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The partial characterization of the epithelial glycoprotein from normal and diseased colons Tsang, Wai-Chiu 1975

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THE  PARTIAL CHARACTERIZATION OF THE E P I T H E L I A L GLYCOPROTEIN FROM NORMAL AND DISEASED COLONS  by WAI-CHIU B.Sc.  University  TSANG of B r i t i s h  Columbia  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  t h e Department  We a c c e p t the  this  required  THE  thesis  of  PATHOLOGY  as c o n f o r m i n g t o  standard  UNIVERSITY OF B R I T I S H COLUMBIA  April,  1975.  In  presenting this  thesis  an advanced degree at the L i b r a r y I  further  fulfilment  the U n i v e r s i t y of  s h a l l make it  agree  in p a r t i a l  freely  of  the  requirements  B r i t i s h Columbia, I agree  available  for  this  thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department  of  this  thesis for  It  financial  of  g a i n s h a l l not  PATHOLOGY  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  February, 1975  or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  that  r e f e r e n c e and s t u d y .  t h a t p e r m i s s i o n for e x t e n s i v e copying o f  by h i s r e p r e s e n t a t i v e s .  for  be allowed without my  ii  ABSTRACT  This and  study reports  the i s o l a t i o n ,  chemical characterization  undegraded and  epithelial  o f a p p a r e n t l y homogenous and  glycoproteins  from p a t i e n t s w i t h u l c e r a t i v e  ischaemic  colitis  of the colon.  removed f r o m e a c h  specimen  fractionation  from normal  colitis, Colonic  Crohn's  pooled yielded gel DEAE and  fraction  t w i c e as f a s t  liquid  about  as t h a t  that  UV-spectroscopy, hexose  agarose  the  B was  c a r b o h y d r a t e components o f e a c h  phosphorus DNA.  t o be homogeneous  analyses of these  o r no p h o s p h o r u s .  o f gas-  gel electro-  g e l and c e l l u l o s e  the presence of fucose, galactose, and l i t t l e  Application  appeared  revealed  protein  of heparin  analysis,  a t pH 8.8.  Agarose  o f the second  agarose  phoreses  acid,  Chemical  A.  fraction  A glycoproteins  DEAE c h r o m a t o g r a p h y ,  A and B.  the m o b i l i t y  of fraction  Indole assays suggested that  of the  and d i s e a s e d s a m p l e  t h e same as t h a t  a t pH 3.0, c o l o r i m e t r i c  Fraction by  revealed  chromatography,  phoresis and  from each normal  (B) was  were  w i t h 1M N a C l .  chromatography  two a p p a r e n t l y homogeneous f r a c t i o n s  electrophoresis  cells  by s h a k i n g w i t h EDTA and t h e g l y c o -  g e l and DEAE c e l l u l o s e  crude e x t r a c t  d i s e a s e and  epithelial  p r o t e i n s were e x t r a c t e d ' f r o m t h e s o n a t e d c e l l s Combined a g a r o s e  human c o l o n s  acetate electroglycoproteins  hexosamine, The m o l a r  sample was  sialic  ratio of  significantly  iii  different  from t h a t  different  diseases.  4 different quantities sialic C9,  of the normal,  and i t v a r i e d  In each of the g l y c o p r o t e i n s  types o f s i a l i c  a c i d s were  found  and t h e y w e r e : ( 1 ) u n s u b s t i t u t e d  acids,  (ii) sialic  ( i i i )s i a l i c  acids  sialic  acids  liquid  chromatography  among t h e  acids  studied,  in different  o r C9  substituted  s u b s t i t u t e d a t C4 and o r a t  s u b s t i t u t e d a t C7 and o r C8, ( i v )  s u b s t i t u t e d a t C4 and a t C7 and o r C8.  alkaline-labile  revealed  that  O - a c e t y l groups.  