGM)  (II) (D) 2.6.2.  2.7.  25  (Ia)  Chondroitin sulfate  (lb)  Keratan  Hyaluronic  Lipids  sulfate  acid  (HA)  Factors affecting  the composition  30 30 32  of cartilage  33 of  cartilage  33  (A)  E f f e c t o f age  33  (B)  Effect of injury  36  (C)  Effect of joint  disease  Synovial f l u i d 2.7.1.  (KS)  (CS)  Composition  36 38  of synovial fluid  39  -viii-  Page (A)  Soluble  constituents  derived  from t h e b l o o d (B)  Constituents joint (I)  39  Hyaluronic  acid  Products  derived  of joint  Lipids of synovial fluid  2.7.3.  Factors  (A)  39  affecting  tissues  41 41  the composition  of  fluid  Effect  Theory o f c r y s t a l  41  from t h e  2.7.2.  synovial  (HA)  Lubricating glycoproteins  catabolism  2.8.  s e c r e t e d by t h e  tissues  (II) (C)  39  41  of joint  disease  41  growth  42  2.8.1.  Supersaturation  43  2.8.2.  Nucleation  46  (A)  Homogeneous n u c l e a t i o n  46  (B)  H e t e r o g e n e o u s and s e c o n d a r y nucleation  2.8.3.  C r y s t a l growth  54  (A)  Surface  54  (B)  Adsorption (I)  (C) 2.8.4.  51  layer theories  Dislocations  affecting  Effect  crystal  55 57  Diffusion theories  Factors (A)  energy t h e o r i e s  62 growth r a t e s  o f seed c r y s t a l  s i z e and  65  -ix-  Page surface  2.8.5.  2.9.  2.10. 3  area  65  (B)  Effect  o f degree o f s u p e r s a t u r a t i o n  67  (C)  Effect  o f temperature  68  (D)  Effect  o f degree o f a g i t a t i o n  69  (E)  Effect  of impurities  69  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  MSUM s o l u t i o n s  72  2.9.1.  D e g r a d a t i o n o f MSUM s o l u t i o n s  73  2.9.2.  Solubility  75  Nucleation  o f MSUM  and c r y s t a l  g r o w t h o f MSUM  EXPERIMENTAL  77 81  3.1.  Instruments  81  3.2.  Materials  82  3.3.  Methods  84  3.3.1.  Preparation  o f MSUM  84  3.3.2.  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  85  (A)  Ultra-violet  85  (B)  Infra-red  (C)  X-ray d i f f r a c t i o n  (D)  Differential  (E)  Scanning e l e c t r o n microscopy  (F)  Determination o f surface  spectroscopy  spectroscopy  scanning  MSUM s e e d c r y s t a l s  85 85 calorimetery  86 86  area o f 86  -X-  Page  3.3.3.  A n a l y s i s o f monosodium u r a t e  3.3.4.  D e g r a d a t i o n o f monosodium  3.3.5.  88  urate  monohydrate s o l u t i o n s  88  (A)  Non-sterile  88  (B)  Sterile  solutions  solutions  89  Determination of saturation o f monosodium u r a t e  solubility  monohydrate  90  (A)  Effect  o f temperature  90  (B)  Effect  of electrolytes  91  (C)  Effect  of chondroitin sulfate,  hyaluronic  acid,  proteoglycan  monomer, p r o t e o g l y c a n and 3.3.6.  monohydrate  aggregate  albumin  91  C r y s t a l g r o w t h o f monosodium  urate  monohydrate (A)  92  Effect  of  supersaturation  concentration, batch  crystal  and s e e d amount o n t h e  crystal  g r o w t h k i n e t i c s o f mono-  sodium u r a t e (B)  seed  Effect  monohydrate  o f a d d i t i v e s on t h e c r y s t a l  g r o w t h o f monosodium  urate  monohydrate (I)  94  Chondroitin and  albumin  95 sulfate 95  -xi -  (II)  Hyaluronic and  acid  proteoglycan  (III) Proteoglycan (IV)  monomer  aggregate  P h o s p h a t i d y l c h o l i n e and phosphatidylserine  3.3.7.  Determination  o f sodium  potassium content acid,  of hyaluronic  c h o n d r o i t i n s u l f a t e and  proteoglycan 3.3.8.  and/or  Effect  samples  o f sodium and p o t a s s i u m  i o n s o n g r o w t h 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 after  crystal  crystals  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.  A s s a y o f monosodium u r a t e  monohydrate  in solution 3.  D e g r a d a t i o n o f monosodium  urate  monohydrate i n s o l u t i o n 4.  Saturation  solubility  o f MSUM  4.4.1.  Effect  o f temperature  4.4.2.  Effect  o f sodium c h l o r i d e  4.4.3.  Effect  of chondroitin  hyaluronic  acid,  sulfate,  proteoglycans  -xi i -  Page and a l b u m i n 4.5.  121  C r y s t a l g r o w t h o f MSUM 4.5.1.  Determination  of supersaturation  concentration  and s e e d amount f o r  crystal 4.5.2.  rate constant  Effect crystal  4.5.4.  Effect crystal  4.5.5.  growth  124  S e l e c t i o n o f t h e method t o d e t e r m i n e the  4.5.3.  124  Effect  of crystal  o f seed c r y s t a l s  growth  on t h e  growth  147  o f s u p e r s a t u r a t i o n on t h e growth r a t e c o n s t a n t  152  o f s o d i u m and p o t a s s i u m  ions  on t h e g r o w t h r a t e c o n s t a n t s 4.5.6.  152  Effect  o f a d d i t i v e s on t h e c r y s t a l  growth  rate constant  (A)  Effect  156  of hyaluronic  on t h e g r o w t h r a t e (B)  Effect  Effect  Effect  Effect  monomer  growth r a t e  constant  160  aggregate  growth r a t e  of phophatidylcholine  phosphatidylserine  158  constant  of proteoglycan  on t h e c r y s t a l (E)  156  constant  of proteoglycan  on t h e c r y s t a l (D)  acid  of chondroitin s u l f a t e  on t h e g r o w t h r a t e (C)  128  constant and  on t h e c r y s t a l  160  -xiiiPage growth r a t e (F)  Effect  c o n s t a n t o f MSUM  163  o f a l b u m i n o n MSUM c r y s t a l  growth 4.4.7.  Characterization  166 o f MSUM c r y s t a l s  growth experiments  5  after 174  SUMMARY AND CONCLUSIONS  180  i 6  REFERENCES  APPENDIX  188 214  -xiv-  LIST OF TABLES  Table  1  2  Page  S u g g e s t e d mechanisms  related to  i n - v i v o MSUM c r y s t a l  formation.  The e s t i m a t e d content  3  increases  of cartilage  w e i g h t and c o n c e n t r a t i o n proteins 4  5  i n the l i p i d  per year  The r e l a t i o n s h i p between  o f age.  monosodium  urate  Saturation  solubility  sodium u r a t e  o f serum 40  pattern of  monohydrate.  105  ( C s ) o f mono-  monohydrate  at different  temperatures. 6  116  R e l a t i o n s h i p b e t w e e n t e m p e r a t u r e and concentration physiologic saturation  o f sodium c h l o r i d e o r  i o n concentration solubility  on t h e  o f monosodium  u r a t e monohydrate. 7  37  molecular  i n synovial fluid.  Powder X - r a y d i f f r a c t i o n  21  Saturation  solubility  120 o f monosodium  -XV-  urate  monohydrate  additives  i n thepresence o f  a t 37°.  MSUM g r o w t h r a t e c o n s t a n t s , obtained of  from t h e l i n e a r  l o g R versus  growth  C = 5 gL  -  1  regression  l o g (C-Cs)  i n 1 L capacity  of  ).  from t h e l i n e a r  l o g R versus  growth  -  1  of  ).  from t h e l i n e a r  log R versus  -  1  of  -  1  K',  regression  l o g (C-Cs)  i n 50 mL c a p a c i t y  C = 6 gL  apparatus;  constants,  from t h e l i n e a r  l o g R versus  growth  (crystal  ).  MSUM g r o w t h r a t e obtained  K',  regression  l o g (C-Cs)  g r o w t h i n 50 mL c a p a c i t y C = 5 g L  (crystal  apparatus,  MSUM g r o w t h r a t e c o n s t a n t s , obtained  K',  regression  l o g (C-Cs)  i n 1 L capacity  C = 6 gL  (crystal  apparatus;  MSUM g r o w t h r a t e c o n s t a n t s , obtained  K*,  (crystal  apparatus;  ) .  MSUM g r o w t h r a t e c o n s t a n t s ,  K',  -xvi -  Table  Page  obtained program  from t h e n o n - l i n e a r (crystal  growth i n 1 L  capacity apparatus; 13  program  from t h e n o n - l i n e a r (crystal  program  C = 6 g L  from t h e n o n - l i n e a r (crystal  capacity  growth  apparatus,  ).  137  program  computer  i n 50 mL  C = 5 g L "''). -  MSUM g r o w t h r a t e c o n s t a n t s , obtained  (crystal  growth  138  K , 1  from t h e n o n - l i n e a r  c a p a c i t y apparatus,  computer  i n 50 mL  C = 6 g L "*"). -  139  MSUM g r o w t h r a t e c o n s t a n t s , K', obtained  from p l o t s  of theintegrated  form o f t h e second o r d e r (crystal C = 5 g L 17  computer  MSUM g r o w t h r a t e c o n s t a n t s , K', obtained  16  136  growth i n 1 L  c a p a c i t y apparatus,  15  C = 5 g L ^).  MSUM g r o w t h r a t e c o n s t a n t s , K', obtained  14  computer  growth -  1  growth  i n 1 L capacity  equation apparatus,  ) .  143  MSUM g r o w t h r a t e c o n s t a n t s , K', obtained  from p l o t s  of theintegrated  form o f t h e second o r d e r growth  equation  -xvii-  Table  Page (crystal  growth  C = 6 g L 18  -  growth  (crystal  capacity  growth  i n 50 mL  C = 5 g L  -  1  equation  ).  145  MSUM g r o w t h r a t e c o n s t a n t ,  K',  from p l o t s o f t h e i n t e g r a t e d  form o f t h e second o r d e r  growth  (crystal  capacity  growth  apparatus,  i n 50 mL  C = 6 g L  O v e r a l l growth r a t e K  o  (obs),  capacity  O v e r a l l growth r a t e K  o  (obs),  capacity  O v e r a l l growth K  o  (obs),  rate  in1 L ).  148  rate growth  in1 L  C = 6 g L ^). constants,  c a l c u l a t e d from  constants,  growth  constants,  K', ( c r y s t a l  apparatus,  rate  C = 5 g L  c a l c u l a t e d from  constants,  146  constants,  K', ( c r y s t a l  apparatus,  equation  ).  c a l c u l a t e d from  constants,  22  K',  form o f t h e second order  obtained  21  144  from p l o t s o f t h e i n t e g r a t e d  apparatus,  20  apparatus,  ) .  MSUM g r o w t h r a t e c o n s t a n t s , obtained  19  1  i n 1 L capacity  K", ( c r y s t a l  rate growth  i n 50 mL  149  -xvi i i -  capacity apparatus,  C = 5 gL  Overall  constants,  growth r a t e  K (obs), Q  calculated  constants,  from  -  1  ) .  rate  K' , ( c r y s t a l g r o w t h  capacity apparatus, C = 6 g L  i n 50 mL ) .  Sodium  and p o t a s s i u m c o n t e n t o f  Effect  o f sodium and p o t a s s i u m  on MSUM g r o w t h r a t e Effect  Effect  acid  on t h e  o f MSUM.  of chondroitin  on t h e g r o w t h k i n e t i c s Effect  ions  constant.  of hyaluronic  growth k i n e t i c s  sulfate o f MSUM.  o f p r o t e o g l y c a n monomer  on t h e g r o w t h k i n e t i c s  o f MSUM.  Effect  aggregate  of proteoglycan  on t h e g r o w t h k i n e t i c s Effect  o f MSUM.  of phosphatidylcholine  on t h e g r o w t h k i n e t i c s Effect  additives  o f MSUM.  of phosphatidylserine  on t h e g r o w t h k i n e t i c s  o f MSUM.  -xix-  L I S T OF  FIGURES  Figure  1  Page  Chemical urate  formulae of  monohydrate,  (A)  (B)  phosphate d i h y d r a t e ,  Monosodium  Calcium  and  (C)  pyro-  Hydroxy  apatite.  2  7  A view o f the  crystal  MSUM v i e w e d down t h e 3  Possible induced  steps  structure  needle a x i s .  involved  in  12  Pathways o f p u r i n e  5  P o s s i b l e pathways i n v o l v e d  6  joint  Possible  7  Structure  metabolism  and  i n man.  joint  15  crystal  diseases.  22 crystal  diseases.  23  of 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  and  collagen,  (b)  aggregate  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 collagen.  in  r e l a t i o n s h i p s between  deposition  8  crystal-  inflammation.  4  related  of  aggregate  and 26  -XX-  Figure  Page  8  Structure of proteoglycan  9  Structures monomer,  10  11  The  12  Free  of:  acid.  29  solubility-supersolubility  critical Effect  the  (i:-4-;  (b) K e r a t a n s u l f a t e .  energy diagram  diagram.  existence  of  50  supersaturation  on  the 52  C r y s t a l growth w i t h o u t 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  location;  completed  (b)  layer;  (c) s u r f a c e n u c l e a t i o n . 15  44  a  nucleation rate. 14  31  for nucleation  nucleus.  of  27  proteoglycan  (a) C h o n d r o i t i n  sulfate,  explaining  13  (a)  (b) h y a l u r o n i c  Structures ii:-6-)  of:  aggregate.  56  Kossel's  model o f a g r o w i n g  surface:  (A)  (C) k i n k s ;  (D)  growth u n i t s ; and  (F)  flat  surface;  crystal (B)  steps;  surface-adsorbed (E)  edge  vacencies;  surface vacencies.  58  - x x "U  Figure  16  17  Page  Dislocations (a)  an edge d i s l o c a t i o n , a n d  (b)  screw d i s l o c a t i o n .  Development starting  18  i ncrystal:  59  o f a growth s p i r a l  from a screw d i s l o c a t i o n .  Saturation  61  s o l u b i l i t y o f MSUM i n  normal s a l i n e .  76  19  Crystal  93  20  Ultra-violet  growth a p p a r a t u s . s p e c t r u m o f monosodium  u r a t e monohydrate 21  Infra-red  solution.  s p e c t r u m o f monosodium  u r a t e monohydrate. 22  102  X-ray d i f f r a c t i o n p a t t e r n o f monosodium u r a t e m o n o h y d r a t e .  23  101  104  Scanning e l e c t r o n micrograph o f monosodium u r a t e m o n o h y d r a t e .  106  24 DSC  25  s c a n o f monosodium  urate  monohydrate. A standard  curve  107 f o r MSUM s o l u t i o n .  108  -xxi i -  Figure  26  Page  D e g r a d a t i o n o f n o n - s t e r i l e MSUM solutions.  27  Degradation o f s t e r i l e filter)  28  110 (0.22  solutions i nVacutainers.  Degradation o f s t e r i l e  Degradation o f s t e r i l e  containers.  Effect  112  (autoclaving)  MSUM s o l u t i o n s i n V a c u t a i n e r s . 30  I l l  (autoclaving)  MSUM s o l u t i o n s i n a l l - g l a s s 29  pm  113  o f t e m p e r a t u r e on t h e  saturation  solubility  of:  ( a ) MSUM;  (b) MSUM i n t h e p r e s e n c e o f physiological 31  A van't  Hoff  ion concentration. plot  f o r MSUM  solubility  i n water. 32  118  Seeded g r o w t h c u r v e s in  f o r MSUM a t 3 7 °  1 L c a p a c i t y a p p a r a t u s a n d an  initial  supersaturation  of 5 g L  - 1  concentration  .  Seeded g r o w t h c u r v e s in  117  125 f o r MSUM a t 3 7 °  1 L c a p a c i t y a p p a r a t u s and an  initial  supersaturation  concentration  -xxi i i Figure  Page of 6 g L  34  35  36  -  1  .  Second-order  126 kinetic  plots of the  integrated  form o f growth e q u a t i o n  for  seeded  g r o w t h o f MSUM a t 3 7 ° i n  the  50 mL c a p a c i t y a p p a r a t u s  initial  supersaturation  of  - 1  5 g L  concentration  .  141  Second-order  kinetic  plots of the  integrated  form o f growth e q u a t i o n  for  seeded  g r o w t h o f MSUM a t 3 7 ° i n  the  50 mL c a p a c i t y a p p a r a t u s a n d a n  initial  supersaturation  of 6 g L  - 1  concentration  .  142  Seeded g r o w t h k i n e t i c s in  o f MSUM a t 3 7 °  t h e presence o f albumin  c a p a c i t y apparatus  e l e c t r o n micrograph  crystals  after  growth.  5 g L  Scanning  e l e c t r o n micrograph  crystals  after  167  super.  175  o f MSUM  Initial  saturation concentration,  .  o f MSUM  Initial  concentration,  growth.  super-  of 5 g L  Scanning  saturation 38  i n t h e 50 mL  a n d an i n i t i a l  saturation concentration 37  and an  super-  5 g L ^.  176  -xxfv-  Figure  39  40  Page  Scanning  e l e c t r o n micrograph  crystals  a f t e r growth i n t h e presence  of albumin  (50 mg).  saturation  concentration,  super-  5 g L ^.  177  Scanning  e l e c t r o n micrograph  crystals  a f t e r growth i n t h e p r e s e n c e  of chondroitin supersaturation 41  Initial  o f MSUM  o f MSUM  s u l f a t e (20 mg). concentration,  Initial  5 g L ^.  Scanning  e l e c t r o n micrograph  crystals  a f t e r growth i n t h e p r e s e n c e  o f p r o t e o g l y c a n monomer supersaturation  178  o f MSUM  (10 mg).  concentration,  Initial  5 g L ^.  179  -XXV-  SYMBOLS AND  ABBREVIATIONS  s'  Degree o f  C  Concentration  of s o l u t e i n the  at  temperature  Cs  some g i v e n  Equilibrium saturation of  Ci  supersaturation  s o l u t e i n the  Solute  solvent  concentration  solvent at a given  concentration  at the  temperature  crystal-solution  interface W  T o t a l q u a n t i t y o f t h e work r e q u i r e d form a s t a b l e c r y s t a l  W  g  The  work r e q u i r e d  of the The  a Ap  The  s u r f a c e of the  surface  crystal form the  bulk  crystal  surface  particle  per  Surface  area  The  nucleus form the  work r e q u i r e d t o  of the £  to  to  energy of the  pressure  p h a s e and  unit  spherical  area  o f the  particle  d i f f e r e n c e between the  the  interior  v  Volume o f t h e  particle  r  Radius o f the  droplet  pr  Vapour p r e s s u r e  of the  over a l i q u i d  liquid  vapour droplet  droplet of radius  r  -xxvi-  p*  Vapour p r e s s u r e over a f l a t  M  Molecular weight o f the substance  f  Density o f the droplet  T  Absolute temperature  R  Gas c o n s t a n t  G  Overall  excess free  energy energy  G  g  Surface excess free  G  v  Volume  G  Overall  crit  where r  excess free  N  excess free  e n e r g y , G, a t maximum ^ J  r = r size  nucleus  R a t e o f n u c l e a t i o n o r t h e number o f n u c l e i formed p e r u n i t  A  time  Constant o f nucleation  AG  Overall of  AG  c r  ^  AG'  .. crit  e n e r g y change f o r t h e f o r m a t i o n  f r e e e n e r g y change a s s o c i a t e d  Overall  free  e n e r g y change a s s o c i a t e d ^ ^ J  T  ^ of  cs  C  interfacial  t o the i n t e r f a c i a l  energy  energy between t h e s u r f a c e  t h e s e e d and t h e l i q u i d  The i n t e r f a c i a l of  £J ^  e  with  nucleation  A constant related s  with  nucleation  heterogeneous  Si  rate  a partical  Overall  t  free  homogeneous  £f  surface  energy  c ,radius of a c r i t i c a l  c  liquid  e n e r g y between t h e s u r f a c e s  the c r y s t a l l i z i n g  The i n t e r f a c i a l  p h a s e and t h e s e e d  energy between t h e  surface  -xxvii-  crystallizing  p h a s e and t h e  liquid  a^  Area of the i t h face of the  crystal  g^  Surface  area  the b  Q  free  energy per u n i t  i t h face  Burgers vector  Rg  Rate o f c r y s t a l  A  Temperature dependent the  B  growth  growth  area  D  Coefficient  h  Thickness  g  K K  Surface  constant  of  solid  of d i f f u s i o n  of the l i q u i d  film  from t h e b u l k  rate  of solute  o f s o l u t e a t t h e s u r f a c e by  Rate o f i n t e g r a t i o n Transport  S  o f the  Rate o f a r r i v a l diffusion  of  rate  Surface  t  constant  rate  S  R  growth  Temperature dependent the  R  solution  into  the s o l i d  integration  rate  growth  constant  Observed o v e r a l l  K'  Observed growth  rate constant,  K'(add)  Observed growth  rate constant  rate  constant (K'= K  the presence of  additive  K'(control)  Observed growth  rate constant  additive Order o f growth r e a c t i o n  e S^  Effective  surface area  Q  S )  f o r MSUM g r o w t h  in  n  surface  constant  K (obs) Q  of  . a t time t  i n the absence o f  -xxviii-  K  Growth r a t e c o n s t a n t  T  f(o )  Term d e p e n d e n t on  c^  T o t a l mass o f s o l i d  S^/S  Geometric t o t a l  t  C  ,C  Concentrations  +  at Cs  ,Cs  +  the  d e p e n d e n t on  temperature  the s u p e r s a t u r a t i o n a t time  t  s u r f a c e area a t time of anions  and  t  cations  supersaturation concentration  Concentrations at the  of anions  and  cations  saturation concentration  P  Partial  pressure  of  Po  Saturated pressure  adsorbate of  adsorbate 23  Nu  A v o g a d r o ' s number  Vc  Volume o f c a l i b r a t i o n  Pa  Ambient p r e s s u r e  A  S i g n a l area or d e s o r p t i o n count  Acs  Cross  sectional  square "\ d  (6.023 x 10 gas  i n atmosphere  Wavelength o f Spacing  in  X-rays  between t h e  I s an  0  Angle o f the  crystallographic  integer i n equation  Heat o f  incident  solution  PGA  Proteoglycan  PGM  Proteoglycan  HA  molecules  gas  meters  n*  PGs  of c a l i b r a t i o n  area of adsorbate  crystal  AH  )  aggregate monomer  Proteoglycans Hyaluronic  acid  27  X-rays  planes  of  a  -xxix-  CS  Chondroitin  sulfate  PC  Phosphatidylcholine  PS  Phosphatidylserine  MSUM  Monosodium u r a t e  CPPD  Calcium pyrophosphate  HAP  Hydroxy a p a t i t e , A p a t i t e , C a l c i u m h y d r o x y a p a t i t e  monohydrate dihydrate  -XXX-  ACKNOWLEDGEMENTS  I w i s h t o t h a n k Dr. encouragement d u r i n g I am  grateful  questions  and  the  t o Dr.  suggestions  ment t h r o u g h o u t  H.M.  Burt  course A.G. and  f o r her  of t h i s Mitchell  t o Dr.  s u p e r v i s i o n and  study. for h i s valuable  J . M c N e i l l f o r encourage-  this project.  S i n c e r e thanks t o : Dr. and  Mark Adams, D e p a r t m e n t o f M e d i c i n e ,  f o r p r o v i d i n g the Mr.  R.  Mr. computer  f o r the  X-ray  analysis.  M.  electron  suggestions  proteoglycans.  B u t t e r s , Department o f M e t a l l u r g y ,  diffraction Ms.  for his  Mager, D e p a r t m e n t o f M e t a l l u r g y ,  f o r the  scanning  microscopy. R.  Burton,  Faculty of Pharmaceutical  sciences, for  analysis.  Several  friends  (who  must r e m a i n anonymous) f o r  their  friendship. Financial Medical  support  from the U n i v e r s i t y o f B r i t i s h  R e s e a r c h C o u n c i l o f Canada and  gratefully  acknowledged.  Columbia,  S t a n l e y Drug P r o d u c t s  is  -1-  1  The  n u c l e a t i o n and g r o w t h o f c r y s t a l s  solutions example,  i s an i m p o r t a n t  process  from  supersaturated  in biological  systems, f o r  i n the deposition o f calcium hydroxyapatite  b o n e s and t e e t h . calcium  INTRODUCTION  O t h e r compounds s u c h as c a l c i u m  pyrophosphates,  as p a t h o l o g i c a l d e p o s i t s  uric  acid,  sodium u r a t e  i n t h e body.  A crystal  crystals i n  oxalates, e t c . may be  found  deposition  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 with  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 associated with  monohydrate connective the  (MSUM) c r y s t a l s  t h e a p p e a r a n c e o f monosodium u r a t e i n the synovial f l u i d  t i s s u e s of the j o i n t .  Hyperuricemia  development o f gouty a r t h r i t i s .  blood  may r e s u l t  excretion  levels  solubility  and a c u t e  urate  Since  levels i n renal t h e mean  i n h e a l t h y m a l e s a r e n e a r l y t h e same as t h e of urate  c h a n g e s may be a f a c t o r sites  i n other  i s required f o r  d i s o r d e r s , impaired  and i n g e s t i o n o f d r u g s and a l c o h o l .  plasma u r a t e saturation  from: m e t a b o l i c  Increased  and/or  i n plasma,  i n the formation  crystallization  local  temperature  of tophi i n peripheral  i n joints.  - 2-  Hyperuriceraia insufficient  to explain  crystallization, crystals  and temperature  gouty a r t h r i t i s  the observed patterns  such as,  i n connective  v a r i a t i o n s alone,  however, a r e  o f MSUM  (1) t h e s e l e c t i v e d e p o s i t i o n  tissues,  i n the later  involvement o f connective  (2) t h e i n c r e a s e d  years o f l i f e  o f MSUM  incidence o f  and (3)  the  t i s s u e s e x p o s e d t o trauma i n t h i s  disease. T h e r e a r e a number o f b i o c h e m i c a l which occur joints  i n thea 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  as a r e s u l t o f f a c t o r s such as ageing,  preexisting  disease.  predispose  affecting  fluid  fluid of  trauma and  c a u s e d b y a g e i n g and d i s e a s e  these t i s s u e s t o c r y s t a l  Although there  changes  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 m a t r i x and s y n o v i a l may  and m e t a b o l i c  deposition.  h a v e b e e n some s t u d i e s  the s o l u b i l i t y  and n u c l e a t i o n  f a c t o r s w h i c h may i n f l u e n c e  the c r y s t a l  o f the factors  rates  o f MSUM, t h e  g r o w t h o f MSUM a r e p o o r l y  understood.  The  objectives  o f thepresent  1.  To i n v e s t i g a t e t h e c r y s t a l  2.  To d e t e r m i n e t h e e f f e c t o f s u p e r s a t u r a t i o n  amount on t h e MSUM c r y s t a l 3.  study  were:  g r o w t h k i n e t i c s o f MSUM.  and s e e d  growth k i n e t i c s .  To d e t e r m i n e t h e 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  « components on t h e MSUM g r o w t h k i n e t i c s .  fluid  -3-  T h e r e a r e a number o f methods f o r s t u d y i n g t h e k i n e t i c s crystal  growth, but  the  seeded growth t e c h n i q u e  be h i g h l y r e p r o d u c i b l e and Supersaturated characterised temperature  sodium u r a t e seed  and  crystals  agitation.  was  employed  has  of  b e e n shown t o  i n these s t u d i e s .  s o l u t i o n s were s e e d e d w i t h o f MSUM u n d e r c o n d i t i o n s o f  wellconstant  -4-  2.  2.1  LITERATURE REVIEW  CRYSTAL DEPOSITION  DISEASES  Gout as a d i s e a s e h a s b e e n known f o r s e v e r a l The  ancient  a c c o u n t s o f g o u t were summarized b y F r a n c i s Adams i n  1844 i n h i s c o m m e n t a r i e s of  i n the t r a n s l a t i o n  Paulus A e g i n e t a " (Hartung, Hippocrates  accuracy ates  referred  the  spring  1981).  specifically  this  In h i s aphorisms to the hereditary  t h e menopause, t h e immunity  the  of cold  in  t o the f i r s t  Nubia,  Egypt,  urate crystals  The  first  remarkable  on g o u t ,  Hippocr-  p r e d i s p o s i t i o n , the of the disease i n  applications  o f e u n u c h s t o g o u t and  (Hartung,  1957).  century of the c h r i s t i a n  S i m i t h and J o n e s  (Hartung,  era.  Gout h a s b e e n In a  cemetery  (1910) f o u n d a s k e l e t o n o f an  e l d e r l y m a l e whose g r e a t t o e and o t h e r j o i n t s of  books  the r a r i t y b e f o r e puberty, the appearance i n  women a f t e r  traced  disease with  o f acute episodes, the worsening and f a l l ,  benefit  o f "The s e v e n  1957).  (400 BC) d e s c r i b e d  (Pritchard,  periodicity  centuries.  showed l a r g e  tophi  1957).  microscopic description  of the c r y s t a l s  derived  f r o m a g o u t y t o p h u s was g i v e n b y A n t o n i v o n Leewenhoek i n e a r l y 1700  (McCarty,  1970),  b u t i t was o n l y  i n 1797, when W o l l a s t o n  -5-  demonstrated "lithiated uric  that  soda"  the extrusions (Hartung, 1957).  "Lithic  acid"  a s t h e c a u s e o f t h e i n f l a m m a t o r y r e s p o n s e were  a d v o c a t e d by S i r A l f r e d He  stated  to  crystals  reported  that  Baring  G a r r o d i n 1859  acute gouty a r t h r i t i s  o f sodium  urate.  crystalline  deposits  i n and a r o u n d  e x a m i n a t i o n s as e a r l y  inflammation.  j o i n t s was  1921; S a n d s t o r m ,  tophaceous I t was  initiated "crystal  gout  was  not u n t i l  i n this  1938).  described  the e a r l y  field  were c o i n e d  after  crystals  as 1907 b y P a i n t e r .  Hollander, to  1961).  1960's t h a t  (McCarty  T h i s a r t h r o p a t h y was similarities  2.1.1  called  was  active  et al.(1947). research The  was  terms  and " c r y s t a l - i n d u c e d i n f l a m m a t i o n "  light  urate  i n the synovial  microscopy  T h i s method o f s y n o v i a l  i n joints  This  t o these deposits  by B r a n d e n b e r g e r  the d i s c o v e r y o f calcium pyrophosphate  crystals  Evidence  The n a t u r e o f t h e p r e c i p i t a t e s  o f sodium  were o b s e r v e d u s i n g p o l a r i z e d  (1900)  noted i n  by H o l l a n d e r and M c C a r t y .  deposition diseases"  1981).  reaction  F r e u d w e i l e r (1901) and H i s s  f o l l o w e d by r e p o r t s o f i n f l a m m a t i o n r e l a t e d (Schmitt,  (Pritchard,  i s an i n f l a m m a t o r y  e x p e r i m e n t s on c r y s t a l - i n d u c e d  radiological  in  i s now known a s  acid.  Crystals,  of  f r o m g o u t y t o p h i were o f  "pseudogout"  ( M c C a r t y and  fluid  examination l e d  dihydrate  e t a l . , 1962;  fluid  Kohn  because  (CPPD) e t a l . , 1962).  ofi t s  t o gout.  GOUT This  disease  i s c a u s e d by t h e d e p o s i t i o n  o f monosodium  urate  -6-  monohydrate The of and  (MSUM) c r y s t a l s  i n cartilage or i n the joint  c h e m i c a l 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 . MSUM h a v e b e e n e x t r a c t e d examined b y p o l a r i z e d  Hollander,  1961).  These  from gouty t o p h i  light  microscopy  crystals  belong  to thet r i c l i n i c  1963) a n d h a v e an a c i c u l a r  shaped  crystal  The c r y s t a l s  0.5-2 um i n w i d t h  structure and  Rinaudo  crystal  (Dieppe and C a l v e r t ,  and B o i s t e l l e  consists  (1982).  ions which  saline  s o l u t i o n h a s been  (Dieppe and C a l v e r t ,  arearticular  cartilage,  1983).  In acute gout,  et  often within  Weinberger  al.,  1982).  form  o f MSUM p o s s e s s  1981a; B u r t e t a l . , o f these  crystals  i n physio-  f o u n d t o b e 1 um s ^ V ^ cm  periarticular  on t h e e a r and i n tendon  1961;  sheets  molecules  only i n connective tissues.  bursae,  crystals  t h e MSUM  1983).  MSUM c r y s t a l l i z e s sites  mobility  (1976)  (Figure 2 ) .  (Dieppe e t a l . ,  The e l e c t r o p h o r e t i c  logical  The w a t e r  s t u d i e s h a v e shown t h a t t h e c r y s t a l s  a n e t n e g a t i v e charge 1983).  by Mandel and Mandel  bond t o f o u r n e i g h b o u r i n g  bonds w i t h t h e p u r i n e r i n g s  Recent  i n length  The c r y s t a l  They h a v e shown t h a t  u r a t e a n i o n s v i a t h e o x y g e n atoms. hydrogen  1983).  o f urate anions stacked i n p a r a l l e l  i n t e r s p e r s e d w i t h sodium  crystal  o r needle  a r e b e t w e e n 2-20 p  o f MSUM h a s b e e n d e t e r m i n e d  fluid  (McCarty and  (Howell e t a l . ,  and  Crystals  and s y n o v i a l  system  habit.  fluid.  sheaths  the synovial  fluid  1979; A g u d e l o  In chronic  tissues,  ( D i e p p e and C a l v e r t , c o n t a i n s numerous MSUM  t h e l e u c o c y t e s (McCarty  et al•,  soft  The m a j o r  and H o l l a n d e r ,  et al.,  1979; G o r d o n  g o u t a l a r g e number o f MSUM  -7-  l  C  5  H  3°3  4  N  N  *  a  H  2  0  3  (A)  C  a  2 4°7 P  *  2 H  2°  (B)  Ca  1 0  (PO ) (OH) 4  6  2  (C)  Figure 1.  