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Proteoglycan aggregation in human intervertebral disc and bovine nasal cartilage Emes, John Hayward 1975

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PROTEOGLYCAN AGGREGATION IN HUMAN INTERVERTEBRAL DISC AND BOVINE NASAL CARTILAGE by JOHN HAYWARD EMES B.Sc. Hons. U n i v e r s i t y o f Reading, England, 1968 M.Sc. U n i v e r s i t y of Guelph, O n t a r i o , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the department of PATHOLOGY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard The U n i v e r s i t y ^ Of B r i t i s h Columbia March 11, 1975 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I ag ree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada i i ABSTRACT H e r n i a t i o n o f the i n t e r v e r t e b r a l d i s c i s a p a t h o l o g i c a l c o n d i t i o n c h a r a c t e r i z e d by p r o t r u s i o n of the t i s s u e p o s t e r i o -l a t e r a l l y , o f t e n impinging on the s p i n a l chord or nerve r o o t s . The d i s e a s e i s accompanied by a r e d u c t i o n i n the average molecular weight and v i s c o s i t y of the d i s c p r o t e o g l y c a n s , i n excess of t h a t which normally occurs w i t h i n c r e a s i n g age. The p r o t e o g l y c a n s of d i s c however have not been examined i n terms of the modern concepts of c a r t i l a g e matrix s t r u c t u r e . Bovine n a s a l c a r t i l a g e , has been shown to c o n t a i n p r o t e o g l y c a n aggregates, trapped i n the i n t e r s t i c . e s s of a c o l l a g e n network, which can be d i s s o c i a t e d with 4M guanidine h y d r o c h l o r i d e i n t o d i f f u s i b l e p r o t e o g l y c a n s u b u n i t s and a multicomponent " l i n k i n g " f r a c t i o n . A s i m i l a r system was thought to occur i n the i n t e r -v e r t e b r a l d i s c . I t seemed p o s s i b l e t h a t , i f such a system was present i n the d i s c , the r e d u c t i o n i n the m o l e c u l a r weight and v i s c o s i t y of the p r o t e o g l y c a n s w i t h i n c r e a s i n g age and h e r n i a t i o n c o u l d be due to a decrease i n p r o t e o g l y c a n a g g r e g a t i o n . The present study showed t h a t p r o t e o g l y c a n aggregates s i m i l a r t o those of bovine n a s a l c a r t i l a g e are found i n the human i n t e r v e r t e b r a l d i s c , but t h a t they o n l y r e p r e s e n t 5% of the t o t a l p r o t e o g l y c a n s i n the t i s s u e . In c o n t r a s t , bovine n a s a l c a r t i l a g e c o n t a i n e d 70% of the p r o t e o g l y c a n s i n the aggregated form. A novel m o d i f i c a t i o n of the e x t r a c t i o n procedure was d e v i s e d by which i t was p o s s i b l e to assess the degree of i i i proteoglycan aggregation. S e q u e n t i a l e x t r a c t i o n of the t i s s u e w i t h a weak and strong e l e c t r o l y t e (0.4 M and 4M guanidine hydrochloride) s e l e c t i v e l y removed non-aggregated and aggregated proteoglycans r e s p e c t i v e l y . This procedure provides a new and r a p i d method f o r assessing the degree of proteoglycan aggregation i n a v a r i e t y of connective t i s s u e s . The small p r o p o r t i o n of aggregate i n the d i s c was almost e x c l u s i v e l y l o c a t e d i n the annulus f i b r o s u s . Re-aggregation s t u d i e s suggested t h a t both d i s c and c a r t i l a g e c o n t a i n two proteoglycans, only one of which i s capable of forming aggregates. Examination of the proteoglycans i n a l i m i t e d number of d i s c s suggested t h a t the degree of aggregation d i d not change w i t h i n c r e a s i n g age. Since, i n a d d i t i o n , aggregates represent only a small p r o p o r t i o n of the d i s c proteoglycans, i t appeared un-l i k e l y t h a t a decrease i n the degree of aggregation could account f o r the decrease i n molecular weight and v i s c o s i t y of the d i s c proteoglycans observed w i t h i n c r e a s i n g age and/or degenerative d i s c d i s e a s e . i v TABLE OF CONTENTS-page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES i x < ABBREVIATIONS x i l INTRODUCTION 1 REVIEW OF THE LITERATURE 4 A. THE INTERVERTEBRAL DISC 4 1) The anatomy of the normal i n -t e r v e r t e b r a l d i s c 4 2) The chemical c o n s t i t u t i o n of the normal d i s c 7 a) Glycosaminoglycans 7 b) Proteoglycans 9 c) C o l l a g e n 12 3) Changes i n the anatomy of the i n t e r v e r t e b r a l d i s c w i t h age 14 4) Changes' i n the chemical con-s t i t u t i o n of the i n t e r v e r t e b r a l d i s c w i t h age 16 5) Changes i n the anatomy of the i n t e r v e r t e b r a l d i s c w i t h hern-i a t i o n 6) Changes i n the chemical con-s t i t u t i o n of the i n t e r v e r t e b r a l d i s c w i t h h e r n i a t i o n 20 7) The mechanism of h e r n i a t i o n o f the human i n t e r v e r t e b r a l d i s c 2 3 B. THE STRUCTURE OF CARTILAGE PROTEO-GLYCANS C. RATIONALE MATERIAL AND METHODS REAGENTS 1) P r e p a r a t i o n of guanidine hydro-c h l o r i d e 2) Cesium c h l o r i d e 3) Other reagents ANALYTICAL METHODS 1) Hexose 2) Hexuronate 3) P r o t e i n 4) Sulphate 5) Hexosamine 6) Gas chromatography CELLULOSE ACETATE ELECTROPHORESIS HISTOLOGY VISCOSITY PAPER CHROMATOGRAPHY GEL CHROMATOGRAPHY . D E N S T T Y U G R A D I E N T U COLLECTION AND PREPARATION OF SPECIMENS 1) Human i n t e r v e r t e b r a l d i s c 2) Human c o s t a l c a r t i l a g e 3) Bovine n a s a l c a r t i l a g e EXTRACTION OF PROTEOGLYCANS 1) S i n g l e stage technique 2) S e q u e n t i a l e x t r a c t i o n of pro-t e o g l y c a n s RESULTS THE EXTRACTION OF PROTEOGLYCANS FROM HUMAN INTERVERTEBRAL DISC DEMONSTRATION OF PROTEOGLYCAN COMPLEXES GEL CHROMATOGRAPHY OP DISC PROTEOGLYCANS 1) S e l e c t i o n o f the g e l 2) Chromatography of aqueous e x t r a c t s 3) Chromatography of d e n s i t y g r a d i e n t c e n t r i f u g a t i o n f r a c t i o n s of 4M GuCl e x t r a c t s of i n t e r v e r t e b r a l d i s c SEQUENTIAL EXTRACTION OF PROTEOGLYCANS OF DISC AND CARTILAGE HISTOCHEMICAL ASSESSMENT OF SEQUENTIAL EXTRACTION OF DISC AND CARTILAGE SEQUENTIAL EXTRACTION OF ANNULUS FIBROSUS AND NUCLEUS PUEPOSUS THE CHEMICAL COMPOSITION OF DISC PROTEO-GLYCANS . " OTHER PROCEDURES DISCUSSION THE NATURE OF PROTEOGLYCAN AGGREGATION THE EXTRACTION OF PROTEOGLYCANS THE DEMONSTRATION OF PROTEOGLYCAN AGGREGATION GEL CHROMATOGRAPHY OF DISC PROTEOGLYCANS SEQUENTIAL EXTRACTION OF PROTEOGLYCANS THE DISTRIBUTION OF PROTEOGLYCAN AGGREGATES IN THE INTERVERTEBRAL DISC THE CHEMICAL COMPOSITION OF DISC PROTEOGLYCANS CONCLUSIONS BIBLIOGRAPHY APPENDIX APPENDIX I I v i i i LIST OF FIGURES F i g u r e 1. The anatomy of the human i n t e r v e r t e b r a l d i s c 21 The i s o l a t i o n of pr o t e o g l y c a n s from bovine nucleus pulposus 3. Proposed s t r u c t u r e of the p r o t e o g l y c a n of bovine i n t e r v e r t e b r a l d i s c 4. Flow c h a r t f o r the i s o l a t i o n of bovine n a s a l c a r t i l a g e proteoglycans. • 5. Standard form used f o r r e c o r d i n g c l i n i c a l data on autopsy cases 6. The hexuronate (% of t o t a l ) removed from i n t e r -v e r t e b r a l d i s c a f t e r e x t r a c t i o n &6x^various times 7. Hexuronate a n a l y s i s of the washings o b t a i n e d f o l l o w i n g e x t r a c t i o n of the d i s c 8. The e x t r a c t i o n of pro t e o g l y c a n s by v a r i o u s agents 9. Flow c h a r t f o r the f r a c t i o n a t i o n of pro t e o g l y c a n s of d i s c and c a r t i l a g e 10. D e n s i t y g r a d i e n t c e n t r i f u g a t i o n of •4M6j3uG'Me.xtra:cts i n 0i4M GueivapH:.5v8.: " ' " * 11. S c h l i e r e n p a t t e r n s of d e n s i t y g r a d i e n t f r a c t i o n s of a 4M GuCl e x t r a c t o f bovine n a s a l c a r t i l a g e and human i n t e r v e r t e b r a l d i s c . 12. D e n s i t y g r a d i e n t c e n t r i f u g a t i o n of h i g h - d e n s i t y f r a c t i o n s of d i s c and c a r t i l a g e (B) i n 4M GuCl 13. Flow c h a r t f o r the f r a c t i o n a t i o n of pro t e o g l y c a n s of i n t e r v e r t e b r a l d i s c f o r g e l chromatography 14. Column chromatography on B i o - G e l A-15m of f r a c t i o n s o b t ained by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of a 4M GuCl e x t r a c t o f human i n t e r v e r t e b r a l d i s c . 15. Column chromatography on B i o - G e l A-15m of aggregated p r o t e o g l y c a n s obtained by d e n s i t y g r a d i e n t c e n t r i f u g -a t i o n o f a 4M GuCl e x t r a c t o f human i n t e r v e r t e b r a l d i s c X page 16. Column chromatography on B i o - G e l A-50m of f r a c t i o n s o b t a i n e d by d e n s i t y g r a d i e n t c e n t r i -f u g a t i o n of a 4M GuCl e x t r a c t of human i n t e r v e r t e b r a l d i s c 68 17. Column chromatography on B i o - G e l A-50m of aggregated p r o t e o g l y c a n s obtained by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f a 4M GuCl e x t r a c t of human i n t e r v e r t e b r a l d i s c 69 18. Column chromatography of aqueous e x t r a c t s of human i n t e r v e r t e b r a l d i s c on B i o - G e l A-5m and A-15m„ 7 0 19. Flow c h a r t f o r the s e q u e n t i a l e x t r a c t i o n and f r a c t i o n a t i o n of pro t e o g l y c a n s of d i s c and c a r t i l a g e 7 8 20. S c h l i e r e n p a t t e r s o f the s e q u e n t i a l e x t r a c t s of i n t e r v e r t e b r a l d i s c and n a s a l c a r t i l a g e 79 21. S c h l i e r e n p a t t e r s of the d e n s i t y g r a d i e n t f r a c t i o n s of 4M GuCl s e q u e n t i a l e x t r a c t s 8 0 22. Photomicrographs of c r y o s t a t s e c t i o n s of human i n t e r v e r t e b r a l d i s c r a n d bovine n a s a l c a r t i l a g e a f t e r s e q u e n t i a l e x t r a c t i o n w i t h 0.4M and 4M GuCl 8 3 23. Photomicrographs of c r y o s t a t s e c t i o n s of annulus f i b r o s u s and nucleus pulposus s t a i n e d w i t h haematoxylin and e o s i n 86 24. Flow c h a r t f o r the s e q u e n t i a l e x t r a c t i o n of pro t e o g l y c a n s from annulus f i b r o s u s and nucleus pulposus 87 25. S c h l i e r e n p a t t e r n s of s e q u e n t i a l e x t r a c t s of annulus f i b r o s u s and nucleus pulposus 8.9 26. I s o l a t i o n of the l i n k i n g f r a c t i o n from annulus f i b r o s u s f o r a d d i t i o n t o the 0.4M and 4M GuCl e x t r a c t s of nucleus pulposus i n an attempt to i n c r e a s e aggregation 91 27. Flow c h a r t f o r the 4M GuCl e x t r a c t i o n and f r a c -t i o n a t i o n of pr o t e o g l y c a n s from human i n t e r v e r t -e b r a l d i s c f o i . 94 28. C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s of d i s c p r o t e o -glycans s t a i n e d w i t h A l c i a n blue at pH 2.5 95 x i page 29. A l c o h o l f r a c t i o n a t i o n of glycosaminoglycans of pro t e o g l y c a n s e x t r a c t e d from human i n t e r v e r t e -b r a l d i s c f o l l o w i n g treatment w i t h sodium boro-hydride r 30. Flow c h a r t f o r the s e q u e n t i a l . e x t r a c t i o n and f r a c t i o n a t i o n of pro t e o g l y c a n s from whole d i s c annulus f i b r o s u s and nucleus pulposus 31. Proposed model f o r p r o t e o g l y c a n aggregates of bovine n a s a l c a r t i l a g e (Heinegard and H a s c a l l , 1974) .• 32. Proposed models t o account f o r the observed p r o t e o g l y c a n e x t r a c t i o n p r o f i l e s o f bovine n a s a l c a r t i l a g e and human i n t e r v e r t e b r a l d i s c 99 100 106 108 33. Proposed s t r u c t u r e of the p r o t e o g l y c a n o f bovine- i n t e r v e r t e b r a l d i s c (Hopwood & Robinson, 1974), 121 x i i ABBREVIATIONS AF Annulus F i b r o s u s BNC Bovine Nasal C a r t i l a g e GP 1 Mixed G l y c o p r o t e i n F l o a t i n g F r a c t i o n GPL G l y c o p r o t e i n - l i n k GuCl Guanidineur.i H y d r o c h l o r i d e IVD I n t e r v e r t e b r a l D i s c MW Mo l e c u l a r Weight NP Nucleus Pulposus PGS P r o t e o g l y c a n Subunit PPC P r o t e o g l y c a n Aggregates ( P r o t e i n P o l y s a c c h a r i d e Complex) PPL P r o t e i n P o l y s a c c h a r i d e ( I s o l a t e d by s a l t p r e c i p i t a t i o n ) x i i i ACKNOWLEDGEMENTS The author wishes t o thank Dr. R. H. Pearce and Dr. P. E. Reid f o r s u p e r v i s i n g t h i s work and the other members of h i s committee; Dr. P. Bragg, Dr. S. Shim and Dr. P. Vassar f o r t h e i r a s s i s t a n c e . Dr. P. Gofton i s thanked.for h i s h e l p f u l a d v i c e and Mr. C.F.A. C u l l i n g f o r h i s constant i n t e r e s t and encouragement. The author thanks the f o l l o w i n g people f o r t h e i r t e c h -n i c a l a s s i s t a n c e ; Mr. J . Durgo of the Department of Bi o c h e m i s t r y f o r a s s i s t a n c e with the a n a l y t i c a l u l t r a c e n t r i f u g a t i o n , Mr. B. Doogan of the Department of Pathology, Vancouver General H o s p i t a l f o r c o l l e c t i o n of specimens, Mr. J . Walker of the Department of Radiology, Vancouver General H o s p i t a l f o r X-r a y i n g the specimens, Ms. B. Barkoczy f o r p r e p a r a t i o n of h i s t o l o g i c a l s e c t i o n s and Mr. C. Ramey f o r a s s i s t a n c e with the chemical a n a l y s e s . In a d d i t i o n the members of the f a c u l t y and s t a f f of the Department of Pathology are thanked f o r p r o v i d i n g c o n s i s t e n t support over the d u r a t i o n of t h i s study. The author i s indebted t o the M.R.C. f o r f i n a n c i a l support i n the form of s t u d e n t s h i p , and to the U n i v e r s i t y of B.C. f o r a bursary and a U n i v e r s i t y Graduate F e l l o w s h i p . T h i s work was supported by the M.R.C, the F l o r e n c e and George Heighway Fund, a g r a n t - i n - a i d from the U n i v e r s i t y of B. C and by the Canadian A r t h r i t i s and Rheumatism S o c i e t y . "Put not y o u r s e l f i n t o amazement how these t h i n g s should be; a l l d i f f i c u l t i e s are but easy when they are known" Measure Act IV, f o r Measure Scene I I . INTRODUCTION Degenerative changes i n the i n t e r v e r t e b r a l d i s c are a major cause of d i s a b i l i t y i n North America and r e s u l t i n c o n s i d e r -able p a i n and d i s c o m f o r t f o r many people, o f t e n d u r i n g the most p r o d u c t i v e years of t h e i r l i v e s . . The pathogenesis o f the d i s e a s e i s u n c l e a r but the p r e v e l e n c e of the d i s o r d e r i n the lumbar and c e r v i c a l regions' of the s p i n a l column suggests t h a t there i s a mechanical or traumatic component. R e s u l t s of p h y s i c a l and chemical i n v e s t i g a t i o n s have demonstrated changes i n the composition of the d i s c w i t h i n c r e a s i n g age; these changes appear to be accentuated i n d e g e n e r a t i v e d i s c d i s e a s e . Whether t h i s o b s e r v a t i o n r e f l e c t s the pathogenesis of the d i s e a s e or simply a consequence of the degenerative changes i s unknown. , While some s t u d i e s of degenerative d i s c d i s e a s e have conc e n t r a t e d on the mechanical aspects and c o l l a g e n , the r o l e of the glycosaminoglycans i n the pathogenesis and progress of the d i s e a s e has been i n v e s t i g a t e d . In a l l the major chemical s t u d i e s of normal and degenerated i n t e r v e r t e b r a l d i s c s , the components of the g l y c o s a m i n o g l y c a r i - r i c h matrix have been i s o l a t e d by s a l t e x t r a c t i o n of high-speed t i s s u e homogenates. At l e a s t s i x s a l t - s o l u b l e f r a c t i o n s , c o n t a i n i n g most of the glycosaminoglycans were o b t a i n e d . The carbohydrate polymers, p r i n c i p a l l y c h o n d r o i t i n s u l p h a t e and k e r a t a n s u l p h a t e , were found to be c o v a l e n t l y l i n k e d to a p r o t e i n , forming l a r g e " h y d r a t e d , , p r o t e i n - p o l y s a c c h a r i d e s (proteoglycans) . Although the v a r i o u s f r a c t i o n s o b t a i n e d were of s i m i l a r composition, a common s t r u c t u r a l r e l a t i o n s h i p between them c o u l d not be found; I n v e s t i g a t i o n s of other c o n n e c t i v e t i s s u e s showed s i m i l a r multicomponent m a t r i c e s , c o n t a i n i n g the same glycosaminoglycans, although i n d i f f e r e n t p r o p o r t i o n s . Improved e x t r a c t i o n procedures, have demonstrated t h a t i n bovine n a s a l c a r t i l a g e the p r o t e o g l y c a n s e x i s t as hig h m o l e c u l a r weight aggregates and t h a t these aggregates can be r e v e r s i b l y d i s s o c i a t e d i n str o n g s a l t s o l u t i o n s , such as 4M guanidinium c h l o r i d e , i n t o a p r o t e o g l y c a n s u b u n i t and a p r o t e i n - r i c h l i n k i n g f r a c t i o n . Aggregation c o u l d o n l y occur i n the presence o f both "fractions a t a low i o n i c c o n c e n t r a -t i o n o f the medium. F u r t h e r experiments showed.that the f r a c -t i o n s obtained by s a l t - p r e c i p i t a t i o n r e p r e s e n t e d the p r o t e o -g l y c a n i n v a r i o u s aggregation s t a t e s . Based on these and subsequent s t u d i e s a hypothesis was advanced t h a t , i n c a r t i -lage at l e a s t , p r o t e o g l y c a n s e x i s t as hig h molecular weight aggregates trapped i n the i n t e r s t i c e s o f the c o l l a g e n network. In view of the s i m i l a r i t y i n composi.t-ipnbbe^ween-^the-s a l t - p r e c i p i t a t e d f r a c t i o n s of c a r t i l a g e and of i n t e r v e r t e b r a l d i s c the hypothesis t h a t such an o r g a n i z a t i o n of the p r o t e o -glycans e x i s t s i n the matrix of the d i s c d i d not appear un-reasonable. I t was p o s s i b l e t h e r e f o r e t h a t the changes i n the p h y s i c a l p r o p e r t i e s o f the pr o t e o g l y c a n s o f the i n t e r v e r t -e b r a l d i s c observed w i t h i n c r e a s i n g age and/or w i t h degeneration c o u l d be e x p l a i n e d by a l t e r a t i o n of the aggregation s t a t e of the p r o t e o g l y c a n s . The f o l l o w i n g q u e s t i o n s were t h e r e f o r e asked: (1) Do p r o t e o g l y c a n aggregates o f the s o r t d e s c r i b e d 3. f o r a r t i c u l a r c a r t i l a g e e x i s t i n the human i n t e r v e r t e b r a l d i s c ? (2) I f so, c o u l d they account f o r the decrease i n v i s c o s i t y and molecular weight of the p r o t e o -glycans observed w i t h i n c r e a s i n g age and/or with d e g e n e r a t i v e d i s c d i s e a s e ? The r e s u l t s of t h i s study suggest t h a t aggregation of p r o t e o g l y c a n s of the human i n t e r v e r t e b r a l d i s c does occur, but t o a c o n s i d e r a b l y l e s s e r degree than f o r c a r t i l a g e . Some s t r i k i n g d i f f e r e n c e s were observed between the n a t i v e p r o t e o g l y c a n s o f d i s c and c a r t i l a g e and there appeared t o be v a r i a t i o n i n aggr e g a t i o n w i t h i n the d i s c i t s e l f . S i nce the p r o p o r t i o n of aggregated p r o t e o g l y c a n s i n the d i s c i s so s m a l l and a l i m i t e d number of o b s e r v a t i o n s of normal d i s c s over a wide age range showed no decrease i n the degree of a g g r e g a t i o n , i t would appear u n l i k e l y t h a t d i s a g g r e g a t i o n of the pro t e o g l y c a n s c o u l d account f o r the observed changes i n the p h y s i c a l p r o p e r t i e s o f the molecules w i t h i n c r e a s i n g age and/or degenerative d i s c d i s e a s e . 4, REVIEW OF THE LITERATURE A. THE INTERVERTEBRAL DISC The i n t e r v e r t e b r a l d i s c i s a s o f t c o n n e c t i v e t i s s u e p l a t e which l i e s between and j o i n s the bodies of adjacent v e r t e b r a e . I t s f u n c t i o n s are not e n t i r e l y c l e a r but i t app-ears to support the spine ( E y r i n g , 1969). The d i s c undergoes e l a s t i c deformation when s t r e s s e d and thereby cushions the e f f e c t of e x t r i n s i c f o r c e s on the s p i n e . I t s g e l - l i k e pro-p e r t i e s allow the t r a n s m i s s i o n of pr e s s u r e evenly over a l a r g e s u r f a c e area of the v e r t e b r a e and i t s f l e x i b i l i t y f a c i l i t a t e s the a r t i c u l a t i o n of the v e r t e b r a l column. The i n t e r v e r t e b r a l d i s c i s s u b j e c t t o s t r u c t u r a l and co m p o s i t i o n a l changes wi t h i n c r e a s i n g age. D i s c s i n the lumbar r e g i o n of the spine are s u s c e p t i b l e t o p a t h o l o g i c a l degener-a t i v e changes, o f t e n r e s u l t i n g i n d i s c h e r n i a t i o n , p a r t i c u l a r l y between the ages of 30 and 40 y e a r s . The d i s c s l o s e t h e i r e l a s t i c p r o p e r t i e s and bulge or rupture p o s t e r i o r l y under the pressure o f the s p i n a l column. The exudate o f t e n p resses on the s p i n a l cord o r l a t e r a l nerve r o o t s r e s u l t i n g i n severe low back p a i n and, i n some cases, l o s s of f u n c t i o n of p e r i p h e r a l nerves. (1) The Anatomy of the Normal I n t e r v e r t e b r a l D i s c . The twenty-three i n t e r v e r t e b r a l d i s c s of the human s p i n a l column c o n s t i t u t e approximately one-quarter of the h e i g h t o f the v e r t e b r a l column. Although t h e r e i s some v a r i -a t i o n i n the s t r u c t u r e of the d i s c i n d i f f e r e n t r e g i o n s of the s p i n e , the b a s i c anatomy i s common to them a l l . Each d i s c 5. c o n s i s t s of f o u r d i s t i n c t p a r t s : the annulus f i b r o s u s , the nucleus pulposus and two c a r t i l a g e n o u s end p l a t e s ( F i g . 1). The end p l a t e s are composed of h y a l i n e c a r t i l a g e and cover the bone of the centrum and separate the bone of the v e r t e -brae from the nucleus pulposus. The annulus f i b r o s u s forms the l i m i t i n g capsule of the nucleus pulposus and c o n s i s t s of p r o t e o g l y c a n s embedded i n a network of c o l l a g e n f i b r e s i n a Japanese f i n g e r - t r a p arrangement, attached to the v e r t e b r a e by Sharpey's f i b r e s (Coventry, 1969). The nucleus pulposus i s a s e m i g e l a t i n o u s matrix of water and p r o t e i n - p o l y s a c c h a r i d e c o n t a i n i n g a network of c o l l a g e n f i b r i l s , f i b r o b l a s t s and n o t o c h o r d a l c e l l s . The e l e c t r o n microscope and h i s t o c h e m i c a l appearance of the i n t e r v e r t e b r a l d i s c ( S z i r m a i , 1970) suggested t h a t the d i f f e r e n t p r o p e r t i e s of the t h r e e main anatomical r e g i o n s are determined by s p e c i f i c s t r u c t u r a l v a r i a t i o n s of the two major components of the m a t r i x : the c o l l a g e n f i b r i l s and p r o t e o g l y c a n s , r a t h e r than d i f f e r e n c e s i n the chemical composition of the c o n s t i t u e n t s . The d i s c has a f i n e v a s c u l a t u r e a t b i r t h which degen-e r a t e s d u r i n g the f i r s t decade of l i f e but r e t u r n s as the d i s c ages. The nucleus becomes f i b r o s e d and i n s p i s s a t e d with i n c r e a s i n g age and becomes i n d i s t i n c t from the annulus f i b -r o s u s . The normal and p a t h o l o g i c h i s t o l o g y of the i n t e r v e r t -e b r a l , d i s c has been d e s c r i b e d by Coventry et_ a l . (1945) and Inman and Saunders (1947). However, i t i s d i f f i c u l t , except i n severe cases, to d i s t i n g u i s h between the h i s t o l o g i c a l appearance of an aged d i s c and one t h a t i s h e r n i a t e d . 6a. F i g . 1. The anatomy of the human i n t e r v e r t e b r a l d i s c . VB, v e r t e b r a l body; PL, p o s t e r i o r ligament; S C , s p i n a l cord; LNR, l a t e r a l nerve r o o t ; TP, t r a n s v e r s e process; AS, a r t i c u l a r s u r f a c e ; NP, nucleus pulposus; AF, annulus f i b r o s u s ; HC, h y a l i n e c a r t i l a g e ; SF, Sharpey's f i b r e s . 