above d i f f e r e n c e s were  these  Gas-  substituents  were  The s i g n i f i c a n c e o f t h e  discussed.  u-pc  I  1.  II.  1  INTRODUCTION 1.  L i t e r a t u r e Review. A. C h e m i s t r y o f t h e i n t e s t i n a l g l y c o proteins . B. The i n v o l v e m e n t o f g l y c o p r o t e i n s in ulcerative c o l i t i s . C. T h e i n v o l v e m e n t o f g l y c o p r o t e i n s i n Crohn's d i s e a s e .  2.  The P r e s e n t  Investigation  MATERIALS AND METHODS  17  1.  P r e p a r a t i o n o f Samples imetric analyses.  f o r GLC and C o l o r -  2.  Gas L i q u i d C h r o m a t o g r a p h i c A. N e u t r a l sugars, B. O - a c e t y l g r o u p s .  3.  Colorimetric  4.  Electrophoresis  5.  P r e l i m i n a r y E x p e r i m e n t s on t h e I s o l a t i o n of Rat C o l o n i c E p i t h e l i a l Glycoproteins.  Analyses,  Analyses. Studies.  A. I s o l a t i o n o f c o l o n i c e p i t h e l i a l c e l l s . B. E x t r a c t i o n o f c o l o n i c g l y c o p r o t e i n s . C. F r a c t i o n a t i o n o f c o l o n i c g l y c o p r o t e i n s . 6.  I s o l a t i o n o f Human C o l o n i c Glycoproteins.  Epithelial  A. S o u r c e o f s p e c i m e n s . B. I s o l a t i o n o f m u c o s a l c e l l s f r o m human colon. C. E x t r a c t i o n and f r a c t i o n a t i o n o f c o l o n i c epithelial glycoproteins. 7.  Saponification  8.  Digestion  9.  Immunodiffusion  10.  Studies.  Studies  Preparation  with  Neuraminidase.  Studies.  of Antisera.  V  page III.  RESULTS AND  DISCUSSION  33  1.  Isolation  of E p i t h e l i a l  Cells.  2.  Extraction of Colonic Glycoproteins.  3.  Fractionation of Colonic Glycoproteins.  Epithelial Epithelial  A. P r e l i m i n a r y m o d e l e x p e r i m e n t s rat glycoproteins. B. E x p e r i m e n t s on human c o l o n i c epithelial cells.  IV.  4.  Blood  5.  Analyses of Fractions  B.  6.  Analyses  A.  7.  Saponification  8.  D i g e s t i o n Study w i t h Neuraminidase.  9.  GLC a n a l y s e s  Contamination.  10.  General  11.  Further  BIBLIOGRAPHY  APPENDIX  with  of Fractions  Studies. Vibrio  of O-acetyl  Cholera  groups.  Discussion, Work  Proposed. 68  72  vi  L I S T OF  TABLES Page.  1.  P e r c e n t a g e r e c o v e r y o f h e x o s e and s i a l i c a c i d a f t e r c o n c e n t r a t i o n o f t h e 105,000g s u p e r n a t a n t o f i n t e s t i n a l e p i t h e l i a l g l y c o p r o t e i n by t h e various procedures.  35  2.  E l e c t r o p h o r e t i c m o b i l i t i e s o f t h e samples obt a i n e d from the v a r i o u s stages i n the f r a c t i o n a t i o n o f c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s ext r a c t e d f r o m n o r m a l and d i s e a s e d human c o l o n s .  41  3.  Chemical analyses  44  4.  Molar r a t i o s of sugars i n c o l o n i c e p i t h e l i a l glycoproteins (Fractions A).  45  5.  Chemical proteins  46  6.  Percentage side chain s u b s t i t u t i o n i n the s i a l i c acids of colonic e p i t h e l i a l glycoproteins.  51  7.  Substitution patterns of s i a l i c epithelial glycoproteins.  55  8.  Molar r a t i o s o f sugars found i n c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s e x p r e s s e d as p e r c e n t a g e s .  of Fractions  B.  analyses of c o l o n i c e p i t h e l i a l (Fractions A).  acids  glyco-  i n colonic  61  vii  LIST OF FIGURES  page 20  1.  Flow diagram of the method used f o r the i s o l a t i o n of i n t a c t r a t c o l o n i c e p i t h e l i a l c e l l s .  2.  Flow diagram of the method f o r the e x t r a c t i o n o f g l y c o p r o t e i n from r a t c o l o n i c e p i t h e l i a l c e l l s .  