Chemical formulae o f (A) Monosodium u r a t e monohydrate, IB) Calcium pyrophosphate d i h y d r a t e , and (C) Hydroxy a p a t i t e .  -8-  F i g u r e 2.  A view o f the c r y s t a l s t r u c t u r e o f MSUM viewed down the n e e d l e a x i s  - 9-  crystals 2.1.2  are located  i n a tophus  ( D i e p p e and  Calvert,  1983).  PSEUDOGOUT  T h i s d i s e a s e i s c a u s e d by t h e d e p o s i t i o n o f c a l c i u m p y r o phosphate  d i h y d r a t e (CPPD) c r y s t a l s .  CPPD i s shown i n F i g u r e CPPD, one  lb.  the m o n o c l i n i c c r y s t a l  in  the  joints  Jacobelli  1973;  HYDROXY A P A T I T E  hydroxy  articular  connective  al.,  Pritzker 1976).  The  1975),  1976)  common s i t e s  and  s p i n e ( D i e p p e and D o h e r t y ,  The  crystals  um  in size.  o f HAP The  composed o f many HAP  2.1.4  and  the other  1966;  CPPD  um.  and  scanning  1966;  synovial  1982;  electron  cartilage McCarty  fluid  Andres  and  ( D i e p p e and  spherical  Calvert,  1983).  MISCELLANEOUS D i c a l c i u m phosphate  dihydrate (Utsinger,  other  (Dieppe e t  and T r a i n e r ,  small  and  et a l . ,  a r e u s u a l l y between  form  Calcium  i n degenerated  i n tendons,  generally  crystals  lc.  are the shoulder j o i n t , h i p  are hexagonal  crystals  of  are deposited  et a l . ,  i s shown i n F i g u r e  ( M c C a r t y and G a t t e r ,  and L u c ,  and  forms  1981a,b).  forms  DEPOSITION DISEASE  e l e c t r o n probe  ( A l i and W i s b y , tissues  McCarty  o f between 0.1-10  (HAP)  system  these  has been i d e n t i f i e d  c a r t i l a g e by  microscopy  1966;  (HAP)  Both  1962;  c h e m i c a l f o r m u l a o f HAP apatite  polymorphic  crystal  Ellman et a l . ,  c r y s t a l s have dimensions  The  system.  (McCarty e t a l . ,  et a l . ,  chemical formula f o r  T h e r e a r e two  b e l o n g i n g to the t r i c l i n i c  to  2.1.3  The  1977;  Faure  joint 1980). 0.1-2 clusters  -10-  e t a l . , 1977), c o r t i c o s t e r o i d s cholesterol identified crystals  2.2  crystals i n joint  ( Z u c k n e r e t a l • , 1963) h a v e a l s o tissues.  are responsible  CRYSTAL-INDUCED The  physical  (Kahn e t a l . , 1970) and  Uric  f o r renal  c r y s t a l s o f MSUM and CPPD, when i n j e c t e d  inflammatory  The important sized  o f the i n d i v i d u a l  factor  into  i n acute  a rat's  workers r e p o r t e d crystals  1971).  reactive.  that  factor  crystals.  has  been i n v e s t i g a t e d  key  factor  into  human  i s known t o b e an F o r example,  different  c a u s e a v a r y i n g d e g r e e o f edema a f t e r paw very  ( D i e p p e and C a l v e r t , small  whereas c r y s t a l s  the  o f HAP p r o d u c e  intradermally  inflammation.  crystals  basis  by Mandel  i s the surface  These  (<0.1 pm) and  large  o f a b o u t 5 pm i n l e n g t h a r e most  i n determining  The s t r u c t u r a l  1983).  being  i n e f f e c t i v e i n producing  The n a t u r e o f t h e c r y s t a l  most i m p o r t a n t  to the natural  Crystals  crystals  (>20 urn) a r e r e l a t i v e l y  inflammation,  into  1977).  urate c r y s t a l s  injected  response s i m i l a r  r e s p o n s e when i n j e c t e d  (Dieppe,  size  fluid  osteoarthritis.  ( B o y l e and S e e g m i l l e r ,  volunteers  i n the joint  s e e n i n g o u t , p s e u d o g o u t and i n  j o i n t s p r o v o k e an i n f l a m m a t o r y  an  oxalate  stones.  presence o f c r y s t a l s  some c a s e s o f i n f l a m m a t o r y  material  and c a l c i u m  INFLAMMATION  p r o d u c e s an a c u t e a r t h r o p a t h y ,  Synthetic  acid  been  surface  i s t h o u g h t t o be t h e  the inflammatory  effect of  of the inflammatory  (1976).  potential  He c o n c l u d e d t h a t t h e  r o u g h n e s s on an a t o m i c  scale.  Thus  'smooth' c r y s t a l s  like  cysteine are inactive,  many c h a r g e d g r o u p s p r o t r u d i n g  whereas u r a t e ,  from t h e s u r f a c e  with  i s very  phlogistic. The  negative  to be r e s p o n s i b l e crystals.  all  inflammation.  charged.  (1965) p r o p o s e d  crystals  the crystals  charge on t h e c r y s t a l  faces  f o r a p a r t o f the inflammatory  Kellermeyer  charge o f urate induce  surface  could  i s thought  effect  that the negative  initiate  o f causing  They a l s o r e p o r t e d  that  highest  inflammatory p o t e n t i a l ,  surface  charge.  Heating  surface  t h e Hageman f a c t o r a n d  D i e p p e e t a l . (1981a,b) r e p o r t e d  capable  of the  inflammation  were  that negatively  sodium u r a t e , w h i c h h a s t h e  a l s o has thehighest  and g r i n d i n g s i g n i f i c a n t l y  negative reduces t h e  s u r f a c e c h a r g e and c o n s e q u e n t l y  t h e inflammatory p o t e n t i a l  (Dieppe e t a l . , 1981a,b).  e t a l . (1983) s t u d i e d t h e  membranolytic  effect  Burt  o f MSUM c r y s t a l s  and r e p o r t e d  b o t h t h e z e t a p o t e n t i a l and m e m b r a n o l y t i c after heating  Various inflammatory 1983). fluid, studies  pathways i n v o l v e d attack  Following  i n themediation  a r e shown i n F i g u r e  crystals  the release o f crystals  show t h a t MSUM c r y s t a l s  into the synovial surface.  ( K o z i n and McCarty,  In-vitro  adsorb a v a r i e t y o f p r o t e i n s  1976, 1977; H a s s e l b a c h e r ,  a n d Schumacher,  o f an acute  3 (Dieppe and C a l v e r t ,  p r o t e i n becomes bound t o t h e c r y s t a l  Hasselbacher  o f MSUM  and r e h y d r a t i o n .  ( K o z i n and McCarty,  IgG,  effect  a decrease i n  1978,  1978), p a r t i c u l a r l y  1976, 1 9 7 7 ) .  t o change t h e s u r f a c e c h a r a c t e r i s t i c s  1982;  immunoglobulin,  Protein binding of the crystals  i s likely a n d may  -12-  Release of crystal into tissue space  Coating of crystals with protein  Cell-membrane interaction  Phagocytosis Activation of protein/enzyme systems  Release of lysosomal enzymes, activation of chemical mediators of inflammation  Activation of mediators of Increased permeability, chemotaxis etc.  Release of lysosomal ond cytoplasmic enzymes, and mediators of Inflammation, sometimes causes cell rupture  Inflammation  Figure 3.  Possible steps involved i n c r y s t a l - i n d u c e d inflammation  enhance the  i n t e r a c t i o n of the c r y s t a l s with  phagocytosis (Doherty  and  activation of c e l l  e t a l . , 1983;  Giclas  S e v e r a l workers have phagocytic was  cells  proposed  phagocytic reduces  by  McCarty  inflammatory  mechanisms  e t a l . , 1979).  r e p o r t e d the c e n t r a l  (1979).  r o l e p l a y e d by  The  evidence  response  f o r the r o l e  i n experimental  the predominance o f polymorphonuclear  cells  with  i n c r y s t a l - i n d u c e d inflammation  the  A mechanism  i n c l u d e s , (a) d e p l e t i n g t h e p h a g o c y t i c  inflammatory  phagocytosis  membranes,  i n c r y s t a l - i n d u c e d inflammation.  cells  the  free  cell  of  cells  animals  and  (b)  active  ( P h e l p s and  McCarty,  1966).  Several  a u t h o r s h a v e documented t h e e v e n t s  incubation of c r y s t a l s with phagocytic (1975) o b s e r v e d crystals  using cine-microphotography.  Once i n s i d e  the  (phagosome).  cell,  Lysosomes t h e n  both  release of crystals  be 2.3  lysosomal  Polymorphs  HYPERURICEMIA AND Uric  acid  i s the  by  that  crystals. a membrane  f u s e d w i t h t h e phagosome membrane.  rapidly  of  the  phagosomes b u t w i t h  died after  for further  r e l e a s e . Release  the major cause o f  Rajan  demonstrated  surrounded  enzymes were t r a n s f e r e d i n t o effect.  and  He  on  sodium' u r a t e  t o engulf the u r a t e  t h e c r y s t a l was  apparent the  in-vitro•  t h e r e a c t i o n between p o l y m o r p h s and  polymorphs e n g u l f e d or attempted  Lysosomal  cells  t h a t occur  that resulting  inflammatory  lysosomal  enzyme  no in  reaction i s thought  to  inflammation. EPIDEMIOLOGY final  product  of catabolism of the  purines,  -14-  a d e n i n e and detail Nuki,  guanine.  (Boyle 1979).  in  F i g u r e 4.  is  i o n i z e d and  acid  The  purine metabolic  and  Seegmiller,  The  steps  A t p h y s i o l o g i c a l pH  include, Barr  over Munan  (1977); Jones  et  Hall  serum u r a t e  et  Scott  males than urate  than  a l . (1977);  vary with  levels  et  gouty  level  reports Fessel  a l . (1977);  and  Simons  the  geographical  are  generally higher A  sex  area,  observed  where, due  i n normal  d i f f e r e n c e i n the  i n childhood levels  but  Mikelson  appears  et a l . ,  levels  adult  serum  i n c r e a s e more i n  makes f e m a l e s l e s s p r o n e t o g o u t y a t t a c k s t h a n  serum u r a t e  uric  (1982); Glynn et a l .  Lower mean serum u r a t e  t h e menopause age  serum  sex.  i n women ( N i s h i o k a e t a l . , 1974;  reach  acid  the  T h e s e s t u d i e s show t h a t  when serum u r a t e  Munan e t a l . , 1 9 7 7 ) .  in  a l . (1977);  Yano  shown  concentration.  f a c t o r s s u c h as  and  i s u s u a l l y not  adolescence,  et  1978;  uric  plasma or  More r e c e n t  Munan  normal a d u l t females.  levels  during  twenty y e a r s .  e t a l . (1983).  Mean serum u r a t e  the  of the  s t u d i e s have appeared  (1980); A k i z u k i  s t a t u s , age  90%  serum u r a t e  a l . (1976);  levels  social  last  Sturge  (1978);  (1983) and  race,  the  Kelley,  metabolism are  more t h a n  i t i s common t o r e p r e s e n t  c o n c e n t r a t i o n as p l a s m a o r  literature  Wyngaarden and  involved i n purine  Numerous e p i d e m i o l o g i c  and  1971;  p a t h w a y s a r e known i n  men  1965;  generally  males u n t i l  t o unknown f a c t o r s ,  they  t h e mean  i n f e m a l e s r i s e s making them s u s c e p t i b l e t o  arthritis.  N o r m a l serum u r a t e  levels  i n h e a l t h y m a l e s i s between 5.5  mg  R i b o s e - 5 - p h o s p h a t e  •  ATP  PRPPsj  •I  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  (PRPP)  PAT dGTP  r  ;DP dGDP Jl  5 - P h o s p h o r i b o s y l - l - a m i n e  GTP  GDP  /  G l u t a m i n e  N u c l e i c a c i d s \\  / Deoxyn u c l e i c  •  D e o x y n u c l e i c a c i d s  ATP >NL JVDP  G l u c o s e Formate  dATP ^ dADP  r^-~—Glutamine  a c i d s N \ N u c l e i c a c i d s  B i c a r b o n a t e - A s p a r t a t e Formate  t£^~-  APRT G u a n i l i c a- -c- i- d- -  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 a c i d (PRPP)  J  dAMP  XO 5  *NT  5'NT  Guanosine  HGPRT  A  Guanine  8-OH  adenine  XO  NP  Adenosine  k  (PRPP)  NP  ADA I n o s i n e - <  2 , 8 - D i h y d r x y a d e n i n e  NP  I I  NP  ADA  Hypoxanthiner£=5=; d e o x y i n o s i n e XO  Guanase  5'NT  1  Deoxyadenosine  X a n t h i n e nt XO D r i c  a c i d  ADA  =  Adenosine  APRT  -  Adenine  HGPRT  -  H y p o x a n t h i n e - g u a n i n e  NP 5*NT PAT PRPPS XO  = « = = =  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 u c l e o t i d a s e . 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 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 a n t h i n e o x i d a s e .  F i g u r e  4 .  d e a m i n a s e .  p h o s p h o r i b o s y l  Pathways o f p u r i n e ( N u k i , 1979).  t r a n s f e r a s e .  p h o s p h o r i b o s y l  m e t a b o l i s m  i n  n a n  t r a n s f e r a s e .  -16-  dL  t o 7.0  mg  h i g h e r than (Hall  dL  .  normal.  In h y p e r u r i c e m i a Hyperuricemia  e t a l . , 1967).  a t t a c k o f a c u t e gout  The  presence  i s an  and  the u r a t e l e v e l s  are  gout  related  are c l o s e l y  of hyperuricemia  important  diagnostic  However, many i n d i v i d u a l s h a v e h y p e r u r i c e m i a ever having  an a t t a c k o f g o u t y  2.4  OF  CAUSES  2.4.1  of urate. a r e due  a r e known i n p a t i e n t s who However, o n l y a b o u t  1970).  The  i n a l l types of primary  partial  d e f i c i e n c y o f the  ribosyl  t r a n s f e r a s e (HGPRTase), a f a m i l i a l  Lesch  and  recessive t r a i t . e t a l . (1967).  are heterozygotes  The  and  guanine  phospho-  i s inherited  enzyme d e f e c t was  populations of  first as  Another years due  enzyme a c t i v i t y  glycogen  to the d e f i c i e n c y o f the  by  children  fibroblasts,  one  and  (Rosenbloom e t a l . , 1 9 6 7 ) .  c o n d i t i o n w h i c h may  i s the Type-I  an  identified  w i t h a n o r m a l 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 others without  or  neurological disorder  Mothers o f the a f f e c t e d  and h a v e two  Scott,  i s the t o t a l  T h i s d i s o r d e r was  Nyhan i n 1964  primary  (Graham and  enzyme, h y p o x a n t h i n e  associated with hyperuricemia.  Seegmiller  gout  are  A strong family  b e s t known o f t h e s e d e f i c i e n c i e s  d e s c r i b e d by  without  5 percent of a l l  to these d e f i c i e n c i e s .  exists  x-linked  f o r years  arthritis.  history  and  indicator.  METABOLISM  Enzyme d e f i c i e n c i e s  cases o f gout  an  HYPERURICEMIA  I N B O R N ERRORS O F  over producers  during  lead  t o g o u t by  storage disease.  t h e age  of  10  This disease i s  enzyme g l u c o s e - 6 - p h o s p h a t a s e  and  -17-  occurs  i n infancy.  produce  Patients with  free glucose  ketosis. tubular  resulting  this  disease  i n h y p e r l a c t i c a c i d e m i a and  T h i s c o n d i t i o n h a s an i n h i b i t o r y excretion of uric  a r e unable t o  acid  effect  on t h e r e n a l  (Yu e t a l . , 1 9 5 7 ) .  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) h a s b e e n r e p o r t e d  i n conjunction with hyperuricemia i n  negro males  An a u t o s o m a l mode o f i n h e r i t a n c e i s  (Long,  1967).  indicated but the connection  b e t w e e n t h e two a b n o r m a l i t i e s  i s not  understood.  IMPAIRED  2.4.2  EXCRETION  Only t e n percent kidneys to  i s finally  o f the t o t a l  excreted.  urate passing  There i s a general  t h e r e l a t i o n s h i p b e t w e e n g o u t and k i d n e y  review,  Steele  deleterious failed renal  through the disagreement as  function.  (1979) h a s i m p l i e d t h a t h y p e r u r i c e m i a  effect  to detect  on k i d n e y  function.  any h a r m f u l  efficiency.  that hyperuricemia  effect  alone has a  and Yu (1975)  of untreated  However, K l i n e n b e r g may i n d u c e  Berger  In a  hyperuricemia  e t a l . (1975)  on  claimed  r e n a l impairment even i n t h e  a b s e n c e o f symptoms. Moderate h y p e r u r i c e m i a disease  (Gresham e t a l . , 1 9 7 1 ) .  insufficiencies subjects diseases  frequently occurs  a r e found  (Sarre,  only  1964; R i c h e t  However, c h r o n i c  (Emmerson,  renal  renal  i n a small p r o p o r t i o n o f gouty e t a l . , 1965).  a p p e a r t o be a s s o c i a t e d w i t h  lead nephropathy  i n chronic  gout,  A number  f o r example,  1963; Morgan e t a l . , 1 9 6 6 ) ,  of renal chronic  -18-  p o l y c y s t i c kidney disease 1974)  (Newcombe, 1973; M a r t i n e z - M a l d o n a d o ,  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).  A l t h o u g h a c u t e gouty a r t h r i t i s h a s been infrequently  and even q u e s t i o n e d  renal disease,  i ti s believed  reported  as a c o m p l i c a t i o n  that  the major cause o f h y p e r u r i c e m i a  impaired  o f chronic  renal excretion i s  ( C u r r i e , 1979; S o r e n s e n , 1980;  G i b s o n e t a l . , 1980).  2.4.3  DRUGS AND ALCOHOL A number o f d r u g s may i n t e r f e r e w i t h  handling  of urate.  The h y p e r u r i c e m i a  f o u n d t o b e due t o s a l t  and  1969).  Salicylates, reduce urate  phenylbutazone,  The  diuretic  and w a t e r l o s s  sulphinpyrazone  and  the excretion o f urate  (Steele  probenecid  e x c r e t i o n a t low doses, whereas, a t h i g h e r  these drugs i n c r e a s e Kelley,  tubular  associated with  t h e r a p y has been Oppenheimer,  the renal  doses  (Wyngaarden and  1978). ingestion of ethanol  (Wyngaarden a n d K e l l e y , attributed  1976).  This  effect  o f gout  o f a l c o h o l has been  renal clearance  of uric  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  ethanol  metabolism  (Lieber  to decreased  may p r e c i p i t a t e an a t t a c k  and D a v i d s o n ,  1962; L i e b e r  e t a l . , 1962; Beck,  e t a l . , 1957; M a c l a c h l a n and Rodnan, 1 9 6 7 ) . has  a l s o been  (Grunst  etal.,  implicated  i n the increased  1977; D e l b a r r e  etal.,  Alcohol  production  1 9 8 1 ; Yu  intake of uric  1967a,b; F a l l e r and  acid  -19-  Fox,  1982). There  i s a higher prevalence  consume e t h a n o l  regularly  D'Alonzo,  and  1968)  (Saker  2.5  and  Grahame,  DEPOSITION OF  2.5.1  ORIGIN OF  e t a l . , 1967;  populations with  have a h i g h p r o p o r t i o n o f p e o p l e (Gibson  o f g o u t amongst  a higher  who  individuals  Pell  who  and  incidence of  consume e t h a n o l  in  gout  excess  1974).  CRYSTALS  CRYSTALS AND  THEIR RELATIONSHIP TO  JOINT  DISEASE The  r e l a t i o n s h i p between  deposition are present necessary,  joint  i s not w e l l understood. i n the body t o  d i s e a s e s and  A number o f complex  activate  crystal  f o r example i n t h e b o n e s and  and  the  which i n h i b i t abnormal  urinary tract, crystal  develop  r e n a l stones.  local  In  pyrophosphate  sites  cannot be  the b i o l o g i c a l  excretory  s e v e r a l i n s t a n c e s where  d i s e a s e t h e r e may  i s a localized  excess  and  For  be  example,  excess  i n gout, t h e r e  either  may a  i n CPPD  of inorganic i s an  increased  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 e s t a b l i s h e d because of the  system.  i t is  organs,  Patients with hyperuricosuria  joint  joint,  o f serum u r a t e .  various of  i n the  where  inhibit i t  cause a s o l u t e  or a g e n e r a l i z e d s o l u t e excess.  deposition disease there  level  There are  f u n c t i o n i n g o f a s y s t e m may to c r y s t a l l i z a t i o n .  systems  a l l o f w h i c h c o n t a i n compounds  growth.  leading  formation  t e e t h and  where i t i s u n d e s i r a b l e , f o r example i n t h e saliva  crystal  complex  at nature  -20-  Some o f t h e s u g g e s t e d mechanisms r e l a t e d formation are l i s t e d about as  these  factors  except  (Scott,  those  related  1983).  Little  i s known  to supersaturation,  relationships  d i s e a s e have been (1983). related  between c r y s t a l d e p o s i t i o n  studied  by Dieppe  (1982) and D i e p p e and  j o i n t diseases  (Figure  5).  They b e l i e v e  result i n nucleation  activators.  D i e p p e e t a l . (1983) h a v e p r o p o s e d  relationships 6).  between c r y s t a l d e p o s i t i o n These i n c l u d e ,  crystallization, deposits crystal  joint  some  possible  disease  causes  joint disease,  are a by-product o f a process causing deposition  i n crystal  o f some  and j o i n t  (1) j o i n t d i s e a s e  (2) c r y s t a l s c a u s e  that  Calvert  and c r y s t a l g r o w t h b e c a u s e  t h e removal o f t h e i n h i b i t o r s o r i n t r o d u c t i o n  (Figure  and j o i n t  They h a v e p r o p o s e d v a r i o u s pathways i n v o l v e d  damage may a l s o  (3) c r y s t a l  arthritis,  (4)  and j o i n t d i s e a s e a r e i n d e p e n d e n t 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  deposition  together.  2.6  interact  CARTILAGE Joints  ligaments,  contain tendons,  a variety synovial  exception of cartilage, these  of connective tissues membrane and c a r t i l a g e .  little  such as With t h e  i s known a b o u t t h e c o m p o s i t i o n o f  tissues. Almost, a l l v e r t e b r a t e  of  such  temperature.  The  of  i n Table.1.  t o MSUM c r y s t a l  connective  (or s k e l e t a l )  cartilages consists tissue  cells  o f a combination  and e x t r a c e l l u l a r  -21-  Table  1.  1.  S u g g e s t e d mechanisms r e l a t e d t o MSUM i n - v i v o c r y s t a l f o r m a t i o n ( S c o t t , 198  Supersaturation fluid  with  o f serum o r s y n o v i a l  MSUM.  2.  Protein binding  of urate.  3.  Turnover o f proteoglycans.  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  7.  Resorption  8.  A g e i n g and a v a s c u l a r i t y .  concentration.  of extravascular  fluid.  -22-  SOLUTE EXCESS  LOCAL TISSUE CONDITIONS  ACTIVATORS/INHIBITORS  DISSOLUTION^  CRYSTAL FORMATION  INFLAMMATION  .ASYMPTOMATIC DEPOSIT  OTHER MECHANISMS ?  TISSUE DAMAGE  Figure  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 ( D i e p p e and C a l v e r t , 1 9 8 3 ) .  -23-  (1)  X  > JOINT DISEASE  (2)  X  > CRYSTALS  ^^JOINT (3)  X  > CRYSTAL  > JOINT  DISEASE  DISEASE  ~ CRYSTALS  (4)  (5)  Figure  X  > JOINT  Y  > CRYSTAL GROWTH  JOINT DISEASE  6.  DISEASE  <===>  CRYSTAL GROWTH  P o s s i b l e r e l a t i o n s h i p s between 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).  crystal  -24-  substance  (fibrous  Salentijn,  1983).  Articular cells  cartilage  (chondrocytes)  abundant m a t r i x this  matrix  tissue  2.6.1  and  ground  substance)  i s an a v a s c u l a r t i s s u e  are s p a r s e l y d i s t r i b u t e d  (Edwards and  Chrisman,  Articular  consists  mainly  articular  varies  The  water content o f a r t i c u l a r c a r t i l a g e Maroudas,  and  1977;  and  1964  and  Maroudas,  ; Mayne and  and  Sage  comprises  i s about  1 9 7 9 ) . The  65-75%  presence  c o l l a g e n makes  approximately  40-50% o f t h e d r y w e i g h t  f i n e mesh work o f  and  of  localization  of  water  cartilage  i s t h e m a j o r component o f t h e e x t r a c e l l u l a r  a l l cartilages  chains  composition  COLLAGEN  Collagen  a  and  components,  elastic.  (B)  al.,  loads.  The  markedly with depth  c o n j u n c t i o n w i t h p r o t e o g l y c a n s and  weight  joints,  WATER  (Venn and  of  In t h e  1979).  (A)  tough  and  of three  proteoglycan aggregates.  in  in a stiff  1979).  w a t e r , c o l l a g e n and  (Muir,  the  CARTILAGE  cartilage  cartilage  Moss-  i n which  can w i t h s t a n d v e r y h i g h compressive  COMPOSITION OF  (Moss and  Vonder-Mark,  fibrils.  t h e i r molecular  (1980).  The  10%  of the t i s s u e 1983).  wet  (Anderson  et  C o l l a g e n appears  as  nomenclature  o r g a n i z a t i o n was  of the  matrix  f o r the c o l l a g e n  proposed  V a r i o u s t y p e s o f c o l l a g e n s found  by  Bornstein  in different  -25-  t i s s u e s have d i f f e r e n t Proteoglycans (Smith  can  e t a l . , 1967)  chondronectin protein  either  Heinegard,  articular  a protein a cartilage  aggregate  weight  proteoglycan Figure  aggregate  1974).  was  The  Rosenberg  model o f t h e  function of proteoglycan aggregation  c o l l a g e n n e t w o r k and function of  (I)  of  et a l •  (Hardingham e t a l . , 1976).  aggregate  presumably has  the (1975).  proteoglycan-  biological  of the  of  Heinegard,  ultrastructure  acid  size  schematic  i t s regions  hyaluronic  However, t h e  aggregate  immobilizes  The  i s n o t known. them i n t h e  a role peculiar  to  the  cartilage.  PROTEOGLYCAN SUBUNIT  (MONOMER)(PGM)  T h e s e a r e m a c r o m o l e c u l e s w h i c h c o n t a i n one glycosaminoglycan  (GAG)  This  approximately  ( H a s c a l and  r e p o r t e d by  8 shows t h e most a c c e p t e d  of  F i g u r e 7 shows a  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 Muir,  i s present.  net-  o f some 30-40 p r o t e o g l y c a n  a molecular  and h y a l u r o n i c a c i d .  H a r d i n g h a m and  matrix  c o l l a g e n forms a f i b r o u s  r e p r e s e n t a t i o n o f t h e d o m a i n s o f c o l l a g e n and  1974;  as  1981).  a random m a c r o m o l e c u l a r mesh  (monomers) e a c h w i t h  such  (PGA)  cartilage,  m a c r o m o l e c u l a r mesh i s an  million  and  1979,  PROTEOGLYCAN AGGREGATE  work, w i t h i n w h i c h  one  through  ( H e w i t t e t a l . , 1980)  In normal  subunits  molecular organization.  interact with collagen d i r e c t l y  or p o s s i b l y  ( P a u l s s o n and  (C)  c h a i n s and  chains, normally  o r more  attached  to  protein.  -26-  gure 7 .  Structure of 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) R e g i o n s o f i n t e r a c t i o n b e t w e e n p r o t e o g l y c a n a g g r e g a t e and c o l l a g e n .  -27-  HYALURONIC  Figure 8.  ACID  Structure of proteoglycan  aggregate.  - 28-  Different  classes of proteoglycans  s i z e of the core p r o t e i n ,  i n the  d e g r e e o f s u l f a t i o n o f t h e GAG oligosaccharides present of  the  s t r u c t u r e and  published The  (Hascall, cartilage  comprising  about  on  (PGs)  type,  number, a v e r a g e  c h a i n s , and  1981; PGs  Muir,  1980;  and and  other reviews  1980).  a r e composed o f a p r o t e i n the weight  was  first  In t h e c a r t i l a g e  proposed  PGs,  smaller but  core g e n e r a l l y  of the molecule,  The  with  size  of  Several  Roden,  chains are attached  m a j o r GAG  the types  the core p r o t e i n .  l a r g e number o f GAG  (1958).  i n the nature  f u n c t i o n s o f p r o t e o g l y c a n s have been  10-20% o f  s t r u c t u r e o f PG  vary  laterally by  a  (Figure 9a).  Mathews and L o z a i t y t e  chondroitin sulfate  variable  to which  (CS)  amounts o f k e r a t a n  i s the sulfate  (KS) .  Most o f t h e PG-monomers c a n hyaluronic interact  acid  (HA).  w i t h HA  extracted  from  physiological  to  form  an  and  and  and  PGs  non  ( H a r d i n g h a m and  with  these  low m o l e c u l a r  Muir,  PGs  salt  1972a,b; Mayes, Mason and  (Brandt  weight  and  1974;  that can  cannot be  solutions Muir,  of  1974;  are heterogeneous i n  composition  They c o n t a i n l e s s p r o t e i n  Muir,  and  dissociating  T h e s e PGs  chemical  are of r e l a t i v e l y  1969). of  size  aggregate  c o n c e n t r a t i o n ( H a r d i n g h a m and  Hardingham e t a l . , 1 9 7 6 ) . molecular  an a g g r e g a t e  However, t h e r e a r e m i n o r PGs  c a r t i l a g e by ionic  form  less  Brandt  and  Muir,  ( T s i g a n o s and KS and  Griffin,  than  Muir,  the m a j o r i t y  Muir, 1973).  1971)  1969;  Simunek  -29-  Figure  9.  Structures of: (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 .  -30-  (Ia)  CHONDROITIN S U L F A T E  Chondroitin glucuronic CS  group per  s u l f a t e c o n s i s t s of  a c i d and  contains  (CS)  N-acetyl  galactosamine.  a b o u t 25-30 d i s a c c h a r i d e  disaccharide  unit  i n one  10a).  uniform along linkage  of  The the  of  The  biological  group i s not  in  The  B o t h CS  Atkins,  1973;  Atkins,  1977).  are  bovine  I t has  b a s i c groups o f  (Maroudas,  1972)  1970)  equal  ordered  further  to  and  helical  from the  been suggested t h a t PGs  sulfate  chain  (Isaac  and  the  i n t e r a c t more other  proteins.  (KS)  N-acetylglucosamine  (Stockwell,  Bjelle,  approximately  K e r a t a n s u l f a t e i s composed o f d i s a c c h a r i d e  chemical  the  i n human and  in chondroitin-4-sulfate  collagen  and  not  1971).  p o s i t i o n of the  s t r o n g l y w i t h the  of galactose  is  (Mourao e t a l . , 1 9 7 6 ) .  allows  KERATAN S U L F A T E  positions,  Lindahl,  i s o m e r s show h i g h l y  than  of  sulfate  near  M u r a t a and  presence of c h o n d r o i t i n - 6 - s u l f a t e  (Ib)  region  s u l f a t e groups p r o j e c t  chondroitin-6-sulfate  isomeric  (Wasteson and  s i g n i f i c a n c e o f the  clear.  conformations.  isomers  a b o u t one  two  appears to predominate  two  average chain  s u l f a t e residues  1977), w h e r e a s i n g r o w t h c a r t i l a g e t h e r e o f the  of  chondroitin-4-sulfate  c a r t i l a g e (Mourao e t a l . , 1976;  proportions  An  fewer i n the  carbohydrate to p r o t e i n  Chondroitin-6-sulfate articular  being  the  or  d i s t r i b u t i o n of the chain,  units  u n i t s and  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 (Figure  repeating  and  (Figure  10b).  X-ray microprobe  methods i n d i c a t e t h a t  i n the  repeating  units  Histo-  analysis  m i d d l e and  deep  -31-  F i g u r e 10.  Structures of: (a) C h o n d r o i t i n ( i : - 6 - ; (b) Keratan s u l f a t e .  ii:-4-)  sulfate,  -32layers of a r t i c u l a r CS  cartilage  i s more v a r i a b l e t h a n CS  degree of  sulfation  of  thought to e x i s t .  KS  are  disaccharide  contains 1972).  (Muir  and  repeating  The  skeletal  ( R o b i n s o n and sulfated  cartilage  (II)  KS  has  and  single  The  Heinegard,  and  other  to  i n the  free  HA  9b  Lempberg,  weight  of  units Other  heparin  sulfate  s u l f a t e d GAGs and  L a s h and  been i s o l a t e d  Vasan  their  (1983).  and  fully  H a r d i n g h a m and  Muir,  characterized 1974;  1972). GAG  composed o f e q u i m o l a r q u a n t i t i e s o f  shows t h e  plays  an  Electron  show HA  structure of  m a i n l y as  microscopic  to c o n s i s t of  essential role  It i s present form.  s u l f a t e group  (HA)  F e s s l e r , 1966)  Figure  populations  R i o l o , 1972).  N-acetylglucosamine.  ( F e s s l e r and  of proteoglycans.  of various  in  population  disaccharide  r e c e n t l y by  1974;  Lempberg,  a c i d and  chain.  