7. (2) The Chemical C o n s t i t u t i o n of the Normal D i s c (a) Glycosaminoglycans The i n t e r c e l l u l a r m atrix of the i n t e r v e r t e b r a l d i s c i s r i c h i n .glycosaminoglycans, the major components of which have been i d e n t i f i e d as c h o n d r o i t i n sulphate- and keratan s u l p h a t e , (Davidson and Woodhall, 1959) Table I. G a r d e l l and R a s t g e l d i (1954) i s o l a t e d the p o l y s a c c h a r i d e s from human nucleus pulposus by enzymic deg r a d a t i o n and p r e c i p i t a t i o n with alcohol;.o The presence of equimolar amounts of hexuronic a c i d and galactosamine suggested t h a t the p o l y s a c c h a r i d e c o n t a i n e d c h o n d r o i t i n s u l p h a t e . I n f r a - r e d s p e c t r a of the e x t r a c t i d e n t i -f i e d the p o l y s a c c h a r i d e as c h o n d r o i t i n 6-sulphate. Glucosamine and g a l a c t o s e were a l s o present i n equimolar p r o p o r t i o n s which, along w i t h the high sulphate content of the e x t r a c t , p r o v i d e d s t r o n g evidence f o r the presence of keratan s u l p h a t e . Antonopoulos (1965) showed t h a t c h o n d r o i t i n / 6 - s u l p h a t e and keratan sulphate accounted f o r over 90% of the hexosamine i n the i n t e r v e r t e b r a l d i s c but the r a t i o between glucosamine and galactosamine v a r i e d c o n s i d e r a b l y from specimen to specimen. The presence of hyaluronate was d i s c o v e r e d by H a l l et a l . (1957) who i s o l a t e d a component from human nucleus pulposus with an e l e c t r o p h o r e t i c m o b i l i t y s i m i l a r t o t h a t of h y a l u r o n -a t e . Nucleus pulposus was e x t r a c t e d with water by hig h speed homogenization a t 0° C and the s o l u t i o n was d e p r o t e i n i z e d by Sevag's method ( f r a c t i o n A ). A second f r a c t i o n (B) was ob-t a i n e d by e x t r a c t i o n of the r e s i d u e from the aqueous.extract w i t h 10% (w/v) ca l c i u m c h l o r i d e . A f u r t h e r f r a c t i o n (C) was e x t r a c t e d by treatment of the r e s i d u e w i t h 0.5% (w/v) TABLE I S t r u c t u r e of the glycaminoglycans of i n t e r v e r t e b r a l d i s c . H y a l u r o n i c A c i d G l u c u r o n i c a c i d (1-3) N A c e t y l glucosamine C 0 0 ~ " CH O 0 H 4 X > 1 OH ' HllcOCHg C h o n d r o i t i n 6-Sulphate G l u c u r o n i c a c i d (1-3) N A c e t y l galactosamine 6-sulphate COOH C H O 0 S 0 ~ OH H N C O C H 3 Keratan Sulphate N' A c e t y l glucosamine 6-sulphate (1-3) g l a c t o s e CH2OSO"^ • . C H 2 O H H ^ J C 0 C H 3 OH 9. potassium hydroxide. E l e c t r o p h o r e t i c and i n f r a - r e d s t u d i e s suggested t h a t the d e p r o t e i n i z e d aqueous e x t r a c t ( f r a c t i o n A) contained predominantly k e r a t a n s u l p h a t e , the c a l c i u m c h l o r i d e e x t r a c t ( f r a c t i o n B) contained mainly hyaluronate and the a l k a l i e x t r a c t ( f r a c t i o n C) contained a mixture of c h o n d r o i t i n 4-sulphate and c h o n d r o i t i n 6-sulphate. The hy a l u r o n a t e , how-ever, was not p r e c i p i t a b l e w i t h p y r i d i n e and was c l e a r l y d i f f e r e n t from hyaluronate i s o l a t e d from other t i s s u e s . Antonopoulos (1965) i s o l a t e d hyaluronate from human nucleus pulposus by column chromatography. F r e e z e - d r i e d n u c l e i p u l p o s i were d i g e s t e d w i t h papain at.65° C f o r 3 h and the crude p o l y s a c c h a r i d e s were p r e c i p i t a t e d by a d d i t i o n of 1% (w/v) c e t y l . L p y r i d i n i u m c h l o r i d e (CPC) at pH 7.5. The p r e c i p i t a t e was d i s s o l v e d i n 1% (w/v) CPC/n-propanol and added to a c e l l u l o s e column. Hyaluronate was e l u t e d from the column wit h 0. 3M sodium c h l o r i d e . b) P r o t e o g l y c a n s Many glycosaminoglycans do not e x i s t i n a f r e e s t a t e i n s i t u but are l i n k e d to a non-collagenous p r o t e i n as p r o t e i n -p o l y s a c c h a r i d e complexes ( p r o t e o g l y c a n s ) . Lyons e t a l . (1964) e x t r a c t e d the p o l y s a c c h a r i d e s from human i n t e r v e r t e b r a l d i s c s w i t h water and i s o l a t e d a m a t e r i a l c o n t a i n i n g p r o t e i n and equimolar amounts of c h o n d r o i t i n 6-sulphate and keratan s u l p h a t e . Lowther and Baxter (1966) i s o l a t e d a s i m i l a r p r o t e i n - p o l y s a c c -h a r i d e complex from bovine i n t e r v e r t e b r a l d i s c s . Rosenberg e t a l . (1967) i s o l a t e d s e v e r a l f r a c t i o n s c o n t a i n i n g both glycosaminoglycans. and p r o t e i n s from bovine nucleus pulposus ( F i g . 2.). The major f r a c t i o n (PPL-3) c o n s i s t e d of 14% p r o t e i n , 10a. F i g . 2. The i s o l a t i o n of p r o t e o g l y c a n s from bovine nucleus pulposus (Rosenberg e t a_., 1967, 1969), DRIED TISSUE POWDER high speed homogenization with water CRUDE PROTEOGLYCAN RESIDUE PPL ~PPH extract with 2M potassium acetate — I PPLR extract with 2.5M CaCl r PPLRF extract with 2M potassium acetate P P L 3 PPL4 PPL5 PPL6 o 35% keratan sulphate and 45% c h o n d r o i t i n s u l p h a t e . . On e l e c t r o p h o r e s i s and sedimentation v e l o c i t y s t u d i e s i n the a n a l y t i c a l u l t r a c e n t r i f u g e PPL-3 behaved as a s i n g l e component. On zone e l e c t r o p h o r e s i s of the remaining f r a c t i o n s c h o n d r o i t i n sulphate and keratan sulphate moved as a s i n g l e s p e c i e s , suggesting t h a t they occur as p a r t s of the same molecule. Lyons and Singer (1971)showed t h a t p r o t e o g l y c a n s from human pulposus contained both c h o n d r o i t i n sulphate and keratan sulphate chains a t t a c h e d v i a a l k a l i - l a b i l e and a l k a l i - s t a b l e bonds t o a common•protein -core. Hopwood and Robinson (1974 a) proposed t h a t two d i f f e r e n t l i n k a g e s occur between keratan sulphate and p r o t e i n i n the i n t e r v e r t e b r a l d i s c ; an a l k a l i - s t a b l e bond between an unknown, r e s i d u e of keratan sulphate and glutamic a c i d and an a l k a l i - l a b i l e l i n k a g e w i t h the f o l l o w i n g s t r u c t u r e : • I Ac Neu Gal Gal NAc • Threonine I I Keratan Sulphate Robinson and Hopwood (1973) showed t h a t at l e a s t 95% of the c h o n d r o i t i n sulphate chains o f the i n t e r v e r t e b r a l d i s c are attached to p r o t e i n v i a an a l k a l i - l a b i l e bond between x y l o s e and s e r i n e . F o l l o w i n g more e x t e n s i v e i n v e s t i g a t i o n of the s t r u c t u r e and composition of d i s c p r o t e o g l y c a n s Hopwood and Robinson (1974 b) proposed a model f o r the c h o n d r o i t i n s u l p h a t e - k e r a t a n s u l p h a t e - p r o t e i n u n i t i n the bovine i n t e r v e r t e b r a l d i s c ( F i g . 3 ) . The p r o t e o g l y c a n s are. not d i s t r i b u t e d evenly i n the d i s c B u t l e r and Wells (1971) showed, both c h e m i c a l l y and histochem i c a l l y , t h a t the nucleus pulposus of the c a t i n t e r v e r t e b r a l d i s c c o n tained a higher c o n c e n t r a t i o n of a glycosaminoglycans than the annulus f i b r o s u s . M i c r o c h e m i c a l assay of v a r i o u s r e g i o n s of the human i n t e r v e r t e b r a l d i s c ( S z i r m a i , 1970) de-monstrated a d e c r e a s i n g glycosaminoglycan content from the cen t r e of the nucleus pulposus outwards, accompanied by a p r o g r e s s i v e decrease i n the c e t y l p y r i d i n i u m c h l o r i d e s o l u b i l i t y . I n t e r e s t i n g l y , the r a t i o of keratan sulphate t o c h o n d r o i t i n sulphate was r e l a t i v e l y c o n s t a n t , which i s c o n s i s t e n t w i t h t h e i r occurence as p a r t s of the same p r o t e o g l y c a n . Gower and P e d r i n i (1969) found t h a t the r a t i o of glucosamine t o galactosamine. was the same i n the annulus f i b r o s u s ' a n d nucleus pulposus although the y i e l d o f p r o t e o g l y c a n from the annulus was lower than from the nucleus pulposus again suggesting t h a t the glycosamino-glycans are j o i n e d t o a common p r o t e i n , c) C o l l a g e n The annulus f i b r o s u s c o n t a i n s l a r g e q u a n t i t i e s of s p e c i f i c a l l y o r i e n t a t e d c o l l a g e n f i b r e s w h i l e the nucleus pulposus c o n t a i n s mainly p r o t e i n - p o l y s a c c h a r i d e and s m a l l e r amounts of randomly o r i e n t a t e d c o l l a g e n f i b r i l s (Meachim & Cornah 1970). Unusual banded s t r u c t u r e s w i t h a p e r i o d i c i t y a t y p i c a l of c o l l a g e n have been observed i n the nucleus pulposus of human s c o l i o t i c and normal r a b b i t spines but t h e i r f u n c t i o n and o r i g i n are not known (Cornah e t a_l. , 1970) . I t has been p o s t u l a t e d t h a t the d i s c matrix e x i s t s as 13a. F i g . 3. Proposed s t r u c t u r e of the p r o t e o g l y c a n of bovine i n t e r v e r t e b r a l d i s c (Hopwood & Robinson 1974). Proteoglycan P r o t e i n Core "Glu -(^eu) x y l g a l g a l gluA C h o n d r o i t i n 25 galNAc Sulphate 25 gluA 28 s o 4 G l y -P-ro-- • T : hr Fucose gal NAc g a l AcNeu g a l — A c N e u 24 gluNAc 24 g a l 31 s o 4 Marinose Glu •gal—AcNeu ( S e r — P r o ) a p r o t e o g l y c a n / c o l l a g e n / w a t e r system i n which the components ac t s y n e r g i s t i c a l l y to produce a s t a b l e , deformable but i n c o m p r e s s i b l e g e l (Szirmai;1970). Evidence from other c a r t i l a g e n o u s t i s s u e s lends support to t h i s hypothesis... S e r a f i n i - F r a c a s s i n i and Smith (1966) observed a c l o s e assoc-i a t i o n between c o l l a g e n and p r o t e i n - p o l y s a c c h a r i d e s i n bovine n a s a l c a r t i l a g e which was supported by evidence of Mohos and Wagner (1969) from a study of c o l l a g e n damage. Mathews (1969) demonstrated a r e v e r s i b l e b i n d i n g between c h o n d r o i t i n sulphate (MW 50,000) and c o l l a g e n i n v i t r o , and proposed a model f o r the p r o t e i n - p o l y s a c c h a r i d e / c o l l a g e n complex i n v i v o . E l e c t r o n micrographs of c o l l a g e n / p r o t e i n -poiLy s a c c h a r i d e b r i d g e s have been p u b l i s h e d by Myers e t a l . (1969) and E i s e n s t e i n e t a l . (1970). (3) Changes i n the Anatomy of the I n t e r v e r t e b r a l D i s c w i t h Age. The macroscopic.and m i c r o s c o p i c changes i n the s t r u c t u r e of the i n t e r v e r t e b r a l d i s c w i t h age have been adequately d e s c r i b e d (Coventry et. a l . , 1945; H a r r i s and McNab, 1954) and have r e c e n t l y been reviewed by Coventry (1969) . The i n t e r v e r t e b r a l d i s c i s endodermal i n o r i g i n and con-t a i n s n o t o c h o r d a l c e l l s and mesenchymal c e l l s , the l a t t e r d e v e l o p i n g i n t o f i b r o b l a s t s , which are the major c e l l type i n the i n t e r v e r t e b r a l d i s c . The newborn d i s c has a s m a l l round nucleus pulposus and r e l a t i v e l y l a r g e v a s c u l a r cartilagenouseefidppiabes. rThe annulus f i b r o s u s e x h i b i t s a r e g u l a r l a t t i c e of c o l l a g e n f i b r i l s d i s -t i n c t from the nucleus pulposus. The young d i s c bulges an-t e r i o r l y and p o s t e r i o r l y as body weight i n c r e a s e s and more pre s s u r e i s brought to bear on the s p i n a l column, the nucleus pulposus becomes f l a t t e n e d and the p r o p o r t i o n of nucleus r e l a t i v e t o the c a r t i l a g e end-plates i n c r e a s e s . The b l o o d v e s s e l s become c o n s t r i c t e d and degenerate d u r i n g the f i r s t decade of l i f e . The n o t o c h o r d a l c e l l s f o l l o w the same f a t e as t h e i r source of n u t r i e n t s i s l o s t . Coventry!(1969) f e e l s t h a t the d i s c reaches m a t u r i t y at approximately 3 0 years and t h a t any changes which occur a f t e r t h i s age are d e g e n e r a t i v e . The water content of the d i s c was thought t o reach a maximum at puberty, and t h e r e a f t e r t o decrease w i t h age, although t i s s u e a n a l y s i s (Gower and P e d r i n i , 1969) suggests t h a t the water content of the d i s c decreased s t e a d i l y from b i r t h onwards. The mature d i s c bulges p o s t e r i o r l y due to the i n c r e a s e d s t a t i c l o a d i n g and the l o r d o t i c posture of the s p i n a l column. The a n t e r i o r annulus i s s t r e t c h e d , w h i l e the p o s t e r i o r annulus i s compressed and fragmentation o c c u r s . With i n c r e a s i n g age the d i s c becomes f i b r o s e d and l e s s d i s t i n c t from the annulus f i b r o s u s . There i s a g e n e r a l t r a n -s i t i o n of the nucleus from a mobile g e l t o a s o l i d f i b r o u s network which o f t e n develops r a d i a l c l e f t s . The annulus f i b r o s u s l o s e s i t s l a m e l l a r s t r u c t u r e and r a d i a l t e a r s appear wi t h i n c r e a s i n g frequency as the d i s c ages ( R i t c h i e and F a h r n i , 1970). F i s s u r i n g of the c a r t i l a g e p l a t e s occurs w i t h the i n v a s i o n of g r a n u l a t i o n t i s s u e and s m a l l b l o o d v e s s e l s i n t o the nucleus pulposus. The aged d i s c exhib i t s marked, s c l e r o s i s and v a s c u l a r i z a t i o n and degenerative changes are e v i d e n t i n a l l the c e l l u l a r components. (.4) Changes i n the Composition of the I n t e r v e r t e b r a l D i s c w i t h Age Puschel (1930) observed t h a t the water content of the human nucleus pulposus decreased g r a d u a l l y from 88% of the f r e s h weight a t b i r t h to 7 0% by the seventh decade. The annulus f i b r o s u s was found t o c o n t a i n l e s s water (.78% of the fre s h weight) at b i r t h , t h i s decreased t o 70%, u s u a l l y by the end o f the second decade. Gower and P e d r i n i (1969) demonstrated a s l i g h t . b u t n o n - s i g n i f i c a n t decrease i n the water content of the human annulus f i b r o s u s w i t h age. In the nucleus pulposus there was a decrease i n water content from 87% of the f r e s h weight,at 4% years t o 72% at 76 y e a r s . C o s t a l c a r t i l a g e e x h i b i t e d a s i m i l a r g r a d u a l decrease i n the h y d r a t i o n w i t h age. H i r s c h e t a l . (1952) found t h a t the newborn nucleus was 92.5% water c o n f i r m i n g the data of P u s c h e l . H i s t o c h e m i c a l examination of the glycosaminoglycans i n the matrix of the i n t e r v e r t e b r a l d i s c . ( H i r s c h e t al.,1952) showed a decrease i n the i n t e n s i t y of the metrachromasia with age; t h i s f i n d i n g was confirmed by the work of N a y l o r and Horton (1955) and Malinsky (1957). H a l l e n (1958) showed an equimolar rajtiio of hexosamine to e s t e r sulphate i n a c i d h y d r o l y s a t e s of f r e e z e - d r i e d human nucleus pulposus: R e l a t i v e to the dry weight of the d i s c both components decreased g r a d u a l l y with i n c r e a s i n g age. F u r t h e r s t u d i e s ( H a l l e n , 1962) i n d i c a t e d an i n c r e a s e i n the dry weight of both the nucleus pulposus and the annulus f i b r o s u s w i t h age accompanied by a p a r a l l e d i n c r e a s e i n the r a t i o of g l u -cosamine : galactosamine. The i n c r e a s e i n dry weight and non-collagenous p r o t e i n with age was thought t o be due t o the d e p o s i t i o n of an i n s o l u b l e m a t e r i a l and not to a decrease i n the water content of the d i s c . N a y l o r (1962) reviewed the b i o c h e m i c a l changes i n the d i s c a s s o c i a t e d w i t h aging and pr-olapse and observed a r e l a t i v e decrease i n the p o l y -s a c c h a r i d e content o f the nucleus pulposus w i t h age which c o u l d be a t t r i b u t e d i n p a r t to the i n c r e a s e i n the c o l l a g e n content. In 0.15 M KC1 e x t r a c t s of r a b b i t nucleus pulposus the r a t i o of keratan s u l p h a t e t o c h o n d r o i t i n sulphate i n c r e a s -ed w i t h age (Davidson and Small, 1963); no data was presented on e i t h e r the absolute or r e l a t i v e p o l y s a c c h a r i d e content of the t i s s u e . Antonopoulos (1965) separated and i d e n t i f i e d the g l y -cosaminoglycans of human nucleus pulposus by d i l u t i o n of protease d i g e s t e d t i s s u e from c e l l u l o s e columns w i t h c e t y l p p y r d d i n i u m c h l o r i d e (CPC) (Antonopoulos e t a l . , 1961). On a n a l y s i s , the f r a c t i o n e l u t e d with 1% (w/v) CPC appeared to be mainly k e r a t a n sulphate; the 0.3 M NaCl f r a c t i o n , h y a l u r o n i c a c i d ; the 0.6M M g C l 2 f r a c t i o n , c h o n d r o i t i n s u l p h a t e ; and the 2.0 M MgC^ f r a c t i o n , k eratan sulphate which d i f f e r e d from the CPC f r a c t i o n o n l y i n i t s s o l u b i l i t y . 18. The t o t a l hexosamine decreased w i t h age from 13.2% of the dry weight of nucleus pulposus i n the 5 to 10 year o l d range t o 9.5% of the dry weight between 65 and 70 y e a r s . The k e r a t a n sulphate and hyaluronate d i d not change wi t h age, having steady v a l u e s of approximately 6% and 1% of the dry weight, r e s p e c t i v e l y . The c h o n d r o i t i n s u l p h a t e S c o n t a i n i n g f r a c t i o n however decreased from 6.2% to 3.0% of the dry weight over the same 6 0 year p e r i o d . Thus the changing r a t i o of keratan sulphate to c h o n d r o i t i n sulphate w i t h age of the nucleus pulposus can be e x p l a i n e d as a decrease i n the a b s o l u t e amount of c h o n d r o i t i n sulphate content of the t i s s u e . P r o t e i n - p o l y s a c c h a r i d e complexes were e x t r a c t e d from human nucleus pulposus by Lyons e t a_l. (1964) 1 In a subsequent study (Lyons e t a l . , 1966) , the e f f e c t s of age and h e r n i a t i o n on the composition of the i n t e r v e r t e b r a l d i s c were measured. Over an 80 year p e r i o d the y i e l d of the main p r o t e o g l y c a n (PP-L) decreased from approximately 30%. ' t o l l e s s than 15%. The hexuronic a c i d and hexosamine content of the nucleus pulposus showed a s i m i l a r t r e n d while the p r o t e i n content i n c r e a s e d s l i g h t l y . The average molecular weight and the l i m i t i n g v i s c o s i t y of the PP-L f r a c t i o n a l s o e x h i b i t e d a steady d e c l i n e w i t h age. Lyons e t a l . (1966) suggested t h a t the observed changes i n the composition and p r o p e r t i e s o f PP-L were com-p a t i b l e w i t h a decrease i n the l e n g t h of the c h o n d r o i t i n s u l -phate s i d e - c h a i n s a t t a c h e d t o the p r o t e i n core of the complex. S i m i l a r changes were a l s o noted i n the m a t e r i a l e x t r a c t e d from the annulus f i b r o s u s . The minor f r a c t i o n (PP-H) was o b t a i n e d i n much lower y i e l d than PP-L, but decreased o n l y s l i g h t l y w i t h i n c r e a s i n g age. The glycosaminoglycan content of PP-H decreased w i t h age a t a r a t e s i m i l a r to t h a t of PP-L. Meyer e t a l . ' (1971) have suggested t h a t the e x t e n t of h y d r a t i o n o f c o n n e c t i v e t i s s u e i s r e g u l a t e d by the d i s t r i b u t i o n and c o n c e n t r a t i o n of the glycosaminoglycans p r e s e n t i n the matrix. Gower and P e d r i n i (1969) concluded t h a t the a g e - r e l a t e d v a r i a t i o n i n the composition of the p r o t e o g l y c a n s of the i n t e r v e r t e b r a l d i s c may be r e s p o n s i b l e f o r p r o g r e s s i v e des. i c a t i o n of the d i s c w i t h i n c r e a s i n g age. Recently however, Comper and Preston (1974) measured the osmotic p r e s s u r e of i n t e r v e r t e b r a l d i s c p r o t e o g l y c a n s from animals -'-of v a r i o u s ages. The r e s u l t s suggested t h a t the age changes, as r e f l e c t e d by a decrease i n water content of the g e l system, were not a r e s u l t of the change i n osmotic p r e s s u r e o f the p r o t e o g l y c a n s , but r a t h e r r e f l e c t e d v a r i a t i o n i n the t r o p i c i n t e r a c t i o n of the p r o t e o g l y c a n s w i t h other components i n m a t r i x . They envisaged t h a t h y d r a t i o n of the t i s s u e was dependent to a l a r g e degree upon the non-collagenous p r o t e i n s of the d i s c which have been shown to be p r e s e n t i n the t i s s u e (Happey et a l . , 1969): and appearr.to denature as the d i s c ages. (5) Changes i n the Anatomy o f the I n t e r v e r t e b r a l D i s c w i t h H e r n i a t i o n . H e r n i a t i o n of the human i n t e r v e r t e b r a l d i s c occurs when a d i s c i n the lumbar or c e r v i c a l r e g i o n of the spine bulges or r u p t u r e s p o s t e r i o r l y , c a u s i n g p r e s s u r e on the s p i n a l c o r d or the l a t e r a l nerve r o o t s . T h i s c o n d i t i o n o f t e n r e s u l t s , 20. i n severe low-back p a i n and/or l o s s of f u n c t i o n of p e r i p h e r a l nerves (Coventry 1969) . H e r n i a t i o n appears to be an exagg-e r a t i o n o fathe degenerative changes which normally occur w i t h age. The annulus f i b r o s u s l o s e s i t s e l a s t i c i t y and p l i a b -i l i t y and the r a d i a l c l e f t s are o f t e n so severe t h a t the l a t -e r a l p r e s s u r e of the nucleus pulposus r e s u l t i n g from the v e r t i c a l l o a d i n g of the d i s c causes the annulus to r u p t u r e p o s t e r i o r l y where l o r d o s i s has produced the most severe degen-e r a t i o n i n the s t r u c t u r e . Brown e t a l . (1957) showed t h a t the nucleus pulposus protrudes i n t o the bone of the centrum w i t h v e r t i c a l l o a d i n g , but i f combined wi t h t o r s i o n a l f f o r c e s the annulus f i b r o s u s i s more l i k e l y t o r u p t u r e . A f t e r approximately 40 years of age, h e r n i a t i o n becomes p r o g r e s s i v e l y l e s s f requent s i n c e the d i s c has changed from a f l e x i b l e g e l i n t o a n o n - e l a s t i c f i b r o u s b l o c k which i s not s u b j e c t to deformation under p r e s s u r e . (6) Changes i n the Composition of the I n t e r v e r t e b r a l D i s c w i t h H e r n i a t i o n . As the normal d i s c i s s u b j e c t to changes i n composition w i t h i n c r e a s i n g age, i t has been d i f f i c u l t to e s t a b l i s h those changes a s s o c i a t e d d i r e c t l y w i t h h e r n i a t i o n , p a r t i c u l a r l y s i n c e the changes appear to be very s i m i l a r to those a s s o c i a t e d w i t h the aging p r o c e s s . Hendry (1958) i s o l a t e d the nucleus pulposus from s e v e r a l p r o l a p s e d i n t e r v e r t e b r a l d i s c s and found t h a t the t i s s u e con-t a i n e d l e s s water than c o n t r o l specimens. He p o s t u l a t e d t h a t the l o s s of water was due t o degradation or l o s s of the p r o t e i n -p o l y s a c c h a r i d e complex. H a l l e n (1958) observed a r e l a t i v e decrease i n the t o t a l p o l y s a c c h a r i d e content of the human nucleus pulposus (with h e r n i a t i o n ) w hile the i c h r o n d r o i - t i n ^ s u l p h a t e .content, decreased S i m i l a r f i n d i n g s s were obtained by Davidson and Woodall (1959) . U l t r a c e n t r i f u g a t i o n of the c h o n d r o i t i n sulphate f r a c t i o n from h e r n i a t e d d i s c s showed i n c r e a s e d p o l y d i s p e r s i t y ; the keratan s u l p h a t e f r a c t i o n appeared normal. These changes were regarded as an a c c e l e r a t i o n of aging caused by a l e s i o n of c o n n e c t i v e t i s s u e metabolism. Happey e t al_. (1961) found the e s t e r - s u l p h a t e content of p r o l a p s e d nucleus pulposus t o be approximately one-h a l f of the r e p o r t e d value f o r normal d i s c when expressed as a percentage of the dry weight of nucleus pulposus. The decrease was r e l a t i v e o n l y , s i n c e the dry weight of the nucleus was i n c r e a s e d . However, a genuine r e d u c t i o n i n the a b s o l u t e q u a n t i t y of glycosaminoglycan accounts f o r p a r t of the decrease. The r e s u l t s favour Hendry's theory t h a t a r e l a t i v e ^ d e h y d r a t i o n of the nucleus leads to p r o l a p s e . A n . a l t e r n a t i v e h y p o t h e s i s (Happey e t aT., 1961) suggested t h a t the normal d e p o s i t i o n of c o l l a g e n f i b r i l s , which i s u s u a l l y a s s o c i a t e d w i t h aging, d i d not occur when there was a d e f i c i e n c y of glycosaminoglycans; the nucleus thus remained a r e l a t i v e l y unstr.engthened g e l , i n c a p a b l e of s u p p o r t i n g the weight of the s p i n e . Happey b e l i e v e d t h a t a d e f i c i e n c y of glycosaminoglycans i n the annulus f i b r o s u s would r e s u l t i n a more f r i a b l e s t r u c t u r e which would be more l i k e l y t o r u p t u r e . M i t c h e l l et. a l . (1961) demonstrated