22  3.  Flow diagram of the method f o r the f r a c t i o n a t i o n or r a t c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s .  23  4.  Flow diagram o f the procedure f o r the d i g e s t i o n s t u d i e s of c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s o f r a t and human u s i n g v i b r i o c h o l e r a n e u r a m i n i d a s e .  28  5.  Photomicrographs of s e c t i o n s E D T A - t r e a t e d human c o l o n .  31  6.  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 r a t c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s at pH 8 . 8 . Procedure used t o monitor the f r a c t i o n a t i o n p r o c e d u r e .  36  7.  Agarose A15M column chromatography of the 105,000g . supernatant e x t r a c t e d from the i s o l a t e d c o l o n i c e p i t h e l i a l c e l l s of a human sample r e s e c t e d f o r carcinoma of the c o l o n .  38  8.  DEAE c e l l u l o s e ion-exchange column chromatography of the c a r b o h y d r a t e - r i c h f r a c t i o n A l o b t a i n e d from agarose A15M column chromatography o f human c o l o n i c c e l l sap o b t a i n e d from a sample r e s e c t e d f o r carcinoma o f the c o l o n .  40  9.  I l l u s t r a t i o n of a t y p i c a l agarose g e l e l e c t r o p h o r e s i s p a t t e r n of the v a r i o u s f r a c t i o n s i n the f r a c t i o n a t i o n of the 105,000g supernatant o f a human sample.  42  10.  G a s - l i q u i d chromatograph of the t r i m e t h y l s i l y l e t h e r s o f the n e u t r a l sugars of c o l o n i c e p i t h e l i a l g l y c o p r o t e i n e x t r a c t e d from the h i s t o l o g i c a l l y normal p a r t o f s u r g i c a l carcinoma specimens o b t a i n e d from p a t i e n t s undergoing c o l e c t o m y .  47  11.  I l l u s t r a t i o n of a t y p i c a l s a p o n i f i c a t i o n time curve of human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n .  49.  from u n t r e a t e d and  ACKNOWLEDGEMENTS  The  author wishes  for h i s guidance  thanks  are extended  Mr. C.F.C. C u l l i n g ,  and  D r . P. V a s s a r . The a u t h o r  his  assistance with  proof-reading laboratory of and  h i s s u p e r v i s o r D r . P. R e i d  and e n c o u r a g e m e n t o v e r  work. In a d d i t i o n , committee,  t o thank  this  the d u r a t i o n o f t h i s t o members o f h i s  Dr.G. G. S. D u t t o n , i s indebted  some o f t h e c h e m i c a l thesis,  t o the s t a f f s  i n the Department o f Pathology  histological  sections,  Research  Financial  French  t o Mr. C. Ramey f o r a n a l y s e s and i n o f the h i s t o l o g y f o r the p r e p a r a t i o n  and t o o t h e r members o f t h e F a c u l t y  s t a f f o f the Department o f Pathology  co-operation.  D r . S.  for their  constant  s u p p o r t was p r o v i d e d b y t h e M e d i c a l  C o u n c i l o f Canada  i n the form o f a s t u d e n t s h i p .  X  INTRODUCTION  The  research described i n this  the i s o l a t i o n  and c h a r a c t e r i z a t i o n  proteins  n o r m a l and d i s e a s e d human  and  from  histochemical evidence  thesis  concerned  of e p i t h e l i a l  suggested  glyco-  colons.  Chemical  that glycoproteins  1 2 play  a role  that  such  of  in intestinal  a study  the e t i o l o g y  cularly  '  and i t was  could contribute to a better  and p a t h o g e n e s i s  ulcerative The  diseases  colitis  of colonic  felt  understanding  diseases  parti-  and C r o h n ' s d i s e a s e .  c h e m i s t r y o f g l y c o p r o t e i n s has been extensive^-  3 ly  reviewed  as  "conjugated  one  by G o t t s c h a l k  .  