13  H a s c a l l and  composition  In c a r t i l a g e ,  not  one  GAGs i n c l u d e d e r m a t a n s u l f a t e ,  i s a non-sulfated  glucuronic studies  the  has  average molecular  t o about  HYALURONIC A C I D  Hjertquist HA  an  from c a r t i l a g e has  (Hascall  l e n g t h and  1 9 7 5 ) . Two  population  u n i t , while  have been reviewed  HA  i n both chain  Hardingham,  One  Hopwood, 1973;  heparin.  sources  and  relative  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  5000-10,000 c o r r e s p o n d i n g  and  p r o p o r t i o n o f KS  varies. KS  per  the  a  HA.  i n the  aggregation  a component o f  PGAs  -33-  (D)  LIPIDS  Lipid articular normal  OF  CARTILAGE  i s found i n the c e l l s cartilage.  Intracellular  i n t h e m a t r i x o f human lipid  c o n s t i t u e n t o f c a r t i l a g e because  absence o f d e g e n e r a t i v e changes 1965).  Extracellular  articular upwards 1952;  and  cartilage  (Ghadially  Schott,  e t a l . , 1965; Marotti,  nature of the l i p i d s characteristics partly  Pearse,  esters  Little  1965;  i n healthy  FACTORS AFFECTING  Zbinden,  Staining  indicate  that they are  triglycerides,  cholesterol,  (Bonner e t a l . ,  1975;  THE  COMPOSITION OF  articular  c h a n g e s w i t h age  cartilage  (Collins  a decrease i n water  (Ruttner  et a l . ,  1974)  resistance  Other  changes  c o n t e n t o f c a r t i l a g e w i t h age  and a d e c r e a s e i n t h e c o l l a g e n c o n t e n t  w i t h age on a d r y w e i g h t b a s i s  elasticity  shows i n c r e a s e d d e g e n e r a t i v e  and Meachim, 1 9 6 1 ) .  include,  The  CARTILAGE  E F F E C T OF AGE  Adult  (Werner  of cartilage  t o c o m p r e s s i o n and  et a l . ,  i s responsible  i s related  1976).  f o r the  t o t h e c o n t e n t and  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 al.,  life  1968).  (A)  et  of  i s known a b o u t t h e  cartilage.  and p h o s p h o l i p i d s  eta l . ,  i n the second decade  and c h e m i c a l a n a l y s i s fats:  (Collins  identified  Stockwell,  1963).  of articular  comprised of n a t u r a l  cholesterol  2.6.2  l i p i d s have been  a  i t i s found i n the  i n the c e l l s  from i n d i v i d u a l s  1963;  i s c o n s i d e r e d t o be  1972;  Scott,  1973,  1975).  The  resistance  to  the  (Harris  -34-  compression  of the c a r t i l a g e  the a l t e r a t i o n s cartilage  decreases  i n the p r o t e o g l y c a n  ( Schofield  and  with  age,  probably  s t r u c t u r e o f the  Weightman,  1978;  due  to  articular  Armstrong et a l . ,  1979).  Evidence  indicates  human a r t i c u l a r 1978,  1979;  t h a t the composition  cartilage  Venn,  1978;  c h a n g e s w i t h age Inerot  a summary o f t h e e f f e c t s  of the matrix  (Elliot  et a l . , 1978).  of ageing  on  and  The  of  Gardener,  following is  the composition  of  cartilage. (i)  Biochemical  quantity  of c a r t i l a g e matrix  h a v e shown d i f f e r e n t content  of  (1973). GAG at  s t u d i e s o f changes i n the n a t u r e  content  results.  articular  Elliott  and  of the  about 4 y e a r s  by  Gardner  The  o f age  t o a b o u t 7% by i n the t o t a l  hyaluronic acid  increases  little t o 2% and  HA  and  Maroudas  15%  70 y e a r s GAG  During  the  o f age.  This also  White  (1980).  cartilage f i r s t decade  (Elliott  The  extracted with  proportion of proteoglycans 4 molar guanidinium  of  the  HA  and  Gardner,  1979).  (iii)  et a l .  (dry weight)  ten years  60 y e a r s  GAG  i n the  c o n t e n t was  of the  i s detected. After 6% by  from  R o u g h l e y and  content  ageing.  reaches  ageing  changes i n t o t a l  w e r e r e p o r t e d by  s u r f a c e zone c a r t i l a g e  increases gradually with very  o r no  during  (1979) reported a decrease  Inerot et a l . (1978),  (ii)  life,  Little  cartilage  observation of a decrease noted  glycosaminoglycans  and  t h a t can  c h l o r i d e decreases  be with  content  -35-  increasing  age  (Inerot  e t a l . , 1978;  Roughley  and  White,  However, B a y l i s s and A l i ( 1 9 7 8 a , b ) ; Simunek and M u i r Sweet e t a l . (1977) proteoglycan (iv)  adults  Sweet e t a l . , 1 9 7 9 ) .  proteoglycans  Venn, 1 9 7 8 ) .  (1972b)  extractable  weight o f c h o n d r o i t i n s u l f a t e i n d i v i d u a l s t o 16,000 i n  of chondroitin  Garg  Bayliss,  increases  of  with  1976;  chondroitin-6-  age  i n adult  human and b o v i n e c a r t i l a g e ( H j e r t q u i s t and Wasteson,  1972;  Hjertquist  1971;  and P r o n o s k y , 1979;  1972;  The k e r a t a n  proteoglycans et  Sweet  1980;  wih  age  1979).  This  turnover during  o f t h e GAG  of the  and  1981).  extractable  Elliott  and  Gardner,  (Elliott  and  t h o u g h t t o be due  (vi)  i n the c a r t i l a g e surface  1979).  Gardner,  zone c a u s e d by wear joints  1979).  Both i n t r a c e l l u l a r  Inerot  to a faster  the mechanical f u n c t i o n i n g of the s y n o v i a l  and G a r d n e r ,  Bjelle,  zone o f a d u l t c a r t i l a g e seems t o  p e r c e n t a g e o f KS  i n c r e a s e has been  Murata  Lust  ( B a y l i s s and A l i , 1978a,b;  e t a l . , 1977;  During ageing the s u p e r f i c i a l accumulate a h i g h e r  and L i p p i e l l o ,  G a r g and Swan,  s u l f a t e content  increases  a l . , 1978;  Mankin  Lempberg e t a l . , 1974;  R o u g l e y and W h i t e ,  (v)  1972;  and  s u l f a t e i n the  However, t h e r e l a t i v e p r o p o r t i o n  to chondroitin-4-sulfate  1972;  Ageing a l s o r e s u l t s i n a  ( S t r i d e r e t a l . , 1976;  and Lempberg,  and  age.  average molecular  i n the p r o p o r t i o n  extracted  i n the  t h e e l d e r l y ( H j e r t q u i s t and Wasteson,  Swan, 1981;  sulfate  an i n c r e a s e  f r o m a b o u t 20,000 i n young  and  decrease  f r a c t i o n with  The  decreases  reported  1980).  and  extracellular lipid  (Elliott  -36-  concentrations  show a d i s t i n c t  (Ghadially  a l . , 1965;  The al.,  et  estimated 1975;  (B)  and  E F F E C T OF  Studies using that  after  a  After degrading  loss  Peiffer,  injury, the  1963;  Bonner  et  i s shown i n T a b l e 1957;  Ravetto,  2  age al.,  1975).  (Bonner  et  1964).  INJURY  the  articular cartilage  of  the  synthesis  of  first  the  rabbit  showed  change o c c u r e d the  (Meachim,  margin of  in  the  injury  1963).  c h o n d r o c y t e s p r o d u c e enzymes c a p a b l e  cartilage protease  degradation  the  proteoglycans along  enhanced GAG  cathepsin-like cartilage  year  superficial injury,  matrix with a f o l l o w e d by  Marotti,  i n c r e a s e per  Hirsch  increase with advancing  matrix  ( C h r i s m a n and  seems t o be  ( D i n g l e and  the  Fessel,  1962).  enzyme r e s p o n s i b l e  Dingle,  1980;  of  Ziff  A for  et a l . ,  1960).  (C)  E F F E C T OF  JOINT  DISEASE  Primary o s t e o a r t h r i t i s disease of  old  trauma o f t e n  age.  occurs  changes i n the secondary  joint  cartilage elasticity structure  Osteoarthritis i n young and cartilage  osteoarthritis  Kempson e t  al.  decreases of of  i s usually  m i d d l e aged  are  (Inerot  similar et  in osteoarthritic is largely  proteoglycans,  not  exclusively  secondary to  (1971) showed t h a t  cartilage  but  The  i n both primary  and  Chrisman,  elasticity  individuals.  related  i t i s therefore  or  individuals.  a l . , 1978; the  disease  a  to  the  likely  of  1969).  the  Since  the  content  and  that  the  -37-  T a b l e 2.  The estimated i n c r e a s e i n the l i p i d c o n t e n t o f c a r t i l a g e per year o f age (Bonner e t a l . 1975; H i r s h and P e i f f e r , 1957; Ravetto, 1964). f  Lipids  Estimated i n c r e a s e per year (% d r y weight) Superficial layer  Deep layer  0.046  0.03  Triglycerides  0.007  0.006  Cholesterol  0.008  0.003  Phospholipids  0.001  0.004  Total  lipid  -38-  composition  of the proteoglycans  I t has  suggested  been  important  The  Thrasher,  (McDevitt  1975;  (ii)  of cartilage  degenerated  water c o n t e n t  and  Lipshitz  Proteoglycan  Muir,  monomers  smaller than  same age  ( I n e r o t e t a l . , 1978).  cathepsins Pelletier  be  ( A l i and  The  significant)  due  to the  Evans,  total  GAG  Bollet  and  Nance  cartilage  M a n k i n and  has  a  higher  Zarins-  from  degenerated  from  normal c a r t i l a g e  degradation  cartilage of  are  the  of  i n c r e a s e d enzyme a c t i v i t y  content  or n e u t r a l proteases  decreases  ( M a n k i n and  SYNOVIAL  than  is slightly  lower  of  (Martel-  (not  i n normal c a r t i l a g e  S i m i l a r o b s e r v a t i o n s were  (1966) and  In o s t e o a r t h r i t i s i n c r e a s e s and  The  1976;  The  1973)  i n degenerated  4-sulfate  2.7  those  ( I n e r o t e t a l . , 1978).  (iv)  f o l l o w i n g changes  e t a l • , 1984).  (iii)  made by  (Mankin e t  e t a l . , 1976).  t o be  p r o t e o g l y c a n s may  i s an  in osteoarthritis.  osteoarthritic  reported  age  osteoarthritis.  of the proteoglycans  S e v e r a l s t u d i e s have r e p o r t e d the  the composition  (i)  that degradation  in  step i n the development o f o s t e o a r t h r i t i s  a l . , 1971. in  i s changed  M a n k i n and  o f the also  Lippello  (1970).  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 -  the c o n c e n t r a t i o n of keratan  Lippiello,  sulfate  1971).  FLUID  synovial fluid  l u b r i c a t i o n of the  joint  same  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 tissues.  The  s y n o v i a l membrane  and  -39-  regulates the  b o t h t h e volume and t h e m a c r o m o l e c u l a r c o m p o s i t i o n o f  synovial  2.7.1  (Horst  possible  constituents  s o u r c e s (Swan,  The protein relative  D E R I V E D FROM T H E BLOOD  constituents  are derived  directly  proportions  constitutes  depends  o f serum  protein  The  p r i m a r i l y on  The r e l a t i o n s h i p b e t w e e n  3 ( K u s h n e r and S o m e r v i l l e ,  molecular  i n synovial  fluid  is•  1971).  t h e low m o l e c u l a r w e i g h t p r o t e i n o f p l a s m a ,  a b o u t 75% o f t h e t o t a l  CONSTITUENTS  p r o t e i n content of synovial  1958).  SECRETED BY THE JOINT  HYALURONIC ACID  Only a small  TISSUES  (HA)  HA o f t h e s y n o v i a l  cartilage.  from t h e plasma.  of the constituents  ( S a n d s o n and Hamerman,  (I)  the  three  g l u c o s e and a l l b u t a few  Albumin,  fluid  from  components,  i n Table  The  are derived  electrolyte  weight and c o n c e n t r a t i o n  (B)  fluid  1978).  t h e i r molecular weights.  fluid  1980).  FLUID  of synovial  SOLUBLE CONSTITUENTS  given  and W a l i t z a ,  COMPOSITION OF SYNOVIAL  The  (A)  fluid  fluid  i s similar  t o t h e HA o f  amount o f HA i s p r e s e n t  i n the synovial  and i t i s t h o u g h t t o b e m a n u f a c t u r e d b y t h e c e l l  s y n o v i a l membrane  HA i n t h e s y n o v i a l  (Baxter  fluid  maintain the v i s c o s i t y  e t a l . , 1973).  lining of  The f u n c t i o n o f  i s not c l e a r but i t i s thought t o  of the synovial  fluid.  -40-  Table 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 .  Component  Molecular weight (x 10 ) J  a ~acid protein 1  glyco-  Normal plasma concentration (mg. mL~ )  Normal SF/Serum ratio  44  0.75- 1.0  0.23 +0.09  74  2.0 - 3.2  0.24 +0.08  .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  Transferin Ceruloplasmin _  0.045+0.024  -41-  (II)  LUBRICATING  The  purified  GLYCOPROTEINS  lubricating proteins  fluid  represent  a b o u t 0.5%  fluid  (Swan and  Radin,  (C)  arthritis  lymphatic  al.,  s u c h as  Synovial  fluid  fluid  as  rheumatoid  l o s s from the  removes t h e  TISSUES  articular  degradation products  uptake.  Chondroitin  a normal  constituent  LIPIDS  sterol  and  by  sulfate is (Silpananta  et  1  OF  SYNOVIAL  Normal s y n o v i a l  some n e u t r a l  phosphatidylinositol  FLUID  fluid  phosphaphatidylcholine,  contains  lipids.  some p h o s p h o l i p i d s ,  Among t h e  sphingomyelin,  and  cephalins  chole-  phospholipids,  lysolecithin,  have been i d e n t i f i e d  (Bole,  Chung e t a l . , 1 9 6 2 ) .  2.7.3  FACTORS A F F E C T I N G THE  (A)  The  and  synovial  1967).  2.7.2  1962;  synovial  i n the  JOINT  o s t e o a r t h r i t i s and  uptake or phagocytic  in synovial  protein  C A T A B O L I S M OF  r e s u l t i n p e r s i s t a n t PG  cartilage.  present  states  total  i n the  1972).  P R O D U C T S D E R I V E D FROM T H E  Disease  ed  o f the  present  EFFECT  total  OF  JOINT  synovial  in degenerative Vernon-Roberts,  joint  C O M P O S I T I O N OF  SYNOVIAL  FLUID  DISEASE  fluid  protein content  disease  1976).  The  and total  is usually  rheumatoid  arthritis  protein content  increas(Currey  increased  - 4 2 -  from  1.8-2.1 g dL  osteoarthritis (Markowitz, from about arthritis  t o 2.9-3.9  increased  joints  joint  molecular  weight  i n t h e normal  as a r e s u l t  disease  joints  synovial  e t a l . , 1967). fluid  and r h e u m a t o i d  in  The l e v e l s o f  are elevated i n arthritis  (Seppala  lipid  levels  (Bole,  et  increase  in triglyceride  phospholipid  change i n d i s e a s e  a decrease  increase fluid  of phospholipids  i n cephalin  (Bole,  1962).  i n the s y n o v i a l  states.  i n the l e v e l s  The  i s l e s s than the increase i n  and c h o l e s t e r o l c o n t e n t  concentration distinct  content  arthritis  1962; Chung e t a l . ,  1962; S m a l l e t a l . , 1964; Newcombe and Cohen, 1 9 6 5 ) .  The  fluid  also  Chung e t a l . ( 1 9 6 2 )  of phosphatidylcholine  and s p h i n g o m y e l i n  i n o s t e o a r t h r i t i s or rheumatoid  levels  shows  repor-  and an  i n the synovial  arthritis.  THEORY OF CRYSTAL GROWTH The d e p o s i t i o n o f a s o l i d  can  fluid  o f the c a t a b o l i c process  i n o s t e o a r t h r i t i s and r h e u m a t o i d  show g r e a t l y i n c r e a s e d  2.8  proteins  1972; B a r k e r e t a l . , 1 9 6 6 ) .  Diseased  ted  decreased  1983).  s u l f a t e i n the synovial  degenerative  arthritis  t o 42-52% i n r h e u m a t o i d  s u l f a t e i s present  c a r t i l a g e (Silpananta  chondroitin  -  the percentage o f albumin  (Markowitz,  released  g dL ^" i n  i n rheumatoid  - 1  and t h e p e r c e n t a g e o f h i g h  i s probably  al.,  However,  56-63% i n n o r m a l  Chondroitin  articular  joints  and t o 4.2-4.9 g d L  1983).  (globulins)  and  i n normal  only  occur  crystalline  phase from s o l u t i o n  i f some d e g r e e o f s u p e r s a t u r a t i o n  or  supercooling  -43-  has  first  process  been achieved  i n t h e system.  c a n be c o n s i d e r e d  Any c r y s t a l l i z a t i o n  t o be comprised o f t h r e e b a s i c  (A)  Achievement o f s u p e r s a t u r a t i o n  (B)  Formation o f c r y s t a l n u c l e i .  (C)  Growth o f t h e c r y s t a l s .  2.8.1  or supercooling.  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  said  t o be s a t u r a t e d  solution the  containing  with  respect  i n a dust  made t o show a p p r e c i a b l e supersaturation  saturated  This  11.  introduced  supersaturated.  t h e terms  supersaturation.  cooled  slowly,  The s t a t e  'labile'  (unstable)  These terms r e f e r t o s u p e r -  nuclei, will  A solubility-supersolubility  The l o w e r c o n t i n u o u s  f o rthe p a r t i c u l a r s a l t .  upper broken curve  by  a l l solutions are stable.  line  curve,  and w i l l diagram  i s t h e normal  T e m p e r a t u r e s and  at which spontaneous c r y s t a l l i z a t i o n the  represented  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 t h e s o l i d  respectively.  curve  than that  degrees o f s u p e r s a t u r a t i o n .  phase i n t h e absence o f s o l i d  Figure  However, a  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 .  i n 1897  'metastable'  phase i s  f r e e a t m o s p h e r e , c a n r e a d i l y be  Below t h e s a t u r a t i o n s o l u b i l i t y ,  and  solid.  solid  i s s a i d t o be  the solid  solutions i n clean containers,  without disturbance,  Ostwald  t o that  more d i s s o l v e d  saturation solubility  Uncontaminated  of  steps:  occurs  not occur, i s shown i n solubility  concentrations  are represented  by  r e f e r r e d t o as t h e s u p e r s o l u b i l i t y c u r v e .  i s n o t as w e l l  defined  as t h e s o l u b i l i t y  c u r v e and  -44-  TEMPERATURE  Figure 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.  -45-  its  position  the  solution.  is  on  the  Despite  ill-defined,  supersaturated metastable  diagram  there  the  d e p e n d s on  fact  exists  such a  solution,  solute  in solution  supersaturation  reaches  (seed  the  the  is  by  ( l i n e ABC),  induced  by  cooling  to point  D may  particularly  s o l u t i o n by  (line  A B'C)  A coefficient defined  by:  be  necessary  a l s o be  are  introduced  into  concentration  of the  When  of  the  metastable  by  11  point C are  mechanical before  carried  i s cooled  crystallization  spontaneous or shock.  reached.  i t may  Further  crystallization  by  out  removing the at constant  can  be  s u p e r s a t u r a t i o n , s',  can  solvent  temper-  11.  or degree of  be  substances.  achieved  evaporation i n Figure  the  i s a t t a i n e d , where  with h i g h l y soluble can  the  improbable.  the  spontaneous  be  a g i t a t i o n o r by  Supersaturation  ature  limits  conditions represented  seeding,  curve  is  point A i n Figure  p o i n t , c r y s t a l l i z a t i o n may  from the  until  In  of  probable.  At t h i s  induced,  crystals)  solution  represented  loss of solvent until  curve.  saturation solubility.  i s greater than  a region of unstable  cannot occur  solubility  growth o c c u r s  spontaneous c r y s t a l l i z a t i o n  without  supersolubility  spontaneous c r y s t a l l i z a t i o n  crystal  If a solution  degree o f a g i t a t i o n  a region of m e t a s t a b i l i t y i n  However, i f f o r e i g n p a r t i c l e s  region,  t h a t the  r e g i o n above t h e  region,  the  be  -46-  where C i s the given  concentration of the  temperature,  and  Cs  i s the  c o n c e n t r a t i o n o f s o l u t e i n the  d e C o p p e t i n 1872 of  s o l v e n t a t the  produced  2.8.2  to the degree o f  at  some  evidence  same  temperature.  t o show t h a t t h e s o l u t i o n was  length  inversely  supersaturation.  NUCLEATION  Supersaturation begin  in solution  equilibrium saturation  time o f s t a b i l i t y o f a supersaturated  proportional  in  substance  alone  to c r y s t a l l i z e .  solution  i s not  Before  sufficient  crystals  a number o f m i n u t e s o l i d  can  bodies  crystallization,  seeds,  embryos o r n u c l e i .  spontaneously  i t may  be  or  homogeneous and (A)  geneous f l u i d condensation  droplets  of a supersaturated  known as  artificially,  centers  of  occur  r e f e r r e d to  as  any  i s formed w i t h i n a homodegree o f c e r t a i n t y .  vapour t o the  they  a t the  evaporate  i s supersaturated.  until  liquid  rapidly New  The  The  phase i s  droplets.  surface of these  minute  even though  nuclei  the  form w h i l e  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  c o a g u l a t i o n o r under c o n d i t i o n s o f v e r y  supersaturation.  exist  N u c l e a t i o n may  appearance o f m i c r o s c o p i c  vapour p r e s s u r e  vapour  ones e v a p o r a t e , e i t h e r by  the  i s very high,  surrounding  nucleus  i s n o t known w i t h  the  grow t h e r e must  NUCLEATION  a crystal  only possible after However, as  to  heterogeneous n u c l e a t i o n r e s p e c t i v e l y .  HOMOGENEOUS  E x a c t l y how  induced  f o r a system  formed  high  mechanism o f homogeneous n u c l e a t i o n i s  old  -47t h o u g h t t o be  as  follows:  Minute s t r u c t u r e s are two  ions  on,  to  or molecules, then  form c l u s t e r s .  barrier  formation  homogeneous energy  For  for nucleation  The  fluid  i n the  quantity  of  W  the  of the  from a t h i r d  with the  p a i r , and  so  W  vapour,  droplet  the  s  liquid to  the  or  or  the  solid  a c e r t a i n amount  solid  surface.  to  form the  &  i s the  area,Ap  the  interior  of  and  volume,  the  droplet,  surface  pressure the  liquid  work r e q u i r e d so  to  f o r example, e q u a t i o n  2 can  g  bulk  of  in a  be  written  s p h e r i c a l d r o p l e t per  d i f f e r e n c e between the  2  W  as  (3)  energy o f the  a = 4^tr  is  (2)  droplet  then  total  that  of a spherical l i q u i d  r e s p e c t i v e l y , of the  a of  nucleus  form the  v  droplet,  The  surface,  W = a£ - vAp  where  energy  particle within  form a s t a b l e c r y s t a l  quantity),  - W  occur,  expenditure of  the  (a n e g a t i v e  v  to  exceeded.  work r e q u i r e d and  formation  supersaturated  of  required  W =  For  collision  a liquid  (a p o s i t i v e q u a n t i t y ) , particle,  from the  must be  creation of  sum  first  crystallization  requires  o f work, W,  equal to the  the  formed,  and  a and  droplet.  unit  v a p o u r p h a s e and  v are  the  surface  I f r i s the  the  area  radius  of  -48-  v =  -  1Tr  3  26 and  therefore  A.P =  equation  r  3 becomes  7 ^  4  (4)  W = -7{r 3  The i n c r e a s e i n t h e v a p o u r p r e s s u r e o f a l i q u i d its  size decreases  c a n be e s t i m a t e d  from  d r o p l e t as  t h e Gibbs-Thomson  equation pr In  2M^ =  p*  (5) RT/r  where, p r a n d p * a r e t h e v a p o u r p r e s s u r e o v e r radius  r and a f l a t  molecular  weight,f  temperature  liquid  surface, respectively,  droplet  M i s the  , t h e d e n s i t y o f t h e d r o p l e t , T, t h e a b s o l u t e  and R, t h e gas  The t e r m , p r / p * the  a liquid  constant.  i s a measure o f t h e s u p e r s a t u r a t i o n , s', o f  system, so e q u a t i o n  5 becomes  2M^ In  s' =  RT/r  (6)  -492M^ or  r  Substituting  (7)  =  3(RT/ln  for r i n equation  167fe M 3  W  Equation 8 gives the  (a s a t u r a t e d required The  In  degree of  2  work o f n u c l e a t i o n  supersaturation  s o l u t i o n ) , In  s'=  is  0 and  of  the  f r e e energy changes a s s o c i a t e d are  as  between a s m a l l  energy,AG / g  that  particle  i s the  and  the  excess f r e e energy ^ G » v  large p a r t i c l e  the  in  s y s t e m . When  amount o f  s'=l,  energy  infinite. w i t h the  follows.  solid  i n s o l u t i o n i s equal  a very  s' )  a measure o f t h e  for nucleation  energyAG,  o f the  2  (8)  homogeneous n u c l e a t i o n  solute  4  = 3(RT/  terms of  s' )  The  particle  to the  excess  sum  free  overall  of  of the  that  excess  ( r = «0 ) and  and  the  and  the  free  the  surface  energy between  particle,  of  excess  solute  b u l k o f the i s the  process  excess the  free  surface  volume  f r e e energy between  solute  in solution.  ^G  g  2 is  a positive quantity  supersaturated  and  solutionAG  i s proportional v  is a  negative  to  r  .  In  quantity  a proportional  3 to r  .  These r e l a t i o n s h i p s are  increases, value  the  when t h e  overall  excess  shown i n F i g u r e  As  r  f r e e e n e r g y , AG,  nucleus achieves a c r i t i c a l  A G crit  12.  4fl^ ( r )  r e a c h e s a maximum c s i z e ( r ), t h a t i s ,  2  c  3  (9)  -50-  Size of nucleus, r  Figure 12.  Free energy diagram f o r nucleation explaining the existence of a c r i t i c a l nucleus.  -51The  critical  stable nucleus. The unit  size  r » represents  t h e minimum  c  Particles  smaller  dissolve.  N ( t h e number o f n u c l e i formed p e r  rate of nucleation,  time p e r u n i t volume),  than r w i l l c  size of a  i s g i v e n by,  N = A exp (-AG/RT)  where A i s a c o n s t a n t energy o f t h e p a r t i c l e ,  andAG  that  (10)  i s the overall  excess  free  i s , t h e work o f n u c l e a t i o n W,  from  e q u a t i o n s 8 and 1 0 ,  16fli? M N =  A  exp (  2  — ) 3RV/ (ln Z  This on  equation  shows t h e d e p e n d e n c e o f t h e r a t e o f n u c l e a t i o n  the temperature,  interfacial  T, t h e d e g r e e o f s u p e r s a t u r a t i o n ,  supersaturation,  supersaturation  r a t e , N versus  s ' , i s shown i n F i g u r e  rate of nucleation  (B)  i s evident  13.  the degree o f A rapid increase i n  once a c r i t i c a l  level of  i s exceeded.  HETEROGENEOUS AND SECONDARY NUCLEATION  C r y s t a l l i z a t i o n may be i n d u c e d supersaturated be  s', and t h e  tension,& .  A p l o t of the nucleation  the  (11) s" )  s o l u t i o n with  crystallized.  small  by i n o c u l a t i n g particles  or seeding  of the material to  E f f e c t i v e s e e d c r y s t a l s do n o t h a v e t o b e t h e  a  -52-  Theoretical  Experimental  Q>  c o  a  Of  o Z  S u p e r s c t u r a t i o n , s"  Figure 1 3 *  E f f e c t of supersaturation on the nucleation r a t e .  -53-  material will  being  crystallized.  frequently  solution. seeding  F o r example i s o m o r p h o u s  induce c r y s t a l l i z a t i o n  substrate  (Edwards and E v a n s ,  f o r homogeneous n u c l e a t i o n . with the formation  heterogeneous nucleation, A G ' ding  1962).  a t degrees o f s u p e r s a t u r a t i o n  change a s s o c i a t e d  c r  f r e e e n e r g y change, ^ j ^> G  cr  nucleation  i s given  by  charge o f the n u c l e a t i n g  presence o f a s u i t a b l e seeding material  nucleation required  supersaturated  T h e r e i s some e v i d e n c e t o show t h a t n u c l e a t i o n  i s d e p e n d e n t on t h e s u r f a c e  The  of the  compounds  L  £ »  induces  lower than  The o v e r a l l  those  free  of a c r i t i c a l  energy  nucleus f o r  i s l e s s than the correspon-  t  associated  with  homogeneous  by,  (12)  where  is  l e s s than unity,  and i s g i v e n  b y e q u a t i o n 13  (Volmer,1939).  (2 + Cos 9 ) ( 1 - Cos 0 )  2  (13)  4  where  Cos  e  ^ s l  -3  si o f t h e s e e d and t h e l i q u i d , cs  the  interfacial  energy between t h e  -54-  surfaces of the c r y s t a l l i z i n g seed the  2.8.3  CRYSTAL  As  interfacial  size  t o grow i n t o crystal  (c sS  crystals  e n e r g y and suggested  energy between the  phase , Cos  that  and  the l i q u i d ,  and  6)  ENERGY  is particles  size.  they begin  Three t h e o r i e s  of  THEORIES  i s most s t a b l e when i t s s u r f a c e  t h u s , i t s s u r f a c e a r e a i s a minimum.  t h e s u r f a c e e n e r g y was  than the  proposed.  of a l i q u i d  t h a t t h e shape  larger  i n a s u p e r s a t u r a t e d system,  of a v i s i b l e  have been  SURFACE  A droplet  the  GROWTH  a r e formed  growth  (A)  crystal  + c€> l  s o o n as s t a b l e n u c l e i ,  critical  and  surface.  crystallizing  =  phase  o f a growing  a minimum.  Gibbs  c r y s t a l would  The  total  free  be  free  (1928) such  that  energy o f a  i n equilibrium with i t s surroundings at constant  temperature  and  p r e s s u r e i s a minimum  volume f r e e e n e r g y p e r u n i t throughout the c r y s t a l ,  f o r a g i v e n volume.  volume i s assumed t o be c o n s t a n t  then  n  (14)  where a ^ i s t h e a r e a o f t h e i t h f a c e o f t h e bounded by n f a c e s ,  I f the  and g^  area of the i t h face.  i s the surface  If a crystal  free  crystal  energy per  unit  i s a l l o w e d t o grow, i t  -55should  develop  surface  i n t o an e q u i l i b r i u m shape t o e n s u r e minimum  f r e e energy  f o ra given  volume o f t h e c r y s t a l .  (1901) showed t h a t t h e e q u i l i b r i u m shape o f a c r y s t a l to  the free  energies  o f the faces.  He s u g g e s t e d  f a c e s w o u l d 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 surface out  energies.  that  Laue  a l l possible  (1943)  surface  There i s l i t l e  surface  Wulff i s related  that  the c r y s t a l  respective  Wulffs  combinations o f faces  determine which o f t h e o v e r a l l minimum.  modified  total  theory  pointing  must b e c o n s i d e r e d  free energies  represent  to a  q u a n t i t a t i v e evidence t o support the  e n e r g y t h e o r i e s and t h e y h a v e n o t b e e n  generally  accepted.  (B)  ADSORPTION LAYER  A theory adsorbed  of crystal  THEORIES  g r o w t h b a s e d on t h e e x i s t e n c e  l a y e r o f s o l u t e atoms o r m o l e c u l e s was p r o p o s e d b y  Volmer  (1939).  theory  h a v e b e e n made b y B r a n d e s  Kossel  o f an  Contributions  and m o d i f i c a t i o n s  t o Volmer's  (1927); S t r a n s k i  (1928) and  (1934).  Atoms, i o n s o r m o l e c u l e s w i l l crystal  surface  a t t a c h themselves onto t h e  where t h e a t t r a c t i v e  forces  are greatest,  that i s  t h e y m i g r a t e t o w a r d s p o s i t i o n s where a maximum number o f l i k e elements a r e l o c a t e d continue Before  until  a whole p l a n e  the crystal  surface  of c r y s t a l l i z a t i o n " suggested  (Figure  14a). surface  stepwise b u i l d  i s completed  can continue  on  nucleus  the c r y s t a l  (two  surface  up w i l l  (Figure 14b).  t o grow, a n o t h e r  must b e f o r m e d on t h e p l a n e  t h a t a monolayer i s l a n d  n u c l e u s ) was c r e a t e d  This  "center  surface. dimensional  (Figure 14c).  