a r i s e i n the t o t a l p o l y s a c c h a r i d e content of the normal, i n t a c t nucleus between the ages of 30 and 40. In t h e i r view p r o l a p s e occured when the p o l y s a c c h a r i d e content f a i l e d to r i s e a t t h i s age. The subsequent l o s s of i n t r a d i s c t e n s i o n caused deformation of d i s c shape w i t h a r e s u l t a n t s t r a i n on the f r i a b l e annulus f i b r o s u s which was thus more l i k e l y to h e r n i a t e than a normal d i s c . In the f o l l o w i n g year Naylor (1962) proposed t h a t h e r n i a t i o n of the i n t e r v e r t e b r a l d i s c r e s u l t e d from an i n c r e a s e i n the i n t r a d i s c p r e s s u r e caused by a r i s e i n osmotic p r e s s u r e when d e p o l y m e r i z a t i o n of the mucopolysaccharides o c c u r r e d . T h i s theory was c o n t r a r y to the evidence i n s i t u (Hendry, 1958). Smare e t a l . (1968) s p e c u l a t e d t h a t i n c r e a s e d i n t r a d i s c p r e s s u r e was r e s p o n s i b l e f o r the s w e l l i n g of the d i s c and the r e s u l t a n t p a i n . K e l l y (1968) however, s t a t e d t h a t the s e l f e x p a n s i b i l i t y of the d i s c i s a medieval concept with no f a c t u a l b a s i s . De-p o l y m e r i z a t i o n of a p r o t e i n - p o l y s a c c h a r i d e complex c o u l d r e s u l t i n an i n c r e a s e d osmotic pressure i n the t i s s u e : t h i s mechanism seems u n l i k e l y s i n c e f r e e p o l y s a c c h a r i d e i s not found i n the d i s c ( E y r i n g , 1969) . Lyons et_ a l . (1964) found the l i m i t i n g v i s c o s i t y of a p r o t e i n - p o l y s a c c h a r i d e complex from h e r n i a t e d d i s c s t o be lower than t h a t of m a t e r i a l e x t r a c t e d from normal d i s c s i n the same age range suggesting t h a t t h e r e i s a decrease i n the e f f e c t i v e s i z e of the molecules. A n a l y s i s of PPL from the nucleus pulposus and annulus f i b r o s u s of h e r n i a t e d d i s c s showed changes s i m i l a r t o those of aging, but markedly exaggerated or a c c e l e r a t e d . There was a decreased y i e l d of p o l y s a c c h a r i d e , a decreased p o l y s a c c h a r i d e content, a decreased l i m i t i n g v i s c o s i t y and a decreased average molecular weight (Lyons et a l . , 1966). These changes, i m p l i e d t h a t the water b i n d i n g c a p a c i t y and e l a s t i c i t y o f the matrix would f a l l and t h a t the nucleus pulposus would t h e r e f o r e be unable t o support the load on the s p i n a l column. Solheim (1966) i s o l a t e d the glycosaminoglycans from h e r n i a t e d human i n t e r v e r t e b r a l d i s c s by the method of Anton-opoulos e_h al_. (1964) . The changes i n the glycosaminoglycans which occur w i t h h e r n i a t i o n were t y p i c a l of an a c c e l e r a t e d aging p r o c e s s . The s o l u b i l i t y o f the c h o n d r o i t i n s u lphate f r a c t i o n was a l s o c o n s i d e r a b l y reduced. In the l i g h t of the r e c e n t work of Comper and Preston (1974) i t seems u n l i k e l y t h a t the h y d r a t i o n o f the d i s c i s r e l a t e d e n t i r e l y t o the compos •it;lona;andi)ihtegri]+.y6:6ftIt'he p r o t e o g l y c a n s . Thus arguments suggesting t h a t v a r i a t i o n i n the composition of the pro t e o g l y c a n s causes an i n c r e a s e (Naylor, 1962; Smare e t a l . , 1968) or a decrease ( M i t c h e l l e t a l . , 1961; Lyons e t a l . , 1966) i n the osmotic p r e s s u r e of the t i s s u e both appear improbable. I t i s p o s s i b l e t h a t the h y d r a t i o n s t a t e o f the t i s s u e i n both normal and patho-l o g i c a l d i s c specimens may be dependent t o a l a r g e extent on the n o n c o l l a g e n o u s p r o t e i n (Happey e t a l . , 1969) r a t h e r than t o the pr o t e o g l y c a n s p r e s e n t i n the t i s s u e m a t r i x . (7) The Mechanism of H e r n i a t i o n of the Human I n t e r -v e r t e b r a l D i s c . S p e c u l a t i o n about the cause of h e r n i a t i o n of i n t e r v e r t -e b r a l d i s c has centered upon those f a c t o r s which c o u l d cause a l t e r a t i o n s i n the composition of the m a t r i x . Whether the changes i n the composition of the d i s c are the cause o'r the e f f e c t of h e r n i a t i o n has not been determined. I n j u r y alone does not u s u a l l y cause h e r n i a t i o n of the i n t e r v e r t e b r a l d i s c , although d i s c s which are l i k e l y to p ro-lapse w i l l be more sus c e p t a b l e to i n j u r y . Lindblom (1957) suggested t h a t h e r n i a t i o n r e s u l t e d from a p r e s s u r e atrophy of the p o s t e r i o r annulus caused by l o r d o s i s of the human s p i n e . H i r s c h and Schajowicz (1952) and H a r r i s and McNab (1954) sugg-e s t e d t h a t the p a r t i c u l a r combination of p r e s s u r e atrophy and t w i s t i n g i n the p o s t e r i o r annulus f i b r o s u s o f the lumbar v e r t e b r a e makes t h i s area e s p e c i a l l y v u l n e r a b l e t o the d e v e l -opment of c l e f t s and r a d i a l t e a r s . Since h e r n i a t i o n mainly occurs i n lumbar v e r t e b r a e , p r e s s u r e and t w i s t i n g f o r c e s seem l i k e l y t o c o n t r i b u t e to h e r n i a t i o n . Prolonged p r e s s u r e on the spine i s thought'to cause f a t i g u e of the annulus which r e s u l t s i n the d e s t r u c t i o n of the p o s t e r i o r annulus ( R i t c h i e and F a h r n i , 1970). Naylor (1962) proposed a complicated mechanism of d i s c p r o l a p s e . The i n i t i a l change was thought to be a d i s t u r b a n c e of the normal e q u i l i b r i u m between s y n t h e s i s and depolymeriz-a t i o n of the p o l y s a c c h a r i d e s i n favour of d e p o l y m e r i z a t i o n . The change may be i n i t i a t e d by an autoimmune rea'c.tdomorbby endocrine i n f l u e n c e s . The l a t t e r s u g g e s t i o n i s based on the work of F e f f e r (1956) and Naylor and Turner (1961) who t r e a t e d p a t i e n t s s u f f e r i n g from i n t e r v e r t e b r a l disch'ke»rhiationl.with h y d r o c o r t i s o n e or a d r e n o c o r t i c p t r o p h i c hormone. Both i n v e s t -i g a t o r s were p a r t i a l l y s u c c e s s f u l i n producing symptomatic r e l i e f from s c i a t i c p a i n . Naylor (1962) s t a t e d t h a t t h e r e i s no evidence that, h e r n i a t i o n of human i n t e r v e r t e b r a l d i s c s i s g e n e t i c a l l y - d e t e r m i n e d . F a r f a n e t a l . (1970) presented data i n d i c a t i n g t h a t the t o r s i o n a l f o r c e s were r e s p o n s i b l e f o r the gross d e t e r i o r -a t i o n of the annular f i b r e s . Rupture of the i n t e r v e r t e b r a l d i s c induced e x p e r i m e n t a l l y by t o r s i o n produced changes sim-i l a r to those n a t u r a l l y o c c u r r i n g i n d i s c s , suggesting t h a t both were the r e s u l t of the same c a u s a t i v e mechanism. Since the a x i s of r o t a t i o n of the lumbar v e r t e b r a e l i e s o u t s i d e the d i s c the annulus i s s u b j e c t to c o n s i d e r a b l y g r e a t e r t o r -s i o n a l s t r e s s than other r e g i o n s o f the s p i n e . The b i o c h e m i c a l evidence suggests t h a t an a c c e l e r a t e d decrease i n the glycosaminoglycans 6,f the i n t e r v e r t e b r a l d i s c with age i s r e s p o n s i b l e f o r a decrease i n the water - b i n d i n g c a p a c i t y o f the mat r i x of the d i s c . The nucleus pulposus, which bears the major l o a d on the s p i n e , t h e r e f o r e l o s e s water and i s unable to support the load e x e r t e d upon i t . The p r e s s -ure i s t r a n s f e r r e d to the annulus f i b r o s u s which, a l r e a d y under l o r d o t i c s t r e s s , r a p i d l y degenerates i n s t r u c t u r e . The annulus bulges or r u p t u r e s p o s t e r i o r l y and s c i a t i c a r e s u l t s . F e s s l e r (1960) suggested t h a t the p h y s i c a l i n t e r a c t i o n between c o l l a g e n , p r o t e i n - p o l y s a c c h a r i d e and water can form a system r e s i s t a n t to mechanical compression. Large hydrated p r o t e i n - p o l y s a c c h a r i d e molecules trapped i n the i n t e r s t i c e s of a f i n e c o l l a g e n network form a v i s c o e l a s t i c s t r u c t u r e which can withstand compression by h i n d e r i n g the flow of water out of i t . An i n c r e a s e i n the pore s i z e of the random meshwork of c o l l a g e n f i b r i l s , such as occurs w i t h the aging of c o l l a g e n i n the i n t e r v e r t e b r a l d i s c , caused decrease i n the f r i c t i o n a l . i n t e r a c t i o n between the p r o t e i n - p o l y s a c c h a r i d e - w a t e r complex and the meshwork and hence a r e d u c t i o n i n the r e s i s t a n c e to compression of the system. Such a mechanism, coupled w i t h the uneven f o r c e s a s s o c i a t e d w i t h l o r d o s i s of the spine c o u l d account f o r the s t r u c t u r a l f a i l u r e of the lumbar i n t e r v e r t -e b r a l d i s c . B. THE STRUCTURE OF CARTILAGE PROTEOGLYCANS High speed homogenization .with v a r i o u s s o l v e n t s has u n t i l r e c e n t l y , been the u s u a l method of e x t r a c t i n g p r o t e o -glycans from con n e c t i v e t i s s u e (Malawasti and Schubert, 1958) but H a r r i n g t o n and Zimm (1965) and Sajdera and H a s c a l l (1969) demonstrated t h a t these procedures r e s u l t e d i n d e g r a d a t i o n o f the macromolecular c o n s t i t u e n t s . S a l t f r a c t i o n a t i o n o f the homogenate ob t a i n e d by homogenization of the t i s s u e i n low i o n i c s t r e n g t h s o l v e n t s produced a number of heterogen-eous f r a c t i o n s , t o which no s t r u c t u r a l or f u n c t i o n a l s i g n i f -i c a n c e c o u l d be a p p l i e d . Sajdera and H a s c a l l (1969) d e s c r i b e d a method by which p r o t e o g l y c a n s c o u l d be removed from bovine n a s a l c a r t i l a g e i n a c c e p t a b l e y i e l d without u s i n g h i g h speed homogenization, thereby a v o i d i n g the problem of molecular d e g r a d a t i o n under hi g h shear f o r c e s . T h i n s l i c e s of c a r t i l a g e were g e n t l y s t i r r e d f o r 24h w i t h 15 vol^&fe/g. of 4M guanidine h y d r o c h l o r i d e (GuCl) or 3M M g C l 2 . The e x t r a c t , c o n t a i n i n g 85% of the t i s s u e u r o n i c a c i d , was d i a l y z e d t o 0.4 M GuCl and c e n t r i f u g e d a t 100,000 x g f o r 48h i n a:.CsCl d e n s i t y g r a d i e n t . A u r o n i c a c i d - r i c h f r a c t i o n was recovered from the bottom o f the tube and p r o t e i n - r i c h f r a c t i o n f l o a t e d t o the top. A n a l y t i c a l u l t r a c e n t r i f u g a t i o n i n 0.4M GuCl demonstrated t h a t the dense f r a c t i o n c o ntained two d i s t i n c t sedimenting s p e c i e s . T h i s mixture was r e c e n t r i f u g e d i n 4M GuCl f o r 48h at 100,000 x g. A u r o n i c a c i d - r i c h f r a c t i o n was again recov-ered from the bottom o f . t h e tube and a p r o t e i n - r i c h component from the top. A n a l y t i c a l u l t r a c e n t r i f u g a t i o n i n 0 . 4M GuCl showed o n l y one s p e c i e s with a sedimentation v e l o c i t y i d e n t i c a l to the slower sedimenting s p e c i e s p r e v i o u s l y found. No boundary was observed i n the upper f r a c t i o n s . When the two components were mixed i n 0.4M GuCl, the bimodal a n a l y t i c a l u l t r a c e n t r i f . u g a t i o n pattern*'was r e s t o r e d . The f a s t e r - s e d i -menting component was ap p a r e n t l y formed at the expense of the o t h e r . Coupled with v i s c o s i t y measurements, t h i s data was i n t e r p r e t e d as r e p r e s e n t i n g a^new concept i n the s t r u c t u r e of c a r t i l a g e ( H a s c a l l and Sa j d e r a , 1969). The pr o t e o g l y c a n s of bovine n a s a l c a r t i l a g e appeared to e x i s t , at l e a s t i n p a r t , as an aggregate (PPC), d i s s o c i a b l e w i t h 4M GuCl i n t o a high molecular weight p r o t e o g l y c a n s u b u n i t (PGS) and a sm a l l p r o t e i n -r i c h l i n k i n g component (GPL). On recombining both f r a c t i o n s i n 0£4M GuCl the aggregate c o u l d be reformed ( F i g . 4 ) . Sub-sequent work by Rosenberg e t al.(1970) demonstrated the r e l a t i o n s h i p between the s p e c i e s obtained by c e n t r i f u g a t i o n of 4M GuCl e x t r a c t s of c a r t i l a g e and the f r a c t i o n s o b t a i n e d 28a. F i g . 4. Flow c h a r t of the i s o l a t i o n of bovine n a s a l c a r t i l a g e p r o teoglycans f o r the demonstration of aggregate formation ( H a s c a l l and S a j d e r a , 1969). 28b BOVINE NASAL CARTILAGE Grind L a t a p i e m i l l e x t r a c t 24h w i t h -iJM GuCl 15ml/g —RESIDUE MM GuCl EXTRACT C l a r i f y w i t h H y f l o Super-cel add CsCl to d e n s i t y I.69 g/ml - u l t r a * c e n t r i f u g e and cut f r a c t i o n s HIGH DENSITY FRACTION (PPC) LOW DENSITY FRACTIONS, CGPI) Add equal v o l . of 7.5M GuCl add CsCl t o d e n s i t y 1.50 g/ml u l t r a -c e n t r i f u g e and cut i n t o 2 f r a c t i o n s d i a l y z e to O.lJM GuCl I HIGH DENSITY FRACTION (PGS) I _ j LOW DENSITY FRACTION(GPL) I MIXTURE (PGS + G P L ^ P P C ) from the h i g h speed homogenates. F r a c t i o n PPL3 resembled the p r o t e o g l y c a n subunit while f r a c t i o n PPL5 resembled the aggregate. C e h t r i f u g a t i o n measurements and e l e c t r o n micro-scopy confirmed these o b s e r v a t i o n s but suggested t h a t PGS was a dimer of t w o l s m a l l e r components (Rosenberg e t al_. , 1970) . Gregory e t al_. (1970) suggested t h a t 4M GuCl e x t r a c t s p r o t e o -glycans from c a r t i l a g e by d i s s o c i a t i n g high-molecular weight aggregates trapped i n the i n t e r s t i c e s of the c o l l a g e n network i n t o f r e e l y d i f f u s a b l e s u b u n i t s and l i n k i n g p r o t e i n which co u l d be converted back to the aggregate form on lowering the i o n i c s t r e n g t h . Tsiganos e t al;(1972) used g e l chroma-tography on Sepharose 2B to demonstrate the formation of h i g h molecular weight aggregates to p i g l a r y n g e a l c a r t i l a g e (Tsiganos a l . •, 1971) and bovine t r a c h e a l c a r t i l a g e (Heinegard, 1972) suggested t h a t the p r o t e o g l y c a n s are both p o l y d i s p e r s e and heterogeneous and t h a t the p r o p o r t i o n of p r o t e o g l y c a n i n the aggregate form may vary from t i s s u e t o t i s s u e and a l s o w i t h i n each t i s s u e under d i f f e r e n t c o n d i t i o n s . Gregory (1973) showed t h a t aggregate formation r e q u i r e d two components o f d i f f e r e n t d e n s i t i e s . T h e h e a v i e r o f the two had a buoyant d e n s i t y i d e n t i c a l to h y a l u r o n i c a c i d which has been shown t o form aggregates w i t h c a r t i l a g e p r o t e o g l y c a n s (Hardingham and Muir, 1973). A 2.7 s p r o t e i n , s o l u b l e i n 4M GuCl, has a l s o been i m p l i c a t e d i n the formation of p r o t e o -g l y c a n aggregates of bovine a r t i c u l a r c a r t i l a g e s (Rosenberg e t a l . , 1973). Hardingham & Muir (1972) showed t h a t p r o t e o g l y c a n s o f l a r y n g e a l c a r t i l a g e form, aggregates, w i t h hyaluronate.." H a s c a l l and Heinegard (1974a, 1974b) and Heinegard and H a s c a l l (1974) proposed t h a t c a r t i l a g e p r o t e o g l y c a n aggregates c o n t a i n p r o t e o -g l y c a n subunits attached to a hyaluronate core w i t h g l y c o p r o t e i n -l i n k . Tsiganos e_t a l , (1972) suggested t h a t a t l e a s t two d i f f e r e n t p r o t e o g l y c a n s occur i n c a r t i l a g e o n l y one of which i s capable of forming aggregates. The h e t e r o g e n e i t y of the pro-t e o g l y c a n s appeared to be r e l a t e d t o v a r i a t i o n i n the s t r u c t u r e of the p r o t e i n core r a t h e r than i n the l e n g t h of the carbohydrate s i d e - c h a i n s . C. RATIONALE The p r o t e o g l y c a n s of bovine n a s a l c a r t i l a g e have been shown to e x i s t , a t l e a s t i n p a r t , as macromolecular aggregates ( H a s c a l l and S a j d e r a , 1969). Gregory (1973) showed t h a t aggregation r e q u i r e d two " l i n k i n g " f r a c t i o n s one of which was p r o t e i n - r i c h and the other appeared to be h y a l u r o n i c a c i d . The a g g r e g a t i o n o f p r o t e o g l y c a n s i n the human i n t e r v -e r t e b r a l d i s c has not y e t been demonstrated. Rosenberg e t a l . (1967), however,showed t h a t the chemical composition of d i s c and c a r t i l a g e p r o t e o g l y c a n s were s i m i l a r and i n the a n a l y t i c a l u l t r a c e n t r i f u g e d i s c p r o t e o g l y c a n s show two peaks, s i m i l a r to those of bovine n a s a l c a r t i l a g e p r o t e o g l y c a n s . S i n c e , as suggested by H a s c a l l and Sajdera (1969), the e x i s t e n c e of aggregates i n the i n t e r v e r t e b r a l d i s c d i d not appear un-reasonable, the f o l l o w i n g q u e s t i o n was t h e r e f o r e asked: Do p r o t e o g l y c a n aggregates of the type d e s c r i b e d by H a s c a l l and Sajdera (1969) i n bovine n a s a l c a r t i l a g e , occur i n the i n t e r v e r t e b r a l d i s c ? Lyons et a l . (1966) and Gower and P e d r i n i (1969) showed v a r i a t i o n i n the composition of the p r o t e o g l y c a n s of the i n t e r v e r t e b r a l d i s c w i t h i n c r e a s i n g age. The y i e l d of p r o t e o g l y c a n s decreased and was accompanied by a r e d u c t i o n i n the h y d r a t i o n of the d i s c and decreases, i n the v i s c o s i t y and average molecular weight of the p r o t e o g l y c a n s . Chemical a n a l y s i s showed a r e d u c t i o n i n the galactosamine and g l u c u r o n i c a c i d of the p r o t e o g l y c a n s w i t h age, although the glucosamine and g a l a c t o s e content remained r e l a t i v e l y c o nstant; the p r o t e i n content Cpfir.feheeproteoglycans i n c r e a s e d s t e a d i l y with age. These r e s u l t s were i n t e r p r e t e d as r e p r e s e n t i n g a decrease i n the number and/or the l e n g t h of the c h o n d r o i t i n s u l p h a t e -chates l e a d i n g to a r e d u c t i o n i n h y d r a t i o n of the m a t r i x . P r o t e o g l y c a n s e x t r a c t e d from surgie.alsspec-imensoo'fhher.niated d i s c e x h i b i t e d s i m i l a r but more exaggerated changes. H e r n i a t i o n has t h e r e f o r e been l i k e n e d t o an a c c e l e r a t i o n of the normal aging process (Lyons et a_l. , 1964; 1966; Gower and P e d r i n i , 1969). In a l l of the major chemical s t u d i e s , of aging and h e r n i a t e d d i s c s , the p r o t e o g l y c a n s were e x t r a c t e d by procedures r e q u i r i n g h i g h speed homogenization of the t i s s u e w i t h s o l u t i o n s . o f low i o n i c s t r e n g t h , s i m i l a r t o t h a t d e s c r i b e d by Rosenberg e t a l . (1967). Thus i t was not known i f the pro t e o g l y c a n s p r e s e n t i n e i t h e r normal or h e r n i a t e d d i s c s were i n the aggregated form, I t was p o s s i b l e t h a t the observed r e d u c t i o n i n molecular weight a n d . v i s c o s i t y of the p r o t e o g l y c a n s r e f l e c t e d a decrease i n the p r o p o r t i o n of aggregated to non-aggregated p r o t e o g l y c a n s i n a d d i t i o n to changes i n the chon-d r o i t i n sulphate chains . F e s s l e r (I960) .^suggested t h a t hydrated p r o t e o g l y c a n s , trapped i n the i n t e r s t i c i e s of a c o l l a g e n meshwork form a system r e s i s t a n t to mechanical compression. A r e d u c t i o n i n p r o t e o g l y c a n a g g r e g a t i o n might t h e r e f o r e r e s u l t i n the l o s s of t h i s p r o p e r t y , l e a d i n g t o a f a i l u r e o f the d i s c . The second p a r t of the proposed i n v e s t i g a t i o n t h e r e f o r e asked the f o l l o w i n g question:' I f p r o t e o g l y c a n aggregates are pr e s e n t i n d i s c , c o u l d a decrease i n aggregation account f o r the r e d u c t i o n i n v i s c o s i t y and mol e c u l a r weight of the pr o t e o g l y c a n s observed i n aged and h e r n i a t e d d i s c s ? MATERIAL AND METHODS REAGENTS 1) P r e p a r a t i o n of guanidine h y d r o c h l o r i d e (GuCl) Guanidine h y d r o c h l o r i d e was prepared from t e c h -n i c a l grade guanidine carbonatec by the procedure of Kawahara and Tanford (1966). Guanidine carbonate (2kg) was p u r i f i e d by d i s s o l v i n g i t i n a minimum volume of water at 4 0 ° C , f i l * t e r i n g (Whatman No.l f i l t e r p a per), c o o l i n g at 4°C and adding 2 volumes of e t h a n o l . The c r y s t a l s o b tained were f i l t e r e d , washed wi t h c o l d a b s o l u t e e t h a n o l and d r i e d i n a vacuum oven at 40°C. The h y d r o c h l o r i d e was formed by mix-i n g the r e c r y s t a l i z e d carbonate w i t h water t o form a paste and adding c h i l l e d HCl to pH 5.4. The s o l u t i o n was f i l t e r e d and the f i l t r a t e c o n c e n t r a t e d t o dryness by vacuum e v a p o r a t i o n below 40°C. The m a t e r i a l was r e c r y s t a l i z e d twice from meth-anol and d r i e d i n a vacuum d e s i c c a t o r over P^^-2) Cesium C h l o r i d e For d e n s i t y g r a d i e n t c e n t r i f u g a t i o n sequanal grade cesium (Pearce Chemical Co.) or t e c h n i c a l grade (Kawecki B e r y l c o I n d u s t r i e s Inc.) was used. 3) Other Reagents A l l o ther reagents used were of reagent grade except where s t a t e d i n the t e x t . ANALYTICAL METHODS 1) Hexose Hexose was determined by the phenol s u l p h u r i c a c i d procedure (Dubois e t a l . , 1956). An a p p r o p r i a t e c o r r -e c t i o n was made f o r the c o n t r i b u t i o n of hexuronate to the chromagen. Using sodium glucuronate and g a l a c t o s e as s t a n -dards i t was found t h a t hexuronate gave 47% of the c o l o u r y i e l d of the mole e q u i v e l e n t of g a l a c t o s e . 2) Hexuronate Hexuronate was estimated a c c o r d i n g to. B i t t e r and Muir (1962). 3) P r o t e i n Chromatographic column f r a c t i o n s and d e n s i t y g r a d i e n t c e n t r i f u g a t i o n f r a c t i o n s were screened f o r p r o t e i n by measurement of the absorbance at 280 or 220nm u s i n g 10mm c e l l s i n a Beckman D.U. u l t r a v i o l e t spectrophotometer. For d e t e r m i n a t i o n of the p r o t e i n content of s p e c i f i c f r a c t i o n s the method of Lowry e t a l . (1951) was used. 4) Sulphate The s u l p h a t e content o f a c i d h y d r o l y z e d p r o t e o -glycans was determined by the t u r b i d i m e t r i c method of Dodgson (1961). Samples c o n t a i n i n g from 10-80 mg sulphate were hydr o l y z e d i n s e a l e d tubes w i t h 25% f o r m i c a c i d f o r 24h a t 100°C i n a t o t a l volume of 1 ml (Antonopoulos,1962). A f t e r h y d r o l y s i s 3ml of 4% t r i c h l o r o a c e t i c a c i d was added f o l l -owed by 1 ml of f r e s h l y prepared 0.5% (w/v) B a C l 2 i n 0.5% (w/v) g e l a t i n ( F i s h e r D.S.P.). The reagents were mixed and the c l o u d s t a b i l i z e d by sta n d i n g f o r 20 min at room temper-a t u r e . The e x t i n c t i o n was measured a g a i n s t a reagent blank i n 10mm c e l l s a t 36-Onm. A standard curve was prepared w i t h s o l u t i o n s of K^SO^ c o n t a i n i n g from 5 t o 100 mg. su l p h a t e . When onl y s m a l l samples c o u l d be obtained the assay was min-a t u r i z e d by a f a c t o r of f i v e . 5) Hexosamine Hexosamine was determined by a m o d i f i c a t i o n of the procedure of Pearson (1963). The sample c o n t a i n i n g bound hexosamine was hy d r o l y z e d f o r 16h a t lOO^C wi t h 4M HC1 i n vacuo and the h y d r o l y z a t e was d r i e d i n vacuo over f l a k e sodium hydroxide. The i n t e r f e r e n c e o f amino a c i d s and n e u t r a l sugars was avoided by p a s s i n g the h y d r o l y z a t e through a c a t i o n ex-changer and e l u t i n g the hexosamine wi t h d i l u t e a c i d . In l a t e r s t u d i e s the c a t i o n exchanger step was omitted s i n c e almost i d e n t i c a l r e s u l t s were ob t a i n e d without removal of the n e u t r a l sugars and amino a c i d s . The chromagen was dev-eloped by treatment of the hexosamine wi t h a c e t y l acetone i n d i l u t e a l k a l i and E h r l i c h ' s -.'reagent. Glucosamine was used to prepare a standard curve. 