I t was  -56-  F i g u r e 14.  C r y s t a l growth w i t h o u t 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 ; (tO completed l a y e r ; (c) surface nucleation.  -57-  However, a h i g h type  degree of  o f two  dimensional  Kossel  (1934) p r o p o s e d  (Figure  15).  The  flat  monatomic s t e p and one  the  o r more k i n k s .  u n i t s on steps.  the  In  crystal  Growth  face  and  a fresh step  step  entirely  covered  (I)  and  the  with  dislocations  are  r e g i o n where t h e There are  at a kink  kink  along  the  site. step  According  to  are  kinks.  two  contain d i s l o c a t i o n s .  one  dimensional  atoms a r e  types  not  properly  of d i s l o c a t i o n ,  This  which  i s any  forms a c l o s e d  i s zero.  closure  dislocations dislocation  I f the  failure the and  the  can  the  s u r r o u n d e d by  be  vector  t o complete  (b ). Q  i s perpendicular  aid  the of  made i n a  circuit  encloses  and  the  I f the  Burgers vector  Burgers v e c t o r  defined with  is a  neighbours.  edge d i s l o c a t i o n  Burgers c i r c u i t  i s the  or  A dislocation  atom-to-atom p a t h  loop.  dislocation-free crystal,  Line defects  defects.  a Burgers c i r c u i t .  the  incorporated  growth and  grow f a s t e s t when i t s f a c e s  A dislocation  circuit  surface  surface nucleation.  screw d i s l o c a t i o n .  ideal  i n the  a  showing  l o o s e l y adsorbed  movement o f t h e  should  face  r e g i o n s by  incomplete,  are  this  DISLOCATIONS  Most c r y s t a l s  crystal  be  vacancies  more e a s i l y  i s c r e a t e d by  a crystal  may  addition there  surface  crystal  i s d i v i d e d i n t o two  itself  for  ( M u l l i n , 1961).  a model o f a g r o w i n g  surface  i s c o m p l e t e d by  theory,  i s necessary  n u c l e a t i o n to occur  u n i t s are  The  this  supersaturation  i s made i n  a  the  dislocation,  In  edge  to  the  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  an  (Figure  16)  -53-  F i g u r e 15-  K o s s e l ' s model o f 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 s ; (B) s t e p s ; (C) k i n k s ; (D) s u r f a c e - a d s o r b e d growth u n i t s ; (E) edge v a c a n c i e s ; and ( F ) s u r f a c e v a c a n c i e s .  -59-  tL  JJ  Dislocation line  Dislocation line  F i g u r e 16.  Dislocations i n crystal: ( 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) p r o p o s e d  arise  during  by  Nabarro  The the  the  (1967) and  surface  are  a crystal  Albon  d i s l o c a t i o n s are  dislocations  to  growth of  influence of  they  s e v e r a l ways i n w h i c h d i s l o c a t i o n s c o u l d  formed d u r i n g and  need  internal important  have been  the  reviewed  growth p r o c e s s  stresses.  under  Screw  for crystal  for surface nucleation  growth,  since  for crystal  growth  continue.  Screw d i s l o c a t i o n s g i v e growth,  first  p o s t u l a t e d by  d i s l o c a t i o n has grow.  Figure  rise  been formed, the  17  (a-c)  Frequently,  grow t o g e t h e r Frank  (Verma, 1953;  calculate Cabrera  and  where R^  i s the  Read, 1 9 5 3 ) . theory  = h(T  2  a  2 t o 9" ,  low  tanh  g r o w t h r a t e , fT  but  supersaturation at high  continue  stages  in  to  the  dislocation.  Burton, b a s e d on  g r o w t h and  Cabrera the  to  The  Burton,  by  (B/<y)  = s ' - l and  (15)  s'=  C/Cs.  A and  B  constants.  concentrations,  . supersaturation  and  screw  were a b l e  supersaturation.  complex t e m p e r a t u r e d e p e n d e n t At  can  (B.C.F) r e l a t i o n s h i p i s g i v e n  R  screw  d e v e l o p when s e v e r a l d i s l o c a t i o n s  g r o w t h r a t e a t any  Frank  face  from a screw  mechanism o f c r y s t a l  the  Once a  successive  (1951) d e v e l o p e d a k i n e t i c  dislocation  (1949). crystal  shows t h e  complex s p i r a l s  t o a p a r t i c u l a r mode o f  Frank  development o f a growth s p i r a l  are  these  (1963).  considered  e l i m i n a t e the  and  R^  i s proportional  . concentrations  R  . is  -61-  Figure 1 7 .  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 .  -62-  proportional (square)  to  or the relationship  growth law t o a l i n e a r  growth  changes from a p a r a b o l i c law as t h e s u p e r s a t u r a t i o n  increases.  (C)  DIFFUSION THEORIES Noyes a n d W h i t n e y  solid  (1897) p r o p o s e d t h a t t h e d e p o s i t i o n o f  on t h e f a c e o f a g r o w i n g c r y s t a l  d i s s o l u t i o n process According  and was e s s e n t i a l l y  t o them t h e r a t e s o f c r y s t a l  was t h e r e v e r s e o f t h e a diffusion  process.  growth o r d i s s o l u t i o n  were  governed by t h e d i f f e r e n c e between t h e c o n c e n t r a t i o n s a t t h e solid  s u r f a c e and i n t h e b u l k  R where the  (16)  C i s the solute concentration i n the supersaturated Cs i s t h e s a t u r a t i o n s o l u b i l i t y  transport  rate  Nernst  t  i s the  t h e Noyes-Whitney e q u a t i o n w i t h t h e  t h a t t h e r e was a t h i n  modified  and K  constant.  (1904) m o d i f i e d  t o t h e growing The  i n t h e form  S i s the surface area o f  solution,  assumption  given  = K. S (C-Cs) t  g  i s t h e growth r a t e ,  solid,  of the solution,  face, through  stagnant  film  of liquid  which s o l u t e molecules  adjacent  diffused.  r e l a t i o n s h i p was: D R  He  concluded  diffusion  g  (17)  S (C - C s ) h  t h a t K. = ( D / h ) ,  where D = c o e f f i c i e n t o f  o f s o l u t e and h = t h i c k n e s s o f t h e l i q u i d  film.  -63-  The  thickness  solid-liquid (1908,  velocity,  1909a,b,  1910)  i n d e f i n a t e l y with limiting  value.  necessarily alone  the  fluid  by  the  phase t o the  constant,  by 1  R  diffusion  integration  concentration  t  s  =  K  =  K  diffusion),  R^  K  g  driving  1924)  growth  Firstly,  transported  are  and a from  f o l l o w e d by incorporated  under  a into  the  f o r c e s can  be  s  S  (C-Ci)  (18)  S  (Ci-Cs)  (19)  i s the  (coefficient  surface  rate of a r r i v a l  from the b u l k into'the solid 18  steps.  occuring  solute concentration  i s the  Equations  steps,  diffusion  of  surface,  transport rate constant  C i i s the  interface,  film  equations:  and  t r a n s f e r by  solid  some  i s not  of c r y s t a l  solute molecules  T h e s e two  R  i s the  reached  (1923a,b;  whereby s o l u t e m o l e c u l e s a r e  lattice.  represented  theory  Marc  increase  that  Valeton  g r o w t h i n v o l v e d two  influence of d i f f e r e n t  where  (1912) and  r e a c t i o n where  crystal  d i d not  that c r y s t a l l i z a t i o n  diffusion  relative  degree o f a g i t a t i o n .  t o e x p l a i n t h e mechanism  the  that crystal  order  the  o f d i s s o l u t i o n and  Berthoud  d i f f u s i o n process,  first  i s on  suggested  modified  of the  f i l m d e p e n d s upon t h e  observed that K  reverse  crystallization.  the bulk  that  sufficient  consequently  liquid  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 He  the  i s not  suggested  of the  and  19  lattice  are  of  reaction rate i at the  crystal-solution  of s o l u t e at the  s o l u t i o n and  R  o f mass  s  i s the  rate  surface of  (surface r e a c t i o n ) .  little  use  i n p r a c t i c e because  -64-  th e concentration eliminate  Ci is difficult  the term C i s i n c e  t o measure.  I t i s possible to  a t steady rate,  = R t  1  = R . g  s  Then t h e g r o w t h r a t e c a n be w r i t t e n a s :  R  g  = K ( o b s ) S (C-Cs) o  (20)  where K ( o b s ) = o b s e r v e d o v e r a l l g r o w t h r a t e Q  and  Equation consecutive surface  1 = K (obs) o  20 d e s c r i b e s  reactions,  r e a c t i o n step,  For  1 +  -  1 K s  t  crystal  transport  g r o w t h when t h e two  o f s o l u t e b y d i f f u s i o n and a  a r e o f comparable v e l o c i t y .  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  aqueous s o l u t i o n , t h e two c o n s e c u t i v e g r o w t h may n o t e x p l a i n several processes processes  constant,  step  f r o m an  mechanism o f c r y s t a l  t h e growth because o f t h e involvement o f  simultaneously  ( M u l l i n , 1972).  Some o f t h e s e  include:  -  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 adsorption  Surface  d i f f u s i o n of solvated  Partial  or t o t a l  Integration -  desolvation  o f ions  ions.  of ions.  into 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 layer.  or unsolvated  layer.  water through t h e a d s o r p t i o n  -65-  Counter d i f f u s i o n Any  through the boundary  o f t h e s e p r o c e s s e s may  render the c r y s t a l Marc  growth  r a t e on  and  be w r i t t e n  = K o  g  (obs) S  greater than f i r s t  FACTORS AFFECTING  (A)  The  (C-Cs)  is and  constant K  e x p e c t e d t o be therefore  behaves  CRYSTAL  g  (21)  n  growth p r o c e s s .  S I Z E AND  f o r the surface  SEED SURFACE  i s independent o f c r y s t a l  as a n o r m a l mass t r a n s f e r size  size.  solution  i n the  solution,  However,  coefficient  and  AREA  integration process  i n d e p e n d e n t o f mass t r a n s f e r  show d e p e n d e n c e on c r y s t a l  order.  GROWTH R A T E S  E F F E C T OF SEED CRYSTAL  rate  of a  as:  where n = t h e o r d e r o f t h e o v e r a l l 2.8.4  complex.  i n many c a s e s t h e d e p e n d e n c e o f  s u p e r s a t u r a t i o n was  R  and  the rate o f c r y s t a l l i z a t i o n  found t h a t  Thus e q u a t i o n 20 may  layer.  become r a t e - c o n t r o l l i n g  phenomenon v e r y  (1908) i n v e s t i g a t e d  number o f s a l t s growth  o f water  K, t  and t h u s s h o u l d velocity  (Phillips,  1973). In the seeded growth seed c r y s t a l s  would  During c r y s t a l o f the seeds effect  result  growth  technique, reduction i n an  i n c r e a s e o f t h e growth  of  rate.  i n the presence o f seeds the s u r f a c e a r e a  increases progressively.  of that  i n the s i z e  i n c r e a s e on t h e c r y s t a l  Little  i s known a b o u t  growth  kinetics.  In a  the  -66-  recent  report,  between  B a r o n e e t a l . (1983) d i s c u s s e d  the increase  growing c r y s t a l .  i n surface  area  the r e l a t i o n s h i p  and mass d e p o s i t e d  The f o l l o w i n g i s a b r i e f  on t h e  summary o f t h e i r  discussion.  The  rate of crystal  growth  f o r a s e e d e d s y s t e m c a n be  interpreted  i n terms o f e m p i r i c a l  theoretical  expressions  (Nancollas,  (O'Hara and Reed,  1978) o r  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 , 1 9 8 0 ) . form o f t h e r a t e e q u a t i o n growing  i n an aqueous  8^.(1983)  (equation  The f u n c t i o n a l  20) f o r many i o n i c  compounds  s o l u t i o n has been d e t a i l e d by Barone e t  and c a n be w r i t t e n a s ,  dc R  where rate  constant  effective the  = (T) X  t  g  K  dt  c^ i s the t o t a l  (22)  <°t>  f  mass  of solid  d e p e n d e n t on t h e t e m p e r a t u r e T,  surface  area  supersaturation,  concentrations  at time  t,  K  (  a t t i m e t , and f ( o ) a t e r m d e p e n d e n t t  o^ =  (C - C s ) / C s , where C and Cs a r e t h e  of precipitating  geometric  effective total  surface  surface  geometry o f t h e c r y s t a l for  example  j  ions or molecules i n s o l u t i o n at  area,  area  S., i s d i f f e r e n t t  ( S ^ ) w h i c h depends  and i s e q u a l  b y BET g a s a d s o r p t i o n .  e  from  only  on t h e  t o the quantity S . depends  a  S^. t h e t o t a l  t i m e 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  T  on t h e  measured,  on  -67-  dimensional  type  o f t h e g r o w t h , and i s t h e q u a n t i t y  determines the rate o f r e a c t i o n . cubic  shaped c r y s t a l s ,  faces  resulting  • Similarly,  For 1 - d i m e n s i o n a l growth o f  growth o c c u r s  i n no i n c r e a s e  only  along  in effective  . f o r 3 - d i m e n s i o n a l g r o w t h S^ =  dimensional  e S  on t h e r a t e o f c r y s t a l ( B a r o n e and N a n c o l l a s , 1970).  changes as t h e s u r f a c e a r e a  ( the  and  f o r two  o f an i n c r e a s e i n  growth i s a p p r e c i a b l e f o r 1978)  In cases  and  negligible  for others  where t h e r a t e o f g r o w t h  increases the r e l a t i v e  increase i n  area  (S./S ) o r e f f e c t i v e t o t a l s u r f a c e a r e a t o ) has a d i r e c t r e l a t i o n s h i p which i s dependent  S./ S t o type  c  o f growth dimension o f the c r y s t a l .  dimensional  S  fc  area.  t  ( L i u and N a n c o l l a s ,  surface  surface  parallel  g r o w t h S > S^.  area  some s a l t s  two  e  As t h e s e e d c r y s t a l s grow, t h e e f f e c t surface  which  t/ o S  For uniform  three  growth  -  where m  %/% =  (  V o m  ) 2 / 3  ( 2 3  and m. a r e t h e t o t a l t  o  >  s e e d masses a t z e r o  time  and a t t i m e , t . (B)  EFFECT  OF DEGREE OF  From t h e g r o w t h e q u a t i o n , proportional confirmed  crystal  to supersaturation.  the d i r e c t  growth r a t e .  SUPERSATURATION  growth r a t e i s d i r e c t l y  Schott  r e l a t i o n s h i p between  (1980)  studied  and  supersaturation  Theoretical calculations indicate that for  and  on  -68-  dis.location the  c o n t r o l l e d growth, the  square of  controlled  the  1961).  The  degree  crystal.  allows  of  A high  preferential leading  for a  when a h i g h  (C)  supersaturation  degree o f  formation  fast  degree of  EFFECT  OF  equation  (Arrhenius,  absolute  studied  and  determined.  Phillips  and  controlled  Epstein  w i t h an by  the  whereas a t h i g h controlled  by  cause  of  a  the  particular direction,  i s present  growth r a t e  temperature  the  This  habit  solid  phase  (Mullin,  1961).  constant,  i s given  by  the  Some o f (1974).  increase surface  crystal  Arrhenius  g r o w t h r a t e has  energy of a c t i v a t i o n f o r the the  studies  have been  The  growth r a t e o f c r y s t a l s  i n t e m p e r a t u r e and  is  r e a c t i o n mechanism a t  temperatures  the  i n one  can  habit  1880).  reaction  increases  a l s o a f f e c t the  o f n e e d l e shaped c r y s t a l s .  e f f e c t o f t e m p e r a t u r e on  extensively  can  dependence  TEMPERATURE  and  The  exponential  supersaturation  to  surface-nucleation  supersaturation  r e l a t i o n s h i p between the the  is proportional  r a t e o f h e a t d i s s i p a t i o n from the  K (obs), Q  r e s u l t i n an  growth o f a c r y s t a l  to the  The  d r i v i n g force while  growth w i l l  (Mullin,  growth r a t e  the  crystallization  d i f f u s i o n process  (Mullin,  been growth  referenced  by  generally low is  1961).  temperatures, generally  -69-  (D)  The  EFFECT  OF DEGREE OF A G I T A T I O N  r a t e a t which a c r y s t a l  under c o n s t a n t seed c r y s t a l s  conditions of supersaturation  relative  conditions An  v e l o c i t y between c r y s t a l  o f the film  controled  growth.  of crystal  i n the i n i t i a l and l i q u i d  The  stages  as  increases, but  when f u r t h e r a g i t a t i o n h a s no  effect.  on t h e c r y s t a l  Therefore  at high  g r o w t h depends s o l e l y  surface  for diffusion  rates of a g i t a t i o n the rate  on t h e r a t e o f t h e s u r f a c e  process.  (E)  The  and s u r f a c e a r e a o f  i n the a g i t a t i o n rate o f the s o l u t i o n decreases the  thickness  reaction  considerably  a r e soon reached  increase  temperature  c a n be a l t e r e d b y a g i t a t i n g t h e s o l u t i o n .  r a t e o f growth i n c r e a s e s the  grows a t a g i v e n  EFFECT  OF IMPURITIES  p r e s e n c e o f i m p u r i t i e s can have a p r o f o u n d  growth o f a c r y s t a l .  Some i m p u r i t i e s s u p p r e s s g r o w t h ,  enhance g r o w t h and o t h e r s Impurities equilibrium  interface.  layers across  of the adsorption  effect  the faces.  some  habit.  They c a n change t h e  layer at the c r y s t a l - s o l u t i o n f a c e s and  and t h u s d i s r u p t t h e f l o w o f g r o w t h T h e y may be b u i l t  chemically with  Impurities  on the  c a n change t h e p r o p e r t i e s o f t h e s o l u t i o n o r t h e  T h e y may be a d s o r b e d o n t o t h e c r y s t a l  exert a blocking  may i n t e r a c t  may m o d i f y t h e c r y s t a l  saturation concentration.  characteristics  effect  i n t o the c r y s t a l s or  the c r y s t a l s .  t h a t h a v e a common i o n a c c e l e r a t e t h e g r o w t h  -70-  p r o c e s s by action. low  reducing  Impurities  Impurities onto the  a common i o n  impurities  origin  to the  can  law  o f mass  accelerate  growth  concentrations.  a c t as  r e t a r d the  It  at  has  diffusion retarders.  g r o w t h by  being  adsorbed  faces.  been suggested t h a t  the  action of  impurities  is  to:  (1)  The  electric  c h a r g e and (2)  (3)  (Mullin,  2.8.5  ions  ( g o v e r n e d by  i n t e r a c t i o n between t h e  crystal,  or  formation  ionic  o f complex  i n a decrease  aquo  i n the  impurity  and  the  ions, growth r a t e o f c r y s t a l s  1972).  (A)  C R Y S T A L GROWTH R A T E  F A C E GROWTH R A T E S  The  d i f f e r e n t faces  different  rates  crystal  orientation. around  o f the  radius),  D E T E R M I N A T I O N OF  small  field  chemical  resulting  face  according  reduce i t at high  these  of organic  I t has due  but  that  crystal  solubility  without  concentrations  been suggested  the  the  of  under i d e n t i c a l  i s mounted on A  a non-isotropic  c r y s t a l may  environmental conditions.  a tungsten wire  and  the  rate of  A  in a particular  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  mounted c r y s t a l  grow a t  is circulated  advance o f  i s observed through a t r a v e l l i n g microscope  the  (Mullin,  crystal 1972).  (B)  OVERALL GROWTH RATES  The  measurement o f c r y s t a l  mass d e p o s i t e d convenient. on  per  This  u n i t time per can  a known mass o f  conditions. information  In the suspended  Two on  growth r a t e s  be  done by  fluidised  growth  and  can  bed  A  be  determining  method t h e  given  density by  s e c o n d method  glass  u s e d by  kinetics  of  Mullin  involves  this  D a v i e s and  silver  have appeared  mass  deposited  kept the  Solution  i n t e r v a l s or  continuously  details  of  the  by  apparatus  (1972).  the  Jones  chloride  i n the  technique to  is  reliable  controlling  column.  m e t e r . 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 first  the  surface  seed c r y s t a l s are  s o l u t i o n by  determined at  recording  method a r e  of c r y s t a l  the  kinetics.  i n a supersaturated  a  function of  methods h a v e b e e n u s e d t o o b t a i n  solution velocity in a vertical  means o f  a  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  crystal  concentrations  u n i t area  as  use  of  a stirrer  solution.  to  suspend  T h i s method  the  was  (1949) t o s t u d y t h e p r e c i p i t a t i o n  from aqueous  literature  s o l u t i o n s . Many  over the  last  reports  s e v e r a l years  study the k i n e t i c s of c r y s t a l  using  g r o w t h o f many  electrolytes.  D a v i e s and and  suggested  Jones  t h a t the  a n i o n s were d i r e c t l y crystals. arrival  (1949) p r o p o s e d a model f o r c r y s t a l rates of adsorption  p r o p o r t i o n a l t o the  They p r o p o s e d  o f p o s i t i v e and  that  i n the  negative  f o r the  surface  event of  ions a t the  growth  cations  area  of  and  seed  simultaneous growth s i t e ,  the  -72-  r a t e o f growth i s g i v e n  R  and  anions  concentrations this  (obs)  , Cs + , C  where C cations  = K o  g  equation  by:  S  (C  and  a t the  o f the reduces  - Cs  +  Cs  )(C  are the  - Cs  concentrations  s u p e r s a t u r a t i o n and  electrolyte.  (24)  )  For  of  the s a t u r a t i o n  equal  ion concentrations,  to:  (25)  The  rate constant  generally fluid  found  t o be  f o r the  independent of the  dynamics under e x p e r i m e n t a l  1970).  Thus t h e  solid  d e p o s i t e d may  weight o f seed considerable  crystals  growth d a t a w i t h o u t  equation  is sufficient  the e f f e c t i v e  r a t e and  During  these  experiments,  surface area  Therefore during  appears t o adequately  the is a  experiments.  describe  the  f o r the  i n t o the  rate  equation.  evidence  t o show t h a t i n many s y s t e m s  g r o w t h a r e a becomes c o n s t a n t  the  t h e mass  there  the  the  i s not  to three times  initially.  been  Nancollas,  diffusion  i n t r o d u c i n g a term t o account  i n surface area  There  added  compounds h a s  stirring  s o l u t e by  as much as two  i n c r e a s e i n the  However, t h e k i n e t i c  increase  be  ionic  c o n d i t i o n s ( L i u and  transport rate of  r a t e c o n t r o l l i n g mechanism. of  growth o f  a t some s t a g e  in  the  -73-  growth p r o c e s s (Nancollas  even though t h e c r y s t a l s  and P u r d i e ,  1961).  The r a t e  however, d i r e c t l y p r o p o r t i o n a l (Doremus,  Two  i n size  constant  (K )i s , 1  t o t h e amount o f s e e d  crystals  1958).  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  i o n s combine t o form a n e u t r a l to  increase  t h e growth s t e p .  The r a t e  salt  t o the f i r s t  model,  molecule which then  adsorbed diffuses  o f formation o f the surface 3  m o l e c u l e s was p r o p o r t i n a l electrolytes  and ( C - C s )  model, t h e o p p o s i t e l y crystal a kink  for  4  charged  i n a growth s t e p . 2  (C-Cs)  for  includes  molecules  t o (C-Cs)  into  into the layer at  o f c r y s t a l growth i s then  1:1 a n d 2:2 e l e c t r o l y t e s a n d  The r a t e  constant  s u c h as p r o b a b i l i t y  the c r y s t a l  In t h e second  d i r e c t l y from t h e adsorbed  2:1 e l e c t r o l y t e s . a l l factors  1:1 a n d 2:2  ions are incorporated  The r a t e for  for  2:1 e l e c t r o l y t e s .  surface alternately,  proportional 3  25  t o (C-Cs)  lattice  i n equation  o f incorporation  of  and f r e q u e n c y o f c o l l i s i o n  w i t h growth s i t e s . 2.9 MSUM SOLUTIONS Uric and  acid  Dikstein,  obtained  i sa dibasic  1955). A l k a l i n e  forming the s a l t  formed when s o l u t i o n s 50°)  a r e mixed  partially  acid  (pKa 5.75 a n d 10.3)  solutions  of uric  acid  of uric  acid  and a l k a l i .  o f sodium h y d r o x i d e and u r i c  and l e f t  t o stand.  i n aqueous s o l u t i o n  Monosodium  (Bergmann  MSUM i s acid  urate  t o sodium and u r a t e  are readily  (pH 8.9, dissociates  i o n s a n d shows  -74-  typical 2.9.1  reactions  of uric  acid  in alkaline  conditions.  D E G R A D A T I O N O F MSUM S O L U T I O N S  U r a t e s a r e decomposed b y a number o f b a c t e r i a , m o l d s , plants  and a number o f a n i m a l  responsible  for this  tissues.  An enzyme u r i c a s e 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 Cl.acidiurici  convert uric  carbon d i o x i d e identified  1970).  1933; M i k h l i n  In-vitro  reaction. acid  h a v e shown t h a t  alkalis  cause  acid  (pH 7.2-8.5)  (Cananllakis  decomposition  and May,  enzymes p r e s e n t i n o r g a n  pteridines.  i n part  anaerobic  1934).  Soybean  into allantoin  t h e end p r o d u c t s o f  1911).  extracts.  uricase  of uric This  of the  oxidizes  uric  a t pH 7.2 u r e a and  and Cohen, acid  1955). (Austin,  decomposition  t o be s i m i l a r t o t h e d e c o m p o s i t i o n  acid,  et a l . ,  a c i d depends on t h e c o n d i t i o n s  buffer  a c i d a r e formed  1911; S t e v e n s  reported  uric  ( M i k h l i n and R u i z o v a ,  has been  ( T r u s z k o w s k i and  and k i d n e y t i s s u e s c a u s e  t o a l l a n t o i n , whereas i n b o r a t e b u f f e r  All May,  of uric  In phosphate  alloxanic  Uricase  1937).  experiments  oxidation  ammonia,  1934; F a n e l l i  u r i c a s e which c o n v e r t s u r i c  ( E c h e v i n and B r u n n e l ,  f o r example,  into  ( B a r k e r , 1938).  and R u i z o v a ,  from b r e a s t  and f o r m u r e a  contain  uricase  acid anaerobically  and a c e t i c a c i d  Extracts  uricolysis seeds  genus,  i n t h e t i s s u e s o f a number o f a n i m a l s  Goldmanowna,  some  caused  Water a t 2 0 0 ° ,  t o a m i x t u r e o f p t e r i d i n e s and  by  1903;  was uricolytic  converts pyrimido(5,4g)  -75-  Uric  acid  allantoin,  i nalkaline  oxonic acid,  on t h e c o n d i t i o n s 2.9.2  solution oxidizes  rapidly to  allantoxadin or oxaluric  (Dalgliesh  and Neuberger,  acid  depending  1954).  S O L U B I L I T Y OF MSUM  The several  solubility studies.  equilibrium  o f MSUM i n w a t e r h a s b e e n t h e s u b j e c t o f  The s o l u b i l i t y  s o l u b i l i t y method  (Wilcox and K h a l a f ,  o f MSUM was d e t e r m i n e d b y t h e  ( L o e b , 1972), h o t s t a g e m i c r o s c o p y  1975) a n d b y s e e d i n g a 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 f o l l o w e d b y r e p e a t e d a n a l y s i s o f t h e s o l u t i o n until  a constant result  solubility of  was o b t a i n e d ( F i d d i s  et al.,  o f MSUM i s a f u n c t i o n o f t e m p e r a t u r e  a physiological  sodium  ion concentration.  1983). The  i n the presence  Figure  18 shows t h e  r e l a t i o n s h i p between t h e temperature and t h e s o l u b i l i t y determined by v a r i o u s groups o f workers. Amorphous 18°.  MSUM h a s a n a p p a r e n t  On s t o r a g e o f t h i s  solution  solubility  t h e apparent  d e c r e a s e s a n d becomes t h a t o f t h e c r y s t a l l i n e a b o u t 0.85 g L  -  1  a t 18° (Barkan,  Supersaturated solutions  solutions  containing  MSUM was a u t o c l a v e d  1922).  converted  into  The c o l l o i d a l a stable  solubility form, w h i c h i s  t o 70° followed by c o o l i n g  (Kohler  1913).  However, when  a t 1 2 1 ° f o r 30 m i n u t e s t h e  formation o f a supersaturated (Schade,  1  o f MSUM p r e p a r e d b y h e a t i n g  solutions  i n water  -  1922).  excess s o l i d  were f o u n d t o b e t r u e  o f 2.03 g L  colloidal  dispersion  form o f sodium  granular  form a f t e r  urate  resulted i n water i s  s t o r a g e (Barkan,  at  -76-  1924).  MSUM i n a s u p e r s a t u r a t e d  metastable  condition,  take p l a c e .  i n which  The u p p e r l i m i t  s o l u t i o n may e x i s t i n a spontaneous p r e c i p i t a t i o n does n o t  o f t h i s metastable  s o l u t i o n o f MSUM was f o u n d t o be a b o u t concentration soluble  concentration  The  state  saturation  and  synovial  solubility  difference  difference  fluid  i s 5 times t h e s a t u r a t i o n  o f MSUM i n p l a s m a and s y n o v i a l p a t i e n t s has been  i n the s o l u b i l i t y  i nhealthy  i sgreater  s o l u t i o n t h e upper l i m i t o f  solution.  from normal and a r t h r i t i c  There i s a small  i s t e n t i m e s more  i t i s i n a 1% s o l u t i o n o f s o d i u m  f o r sodium u r a t e  for this  f o r a pure  2.5 t i m e s t h e s a t u r a t i o n  Sodium u r a t e  b u t i n a 1% s o d i u m c h l o r i d e  metastable  fluid  1913).  i n pure water than  chloride, the  (Kohler,  state  individuals.  determined.  o f MSUM i n p l a s m a  However,  this  i n patients with various a r t h r i t i d e s  (Dorner e t a l . , 1981).  Bovine nasal solubilizing Schubert sulfate the  e f f e c t o n MSUM i n b u f f e r  (1970) s t u d i e d  the  1978).  whereas c h o n d r o i t i n  increase  i nsolubility  effect of chondroitin  chondroitin that  amounts o f  s u l f a t e and albumin p r o d u c e d a  e v e n when p r e s e n t  i n large  s u l f a t e on t h e s o l u b i l i t y  disagreement with Laurent solubility  Katz and  o f MSUM a n d r e p o r t e d  MSUM s o l u b i l i t y was g r e a t l y e n h a n c e d b y s m a l l  slight  in  (Katz,  the e f f e c t o f proteoglycans,  a n d a l b u m i n on t h e s o l u b i l i t y  proteoglycan,  The  c a r t i l a g e h a s b e e n shown t o h a v e an a p p r e c i a b l e  (1964) who r e p o r t e d  amounts.  o f MSUM was  a decrease i n  o f MSUM i n t h e p r e s e n c e o f c h o n d r o i t i n  sulfate.  -77-  Perricone urate  and B r a n d t  solubility  proteoglycan  by connective  aggregate  o f proteoglycan  digestion  This  proteoglycan (Perricone  on u r a t e  t h e d i s s o l u t i o n o f a b o u t 2.5  solubility  a n enzyme h y a l u r o n i c  solutions after  aggregate  They a l s o r e p o r t e d  a g g r e g a t e d i d n o t s u s t a i n sodium u r a t e  that the  concentration i n  24 h o u r s .  Perricone  and B r a n d t  (1979) r e p o r t e d  t h a t t h e enhancement o f s o d i u m u r a t e  by  aggregate  proteoglycan  (prepared  i o n s ) was due t o t h e c a t i o n i c ions.  Potassium entered  more s o l u b l e t h a n in  sodium u r a t e  2.10  solubility  i n thepresence o f potassium  exchange o f p o t a s s i u m w i t h  sodium  t h e s o l u t i o n and p o t a s s i u m u r a t e  sodium u r a t e  r e s u l t e d i n t h e observed  being  increase  solubility.  NUCLEATION AND CRYSTAL GROWTH OF MSUM Khalaf  and W i l o x  (1973) a n d W i l c o x a n d K h a l a f  developed the h o t stage  microscope technique  n u c l e a t i o n o f MSUM a n d f o u n d t h a t c a l c i u m increased for  a c i d B l-> 3  s o l u b i l i t y the  as a l a r g e macromolecular 1978).  This  was a b o l i s h e d b y  indicated that t o increase urate  and Brandt,  that  than non-aggregated p r o t e o g l y c a n s .  must e x i s t  supersaturated  t h e enhancement o f  t i s s u e components a n d f o u n d  o f the aggregate with  hydrolase.  1979) s t u d i e d  facilitated  t i m e s more s o d i u m u r a t e effect  (1978,  t o observe t h e  and h y d r o g e n  ions  t h e n u c l e a t i o n r a t e o f MSUM a s d e t e r m i n e d b y t h e t i m e  t h e appearance o f observable  to have a mixed e f f e c t fluid  (1975)  nuclei.  i o n s were  o n t h e number o f n u c l e i f o r m e d .  from gouty p a t i e n t s i n c r e a s e d  However, s y n o v i a l f l u i d  Cupric  found  Synovial  t h e number o f n u c l e i f o r m e d .  f r o m r h e u m a t o i d p a t i e n t s was f o u n d t o  -78-  inhibit  nucleation.  Tak e t a l . (1980) s t u d i e d  o f MSUM u n d e r p h y s i o l o g i c a l c o n d i t i o n s ionic  concentration  greatly  enhanced by s y n o v i a l  synovial  fluid  moderately arthritis also  had  from d e g e n e r a t i v e  patients  no e f f e c t .  nucleation Nucleation  more s e n s i t i v e t o u r a t e and ions  slightly  The has  disease  and f l u i d  from  a c i d and p u r i n e s  and o t h e r  rheumatoid Tak e t a l .  agents.  tissue  (Tak  a n d W i l c o x , 1980)  q u a n t i t i e s o f p o t a s s i u m a n d magnesium o f MSUM.  1965a).  were  shown t o i n h i b i t  solutions  that the  c r y s t a l s was i n h i b i t e d b y s u r f a c e g r o w t h r a t e o f MSUM c r y s t a l s i n t h e  p r e s e n c e o f 0.1% b e n z a l k o n i u m c h l o r i d e was r e d u c e d al.,  components  h a s b e e n shown t o b e  A l l e n e t al.(1965a,b) reported  The l i n e a r  (1980)  had a minimal  g r o w t h o f MSUM c r y s t a l s f r o m s u p e r s a t u r a t e d  growth o f t h e needle-shaped  whereas  patients  connective  o f sodium u r a t e  inhibited the nucleation  been s t u d i e d .  active  joint  ion concentration  addition of physiologic  o f MSUM was  from g o u t y p a t i e n t s ,  inhibited the nucleation.  that hyaluronic  on u r a t e  o f t e m p e r a t u r e , pH a n d  the nucleation  fluid  enhanced n u c l e a t i o n  reported  effect  and found t h a t  the nucleation  (Allen et  Dyes s u c h a s b i s m a r c k brown a n d m e t h y l e n e the crystal  growth  o f MSUM  blue  (Allen eta l . ,  1965b). The  s e e d e d g r o w t h t e c h n i q u e was u s e d b y E r w i n a n d N a n c o l l a s  (1981) t o d e t e r m i n e t h e r a t e o f g r o w t h o f MSUM and t h e e f f e c t o f additives  on t h e g r o w t h o f MSUM.  MSUM f o l l o w e d controlled  t h e square  law w h i c h  growth p r o c e s s .  They showed t h a t  t h e growth o f  indicated a surface  Methylene blue  reaction  , a c a t i o n i c dye,  -79-  significantly  reduced the r a t e o f c r y s t a l  ppm, w h e r e a s ,  organic  negligible  They  p h o s p h o n a t e and s o d i u m h e p a r i n  e t a l . (1983)  studied  thepoisoning  effect of  r e d d y e a n d serum a l b u m i n o n t h e c r y s t a l  determined the c r y s t a l l i z a t i o n  of the additives.  solution increased  -  1  n e u t r a l r e d t o a 70  the c r y s t a l l i z a t i o n  t i m e b y 50 m i n u t e s . A l b u m i n a t a c o n c e n t r a t i o n thec r y s t a l l i z a t i o n  of the l i n e a r  inhibitor  F i d d i s e t a l . (1983)  reported  _  1  Heparin  o f urate t h e dependence  g r o w t h r a t e o f MSUM on t h e s u p e r s a t u r a t i o n .  Expressed  a s a power law, t h e l i n e a r  vary  supersaturation  with  o f 10 g L  t i m e b y a f a c t o r o f 4.  (0.01%) was shown t o be a n e f f e c t i v e cystallization.  g r o w t h o f MSUM.  time a t 37° i n thepresence  A d d i t i o n o f 33 mmol L  mmol L ^ s o d i u m u r a t e  increased  had a  effect.  Fiddis neutral  g r o w t h o f MSUM a t 13  g r o w t h r a t e was f o u n d t o  defined as:  ((Na+)(HU-))  1/2  4.5  1/2 •( (Na+) (HU-) )  which they proposed f i t t e d growth.  t h e s c r e w d i s l o c a t i o n model  of crystal  -80-  •• <9  •  O  Oi  0.03  0.06  SODIUM URATE CONCENTRATION,  Figure 18.  Solubility  0.09 mmol L  of sodium urate i n normal  saline.  (•) Wilcox et a l . , 1975 by hot stage; (o) Wilcox et a l . . 1975; (•) A l l e n et a l . . 1965; (o) Erwin and Nancollas, 1981; and  (*) F i d d i s et a l . . .1983-  -81-  3 3.1.  EXPERIMENTAL  INSTRUMENTS A u t o c l a v e , AMSCO G e n e r a l p u r p o s e , Atomic  American  Sterilizer.  A b s o r p t i o n Flame E m i s s i o n S p e c t r o m e t e r ,  Cahn E l e c t r o b a l a n c e ,  Gram V e n t r o n C o r p o r a t i o n .  C o n s t a n t Temperature  Water C i r c u l a t o r ,  Differential Electronic  Stroboscope, Flashtac  r e d Spectrophotometer,  Oven,  Isotemp,  Meter,  Fisher  IR 10, Beckman.  Scientific.  Accumet, F i s h e r  Scientific.  S c a n n i n g E l e c t r o n M i c r o s c o p e , ETEC Fisher  Stedi  Elmer.  Electronic Corporation.  R o t a r y Vacuum E v a p o r a t o r , R o t a v a p o r ,  Stirrer,  Haake F T .  S c a n n i n g C a l o r i m e t e r , DSC-1B, P e r k i n  Infra  pH  J e r a l d Ash.  Speed  Laboratories.  Autoscan.  Stirrer,  S u r f a c e Area A n a l y z e r , Quantasorb  Buchi  Fisher  Scientific.  , Quantachrome  Corporation U.V  Spectrophotometer,  Ultrasonic  Becman Model 24.  Cleaner, Mettler  Water Bath Shaker,  Electronics.  Aquatherm, New B r u n s w i c k  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 , Brunswick  Scientific  F r i d g e d f l o w Bath C i r c u l a t o r , Co. I n c .  X-Ray D i f f r a c t o m e t e r , Wide A n g l e ,  Philips.  New  -82-  3 . 2 .  MATERIALS  Albumin,  f r a c t i o n - V , from b o v i n e  Aseptic  filtration  Calcium  c h l o r i d e , B.D.H.  Chondroitin  Disposable  unit, Millipore  sulfate,  cartilage  sodium s a l t ,  (99%),  Sigma  filtration  units,  acid, salt  (0.1%  Human U m b i l i c a l C o r d , Membrane f i l t e r s ,  Phosphatidylserine  (40%), (80%),  Corporation.  f r o m Whale o r S h a r k  (Millex-GS,0.22um, Corporation.  sodium,  Sigma  Millipore  Phosphatidylcholine  Chemicals.  Chemicals.  M i l l e x - H A , 0 . 8 urn), M i l l i p o r e Hyaluronic  serum, Sigma  9.7% p o t a s s i u m ) ,  from  Chemicals.  Corporation. from Soybean, Sigma  from Bovine B r a i n ,  Chemicals.  Sigma  Chemicals. Potassium c h l o r i d e , Analar,  B.D.H.  *  Proteoglycan  aggregate,  Proteoglycan  monomer, (A1D1 f r a c t i o n ) .  (*) o b t a i n e d  f r o m D r . Mark Adams, F a c u l t y o f M e d i c i n e ,  *  U n i v e r s i t y o f B.C. Appendix.  (Alfraction) .  Method o f p r e p a r a t i o n  given i n  -83-  Sodium c h l o r i d e ,  ACS,  Fisher  Sodium h y d r o x i d e , ACS, Sterile  evacuated  glass  Scientific.  Fisher tubes,  Scientific. red top Vaccutainer, Becton  Dickinson. Uric  acid  ( 9 9 % ) , Sigma  Chemicals.  -84-  3.3  METHODS  3.3.1  P R E P A R A T I O N O F MSUM  Crystals  o f MSUM were grown a c c o r d i n g t o t h e method  d e s c r i b e d b y Denko and W h i t e h o u s e acid was  (6.0 g L ) a n d 1 M s o d i u m h y d r o x i d e 1  left  formed  hours  times  with  cold  a t 5 5 ° a n d pH 8.9  filtration  distilled  water  i n a c i r c u l a t i n g h o t a i r oven.  thepreparation of solutions  seed  A solution of uric  t o s t a n d o v e r n i g h t a t room t e m p e r a t u r e .  were s e p a r a t e d b y s u c t i o n  several  for  (1976).  crystals  (batch A seed  Batch  i n thepreliminary  The c r y s t a l s  a n d were  and d r i e d  rinsed a t 6 0 ° f o r 12  T h e s e c r y s t a l s were  i n v a r i o u s experiments crystal  growth  and as  experiments  crystals).  B seed  supersaturation  crystals,  and p r e s e n c e  used  t o study t h e e f f e c t o f  o f a d d i t i v e s on c r y s t a l  g r o w t h , was  p r e p a r e d b y a m o d i f i c a t i o n o f t h e method d e s c r i b e d a b o v e . solution of uric  acid  (6.0 g L  through  membrane f i l t e r  The f i l t r a t e  with cold, hours  while hot.  distilled  was l e f t  w a t e r and d r i e d  t o stand  several  times  a t 6 0 ° f o r 12  i n a c i r c u l a t i n g h o t a i r oven.  D r y i n g o f MSUM samples r e s u l t e d cake.  a 0.22 um M i l l i p o r e  The c r y s t a l s were s e p a r a t e d , r i n s e d filtered,  A  ) and 1 M sodium h y d r o x i d e a t  5 5 ° a n d pH 8.9 was f i l t e r e d  overnight.  used  Samples were t h e r e f o r e g r o u n d  i n the formation o f a hard i n a g l a s s m o r t a r and  -85-  pestle prior  t o t h e i r use i n v a r i o u s  experiments.  The g r o u n d  b a t c h B s e e d c r y s t a l s were p a s s e d t h r o u g h a s e t o f s i e v e s (# 50/80, US s t a n d a r d ) o f mesh s i z e  180 jim t o 300 jam i n an a t t e m p t  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  (A)  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  ULTRA-VIOLET  Approximately in  SPECTROSCOPY  10 mg o f g r o u n d MSUM c r y s t a l s were  100 mL o f d i s t i l l e d  solution  water.  A suitable  dissolved  dilution of this  was s c a n n e d f r o m 320 nm t o 230 nm on an u l t r a - v i o l e t  spectrophotometer. (B)  INFRA-RED  SPECTROSCOPY  Approximately approximately of  2 mg o f a n MSUM sample was m i x e d  200 mg o f a n h y d r o u s p o t a s s i u m b r o m i d e .  t h i s m i x t u r e was c o m p r e s s e d  u s i n g a d i e and p u n c h was  into  a disc  with A portion  a t 10 t o n p r e s s u r e  i n a hydraulic press.  The c o m p r e s s e d  disc  s c a n n e d i n t h e 625-3800 cm ^ r a n g e u s i n g a n i n f r a r e d  spectrophotometer.  (C)  X-RAY  DIFFRACTION  Approximately packed to  i n a glass  CuKCC r a d i a t i o n  100 mg o f a g r o u n d MSUM sample was t i g h t l y sample h o l d e r a wide  ( t h i c k n e s s = 0.1cm) and e x p o s e d  angle X-ray diffTactometer a t a  s c a n n i n g r a t e o f 2 d e g r e e s o f 20 p e r m i n u t e . The  location  and i n t e n s i t y o f t h e p e a k s a t d i f f e r e n t  values  -86-  of  (D)  29 were  studied.  D I F F E R E N T I A L SCANNING  Ground scanning  s a m p l e s o f MSUM were a n a l y s e d  calorimeter.  minute  and a n a l y s e d  a differential  a t a scanning  i n open aluminum sample p a n s .  water o f h y d r a t i o n  t h e pan a f t e r  e n d o t h e r m i c peak, and t h e p e r c e n t  rate  o f20°  Vaporization o f the  f r o m t h e open pans was  q u a n t i t a t i v e l y by weighing  (E)  using  Samples o f 1-5 mg were w e i g h e d on a  Cahn-Gram e l e c t r o b a l a n c e per  CALORIMETERY  estimated  t h e appearance o f t h e  water l o s s c a l c u l a t e d .  SCANNING E L E C T R O N M I C R O S C O P Y  A small dispersed  q u a n t i t y o f a n MSUM s e e d c r y s t a l  on an SEM sample h o l d e r .  spectrographic  graphite  sample was  The sample was c o a t e d  u n d e r vacuum.  Scanning  with  electron  m i c r o g r a p h s were t a k e n a t 4000 x o r 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)  D E T E R M I N A T I O N O F S U R F A C E A R E A O F MSUM S E E D  The  surface  areas  were d e t e r m i n e d u s i n g Three p o i n t  CRYSTALS  o f t h e two b a t c h e s o f MSUM s e e d the Quantasorb surface  BET s u r f a c e  area  0.072%, 0.104% a n d 0.184%  determinations  area  crystals  analyser.  were made  mole f r a c t i o n k r y p t o n  using  i n h e l i u m gas  mixtures. An  a c c u r a t e l y weighed  in a g l a s s  cell  sample  (approximately  and degassed a t t h e o u t g a s s i n g  O.lg) port  was p l a c e d  f o r one h o u r  -87-  at  60° under  a slow  stream of n i t r o g e n gas.  transfered  t o t h e a d s o r b i n g gas p o r t  nitrogen.  Krypton  from one  and  was  desorbed  bringing water The  the c e l l  the l i q u i d  p r o c e d u r e was  The  f o r 30  nitrogen  t o room t e m p e r a t u r e  a t room t e m p e r a t u r e .  was liquid  of the three krypton-helium mixtures  a l l o w e d t o a d s o r b o n t o t h e MSUM sample removing  cell  cooled with  was  by  The  repeated t h r e e times  flask  by d i p p i n g  desorption  min. and  Krypton quickly  the c e l l  c o u n t was  recorded.  f o r each o f the t h r e e  krypton-helium mixtures.  The  the  ( a t each k r y p t o n c o n c e n t r a t i o n )  mean d e s o r p t i o n c o u n t  calibrated  s u r f a c e a r e a was  calculated  with the d e s o r p t i o n counts accumulated  v o l u m e s o f n i t r o g e n gas  S  where P  =  (1  u s i n g the f o l l o w i n g  P A ) (—) Po Ac  = partial  Nu  Acs  Vc(  in  from  from measured  equation  Pa )  metre square  (26)  RT  pressure of adsorbate,  Po = s a t u r a t e d p r e s s u r e o f a d s o r b a t e , 23 Nu  = A v o g a d r o s number = 6.023 x 10  R  = gas  c o n s t a n t = 82.1  cc  Vc = volume o f c a l i b r a t i o n Pa = a m b i e n t A  = signal  ,  atm./mole.degree, gas,  p r e s s u r e i n atmosphere, area  ( d e s o r p t i o n c o u n t o f sample  Ac = a r e a o f c a l i b r a t i o n calibration  (desorption count  ),  of  gas,  Acs= c r o s s s e c t i o n a l a r e a of adsorbate molecules i n s q u a r e m e t e r s ( f o r k r y p t o n , 19.5 x l O _5 m e t e r  -88-  square p e r atm.),  T  = temperature temperature  of calibration i n degree  A N A L Y S I S O F MONOSODIUM U R A T E  3.3.3  volume f o r  ambient  Kelvin. MONOHYDRATE  U r a t e 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 a n a l y z e d b y m e a s u r i n g the absorbances  a t 292 nm u s i n g  an u l t r a - v i o l e t  recording  spectrophotometer. A u r a t e s t a n d a r d c u r v e was p r e p a r e d b y d i s s o l v i n g an a c c u r a t e l y weighed, volumetric  flask  200 mg sample o f MSUM i n w a t e r  in a  a n d m a k i n g up t o 100 mL w i t h w a t e r .  further dilution,  t h e absorbances  After  were i m m e d i a t e l y r e a d a t 292  nm.  D E G R A D A T I O N O F MONOSODIUM U R A T E  3.3.4  (A)  NON-STERILE  SOLUTIONS  A  solution  0.2 g L  1  mL o f t h e s o l u t i o n flasks in  fitted  temperature equilibrated  added  and  t o each glass  of five,  stoppers.  SOLUTIONS  was p r e p a r e d 150 mL g l a s s  a n d 125  Erlenmeyer  One f l a s k was s t o r e d  ( 4 ° ) , one f l a s k was k e p t a t room  ( 2 2 ° ) , one f l a s k was s t o r e d  i n a water  bath  a t 4 5 ° and r e m a i n i n g f l a s k s were s t o r e d  c i r c u l a t i n g h o t a i r oven of  o f MSUM i n w a t e r  w i t h ground  the refrigerator  MONOHYDRATE  a t 3 5 ° and 65°.  s o l u t i o n were w i t h d r a w n 96 h o u r s , a n d a f t e r  from each  flask  further dilution,  s o l u t i o n s was r e a d a t 292 nm.  Aliquots after  in a  o f 3 mL  6, 24, 48, 72,  t h e absorbance  of the  -89-  (B)  STERILE SOLUTIONS All  s o l u t i o n s were p r e p a r e d w i t h f r e s h l y  d e i o n i z e d water 121°  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  f o r 30 m i n u t e s .  A 0.2 g L approximately Millipore into  which  distilled,  s o l u t i o n o f MSUM i n w a t e r 10 mL o f s o l u t i o n  f i l t r a t i o n unit  each o f s i x s t e r i l e ,  Dickinson).  filtered  fitted  through a glass  w i t h a 0.22 jam f i l t e r  r e d t o p Vacutainer tubes  The t u b e s were s t o r e d  at 4°.  mL sample was removed f r o m one t u b e  After  solution  Samples were a l s o t a k e n a t t i m e  72,  a n d 96 h o u r s . The s a m p l e s  the  s o l u t i o n s was r e a d a t 292 nm.  for  tubes  stored  also  described  f i l t r a t i o n unit  directly  above.  were d i l u t e d  a 3  o f 24, 48,  and t h e absorbance o f  T h i s p r o c e d u r e was r e p e a t e d  fitted  through a l a r g e  w i t h a 0.22 urn f i l t e r a n d  into  stoppers. sterile,  The f l a s k s  t u b e s were a l l o w e d t o c o o l  r e d t o p V a c u t a i n e r tubes as  a n d t u b e s c o n t a i n i n g MSUM  t o room t e m p e r a t u r e  one o f e a c h o f t h e f l a s k s  sterile  The MSUM s o l u t i o n was  were t h e n a u t o c l a v e d a t 1 2 1 ° f o r 35 m i n u t e s .  in  6 hours,  intervals  (0.2 g L ^) was f i l t e r e d  flasks with glass  filtered  (Becton  i n t h e tube  125 mL o f t h e s o l u t i o n p l a c e d i n t o e a c h o f f i v e , Erlenmeyer  directly  a t 2 2 ° , 3 5 ° , 4 5 ° and 6 5 ° .  An MSUM s o l u t i o n scale Millipore  syringe  using a s t e r i l e disposable  s y r i n g e and n e e d l e and t h e r e m a i n i n g discarded.  was p r e p a r e d and  solution  The f l a s k s a n d and t h e s o l u t i o n s  and V a c u t a i n e r tubes  were  -90-  immediately tubes and  analyzed  a n d f l a s k s were s t o r e d  a t 22°,  and n e e d l e s ,  were f u r t h e r  3 5 ° , 4 5 ° , and 6 5 °  diluted  after  disposable  12, 24, 48, 72 a n d 96 h o u r s .  and t h e absorbance  Samples  o f t h e s o l u t i o n s was r e a d  292 nm.  D E T E R M I N A T I O N 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  3.3.5  URATE  (A)  EFFECT  MONOHYDRATE  OF  I n t o each  TEMPERATURE  o f t h r e e 125 mL E r l e n m e y e r  f r e s h l y washed a n d d r i e d distilled The  g l a s s marbles,  water and t h e f l a s k s  flasks  bath  were e q u i l i b r a t e d  shaker  fitted  fitted  flasks containing  was p l a c e d 100 mL o f with  ground  a t a g i v e n temperature  w i t h h e a t i n g and c o o l i n g  units.  (200-500mg sample) o f MSUM was added t o e a c h shaken v i g o r o u s l y . from e a c h filtered the  The r e m a i n i n g  s a m p l e s removed a s d e s c r i b e d above u s i n g s t e r i l e ,  syringes  at  for the urate concentration.  flask  after  through  Sampling  gave i d e n t i c a l 9.0°, 41.8°,  15.1°, 51.0°  intervals,  An e x c e s s  o f t h e f l a s k s and  o f approximately  v a r i o u s time  2 mL were  withdrawn  immediately  f o r t h e MSUM c o n c e n t r a t i o n a f t e r  u n i t and suitable  was t e r m i n a t e d when t h r e e c o n s e c u t i v e a s s a y s  results.  24.7°,  stoppers.  i n a water  a 0.22 um M i l l e x - G S d i s p o s a b l e f i l t e r  f i l t r a t e assayed  dilution.  Aliquots  glass  Solubilities  29.9°,  and 5 5 . 0 ° .  35.0°,  were d e t e r m i n e d  37.0°,  40.0°,  a t 4.4°,  -91-  (B)  E F F E C T O F E L E C T R O L Y T E S ON T H E S A T U R A T I O N S O L U B I L I T Y O F MONOSODIUM U R A T E MONOHYDRATE  IN  WATER  To d e t e r m i n e t h e e f f e c t o f sodium e l e c t r o l y t e s p r e s e n t i n plasma solubility (0.2,  o f MSUM, v a r i o u s  and s y n o v i a l  concentrations  0.4, 0.6 a n d 1.0 % w/v, f i n a l  m i x t u r e o f sodium  chloride  % w/v) a n d c a l c i u m concentrations) previously  chloride  described flasks  chloride  ), and a  p o t a s s i u m c h l o r i d e (0.03  t o 125 mL E r l e n m e y e r glass  marbles  MSUM samples  vigorously.  flasks  containing  a n d 100 mL o f d i s t i l l e d temperature as  (200mg) were added  t o these  Sample w i t h d r a w a l and a n a l y s e s  were c a r r i e d o u t as d e s c r i b e d were d e t e r m i n e d a t 4 . 4 ° ,  on t h e  o f sodium  e q u i l i b r a t e d a t a given  previously.  and shaken  fluid  (0.035% w/v) ( a l l a r e f i n a l  and d r i e d  water which h a d been  and o t h e r  concentration  (0.78% w/v),  were added  washed  chloride  i n section  3.3.5.(A).  10.4°, 17.6°, 24.7°,  Solubilities  29.9°,  41.8° and 5 1 . 1 ° .  (C)  E F F E C T O F CHONDROITIN S U L F A T E , PROTEOGLYCAN  HYALURONIC A C I D ,  ALBUMIN,  MONOMER A N D P R O T E O G L Y C A N A G G R E G A T E ON T H E  S A T U R A T I O N S O L U B I L I T Y O F MSUM  The  saturation  determined  100  o f MSUM i n w a t e r  i n the presence o f these a d d i t i v e s  concentrations. flasks  solubility  containing  The a d d i t i v e was added previously  mL o f d i s t i l l e d  water.  a t 3 7 ° was  at several  t o 125 mL E r l e n m e y e r  washed and d r i e d g l a s s  m a r b l e s and  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  described  i n section  disposable  and t h e s o l u b i l i t y  3.3.5(A).  Either  determined as  0.45 urn o r 0.8 pm M i l l e x  f i l t e r s were u s e d t o f i l t e r  solutions  c o n t a i n i n g HA  and PGs. The  following  chondroitin hyaluronic albumin  a d d i t i v e s were  sulfate acid  (20, 40, and 60 mg d L ^)  (10, 20 a n d 40 mg dL ^)  (10, 20, 40, 60 a n d 100 mg d L  p r o t e o g l y c a n monomer  )  (20, 40 a n d 100 mg d L ) 1  of thecrystal  growth  apparatus  The s u p e r s a t u r a t e d MSUM s o l u t i o n  placed  temperature  i n a water water  with a cover t o minimize stirrer  1 L vessel  solvent  t o a constant depth  temperature  through a  (2.5 cm f r o m t h e b o t t o m  f o r the  f o r t h e 50 mL r e a c t i o n  C o n s t a n t checks on t h e r o t a t i o n using  An a l l g l a s s  into the solution  a t a c o n s t a n t speed  a l l the experiments  v e s s e l and  a constant  evaporation.  a n d 1 cm f r o m t h e b o t t o m  v e s s e l ) and r o t a t e d  reaction  The r e a c t i o n v e s s e l was f i t t e d  w i t h two p a d d l e s was l o w e r e d  central port  motor.  bath a t 37° using  circulator.  i s shown i n F i g u r e  (1000 mL o r 50 mL) was  i n t h e 1000 mL o r 50 mL c a p a c i t y g l a s s  equilibrated  out  )  CRYSTAL GROWTH OF MONOSODIUM URATE MONOHYDRATE A diagram  19.  - 1  (10, 20, 40 a n d 100 mg dL  proteoglycan aggregate  3.3.6  used:  (200 rpm) b y means o f a s p e e d were made t h r o u g h -  an e l e c t r o n i c  stroboscope.  o f t h e s u p e r s a t u r a t e d s o l u t i o n was m o n i t o r e d  The  Figure 19.  C r y s t a l growth apparatus  -94-  continuously the  second  u s i n g a thermometer  port.  s t o p p e r and was  The used  third  p o r t was  f o r sample  Solutions of d i f f e r i n g p r e p a r e d by g,  5.0  heat  g,  dissolving  6.0  g)  (85 t o 90°)  cooled  s o l u t i o n was transfered  (A)  degrees  filtered  to the r e a c t i o n  samples  (950 mL)  4 5 ° , t h e pH was  through  w i t h a ground  through glass  o f s u p e r s a t u r a t i o n were  constant s t i r r i n g .  t o 1 L w i t h pH  placed  withdrawal.  i n d i s t i l l e d water and  fitted  a c c u r a t e l y weighed  s l o w l y t o about  volume made up  r e a d i n g t o 0.1  o f MSUM  (4.0  with the a i d of  The  solution  was  a d j u s t e d t o 7.4  and  the  7.4-adjusted d i s t i l l e d water. a 0.22  pm  membrane f i l t e r  The  and  vessel.  E F F E C T OF  SUPERSATURATION CONCENTRATION, SEED CRYSTAL  BATCH AND  SEED AMOUNT ON  THE  CRYSTAL GROWTH KINETICS  OF  MONOSODIUM URATE MONOHYDRATE Initial of  crystal  g r o w t h s t u d i e s were c a r r i e d  a supersaturated solution  determine  the e f f e c t  were c a r r i e d  300  mg  mg,  500  solution  in a 1 L reaction vessel.  o f c o n c e n t r a t i o n o f seed  experiments  o u t by  adding e i t h e r  o r 1 g o f seed c r y s t a l s  i n the r e a c t i o n v e s s e l .  filtered  disposable described These  filter  immediately unit,  in section experiments  100  and  mg,  200  mg,  to the supersaturated  time  t h r o u g h a 0.22  diluted  To  crystals,  A l i q u o t s o f 1 mL  medium were w i t h d r a w n a t p r e d e t e r m i n e d minutes,  out u s i n g 1 L  o f growth  intervals  up t o  360  urn M i l l e x - G S  a n a l y z e d f o r MSUM c o n t e n t  as  3.3.5.(A). were r e p e a t e d u s i n g a s m a l l e r s c a l e  6  - 9 5 -  apparatus. L  Supersaturated solutions  MSUM were p r e p a r e d a s d e s c r i b e d  mL g l a s s  reaction  added e i t h e r crystals. 0.22 for  (B)  vessel.  o f 0.3 mL were w i t h d r a w n ,  MSUM c o n t e n t  (see s e c t i o n  (I)  filter  small  scale  were  filtered  through a  d i l u t e d and a n a l y s e d  3.3.5 ( A ) ) .  GROWTH O F  MONOHYDRATE  (50 mL) g r o w t h a p p a r a t u s was u s e d  fora l l  experiments. CHONDROITIN S U L F A T E  Accurately chondroitin  AND A L B U M I N  weighed q u a n t i t i e s  o f 10 mg, 20 mg, o r 30 mg o f  s u l f a t e o r 10 mg, 50 mg, 100 mg, o r 200 mg, o f  a l b u m i n were d i s s o l v e d  i n 5 mL o f d i s t i l l e d w a t e r  t h r o u g h a 0.22 um M i l l e x - G S f i l t e r reaction  vessel.  supersaturated 5.0  g L  for  30 m i n u t e s ,  rpm.  unit,  O F A D D I T I V E S ON T H E C R Y S T A L  MONOSODIUM U R A T E  the  i n a 50  To t h e s u p e r s a t u r a t e d s o l u t i o n s  um M i l l e x - G S d i s p o s a b l e  The  above a n d p l a c e d  5 or 6 g  5 mg, 10 mg, 20 mg, 30 mg, 40 mg, o r 50 mg o f s e e d  Aliquots  EFFECT  (50 mL) c o n t a i n i n g  Seed  To t h e r e a c t i o n  solution  such t h a t  unit directly vessel a final  was o b t a i n e d . T h e s o l u t i o n stirring  crystals  constantly  described  filtered  i n section  immediately 3.3.5 ( A ) .  MSUM c o n c e n t r a t i o n was e q u i l i b r a t e d speed  of a t37°  o f 200  a d d e d a n d 0.3 mL a l i q u o t s o f  g r o w t h medium w i t h d r a w n a t p r e d e t e r m i n e d S a m p l e s were  into the  were added 45 mL o f a  at a stirrer  (40mg) w e r e t h e n  and f i l t e r e d  t i m e s up t o 360 m i n u t e s .  and a s s a y e d  f o r MSUM c o n t e n t a s  -96-  (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  A  MONOMER  5 mg, 10 mg o r 20 mg sample o f h y a l u r o n i c a c i d  10 mg o f p r o t e o g l y c a n monomer  ( A l DI f r a c t i o n  c a r t i l a g e ) was added t o t h e r e a c t i o n v e s s e l filtered in  (0.22  the water.  ""1  minutes seed  solution  o r 6 . 0 g L  —1  and a s t i r r e r  crystals  assayed  (III)  to obtain a final  .  After  speed  were a d d e d .  equilibrating  Aliquots time  intervals,  (see s e c t i o n  filtered  3.3.5 ( A ) ) .  (10 mg t o 50 mg) was  (0.22 jim f i l t e r ) d i s t i l l e d w a t e r i n  (0.22 jim f i l t e r )  supersaturated solution of  c o n c e n t r a t i o n o f 5.0 g L  was 200 rpm and t h e m i x t u r e was e q u i l i b r a t e d  30 m i n u t e s .  diluted  To t h e g r o w t h c e l l were added 45 mL o f a  MSUM t o o b t a i n a f i n a l speed  filtered,  AGGREGATE  i n 5 mL o f f i l t e r e d  previously  o a t 37 f o r 30  o f 0.3 mL o f g r o w t h medium  A sample o f p r o t e o g l y c a n a g g r e g a t e  the growth c e l l .  MSUM c o n c e n t r a t i o n o f  o f 200 rpm, 30 mg, 40 mg, o r 50 mg o f  f o r MSUM c o n t e n t  PROTEOGLYCAN  dispersed  c o n t a i n i n g 5 mL  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  were removed a t p r e d e t e r m i n e d and  bovine  To t h e r e a c t i o n v e s s e l were added 45 mL o f a  supersaturated 5.0gL  urn M i l l e x - G S  from  o r 5mg o r  . The ^ s t i r r e r a t 37° f o r  An a c c u r a t e q u a n t i t y (40 mg) o f s e e d c r y s t a l s  were  added a n d a l i q u o t s  o f 0.3 mL o f g r o w t h medium were w i t h d r a w n a t  predetermined  intervals.  filtered for  time  through  The samples were  immediately  0.8 um M i l l e x - H A f i l t e r s , d i l u t e d  MSUM c o n t e n t a s d e s c r i b e d p r e v i o u s l y  (section  and a s s a y e d 3.3.5 ( A ) ) .  -97-  (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  10 mg, 20 mg o r 30 mg sample o f p h o s p h a t i d y l  mg o r 20 mg o f p h o s p h a t i d y l  serine  c h l o r o f o r m - methanol mixture flask. using  The s o l v e n t a rotary  was d i s s o l v e d  vacuum e v a p o r a t o r .  The t h i n  film  of  o f t h e f l a s k was d i s p e r s e d  filtered  distilled  sonicator. reaction  The p h o s p h o l i p i d  vessel  added t o o b t a i n equilibrating rpm,  water u s i n g  i n 5 mL  s u s p e n s i o n was t r a n s f e r r e d  concentration  -  40 mg o r 50 mg o f s e e d  into the  (45 mL) was  o f 5.0 g L "^.  a t 3 7 ° f o r 30 m i n u t e s a n d s t i r r e r  After  s p e e d o f 200  c r y s t a l s were a d d e d . A l i q u o t s  mL o f g r o w t h medium were f i l t e r e d , c o n t e n t as d e s c r i b e d  phospholipid  an u l t r a -  and a s u p e r s a t u r a t e d MSUM s o l u t i o n a final  bottomed  u n d e r vacuum a t 6 0 °  formed on t h e i n s i d e w a l l (0.22 pm f i l t e r )  i n section  d i l u t e d and a s s a y e d  3.3.5  o r 10  i n 10 mL o f a  (2:1) i n a 50 mL r o u n d  m i x t u r e was e v a p o r a t e d  choline  o f 0.3  f o r MSUM  (A).  D E T E R M I N A T I O N O F SODIUM A N D / O R P O T A S S I U M CONTENT O F  3.3.7  HYALURONIC A C I D ,  CHONDROITIN S U L F A T E AND PROTEOGLYCAN  MONOMER S A M P L E S  An  accurately  potassium c h l o r i d e volumetric potassium solutions  weighed  100 mg q u a n t i t y  was d i s s o l v e d  o f sodium c h l o r i d e o r  i ndistilled  water i n a  f l a s k a n d t h e v o l u m e was made up t o 100 mL. S o d i u m o r standard  solutions  t o obtain  a concentration  mg d L ^ o f s o d i u m c h l o r i d e emission o f these  were made b y f u r t h e r  solutions  range  diluting  these  o f 0.2 mg dL ^ t o 2.0  o r potassium c h l o r i d e .  