6) Gas Chromatography The n e u t r a l hexoses of the pr o t e o g l y c a n s were i s o l a t e d by the r e s i n h y d r o l y s i s procedure of Lehnhardt and Winzler (1968) and analyzed by gas chromatography as t h e i r t r i m e t h y l s i l y l e t h e r s on a Hewlett Packard 7610 A d u a l column gas chromatograph f i t t e d w i t h flame i o n i z a t i o n d e t e c -t o r s and on-column i n j e c t i o n (Reid e t a l . , 1970). Chromat-ography was c a r r i e d out i s o t h e r m a l l y a t 195°C on 250cm x 6mm O.D. copper columns c o n t a i n i n g 10% (w/w) SE 52 on 80-100 mesh Di a p o r t S u s i n g helium as the c a r r i e r gas. Peak areas were recorded w i t h a 3370B e l e c t r o n i c i n t e g r a t o r . ' Analyses were c a r r i e d out by an i n t e r n a l standard procedure u s i n g ^mannitol as the standard. CELLULOSE ACETATE ELECTROPHORESIS E l e c t r o p h o r e s i s o f t t h e p r o t e o g l y c a n s was c a r r i e d out i n a Gelman e l e c t r o p h o r e s i s chamber, u s i n g TRIS b a r b i t a l -sodium b a r b i t a l b u f f e r , pH 8.6, at 300v f o r 40min a t room temperature on Sepraphore I I I 2.5cm x 15cm c e l l u l o s e a c e t a t e s t r i p s . The s t r i p s were s t a i n e d with a l c i a n blue 8GS (Fisher) at pH 1.0 and pH 3.0 or wi t h h i g h i r o n diamine, pH 1.5, by the method of Reid e t a l . (1972). HISTOLOGY C r y o s t a t and paraffin-embedded s e c t i o n s of d i s c and car i l a g e , 5-8 p i n t h i c k n e s s , were prepared by standard methods. S e c t i o n s were s t r a i n e d w i t h A l c i a n blue a t pH 1.0 and/ppH 2.5 or w i t h haematoxylin and e e s i n ( C u l l i n g , 1974) . VISCOSITY The v i s c o s i t y was measured i n a semi-micro viscometer w i t h a flow time f o r water c l o s e to 60s (Model A-100, Cannon Instrument Co., S t a t e C o l l e g e , Pa., U.S.A.). PAPER CHROMATOGRAPHY The hexoses and hexosamines of the p r o t e o g l y c a n s were i s o l a t e d by the r e s i n h y d r o l y s i s procedure m o d i f i e d from Pearson (1963) and Boas (1953) used f o r the c o l o r i m e t r i c d e t e r m i n a t i o n of hexosamine. The n e u t r a l sugars were separ-ated by descending flow paper chromatography on Whatman No. 1 paper u s i n g e t h y l a c e t a t e : p y r i d i n e : water, 8:2:1 (v/v/v) as the mobile phase. Galactose., mannose, fucose and x y l o s e were used as standards and the r e d u c i n g sugars were sta'iiried^vwith s i l v e r ( F i s h e r and Nebel, 1955). Hexosamines were separated by the same procedure u s i n g e t h y l a c e t a t e : p y r i d i n e : water, 40:11:6 (v/v/v) as the - s t a t i o n a r y phase and e t h y l a c e t a t e : p y r i d i n e : water: a c e t i c a c i d , 5:5:3:1 (v/v/v/v) as the mobile phase. The r e d u c i n g sugars were st/ainej£tyith s i l v e r and amino n i t r o g e n was d e t e c t e d w i t h n i n h y d r i n . Glucosamine, g a l a c t o s -amine and g a l a c t o s e were used as standards. GEL CHROMATOGRAPHY The macromolecular components of aqueous and 4M GuCl e x t r a c t s of human i n t e r v e r t e b r a l d i s c s were separated by g e l chromatography on agarose g e l s (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) i n 100 cm x 2.5cm I.D. pharmacia columns f i t t e d w ith flow adaptors. F r a c t i o n s were c o l l e c t e d w i t h an LKB Radi Rac f r a c t i o n c o l l e c t o r f i t t e d with a syphon. B i o - G e l A-15m ( e x c l u s i o n l i m i t 15 x 10 ) and'Bio-Gel A-50m ( e x c l u s i o n l i m i t 50 x 10 ) were g e n e r a l l y used. A l l g e l s were p r e s w o l l e n 100-200 mesh beads c o n t a i n i n g 0.01% sodium a z i d e to prevent b a c t e r i a l contamination.. A l l columns were e l u t e d with 0. 5M a c e t a t e b u f f e r , pH 5.8, d e l i v e r e d from a M a r i o t t e f l a s k w i t h a maximum h y d r o s t a t i c p r e s s u r e of 50cm of water. Columns were e q u i l i b r a t e d by e l u t i o n w i t h 2 column volumes of b u f f e r b e f o r e a p p l i c a t i o n of the sample. F l u s h i n g the column wi t h b u f f e r a l s o removed sodium a z i d e which i n t e r f e r e s w i t h the hexuronate a n a l y s i s . The column e f f l u e n t was monitored f o r p r o t e i n by measurement of the absorbance at 220nm and the hexuronate content of the f r a c t i o n was determined by the procedure of B i t t e r and Muir (1962). DENSITY GRADIENT CENTRIFUGATION The s i n g l e and double stage procedures d e s c r i b e d by H a s c a l l and Sajdera (1969) were f o l l o w e d . A s s o c i a t i v e c e n t r i f u g a t i o n was performed i n 0.4M guanidinium c h l o r i d e , 0.05M ammonium a c e t a t e , pH 5.8, a t a l o a d i n g d e n s i t y of 1.50 g/ml. The l o a d i n g d e n s i t y was a d j u s t e d by a d d i t i o n of s o l i d , sequanal grade cesium c h l o r i d e . A l l samples were ^ c e n t r i f u g e d i n a Spinco Model L p r e p a r a t i v e u l t r a c e n t r i f u g e w i t h a type 50 r o t o r and c e l l u l o s e n i t r a t e tubes at 40,000 rev/min (£r a. V 96500 x g) f o r 44-48h at 20°C. F r a c t i o n s were c o l l e c t e d by puncture of the bottom of the tube and displacement of i t s contents from above wi t h kerosene d e l i v e r e d from a s y r i n g e . The d e n s i t i e s of the f r a c t i o n s were determined by weight u s i n g a 500 j j l k i r k m i c r o p i p e t t e . ANALYTICAL ULTRACENTRIFUGATION Sedimentation v e l o c i t y a n a l y t i c a l u l t r a c e n t r i f u g a t i o n was performed i n a Spinco Model E ^ a n a l y t i c a l u l t r a c e n t r i f u g e . 39. A l l samples were prepared i n 0.4M GuCl, 0.05M ammonium a c e t a t e , pH 5.8; the r e f e r e n c e s e c t o r contained b u f f e r s o l u t i o n alone. A double s e c t o r c e l l w i t h a 12mm c h a r c o a l - f i l l e d - e p o n c e n t r e -p i e c e and sapphire windows was used. Samples were c e n t r i f u g e d at 4 8 000 rev/min and t i m i n g was begun on r e a c h i n g maximum speed. R e s u l t s were recorded on 10 x 25cm kodak m e t a l l o g r a p h i c p l a t e s u s i n g S c h l i e r e n o p t i c s . Measurements were made wit h c a l i p e r s from c o n t a c t p r i n t s o:-f, the p l a t e s . . COLLECTION AND PREPARATION OF SPECIMENS 1) Human Lumbar D i s c s I n t e r v e r t e b r a l d i s c s were obtained from the morgue of the Vancouver General H o s p i t a l , c o u r t e s y of the Dept. of Pathology. Specimens were from males who had d i e d of some acute episode and who had no known, h i s t o r y of d i s e a s e a f f e c -t i n g the spine or c o n n e c t i v e t i s s u e s . C l i n i c a l data f o r each case was was recorded on a standard 'form ( F i g . 5 ) . The lumbar v e r t e b r a l bodies and d i s c s were removed as a s i n g l e specimen, w i t h i n 24h of death, w i t h a S t r y k e r bone saw. Some d i f f i c u l t y was found i n removing i n t a c t , the d i s c between the f i f t h lumbar v e r t e b r a e and the Sacrum; t h i s was p a r t l y overcome by u s i n g a Black and Decker j i g saw i n s t e a d of a bone saw. Those specimens processed immediately were c h i l l e d to 0°C, d i s s e c t e d f r e e of adhering c o n n e c t i v e t i s s u e and phot-ographed. The d i s c s were removed wi t h a s c a l p e l , photographed and s t o r e d a t -20°C. Those specimens which c o u l d not be pro-, cessed immediately were s t o r e d a t -20°C; they were subsequently p a r t i a l l y thawed, cleaned of adhering t i s s u e photographed and 40a. F i g . 5. Standard form used f o r r e c o r d i n g c l i n i c a l data on autopsy cases. 40b. Code No, P a t i e n t Name . Sex Age U n i t No. . . ' S u r g i c a l / A u t o p s y No P h y s i c i a n , Presumptive Diagnosis Death/Operation Date Time T i s s u e C o l l e c t e d Date Time P r o c e s s e d by Comments 4P 4 1 . X-rayed. The d i s c s were removed f o l l o w i n g X-ray and s t o r e d at -20°C, or l e f t a t tached to the v e r t e b r a l bodies and d i s s -ected immediately before, use. In a l a t e r study nucleus pulposus and annulus f i b r o s u s were analyzed s e p a r a t e l y . The d i s c s were obtained as d e s c r i -bed above and the nucleus pulposus was removed^,with d i s s e c t i n g s c i s s o r s . The separated t i s s u e s were photographed and samples • were taken f o r h i s t o l o g i c a l examination. 2) Human C o s t a l C a r t i l a g e Samples of human c o s t a l c a r t i l a g e ' were obtained from a l i m i t e d number of a u t o p s i e s at the same time as removal of the lumbar s p i n e . A l l specimens were co o l e d a t 0°C, d i s s -ected f r e e of extraneous t i s s u e , d i c e d i n t o 2 t o 5mm cubes and s t o r e d at -20°C. 3) Bovine Nasal C a r t i l a g e Bovine n a s a l c a r t i l a g e was ob t a i n e d from a l o c a l a b a t t o i r . The t i s s u e was e x c i s s e d immediately a f t e r death and removed to the l a b o r a t o r y packed i n i c e where i t was d i s s e c t e d f r e e of extraneous m a t e r i a l , d i c e d and s t o r e d at -20°C. When r e q u i r e d a l l specimens were p a r t i a l l y thawed and ground i n a c h i l l e d L a t a p i e m i l l . EXTRACTION OF PROTEOGLYCANS 1) S i n g l e Stage Technique Weighed, ground samples of t i s s u e were a g i t a t e d g e n t l y f o r 24h wit h one of the v a r i o u s s o l v e n t s examined (15 ml per g f r e s h wt. of t i s s u e ) at room temperature (Sweeny e_t a l . , 1963). A l l s o l v e n t s c o ntained 0.05M a c e t i c a c i d and were a d j u s t e d to pH 5.8 wi t h ammonium hydroxide. The r e s i d u e was separated by c e n t r i f u g a t i o n at 2000 x g f o r 15min at room temperature and the s o l v e n t phase decanted. In experiments to determine the e f f i c i e n c y of e x t r a c t i o n of the r e s i d u e was washed t w i c e with 5 volumes of s o l v e n t f o r 2h and the washings and r e s i d u e was subsequently analyzed. The e x t r a c t s were c l a r i f i e d by vacuum f i l t r a t i o n through H y f l o - S u p e r c e l and the f i l t e r cakes were d i s c a r d e d without washing. 2) S e q u e n t i a l E x t r a c t i o n of Proteoglycans Weighed, ground samples of t i s s u e were a g i t a t e d g e n t l y f o r 24h at room temperature (Sweeny e t a_l. , 1963) with 0.4M GuCl (15ml per g f r e s h wt. of t i s s u e ) . The r e s i d u e was separated by c e n t r i f u g a t i o n a t 2000 x g f o r 15min a t room temperature and the s o l v e n t phase was decanted. The r e s i d u e was washed twice f o r 2 h a t room temperature w i t h the same s o l v e n t (5ml per g f r e s h wt.) ; the washings being r e t a i n e d f o r a n a l y s i s . The second e x t r a c t a n t , 4M GuCl i n 0.05M a c e t a t e b u f f e r , pH 5.8, was then added and the e x t r a c t i o n procedure repeated. The r e s i d u e was separated by c e n t r i f u g a t i o n at 2000 x g f o r 15 min at room temperature and the s o l v e n t phase was decanted; the r e s i d u e being d i s c a r d e d without f u r t h e r washing. A l l e x t r a c t s were c l a r i f i e d by vacuum f i l t r a t i o n through H y f l o -S u p e r c e l and the f i l t e r cakes were d i s c a r d e d without washing. RESULTS THE EXTRACTION OF PROTEOGLYCANS FROM HUMAN INTERVERTEBRAL DISC. Proteoglycans have been e x t r a c t e d from a v a r i e t y of c o n n e c t i v e t i s s u e by high speed homogenization of f i n e l y d i v i d e d t i s s u e w i t h water or weak e l e c t r o l y t e s (Gerber e t a l . , 1960; Lyons e t aJL. , 1966). Such techniques however have been shown, to degrade the macromolecules (Harrington and Zimm, 1965; Sajdera and H a s c a l l , 1969). Sajdera and H a s c a l l (1969) showed t h a t p r o t e o g l y c a n s c o u l d be e x t r a c t e d i n high y i e l d from bovine n a s a l c a r t i l a g e . w i t h 4M GuCl or 3M M gCl 2 without use of h i g h speed homogenization. Since t h i s technique had not been a p p l i e d to i n t e r v e r t e b r a l d i s c an experiment was c a r r i e d out to determine the most e f f i c i e n t e x t r a c t a n t of d i s c p r o t e o g l y c a n s under the c o n d i t i o n s d e f i n e d by S a j d e r a and H a s c a l l (1969). Although an e x t r a c t i o n time of 24h had been used f o r the i s o l a t i o n of c a r t i l a g e p r o t e o g l y c a n s , samples of pooled lumbar i n t e r v e r t e b r a l d i s c s from three s u b j e c t s between 40 and 60 years o l d were e x t r a c t e d w i t h water, 0.15M NaCl and 4M GuCl f o r 24h, 48h and 72h to determine i f a 24h e x t r a c t i o n p e r i o d was s u i t a b l e f o r i n t e r v e r t e b r a l d i s c . The r e s u l t s showed t h a t over 75% of the hexuronate was e x t r a c t e d i n the f i r s t 24h r e g a r d l e s s of the s o l v e n t used. A f u r t h e r 5% c o u l d be removed by extending the e x t r a c t i o n p e r i o d to 48h ( F i g . 6). An e x t r a c t i o n p e r i o d of 24h was s e l e c t e d however i n order to compare the r e s u l t s w i t h p u b l i s h e d data and to reduce the. p r e p a r a t i o n time and hence 44a. F i g . 6. The hexuronate (% of v e r t e b r a l d i s c a f t e r w ith water, • ; 0.15M t o t a l ) removed from i n t e r ^ e x t r a c t i o n f o r v a r i o u s times NaCl, O; and 4M GuCl, #. 44b. lOOr 24 48 72 96 HOURS 45a. F i g 7. Hexuronate a n a l y s i s of the washings obtained f o l l o w i n g e x t r a c t i o n of i n t e r v e r t e b r a l d i s c with water, •; 0.15M NaCl, O; and 4M GuCl, • . t 2 3 EXTRACT WASHINGS 46a. F i g . 8. The e x t r a c t i o n of p r o t e o g l y c a n s by v a r i o u s reagents. The f r a c t i o n of the p r o t e o g l y c a n e x t r a c t e d was expressed as the percentage of the t o t a l hexuronate of the t i s s u e ( e x t r a c t + residue) found i n the e x t r a c t . A, i n t e r v e r t e b r a l d i s c ; B, bovine n a s a l c a r t i l a g e ; C, c o s t a l c a r t i l a g e . K C l , •; MgC^/ • ; GuCl, • ; Urea, 0. 47. the r i s k of p r o t e o g l y c a n d e g r a d a t i o n and b a c t e r i a l contamin-a t i o n which were more l i k e l y t o occur with longer, e x t r a c t i o n times at room temperature. To determine the number of washings necessary to remove, the s o l u b l e p r o t e o g l y c a n remaining i n the r e s i d u e a f t e r ex-t r a c t i o n , d i s c samples were e x t r a c t e d f o r 24h with water, 0.15M NaCl and 4M GuCl. The r e s i d u e s were washed th r e e times w i t h 5 volumes of s o l v e n t f o r 2h and the hexuronate content of the washings was determined ( F i g . 7). Two washings removed over 90% of the remaining hexuronate r e g a r d l e s s of the s o l v e n t used. A n e g l i g a b l e ammount of hexuronate was removed by a t h i r d washing. The f o l l o w i n g e x t r a c t i o n procedure was t h e r e f o r e used. .Human i n t e r v e r t e b r a l d i s c was e x t r a c t e d f o r 24h at room temp-e r a t u r e w i t h water, 0.15M NaCl and v a r i o u s c o n c e n t r a t i o n s of KC1, MgCl 2, GuCl and urea. Human c o s t a l c a r t i l a g e from the same p a t i e n t s and bovine n a s a l c a r t i l a g e were e x t r a c t e d i n p a r a l l e l f o r the purpose of comparison. The r e s i d u e was washed twice with 5 volumes.' of s o l v e n t f o r 2h. The hexuronate content of the e x t r a c t s and r e s i d u e s was estimated and the e f f i c i e n c y of the e x t r a c t i o n procedure was determined from the percentage of the t o t a l hexuronate e x t r a c t e d . The r e s u l t s o b t a i n e d are shown i n F i g . 8. For pooled bovine n a s a l c a r t i l a g e the r e s u l t s were s i m i l a r t o the p u b l i s h e d data (Sajdera and H a s c a l l , 1969; Mason and Mayes, 1973); weak e l e c t r o l y t e s e x t r a c t e d l e s s than h a l f the hexuronate removed from the t i s s u e by a s i m i l a r e x t r a c t i o n w i t h 4M GuCl. In c o n t r a s t t o c a r t i l a g e over 70% of the hexuronate was e x t r a c t e d from human i n t e r v e r t e b r a l d i s c whichever solvent.was used. H a l l e n (1970) e x t r a c t e d human i n t e r v e r t e b r a l d i s c s w i t h a v a r i e t y of s o l v e n t s under the same c o n d i t i o n s and obtained s i m i l a r r e s u l t s to those r e p o r t e d above.. When human c o s t a l c a r t i l a g e from the- same, three p a t i e n t s was e x t r a c t e d w i t h v a r i o u s s o l v e n t s the e x t r a c t i o n p r o f i l e o b t a i n e d was q u i t e d i f f e r e n t from t h a t o f e x t r a c t s of i n t e r v e r t e b r a l d i s c s obtained from the same i n d i v i d u a l s ; the p a t t e r n , however, c l o s e l y resembled the e x t r a c t i o n p r o f i l e of bovine n a s a l c a r t i l a g e e x t r a c t s . Since both i n t e r v e r t e b r a l d i s c 5and c a r t i l a g e c o n t a i n p r o t e o g l y c a n s of s i m i l a r composition (Rosenberg e t a l . , 1967), the l a r g e d i f f e r e n c e found between the e x t r a c t i o n p r o f i l e s suggested t h a t the macromolecular o r g a n i z a t i o n of the p r o t e o -glycans i n the t i s s u e s c o u l d not be the. same. The s i m i l a r i t y of the e x t r a c t i o n p r o f i l e s of bovine n a s a l c a r t i l a g e and human c o s t a l c a r t i l a g e suggested t h a t the d i f f e r e n c e between the p r o p e r t i e s of human d i s c and bovine n a s a l c a r t i l a g e was r e l a t e d t o the nature of the t i s s u e r a t h e r than the s p e c i e s s e l e c t e d f o r i n v e s t i g a t i o n . DEMONSTRATION OF PROTEOGLYCAN COMPLEXES Evidence f o r the presence of proteoglycan,- aggregates i n d i s c wa s£'"sought,us.ingfLithep pro,ced:u-re5,dev-isedt-by£ Hasea 11 and Sajdera (1969) f o r bovine n a s a l c a r t i l a g e ( F i g . 9) (Ernes and Pearce, 1975). D i s c s from t h r e e i n d i v i d u a l s o f wid e l y d i f f e r i n g ages and as bovine n a s a l c a r t i l a g e were, e x t r a c t e d w i t h b u f f e r e d 4M GuCl. The c l a r i f i e d e x t r a c t s were a d j u s t e d to a s s o c i a t i v e c o n d i t i o n s by d i a l y s i s a g a i n s t nine volumes of 0.05M ammonium a c e t a t e b u f f e r , pH 5.8, and c e n t r i f u g e d i n a cesium c h l o r i d e g r a d i e n t ( F i g . 10). With both t i s s u e s , a p r o t e i n - r i c h f r a c t i o n rose to the top and a h e x u r o n a t e - r i c h f r a c t i o n sank t o the bottom of the tube. The g e l which formed at the top of the tube was assumed to. comprise a c o l l a g e n . The bottom two f r a c t i o n s were pooled f o r each, t i s s u e and examined i n the a n a l y t i c a l u l t r a c e n t r i f u g e a f t e r d i a l y s i s a g a i n s t b u f f e r e d 0.4M GuCl t o remove, cesium c h l o r i d e ( F i g . 11). The bimodal p a t t e r n r e p o r t e d f o r c a r t i l a g e by H a s c a l l and Sajdera (1969) was confirmed. The d i s c e x t r a c t s a l s o showed a bimodal p a t t e r n ; the more, r a p i d l y sedimenting peak was very much l e s s prominent and separated p o o r l y from the more slo w l y sedimenting component. Both peaks of the d i s c e x t r a c t migrated more slo w l y than those o f c a r t i l a g e s u g g e s t i n g t h a t the pro-teoglycans are of lower molecular weight. On the b a s i s of these o b s e r v a t i o n s the d i s c appeared t o c o n t a i n a s m a l l amount of p r o t e o g l y c a n aggregate s i m i l a r to t h a t of bovine n a s a l c a r t i l a g e . 50. The p o s s i b l e r e v e r s i b i l i t y of aggregation was examined f u r t h e r b y . d e n s i t y g r a d i e n t c e n t r i f u g a t i o n under d i s s o c i a t i v e c o n d i t i o n s ( H a s c a l l and S a j d e r a , 1969). An equal volume of b u f f e r e d 7. 5M GuCl was added to a p o r t i o n of f r a c t i o n B ( F i g . 9) and the d e n s i t y a d j u s t e d by the a d d i t i o n of cesium c h l -o r i d e . A f t e r c e n t r i f u g a t i o n to e q u i l i b r i u m , both, t i s s u e s y i e l d e d a h e x u r o n a t e - r i c h h i g h - d e n s i t y f r a c t i o n (C) ( F i g . 12). Again, the y i e l d of hexuronate from d i s c was about one-eighth of t h a t from c a r t i l a g e . The two bottom f r a c t i o n s were pooled f o r each t i s s u e , d i a l y s e d a g a i n s t b u f f e r e d 0.4M GuCl, and examined i n the a n a l y t i c a l u l t r a c e n t r i f u g e ( F i g . 11C). As r e p o r t e d by H a s c a l l and Sajdera (1969),, c a r t i l a g e showed a s i n g l e symmetrical peak sedimenting l i k e the more slo w l y sed-imenting peak of the s t a r t i n g m a t e r i a l . Although the r e s u l t s w i t h d i s c were s i m i l a r , the peak sedimented more sl o w l y and was broader at a c o r r e s p o n d i n g p o s i t i o n i n the c e l l , s u ggesting the the p r o t e o g l y c a n of d i s c was more p o l y d i s p e r s e and of lower molecular weight than t h a t of c a r t i l a g e . The low-density f r a c t i o n s remaining i n the upper p a r t of the tubes were a l s o d i a l y s e d a g a i n s t b u f f e r e d 0.4M GuCl and examined i n the ana-l y t i c a l u l t r a c e n t r i f uge ( F i g . lit).) . No c l e a r peak was seen w i t h e i t h e r t i s s u e , e x c l u d i n g the p o s s i b i l i t y t h a t the r a p i d l y -sedimenting peak seen i n F i g . 11B r e p r e s e n t e d a separate p r o t e i n -r i c h macro-molecular component. S i m i l a r p a i r of tubes was r e -mixed a f t e r c e n t r i f u g a t i o n i n 4M GuCl, d i a l y s e d a g a i n s t b u f f e r e d 0.4M GuCl and examined i n the a n a l y t i c a l u l t r a c e n t r i f u g e ( F i g . l l E ) . Although recombination of the h i g h - and low-density f r a c t i o n s of both t i s s u e s r e s u l t e d i n the r e g e n e r a t i o n of the bimodal p a t t e r n , the p r o p o r t i o n of the more, r a p i d l y sedimenting com-ponent was l e s s than i n the i n i t i a l u l t r a c e n t r i f u g a t i o n ( c f . F i g . 11B). The low-density f r a c t i o n of d i s c p r o t e o g l y c a n appeared t o be e s s e n t i a l f o r the formation of the aggregate as r e p o r t e d f o r c a r t i l a g e ( H a s c a l l and S a j d e r a , 1969); the i n -completeness of r e v e r s i b i l i t y has a l s o been r e p o r t e d by t h i s group (Gregory e t a_l. , 1970) . In a d d i t i o n , , the formation o f complex was examined v i s -c o m e t r i c a l l y (Table I ) . Much of the v i s c o s i t y of the complex of c a r t i l a g e ( f r a c t i o n B) was l o s t when i t was d i s s o c i a t e d i n t o i t s h i g h - and low-density components, f r a c t i o n C and D, r e s p e c t i v e l y . Recombination of these components ( f r a c t i o n s E) r e s t o r e d most, but not a l l , of the i n i t i a l v i s c o s i t y . With i n t e r v e r t e b r a l d i s c , the i n i t i a l v i s c o s i t y of f r a c t i o n B was much l e s s a t e q u i v a l e n t c o n c e n t r a t i o n . A f t e r d i s s o c i a t i o n , a much g r e a t e r v i s c o s i t y was found i n the h e x u r o n a t e - r i c h component, f r a c t i o n E, wit h l i t t l e i n the p r o t e i n - r i c h comp-onent, f r a c t i o n F. T h e i r v i s c o s i t i e s were comparable to the corresponding f r a c t i o n of c a r t i l a g e . Recombination of the components of d i s c ( F r a c t i o n E) y i e l d e d a v i s c o s i t y i n t e r m e d i a t e to f r a c t i o n B and C. Since the v i s c o s i t i e s a s s o c i a t e d w i t h the h i g h - and low-density components of d i s c and of c a r t i l a g e ( f r a c t i o n C and D, r e s p e c t i v e l y ) were comparable, the l e s s e r v i s c o s i t i e s found i n the f r a c t i o n of d i s c showing complex formation ( f r a c t i o n s B and E) suggested t h a t a much s m a l l e r p r o p o r t i o n of the d i s c p r o t e o g l y c a n was i n v o l v e d i n complex TABLE I I . V i s c o s i t i e s of d e n s i t y g r a d i e n t f r a c t i o n s of a 4M GuCl e x t r a c t of i n t e r v e r t e b r a l d i s c .and n a s a l c a r t i l a g e The o r i g i n of the f r a c t i o n s i s d e s c r i b e d i n F i g . 