The p e r c e n t  was m e a s u r e d on a f l a m e p h o t o m e t e r a t  -98-  t h e maximum w a v e l e n g t h o f 585 nm f o r s o d i u m and 765 n>m f o r potassium.  A calibration  curve  o f percent  c o n c e n t r a t i o n o f sodium i o n o r p o t a s s i u m  emission  versus  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 proteoglycan distilled mL.  monomer o r p r o t e o g l y c a n  water  The p e r c e n t  proteoglycan percent  emission  EFFECT  A  was m e a s u r e d a t 765 nm a n d 585 nm.  from t h e s t a n d a r d  sodium s a l t  representing  supersaturated  potassium  s o d i u m i n a 30 mg sample o f was added t o 45 mL o f a T h e volume o f t h e water  MSUM c o n c e n t r a t i o n o f t h e  s o l u t i o n was 5.0 g L ^.  and e q u i l i b r a t e d  (40 mg) were added  as d e s c r i b e d  ( e q u i v a l e n t t o 1.6  t o 50 mL b y t h e a d d i t i o n o f d i s t i l l e d  t h r o u g h a 0.22 um M i l l e x - G S  growth c e l l  This  5.6% w/w  s o l u t i o n o f MSUM (pH 7 . 4 ) .  7.4) s o t h a t t h e f i n a l  crystals  The  curve.  of chondroitin sulfate,  s o l u t i o n was a d j u s t e d  filtered  acid,  i o n i n t h e samples  0.7 mL v o l u m e o f 0.1 M s o d i u m h y d r o x i d e  supersaturated  the  f o r c h o n d r o i t i n s u l f a t e and  O F SODIUM AND P O T A S S I U M I O N S ON GROWTH O F MSUM  mg s o d i u m i o n )  (pH  f l a s k a n d t h e volume made t o 100  o f sodium i o n a n d p o t a s s i u m  were d e t e r m i n e d  3.3.8  emission  a g g r e g a t e was d i s s o l v e d i n  was m e a s u r e d a t 585 nm a n d f o r h y a l u r o n i c  concentrations  the  i n a volumetric  acid,  The s o l u t i o n was  disposable  filter  a t 3 7° f o r 30 m i n u t e s .  and t h e growth  experiment  unit  into  Seed carried out  i n s e c t i o n 3.3.6.  e x p e r i m e n t was r e p e a t e d hydroxide  solution  using  0.34 mL o f a 0.1 M  ( e q u i v a l e n t t o 1.3 mg p o t a s s i u m i o n )  -99-  representing hyaluronic  acid.  s o l u t i o n was 3 . 3 . 9  6.5%  5.0  w/w  of potassium  The  concentration  g L ^  and  CHARACTERIZATION  Impurities  and  OF  50 mg  i n a 20 mg  o f s e e d c r y s t a l s were  a d d i t i v e s i n t h e growth or c r y s t a l  determine whether t h e a d d i t i v e s had  caused  AFTER  used.  CRYSTAL  obtained  GROWTH  medium c a n r e s u l t  structure. any  in  To  change i n t h e  or s t r u c t u r e , scanning e l e c t r o n microscopy  powder X - r a y d i f f r a c t i o n crystals  supersaturated  MSUM C R Y S T A L S  a change i n c r y s t a l m o r p h o l o g y  crystal habit  of the  sample o f  and  s t u d i e s were c a r r i e d o u t on t h e MSUM  after various  crystal  growth  experiments.  Where e v e r p o s s i b l e , t h e s u p e r s a t u r a t e d  solution containing  the  c r y s t a l s was  membrane f i l t e r  the  filtered  t h r o u g h a 0.22  crystals  on t h e f i l t e r  at  The  60°.  mortar  and  and  were d r i e d i n a c i r c u l a t i n g h o t a i r oven  d r i e d c r y s t a l s were g r o u n d  subjected  as d e s c r i b e d  um  t o SEM  i n a glass pestle  and powder X - r a y d i f f r a c t i o n  i n s e c t i o n 3.3.2(E) and  3.3.2(C).  and analyses  -100-  4  4 . 1  RESULTS  CHARACTERIZATION  Ultra-violet  OF  AND  DISCUSSION  MONOSODIUM  and i n f r a - r e d  URATE  MONOHYDRATE  absorption spectroscopy  a r e u s e d t o d e t e r m i n e and c o n f i r m t h e f u n c t i o n a l i n known compounds. s o l u t i o n o f MSUM  The u l t r a - v i o l e t  ( F i g u r e 20) showed t h e ^ m a x  nm.  T h e s e two v a l u e s  uric  acid  salt  of uric  The 3580 c m  (West,  - 1  900 that  ;  1  and  a t 235 and a t 292  t h e ^max  values  the formation  1  (=CH); 1750 c m  of ionised  o f t h e sodium  - 1  (=C=0);  1  - 1  ;  1390 c m  - 1  ;  1250 c m  - 1  ;  1000 c m  - 1  and  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 i n the l i t e r a t u r e  (Dieppe  and C a l v e r t , 1 9 8 3 ) .  r e l a t i o n s h i p between t h e wavelength o f t h e X - r a y s ,  t h e s p a c i n g between t h e c r y s t a l l o g r a p h i c  crystal  f o r an aqueous  (=C=C=, =C=N-,-COO~); 1610 cm" ; 1525  - 1  reported The  present  s p e c t r u m o f MSUM ( F i g u r e 21) showed p e a k s a t  (-OH); 2850-3150 cm"  1425 c m  cm .  groups  acid.  1750-1650 c m cm  c o i n c i d e with  1970) i n d i c a t i n g  infra-red - 1  spectrum  methods  planes,  d, o f a  i s g i v e n by B r a g g s law: 1  n ^  = 2d Sine  where n i s an i n t e g e r and 9 i s t h e a n g l e  (27)  of the incident  X-rays.  -101-  F i g u r e 20.  Ultra-violet  spectrum o f  u r a t e monohydrate  monosodium  solution.  W<*.l«ngth(um)  100-  5  1  F i g u r e 21.  Infra-red  7  6  '  1  0  9  »  1  10 1  12 1  K 1  £  j  spectrum o f monosodium u r a t e monohydrate.  -103-  Since  s i n Q must be b e t w e e n 0 a n d 1 t h e r e a r e u s u a l l y  no more  t h a n one o r two d i f f r a c t i o n o r d e r s i f ^ i s c o m p a r a b l e t o d . The Figure  powder X - r a y  22;  d i f f r a c t i o n p a t t e r n o f MSUM i s shown i n  The c a l c u l a t e d  d - v a l u e s a r e shown i n T a b l e 4.  d - v a l u e s were c h a r a c t e r i s t i c (Selected  o f monosodium u r a t e m o n o h y d r a t e  Powder D i f f r a c t i o n D a t a  Boistelle,  The  f o r M i n e r a l s , 1974; R i n a u d o and  1982).  A scanning electron monohydrate c r y s t a l s long, well-formed  micrograph  o f t h e monosodium u r a t e  i s shown i n F i g u r e 23.  needle-shaped  The c r y s t a l s h a d a  or acicular  crystal habit.  A t y p i c a l DSC s c a n o f an MSUM sample i s shown i n F i g u r e 24. A broad  endothermic  the l o s s  peak b e t w e e n 1 8 0 ° t o 2 4 0 ° a c c o u n t e d  o f 8.78 % w/w o f w a t e r o f h y d r a t i o n .  loss  corresponded  to the loss  that  t h e c r y s t a l s were t h e m o n o h y d r a t e .  of p e r c e n t water l o s s 4.2  The p e r c e n t w a t e r  o f one mole o f w a t e r ,  confirming  The t h e o r e t i c a l  value  f o r MSUM i s 8.65% w/w.  ASSAY OF MONOSODIUM URATE MONOHYDRATE I N SOLUTION The  Figure  standard curve  25.  f o r an MSUM s o l u t i o n  i n w a t e r i s shown i n  The p l o t was l i n e a r o v e r a c o n c e n t r a t i o n r a n g e o f  0.002 g L ^ t o 0.034 g L ^, t h e c o r r e l a t i o n  coefficient,  2 r  for  , was 0.999 a n d t h e s l o p e  concentration/cell  width)  (absorptivity,  absorbance/  was 56.8 L g ^cm ^.  F i g u r e 22.  X-ray diffraction  p a t t e r n o f monosodium u r a t e monohydrate  -105-  4.  Powder X - r a y d i f f r a c t i o n p a t t e r n o f monosodium u r a t e m o n o h y d r a t e .  Interplanar distance ( d - s p a c i n g , 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 strong strong medium s t r o n g medium W = weak  VS S MS M  Relative intensity  -106-  Figure 23.  Scanning electron micrograph of monosodium urate monohydrate.  -107-  195  F i g u r e 24.  DSC  scan  #  of monosodium u r a t e  monohydrate.  -108-  Figure 25.  A standard curve f o r MSUM s o l u t i o n  (n = 5; r = 0.999)  -109-  4.3  DEGRADATION OF MONOSODIUM URATE MONOHYDRATE I N SOLUTION The  r e s u l t s o f degradation  studies are given  26-29 a n d a r e p l o t t e d a s c o n c e n t r a t i o n s o l u t i o n versus Figure solutions. showing 35°,  MSUM  solutions  stored  o f n o n - s t e r i l e MSUM  a t 4 ° were  c h a n g e i n MSUM c o n c e n t r a t i o n  4 5 ° and 6 5 ° , t h e r e  concentration  o f MSUM r e m a i n i n g i n  time o f i n c u b a t i o n .  26 shows t h e d e g r a d a t i o n  little  was a g r a d u a l  decrease  relatively  stable,  with  At 22°,  o f MSUM a s t h e t i m e o f i n c u b a t i o n was i n c r e a s e d .  MSUM o c c u r e d more  r a p i d l y , with  a t 4 5 ° , w h i c h were more  (Figure 27).  showed a smooth d e c l i n e sterile  22°,  concentration,  3 5 ° and 6 5 ° .  times,  filters  each time  stored  by a s e p t i c stoppered  However, w h e r e a s n o n - s t e r i l e s o l u t i o n s  i n Vacutainers particularly  showed  with  time, t h e  some f l u c t u a t i o n s  f o r solutions stored a t  T h e s e e x p e r i m e n t s were r e p e a t e d  and t h e s o l u t i o n s s t e r i l i z e d  Vacutainers  i n t o rubber  i n MSUM c o n c e n t r a t i o n  solutions stored  in urate  of solutions  by s o l u t i o n s s t e r i l i z e d  through M i l l i p o r e  Vacutainers  the exception  degradation o f  s t a b l e than s o l u t i o n s stored a t 35°.  r e s u l t s were g i v e n  filtration  time.  i n the  As t h e t e m p e r a t u r e o f i n c u b a t i o n was i n c r e a s e d ,  Similar  i n Figures  by f i l t r a t i o n  a l w a y s showed v a r i a b i l i t y  i n urate  several and s t o r e d i n  concentration at  interval.  During  the course o f these  presence o f rubber o r p l a s t i c c a u s e a l o s s o f MSUM f r o m  studies,  containers  i t was f o u n d t h a t t h e or closures  s o l u t i o n and t h a t  this  appeared t o  e f f e c t was more  -110-  F i g u r e 26.  Degradation  o f 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 (•)  -Ill-  F i g u r e  27.  D e g r a d a t i o n of MSUM s o l u t i o n s  4°C  (O):  s t e r i l e (0.22 um f i l t e r ) i n V a c u t a i n e r s . _  22°C ( • ) ;  3^°C  45°C  (V);  -112-  TIME, hours  Figure 28.  Degradation of s t e r i l e (by autoclaving) MSUM s o l u t i o n s i n 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 the  a t higher  degradation  autoclaving)  temperatures.  of sterile  was s t u d i e d  s o l u t i o n s o f MSUM  i nall-glass  with  rubber c l o s u r e s .  The r e s u l t s  30.  After autoclaving  a t 121°  concentration decreased  f r o m 0.198 g L  22°  1  1  t o 0.165 g L  -  1  _  ( F i g u r e 28)  1  stored  i n glass  little  were r e l a t i v e l y  stable.  gradually decreased  rubber  stoppers  A t 4 5 ° and 65°,  t o 0.147 g L  -  1  and  flasks at  change i n t h e  t o 0.168 g  a t 2 2 ° and 3 5 °  MSUM  a n d 0.092 g L  At 35°  Solutions  1  with  vessels  for solutions i n the  o f MSUM i n s o l u t i o n f r o m 0 t o 96 h o u r s .  i n Vacutainers  29 a n d  i nall-glass  a n d 0.117 g L , r e s p e c t i v e l y , a t 96 h o u r s .  decreased  and V a c u t a i n e r s  f o r 35 m i n u t e s , t h e  stable with  6 5 ° t h e MSUM c o n c e n t r a t i o n  stored  observation,  a r e shown i n F i g u r e s  Solutions  and 4 5 ° were r e l a t i v e l y  and  containers  t o 0.183 g L  1  ( F i g u r e 29).  concentration  L  -  _  this  ( s t e r i l i z e d by  o f MSUM i n s o l u t i o n s c o n t a i n e d  f r o m 0.198 g L  Vacutainers  To c o n f i r m  -  1  concentrations  , r e s p e c t i v e l y , a t 96  hours.  Heating  t h e s o l u t i o n s i n an a u t o c l a v e  MSUM c o n c e n t r a t i o n . stored los^  The solutions with  T h i s d e c r e a s e was g r e a t e r  i n Vacutainers,  by a b s o r p t i o n  data  decreased  indicating  that  i n t o the rubber  i n Figures  and s t e r i l e  time.  gave a d e c r e a s e i n for solutions  some u r a t e may h a v e b e e n  stoppers.  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 o f MSUM u n d e r g o  decomposition  I n 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 i n two ways, b y b a c t e r i a l  may h a v e  c o n s u m p t i o n and c h e m i c a l  -115-  degradation. stored reason  Forboth  a t 4 5 ° were  more  for this behavior  done t o d e t e r m i n e  The may  non-sterile  without  stable  than  those  i s not understood.  thedegradation products  degradation  be conducted  and s t e r i l e  studies indicate  under n o n - s t e r i l e  any a p p r e c i a b l e decrease  systems,  solutions  s t o r e d a t 3 5 ° . The No s t u d i e s were  o f MSUM.  that  crystal  growth s t u d i e s  c o n d i t i o n s f o r about  8 hours  i n MSUM c o n c e n t r a t i o n due t o  decomposition.  4.4  S A T U R A T I O N S O L U B I L I T Y O F MSUM  The such  solubility  as temperature,  impurities. prepared (about  o f MSUM i n - v i t r o the presence  The p r e s e n c e  i s i n f l u e n c e d by f a c t o r s  o f i o n s and t h e p r e s e n c e o f  o f amorphous m a t e r i a l i n c o m m e r c i a l l y  MSUM h a s b e e n r e p o r t e d t o show a n i n c r e a s e d  50% more) o f MSUM ( K i p p e n e t a l . , 1 9 7 4 ) .  The  MSUM p r e p a r e d  i n o u r l a b o r a t o r y was u s e d  experiments  t o determine  temperature  and a d d i t i o n o f s o d i u m c h l o r i d e ,  chondroitin  sulfate,  solubility 4.4.1  solubility  such as  hyaluronic acid,  p r o t e o g l y c a n s and a l b u m i n  o n t h e aqueous  O F TEMPERATURE  saturation  temperature  of factors  o f MSUM.  EFFECT  The  the effect  solubility  i s given i n Table  (Cs) o f MSUM a s a f u n c t i o n o f 5 and F i g u r e 30. A v a n ' t H o f f  (shown i n F i g u r e 31) o f l o g a r i t h m o f s a t u r a t i o n reciprocal  in a l l  o f absolute temperature  should y i e l d  solubility a straight  plot  versus line,  Table  5.  S a t u r a t i o n s o l u b i l i t y (Cs) o f monosodium u r a t e at d i f f e r e n t temperatures.  monohydrate  Temperature 4.4  9.0  15.1  24.7  29.9  35.0  37.0  40.0  41.8  51.0  55.0  (°C)  Saturation solubility (g  L" ) 1  0.476 0.560 0.605 0.998 1.050 1.300 1.346 1.617 1.688 1.900 2.493  -117-  Figure 30.  E f f e c t of temperature on the saturation s o l u b i l i t y of: (a) MSUM; (b) MSUM i n the presence of p h y s i o l o g i c a l ion concentration.  -118-  Figure 31.  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.  -119-  i s - A H / 2 . 3 0 3 R , where  t h e s l o p e o f which solution. the  However, a r e q u i r e m e n t o f t h i s  temperature  constant. AH  range  i s not too large,  Assuming t h a t  f o r MSUM i n w a t e r  over the 50° temperature  Allen  1  slope o f t h e van't Hoff p l o t to  b e - 1132 ° K .  determined  i s that  remains a  range, t h e  i s constant, the slope o f t h e van't  o r + 24.3 K J m o l " .  - 1  relationship  so t h a t A H  p l o t was f o u n d t o be - 1267.7 ° K , g i v i n g mol  A H i s the heat o f  a ^.H o f + 5.8 K c a l  e t a l . (1965b) f o u n d t h e  for the s o l u b i l i t y  The s a t u r a t i o n  by i n t e r p o l a t i o n  Hoff  solubility  o f MSUM i n w a t e r  a t 37°,  o f t h e v a n ' t H o f f p l o t was 1.379 g  L" . 1  It  i s well  documented t h a t  effect  on MSUM s o l u b i l i t y  Wilcox  e t a l . , 1972; F i d d i s  variations  methods u s e d  4.4.2  1972; A l l e n  e t a l . , 1983).  et al.,  There  1965a,b;  a r e some  temperatures.  T h i s may be due t o t h e d i f f e r e n t  i n thedetermination of the saturation  solubility  other  (Loeb,  has a s i g n i f i c a n t  i n t h e r e p o r t e d v a l u e s o f t h e 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  Our  temperature  data i swithin  t h e range  solubility.  o f v a l u e s r e p o r t e d by  workers.  EFFECT OF SODIUM CHLORIDE The  differing  saturation  s o l u b i l i t y o f MSUM i n t h e p r e s e n c e o f  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 t h e p h y s i o l o g i c  concentrations of chloride,  s o d i u m , c a l c i u m and p o t a s s i u m  i n plasma  was s t u d i e d  ture.  or synovial  The r e s u l t s  fluid  a r e g i v e n i n T a b l e 6.  as a f u n c t i o n  present  o f tempera-  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 b e t w e e n t e m p e r a t u r e 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 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 t h e s a t u r a t i o n s o l u b i l i t y o f monosodium u r a t e m o n o h y d r a t e .  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  (g L  )  Temperature ( C)  0.2% 0.4% sodium sodium chloride chloride  0.6% 1.0% sodium sodium chloride chloride  0.88% N a C l 0.032%CaCl 0.03% KC1  4.4  0.047  0.023  0.018  0.012  0.015  10.4  0.664  0.035  0.031  0.013  0.021  17.8  0.106  0.053  0.032  0.022  0.030  24.7  0.143  0.064  0.042  0.027  0.035  29.9  0.240  0.116  0.072  0.042  0.063  41.8  0.471  0.229  0.155  0.096  0.139  51.1  0.599  0.336  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-  b e t w e e n MSUM s a t u r a t i o n the  p r e s e n c e and absence  The even  saturation  i n water  of electrolytes  solubility  and temperature i n  i s shown i n F i g u r e 30.  o f MSUM d e c r e a s e d  significantly  i n t h e p r e s e n c e o f l o w c o n c e n t r a t i o n s o f sodium  (Kippen e t a l . ,  1974).  s p e c i e s i n plasma  these  ions  a l s o caused  and s y n o v i a l  fluid  a large decrease  each  temperature.  K h a l a f and W i l c o x  calcium  ions reduced  t h e MSUM s o l u b i l i t y  potassium 4.4.3  and c u p r i c  chloride  The c o n c e n t r a t i o n s o f t h e d i f f e r e n t  ionic  at  solubility  ions  increased  a r e v e r y s i m i l a r and  i n the s o l u b i l i t y (1973) showed i n water,  o f MSUM  that  whereas  the s o l u b i l i t y .  E F F E C T OF CHONDROITIN SULFATE, HYALURONIC ACID, PROTEOGLYCANS AND ALBUMIN The  the  saturation  solubility  presence o f these a d d i t i v e s  subsequently crystal  used  o f MSUM i n w a t e r a t 37°.  These  v a l u e s were  i n the determination of rate  growth i n t h e p r e s e n c e o f a d d i t i v e s .  was d e t e r m i n e d i n  c o n s t a n t s f o r MSUM  The r e s u l t s a r e  shown i n T a b l e 7. The  p r e s e n c e o f 60 mg dL  solubility L  1  .  o f MSUM f r o m  Laurent  (1964) a l s o  K a t z and S c h u b e r t solubility  CS d e c r e a s e d t h e s a t u r a t i o n  1.379 g L  -  1  (no a d d i t i v e )  showed a s i m i l a r  effect.  (1970) r e p o r t e d a s m a l l i n c r e a s e  t o 1.329 g However, i n MSUM  i n t h e p r e s e n c e o f CS.  Chondroitin of  1  sulfate  o b t a i n e d c o m m e r c i a l l y i s t h e sodium  a mixture o f chondroitin-6-sulfate  salt  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 t h e p r e s e n c e o f a d d i t i v e s a t 37 .  Saturation  solubility  o f MSUM  a  (g L ) 1  Additive A d d i t i v e c o n c e n t r a t i o n s (mg dL ) 10 20 40 60 100 1  Chondroitin sulfate  1.358  1.347 1.329 1.401  Hyaluronic acid  1.392  1.387  Albumin  1.382  1.391 1.397  1.395  Proteoglycan monomer  1.402  1.397 1.399  1.401  1.408  1.403  Proteoglycan aggregate  a: mean o f t h r e e  determinations.  1.397  -123-  containing  about  5.6% w/w o f s o d i u m .  saturation  solubility  The d e c r e a s e  o f MSUM i n w a t e r  i n the  i n the presence o f  chondroitin  s u l f a t e may b e due t o t h e p r e s e n c e o f sodium i n  chondroitin  sulfate  Hyaluronic can aggregate  sample  acid,  (common i o n e f f e c t ) .  albumin,  caused v e r y s l i g h t  p r o t e o g l y c a n monomer increases  and p r o t e o g l -  i nthe solubility of  MSUM.  Katz and Schubert solubility observed  (1970) r e p o r t e d a s l i g h t  o f MSUM i n t h e p r e s e n c e o f p r o t e o g l y c a n s .  enhancement o f MSUM s o l u b i l i t y  amounts o f p r o t e o g l y c a n s . found the  t o cause  solubility solubility  and B r a n d t  increase  solubility (prepared  i n MSUM s o l u b i l i t y  were  even i n  additives. i n urate  o f PGA. However, t h e i n c r e a s e  i n MSUM  was n o t s u s t a i n e d b e y o n d 4 h o u r s . and Brandt  (1979) r e p o r t e d a g r e a t l y  o f MSUM i n t h e p r e s e n c e  increase  exchange o f p o t a s s i u m i o n s o f MSUM.  increased  o f proteoglycan aggregate  i n t h e presence o f potassium  the observed  sodium  They  and albumin  (1978) r e p o r t e d a n i n c r e a s e  i n thepresence  Perricone  that  Chondroitin sulfate  only a slight  i n the  i n the presence o f small  presence o f large concentrations o f these  Perricone  increase  ions).  i nsolubility  They c o n c l u d e d  was due t o t h e c a t i o n i c  ions o f t h e p r o t e o g l y c a n aggregate  with  -124-  4.4  C R Y S T A L GROWTH O F MSUM  4.4.1  D E T E R M I N A T I O N O F SUPERSATURATION CONCENTRATION AND S E E D AMOUNT F O R C R Y S T A L GROWTH  Preliminary no L  crystal ^.  experiments conducted  growth a t a s u p e r s a t u r a t i o n  The r e s u l t s  supersaturation  supersaturation was 100 mg. tion  32 a n d 33.  concentation  of 5 g L  curve.  of 4 g out a t are  1  was 5 g L  -  1  and t h e seed  there  was s l o w l i n e a r  amount  supersaturagrowth  g r o w t h showing a p a r a b o l i c  S i m i l a r l y when t h e s u p e r s a t u r a t i o n  o f MSUM was 6 g L , t h e a d d i t i o n o f 200 mg s e e d s 1  showed s l o w g r o w t h  f o r about  2 hours  followed by a r a p i d  g r o w t h p h a s e . A d d i t i o n o f 500 mg a n d 1000 mg s e e d s t o  supersaturated  concentration the  1  non-linear  concentration-time  the  and 6 g L  1  I n t h e p r e s e n c e o f 200 mg s e e d s a n d a  by r a p i d  non-linear  of 5 g L  showed  No g r o w t h was o b s e r v e d when t h e  concentration  concentration  followed  concentration  o f some o f t h e e x p e r i m e n t s c a r r i e d  concentrations  shown i n F i g u r e s  i n the IL apparatus  solutions,  versus  resulted  time curves  i ntypical  non-linear  throughout t h e time course o f  experiment.  Similar  r e s u l t s were o b t a i n e d  Supersaturation 5 g L  1  concentrations  and g r e a t e r .  required  t o give  Greater  using  necessary  t h e 50 mL for crystal  apparatus. g r o w t h were  t h a n 30 mg s e e d c r y s t a l s  the non-linear  growth  were  curves.  I n t h e s e e d e d g r o w t h s t u d i e s o f MSUM b y E r w i n a n d N a n c o l l a s (1981) no p e r i o d  o f s l o w g r o w t h o r i n d u c t i o n p e r i o d was o b s e r v e d .  -125-  <  CL l±J O  z o  TIME, min. F i g u r e  32.  37°»  Seeded growth c u r v e s f o r MSUM a t i n 1 L c a p a c i t y a p p a r a t u s 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 - l . Added seed amount: 100 m g (v); 200 mg (•); mg ( A ) ; mg ( • ) ; mg  300  500  1000  (o).  -126-  < LU O  o o  120  240  360  TIME, min. F i g u r e  33.  Seeded g i n 1 1 c s u p e r s a t Added se mg  1000  rowth c u r v e s f o r MSUM a p a c i t y a p p a r a t u s and u r a t i o n c o n c e n t r a t i o n ed amount: 200 mg ( • ) ; (O).  at 37? an i n i t i a l of 6 g L" . 500 mg (•); 1  -127-  At L L  an  -1 1  initial  urate they  nship  supersaturation  and  seed c o n c e n t r a t i o n  observed  between  the  urate  typical  and  1  o f 0.18  second  concentration  show t h a t a v e r y h i g h g L  concentration  initial  l a r g e amount o f  the  van  crystallization  either given  i n c r e a s i n g the s e e d amount o r  initial  initial  i n d u c t i o n p e r i o d . He process.  showed t h a t t h e  c h r o m a t e were p r o b a b l y  growth o f occured  product. silver  due  proportional  to the  been observed.  Nancollas  and  Purdie  (1957)  i n the  quantity of  In the  i n seeded  showed  that  length  to a  for a  of  the  nucleation  subsequently silver by  a seeded  crystallization  s e e d c r y s t a l s were added  i n d u c t i o n p e r i o d was seed  in  definite  the  growth o f  found t h a t d e l a y e d  The  5  mg)  concentration  decreased  to  inversely  added.  E v e n when i m p u r i t i e s were a b s e n t , still  He  D a v i e s e t a l . (1955) s t u d i e d t h e  solutions.  of  supersaturated  to surface contamination  c h l o r i d e and  however  induction periods  s e e d amount a t a  Nancollas  when s u r f a c e - c o n t a m i n a t e d  supersaturated  results,  his observations  induction periods  relatio-  curves.  seed c r y s t a l s .  attributed  g  induction effects  concentration  2 g  concentration  supersaturation  However, Howard and  hydrolysis  growth  i n c r e a s i n g the  supersaturation  Our  c h r o m a t e when  s o l u t i o n s were i n o c u l a t e d w i t h  0.54  ( g r e a t e r t h a n 200  (1940) o b s e r v e d  of s i l v e r  and  non-linear  time.  seed c r y s t a l s  Hook  -1  order,  and  s t u d i e s have demonstrated  growth systems.  g L  supersaturation  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  Other  of approximately  induction periods  s e e d e d g r o w t h o f magnesium  (1961) showed t h a t t h e  have oxalate,  rate constant  for  -128-  g r o w t h d e p e n d e d o n t h e number o f c r y s t a l l i z a t i o n the  i n o c u l a t i n g seed c r y s t a l s .  the  i n d u c t i o n p e r i o d was  supersaturation during  gradients  tended  present i n  They f o u n d t h a t t h e d u r a t i o n o f  i n v e r s e l y p r o p o r t i o n a l to the  concentration  the time l a g .  sites  and b u l k  n u c l e a t i o n was  I t was t h o u g h t t h a t t h e h i g h  to build  up s o l u t e b y a d i f f u s i o n  observed  concentration process  faster  t h a n i t c o u l d be accommodated on t h e a v a i l a b l e g r o w t h s i t e s . 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 , inoculating enabling  Our  increased,  second order  observation  inoculating reduced  s e e d was  and f i n a l l y  growth t o b e g i n  abolished  achievement o f a c r i t i c a l  the  seed c r y s t a l s .  number order  i n t h e amount o f degree o f s u p e r s a t u r a t i o n ,  t h e i n d u c t i o n p e r i o d may be due t o number o f a c t i v e g r o w t h s i t e s  As t h e amount o f s e e d c r y s t a l s  solution i s increased,  o f a c t i v e growth s i t e s  disappeared,  immediately.  t h a t an i n c r e a s e  the  supersaturated  o r when t h e amount o f  the induction period  seed m a t e r i a l a t a g i v e n  At  achievement  added t o t h e of the c r i t i c a l  would t h e n l e a d t o immediate  growth o f t h e seed c r y s t a l s  with  the t y p i c a l  on  second  non-linear  curves. 4.4.2  SELECTION OF THE METHOD TO DETERMINE THE RATE CONSTANT OF CRYSTAL GROWTH The  rate constants  for crystal  s e v e r a l d i f f e r e n t methods. a d d i t i v e s were u s e d An  equation  for this  g r o w t h were d e t e r m i n e d  The d a t a  from e x p e r i m e n t s  purpose.  o f the e m p i r i c a l  form:  using  without  •129-  =  K ' ( C - C s )n  (28)  where Rg  = t h e r a t e o f growth unit  K'(the  observed  Q  i n concentration per  time),  rate constant  K (obs)=  (decrease  of the reaction) =  overall  K (obs) S o  rate constant  S = s u r f a c e a r e a o f t h e seed  and was  saturation solubility  solution,  o f MSUM a t 3 7 ° ,  n = order o f reaction, used t o c a l c u l a t e Logarithms  Linear  t h e growth r a t e c o n s t a n t  o f equation  log  log  least  R  g  28 were t a k e n  = l o g K' + n l o g  square  fits  K', was o b t a i n e d  as f o l l o w s .  to give:  (C-Cs)  (29)  were p e r f o r m e d o n l o g R  (C - C s ) o n t h e UBC c o m p u t e r .  reaction,  The r a t e c o n s t a n t  from t h e i n t e r c e p t  the rate constants,  along with Tables  g  versus  o f the  o f the f i t .  s l o p e o f t h e f i t gave n, t h e o r d e r o f t h e r e a c t i o n . of  growth,  crystals,  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 Cs=  of crystal  The  The v a l u e s  K', and t h e o r d e r o f t h e r e a c t i o n , n,  the correlation  coefficients  o f the f i t s  aregiven i n  8 t o 11  I n most o f t h e e x p e r i m e n t s , gave p o o r c o r r e l a t i o n  the linear  coefficients  resulting  least-square i n rate  for a given  seed  fits  constant  values  w h i c h were i n p o o r agreement  amount. The  values  o f n, t h e o r d e r o f t h e r e a c t i o n , v a r i e d b e t w e e n 0.4 t o  -130-  Table  8.  (crystal  MSUM g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d f r o m t h e 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).  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 batch  Seed amount (mg)  Correlation coefficient  K'x 10 (L g  min  Reaction order  )  ———  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 g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d f r o m t h e 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). a  (crystal  growth  i n IL c a p a c i t y apparatus; C = 6 g L  )  Growth r a t e constant, E x p e r i m e n t Seed batch #  Seed amount (mg)  K'x 10 IT  (L g  c  Correlation coefficient  "I • "I \ mm )  Reaction order (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-  Table  10.  MSUM g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d f r o m 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 g r o w t h i n 50 mL c a p a c i t y a p p r a t u s ; s e e d c r y s t a l s f r o m b a t c h B)  C = 5 g  L  Growth r a t e constant Seed E x p e r i m e n t amount (mg) #  K'x  10  (L g  c  mxn  Correlation Coefficient  Reaction order (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.  the  -133-  Table  11.  MSUM g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d f r o m t h e 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 g r o w t h i n 50 mL c a p a c i t y a p p a r a t u s ; s e e d c r y s t a l s f r o m b a t c h B)  C = 6 g L  ;  Growth r a t e constant, Experiment #  Seed amount (mg)  Correlation K'x 10 coefficient _ _ (L g m i n ) 1  1  Reaction order (n)  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 t h e  (>0.95), n was Due was  not  rate  between 2 t o  to the  3.  poor c o r r e l a t i o n of the  used to determine the  constant  Marc highly  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  e f f e c t of additives  t h i s method  on  the  growth  f o r MSUM.  (1908) o b s e r v e d t h a t  and  data points  moderately  the  soluble  rate  of  c r y s t a l growth  s u b s t a n c e s was  usually  of  proportional  2 to  (C - Cs)  .  A  similar observation  Nancollas  (1955).  is  spiral  due  to  (Nielsen,  I t has step  was  made by  been suggested t h a t  rate R  of  = K'  to  c r y s t a l growth,  (C - C s )  and  this relationship  c o n t r o l l e d growth c l o s e  1 9 6 4 ) . The  Davies  equilibrium  R,  then  i s given  by  (30)  2  g  A number o f follow  the  1:1  and  2:2  same g r o w t h law.  ( D a v i e s and  Nancollas,  e l e c t r o l y t e s h a v e b e e n shown These i n c l u d e ,  1 9 5 5 ) , magnesium o x a l a t e  Purdie,  1961), c a l c i u m  1970).  A number o f o t h e r e l e c t r o l y t e s w h i c h  g r o w t h law recently, crystal the of  growth o f  same e q u a t i o n 0.9  to  parabolic  sulfate dihydrate  have been r e f e r e n c e d E r w i n and  3.0. rate  Nancollas  Further law  by  was  Nancollas  and  a range of evidence shown by  reported  follow  the  the  (1979). the the  and  Nancollas, same  More  dissolution  and  applicability  supersaturation, f o r the  chloride  (Nancollas  ( L i u and  (19.81) s t u d i e d  sodium u r a t e over  silver  to  s',(s'=  applicability  independence of the  of  of  C/Cs),  the  rate  of  -135-  crystallization  on  the  stirring  c o n t r o l l e d mechanism f o r t h e The years  dynamics,  crystallization  i n t e g r a t e d form o f e q u a t i o n  t o determine the  compounds.  indicating  30 h a s  of  30  sodium  surface urate.  been used over  r e a c t i o n rate constant  Integration of equation  a  the  for various  gives the  following  equation:  = K' (C-Cs) where C i s t h e Ci  i s the  initial  Equation  31  t  (31)  (Ci-Cs)  c o n c e n t r a t i o n at v a r i o u s time  supersaturation c a n be  intervals  and  concentration.  rearranged  to equation  32:  1 C =  + Cs  (32)  1 K't  + (Ci-Cs)  This and  t.  equation  A computer program  used t o e s t i m a t e K' fit  and  shows t h e  the  the  If  the  coefficients  i n Tables  (>0.98) were o b t a i n e d  order  (non-lin, Metzler  r e a c t i o n rate constant,  correlation  are given  n o n - l i n e a r r e l a t i o n s h i p between  by  12  t o 15.  this  obtained  e t a l . , 1974) K*. by  The this  Good c o r r e l a t i o n  C  was  values  of  non-linear coefficients  method.  o f t h e g r o w t h r e a c t i o n , n,  i s 2 then  a plot  of  -136-  Table  (crystal  12.  MSUM g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d f r o m t h e n o n - l i n e a r computer p r o g r a m .  growth  Experiment #  i n IL c a p a c i t y apparatus;  Seed batch  C = 5 g L  Growth Rate constant, Seed amount (  m  g  )  K'xlO  Correlation coefficient  fr • ") I i (L g - min 1  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  )  -137-  Table  (crystal  13.  MSUM g r o w t h r a t e c o n s t a n t s o b t a i n e d from t h e n o n - l i n e a r computer program.  growth i n IL c a p a c i t y a p p a r a t u s ;  C = 6 g L  )  Growth r a t e constant, Experiment #  Seed batch  Seed amount (mg)  K'x 1 0  5  Correlation coefficient  IT • " I )\ (L g " Imin  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  -138-  Table  14. MSUM g r o w t h r a t e c o n s t a n t s obtained from t h e n o n - l i n e a r c o m p u t e r 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 a p p a r a t u s ; C = 5 g L ; s e e d c r y s t a l s f r o m b a t c h B)  Experiment #  Seed amount (  m  g  )  Growth r a t e constant, Correalation K'xlO coefficient ij  -  (L g  •  1  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 g r o w t h r a t e c o n s t a n t s o b t a i n e d t h e n o n - l i n e a r computer p r o g r a m .  ( 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 a p p a r a t u s ; C = 6 g L ; s e e d s f r o m b a t c h B)  Growth r a t e constant, Experiment #  Seed amount (mg)  K'xlO , (Lg m i n 1  Correlation coefficient 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  from  -140-  versus t (C-Cs)  (Ci-Cs)  should give a s t r a i g h t for  some o f t h e e x p e r i m e n t s  Linear  regression  (l/(Ci-Cs)) Excellent K'  l i n e w i t h a s l o p e o f K'.  and  and  a r e shown i n F i g u r e s 34  a n a l y s e s on  the values of  t were p e r f o r m e d  correlation  on  Both  are given i n Tables  experiments Since  gave good c o r r e l a t i o n  the  added  seed  linear  values of  regression  and  coefficients  amount v a r i e d  growth  T h e r e f o r e the  of  16-19  i n most o f  the  overall from  crystals.  The  crystal K'/S,  i n each  was  corrected  experiment.  The was  Q  where S i s t h e s u r f a c e a r e a o f added  f o r the weight  t h e s e v a l u e s were u s e d overall  c o n s t a n t , K',  o f seeds  from b a t c h e s  taken  i n each  as v a l u e s o f S t o d e t e r m i n e  growth r a t e  i s evident that  crystals  constant,  then  also  growth r a t e c o n s t a n t , K ( o b s ) ,  s u r f a c e a r e a s o f seed  B were c o r r e c t e d  between e x p e r i m e n t s ,  f o r g r o w t h was  rate  s u r f a c e a r e a o f added s e e d s  determined  It  The  (> 0 . 9 9 ) .  different.  observed  -  for different  ( T a b l e s 12-15  available  and  35.  calculator.  coefficients  the s u r f a c e a r e a o f seeds  observed  plots  16-19.  (l/(Ci-Cs)) versus t  respectively)  for the  a pocket  t h e methods, n o n - l i n p r o g r a m and  (1/(C-Cs)) -  and  (l/(C-Cs))  c o e f f i c i e n t s were o b t a i n e d .  the r e s p e c t i v e c o r r e l a t i o n  experiments  Typical  seed A  and  experiment the  K (obs). Q  f o r a s e t o f experiments  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 b a t c h o f s e e d  where t h e crystals  same  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 form of gro of MSUM a t and an i n i t Seed amount mg, exp exp  20  r k i wth 3 7 ° i a l : 5 (  38 3 4 (O; 5 0  n e t i c p l o t s of t h e i n t e g r a t e d e q u a t i o n f o r t h e seeded growth i n t h e 5 0 mL c a p a c i t y a p p a r a t u s 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 S mg, e x p 41 (o); 1 0 m g , exp 3 9 (A); o ) ; mg, exp (•;; 40 mg, mg, exp  30  32  (•).  36  t  'JJ«  I [ j  142-  0.4 1  TIME, F i g u r e 35.  min  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 of growth e q u a t i o n f o r the seeded growth o f MSUM at 3 7 ° i n the 5 0 mL c a p a c i t y a p p a r a t u s 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  (crystal  16.  MSUM g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d from p l o t s o f t h e i n t e g r a t e d form o f the second o r d e r growth e q u a t i o n .  growth i n I L c a p a c i t y apparatus;  Experiment #  seed batch  Seed amount (  m  g  )  C = 5 g L~ )  Growth r a t e constant, ^ Correlation K'xlO coefficient tr (L g  1  • 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  (crystal  17.  MSUM g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d from p l o t s o f t h e i n t e g r a t e d f o r m o f the second o r d e r growth e q u a t i o n .  growth i n I L c a p a c i t y apparatus;  Experiment #  Seed batch  Seed amount (mg)  C = 6 g L  )  Growth r a t e constant, j. Correlation K'x 10 coefficient -1  IT  (L g  • "1\ min )  9  A  200  3  A  500  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  44.8 0.998 ( u p t o 180 min) 68.6 0.991  -145-  Table  18.  MSUM g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d from p l o t s o f t h e i n t e g r a t e d f o r m o f t h e second o r d e r growth e q u a t i o n .  ( c r y s t a l g r o w t h i - 50 mL c a p a c i t y a p p a r a t u s ; C = 5 g L s e e d c r y s t a l s f r o m b a t c h B) n  Growth r a t e constant, Experiment #  Seed amount (mg)  K'x 10 IT  (L g  c  Correlation coefficient  " I • ~1\ min ) 7.7  0.946  41  5  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 g r o w t h r a t e c o n s t a n t s , K', o b t a i n e d from p l o t s o f t h e i n t e g r a t e d f o r m o f t h e second o r d e r growth e q u a t i o n .  (crystal g j i - 50 mL c a p a c i t y a p p a r a t u s ; C = 6 g L ; s e e d c r y s t a l s from b a t c h B) o  w  t  n  n  Growth r a t e constant, Experiment #  Seed amount (mg)  K'xlO (L g  Correlation coefficient  5  min  )  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 (obs)  v a l u e s s h o u l d be  Q  The  values of K  similar.  (obs) shown i n T a b l e s 20 t o 23  were  o calculated  f r o m t h e v a l u e s o f K'  g e n e r a t e d by  t h e two  different  methods. The  observed o v e r a l l  K (obs)  e s t i m a t e d from  Q  variation  c r y s t a l growth  the  non-lin  rate constants,  computer  program  i n values f o r a given set experiments.  K (obs)  determined  similar  values w i t h i n a given set of experiments.  crystal  growth  Q  linear  on t h e e f f e c t  EFFECT  OF  growth  SEED CRYSTALS  t o 1.179  t o 1.548  experiments. was  m  seed c r y s t a l s  2  g  was m  2  g  1.0447 m -1  ) and  gave Therefore the  versus t f o ra l l  o f a d d i t i v e s on  the growth  rate  1.4521 m —1  ).  showed d i f f e r e n t  The g  —1  2—1 g  CRYSTAL  A and  GROWTH  B were u s e d  mean s u r f a c e (n = 3; r a n g e  (n = 3; r a n g e :  results  i n the  area of Batch 0.908 m  seeds  1.417  of the e f f e c t  o f t h e s e two  The  growth  methods u s e d  2  The  shown i n T a b l e 20.  The  ON T H E  A  2  t h e mean s u r f a c e a r e a o f B a t c h B  r a t e c o n s t a n t s f o r growth are  (l/(Ci-Cs))  b a t c h e s o f seed c r y s t a l s  seed c r y s t a l s  —1  plot  were d e t e r m i n e d by  Q  experiments  crystal  g  constants, K (obs) (l/(C-Cs)) -  Two  -1  reciprocal  However,  MSUM.  4.4.3  g  rate  regression of  further of  from t h e l i n e a r ,  show a w i d e  on  m  the  b a t c h e s o f seed  f r o m t h e two  2  crystals  different  batches  rate constants.  i n t h e p r e p a r a t i o n o f t h e two  batches  -148-  Table  (crystal  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 , .  growth i n IL c a p a c i t y  apparatus;  Overall Experiment #  Seed batch  Seed amount (mg)  C = 5 g L  growth r a t e  3 (obs) x 10 _^ _^ ^ (L g min m ) K  (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 c o n s t a n t from non-1in program •  obtained  (b)  C a l c u l a t e d from r a t e c o n s t a n t 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 (1/(C-Cs) - l / ( C i - C s ) ) v e r s u s t .  1  )  constant,  -149-  Table  (crystal  21.  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 ,  growth i n IL c a p a c i t y  a p p a r a t u s ; C = 6 gL  Overall Experiment #  Seed batch  Seed amount (mg)  9  A  200  3  A  10  (obs), R'.  growth r a t e  3 (obs) x 10 _ _^ _ (L g min m~ ) (a) (b) K  1  2  500  1.6 ( u p t o 180 1.0  min) 1.3  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 c o n s t a n t s from non-1in program  (b)  c a l c u l a t e d from r a t e c o n s t a n t s 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  obtained obtained  )  constant  -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 c a l c u l a t e d from r a t e c o n s t a n t s ,  (obs), 8'.  ( c r y s t a l _ c | r o w t h i n 50 mL c a p a c i t y a p p a r a t u s ; C = 5 gL ; s e e d c r y s t a l s f r o m b a t c h B) O v e r a l l growth r a t e Experiment #  Seed amount (mg)  K  (obs)  x 10  constant  3  ft  (L g (a)  min  m- ) (b) 2  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 c o n s t a n t s o b t a i n e d from non-1in program.  (b)  constants obtained C a l c u l a t e d from r a t e 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 ' .  ( c r y s t a l _ t j j r o w t h i n 50 mL c a p a c i t y a p p a r a t u s ? C = 6 gL ; s e e d c r y s t a l s from b a t c h B)  O v e r a l l growth Experiment #  Seed amount (mg)  K  x  10  rate constant,  3  -1 . -1 -2 \ (L g" 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 o n s t a n t s o b t a i n e d from n o n - 1 i n program.  (b)  c a l c u l a t e d from r a t e c o n s t a n t s 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 .  -152-  varied  i n several aspects  likely  that these  differences  (see  variations i npreparation  i n t h e type  content  of crystals  related  (Van  and t h e i r  Batchelder  differences  showed t h a t different  4.4.4  seed c r y s t a l s  i n t h e two  growth r a t e s a r e c l o s e l y Brooks e t a l . , rate  f r o m B a t c h e s A a n d B may b e due t o t h e  were r e p o r t e d  growth r a t e  led to significant  The d i f f e r e n c e s i n g r o w t h  i n defect content  observations  crystal  a n d Vaughan, 1967;  shown b y s e e d s  i t i s  I t i s w e l l documented t h a t t h e d e f e c t  P a t e l a n d Rao, 1 9 7 9 ) .  constants  Therefore  a n d number o f d e f e c t s p r e s e n t  b a t c h e s o f MSUM c r y s t a l s .  1968;  s e c t i o n 3.3.1).  o f t h e seed c r y s t a l s .  b y E r w i n and N a n c o l l a s  Similar  (1981) who  o f MSUM f r o m d i f f e r e n t b a t c h e s h a d  constants.  E F F E C T OF SUPERSATURATION ON THE CRYSTAL GROWTH RATE CONSTANT A  change  to 6 g L  1  i n the supersaturation  i n t h e 50 mL a p p a r a t u s ,  crystals  f r o m t h e same b a t c h  constant  values  by about  concentration  from  5 g L  i n t h e p r e s e n c e o f seed  increased  the crystal  growth  rate  3 to 4 fold.  The g r o w t h r a t e c o n s t a n t _3 v a l u e , K ( o b s ) , i n c r e a s e d from a p p r o x i m a t e l y 7 x 10 L -1 -1 -2 -3 -1 -1 g min m t o approximately 24 x 10 L g min Q  _2 m L  when t h e s u p e r s a t u r a t i o n - 1  to 6 g L  4.4.5  _  1  (Tables  concentration  was c h a n g e d  from 5  22 a n d 2 3 ) .  E F F E C T OF SODIUM AND POTASSIUM IONS ON THE GROWTH RATE CONSTANTS Chondroitin  sulfate,  hyaluronic  acid, proteoglycan  monomer  -153-  and  proteoglycan  are  introduced  aggregate c o n t a i n  during  potassium contents by  their  preparation.  o f these  cartilage  f l a m e p h o t o m e t r y and a r e shown  The tions  effect  a concentration  used  urated  s o l u t i o n (5.0 g L a n d a r e shown  1  ) .  o f the concentra-  i n t h e maximum q u a n t i t i e s o f  to the highest  Rate c o n s t a n t s ,  concentration  constant . with  ranged  a mean o f 44.1 x 10  not s i g n i f i c a n t l y  of  sodium i o n s .  K', were  ( i n 50 mL) o f s o d i u m t h e r a t e  f r o m 42.5 x 10 ^ L g  is  -5  different  Similarly  L g  1  -1  min  1  t o 48.5 x  -1 min .  This  from t h a t o b t a i n e d  value  i n t h e absence  i n t h e p r e s e n c e o f 1.3 mg  ( i n 50 mL)  -5 o f p o t a s s i u m , a mean r a t e c o n s t a n t min  1  was  a d d i t i o n o f sodium i o n s MSUM, b y t h e common  L g  ( s e c t i o n 4.4.2) t h a t t h e  suppresses the s a t u r a t i o n s o l u b i l i t y o f  ion effect.  However,  i t i s apparent  that the  a d d i t i o n a l c o n t r i b u t i o n made b y t h e s o d i u m o r p o t a s s i u m  ions present of  o f 44.3 x 10  -1  obtained.  I t h a s b e e n shown p r e v i o u s l y  small  supersat-  i n T a b l e 25.  In t h e p r e s e n c e o f 1.6 mg  -5  determined  i n any o f t h e a d d i t i v e s t u d i e s were added t o a  determined  10  components were  g r o w t h e x p e r i m e n t s was d e t e r m i n e d .  Sodium o r p o t a s s i u m i o n s e q u i v a l e n t present  The s o d i u m a n d  on t h e g r o w t h r a t e c o n s t a n t s  i n crystal  which  i n T a b l e 24.  o f sodium o r p o t a s s i u m p r e s e n t  additives  o f ions  i n the additives to the o v e r a l l high  sodium i o n s  i n the supersaturated  t o c a u s e any s i g n i f i c a n t  concentration  s o l u t i o n s , was  change i n t h e r a t e c o n s t a n t  insufficient f o r growth.  -154-  Table  24.  Sodium and p o t a s s i u m content o f additives.  ADDITIVE  CHONDROITIN SULFATE  SODIUM (% w/w)  POTASSIUM (% w/w)  5.6  HYALURONIC ACID PROTEOGLYCAN MONOMER PROTEOGLYCAN AGGREGATE  0.11  5.8 5.2  6.3  -155-  Table  2&.  (crystal  Experiment #  E f f e c t o f s o d i u m and p o t a s s i u m i o n s on MSUM c r y s t a l g r o w t h r a t e c o n s t a n t . g r o w t h i n 50 mL  Seed amount (mg)  apparatus;  Sodium/ potassium added (mg)  C = 5 gL  Growth r a t e constant,K', xlO _ _^ (L g min ) 1  33  40  42.6  34  "  48.4  101  "  102  "  "  48.5  103  "  "  43.3  104  "  105  "  1.6(Na ) +  1.3(K ) +  "  42.5  44.9 45.7  -156-  4.4.6  E F F E C T OF ADDITIVES ON CRYSTAL GROWTH RATE CONSTANT The  f o l l o w i n g method was u s e d  generated by c r y s t a l  for the a n a l y s i s o f the data  growth experiments  i n the presence o f the  additives, hyaluronic  acid, chondroitin sulfate,  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  atidylserine.  The o b s e r v e d  rate constant,  proteoglycan  K'(add),  and p h o s p h for the  g r o w t h o f MSUM i n t h e p r e s e n c e o f a d d i t i v e s , was c a l c u l a t e d f r o m equation  31 a s d i s c u s s e d  previously  taken t o be t h e s a t u r a t i o n s o l u b i l i t y the  same c o n c e n t r a t i o n  The  ratio  additive,  o f observed  rate constant  i s t h e c o n t r o l growth r a t e c o n s t a n t  supersaturation  potassium was  either  than o r l e s s  increased  altered  concentration,  ion concentration.  greater  i n the presence o f  was c a l c u l a t e d , where  absence o f any a d d i t i v e s , determined of  o f MSUM i n t h e p r e s e n c e o f  o f a d d i t i v e (see T a b l e 7 ) .  K'(add) t o K ' ( c o n t r o l )  K'(control)  ( s e c t i o n 4 . 4 . 2 ) , where Cs was  f o r MSUM i n t h e  under i d e n t i c a l  conditions  s e e d amount a n d s o d i u m o r  I f the ratio  K'(add)/  K"(control)  than u n i t y , t h e a d d i t i v e under  o r decreased  t h e growth r a t e c o n s t a n t  study and h e n c e  t h e growth r a t e .  Statistical of variance  analyses  were p e r f o r m e d  using  f o l l o w e d b y a Neuman-Keul's t e s t .  a o n e way a n a l y s i s 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)  E F F E C T OF HYALURONIC ACID ON THE GROWTH RATE CONSTANT The  effect  shown i n T a b l e  o f HA on t h e g r o w t h r a t e c o n s t a n t 26.  o f MSUM i s  I n t h e p r e s e n c e o f HA (5 mg t o 20 mg) i n t h e  -157-  Table  26.  Experiment #  (a)  (b)  a:  E f f e c t o f h y a l u r o n i c a c i d on t h e g r o w t h k i n e t i c s o f MSUM i n 50 mL a p p a r a t u s .  Hyaluronic a c i d (mg)  I n i t i a l supersaturation  K'(add)/K'(control)  concentration  82  5  1.148  83  5  0.987  81  10  1.060  84  10  0.876  80  20  0.867  85  20  1.010  I n i t i a l supersaturation  concentration  87  5  0.783  88  5  0.778  86  10  0.742  89  10  0.762  a  = 5 g L  1  .  = 6 g L  1  .  Ratio o f the observed rate constant i n the p r e s e n c e o f HA t o t h e o b s e r v e d r a t e c o n s t a n t i n t h e a b s e n c e o f HA.  -158-  50 5  mL a p p a r a t u s w i t h g L  and  K'(control)  ever, L  1  and seed  1  supersaturation  amount o f 50 mg t h e r a t i o s showed o n l y  when t h e i n i t i a l  concentration of  between t h e K'(add)  s m a l l v a r i a t i o n s from u n i t y .  supersaturation  concentration  a n d t h e s e e d amount was 30 mg t h e K ' ( a d d ) /  decreased for  an i n i t i a l  How-  was 6 g  K'(control)  t o 0.78 (±0.003) f o r 5 mg o f HA a n d t o 0.752 (± 0.010)  10 mg o f HA.  The  analysis of variance  significant  d i f f e r e n c e i n growth r a t e c o n s t a n t s  o f HA f r o m t h o s e significance, 5 g L . 1  and Neuman-Keul's t e s t  of the controls  i n the presence  ( a b s e n c e o f HA) a t t h e l e v e l o f  p< 0.05 when t h e s u p e r s a t u r a t i o n  However, t h e same s t a t i s t i c a l  significant  showed no  decrease i n the c r y s t a l  c o n c e n t r a t i o n was  a n a l y s i s showed a  growth r a t e c o n s t a n t  p r e s e n c e o f 5 a n d 10 mg o f HA when t h e s u p e r s a t u r a t i o n  i n the concentra-  t i o n was 6 g L . 1  Hence, a l t h o u g h MSUM g r o w t h entrations, higher  (B)  a t both  HA d e c r e a s e d 5 g L  the decrease  1  t h e growth r a t e c o n s t a n t  and 6 g L  1  supersaturation  f r o m c o n t r o l was s i g n i f i c a n t  for conc-  only  a t the  degree o f s u p e r s a t u r a t i o n .  EFFECT  O F C H O N D R O I T I N S U L F A T E ON T H E GROWTH R A T E  CONSTANT  The  ratios  chondroitin additive  o f K'(add)  (rate constant  sulfate) t o K'(control)  aregiven  i n Table  27.  i n the presence o f  for different  The r a t i o  amounts o f  increased  f r o m 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 t h e g r o w t h k i n e t i c s o f MSUM i n 50 mL apparatus. I n i t i a l supersaturation concentration, 5 g L  Experiment #  a:  Chondroitin sulfate (mg)  K'(add)/K'(control)  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  R a t i o o f the o f CS t o t h e o f CS.  observed rate constant observed rate constant  i n the i n the  3  presence absence  -160-  mg  chondroitin  The  analysis  significant in  sulfate.  o f v a r i a n c e and Newman-Keul's t e s t  increase  i n the r a t e  the presence o f c h o n d r o i t i n  level  of significance,  (C)  EFFECT RATE  The  OF  p, o f  PROTEOGLYCAN  c o n s t a n t f o r MSUM c r y s t a l  sulfate  over  that  of controls  MONOMER ON T H E  CRYSTAL  ratios  Since,  o f K'(add)  the r a t i o  (rate  o f PGM  rate  i s evident.  t h e Newman-Keul s t e s t 1  the c o n t r o l  and  10 mg  experiments  showed no and  PROTEOGLYCAN  small  significant  level  of  difference  experiments  and and  between  i n the  significance.  A G G R E G A T E ON T H E  CRYSTAL  GROWTH  CONSTANT  ratios  proteoglycan  o f K'(add)  aggregate)  ( r a t e c o n s t a n t i n the presence o f  to K'(control)  are given i n Table  ( ± 0 . 0 5 0 ) , 0.987  at d i f f e r e n t  added  amounts  29.  I n t h e p r e s e n c e o f 10 mg, were 0.966  greater than unity a  t h e growth  (D)  o f PGA  28.  However, t h e a n a l y s i s o f v a r i a n c e  a t t h e 5%  E F F E C T OF  added  c o n s t a n t i n t h e p r e s e n c e o f 5 mg  p r e s e n c e o f PGM  The  GROWTH  constant i n the presence of  is slightly  i n the growth  RATE  at a  CONSTANT  amounts o f p r o t e o g l y c a n monomer a r e g i v e n i n T a b l e  10 mg  growth  <0.05.  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  increase  showed a  20mg and  (± 0.140) and  50 mg 1.163  o f PGA  the  (± 0.025)  ratios  -161-  Table  28.  Experiment #  a:  E f f e c t o f p r o t e o g l y c a n monomer on t h e g r o w t h k i n e t i c s o f MSUM i n 50 mL apparatus. I n i t i a l supersaturation concentration , 5 g L  Proteoglycan monomer (mg)  K'(add)/K'(control)  121  5  0.965  122  5  1.028  123  5  1.047  124  10  1.071  125  10  1.027  Ratio o f the observed o f PGMto t h e o b s e r v e d o f PGM.  rate constant rate constant  a  i n the presence i n t h e absence  -162-  Table  29.  E f f e c t o f p r o t e o g l y c a n a g g r e g a t e on t h e g r o w t h k i n e t i c s o f MSUM i n 50 mL apparatus. I n i t i a l supersaturation c o n c e n t r a t i o n , 5 g L~ .  Experiment #  a:  Proteoglycan aggregate (mg)  K'(add)/K'(control)  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 t h e o b s e r v e d r a t e c o n s t a n t i n t h e a b s e n c e of PGA.  -163-  respectively.  The one way a n a l y s i s o f v a r i a n c e  Neuman-Keul's t e s t a t t h e 5% l e v e l  (E)  showed no s i g n i f i c a n t  followed  by t h e  d i f f e r e n c e from c o n t r o l  of significance.  EFFECT OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLSERINE ON THE CRYSTAL GROWTH RATE CONSTANT OF MSUM The  and  results  of theeffects  phosphatidyl  constant  of thephospholipids,  serine expressed  as r a t i o s  ph-choline  o f K'(add)  (rate  i n t h e p r e s e n c e o f e i t h e r o f t h e two p h o s p h o l i p i d s ) t o  K'(control)  on t h e MSUM c r y s t a l  g r o w t h a r e shown i n T a b l e s  30 a n d  31.  Phosphatidylcholine constant  caused  an i n c r e a s e  i n most o f t h e e x p e r i m e n t s .  K ' ( c o n t r o l ) ) were 1.120 (± 0.070),  i n t h e growth  The r a t i o s  rate  (K'(add)/  1.325 (± 0.220) a n d 1.070 (±  0.220) i n t h e p r e s e n c e o f 10 mg, 20 mg and 30 mg o f phohphatidylcholine As constant  i s evident values  r e s p e c t i v e l y (Table from t h e r e s u l t s ,  were o b s e r v e d  phosphatidylcholine choline  (label  supersaturated impurities. variability observed  used  claim).  30).  large v a r i a t i o n s i n the rate  i n the presence  i n this  Therefore  s o l u t i o n contained  study  was o n l y  increase  substantial levels of the h i g h  degree o f  I t i salso possible that the  i n t h e growth r a t e c o n s t a n t  phosphatidylcholine atidylcholine  experiments.  80% p h o s p h a t i d y l -  t h e samples added t o t h e  T h e s e i m p u r i t i e s may h a v e c a u s e d i n these  o f p h - c h o l i n e . The  i n the presence o f  was due t o a c o m b i n a t i o n o f b o t h t h e p h o s p h -  and o t h e r  impurities present  i n the phosphatidyl-  -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 t h e g r o w t h k i n e t i c s o f MSUM i n 50 mL apparatus. I n i t i a l supersaturation concentration, 5 g L  Experiment #  a:  Phosphatidylcholine (mg)  K'(add)/K'(control)  53  10  1.050  54  10  1.190  55  20  1.293  56  20  1.357  57  30  1.299  58  30  0.862  R a t i o of the o f PC t o t h e o f PC.  observed observed  rate constant rate constant  i n the i n the  a  presence absence  -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 t h e g r o w t h k i n e t i c s o f MSUM i n 50 mL apparatus. I n i t i a l supersaturation concentration, 5 g L  Experiment #  a:  Phosphatidylserine (mg)  K'(add)/K (control) 1  71  10  1.171  75  10  1.071  72  20  1.027  73  20  1.047  74  20  1.028  Ratio of the o f PS t o t h e o f PS.  observed rate constant observed rate constant  i n the i n the  a  presence absence  -166-  choline  sample.  Phosphatidylserine growth  rate  constant.  unity,  i n the ratios,  showed a v e r y This  small  i s evidenced  increase  by a small  K* ( a d d ) / K ' ( c o n t r o l )  p r e s e n c e o f 10 mg and 20 mg o f p h - s e r i n e  (Table  i n the increase  31).  from  In the  t h e r a t i o s were 1.120  (± 0.050) a n d 1.030 (± 0 . 0 1 0 ) .  The results rate  analysis of variance showed a s i g n i f i c a n t  constant  However,  on t h e  over  that  of control.  o f s i g n i f i c a n c e t h e r e was no i n the  over that o f the c o n t r o l .  EFFECT OF ALBUMIN ON MSUM CRYSTAL GROWTH The  in  test  (p < 0.05) i n t h e g r o w t h  d i f f e r e n c e i n t h e growth r a t e c o n s t a n t  presence o f ph-serine  (F)  increase  i n the presence o f ph-choline  a t t h e same l e v e l  significant  a n d t h e Neuman-Keul's  effect  Figure  o f a l b u m i n on t h e c r y s t a l  36. T h e g r o w t h  rate  constants  g r o w t h o f MSUM i s shown f o r MSUM i n t h e p r e s e n c e  o f a l b u m i n a r e n o t g i v e n b e c a u s e v e r y p o o r c o r r e l a t i o n s were obtained  when t h e c o 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  e q u a t i o n 31.  At the  an i n i t i a l  supersaturation  concentration-time  concentration albumin.  with  showed a l i n e a r  of 5 gL  -  1  ,  decrease i n  i n t h e p r e s e n c e o f 10 a n d 50 mg o f  The g r o w t h r a t e  a l b u m i n was l e s s albumin  time  plot  concentration  i n t h e p r e s e n c e o f 10 mg and 50 mg o f  t h a n t h e g r o w t h r a t e o f MSUM i n t h e a b s e n c e o f  (control).  Complete  inhibition  of crystal  s e e n i n t h e p r e s e n c e o f 200 mg o f a l b u m i n .  g r o w t h was  -167-  6i  o  OH 0  1  120  r240  r 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); c o n t r o l ( • ) .  -168-  Th e p r e s e n c e o f i o n s o r m o l e c u l e s o t h e r material being  c r y s t a l l i z e d can  growth k i n e t i c s  o r on  the  Atoms o r m o l e c u l e s may the  formation  specifically inhibit  the  of defects  i n the  growth o f the  ions  face,  For  G r o n and  was  s u c h as  Hay  Cr  3 +  ,  Fe  The  f o u n d t o be saliva.  crystals.  with  the  the  a b o u t two  However, no  and  can  and crystal  a c t i v e agents are  +  and  commonly  inhibitory  effect  of dicalcium  saliva,  more p r o t e i n  which c o n t a i n s  t i m e s more i n h i b i t o r y effect  on  than  the  o b s e r v e d when serum  r a n g e 0.6-5.5 mg  mL ) -1  of  phosphate  fraction  d i c a l c i u m p h o s p h a t e was  to  1980).  precipitation  inhibitory  2.8.4).  lead  macromolecular p r o t e i n  stimulated  (concentration  (Mullin,  3  the  section  t o a change i n  A l  the  Some compounds  surface and  on  face of a c r y s t a l  leading  3 +  (see  lattice  (1976) showed t h a t t h e  s e c r e t i o n s on  associated  saliva.  