9. A l l measurements were made i n 0.4M GuCl, 0.05M-acetate, pH 5.8 at 20° C. T i s s u e and F r a c t i o n Hexuronate pmol/ml S p e c i f i c V i s c o s i t y Bovine n a s a l c a r t i l a g e (B) 4.6 2.64 CCI 4.0 0.80 CD) 0.7 0.26 . CE) 4.7 1.90 ;7 Human I n t e r v e r t e b r a l d i s c (B) 4.3 0.95 CO 4.6 0.84 (D) 0.8 0.22 CE) 5.4 0.90 formation, a c o n c l u s i o n conforming t o the f i n d i n g s on ana-l y t i c a l u l t r a c e n t r i f u g a t i o n . The c a r t i l a g e data confirmed t h a t of H a s c a l l and S a j d e r a (1969) . Considered both as a whole 1 and i n r e l a t i o n to the work of H a s c a l l and Sajdera (1969), the data suggests t h a t the two components found by u l t r a c e n t r i f u g a t i o n of the d i s c e x t r a c t r e p r e s e n t a p r o t e o g l y c a n monomer and an aggregate formed by i t s r e v e r s i b l e a s s o c i a t i o n w i t h other components of the t i s s u e which had lower d e n s i t y i n a cesium c h l o r i d e g r a d i e n t . The complex was more d i f f i c u l t to demonstrate w i t h d i s c than w i t h c a r t i l a g e because i t r e p r e s e n t e d a much s m a l l e r p r o p o r t i o n of the t o t a l p r o t e o g l y c a n . D i s c s from a s t i l l b i r t h and two men, aged 23 and 58 y e a r s , were used i n t h i s study. The d i s c between the f i r s t and second lumbar v e r t e b r a e of the two a d u l t s was used; the f i v e lumbar d i s c s of the s t i l l b i r t h were pooled. The e x t r a c -t i o n data was s i m i l a r f o r the three specimens examined although s l i g h t l y more m a t e r i a l was o b t a i n e d from the s t i l l b i r t h . . There was no observable d i f f e r e n c e i n the degree of p r o t e o -g l y c a n aggregation between any of the t h r e e specimens examined, suggesting t h a t the degree of aggregation was not a g e - r e l a t e d . However, s i n c e the p r o p o r t i o n of aggregate i n the d i s c was so s m a l l and t h i s study was based on o n l y three specimens i t was d i f f i c u l t to q u a n t i t a t i v e l y assess the r e s u l t s . On the b a s i s of v i s c o s i t y measurements H a s c a l l and Sajdera (1969) showed t h a t pH 5.9 was o p t i m a l f o r the aggregation of bovine n a s a l c a r t i l a g e p r o t e o g l y c a n s . I t i s p o s s i b l e however t h a t t h i s pH i s not o p t i m a l f o r aggregation of i n t e r v e r t e b r a l 54a. F i g . 9. Flow c h a r t f o r the f r a c t i o n a t i o n of proteoglycans of d i s c and c a r t i l a g e . The l a b e l s A through E i d e n t i f y the corresponding f r a c t i o n s on the S c h l i e r e n diagram ( F i g . 11) and v i s c o s i t y data (Table I I ) . 54b SPECIMEN G r i n d L a t a p i e m i l l e x t r a c t 24h w i t h 4M GuCl 15ml/g •RESIDUE 4M GuCl EXTRACT (A) HIGH DENSITY FRACTION(B) C l a r i f y w i t h H y f l o S u p e r c e l add C s C l t o d e n s i t y 1,69 g/ml u l t r a -c e n t r i f u g e and cut f r a c t i o n s LOW DENSITY FRACTIONS 1 Add e q u a l v o l . o f 7.5M GuCl add C s C l t o d e n s i t y 1.50 g/ml u l t r a -c e n t r i f u g e and cut i n t o , 2 f r a c t i o n s d i a l y z e t o 0.4M GuCl / HIGH DENSITY FRACTION (C) I MIXTURE (E) 1 LOW DENSITY FRACTION (D) I 55a. F i g . 10. Densi t y g r a d i e n t c e n t r i f u g a t i o n of 4M GuCl e x t r a c t s i n 0.4M GuCl, pH 5.8. A f t e r c e n t r i f u g a t i o n 2ml f r a c t i o n s were c o l l e c t e d ; d e n s i t y , O; hexuronate, •; and absorbance at 280nm, •; were measured on each. 56a. F i g . 11. S c h l i e r e n p a t t e r n s of d e n s i t y - g r a d i e n t f r a c t i o n s of a 4M GuCl e x t r a c t r o f bovine n a s a l c a r t i l a g e and human i n t e r v e r t e b r a l d i s c . The d i r e c t i o n of sedimentation i s from l e f t to r i g h t . The v e r t i c a l l i n e r e p r e s e n t s the s o l v e n t - s o l u t e meniscus. The samples were c e n t r i f u g e d i n 0.4M GuCl, 0.05M a c e t a t e , pH 5.8 a t a bar angle of 50°. Photographs were taken 20 minutes a f t e r r e a c h i n g top speed. The o r i g i n of the f r a c t i o n s i s d e s c r i b e d i n F i g . 9. C o n c e n t r a t i o n i s expressed i n ^nmol of hexuronage/ml. P a t t e r n s i n the upper row are of bovine n a s a l c a r t i l a g e e x t r a c t s , lower row pa t t e r n s are of d i s c e x t r a c t s . Bovine n a s a l c a r t i l a g e B, 2.7 . |Amol/ml; C, 5.2 p.mol/ml; D, 0.8 |A,mol/ml; E, 4.0 jAmol/ml. Human i n t e r v e r t e b r a l d i s c B, 3.2 pmol/ml; C, 2.7 |*.mol/ml; D, 0*7 jA.mol/ml; E, 3.0 f*mol/ml. 57a. F i g . 12. Densi t y g r a d i e n t c e n t r i f u g a t i o n of h i g h - d e n s i t y f r a c t i o n s of d i s c and c a r t i l a g e (b) i n 4M GuCl. The two h i g h e s t - d e n s i t y f r a c t i o n s were pooled and c e n t r i f u g e d i n a cesium c h l o r i d e g r a d i e n t . Two ml f r a c t i o n s were c o l l e c t e d and analyzed as d e s c r i b e d i n F i g . 9. FRACTION NUMBER 58. d i s c p r o t e o g l y c a n s . A c c o r d i n g l y the optimum pH f o r aggregation of bovine n a s a l c a r t i l a g e p r o t e o g l y c a n s was reexamined i n the a n a l y t i c a l u l t r a c e n t r i f u g e over the rage pH 4 to pH8. Maxi-mum aggregation was found a t pH 6.0 i n c l o s e agreement wi t h data of H a s c a l l and Sajdera (1969). Aggregation of d i s c p r o t e o g l y c a n s appeared t o be independent of pH over the range pH 4 to pH 8. The optimum pH f o r aggregation of c a r t i l a g e p r o t e o g l y c a n s (pH 5.8 - 5.9) was t h e r e f o r e used i n a l l sub-sequent s t u d i e s of d i s c and c a r t i l a g e p roteoglycans, (Appendix II] To determine i f the g l y c o p r o t e i n - l i n k component l i m i t e d a g gregation i n the i n t e r v e r t e b r a l d i s c , the,component f r a c t i o n s were separated by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n and remixed i n d i f f e r e n t p r o p o r t i o n s . The degree of aggregation c o u l d not be i n c r e a s e d by r a i s i n g the p r o p o r t i o n of l i n k f r a c t i o n i n the mixture, suggesting t h a t the t i s s u e c o n t a i n s p r o t e o g l y c a n s that: . are c i n c a p a b l e of forming aggregates. GEL CHROMATOGRAPHY OF DISC PROTEOGLYCANS The r e s u l t s of d e n s i t y g r a d i e n t c e n t r i f u g a t i o n and ana-l y t i c a l u l t r a c e n t r i f u g a t i o n of 4M G u C l ' e x t r a c t s of d i s c and c a r t i l a g e suggested t h a t o n l y a s m a l l p r o p o r t i o n of the d i s c , p r o t e o g l y c a n s e x i s t as aggregates. However, the s e p a r a t i o n of the macromolecules by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n d i d not appear t o be complete; g e l chromatography was t h e r e f o r e employed as an a l t e r n a t i v e method of examining such macromol-e c u l e s , i n an attempt t o achieve a more complete s e p a r a t i o n of the prot e o g l y c a n s and to assess the degree of aggr e g a t i o n . S e v e r a l attempts have been made to separate the pr o t e o -glycans of c a r t i l a g e on Sepharose columns (Brandt and Muir, 1969,1971; Heinegard, 1972) but, s i n c e a hig h p r o p o r t i o n of the p r o t e o g l y c a n s was probably excluded from the g e l , the r e s u l t s were u n s a t i s f a c t o r y . I t was decided to continue t h i s work because Bio-Rad L a b o r a t o r i e s now o f f e r a range of agarose g e l s capable o f s e p a r a t i n g molecules with m o l e c u l a r weights 6 of up to 150 x 10 . They are a p p a r e n t l y s u i t a b l e f o r e l u t i o n w ith a wide range of s o l v e n t s and t h e r e f o r e appeared to be i d e a l f o r the column chromatography of d i s c p r o t e o g l y c a n s i n the d i s s o c i a t i v e s o l v e n t s "I4M.GuCl or 3M MgC^. While the manufacturers c l a i m t h a t p r o t e i n s and n u c l e i c a c i d s can be e l u t e d s a t i s f a c t o r i l y with s o l v e n t s such as 6M urea and 6M GuCl, the f o l l o w i n g experiment showed t h a t even s o l v e n t con-c e n t r a t i o n s as low as 3M MgCl^ or 4M GuCl were u n s u i t a b l e as column e l u e n t s , s i n c e they e l u t e d a h e x u r o n a t e - p o s i t i v e m a t e r i a l from the g e l s . P r e swollen agarose g e l s (Sepharose 4B and B i o - G e l A-5m) were mixed with an equal volume of water and 1ml samples were added to 5ml of the f o l l o w i n g s o l u t i o n s : 0.05M a c e t a t e b u f f e r , pH 5.8; 0.05M ac e t a t e b u f f e r , pH 5.8, c o n t a i n i n g e i t h e r 3M MgCl 2 or 4M GuCl; and water. Reagent blanks were prepared c o n t a i n i n g the s o l u t i o n l i s t e d above without the a d d i t i o n of the g e l s . A f t e r s t a n d i n g o v e r n i g h t the agarose was s e d i -mented ^ by c e n t r i f u g a t i o n at 2000 x g f o r 5 min and samples of the supernates and blanks were withdrawn f o r hexuronate ana-60a. F i g . 13. Column chromatography of aqueous e x t r a c t s of human i n t e r v e r t e b r a l d i s c on I, B i o - G e l A-5m and I I , A-15m. Column e f f l u e n t s were monitored f o r hexuronate (———) and p r o t e i n (- ) . 61. l y s i s . The r e s u l t s obtained are presented i n Table I I I . I t can be seen t h a t s t r o n g e l e c t r o l y t e s r e l e a s e d a hexuronate-p o s i t i v e m a t e r i a l from both g e l s . On the b a s i s of these r e s u l t s s t r o n g e l e c t r o l y t e s were not used f o r the e l u t i o n o f proteoglycans from agarose columns. In a l l subsequent experiments 0.05M+ acetate', pH 5.8, was used as the e l u e n t . S e l e c t i o n of the g e l Proteoglycans are thought to e x i s t i n s o l u t i o n as roughly s p h e r i c a l , hydrated macromolecules w i t h hydrodynamic volumes very much g r e a t e r than p r o t e i n s of a s i m i l a r molecule weight ( H a s c a l l and Sa j d e r a , 1970). Since column chromatography on agarose g e l s separates mixtures of molecules on the b a s i s of t h e i r hydrodynamic volumes^rather than t h e i r molecular weights, onl y g e l s with a l a r g e p o r e - s i z e can be used f o r the s e p a r a t i o n of d i s c p r o t e o g l y c a n s . Aqueous and 4M GuCl e x t r a c t s o f d i s c appeared to be i n -cluded on B i o - G e l A-150M, ( e x c l u s i o n l i m i t 150 x 10^) but the 6 sm a l l e r p o r e - s i z e g e l s , A-15m ( e x c l u s i o n l i m i t 15 x 10 ) and A-50m ( e x c l u s i o n l i m i t 50 x 10 ) were g e n e r a l l y used i n the f o l l o w i n g s t u d i e s s i n c e the columns were more s t a b l e and e l u -t i o n times were c o n s i d e r a b l y s h o r t e r than w i t h A-150m columns. Chromatography of aqueous e x t r a c t s Aqueous e x t r a c t s of human pooled lumbar i n t e r v e r t e b r a l d i s c s from two a d u l t s between 30 and 60 years o f a g e w e r e con-c e n t r a t e d by u l t r a f i l t r a t i o n w i t h an Amicon UM 10 f i l t e r and chromatographed on B i o - G e l A-5m and A-15m i n an attempt to separate the p r o t e o g l y c a n s p e c i e s and to estimate the degree of Table I I I . The e f f e c t of strong e l e c t r o l y t e s on the release of a hexuronate-positive material from agarose gels. Hexuronate chromagen Solvent water 0.05M acetate 3M MgCl 2 4M GuCl No gel - - 1 +•; Sepharose 4B - - +++ +++ Bio-Gel A--5m - - ++ ++ 1. The weak p o s i t i v e r e s u l t was probably due to MgSO preci p i t a t e d during analysis. of p r o t e o g l y c a n a g g r e g a t i o n . The r e s u l t s o b tained ( F i g . 13) showed a bimodal e l u t i o n p r o f i l e ; a hypersharp peak e l u t e d at the v o i d volume of the column followedfoby a second, o v e r l a p p i n g , broad peak. Presumably the former was predominantly a r t i f a c t u a l , caused by the accumu-l a t i o n of excluded molecules a t the v o i d volume of the column. Although t h i s phenomenon prevented the assessment of the degree of aggregation the l o g i c a l p r o g r e s s i o n to even l a r g e r p o r e - s i z e g e l s was not f o l l o w e d , because the apparent p o l y d i s p e r s i t y would not allow the complete s e p a r a t i o n of the molecules. S i m i l a r problems of p o l y d i s p e r s i t y and g e l e x c l u s i o n were encountered by Tsiganos and Muir (1970) who examined the h e t e r o g e n e i t y of c a r t i l a g e p r o t e o g l y c a n s . I t thus appeared I m p r a c t i c a l to demonstrate the e x i s t e n c e of p r o t e o g l y c a n aggregate of d i s c d i r e c t l y , so an i n d i r e c t method, based on the procedure des-c r i b e d by Sajdera and H a s c a l l (1969), f o r the i s o l a t i o n of c a r t i l a g e p r o t e o g l y c a n s , was used. Chromatography of d e n s i t y g r a d i e n t c e n t r i f u g a t i o n f r a c t i o n s of 4M GuCl e x t r a c t s of i n t e r v e r t e b r a l d i s c . P r o t e o g l y c a n aggregation i n bovine n a s a l c a r t i l a g e and human i n t e r v e r t e b r a l d i s c has heen demonstrated by v i s c o s i t y and a n a l y t i c a l u l t r a c e n t r i f u g a t i o n of f r a c t i o n o b t a i n e d by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of 4M GuCl e x t r a c t s a c c o r d i n g to the procedure of Sajdera and H a s c a l l (1969) as d e s c r i b e d i n F i g . 14. The d i s c appears to c o n t a i n c o n s i d e r a b l y l e s s aggregate than c a r t i l a g e , so t h a t the v i s c o m e t r i c data and s c h l e i r e n p a t t e r n s from d i s c f r a c t i o n s d i d not .show c l e a r l y the formation of d i s c aggregates. The g e l chromatographic, " e l u t i o n p r o f i l e s of f r a c t i o n s o b tained by the d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of d i s c e x t r a c t s ( F i g . 14), were compared with the p r o f i l e o b t a i n e d by mixing the f r a c t i o n s t o g e t h e r , and t h a t p r e d i c t e d by summating the p r o f i l e s of the i n d i v i d u a l f r a c t i o n s . P r e d i c t e d p r o f i l e s were c a l c u l a t e d by summation of the hexuronate content of comparable e f f l u e n t f r a c t i o n s , making an a p p r o p r i a t e c o r r e c t i o n t o permit comparison of the p r o f i l e s . 4M GuCl e x t r a c t s of lumbar d i s c s from f o u r ^ i n d i v i d u a l s between 30 and 65 years o l d (A) were separated by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n t o a dense p r o t e o g l y c a n - r i c h f r a c t i o n (B) and a p r o t e i n - r i c h f l o a t i n g component ( F i g . 14). A p o r t i o n of F r a c t i o n B was r e t a i n e d f o r chromatography and the remainder was r e c e n t r i f u g e d , i n d i s s o c i a t i v e c o n d i t i o n s , and d i v i d e d i n t o t hree equal p o r t i o n s , (C, M and D). Each f r a c t i o n was d i a l y z e d a g a i n s t 90 volumes of 0.05M a c e t a t e , pH 5.8, and e l u t e d from e i t h e r B i o - G e l A-15m or A-50m columns. P o r t i o n s of these f r a c t i o n s were remixed and e l u t e d from the g e l i n order to demonstrate the formation of aggregate. The e l u t i o n p r o f i l e of the mixture was compared with the p r o f i l e of the aggregate f r a c t i o n (B) and t h a t p r e d i c t e d . The r e s u l t s are presented i n F i g s . 15 to 18. The sedimentation of the pr o t e o g l y c a n s by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n appeared to be incomplete s i n c e , s u r p r i s i n g l y - , a h e x u r o n a t e - p o s i t i v e excluded peak was observed i n the low d e n s i t y f r a c t i o n (D). T h i s d i s p r o p o r t i o n a t e l y l a r g e excluded peak c o u l d p o s s i b l y r e p r e s e n t aggregate formed by a combination of contaminating p r o t e o g l y c a n s u b u n i t s w i t h a " l i n k i n g " f r a c t i o n . TheiA-15m e l u t i o n p r o f i l e of the mixture o f f r a c t i o n s C + D was d i f f e r e n t from t h a t p r e d i c t e d by summation of the components; the p r o f i l e showed an apparent s h i p towards the l e f t , i n d i c a t i o n o f the formation of a higher molecular weight s p e c i e s ( F i g . 16), and was s i m i l a r t o t h e . p r o f i l e of the aggregate f r a c t i o n (B). S i m i l a r l y , e l u t i o n of the pr o t e o g l y c a n s from A-50m showed t h a t recombination of the three f r a c t i o n (C+M+D) r e s u l t e d i n a higher average molecular weight o f the prot e o g l y c a n s than t h a t p r e d i c t e d by summation of the p r o f i l e s of the components ( F i g . 18). When o n l y f r a c t i o n s C and M were r e -combined, a s i m i l a r t r e n d was observed, s u g g e s t i n g t h a t " l i n k i n g a c t i v i t y was not r e s t r i c t e d to the low d e n s i t y f r a c t i o n (D). 66a. F i g . 14. Flow c h a r t f o r the f r a c t i o n a t i o n of p r o t e o g l y c a n s of i n t e r v e r t e b r a l d i s c f o r g e l chromatography. The l a b e l s A through E i d e n t i f y the corresponding f r a c t i o n s on the column e l u t i o n p r o f i l e s ( F i g s . 15 t o 18). 66b. SPECIMEN 4M GuCl EXTRACT (A) G r i n d L a t a p l e m i l l , e x t r a c t 24h w i t h 4M G u C l , 15ml/g RESIDUE C l a r i f y w i t h H y f l o s u p e r c e l , add CsO.l t o d e n s i t y 1.69g/ml, u l t r a -c e n t r i f u g e and cut f r a c t i o n s . HIGH DENSITY FRACTION (B) LOW DENSITY FRACTIONS-Add,...equal v o l . of 7. 5M G u C l , add CsC'i\to d e n s i t y 1.50g/ml, u l t r a -c e n f - r i f u g e and cut f r a c t i o n s d i a l y z e t o 0.4M GuCl HIGH DENSITY MIDDLE DENSITY LOW DENSITY FRACTION (C) FRACTION (M) FRACTION (D) I ' i MIXTURE (E) 67a. F i g . 15. Column chromatography on B i o - G e l A-15m of f r a c t i o n s obtained by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of a 4M GuCl e x t r a c t of human i n t e r v e r t e b r a l d i s c . Column e f f l u e n t s were monitored f o r hexuronate ( ) and p r o t e i n ( ). 1, h i g h d e n s i t y ] f r a c t i o n (C); I I , low d e n s i t y f r a c t i o n D. EFFLUENT VOL. (ml). 68a. F i g . 16. Column chromatography on B i o - G e l A-15m of aggregated pr o t e o g l y c a n s obtained by d e n s i t y g r a d i e n t c e n t r i -f u g a t i o n of a 4M GuCl e x t r a c t of human i n t e r v e r t e b r a l d i s c . F r a c t i o n A (————•); f r a c t i o n E (C+D) ( ) ; p r e d i c t e d p r o f i l e by summation o f the p r o f i l e s of C and D (- -),.'' 50 100 150 EFFLUENT VOL. (ml). 69a. F i g . 17. Column chromatography on B i o - G e l A-5 0m of f r a c t i o n s o b tained by d e n s i t y g r a d i e n t c e n t r i -f u g a t i o n of a 4M GuCl e x t r a c t of human i n t e r v e r t e b r a l d i s c . Column e f f l u e n t s were monitored f o r hexuronate ( ) and p r o t e i n ( — ) . I, densest f r a c t i o n (C); I I middle f r a c t i o n (M); I I I , l e a s t dense f r a c t i o n . 69b EFFLUENT VOL. (ml). 70a. F i g . 18. .Column chromatography- on B i b - G e l A-50m of aggregated p r o t e o g l y c a n s obtained by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f a 4M GuCl e x t r a c t of human i n t e r v e r t e b r a l d i s c . F r a c t i o n B ( -).;• (C + M + D ) ( ) ; p r e d i c t e d p r o f i l e by summation of the p r o f i l e s of f r a c t i o n s C, M and D ( ) . 0-3 50 TOO 150 EFFLUENT VOL. (ml). S i m i l a r , but more,dramatic s h i f t s i n the e l u t i o n p r o f i l e were observed when bovine n a s a l c a r t i l a g e was analyzed by the same procedure (Tsiganos e_t ajL. , 1971). " L i n k i n g " a c t i v i t y was a l s o found i n the middle f r a c t i o n .(M) . F u r t h e r i n v e s t i -g a t i o n of c a r t i l a g e p r o t e o g l y c a n aggregation by Gregory (1973) suggested t h a t two components were r e q u i r e d f o r p r o t e o g l y c a n a g g r e g a t i o n , one of which was recovered from the middle f r a c -t i o n and appeared to be h y a l u r o n a t e . The r e s u l t s are c o n s i s t e n t with the e x i s t e n c e of p r o t e o g l y c a n aggregates i n the human i n t e r v e r t e b r a l d i s c s i m i l a r t o those d e s c r i b e d f o r c a r t i l a g e by H a s c a l l and S a j d e r a (1969) . The e x c l u s i o n of l a r g e molecules from the g e l and the apparent p o l y d i s p e r s i t y of the p r o t e o g l y c a n s made e s t i m a t i o n s of the degree of aggregation i m p o s s i b l e , but the r e s u l t s suggested t h a t 4M GuCl d i s s o c i a b l e aggregates r e p r e s e n t o n l y a very s m a l l p r o p o r t i o n of the t o t a l p r o teoglycans i n the t i s s u e . S ajdera e_t a_l. (1970) suggested t h a t the p r o t e o g l y c a n aggregates of bovine n a s a l c a r t i l a g e are trapped i n the i n t e r -s t i c i e s of a c o l l a g e n network and can o n l y be l i b e r a t e d a f t e r d i s s o c i a t i o n i n t o s m a l l e r d i f f u s i b l e s u b - u n i t s , w i t h 4M GuCl. I f a s i m i l a r phenomenon occurs i n the d i s c i t would be expected t h a t the aqueous e x t r a c t s would r e p r e s e n t non-aggregated s p e c i e s and would t h e r e f o r e show a s h i f t towards lower- molecular weight s p e c i e s . Although crude 4M GuCl e x t r a c t s of d i s c were not chromatographed, a comparison of the e l u t i o n p r o f i l e of an aqueous e x t r a c t w i t h the aggregate f r a c t i o n (B), prepared from a 4M GuCl e x t r a c t , showed no s i g n i f i c a n t d i f f e r e n c e between the p r o f i l e s . This i n d i c a t e d that e i t h e r l i t t l e or np proteo glycan aggregation occurs or tha t the aggregates are not tr a p ped w i t h i n the c o l l a g e n network of the d i s c as they are i n c a r t i l a g e . SEQUENTIAL EXTRACTION OF PROTEOGLYCANS OF DISC AND CARTILAGE I f i ' d i s s o c i a t i o n of the complex i s necessary f o r the e x t r a c t i o n of aggregated PGS (Gregory, et a l . , 1 9 7 0 ) , p r i o r e x t r a c t i o n w i t h a n o n - d i s s o c i a t i v e solvent should remove the free proteoglycan from a t i s s u e . Subsequent treatment w i t h a d i s s o c i a t i v e solvent would then be expected to remove the aggregated PGS s e l e c t i v e l y . To t e s t t h i s hypothesis, samples of bovine nasal c a r t i l a g e and jthe. lumbar d i s c s obtained from three i n d i v i d u a l s ; a s t i l l b i r t h and two a d u l t s aged 23 and 58 years o l d were e x t r a c t e d c o n s e c u t i v e l y w i t h an a s s o c i a t i v e s o l v e n t , buffered 0.4M NaCl, and a d i s s o c i a t i v e s o l v e n t , buffered 4M GuCl ( F i g . 19; Table IV) (Ernes and Pearce, 1975)!. The q u a n t i t e s of hexuronate e x t r a c t e d from the t i s s u e s w i t h 0.4M GuCl were comparable. Two washes w i t h t h i s e x t r a c t a n t removed very l i t t l e f u r t h e r hexuronate, suggesting t h a t the i n i t i a l e x t r a c t i o n s had been e s s e n t i a l l y complete. Sub-sequent treatment w i t h 4M GuCl e x t r a c t e d t w e n t y - f i v e times more hexuronate from c a r t i l a g e than from d i s c . In ge n e r a l , the r a t i o s of hexuronate i n the 0.4M to t h a t i n the 4M GuCl e x t r a c t s corresponded to the r e l a t i v e p r oportions of the slo w l y - and rap i d l y - s e d i m e n t i n g components of the two t i s s u e s i n conformity w i t h the hypothesis. Table IV. Se q u e n t i a l E x t r a c t i o n of T i s s u e Hexuronate Each value r e p r e s e n t s the mean of 3 experiments. Hexuronate i s expressed as mol/g wet wt. of t i s s u e s and as a percentage '-.of the t o t a l hexuronate e x t r a c t e d . HUMAN INTERVERTEBRAL DISC BOVINE NASAL CARTILAGE EXTRACT mol HEXURONATE/g mol HEXURONATE/g 0 . 4 M - G U C 1 Wash 1 Wash 2 67.5 4.5 1.3 85.3 5.7 1.6 72.9 4.3 1.5 31. 0 1.7 0.6 .4.0M-GUC1 T o t a l 5.7 79.0 7.4 100.0 156.1 234.8 66.7 100.0 CO The f r a c t i o n s were examined i n the a n a l y t i c a l u l t r a -c e n t r i f u g e a f t e r d i a l y s i s a g a i n s t b u f f e r e d 0. 4M GuCl ( F i g . 20). The 0.4M GuCl e x t r a c t of c a r t i l a g e (A) showed two components, t h a t which sedimented more r a p i d l y b e i n g the more prominent, and resembling the f r e e p r o t e o g l y c a n of c a r t i l a g e (PGS). The l e s s abundant, more slowly-sedimenting component resembled f r a c t i o n GP-1 (Gregory e_t al. , 1970) . The 4M GuCl e x t r a c t of c a r t i l a g e (B) contained the same two components i n much sm a l l e r amounts, p a r t i c u l a r l y with r e s p e c t to the PGS-like f r a c t i o n , and an a d d i t i o n a l s u b s t a n t i a l sharp peak corresponding i n u l t r a c e n t r i f u g a l 4 b e h a v i o u r of the aggregate, PPC. In a l l these r e s p e c t s , the r e s u l t s agreed w i t h those, of Gregory e t a l . , (1970). On the other hand, i n the 0.4M GuCl e x t r a c t of d i s c (A), the more sl o w l y sedimenting component was the more promi-nent and behaved l i k e the f r e e p r o t e o g l y c a n ( F i g . 