crystal  example,  used t o change c r y s t a l h a b i t s  salivary  the  morphology  adsorb onto a p a r t i c u l a r  morphology or h a b i t . trivalent  h a v e number o f e f f e c t s  crystal  enter  than those of  of was  unstimulated  precipitation  of  proteins  were i n c l u d e d  in  the  g r o w t h medium. Moreno of calcium salivary  a l . (1979)  hydroxyapatite  proteins.  growth r a t e blocking  et  induced  of the  crystals.  observed  precipitation  They s u g g e s t e d by  crystal  macromolecules onto the seed  also  the  a reduction  i n the  t h a t the  in  produced  growth s i t e s through a d s o r p t i o n surface  of the  calcium  rate  p r e s e n c e o f human  reduction  m a c r o m o l e c u l e s was  i n the  the  by of  the  hydroxyapatite  -169-  E r w i n and N a n c o l l a s  (1981) s t u d i e d t h e e f f e c t  s o d i u m on t h e g r o w t h r a t e o f MSUM and r e p o r t e d  of heparin  no e f f e c t  of this  m a c r o m o l e c u l e a t l e v e l s o f 0.001-10 ppm.  In our s t u d i e s both  increased  and d e c r e a s e d  growth r a t e s o f  MSUM were o b s e r v e d when t h e m a c r o m o l e c u l e s , h y a l u r o n i c chondroitin  sulfate,  proteoglycan  and  a l b u m i n were i n c l u d e d  and  albumin  inhibited  proteoglycan  monomer, p r o t e o g l y c a n  i n t h e g r o w t h medium.  aggregate  Hyaluronic  acid  MSUM g r o w t h , c h o n d r o i t i n s u l f a t e and  monomer a c c e l e r a t e d MSUM g r o w t h a n d p r o t e o g l y c a n  a g g r e g a t e h a d no e f f e c t  o n MSUM g r o w t h  kinetics.  Although chondroitin s u l f a t e s i g n i f i c a n t l y growth r a t e c o n s t a n t , content  acid,  this  increased the  i n c r e a s e was n o t due t o t h e s o d i u m  o f t h e CS sample a s shown b y t h e r e s u l t s  i n s e c t i o n 4.4.5  (Table 25). MSUM h a s b e e n shown t o a v i d l y b i n d ( K o z i n and McCarty, negative  surface potential,  negatively  charged p r o t e i n s  Chondroitin the  1976) a n d a l t h o u g h  negative  they  bind  with  MSUM c r y s t a l s h a v e a h i g h significant  amounts o f  such as t h e immunoglobulin IgG.  sulfate i s a highly negatively charge a r i s i n g  a number o f p r o t e i n s  charged  macromolecule,  f r o m t h e s u l f a t e (SO^) g r o u p s on a  the  molecule.  I t i s p o s s i b l e t h a t CS a d s o r b s  the  MSUM s e e d  crystals  an  and t h a t  accelerator of crystal  growth.  onto  following adsorption,  sodium  ions  CS a c t s a s  I f t h e SO^ g r o u p s on t h e CS  m o l e c u l e p r o j e c t e d o u t w a r d s away f r o m t h e c r y s t a l groups might a t t r a c t  the surface o f  surface  from t h e s u p e r s a t u r a t e d  these solution  -170-  and  these centers  subsequent lattice.  incorporation o f urate  In t h i s  This  would then a c t as " c a t a l y s t s " f o r t h e  way, CS m i g h t  proposed  proteoglycans calcifies,  function  i n the c a l c i f i c a t i o n  matrix  i n t o t h e MSUM as a growth  crystal accelerator.  f u n c t i o n o f CS i s s i m i l a r t o t h e r o l e o f  i t must b e c o n v e r t e d  calcifiable  ions  (Urist,  o f bone.  Before a t i s s u e .  from a n o n - c a l c i f i a b l e t o a  1976).  I t i s thought that the 2+  proteoglycans o f c a r t i l a g e bind attracts  phosphate  ions  center.  I t i salso believed  calcium  ions  (PO^) a n d f o r m s that  ) which  an i n o r g a n i c  thenegatively 2+  phospholipids  o f matrix v e s i c l e s bind  electrostatic  i n t e r a c t i o n which then allows  i n t e r a c t w i t h phosphate ions  (Ca  Ca  incidence the  o f gouty  proportion  decreases,  arthritis  the calcium 1976).  i ncartilage.  i n o u r growth experiments,  CS, c o u l d  was u s e d .  have q u i t e  and a l s o t h a t t h e advancing  age.  Since  that  and c h o n d r o i t i n - 6 -  t h e two i s o m e r s  d i f f e r e n t e f f e c t s o n MSUM c r y s t a l  chondroitin-6-sulfate  be noted  a m i x t u r e o f unknown c o m p o s i t i o n o f  I t i spossible that  c o r r e l a t e with the observed  i n the  be a m a j o r c a u s e o f  However, i t s h o u l d  two i s o m e r s o f CS, c h o n d r o i t i n - 4 - s u l f a t e  sulfate,  such as  t o e x p l a i n how a d e c r e a s e  amounts o f t h e g r o w t h a c c e l e r a t o r ,  the  with  ions t o  o f CS i n b o t h a g e i n g a n d o s t e o a r t h r i t i c c a r t i l a g e  i t becomes d i f f i c u l t  MSUM d e p o s i t i o n  deposition  increases  acidic  through a two-point  i n solution (Urist,  to crystal  nucleation  charged,  T h e r e i s some e v i d e n c e t h a t p r e e x i s t i n g d i s e a s e o s t e o a r t h r i t i s may l e a d  then  could  growth which might  alterations i n the ratios of  to chondroitin-4-sulfate  i n aged a n d  -171-  osteoarthritic Crystals vial  fluid.  arthritis  cartilage. o f MSUM c a n a l s o p r e c i p i t a t e  CS i s f o u n d  (Barker  directly  i n the synovial f l u i d  e t a l . , 1966)  but there  of patients  i s controversy  w h e t h e r CS i s a n o r m a l 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 al., low  1966; S i l p a n a n t a e t a l . , 1 9 6 7 ) . levels  two p h o s p h o l i p i d s  phosphatidylserine, MSUM. However, used  is  an a c i d i c  negatively quaternary these  (Barker e t  fluid.  a l s o i n c r e a s e d t h e growth r a t e c o n s t a n t o f  a t the concentration  levels  of phosphatidylserine i n t h e growth  a net negative  pH, w h e r e a s p h o s p h a t i d y l c h o l i n e  charge a t  c a r r i e s both a  s e c o n d a r y p h o s p h a t e and a p o s i t i v e l y  amine a n d i s i s o e l e c t r i c  phospholipids  rate  Phosphatidylserine  phospholipid, bearing  charged  growth l e a d i n g t o  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  was n o t s i g n i f i c a n t .  physiologic  the  i n the synovial  i n t h e growth experiments, t h e i n c r e a s e  constant  as t o  I t i s possible that the  a c c e l e r a t i o n o f MSUM c r y s t a l  appearance o f c r y s t a l s  The  with  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 the  i n t h e syno-  charged  i n t h e pH r a n g e 3-10.  Hence,  may f u n c t i o n a s MSUM g r o w t h a c c e l e r a t o r s b y  same mechanisms a s 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  extracellular  lipid  extracellular  lipids  content  i n c r e a s e i n t h e i n t r a c e l l u l a r and of articular  a r e prominent  c a r t i l a g e with  i n the surface layers o f  cartilage.  These l i p i d s  cholesterol  or cholesterol esters, phospholipids  Normal s y n o v i a l f l u i d  a r e comprised  contains  age and  of triglycerides, and g l y c o l i p i d 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  arthritis  fluid  from p a t i e n t s w i t h  a n d o s t e o a r t h r i t i s show i n c r e a s e d  lipids,  c h o l e s t e r o l and n e u t r a l  osition  o f normal s y n o v i a l  with phosphatidylcholine  lipids.  fluid  being  amounts o f p h o s p h o -  The p h o s p h o l i p i d  i s similar t o that  that  the raised l e v e l s o f phospholipids  diseased  c a r t i l a g e and s y n o v i a l  fluid  could  growth r a t e  constant,  significant  i n aged o r  decreases  o f t h e a c t i v e growth s i t e s  surface  with  on t h e c r y s t a l  i n t h e MSUM o f these  subsequent  surface.  found t o be r a i s e d i n t h e s y n o v i a l  gouty p a t i e n t s .  Thus i n c r e a s e d  not account  l e v e l s o f t h e growth  fluids of inhibitor  f o r t h e a p p e a r a n c e o f MSUM c r y s t a l s i n  gouty s y n o v i a l  fluids.  with advancing  age a n d a l t h o u g h HA h a s b e e n shown t o b e a  crystallization significant  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  inhibitor,  poisoning  Albumin  l e v e l s have been  albumin could  t h e growth  i n these t i s s u e s .  p r o b a b l y due t o a d s o r p t i o n  m o l e c u l e s o n t o t h e MSUM c r y s t a l  It i s  accelerate  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  comp-  o f plasma  t h e major c o n s t i t u e n t .  possible  A l b u m i n a n d HA c a u s e d  rheumatoid  i t i sunlikely that  factor i n the c r y s t a l l i z a t i o n  increase  HA i s a  o f MSUM i n a r t i c u l a r  cartilage.  At mL  1  concentrations  and p r o t e o g l y c a n  significant  effect of  of proteoglycan aggregate,  the  o f bovine nasal  0.2-1.0 mg mL  1  , we f o u n d n o  e i t h e r PG monomer o r PG a g g r e g a t e on t h e  g r o w t h k i n e t i c s o f MSUM. effect  monomer b e t w e e n 0.1-0.2 mg  B l u m e n t h a l e t a l . (1979) s t u d i e d t h e  c a r t i l a g e PG a g g r e g a t e and PG monomer o n  d i r e c t p r e c i p i t a t i o n o f hydroxyapatite  from l o w c o n c e n t r a t i o n  -173-  calcium phosphate concentration, (induction PG-free  t h e aggregate  more e f f e c t i v e  was  affected  formation  by proteoglycans,  compared t o t h e  formation.  on a w e i g h t b a s i s than Although  o f onset  was m e a s u r e d  o f hydroxyapatite  of apatite.  that with increasing  i n c r e a s e d t h e time  The i n d u c t i o n t i m e  t o onset  formation  They f o u n d  time) o f h y d r o x y a p a t i t e  control.  reagents  solutions.  from m i x i n g o f  The aggregate  was  t h e monomer i n d e l a y i n g t h e  t h e onset  of apatite  t h e subsequent growth  formation kinetics  were n o t a f f e c t e d .  The  latter  observation  i s i n agreement w i t h o u r f i n d i n g s  that proteoglycans  did not s i g n i f i c a n t l y  kinetics  The PG c o n c e n t r a t i o n s u s e d b y B l u m e n t h a l e t  al.  o f MSUM.  1  i n cartilage  (1975) t o b e i n t h e r a n g e o f 15-60 mg m L (1979) p r e d i c t e d t h a t s u c h h i g h  have a p o t e n t  inhibitory  possible  that these high  addition  to inhibiting  affect  confirm It  - 1  Since the  was f o u n d  by Maroudas  , Blumenthal e t  PG c o n c e n t r a t i o n s  would  on a p a t i t e d e p o s i t i o n .  PG c o n c e n t r a t i o n s  the onset  It i s  i ncartilage, i n  o f a p a t i t e formation  o f MSUM.  o f MSUM e m p l o y i n g h i g h  could also  S t u d i e s o f t h e growth  PG c o n c e n t r a t i o n s a r e needed t o  this. i sevident that the effects  synovial fluid  supersaturated suggest  effect  t h e growth k i n e t i c s  kinetics  and  t h e growth  (1979) were i n t h e r a n g e o f 0.1-1.5 mg mL .  concentration o f proteoglycans  al.,  alter  of the different  cartilage  components o n t h e s e e d e d g r o w t h o f MSUM f r o m  solutions arevery  that alterations  complex.  Our r e s u l t s  would  i n t h e 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 metabolism crystals  o f ageing, p r e e x i s t i n g could play  i n joints.  involving  significant However,  disease or altered roles  crystal  the interplay of several  alterations  matrix  i n t h e g r o w t h o f MSUM  d e p o s i t i o n may b e a p r o c e s s  factors.  i n more t h a n one c a r t i l a g e  For instance,  or synovial  fluid  component may b e n e c e s s a r y b e f o r e MSUM g r o w t h p r o c e e d s . such as l o c a l  temperature  a n d pH, c o n c e n t r a t i o n s o f i o n s s u c h a s  magnesium, c a l c i u m , sodium, p y r o p h o s p h a t e cartilage  and o t h e r c a r t i l a g e  as g l y c o p r o t e i n s deposition  4.4.7  a n d g r o w t h o f MSUM  absence  t o date  such  influence the  crystals.  after  growth experiments  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  Scanning  and X - r a y  figures retained The  show t h a t  the needle  obtained  shaped  crystal habit  t h e r e was no a l t e r a t i o n  after  growth  o f MSUM i s  experiments. after  growth e i t h e r  o f a d d i t i v e s were i d e n t i c a l  for theoriginal  growth.  using scanning  a r e shown i n F i g u r e s 37 t o 4 1 . The  f o r a l l MSUM s a m p l e s  p r e s e n c e o r absence  i n the presence  o f MSUM c r y s t a l s  on c o m p l e t i o n o f t h e g r o w t h d-values  both  diffraction.  e l e c t r o n micrographs  the presence o f a d d i t i v e s  after  components n o t s t u d i e d  content o f  CHARACTERIZATION OF MSUM AFTER GROWTH EXPERIMENTS  e l e c t r o n microscopy  in  e t c . , water  a n d k e r a t a n s u l f a t e may a l s o  MSUM c r y s t a l s and  Factors  MSUM s e e d c r y s t a l s ,  i n the c r y s t a l l i n e  i n the  t o d-values  confirming that  s t r u c t u r e o f MSUM  -175-  Scanning electron micrograph of MSUM c r y s t a l s a f t e r growth. I n i t i a l supersaturation concentration 5 g iTi  -176-  Figure 38.  Scanning electron micrograph of MSUM crystals after growth. I n i t i a l supersaturation concentration, 6 g L ~ l .  -177-  Figure  39.  Scanning electron micrograph of MSUM c r y s t a l s a f t e r growth i n the presence o f albumin (50mg). I n i t i a l supersaturation concentration, 5 g L ~ i .  -178-  Figure 40.  Scanning electron micrograph of MSUM c r y s t a l s a f t e r growth i n the presence of chondroitin s u l f a t e (20 mg). I n i t i a l supersaturation concentration, 5 g IT*-.  -179-  Figure 41.  Scanning electron micrograph of MSUM c r y s t a l s a f t e r growth i n the presence of proteoglycan monomer (10 mg). I n i t i a l supersaturation concentration, 5 g L-'-. -  -180-  5  (A) studied  SUMMARY AND CONCLUSIONS  T h e d e g r a d a t i o n o f MSUM i n aqueous s o l u t i o n s was under n o n - s t e r i l e  and s t e r i l e  conditions.  solutions  were p r o d u c e d b y ( i ) f i l t r a t i o n  Millipore  filters  autoclaving  i nall-glass  Vacutainers.  glass 35°,  r e s u l t s were  MSUM s o l u t i o n s  at different obtained:  stored  s t o p p e r s a t 4 ° were r e l a t i v e l y  i n glass  stable,  increased,  o f MSUM.  results  was  d e g r a d a t i o n o f MSUM o c c u r e d more r a p i d l y w i t h t h e  solutions  stored  stored  filters  sterilized  and s t o r e d  t o the non-sterile  determinations variability  a t 4 5 ° , w h i c h were more  stable  a t 3 5°.  MSUM s o l u t i o n s  Millipore  with  i n the  As t h e t e m p e r a t u r e o f i n c u b a t i o n  exception o f solutions  2.  flasks  whereas a t 2 2 ° ,  4 5 ° a n d 6 5 ° , t h e r e was a g r a d u a l d e c r e a s e  concentration  than  and ( i i i ) a u t o c l a v i n g i n  were i n c u b a t e d  The f o l l o w i n g  Non-sterile  t h r o u g h 0.22 um  stoppered Vacutainers ( i i )  containers  The s o l u t i o n s  temperatures. 1.  i n t o rubber  Sterile  showed t h a t  by f i l t r a t i o n  i n Vacutainers  solutions. these  showed  However,  solutions  t h r o u g h 0.22 um similar  repeated  d e m o n s t r a t e d marked  i n u r a t e 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°,  3 5 ° and 6 5 ° .  3.  MSUM s o l u t i o n s s t e r i l i z e d  decrease The  i n urate  decrease  stored  concentration  This  probably  autoclaving.  for solutions  was some u r a t e  loss  due t o a b s o r p t i o n  i n the  into the  stoppers.  MSUM s o l u t i o n s i n a l l - g l a s s  2 2 ° , 3 5 ° a n d 4 5 ° were r e l a t i v e l y  after  after  for solutions stored i n a l l - g l a s s  stoppers,  4. A u t o c l a v e d at  than  indicated that there  presence o f rubber rubber  immediately  i n MSUM c o n c e n t r a t i o n was g r e a t e r  i n Vacutainers  containers.  b y a u t o c l a v i n g showed a  an i n i t i a l  decrease  i n urate  containers  stored  s t a b l e u p t o 96 h o u r s concentration  during  a u t o c l a v i n g , w h e r e a s s o l u t i o n s s t o r e d a t 6 5 ° showed a g r a d u a l decrease  i n urate  autoclaved 45°  concentration with  MSUM s o l u t i o n s were r e l a t i v e l y  u p t o 96 h o u r s a f t e r  during  time.  an i n i t i a l  loss  In V a c u t a i n e r s , t h e s t a b l e a t 2 2 ° and  i n urate  concentration  a u t o c l a v i n g , w h e r e a s s o l u t i o n s s t o r e d a t 3 5 ° and 6 5 °  showed a g r a d u a l  decrease  i n urate  concentration  f r o m 0 t o 96  hours. 5.  N o n - s t e r i l e and s t e r i l e  decomposition  with  time.  I n 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 h a v e d e c r e a s e d and  chemical  decrease  degradation,  MSUM s o l u t i o n s u n d e r w e n t  by both  bacterial  whereas i n s t e r i l e  i n MSUM c o n c e n t r a t i o n was p r o b a b l y  consumption  solutionsthe due o n l y t o c h e m i c a l  degradation. It  was e v i d e n t  from t h e s e  experiments t h a t the c r y s t a l  -182-  g r o w t h o f MSUM c o u l d conditions (B)  be s t u d i e d  upto 8 h o u r s under n o n - s t e r i l e  a t 37° w i t h o u t a p p r e c i a b l e The s a t u r a t i o n s o l u b i l i t y  at d i f f e r e n t  degradation  o f MSUM.  o f MSUM i n w a t e r was s t u d i e d  temperatures and i n t h e p r e s e n c e o f d i f f e r i n g  concentrations  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 ,  proteoglycans,  hyaluronic  a c i d and a l b u m i n .  The f o l l o w i n g i s a  summary o f t h e r e s u l t s : 1.  The h e a t o f s o l u t i o n d e t e r m i n e d  from a v a n ' t H o f f  plot  o v e r a t e m p e r a t u r e r a n g e o f 4 . 4 ° t o 55° was 5 . 8 K c a l .  -1  mole 2. tly  probably  significan-  Chondroitin  s u l f a t e decreased the s o l u b i l i t y  due t o a common i o n e f f e c t  o f MSUM,  from t h e sodium p r e s e n t i n  chondroitin sulfate. 4.  Hyaluronic  acid, proteoglycan  a g g r e g a t e and a l b u m i n caused v e r y saturation (C)  solubility The c r y s t a l  seeded growth The and  o f MSUM d e c r e a s e d  i n t h e p r e s e n c e o f sodium c h l o r i d e . 3.  the  The s a t u r a t i o n s o l u b i l i t y  slight  proteoglycan  increases  i n the  o f MSUM a t 3 7 ° . g r o w t h k i n e t i c s o f MSUM was s t u d i e d b y t h e  technique.  effects o f supersaturation  theadditives, chondroitin  proteoglycans  monomer,  concentration,  sulfate, hyaluronic  (monomer a n d a g g r e g a t e ) ,  (phosphatidylcholine  s e e d amount acid,  a l b u m i n and p h o s p h o l i p i d s  and p h o s p h a t i d y l s e r i n e )  on t h e growth  183  kinetics  o f MSUM were  studied. A brief  summary o f  the  results  is  g i v e n below:  1.  T h r e e methods were u s e d t o d e t e r m i n e t h e  constant,  K'  (K'=  K (obs) S  growth r a t e c o n s t a n t , crystals). was  used  square  fits  gave p o o r c o r r e l a t i o n  the  curves  correlation  obtained  from the  experiments the coefficients  v a r i e d between 0.4 coefficient  crystal  equation defined  with the  1:1  was  good  2:2  least  resulted  rearranged  al.,  1974)  However, when  of the  and  n  the fits  constant amount.  slope  t o o b t a i n an  rate constants,  was  growth  concentration  and  ( M e t z l e r e_t K'.  method.  assumed t o  growth e q u a t i o n K",  order  which  the growth r a t e c o n s t a n t ,  growth p r o c e s s  3.  second  equation  this  The  of  between 2 t o  i n t e g r a t e d form o f the  i n t e g r a t e d form o f the  the growth  (C-Cs).  the  f o l l o w the  c o e f f i c i e n t s were o b t a i n e d by  the order of the  second, p l o t s  seed  A n o n - l i n e a r computer p r o g r a m  used t o e s t i m a t e  Good c o r r e l a t i o n  square  from the  (>0.95), n was  The  of  i n rate  for a given  electrolytes  log  intercept  n o n - l i n e a r r e l a t i o n s h i p between the  (equation 32).  constructed  and  n = 2 was  time  Since,  linear  and  t o 7.2.  growth e q u a t i o n .  was  (C-Cs)  log R versus  the order of r e a c t i o n , obtained  A number o f (n=2)  was  w h i c h were i n p o o r agreement  v a l u e s o f n,  seed  e m p i r i c a l f o r m : R = K'  w e r e p e r f o r m e d on  K',  I n most o f t h e  values  of the  added  purpose.  rate constant,  fit.  overall  Q  S = s u r f a c e area o f the  equation  for this  Least The  An  ; K ( o b s ) = observed  Q  growth r a t e  be were  were o b t a i n e d  from  -184-  th e slopes  o f these  coefficients The K'/S.  were  straight  line  plots.  K (obs), Q  The g r o w t h r a t e c o n s t a n t s , computer program  equation  initial  supersaturation  However, K ( o b s ) , o  values  within a given  method e m p l o y i n g t h e l i n e a r p l o t  o f t h e growth e q u a t i o n experiments  from t h e  estimated  p l o t s o f t h e i n t e g r a t e d form o f t h e growth  gave s i m i l a r  The  estimated  Q  from  showed w i d e v a r i a t i o n s i n t h e v a l u e s  a n d s e e d amount.  from t h e l i n e a r  was d e t e r m i n e d  K (obs),  for a s e t o f experiments a t a given concentration  correlation  obtained.  growth r a t e c o n s t a n t ,  non-linear  Excellent  s e t o f experiments.  of the integrated  t o d e t e r m i n e K ( o b s ) was u s e d Q  involving the effect  form  in all  o f a d d i t i v e s o n MSUM g r o w t h  kinetics. 2. of  The g r o w t h o f MSUM s e e d  supersaturation  crystal 4 g L . 1  tion  crystals  and a c r i t i c a l  g r o w t h was o b s e r v e d  degree No  concentration of  I n t h e 1 L c a p a c i t y a p p a r a t u s when t h e s u p e r s a t u r a -  concentration  seeds t h e r e  was 5 g L , no g r o w t h was o b s e r v e d 1  was a s l o w l i n e a r  rapid non-linear  concentration hours  a.high  q u a n t i t y o f seed c r y s t a l s .  at a supersaturation  p r e s e n c e o f 100 mg s e e d c r y s t a l s .  by  required  Similar  I n t h e p r e s e n c e o f 200 mg o f  growth f o r about  3 hours  followed  S i m i l a r l y when t h e s u p e r s a t u r a t i o n  was 6 g L , a s l o w g r o w t h p e r i o d 1  followed  mg s e e d s were  growth.  i n the  by r a p i d n o n - l i n e a r  f o r about 2  g r o w t h was o b s e r v e d when 200  present. r e s u l t s were o b t a i n e d  using  t h e 50 mL c a p a c i t y  -185-  apparatus. observed  A period  i n t h e p r e s e n c e o f 5 mg  supersaturation of  5 mg  6 g L  concentration  t o 10 mg  (induction period)  t o 20 mg  was  5 g L  i t was  i n t h e added  increase  4  and i n t h e p r e s e n c e concentration  s e e d amount a t a g i v e n  was  degree o f  decreased the length o f the induction  abolished  at a certain  "critical"  In t h e p r e s e n c e o f seed c r y s t a l s  1  1  .  1  supersaturation  L  -  was  s e e d s when t h e  s e e d s when t h e s u p e r s a t u r a t i o n  An i n c r e a s e  until  o f slow growth  i n supersaturation  i n t h e 50 mL  capacity  added  period seed  amount.  f r o m t h e same b a t c h ,  concentration apparatus  from 5 g L  increased  1  an  to 6 g  K (obs)  by  Q  3 to  fold. 3.  crystal  The two d i f f e r e n t b a t c h e s o f s e e d c r y s t a l s growth experiments  constants.  showed d i f f e r e n t  The g r o w t h r a t e c o n s t a n t s ,  s e e d s were h i g h e r seed c r y s t a l s .  growth  K (obs), Q  used  i n the  rate  from b a t c h  than t h e growth r a t e c o n s t a n t s  from b a t c h  These d i f f e r e n c e s i n K ( o b s ) v a l u e s Q  A B  were  t h o u g h t t o be due t o t h e d i f f e r e n c e s i n t h e t y p e and number o f defects 4. 50 mL  present  i n t h e two b a t c h e s o f s e e d  crystals.  I n t h e p r e s e n c e o f s o d i u m and p o t a s s i u m  and 1.3 mg  i n 50 mL  r e s p e c t i v e l y ) there  change i n t h e g r o w t h r a t e c o n s t a n t  decreased  at both  (1.6 mg i n  no  significant  acid  the growth  was  values.  I n t h e p r e s e n c e o f 5 t o 20 mg o f h y a l u r o n i c rate constant  ions  5 g L  1  and 6 g L  1  -185-  supersaturation  concentrations.  c o n t r o l was s i g n i f i c a n t concentration. increased the  only  Chondroitin  10 mg o f p r o t e o g l y c a n constant  was o b s e r v e d  monomer.  aggregate  This  increase  i n t h e growth  rate  of significance.  In the presence o f the  and p h o s p h a t i d y l s e r i n e , a n was o b s e r v e d .  T h e r e was a  whereas  for phosphatidylserine  the increase  not s i g n i f i c a n t .  Albumin At  increase i n  i n t h e growth r a t e caused by  phosphatidylcholine, was  A small  (5 mg t o 20 mg) showed no s i g n i f i c a n t  phosphatidylcholine  increase  significantly  i n t h e p r e s e n c e o f 5 mg t o  a t t h e 5% l e v e l  i n t h e growth r a t e c o n s t a n t  significant  from t h e  supersaturation  increase  change i n MSUM g r o w t h r a t e c o n s t a n t . phospholipids,  1  o f MSUM g r o w t h .  was n o t s i g n i f i c a n t  Proteoglycan  at 6 g L  s u l f a t e (10-30 mg)  the rate constant  growth r a t e c o n s t a n t  However, t h e d e c r e a s e  (10 mg t o 200 mg) c a u s e d  lower c o n c e n t r a t i o n s  g r o w t h r a t e was o b s e r v e d Complete  inhibition  i n h i b i t i o n o f MSUM g r o w t h .  (10 mg a n d 50 mg), a d e c r e a s e from t h e c o n c e n t r a t i o n - t i m e  o f MSUM c r y s t a l  i n the plot.  g r o w t h was s e e n i n t h e  p r e s e n c e o f lOOmg and 200 mg o f a l b u m i n . In these  studies both  increased  and d e c r e a s e d  o f MSUM were o b s e r v e d when t h e m a c r o m o l e c u l e s : chondroitin  sulfate,  a l b u m i n were i n c l u d e d indicated  hyaluronic  rates acid,  (monomer a n d a g g r e g a t e ) a n d  i n t h e g r o w t h medium.  that the process  supersaturated complex.  proteoglycans  growth  of crystallization  These  results  o f MSUM f r o m  s o l u t i o n s i n the presence o f these  a d d i t i v e s was  I t i sp o s s i b l e that a l t e r a t i o n s i n t h e composition o f  -187-  synovial  fluid  or c a r t i l a g e  as a r e s u l t o f a g e i n g  or  preexisting  disease or a l t e r e d  metabolism o f the c a r t i l a g e matrix could  a significant role  i n the growth  o f MSUM c r y s t a l s .  play  -188-  6  REFERENCES  A g u d e l o , C.A., W e i n b e r g e r , A., Schumacher, H.R., T u r n e r , R. and M o l i n a , J . 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(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 m u c o p r o t e i n . J . C l i n . I n v e s t . , 39: 400-12.  Z u c k n e r , J . , U d d i n , J . , G a n t n e r , G . E . J r . and D o r n e r , 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 The of  M u i r and  from the The slices. buffer  chloride density gradient  Hardingham  (1975) was  bovine nasal  c a r t i l a g e was  The  g l a s s wool.  s m a l l and  thin  rolled  f o r 24 h o u r s  cartilage  i n the  cold  filtration  room  rolled  the  r e s i d u e by  t h e n added t o t h e r e s i d u e and  f o r 3 hours  at 4°.  filtration  were p o o l e d and were k e p t e x t r a c t was  e x t r a c t was  through g l a s s wool. frozen t i l l  further  placed i n a dialysing  a l a r g e volume o f 0.5  extraction buffer.  The  The  foaming,  f o r 48 h o u r s  two  from  extracts  treatment. dialysed  overnight.  g mL  The m i x t u r e was  p l a c e d i n the u l t r a c e n t r i f u g e  ultracentrifuged  The  cylinder  a d j u s t e d t o 1.69  chloride.  separated  t u b e and  dialysed  p l a c e d i n a weighed measuring  a d d i t i o n o f cesium  mixture  M guanidinium h y d r o c h l o r i d e  m i x t u r e was  d e n s i t y o f t h e m i x t u r e was  the  a t 100,000g a t  1  and by  The the  the  mixed g e n t l y tubes 20°.  and  and  through  F i v e volumes o f the 4 M g u a n i d i n i u m h y d r o c h l o r i d e  was  e x t r a c t was  cut into  s e p a r a t e d from the r e s i d u e by  e x t r a c t i o n b u f f e r was  avoid  to i s o l a t e proteoglycans  were added t o e a c h gram o f t h e  m i x t u r e was  e x t r a c t was  against  method  volumes o f 4 M g u a n i d i n i u m h y d r o c h l o r i d e e x t r a c t i o n  ( s e e page 211)  The  used  centrifugation  cartilage.  Ten  slices. the  cesium  PROTEOGLYCANS FROM BOVINE NASAL CARTILAGE  to  -211-  Th e c e n t r i f u g e  To h a l f 7.5  t u b e s were t h e n c u t i n t o two  (1)  bottom  1/4  (2)  t o p 3/4  portion = Al  portion =  o f t h e A l f r a c t i o n was  A2-4  added e q u a l v o l u m e s o f  M guanidinium hydrochloride buffer g mL  centrifuged  a t 100,000 g and  2 0 ° f o r 48 h o u r s .  into  The  mixture  this  was  The  t u b e s were  three portions: (1)  bottom  1/4  portion =  (2)  middle  2/4  p o r t i o n = A1D2-3  (3)  t o p 1/4  portion =  A1D1  A1D4  remaining h a l f of the A l f r a c t i o n  were d i a l y s e d  against  t h r e e c h a n g e s o f 0.2  the p r o d u c t s l y o p h i l i z e d . aggregate  and A1D1  Extraction 4.0  The  the d e n s i t y o f  a d j u s t e d t o 1.69  1  .  and  m i x t u r e was  cut  portions:  The  f r a c t i o n was  and  M sodium  A l f r a c t i o n was  buffer:  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  0.10  M Ethylamino c a p r o l i c  EDTA acid.  0.005 M B e n z a m i d i n e h y d r o c h l o r i d e M Phenylmethyl  0.004 M n - e t h y l  sulfonyl  maleimide  flouride  fraction  chloride  proteoglycan  p r o t e o g l y c a n monomer.  M Guanidinium h y d r o c h l o r i d e  0.001  t h e A1D1  and  

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