20). A f a s t e r minor component resembled the aggregate seen i n d i s c ( c f . F i g . 19). The presence of complex i n such e x t r a c t s has been a t t r i b u t e d to d i f f u s i b l e and s u r f a c e aggregates (Gregory, e t a l . , 1970). The 4M GuCl e x t r a c t of d i s c (B) showed o n l y two components i n amounts t h a t were approximately equal but i n too. low c o n c e n t r a t i o n , to permit c l e a r i n t e r p r e t a t i o n . In an e f f o r t to e l u c i d a t e the composition of the crude e x t r a c t s , they were d i a l y s e d a g a i n s t e d B u f f e r e d 0.4M GuCl, cesium c h l o r i d e was added and they were c e n t r i f u g e d to e q u i l -i b r i u m and f r a c t i o n a t e d . The bottom two•-2ml f r a c t i o n s were pooled, d i a l y z e d f r e e of cesium c h l o r i d e and examined by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n ( F i g . 20). The 0.4M GuCl e x t r a c t of c a r t i l a g e (C) showed a s i n g l e peak of p r o t e o g l y c a n monomer ( F i g . 20); peaks of monomer and of aggregate were seen i n the 4M GuCl e x t r a c t (D) c o r r o b o r a t -i n g the o b s e r v a t i o n s o f Gregory e t a l . , (1970). The r e s u l t s with d i s c were s i m i l a r . The 0.4M GuCl e x t r a c t contained a s i n g l e component resembling p r o t e o g l y c a n monomer and the 4M GuCl e x t r a c t (D) showed two components corre s p o n d i n g t o monomer and aggregate. Thus, as p r e d i c t e d , p r i o r e x t r a c t i o n w i t h 0.4M GuCl appeared to have removed the bulk of the pro-t e o g l y c a n monomer from the t i s s u e and to have p e r m i t t e d a c l e a r e r demonstration of the presence of aggregate than had been p o s s i b l e w i t h d i r e c t e x t r a c t i o n . The presence of two components i n the 4M GuCl e x t r a c t can be i n t e r p r e t e d i n s e v e r a l ways: the f a i l u r e of the f i r s t e x t r a c t a n t t o remove completely the non-aggregated p r o t e o g l y c a n of the t i s s u e ; the incomplete r e v e r s i b i l i t y of aggrega t i o n , as suggested by Gregory et_ a l . (1970); or d e n a t u r a t i o n or l o s s of some component e s s e n t i a l f o r a ggregation (Gregory, 1973; Hardingham and Muir, 1972, 1974; H a s c a l l and Heinegard, 1974 a,b; Heinegard and H a s c a l l , 1974). F u r t h e r evidence f o r the presence of both monomeric and aggregated p r o t e o g l y c a n i n the 4M GuCl e x t r a c t ( f r a c t i o n B, F i g . 20) was sought by demonstration of r e v e r s i b l e d i s s -o c i a t i o n ( F i g . 19). To the remainder o f the h e x u r o n a t e - r i c h f r a c t i o n s ( f r a c t i o n s D, F i g . 19) an equal volume of 7.5M GuCl was added, the s o l u t i o n u l t r a c e n t r i f u g e d t o e q u i l i b r i u m and the contents of the tube separated i n t o h i g h - d e n s i t y (lower 40%) and low-density (upper 60%) f r a c t i o n s , as b e f o r e . A f t e r d i a l y s i s a g a i n s t b u f f e r e d 0.4M GuCl, the h i g h - d e n s i t y f r a c -t i o n s of both c a r t i l a g e and d i s c (E) c o n t a i n e d a s i n g l e peak ( F i g . 21); the d i s c p r o t e o g l y c a n sedimented more s l o w l y than t h a t of c a r t i l a g e and showed a broader peak. No peak was seen w i t h e i t h e r low-density f r a c t i o n ( F). Re-mixing a rep-l i c a t e tube before d i a l y s i s demonstrated the p a r t i a l reform-a t i o n of aggregate (G). Thus, the two peaks seen i n F i g . 20 r e p r e s e n t e d f r e e p r o t e o g l y c a n and aggregate as a n t i c i p a t e d from p r e v i o u s r e s u l t s . I t was c l e a r from the r e s u l t s however, t h a t p r o t e o g l y c a n aggregates of human i n t e r v e r t e b r a l d i s c r e p resented o n l y a s m a l l p r o p o r t i o n of the t o t a l p r o t e o g l y c a n i n the t i s s u e . C a l c u l a t i o n of the percentage of aggregate from the e x t r a c t i o n data and the a n a l y t i c a l u l t r a c e n t r i f u g a t i o n p a t t e r n s showed t h a t about 70% of the p r o t e o g l y c a n s of c a r t -i l a g e e x i s t as a g g r e g a t e s w h i l e l e s s than 5% of t h e , p r o t e o -g l y c a n s are aggregated i n the i n t e r v e r t e b r a l d i s c . There appeared to be no d i f f e r e n c e i n the degree of a g g r e g a t i o n with the age of the d i s c based on the three cases examined. Subsequent examination of f i v e cases l y i n g w i t h i n a narrower age range (40-60)years of age) showed no d e t e c t a b l e change i n the degree of p r o t e o g l y c a n a g g r e g a t i o n . The s e q u e n t i a l e x t r a c t i o n procedure appeared to T o f f e r a r a p i d and novel method f o r a s s e s s i n g the degree of aggre-g a t i o n o f the p r o t e o g l y c a n s i n a v a r i e t y of c o n n e c t i v e t i s s u e s . Human c o s t a l c a r t i l a g e , f o r example, was shown to have a s i m i l a r e x t r a c t i o n p r o f i l e to t h a t of bovine n a s a l c a r t i l a g e and a p r e d i c t e d degree of p r o t e o g l y c a n a g g r e g a t i o n of approx-i m a t e l y 50%. The a n a l y t i c a l u l t r a c e n t r i f u g a t i o n data y i e l d e d a v a l u e of 50-60% ag g r e g a t i o n , i n c l o s e agreement wi t h the p r e d i c t e d v a l u e . T h i s method c o u l d p r o v i d e a u s e f u l s c r e e n i n g t e s t f o r a s s e s s i n g the degree of a g g r e g a t i o n o f p r o t e o g l y c a n s i n c onnective t i s s u e , although, without f u r t h e r work the r e s u l t s would be open to other i n t e r p r e t a t i o n s . 78a. F i g . 19. Flow c h a r t f o r the s e q u e n t i a l e x t r a c t i o n and fract-i o n a t i o n of p r o t e o g l y c a n s of d i s c and c a r t i l a g e . The l a b e l s A through G i d e n t i f y the corresponding f r a c t i o n s oh the S c h l i e r e n diagrams ( F i g s . 20 and 21). SPECIMEN Grind Latapie Mill Extract with 0.4M-GuCl, 15ml/g 2 4 hours, 20 C R E S I D U E Wash twice with 0.4M-GuCl 0.4M-GuCl EXTRACT (A) 5rol/g W A S H r N G S Extract with 4M-GuCl, 15ml/g 24 hours, 20 C R E S I D U E Clarify with Hyflo supercel add CSC1 to density 1.69 g/ml ultra centrifuge and cut fractions. Dialize to 0.4M-GuCl. 4M-GUC1 EXTRACT (B) Clarify with Hyflo supercel add CSCl to density 1.69 g/ml ultracentrifuge ' and cut fractions. i LOW DENSITY FRACTIONS HIGH DENSITY FRACTION (C) LOW DENSITY FRACTIONS HIGH DENSITY FRACTION (D) Add equal Vol. 7.5M-GuCl. add CSCl to density 1.50 g/ml ultracentrifuge and cut fractions. Dialize to 0.4M-GUC1. HIGH DENSITY FRACTION LOW DENSITY FRACTION (F) (E) MIXTURE (G) 79a. F i g . 20. S c h l i e r e n p a t t e r n s of the s e q u e n t i a l e x t r a c t s of i n t e r v e r t e b r a l d i s c and n a s a l c a r t i l a g e . The o r i g i n of the f r a c t i o n s i s d e s c r i b e d i n F i g . 19. Photographs were taken 20 mins. a f t e r r e a c h i n g top speed at a bar angle of 50°. C o n c e n t r a t i o n i s expressed i n jjimol of hexuronate/ml; upper, row: A, 4.3 fimol/ml; B, 3.2 jimol/ml; C, 5.4 |jimbl/ml; D, 519 j<mol/ml. Lower row: human i n t e r v e r t e b r a l d i s c A, 4.1 jAmol/ml; B, 0.9 |*mol/ml; C, 4.2 pmol/ml; D, 1.6 jA.mol/ml. 79b. 80a. F i g . 2 1 . f S c h l i e r e n p a t t e r n s of the d e n s i t y g r a d i e n t f r a c t i o n s of 4M GuCl s e q u e n t i a l e x t r a c t s . The o r i g i n of the f r a c t i o n s i s d e s c r i b e d i n F i g . 19. Photographs were taken 20 mins. a f t e r r e a c h i n g top speed at a bar angle of 50°, C o n c e n t r a t i o n i s expressed i n mol of hexuronate/ml. Upper row:, bovine n a s a l c a r t i l a g e E, 5.4 yumol/ml; F, 0.4 pmol/ ml; ^mol/ml; G, 4.8 y*mol/ml. Lower row; i n t e r -v e r t e b r a l d i s c E, 5.9^*mol/ml; F, 0.6 yumol/ml; G, 1. 7 ^ «mol/ml. . * 80b. 4. HISTOCHEMICAL ASSESSMENT OF SEQUENTIAL EXTRACTION OF CARTILAGE AND DISC Although t h i s i n v e s t i g a t i o n was mainly chemical i n nature i t was decided t o u t i l i z e h i s t o c h e m i c a l methodology i n an attempt t o c o n f i r m and/or extend the former f i n d i n g s . I t was f e l t t h a t the s s t ^ i n i n g o r s e c t i o n s of r e l e v a n t t i s s u e , which had been e x t r a c t e d s e q u e n t i a l l y by the method p r e v i o u s l y d e s c r i b e d would d e f i n e those areas from which m a t e r i a l was removed, as w e l l as the amount of m a t e r i a l removed. Un f i x e d , c r y o s t a t s e c t i o n s were e x t r a c t e d s e q u e n t i a l l y w i t h 0. 4M and 4M.GuCl. S e c t i o n s were s t a i n e d w i t h A l c i a n blue at pH 1.0 and pH 2.5; i t being g e n e r a l l y accepted t h a t A l c i a n Blue at pH 1.0 s e l e c t i v e l y s t a i n s sulphate r e s i d u e s , w h i l e A l c i a n blue a t pH 2.5 s t a i n s predominantly c a r b o x y l groups, some phosphate, and of course sulphate r e s i d u e s ( C u l l i n g , 1974). A number of c r y o s t a t s e c t i o n s (5-7^0 of human i n t e r v e r t -e b r a l d i s c s from t h r e e i n d i v i d u a l s between 40 and 60 years of age and of bovine n a s a l c a r t i l a g e were e x t r a c t e d f o r 24 h a t room temperature w i t h a l a r g e excess of b u f f e r e d 0.4M GuCl. The s e c t i o n s were then washed twice w i t h s o l v e n t f o r 2h a t room temperature and r e p r e s e n t a t i v e s e c t i o n s were removed f o r s t a i n i n g . The remaining s e c t i o n s were then t r e a t e d w i t h b u f f e r e d 4M GuCl f o r a f u r t h e r 24h a t room, temperature. Untreated s e c t i o n s were used as c o n t r o l s . S e c t i o n s were s t a i n e d s i m u l t a n e o u s l y w i t h A l c i a n blue at pH ls'O and pH 2.5 ( F i g . 22). A l c i a n blue s t a i n i n g at both Phs gave i d e n t i c a l r e s u l t s f o r i n t e r v e r t e b r a l d i s c - a n d bovine n a s a l c a r t i l a g e . C o n t r o l specimens showed a r e l a t i v e l y uniform d i s t r i b u t i o n of A l c i a n b l u e s t a i n i n g i n the ma t r i x o f both c a r t i l a g e and d i s c . No obvious d i f f e r e n c e s were observed between the annulus f i b r o s u s and nucleus pulposus. Approximately 70-80% of the A l c i a n blue s t a i n i n g was removed from the matrix of the d i s c a f t e r i t had been t r e a t e d with 0.4M GuCl; the r e s i d u a l s t a i n i n g appeared to be r e s t r i c t e d t o the p e r i l a c u n a r a r ea. In c o n t r a s t 0.4M GuCl removed o n l y 10-20% of the s t a i n a b l e m a t e r i a l from the matrix of bovine n a s a l c a r t i l a g e . Subsequent treatment of s e c t i o n s w i t h 4M GuCl d i d not appear to remove any more s t a i n a b l e m a t e r i a l from the matrix of the d i s c ; s i m i l a r treatment of c a r t i l a g e however, r e s u l t e d i n an'almost u n s t a i n e d s e c t i o n , the s m a l l , r e s i d u a l A l c i a n blue s t a i n i n g being r e s t r i c t e d to the p e r i l a c u n a r r e g i o n of the matrix. The r e s u l t s of t h i s study demonstrated d r a m a t i c a l l y the d i f f e r e n c e between the s u b s e q u e n t i a l e x t r a c t i o n o f d i s c and t h a t of c a r t i l a g e . S u b s e q u e n t i a l e x t r a c t i o n suggested t h a t pro-t e o g l y c a n ^ aggregates o f c a r t i l a g e were not r e s t r i c t e d t o any par i c u l a r l o c a t i o n i n the t i s s u e , but i n the d i s c there appeared to be l i t t l e a g g r e g a t i o n i n e i t h e r the-'nucleus pulposus or annulus f i b r o s u s . 83a. F i g . 22, Photomicrographs of c r y o s t a t s e c t i o n s of bovine n a s a l c a r t i l a g e (upper row) and human I n t e r v e r t e -b r a l d i s c (lower row) a f t e r s e q u e n t i a l e x t r a c t i o n w i t h 0.4M and 4M GuCl. S e c t i o n s were s t a i n e d w i t h A l c i a n blue at pH 2.5. A, u n t r e a t e d s e c t i o n s ; B, s e c t i o n s t r e a t e d w i t h 0.4M GuCl; C, s e c t i o n s t r e a t e d w i t h 0. 4M and 4M GuCl. M a g n i f i c a t i o n approximately 6 00 x. SEQUENTIAL EXTRACTION OF ANNULUS FIBROSUS AND NUCLEUS PULPOSUS, The annulus f i b r o s u s and nucleus pulposus are s t r u c t u r a l l y d i f f e r e n t from one another; the former c o n s i s t i n g of p r o t e o g l y c a n and a dense r e g u l a r network of c o l l a g e n f i b r e s , w h i l e the l a t t e r c o n t a i n s a l o o s e , a p p a r e n t l y unorganized, c o l l a g e n network i n a p r o t e o g l y c a n r i c h m atrix (Coventry, 1969). The composition of the p r o t e o g l y c a n s from the annulus and nucleus of the human i n t e r v e r t e b r a l d i s c appeared to be i d e n t i c a l (Gower and P e d r i n i , 1969X. I t was p o s s i b l e however, t h a t the s t r u c t u r e of the c o l l a g e n matrix c o u l d be r e s p o n s i b l e f o r a d i f f e r e n c e i n the e x t r a c t a b i l i t y of p r o t e o g l y c a n s from the annulus and nucleus even though t h e i r chemical compositions were the same. Although h i s t o c h e m i c a l l y t h e r e appeared to be no d i f f e r e n c e i n the q u a n t i t y of p r o t e o g l y c a n e x t r a c t e d from the annulus and nucleus, the techniques employed would not have shown s m a l l d i f f e r e n c e s i n the amount of m a t e r i a l removed from the t i s s u e . A q u a n t i t a t i v e , s e q u e n t i a l e x t r a c t i o n study, s i m i l a r t o t h a t p r e v i o u s l y d e s c r i b e d f o r whole t i s s u e was t h e r e -f o r e performed. Human i n t e r v e r t e b r a l d i s c was separated i n t o annulus f i b r o s u s and nucleus pulposus and samples taken f o r h i s t o l o g y . C r y o s t a t s e c t i o n s s t a i n e d w i t h haematoxylin and eQ-Sjih ( F i g . 23) showed t h a t n e i t h e r s e c t i o n appeared to be s e r i o u s l y contam-inated;.'with the o t h e r . The remaining t i s s u e was ground i n a c h i l l e d l a t a p i e m i l l and e x t r a c t e d as d e s c r i b e d i n F i g . 24. The e x t r a c t i o n data i s presented i n Table V. T a b l e V . S e q u e n t i a l E x t r a c t i o n of Annulus F i b r o s u s and Nucleus P u l p o s u s . Each v a l u e r e p r e s e n t s the mean r e s u l t from t h r e e specimens. Hexuronate i s expressed as ymol per g wet wt. and as a percentage of the t o t a l hexuronate e x t r a c t e d EXTRACT ANNULUS FIBROSUS ymol hexuronate/g % NUCLEUS PUEP'OSUSV ymol hexuronate/g % 0.4M GuCl .. 58.8 79 .5 95.3 86 .9 wash 1 5.3 7 .1 6.8 6 .2 wash 2 1.3 1 .7 1.4 1 .3 4M GuCl 8.6 11 .7 6.2 5 .6 T o t a l 74.0 . 100 .0 109.7 100 .0 86a. F i g . 23. Photomicrographs of c r y o s t a t s e c t i o n s of annulus f i b r o s u s and nucleus pulposus s t a i n e d w i t h haematoxylin and e o s i n . M a g n i f i c a t i o n approximately 600 x. Nucleus pulposus 87a. F i g . 24. Flow c h a r t f o r the s e q u e n t i a l e x t r a c t i o n of pro-t e o g l y c a n s from annulus f i b r o s u s and nucleus pulposus. The l a b e l s A through D i d e n t i f y the corr e s p o n d i n g f r a c t i o n s oh the S c h l i e r e n diagram ( F i g . 25). 87b SPECIMEN Grind Latapie Mill Extract with 0.4M-GuCl, 15ml/g 2 4 hours, 20°C 0.4M-GUC1 EXTRACT (A) •RESIDUE Wash twice with 0.4M-GuCl 5ml/g WASHINGS Extract with 4M-GuCl, 15ml/g 24 hours, 20°C RESIDUE Clarify with Hyflo supercel add CSCl to density 1.69 g/ml ultra-centrifuge and cut fractions. Dialize to 0.4M-GuCl. 4M-GuCl EXTRACT (B) Clarify with Hyflo supercel add CSCl to density 1.69 g/rnl ultracentrifuge and cut fraction^. LOW DENSITY FRACTIONS HIGH DENSITY FRACTION (C) LOW DENSITY FRACTIONS HIGH DENSITY FRACTION (D) Expressed as a percentage of the t o t a l hexuronate e x t r a c t e d , 0. 4M GuCl removed on l y s l i g h t l y more hexuronate from the nucleus than from the annulus; i n c o n t r a s t 4M.GuCl subsequently removed more than double the percentage of hexuronate from the annulus than from the n u c l e u s . Two e x p l a n a t i o n s of these r e s u l t s seemed p l a u s i b l e . The g r e a t e r q u a n t i t y of hexuronate e x t r a c t e d from the annulus by 4M GuCl.could simply be due to l a r g e r p r o p o r t i o n of aggregated m a t e r i a l i n the t i s s u e . A l t e r n a t i v e l y the o b s e r v a t i o n c o u l d be due to a d i f f e r e n c e i n the arrangement.of the c o l l a g e n f i b r e s s i n c e i t i s p o s s i b l e t h a t the mesh-size of the c o l l a g e n network c o u l d a f f e c t the e x t r a c t i o n c h a r a c t e r i s t i c s of the p r o t e o g l y c a n s . The e x t r a c t s were therefore, examined i n the a n a l y t i c a l u l t a -c e n t r i f u g e , b e f o r e and a f t e r d e n s i t y g r a d i e n t . c e n t r i f u g a t i o n to assess the degree of aggregation of the p r o t e o g l y c a n s ( F i g . 24). The r e s u l t s o b t a i n e d are shown i n F i g . 25. The 0.4M GuCl e x t r a c t of the annulus f i b r o s u s (A A) ex-h i b i t e d two components, a small, r a p i d l y - s e d i m e n t i n g peak and a l a r g e r , more sl o w l y - s e d i m e n t i n g peak. The 0.4M GuCl e x t r a c t (B ) contained two components with sedimentation r a t e s s i m i l a r t o those of (A ) but the p r o p o r t i o n of the r a p i d l y - s e d i m e n t i n g aggregate peak was l a r g e r , suggesting t h a t non-aggregated pro-t e o g l y c a n s were s e l e c t i v e l y removed by e x t r a c t i o n w i t h 0.4M GuCl. A f t e r c e n t r i f u g a t i o n i n a cesium c h l o r i d e g r a d i e n t , the high d e n s i t y f r a c t i o n s ( C A And D^) showed s i m i l a r p a t t e r n s to the c l a r i f i e d e x t r a c t i o n ( A A and B^), although the aggregate peak was d i m i n i s h e d i n area. A s i m i l a r d i m i n u t i o n i n the area 89a. F i g . 25. S c h l i e r e n p a t t e r n s of s e q u e n t i a l e x t r a c t s of annulus f i b r o s u s and nucleus pulposus. The d i r e c t i o n of sedimentation i s from l e f t t o r i g h t . Photographs were taken 25 minutes a f t e r r e a c h i n g top speed at a bar angle of 5 0°. The o r i g i n of the f r a c t i o n s i s d e s c r i b e d i n F i g . 24. C o n c e n t r a t i o n i s expressed i n mol of hexuronate per ml. P a t t e r n s i n the upper row are of annulus f i b r o s u s , lower row p a t t e r s are of nucleus pulposus. Annulus f i b r o s u s A, 3.9 ^.mol/ml; B, 0i 61 y^mol/ml; C, 4 . 6 mol/ml; X , 4 . 6 ym mol/ml; D, 0 . 72 pmol/ml; Nucleus pulposus A, 6.9yx mol/ml; B, 0.41 j*. mol/ml; C, 5.6 umol/ml; D, 0.8 u mol/ml. p ( ( s -< J 89b. 1 i pq -* of the aggregate peak was found when whole d i s c e x t r a c t s were examined, (see p. 51 ). In c o n t r a s t , s c h l i e r e n p a t t e r n s of 0. 4M and 4M GuCl e x t r a c t s of nucleus pulposus, both b e f o r e (A^, B N) and a f t e r ( C ^ , ) d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , showed a very much sm a l l e r p r o p o r t i o n o f / t h e aggregate peak. A.comparison of the r e l a t i v e areas of the peaks ( B a r t l e t and Smith 1960) i n d i c a t e d t h a t approximately 10 to 20% of the p r o t e o g l y c a n s of the annulus f i b r o s u s e x i s t as aggregates w h i l e l e s s than 5% of the p r o t e o g l y c a n s are aggregated i n the nucleus pulposus. Hardingham and Muir (1972) suggested t h a t two p o p u l a t i o n s of p r o t e o g l y c a n s e x i s t i n c a r t i l a g e , one of which i s capable, and the other i n c a p a b l e , of forming aggregates. I f two such p o p u l a t i o n s : a r e present i n the i n t e r v e r t e b r a l d i s c , the aggregation data presented above c o u l d be e x p l a i n e d i n two ways. E i t h e r the c o n c e n t r a t i o n of the l i n k i n g components l i m i t s the aggregation of the p r o t e o g l y c a n s i n the nucleus, or t h e r e i s a l a r g e r r a t i o of non-aggregatable to aggregatable p r o t e o g l y c a n s i n the nucleus than i n the annulus. In an attempt, to e l u c i d a t e t h i s problem the f o l l o w i n g experiment was performed. The p r o t e o g l y c a n l i n k i n g f r a c t i o n of the annulus f i b -rosus (F ) was i s o l a t e d by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of the s e q u e n t i a l 4M GuCl e x t r a c t of the t i s s u e ( F i g . 26) and added to the 0.4M and 4M GuCl s e q u e n t i a l e x t r a c t s of nucleus pulposus (Ajq/ • T n e mixtures were then examined i n the a n a l y t i c a l u l t r a c e n t r i f u g e t t o n a s s e s s the degree of p r o t e o g l y c a n aggregation. The p r o t e o g l y c a n f r a c t i o n s C.T and D were not used, s i n c e i t has been shown t h a t cesium 91a. F i g . 26. I s o l a t i o n of the l i n k i n g f r a c t i o n from annulus f i b r o s u s f o r a d d i t i o n to the 0. 4M and. 4M GuCl e x t r a c t s of nucleus pulposus i n an attempt to i n c r e a s e a g g r e g a t i o n . The o r i g i n of the f r a c t i o n s i s d e s c r i b e d i n F i g . 24. 91b. ANNULUS FIBROSUS FRACTION D n Add e q u a l volume o f 7 . 5 M GuCl a d j u s t d e n s i t y t o 1.50 g/ml w i t h CsCL c e n t r i f u g e and cut f r a c t i o n s HIGH DENSITY FRACTION E A LOW DENSITY FRACTION 23 D i a l y z e - t o 0 . 4M GuCl pH 5.8 F A m i x e d w i t h f r a c t i o n s A N o r B ^ F A m l x e d w i t h f r a c t i o n E^ t o c o n f i r m a ggregate f o r m a t i o n . 92. c h l o r i d e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n causes a p a r t i a l d i s s o c i a t i o n o f p r o t e o g l y c a n s aggregates; the same phenomenon has been observed i n bovine n a s a l c a r t i l a g e (Gregory, 1973) . THE COMPOSITION OF DISC PROTEOGLYCANS 0nr.anaiy.sis8thee.purified 4M GuCl e x t r a c t of the human i n t e r v e r t e b r a l d i s c ( f r a c t i o n B, F i g . 27.) and the i s o l a t e d p r o t e o g l y c a n s u b u n i t ( f r a c t i o n C, F i g . 27) were found to c o n t a i n hexose, hexuronate, hexosamine, p r o t e i n and sulphate (Table V I ) . The hexose and p r o t e i n contents of both f r a c t i o n s were lower than those values o b t a i n e d by ot h e r workers u s i n g d i f f e r e n t i s o l a t i o n procedures. . I t was p o s s i b l e t h a t the r e d -uced p r o t e i n content r e f l e c t e d a more complete s e p a r a t i o n of the p r o t e o g l y c a n from contaminating p r o t e i n . On e l e c t r o p h o r -e s i s the p r o t e o g l y c a n s migrated as a s i n g l e band stainedx, w i t h A l c i a n blue a t pH 2.5 ( F i g . 28) suggesting the presence of a c i d groups; A l c i a n blue a t pH 1.0, sugges t i n g the presence of su l p h a t e , ( C u l l i n g , 1974); h i g h - i r o n diamine, suggesting the presence o f O-sulphate e s t e r (Reid e t al_, , 1972), and weakly with ponceau S suggesting the presence of p r o t e i n . T h e ^ a l k a l i l a b i l e p o l y s a c c h a r i d e s were l i b e r a t e d and reduced by t r e a t i n g the i s o l a t e d p r o t e o g l y c a n s w i t h 0.02 M sodium borohydrideC: i n 0. 5M KOH f o r 10 days . a t 4°C. At the end of the r e a c t i o n the excess borohydride was de s t r o y e d by a c i d i f i c a t i o n t o pH 5.0 wi t h a c e t i c a c i d . The glycosamino-glycans were p r e c i p i t a t e d from the mixture by a d d i t i o n of 2.5 volumes o f ethanol;and s t a n d i n g o v e r n i g h t a t 4C°C. The p r e c -i p i t a t e was washed wi t h ethanol,. d r i e d i n a vacuum d e s s i c a t o r TableVI?. The composition of the proteoglycans extracted from intervertebral disc isolated by different methods. Data i s presented as is described in f i g . 26. Reference Gower & Pedrini (1969). Gower & Pedrini (1969). Lyons et a l . (1966). Comper & Preston (1974). Robinson & Hopwood (1973). Rosenberg ^ t a l . (1967). 4M GuCl extract (Fraction B). 4M GuCl extract (Fraction C). i a percentage of the dry weight of tissue. The origin of fractions B and C Animal Tissue HexNH,1 2 Hexose HexCOOH Protein Sulphate Human ft NP 17.5 11.7 14.9 25.7 -Human ft AF 14.4 11.1 11.2 38.8 -Human ft NP 32.2 - 17.9 32.1 -Bovine/Equine Disc 22.3 21.3 11.1 15.8 14.1 Bovine Disc 28.1 18.7 15.2 9.2 • -Bovine NP (PPL) 17.4 14.9 10.9 23.9 -Human * Disc 17.8 5.75 14.8 5.5 9.6 Human * Disc 17.2 9.4 16.9 8.7 13.7 * Tissue of comparable ages, (21-30 years). 1. Free base. 2. Free acid. F i g . 27. Flow c h a r t f o r the 4M GuCl e x t r a c t i o n and f r a c t i o n -a t i o n of p r o t e o g l y c a n s from human i n t e r v e r t e b r a l d i s c . The l e t t e r s A through to E i d e n t i f y the f r a c t i o n s analysed c h e m i c a l l y and i n the a n a l y t i c a l u l t r a c e n t r i f u g e . 94b. SPECIMEN Grind L a t a p i e m i l l e x t r a c t 24h w i t h 4M GuCl 15ml/g •RESIDUE 4M GuCl EXTRACT (A) C l a r i f y w i t h H y f l o Supercel add CsCl t o d e n s i t y 1.69 g/ml u l t r a -c e n t r i f u g e and cut f r a c t i o n s HIGH DENSITY FRACTION(B) LOW DENSITY FRACTIONS I Add equal v o l . of 7-5M GuCl add CsCl t o d e n s i t y 1.50 g/ml u l t r a -c e n t r i f u g e and cut into, 2 f r a c t i o n s d i a l y z e t o 0.4M GuCl / I HIGH DENSITY FRACTION (C) r ~ MIXTURE (E) 1 LOW DENSITY FRACTION (D) I 1 95a. F i g . 28. C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s of d i s c p r o t e o -glycans s t a i n e d w i t h A l c i a n b l u e at pH 2.5. 1. C h o n d r o i t i n 4-sulphate 2. H y a l u r o n i c a c i d 3. Keratan sulphate 4.. 4M GuCl e x t r a c t ( F r a c t i o n A, F i g . 27). 5. 4M GuCl e x t r a c t - p u r i f i e d ( F r a c t i o n B, F i g . 27). 6. 4M GuCl e x t r a c t - d i s s o c i a t e d ( F r a c t i o n C, F i g . 27). 7. 0.4M GuCl e x t r a c t ( F r a c t i o n A, F i g . 30). 8. 4M GuCl e x t r a c t - s e q u e n t i a l ( F r a c t i o n B, F i g . 30). 9. 4M GuCl e x t r a c t pptd w i t h a l c o h o l . 10. NaBH^/NaOH hy d r o l y z e d p r o t e o g l y c a n pptd i n 75% a l c o h o l . 11. NaBH^/NaOH hy d r o l y z e d pptd i n 40% a l c o h o l . and?*dissolved i n 1ml of water. An equal volume of 10% w/v c a l c i u m a c e t a t e i n 1M a c e t i c a c i d was added and the g l y c o s -aminoglycans were p r e c i p i t a t e d by stepwise a d d i t i o n of a l c o h o l . The f r a c t i o n s were screened f o r hexuronate and hexose and the r e s u l t s are presented i n F i g . 29. The hexuronate was almost e n t i r e l y r e c o v e r e d from the f r a c t i o n p r e c i p i t a t e d below 40% a l c o h o l ; the m a j o r i t y of the hexose was found, i n the f r a c t i o n subsequently p r e c i p i t a t e d i n 75% e t h a n o l . On e l e c t r o p h o r e s i s the former behaved l i k e c h o n d r o i t i n sulphate and the l a t t e r behaved l i k e k e ratan sulphate ( F i g . 28). Both p r e c i p i t a t e d f r a c t i o n s were washed wi t h a l c o h o l , d r i e d i n a vacuum d e s s i c a t o r , h y d r o l i z e d f o r 16h. at 100°C wi t h 0.01M HC1 and examined by paper chromatography to i d e n t i f y the hexoses and hexosamines. The h e x u r o n a t e - r i c h peak co n t a i n e d mainly galactosamine and no d e t e c t a b l e hexose, while the h e x o s e - r i c h peak contained glucosamine, g a l a c t o s e and t r a c e s of g a l a c t o s -amine. The data suggested t h a t the d i s c p r o t e o g l y c a n s c o n t a i n e d both keratan sulphate and c h o n d r o i t i n s u l p h a t e . Gas l i q u i d chromatography of the n e u t r a l sugars of the d i s c p r o t e o g l y c a n s e x t r a c t e d w i t h 4M GuCl showed the presence of g a l a c t o s e , x y l o s e and p o s s i b l y a t r a c e of mannose, although the peak was obscured by the f i r s t peak of g a l a c t o s e . The mat-e r i a l e x t r a c t e d from human i n t e r v e r t e b r a l d i s c by 4M GuCl, and which sedimented by c e n t r i f u g a t i o n i n a cesium c h l o r i d e g r a d i e n t , appeared to be a p r o t e o g l y c a n c o n t a i n i n g keratan s u l p h a t e , c h o n d r o i t i n s u l p h a t e , p r o t e i n and traces, of xylose, and mannose, which i s c o n s i s t e n t with.the known, composition of d i s c proteoglycans'. Analyses of the f r a c t i o n s o b t ained by d e n s i t y g r a d i e n t c e n t r i f l i g a t i o n of s e q u e n t i a l e x t r a c t s of whole d i s c , annulus f i b r o s u s and nucleus pulposus were performed to determine i f the p r o t e o g l y c a n s o b t a i n e d were c h e m i c a l l y d i f f e r e n t from one another. The r e s u l t s , expressed as mole r a t i o s , are presented i n Table V I I . The o r i g i n of the f r a c t i o n s i s d e s c r i b e d i n F i g s . 27 and 30. The 0.4M and 4M GuCl s e q u e n t i a l e x t r a c t s of whole d i s c d i d not appear to be g r o s s l y d i f f e r e n t from one another. However the p r o t e o g l y c a n s i s o l a t e d from the annulus f i b r o s u s by the s e q u e n t i a l e x t r a c t i o n procedure were found to have a lower r a t i o of hexosamine to hexuronate • and hexose to hexuronate than the corresponding f r a c t i o n from the i n t e r v e r t e b r a l d i s c . A s i m i l a r t r e n d was observed i n the f r a c t i o n s from both thai 0. 4M and s e q u e n t i a l . 4M GuCl e x t r a c t s suggesting t h a t the d i f f e r e n c e s was not due to the s t r u c t u r e of the aggregating and non-aggregating p r o t e o g l y c a n s . TABLE VII. The mole ratios of the constituents of the proteoglycans isolated from human intervertebral disc by the dissociative procedure adapted from Hascall & Sajdera (1969). The origin of the fractions B through E is described in figs. 27 and 30. Mole Ratios Reference Tissue Protein/HexCOOH* HexNH2/HexCOOH Hex/HexCOOH S04~/HexCOOH Gower & Pedrini (1969). NP 1. 72 1.14 0.73 -Gower & Pedrini (1969) . AF 3. 47 . 1.25 0.92 -Lyons et al . (1966) . NP 1. 79 1.75 - -Fraction C, f i g . 30 Disc 0. 36 1. 30 0.54 1.16 D, f i g . 30 Disc 0. 29 0.94 . 0.87 1.45 E, f i g . 30 Disc 0. 39 0.95 0.94 1.61 B, f i g . 27 Disc 0. 35 1.14 0.40 1.31 c, f i g . 27 Disc 0. 60 1.10 0.84 1.64 c, f i g . 30 AF 0. 38 1.70 0.75 1.30 c, f i g . 30 NP 0. 32 2.20 0.88 1.52 " D ' f i g . 30 AF 0. 44 1.43 0.64 1.13 D, f i g . 30 NP 0. 42 2.44 0.93 1.00 VD 00 99a. F i g . 29. A l c o h o l f r a c t i o n a t i o n of glycosaminoglycans-of p r o t e o g l y c a n s e x t r a c t e d from human i n t e r v e r t e b r a l d i s c f o l l o w i n g treatment w i t h sodium bor o h y d r i d e . Hexose (0) v a l u e s were a d j u s t e d f o r the c o n t r i b u t i o n of hexuronate ( • ) to the chromagen. 100a. F i g . 30. Flow c h a r t f o r the s e q u e n t i a l e x t r a c t i o n and f r a c -t i o n a t i o n of p r o t e o g l y c a n s from whole d i s c , annulus f i b r o s u s and nucleus pulposus. The l e t t e r s A through to G i d e n t i f y the f r a c t i o n s examined c h e m i c a l l y a n d i i n the a n a l y t i c a l u l t r a c e n t r i f u g e . 100b SPECI MSN Grind Latapie Mi l l Extract with 0.4M-GuCl, 15ml/g 24 hours, 20 C 0.4M-GuCl EXTRACT (A) RESIDUE Wash twice with 0.4M-GuCl 5ml/g WASHINGS Extract with 4M-GuCl, 15ml/g 24 hours, 20 C RESIDUE Clarify with Hyflo supercel add CSCl to density 1.69 g/ml ultra -centrifuge and cut fractions. Dialize to 0.4M-GuCl. LOW DENSITY FRACTIONS 4M-GUC1 EXTRACT (B) Clarify with Hyflo supercel add CSCl to density 1.69 g/ml ultraeentrifuge • and cut fraction^. HIGH DENSITY FRACTION (C) LOW DENSITY FACTIONS (D) HIGH DENSITY FRACTIO^ Add equal Vol. 7.5M-GuCl. add CSCl to density 1.50 g/ml ultraeentrifuge and cut fractions. Dialize to 0.4M-GuCl. HIGH DENSITY FRACTION LOW DENSITY FRACTION (F) I .(E) I MIXTURE (G) OTHER PROCEDURES S e v e r a l other methods were attempted i n order t o i d e n t i f y and separate the aggregated and non-aggregated pro-teoglycans of the human i n t e r v e r t e b r a l d i s c . These were as f o l l o w s : 1. DEAE c e l l u l o s e chromatographysseparated the pro-teoglycans from much of the contaminating p r o t e i n but the former was e l u t e d from the column as a s i n g l e , narrow band which showed noi.evidence of s e p a r a t i o n i n t o aggregated and non-aggregated s p e c i e s 2. I s o e l e c t r i c f o c u s s i n g of the proteoglycans of d i s c •< • i n a sucrose d e n s i t y g r a d i e n t produced a complex o v e r l a p p i n g s e r i e s of bands which c o u l d not be i d e n t i f i e d or separated f u r t h e r . 3. E l e c t r o n microscopy of t h i n f i l m s c o n t a i n i n g a mixture of cytochrome c and d i s c p r o teoglycans f a i l e d t o show the presence of aggregates as had been demonstrated i n bovine n a s a l c a r t i l a g e (Rosenber et a l . , 1970). However s i n c e bovine n a s a l c a r t i l a g e was not compared w i t h d i s c as a p o s i t i v e c o n t r o l the r e s u l t s were i n c o n c l u s i v e . 4. Chromatographic columns c o n t a i n i n g l a r g e p o r e - s i z e g l a s s beads were a l s o examined as a p o s s i b l e t e c h -nique f o r s e p a r a t i n g the p r o t e o g l y c a n s p e c i e s . These are s u i t a b l e f o r the s e p a r a t i o n of very l a r g e mole-c u l e s and f o r e l u t i o n with the d i s s o c i a t i v e s o l v e n t , 4M GuCl. However, the proteoglycans appeared to b i n d t o the column packing even a f t e r they had been coated w i t h p o l y e t h y l e n e g l y c o l and the p r o t e o g l y c a n s ' e l u t e d as a s i n g l e symmetrical peak. DISCUSSION The b a s i s of t h i s work was the hypothesis t h a t the proteoglycans of the human i n t e r v e r t e b r a l d i s c occur as aggre-gates, these aggregates being r e v e r s i b l y d i s s o c i a b l e by 4M GuCl. Such a system has been shown to occur i n bovine n a s a l c a r t i l a g e ( H a s c a l l and Saj d e r a , 1969), bovine t r a c h e a l c a r t i l a g e (Heinegard, 1972), bovine humoral a r t i c u l a r c a r t i l a g e (Rosenberg e t a_l. , 1973) and p o r c i n e laryngeal-^ • c a r t i l a g e (Hardingham and Muir, 1972). Furthermore H a s c a l l and Sajdera (1969) proposed t h a t the aggregation of pro t e o g l y c a n s would be s i m i l a r i n a v a r i e t y of c a r t i l a g e n o u s t i s s u e s , i n c l u d i n g the i n t e r v e r t e b r a l d i s c . That i n t e r v e r t e b r a l d i s c c o ntained proteoglycans of a s i m i l a r s t r u c t u r e t o those of c a r t i l a g e was demonstrated by Rosenberg e t al.(1967) and i n 1969, H a s c a l l and Sajdera u s i n g a n a l y t i c a l u l t r a c e n t r i f u g a t i o n showed t h a t these proteoglycans sedimented as two s p e c i e s , w i t h a s e d i -mentation p r o f i l e s i m i l a r to t h a t of c a r t i l a g e . The bimodal c e n t r i f u g a t i o n p a t t e r n of p r o t e o g l y c a n p r e p a r a t i o n s of both d i s c and c a r t i l a g e c o u l d be a b o l i s h e d by treatement with d i t h i o t h r e i t o l . T h i s reagent r e d u c t i v e l y c l e a v e s d i s u l -phide bonds of c y s t i n e to c y s t e i n e . T h i s again suggested t h a t the d i s c c o n t a i n s aggregates s i m i l a r t o those d e s c r i b e d i n c a r t i l a g e (Hashimoto and Ludowieg, 1968). The i n t e n t i o n of t h i s study, was to seek evidence t h a t would prove or d i s p r o v e the e x i s t e n c e of such p r o t e o g l y c a n 104. aggregates i n human i n t e r v e r t e b r a l d i s c and i f presen t t o determine t h e i r r o l e i n aging, and/or degenerative d i s c d i s e a s e . THE NATURE OF PROTEOGLYCAN AGGREGATION Sajdera and H a s c a l l (1969) showed t h a t p r o t e o g l y c a n s c o u l d be e x t r a c t e d from c a r t i l a g e i n hig h y i e l d by the. use of s t r o n g e l e c t r o l y t e s , such as 4M GuCl or 3M MgC^- The examination of t h e i r v a r i o u s f r a c t i o n s , o b t a i n e d by c e n t r i -f u g a t i o n of the e x t r a c t s i n a cesium c h l o r i d e g r a d i e n t , sugg-ested t h a t the pr o t e o g l y c a n s e x i s t e d as aggregates, which were d i s s o c i a b l e by 4M GuCl i n t o two major f r a c t i o n s . These f r a c t i o n s , were a p r o t e i n - r i c h l i n k i n g f r a c t i o n (GPL) and. a hexuronate-r i c h p r o t e o g l y c a n (PGS) ( H a s c a l l and, S a j d e r a , 1969). The l i n k i n g component (GPL) was a very much s m a l l e r p r o p o r t i o n of the aggregate than the PGS s i n c e i t re p r e s e n t e d l e s s than 3% of the t o t a l weight of the p r o t e o g l y c a n s . Heinegard (1972) showed t h a t the GPL f r a c t i o n c o n tained two components both of which were i n c l u d e d on Sephadex G-200. GPL vwas r i c h i n hexosamine and p r o t e i n , p a r t i c u l a r l y i n the amino a c i d c y s t i n e . Aggregation was prevented by r e d u c t i o n with d i t h i o t h r e i t o l s u g g e s t i n g t h a t d i s u l p h i d e bonds were r e q u i r e d t o ma i n t a i n the i n t e r g r i t y of the g l y c o p r o t e i n l i n k ( H a s c a l l and Sa j d e r a , 1969). The aggregation of c a r t i l a g e p r o t e o g l y c a n s was found to be op t i m a l at pH 5.8: d i s a g g r e g a t i o n o c c u r i n g to a g r e a t e r degree at h i g h e r or lower pHs. Rosenberg e t a_l. (1970) ob t a i n e d e l e c t r o n micrographs of c a r t i l a g e p r o t e o g l y c a n subunits and aggregates and proposed t h a t feven s e v e r a l p r o t e o g l y c a n subunits were c l u s t e r e d around a s m a l l , c e n t r a l GPL component to form an aggregate. Gregory (1973) showed t h a t c a r t i l a g e p r o t e o g l y c a n s r e -q u i r e d two components of d i f f e r e n t boujyant d e n s i t y f o r maximum aggregation. The low-density component was s i m i l a r to t h a t d e s c r i b e d by H a s c a l l and Sajdera (1969) but the h i g h - d e n s i t y component had a bouyant d e n s i t y of 1.46, the same as t h a t of h y a l u r o n i c a c i d . At the same time Hardingham and Muir (1972) d e s c r i b e d the i n t e r a c t i o n of p r o t e o g l y c a n from p i g l a r y n x with h y a l u r o n i c a c i d i s o l a t e d from v a r i o u s t i s s u e s . The s t o i c h e i o m e t r y of the i n t e r a c t i o n suggested t h a t 10-3 0 p r o t e o g l y c a n molecules c o u l d bind to a s i n g l e molecule of hyaluronate. The system was found to be pH dependent and r e v e r s i b l y d i s s o c i a b l e with 4M GuCl l i k e the aggregates of bovine n a s a l c a r t i l a g e . H a s c a l l and Heinegard (1974a, 1974b) and Heinegard and H a s c a l l (1974) p u b l i s h e d a s e r i e s of three papers which showed t h a t the p r o t e o g l y c a n aggregates of bovine n a s a l c a r t i l a g e c o n t a i n p r o t e o g l y c a n s u b u n i t , h y a l u r o n i c a c i d and g l y c o p r o t e i n - l i n k ; f i g . 29 shows t h e i r p o s t u l a t e d model of p r o t e o g l y c a n aggregates. Such a system appeared p o s s i b l e i n d i s c s i n c e H a l l e t al_. (1957) had shown t h a t the t i s s u e contained a s m a l l amount of h y a l u r o n i c a c i d . The aggregation of d i s c p r o t e o g l y c a n s was t h e r e f o r e i n v e s t i g a t e d by s e v e r a l d i f f e r e n t procedures.-Bovine n a s a l c a r t i l a g e was t r e a t e d i d e n t i c a l l y i n order to demonstrate t h a t any d i f f e r e n c e s observed were r e a l and not due to f a u l t y technique. In a l l the experiments conducted bovine n a s a l c a r t i l a g e behaved i n a manner c o n s i s t e n t w i t h the p u b l i s h e d data. F i g . 31. Proposed model f o r p r o t e o g l y c a n aggregates of bovine n a s a l c a r t i l a g e (Heinegard and H a s c a l l , 1974). 107 THE EXTRACTION OF PROTEOGLYCANS The e x t r a c t i o n of pro t e o g l y c a n s from d i s c -and c a r t i l a g e with v a r i o u s s o l v e n t s y i e l d e d s u r p r i s i n g r e s u l t s . The p r o t e o -g l y c a n of d i s c c o u l d be e x t r a c t e d r e a d i l y w i t h e i t h e r weak or strong e l e c t r o l y t e s ; S i m i l a r r e s u l t s were obtained by H a l l e n (1970) using fewer s o l v e n t s . In c o n t r a s t , weak e l e c t r o l y t e s , removed o n l y 30% of the m a t e r i a l from c a r t i l a g e t h a t was r e -moved with 4M GuCl. I f ' t h e p r o t e o g l y c a n aggregates of i n t e r -v e r t e b r a l d i s c are trapped i n a c o l l a g e n network, and can on l y be l i b e r a t e d a f t e r d i s s o c i a t i o n i n t o s m a l l e r d i f f u s i b l e components, as has been suggested f o r bovine n a s a l c a r t i l a g e , then, as i l l u s t r a t e d i n F i g . 32, there are at l e a s t three p o s s i b l e e x p l a n a t i o n s f o r the observed e x t r a c t i o n data. The aggregates c o u l d be s m a l l enough to pass through the c o l l a g e n meshwork without p r i o r d i s s o c i a t i o n ( I ) ; the p o r e - s i z e of the c o l l a g e n meshwork i n the d i s c c o u l d be l a r g e r than t h a t i n bovine n a s a l c a r t i l a g e , a l l o w i n g aggregates of s i m i l a r s i z e t o d i f f u s e out of the t i s s u e ( I I ) ; or the prot e o g l y c a n s are not aggregated and t h e r e f o r e are sm a l l enough to pass through the c o l l a g e n n e t w o r k ( I I I ) . The degree of aggregation of the proteoglycans was t h e r e f o r e assessed by examination o f the e x t r a c t s i n the a n a l y t i c a l u l t r a c e n t r i f u g e . THE DEMONSTRATION OF PROTEOGLYCAN AGGREGATION The r e s u l t s o b tained from the examination o f the crude 4M GuCl e x t r a c t s showed tha-t both the r a p i d l y - and s l o w l y -sedimenting p r o t e o g l y c a n s of d i s c were s m a l l e r and more p o l y -108a. F i g . 32. Proposed models to account f o r the observed proteo-. g l y c a n e x t r a c t i o n p r o f i l e s of bovine n a s a l c a r t i l a g e and human i n t e r v e r t e b r a l d i s c . The s t r a i g h t - l i n e s r e p r e s e n t the c o l l a g e n meshwork and pores. A, the r e v e r s i b l e aggregate model proposed f o r c a r t i l a g e by H a s c a l l and Sajdera (1969). B, models of i n t e r v e r t e b r a l d i s c , I, sm a l l d i f f u s i b l e aggregates; I I , aggregates d i f f u s i b l e due to a l a r g e p o r e - s i z e ; I I I , f r e e l y d i f f u s i b l e non-aggregated p r o t e o g l y c a n s . proteoglycan subunit linking components Aggregate d i s p e r s e than those of c a r t i l a g e . In a d d i t i o n , t h e r a p i d l y sedimenting peak of the d i s c e x t r a c t represented a very much sma l l e r p o r t i o n of the t o t a l than was the case i n c a r t i l a g e e x t r a c t s . While a n a l y t i c a l u l t r a c e n t r i f u g a t i o n demonstrated a bimodal d i s t r i b u t i o n , the r e s u l t s d i d not prove the e x i s t e n c e of a r e v e r s i b l e aggregate-subunit system as found i n bovine n a s a l c a r t i l a g e . To demonstrate the presence or absence of t h i s type of system i n d i s c the p r o t e o g l y c a n s were examined by the two stage d e n s i t y g r a d i e n t procedure of H a s c a l l and Sajdera (1969). The p r o t e o g l y c a n s e x t r a c t e d from both t i s s u e s were p a r t i a l l y p u r i f i e d by c e n t r i f u g a t i o n i n a cesium c h l o r i d e d e n s i t y g r a d i e n t and re-examined i n the a n a l y t i c a l u l t r a c e n t -r i f u g e . The r e s u l t s o b t a i n e d from c a r t i l a g e were i d e n t i c a l w ith the p u b l i s h e d data ( H a s c a l l and S a j d e r a , 1969), whereas the d i s c p r oteoglycans showed a feiniQdal. but more p o l y -d i s p e r s e d i s t r i b u t i o n . The p u r i f i e d p r o t e o g l y c a n s from both t i s s u e s showed, compared to the crude e x t r a c t s , a reduced p r o p o r t i o n of the r a p i d l y - s e d i m e n t i n g peak, suggesting t h a t the aggregates were un s t a b l e i n the cesium c h l o r i d e g r a d i e n t . A s i m i l a r phenomenon had been observed by H a s c a l l and Sajdera (1969). To d i s s o c i a t e p o s s i b l e aggregates the p u r i f i e d p r o t e o -glycans were r e c e n t r i f u g e d i n a cesium c h l o r i d e d e n s i t y g r a d i e n t i n the presence of 4M GuCl, t h i s separates the high d e n s i t y p r o t e o g l y c a n subunit from the low d e n s i t y l i n k i n g components. Again the d e n s i t y g r a d i e n t f r a c t i o n s of c a r t i l a g e 110. were i d e n t i c a l i n composition to those d e s c r i b e d by H a s c a l l and Sajdera (1969)„ A s i m i l a r s e p a r a t i o n was observed i n d i s c although the y i e l d of p r o t e o g l y c a n was o n l y about one-e i g h t h t h a t of c a r t i l a g e and the molecules e x h i b i t e d a l a r g e r range of bouyant d e n s i t i e s suggesting i n c r e a s e d p o l y d i s p e r s i t y . The dense p r o t e o g l y c a n f r a c t i o n of both t i s s u e s behaved as a s i n g l e peak i n the a n a l y t i c a l u l t r a e e n t r i f u g e ; the f l o a t i n g components contained no macromolecular s p e c i e s . On mixing the high and low d e n s i t y f r a c t i o n s together the bimodal p r o f i l e s were r e s t o r e d , although not to the same degree as b e f o r e d i s s o c i a t i o n . ' The c a r t i l a g e p r o t e o g l y c a n s behaved i n a s i m i l a r manner t o those i s o l a t e d from the same type of t i s s u e , by H a s c a l l and Sajdera (1969) c o n f i r m i n g the formation of aggregates. The f r a c t i o n s o b t a i n e d from d i s c e x t r a c t s ' however, showed on l y a t r a c e of aggregate. Gregory e t a l . (1970) have shown t h a t r e a g g r e g a t i b n of the p r o t e o g l y c a n s of c a r t i l a g e i s incomplete, presumably due to the l o s s or damange of some e s s e n t i a l component d u r i n g the i s o l a t i o n procedure. V i s c o m e t r i c measurements showed on l y a very small decrease i n the flow r a t e 1 when the p r o t e o g l y c a n subunit and l i n k i n g f r a c t i o n s of d i s c were remixed; w h i l e i n c a r t i l a g e the flow r a t e was h a l v e d . T h i s r e s u l t was s i m i l a r t o the p u b l i s h e d data on c a r t i l a g e p r o t e o g l y c a n s ( H a s c a l l and S a j d e r a , 1969) suggesting t h a t ^ c o n s i d e r a b l e r e a g g r e g a t i o n had o c c u r r e d . Using the same c r i t e r i a , d i s c p r o t e o g l y c a n s d i d not appear to show s i g n i f i c a n t a ggregation, c o n f i r m i n g the a n a l y t i c a l u l t r a c e n t r i f u g a t i o n data. The r e s u l t s suggested t h e r e f o r e , t h a t i n the i n t e r v e r t e b r a l d i s c aggregates r e p r e s e n t e d a 111. very much s m a l l e r p r o p o r t i o n of the pr o t e o g l y c a n s t h a n i i n bovine n a s a l c a r t i l a g e . Aggregation o f d i s c p r o t e o g l y c a n s d i d not appear i n to be l i m i t e d by a d e f i c i e n c y of a l i n k i n g component, s i n c e i n c r e a s i n g the r a t i o of the l i n k i n g f r a c t i o n t o the p r o t e o g l y c a n subunit d i d not i n c r e a s e the p r o p o r t i o n of aggregate formed: i t was p o s s i b l e however t h a t the c o n d i t i o n s f o r d i s c p r o t e o g l y c a n . aggre g a t i o n were not o p t i m a l i n the mixture. The p r o t e o g l y c a n e x t r a c t s of both d i s c and c a r t i l a g e were t h e r e f o r e examined i n the a n a l y t i c a l u l t r a c e n t r i f u g e at v a r i o u s pHs to determine the optimum pH f o r aggr e g a t i o n . C a r t i l a g e e x t r a c t s e x h i b i t e d an aggregation optimum a t pH 6.0, c l o s e t o the value of pH 5,8 r e p o r t e d f o r the same t i s s u e by H a s c a l l and Saj d e r a (1969). Aggregation of d i s c p r o t e o g l y c a n s appeared t o be independent of pH over a wide range so t h a t i t was u n l i k e l y t h a t the absence of aggregates was due to the^pH of the system used. Hardingham and Muir (1972) found t h a t a p r o p o r t i o n of the p r o t e o g l y c a n s of. p i g l a r y n g e a l ! c a r t i l a g e were unable to form aggregates or i n t e r a c t w i t h h y a l u r o n i c a c i d . I t seem probable t h e r e f o r e , t h a t the human i n t e r v e r t e b r a l d i s c c o ntained two s p e c i e s of p r o t e o g l y c a n s , the major one of which was unable to form p r o t e o g l y c a n aggregates. In c o n t r a s t , the major p r o p o r t i o n of c a r t i l a g e p r o t e o g l y c a n s i s capable of forming aggregates, (Sajdera e t a l . , 1970). 112. One other important f a c t o r has not been c o n s i d e r e d . I t i s p o s s i b l e t h a t the aggregation of d i s c p r o t e o g l y c a n s r e q u i r e s a low molecular weight, component, such as a b i v a l e n t c a t i o n , , which i s l o s t d u r i n g d i a l y s i s of the d i s s o c i a t e d p r o t e o g l y c a n e x t r a c t . However the nature of the methods employed made the i n v e s t i g a t i o n of such an aggregate system extremely d i f f i c u l t and . t i t was t h e r e f o r e not attempted. GEL CHROMATOGRAPHY OF DISC PROTEOGLYCANS Since g e l chromatography separates molecules a c c o r d i n g t o t h e i r effective.hydrodynamic. volume the technique was used to i n v e s t i g a t e the s i z e d i s t r i b u t i o n of pro t e o g l y c a n s from i n t e r v e r t e b r a l d i s c and to demonstrate the p o s s i b l e formation o f proteoglycan-aggregates. Brandt and Muir (1969) showed t h a t p r o t e o g l y c a n s e x t r a c t e d from porcine"!laryngeal, c a r t i l a g e w i t h v a r i o u s s o l v e n t s appeared t o have d i f f e r e n t g e l f i l t r a t i o n p r o f i l e s . They suggested t h a t the pr o t e o g l y c a n s l a y i n a s e r i e s of "compartments" ^ p r o g r e s s i v e l y r e s i s t a n t t o s o l u b i l i z a t i o n i n n e u t r a l s a l t , and t h a t t h e i r molecular s i z e and composition was probably r e l a t e d t o t h e i r s o l u b i l i t i e s . . Tsiganos e t a l . (1972) e x t r a c t e d p r o t e o g l y c a n aggregates from l a r y n g e a l c a r t i l a g e and f r a c t i o n a t e d t h e i r components by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . The f r a c t i o n s o b t a i n e d were chromato-graphed on Sepharose 2B and the e l u t i o n p r o f i l e s were com-pared w i t h those o b t a i n e d by mixing the f r a c t i o n s t o g e t h e r . The formation o f aggregates was demonstrated by a s h i f t i n the d i s t r i b u t i o n of the macromolecules towards excluded s p e c i e s . When aqueous e x t r a c t s of d i s c p r o t e o g l y c a n s were e l u t e d from agarose g e l s they showed an incomplete s e p a r a t i o n of the s p e c i e s , a l a r g e p r o p o r t i o n of which were excluded from the g e l s . I t t h e r e f o r e appeared u n l i k e l y t h a t the degree of aggregation c o u l d be assessed by t h i s procedure, although i t might be p o s s i b l e t o demonstrate t h a t aggregates;-were formed. The f r a c t i o n s o b tained from the two stage d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of 4M GuCl e x t r a c t s of i n t e r v e r t e b r a l d i s c were chromatographed both s e p a r a t e l y and a f t e r r e m i x i n g . The e l u t i o n p r o f i l e p r e d i c t e d by summation of the p r o f i l e s o f the i n d i v i d u a l f r a c t i o n s e x h i b i t e d a s m a l l e r p r o p o r t i o n of excluded molecules than the a c t u a l p r o f i l e o b t a i n e d from e i t h e r the mixed f r a c t i o n s or- o f the o r i g i n a l aggregated s p e c i e s . The excluded molecules appeared to be formed a t the expense of the low mol e c u l a r weight s p e c i e s s t r o n g l y s u g g e s t i n g the formation of pr o t e o g l y c a n s aggregates. SEQUENTIAL EXTRACTION OF PROTEOGLYCANS H a s c a l l and Sajdera (1969) suggested t h a t 4M GuCl ex-t r a c t s p r o t e o g l y c a n s from c a r t i l a g e by d i s s o c i a t i n g n o n - d i f f u s i b l e aggregates i n t o d i f f u s i b l e s u b u n i t s . T h i s p r o p o s a l i m p l i e d t h a t p r i o r t o e x t r a c t i o n w i t h weak e l e c t r o l y t e s would s e l e c t i v e l y remove d i f f u s i b l e aggregates and non-aggregated molecules. P r o t e o g l y c a n s o f the i n t e r v e r t e b r a l d i s c have been shown to be e x t r a c t e d w i t h equal e f f i c i e n c y by e i t h e r weak and strong e l e c t r o l y t e s . Since g e l chromatography and a n a l y t i c a l u l t r a -114 c e n t r i f u g a t i o n of 4M GuCl e x t r a c t s of d i s c showed t h a t o n l y a small p o r t i o n of the proteoglycans i s p r e s e n t i n an aggregated form, i t was f e a s i b l e t h a t the e x t r a c t i o n data r e f l e c t e d the degree of aggregation of the p r o t e o g l y c a n s i n the t i s s u e . The procedure d e s c r i b e d by H a s c a l l and Sajdera (1969) f o r the i s o l a t i o n of proteoglycans was t h e r e f o r e modi-f i e d by i n t r o d u c i n g s e q u e n t i a l e x t r a c t i o n of the t i s s u e with 0.4M+GuCl, f o l l o w e d by 4M,GuCl. The e x t r a c t i o n data obtained showed t h a t the same amount (70 ji mol hexuronate/g) was removed from both, t i s s u e s , but whereas i n human i n t e r v e r t e b r a l d i s c t h i s r e presented over 75% of the t o t a l hexuronate,in bovine n a s a l c a r t i l a g e i t represented o n l y 25% of the t o t a l . Subse-quent treatment of the r e s i d u e s w i t h 4M GuCl removed a f u r t h e r 6 j<mol hexuronate/g from d i s c , but more than 25 times t h a t amount from c a r t i l a g e . A n a l y t i c a l u l t r a c e n t r i f u g a t i o n showed t h a t the 0.4M GuCl e x t r a c t of both t i s s u e s was composed almost e n t i r e l y of the s l o w l y - s e d i m e n t i n g , non-aggregated, p r o t e o g l y c a n s . The s e q u e n t i a l 4M GuCl e x t r a c t of the t i s s u e s showed a s t r i n k i n g d i f f e r e n c e i n t h a t w h i l e c a r t i l a g e c o ntained almost e n t i r e l y aggregated p r o t e o g l y c a n s contaminated w i t h t r a c e s of the sub-u n i t and GPI components; the d i s c e x t r a c t was composed of equal p r o p o r t i o n s of the f a s t - and slowly-sedimenting components. The subunit and l i n k i n g components of the s e q u e n t i a l 4M GuCl e x t r a c t s of both t i s s u e s were then separated by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n and subsequently examined i b y t h e a n a l y t i c a l u l t r a c e n t r i f u g a t i o n . Recombination of these f r a c t i o n s confirmed 115. the e x i s t e n c e of p r o t e o g l y c a n aggregates i n both d i s c and c a r t i l a g e . Since the 0.4M GuCl e x t r a c t of d i s c and c a r t i l a g e con-t a i n e d predominently the non-aggregated s p e c i e s and the 4M GuCl e x t r a c t , aggregated species, i t appeared t h a t t h i s s e q u e n t i a l e x t r a c t i o n data r e f l e c t e d • t h e s t a t e of p r o t e o g l y c a n aggregation i n the t i s s u e . I f t h i s hypothesis i s v a l i d , then the p r o t e o -glycans of human c o s t a l c a r t i l a g e should be predominently aggregated s i n c e the e x t r a c t i o n p r o f i l e was s i m i l a r t o t h a t of bovine n a s a l c a r t i l a g e . A n a l y t i c a l u l t r a c e n t r i f u g a t i o n of the s e q u e n t i a l 0.4M and 4M GuCl e x t r a c t s showed t h a t over 50% of the proteoglycans' were aggregated, thus p r o v i d i n g s t r o n g evidence f o r the v a l i d i t y of t h i s h y p o t h e s i s . A n a l y t i c a l u l t r a c e n t r i f u g a t i o n of the e x t r a c t s a f t e r p a r t i a l p u r i f i c a t i o n by d e n s i t y g r a d i e n t c e n t r i f g u a t i o n showed t h a t t h i s procedure causes some dissoceit.a)^rpR^^^-gk^g:e^^s ^suggesting t h a t the degree of a g g r e g a t i o n i n vivo, was b e t t e r assessed by examination of the crude e x t r a c t s . That s e q u e n t i a l e x t r a c t i o n data p r o v i d e d an approximate method of a s s e s s i n g the degree of a g r e g a t i o n i n the t i s s u e was shown by the f a c t t h a t our ex-t r a c t i o n data gave a value of 67% a g g r e g a t i o n f o r c a r t i l a g e p r o t e o g l y c a n s which compared very f a v o u r a b l y w i t h the 70% a g g r e g a t i o n observed f o r the same t i s s u e by H a s c a l l and Sajdera (1969) and Sajdera e t a l . (1970). The estimated 7% aggregation of d i s c p r o teoglycans i n the crude e x t r a c t was c o n s i s t e n t with-the data o b t a i n e d by other procedures. A c a r e f u l comparison of the peak areas of the S c h l i e r e n p r o f i l e s p e r m i t t e d another, and probably more p r e c i s e assessment of the degree of aggregation. For c a r t i l a g e , aggregated p r o t e o g l y c a n was c a l c u l a t e d to r e p r e s e n t about 65% of the t o t a l , whereas i n d i s c l e s s than 5% of the proteoglycans were b e l i e v e d to be aggregated. The d i s c s used i n t h i s study were s e l e c t e d from three males; a s t i l l b i r t h and two a d u l t s of 23 and 58 years o l d . The p r o t e o g l y c a n s from a l l three showed a s i m i l a r degree of aggregation, the o n l y d i f f e r e n c e observed by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n being apparent increases i n p o l y d i s p e r s i t y of both the aggregate and s u b u n i t , w i t h i n c r e a s i n g age. These r e s u l t s suggest t h a t a r e d u c t i o n i n the degree of aggregation of 'the proteoglycans i s not r e l a t e d to the decrease i n v i s c o s i t y and average molecular weight which occur with i n c r e a s i n g age and h e r n i a t i o n of the i n t e r v e r t e b r a l d i s c . However, t h i s c o n c l u s i o n i s advanced on the r e s u l t s o b t a i n e d from o n l y 3 p a t i e n t s and do not p r o v i d e a r e l i a b l e answer to the q u e s t i o n . Although no d e t e c t a b l e d i f f e r e n c e was found between any of the other cases examined, the samples - a l l f e l l w i t h i n a narrow age range (40-60years) and were not ex-pected to show l a r g e v a r i a t i o n s i n composition or s t r u c t u r e No h e r n i a t e d d i s c s were found i n any of the autopsy cases examined and specimens removed s u r g i c a l l y were not used s i n c e i t was f e l t t h a t they r e p r e s e n t e d the end-product of a p a t h o l o g i c a l process and were not s u i t a b l e m a t e r i a l f o r com-p a r i s o n w i t h normal t i s s u e s . The s e q u e n t i a l e x t r a c t i o n procedure p r o v i d e d a novel and u s e f u l improvement to the d i s s o c i a t i v e e x t r a c t i o n procedure d e s c r i b e d by H a s c a l l and Saj d e r a (1969). F i r s t l y , i t p e r m i t t e d the p a r t i a l s e p a r a t i o n of aggregated and non-aggregated p r o t e o -glycans and p r o v i d e d a simple and extremely r a p i d method of a s s e s s i n g the degree of p r o t e o g l y c a n a g g r e g a t i o n . Secondly, by i n c r e a s i n g the p r o p o r t i o n of aggregate.sm i n the. 4M GuCl e x t r a c t i t was p o s s i b l e to o b t a i n r e l e v e n t data from human i n t e r v e r t e b r a l d i s c which was found t o c o n t a i n o n l y a s m a l l f r a c t i o n of p r o t e o g l y c a n aggregate. F i n a l l y , s e q u e n t i a l ex-t r a c t i o n o f c r y o s t a t s e c t i o n s of t i s s u e i n t r o d u c e d the p o s s i b i l i t y of l o c a t i n g the s i t e of p r o t e o g l y c a n aggregates i n a v a r i e t y of con n e c t i v e t i s s u e s . THE DISTRIBUTION OF PROTEOGLYCAN AGGREGATES IN THE INTERVERT-EBRAL DISC Although i n d i s c aggregated p r o t e o g l y c a n s have been shown t o r e p r e s e n t o n l y 7% of the t o t a l , i f they were concen-t r a t e d i n a s p e c i f i c area r a t h e r than evenly d i s t r i b u t e d , they might c o n s t i t u t e an important s t r u c t u r a l element i n the t i s s u e . T h e r e f o r e an experiment was d e v i s e d to determine the l o c a t i o n of p r o t e o g l y c a n aggregates i n d i s c and c a r t i l a g e based on the p r e f e r e n t i a l e x t r a c t i o n of non-aggregated pr o t e o g l y c a n s by weak e l e c t r o l y t e s ; the aggregated molecules c o u l d subsequently be e x t r a c t e d with a d i s s o c i a t i v e s o l v e n t . C r y o s t a t s e c t i o n s of d i s c and c a r t i l a g e were e x t r a c t e d s e q u e n t i a l l y w i t h 0.4M and 4M GuCl and examined by histochemica methods to show what had been removed from the t i s s u e s . Sec-t i o n s were s t a i n e d w i t h A l c i a n blue a t pH 1.0 f o r sulphate groups and pH 2.5 f o r s u l p h a t e , phosphate and a c i d groups. The r e s u l t s demonstrated d r a m a t i c a l l y the d i f f e r e n c e between the e x t r a c t a b i l i t y of the proteoglycans from d i s c and c a r t -i l a g e . P r o t e o g l y c a n aggregates of bovine n a s a l c a r t i l a g e appeared to be d i s t r i b u t e d evehl'y throughout the t i s s u e . Although the d i f f e r e n c e s were extremely s m a l l , the aggregated p r o t e o g l y c a n s were not l o c a l i z e d i n the d i s c to any observable degree. The d e t e r m i n a t i o n of the d i s t r i b u t i o n of proteoglycans aggregates by h i s t o c h e m i c a l methods f o l l o w i n g s e q u e n t i a l e x t r a c t i o n of c r y o s t a t s e c t i o n s c o u l d be of s i g n i f i c a n t value i n the i n v e s t i g a t i o n of d i s e a s e processes i n a v a r i e t y of connective t i s s u e s . Since a change i n the e x t r a c t i o n charac-t e r i s t i c s , c o u l d have a number of p o s s i b l e o r i g i n s ( c f . F i g . 32) a n a l y t i c a l u l t r a c e n t r i f u g a t i o n o b s e r v a t i o n s would be r e -q u i r e d to i d e n t i f y the l e s i o n . The d i s c was separated i n t o annulus f i b r o s u s and nucleu pulposus and e x t r a c t e d s e q u e n t i a l l y w i t h 0.4M and 4M GuCl to determine i f the aggregate i s o n l y present i n one or other of these r e g i o n s . The e x t r a c t i o n data suggested t h a t the annulus f i b r o s u s c o ntained twice as many aggregates as the 119. nucleus pulposus. When the e x t r a c t s were examined i n the a n a l y t i c a l u l t r a c e n t r i f u g e i t was c l e a r t h a t p r o t e o g l y c a n aggregates were almost e x c l u s i v e l y l o c a t e d i n the annulus f i b r o s u s . E i t h e r some e s s e n t i a l aggregating component was m i s s i n g from the nucleus pulposus or the p r o t e o g l y c a n s present were of non-aggregating s p e c i e s . To t e s t the hypothesis t h a t a component e s s e n t i a l f o r aggregation -twas absent from the nucleus pulposus the l i n k i n g f r a c t i o n of the p r o t e o -glycans from the annulus f i b r o s u s was i s o l a t e d by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n and added to the 4M GuCl e x t r a c t of nucleus pulposus. Since there was no change i n the pro-p o r t i o n of aggregate, the r e s u l t s suggested t h a t the p r o t e o -glycans of the nucleus were of ah non-aggregating v a r i e t y . Recombination of the l i n k i n g and subunit f r a c t i o n s of the annulus r e s u l t e d i n the formation of the aggregates, thus c o n f i r m i n g t h a t the f r a c t i o n was capable of c a u s i n g a g g r e g a t i o n . THE CHEMICAL COMPOSITION OF DISC PROTEOGLYCANS The i n t e r v e r t e b r a l d i s c has been shown to c o n t a i n p r o t e o g l y c a n s of s i m i l a r s t r u c t u r e to those of c a r t i l a g e (Rosen-berg e t a l . , 1967; Rosenverg e j t . a l . , 1970). Hopwood and Robinson (1974) proposed a model of the p r o t e o g l y c a n of bovine i n t e r v e r t -e b r a l d i s c , shown i n f i g . 33, i n which both keratan sulphate and cchondroitin-'Sulphate were attached through s p e c i f i c sugar sequences to the p r o t e o g l y c a n p r o t e i n c o r e . A n a l y s i s of the p a r t i a l l y p u r i f i e d 4M GuCl e x t r a c t of i n t e r v e r t e b r a l d i s c ( f r a c t i o n B, F i g . 27) and the high d e n s i t y p r o t e o g l y c a n ( f r a c t i o n G, F i g . 27) i n d i c a t e d the presence of hexuronate, hexosamine and sulphate i n s i m i l a r c o n c e n t r a t i o n to the p u b l i s h e d p r o t e o g l y c a n composition. The hexose and p r o t e i n contents were c o n s i d e r a b l y lower than the p u b l i s h e d v a l u e s , but the data, p a r t i c u l a r l y the reduced p r o t e i n content, could be due to the s e p a r a t i o n of contaminating p r o t e i n / g l y c o -p r o t e i n i n the d e n s i t y g r a d i e n t u l t r a e e n t r i f u g e . F o l l o w i n g r e d u c t i o n of the p r o t e o g l y c a n w i t h sodium borohydride, the l i b e r a t e d glycosaminoglycans were f r a c t i o n a t e d by p r e c i p i t a t i o n w ith i n c r e a s i n g c o n c e n t r a t i o n s of a l c o h o l . The f r a c t i o n p r e -c i p i t a t e d by 40% e t h a n o l contained mainly hexuronate and galactosamine; the f r a c t i o n subsequently p r e c i p i t a t e d by 75% a l c o h o l contained mainly g a l a c t o s e and glucosamine. The r e s u l t s suggested t h a t the d i s c proteoglycans contained both keratan sulphate and c h o n d r o i t i n sulphate polymers which was confirmed by e l e c t r o p h o r e s i s of the f r a c t i o n s on c e l l u l o s e a c e t a t e . Although q u a n t i t a t i v e v a l u e s were not ob t a i n e d , gas l i q u i d chromatography of the n e u t r a l sugars f o l l o w i n g a c i d h y d r o l y s i s of the proteoglycans showed the presence of g a l a c -tose x y l o s e and perhaps mannose although i t was d i f f i c u l t to determine i f the l a s t of these was prese n t s i n c e i t e l u t e d under the f i r s t peak of g a l a c t o s e . On c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s the i n t a c t p r o t e o g l y c a n s moved as a s i n g l e band with a m i g r a t i o n r a t e s i m i l a r t o t h a t of c h o n d r o i t i n 121 . F i g . 33. P r o p o s a l s t r u c t u r e of the p r o t e o g l y c a n of bovine i n t e r v e r t e b r a l d i s c (Hopwood and Robinson, 1974). Proteoglycan P r o t e i n Core " Glu -(£\|§). •Ser x y l g a l g a l g l u A C h o n d r o i t i n Sulphate 25 galNAc 25 gluA 28 SO,, Gly •P3?-0._- •Thr Fucose gal NAc g a l AcNeu - g a l — A c N e u g a l — A c N e u 24 gluNAc . 2 4 . g a l 31 so 4 Marinose 1 G l u — u ( S e r — P r o ) 1 2 3 . s u l p h a t e . The p r o t e o g l y c a n s s t a i n e d s t r o n g l y with A l c i a n blue at pH 1.0 and pH 2.5, suggesting the presence of sulphate groups, and very weakly with Ponceau S i n d i c a t i n g t h a t o n l y a s m a l l amount of p r o t e i n was p r e s e n t . Viewed overall;/„ the r e s u l t s were c o n s i s t e n t w i t h the expected*.behaviour and ana-l y t i c a l data of a r e l a t i v e l y homogeneous p r o t e o g l y c a n o b t a i n e d from i n t e r v e r t e b r a l d i s c . The development of the s e q u e n t i a l e x t r a c t i o n procedure f o r the s e p a r a t i o n of non-aggregated and aggregated p r o t e o g l y -cans made i t necessary to analyse the f r a c t i o n s o b t a i n e d , . t o determine i f they were a l s o d i f f e r e n t i n chemical c o n s t i t u t i o n . The data presented i n Table VII showed no c o n s i s t e n t l y l a r g e d i f f e r e n c e s between the 0.4M and 4M GuCl e x t r a c t s . A n a l y s i s of the p r o t e o g l y c a n s i s o l a t e d from annulus f i b r o s u s and nucleus pulposus however, suggested t h a t the l a t t e r c o n t a i n e d c o n s i d -e r a b l y more hexosamine, i n both,the 0.4M.and 4M GuCl s e q u e n t i a l e x t r a c t s than d i d the former. The s i g n i f i c a n c e of t h i s obser-v a t i o n i s unknown. The r e s u l t s of t h i s work and s i m i l a r i n v e s t i g a t i o n s of c a r t i l a g e p r o t e o g l y c a n s (Brandt and Muir, 1971) have suggested t h a t connective t i s s u e s c o n t a i n two s p e c i e s of p r o t e o g l y c a n s o n l y one of which i s capable of forming aggregates. While t h i s study does not show any observable d i f f e r e n c e s i n the composition of the f r a c t i o n s , i t would appear l i k e l y t h a t the p r o t e o g l y c a n s must be s t r u c t u r a l l y d i f f e r e n t from one another. One c o u l d s p e c u l a t e t h a t such s t r u c t u r a l changes would probably be i n the p r o t e o g l y c a n p r o t e i n c o r e , i n the r e g i o n where the g l y c o p r o t e i n - l i n k or hyaluronate are a t t a c h e d . A d e t a i l e d c h a r a c t e r i z a t i o n of the two p r o t e o g l y c a n s p e c i e s appears to r e p r e s e n t a promising l i n e o f . f u t u r e r e s e a r c h l e a d i n g to a f u l l e r understanding of the aggregation of c o n n e c t i v e t i s s u e p r o t e o g l y c a n s . CONCLUSIONS T h i s t h e s i s was undertaken to answer the f o l l o w i n g q u e s t i o n s : 1. Do p r o t e o g l y c a n aggregates, s i m i l a r t o those of bovine n a s a l c a r t i l a g e , e x i s t i n the i n t e r v e r t e b r a l d i s c ? 2. I f so, c o u l d they account f o r the observed decrease i n v i s c o s i t y and average molecular weight of the p r o t e o g l y c a n s t h a t occur w i t h i n c r e a s i n g age and/or w i t h degenerative i d i s c d i s e a s e . Gel chromatography, v i s c o s i t y and a n a l y t i c a l u l t r a c e n t r i f u g -a t i o n of the p r o t e o g l y c a n f r a c t i o n s o b t a i n e d from various, d i s c e x t r a c t s suggested t h a t o n l y about 5% of the p r o t e o g l y -cans were prese n t as aggregates; i n c o n t r a s t about 70% of the p r o t e o g l y c a n s of bovine n a s a l c a r t i l a g e were aggregated i n the t i s s u e . In a d d i t i o n , both the aggregate and s u b u n i t of the d i s c p r o t e o g l y c a n s were s m a l l e r and more polydisper.se than those of c a r t i l a g e . Most of the d i s c aggregates were c o n f i n e d t o the annulus f i b r o s u s . (Etsappeared t h a t the r e -maining 95% of the p r o t e o g l y c a n s were i n c a p a b l e of forming aggregates although they appeared s i m i l a r in rcomposition to the aggregating s p e c i e s . . Examination of d i s c s over a wide age range showed no d i f f e r e n c e i n the degree of p r o t e o g l y c a n aggregation; the only change observed was an apparent i n c r e a s e i n p o l y d i s p e r -s i t y of both the aggregated andnnon-aggregated s p e c i e s with i n c r e a s i n g age. Since these o b s e r v a t i o n s were made on a very l i m i t e d number o f samples i t was i m p o s s i b l e to draw v a l i d c o n c l u s i o n s from the r e s u l t s . The answer to the second q u e s t i o n however appeared to be negative s i n c e the d i s c was found to c o n t a i n o n l y 5% of the p r o t e o g l y c a n s i n an aggregated form. I t appeared u n l i k e l y t h e r e f o r e , t h a t the reduced v i s c o s i t y and average molecular weight of the p r o t e o g l y c a n s observed i n . aged and h e r n i a t e d d i s c s c o u l d be accounted f o r by a change i n the degree of p r o t e o g l y c a n a g g r e g a t i o n . 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APPENDIX Age and d i a g n o s i s of the autopsy cases used.for t h i s study. P a t i e n t s with bone or c o n n e c t i v e t i s s u e a b n o r m a l i t i e s i n c l u d i n g severe o s t e o a r t h r i t i s were not used. Number Age 9 Diagnosis D i s c s Removed 1 67 A t h e r o s c l e r o t i c h e a r t d i s e a s e L l ' L 1 ~ L 5 2 37 C r a n i a l pharyngioma L l " s 3 77 Pneumonia L 2-S 4 41 Acromegaly L^-S 5 51 Lymp ho s a r c oma L 1-S 6 58 C i r r h o s i s L 1-S 7 52 A t h e r o - S c h e r o t i c h e a r t d i s e a s e L 1-S 8, 46 Carcinoma of c o l o n L 1 " L 5 9 23 B a c t e r i a l e n d o c a r d i t i s L^-S 10 51 M e s e n t e r i c a r t e r y thrombosis L 1-S 11 49 Pulmonary hemorrhage L^-S 12 34 Ruptured c e r e b r a l aneurism L^-S 13 21 Polycythemia r u b r a v e r a L 1 " L 5 14 57 Pneumonia L 1 " L 5 15 SB Asphyxia C 1 2 - S 16 39 P o r t a l c i r r h o s i s L^-S 17 55 H e p atic c i r r h o s i s L^-S 18 60 A t h e r o s c l e r o t i c h e a r t d i s e a s e V s 19 52 Carcinoma of stomach L^-S 20 56 Pulmonary abscess L r L 5 21 47 C e r e b r a l hemorrhage 22 30 S k u l l f r a c t u r e 23 55 A t h e r o s c l e r o t i c h e a r t d i s e a s e L n -S APPENDIX II S c h l i e r e n p a t t e r n s of 4M GuCl e x t r a c t s of bovine nasa c a r t i l a g e (A through D) and human i n t e r v e r t e b r a l d i s c (E through H) showing the e f f e c t of pH on the degree p r o t e o g l y c a n aggregation. Bovine Nasal C a r t i l a g e . 4.5 5.8 D 9.0 

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