p r o t e i n s c o n t a i n i n g as p r o s t h e t i c  o r more h e t e r o s a c c h a r i d e  branched, lacking  with  Glycoproteins are best defined  a relatively  a serially  ( s ) , the l a t t e r  g r o u p (s)  are usually  low number o f s u g a r r e s i d u e s ,  repeating unit  and b o u n d c o v a l e n t l y t o  4 the p o l y p e p t i d e c h a i n in  animal  tissues  Glycoproteins covalent  acid  D-xylose, various The  of carbohydrate  of protein  that  i n conjugated  of the carbohydrate form w i t h  proteins.  a r e a d i v e r s e g r o u p o f compounds s i n c e t h e  composition,  sugars  A major p o r t i o n  i s found  association  wide range  .  types.  While  occurs  t h e y have no u n i q u e  t h e y do c o n t a i n a c h a r a c t e r i s t i c  include D-galactose,  N-acetyl-D-glucosamine,  derivatives  carbohydrate  with protein  of neuraminic  content o f these  in a amino  group o f  D-mannose, D - g l u c o s e ,  L-fucose,  N-acetyl-D-galactosamine acid  (the s i a l i c  g l y c o p r o t e i n s may  ,  and  acids). vary  from  2  less  than  weight  one p e r c e n t  of the molecule,  may be p r e s e n t  sugar  phomucin,  definition.  protein."  Terms t h a t  o n l y two t e r m s w i l l  macromolecules -  acid  muco-  and g l y c o p r o t e i n .  be u s e d  In t h i s  t o d e s c r i b e these  (a) g l y c o p r o t e i n and  Cb)  sul-  types  glycosaminoglycan.  m a c r o m o l e c u l e s w h i c h c o n t a i n s one o r more o f t h e f o l l o w i n g  carbohydrate tan  polymers:  s u l p h a t e , dermatan  hyaluronic acid,  heparin  sulphate, kera-  s u l p h a t e and c h r o n d r o i t i n  4 and 6 s u l p h a t e  will  be t e r m e d g l y c o s a m i n o g l y c a n s ;  a l l other  will  be d e s i g n a t e d g l y c o p r o t e i n s .  According to this  glycosaminoglycans  glycoproteins are mainly discussed  here  as a m a j o r c a r b o h y d r a t e  normally  found  macromolecules  (with the e x c e p t i o n o f k e r a t a n  contain uronic acid  contain s i a l i c  acid.  i s a diffuse,  non-specific  sulphate)  Glycosaminoglycans the g l y c o p r o t e i n s  a r e assumed t o be o f e p i t h e l i a l  colitis  origin.  i s a d i s e a s e o f unknown inflammatory  classification  constituent while  i n connective tissues while  Ulcerative It  include:  mucosubstance,  s i a l o m u c i n , carboxymucin,  glycosaminoglycan  i s i n accord  have been employed  mucopolysaccharide,  fucomucin,  polysaccharide, thesis,  types  i s considerable confusion i n the terminology  mucin, mucoprotein,  Any  percent of the  t o d e s c r i b e m a c r o m o l e c u l e s whose c o m p o s i t i o n  with this  of  eight  and as many as s e v e n  i n any g i v e n  There used  t o more t h a n  condition  etiology. of the  3.  mucous membrane  of the large  of  an e x u d a t i v e  and v a s c u l a r t y p e  of  granulation tissue  can  occur  for either  in  the t h i r d  of  cancer  five  time  Although  attendance  I t has been e s t i m a t e d  i n patients with ulcerative  and t e n t i m e s  population  ^'^.  g r e a t e r than  disease  at hospital i s that  colitis  the r i s k  the r i s k  i s between  i n the general  Changes i n t h e e p i t h e l i a l g l y c o p r o t e i n s  have b e e n d e m o n s t r a t e d disease ^  both  h i s t o c h e m i c a l l y and c h e m i c a l l y  in  this  or  a c o n s e q u e n c e o f t h e d i s e a s e i s unknown.  Chemistry  this  a t any a g e , t h e peak age o f  s e x on f i r s t  decade.  The i n f l a m m a t i o n i s  and g e n e r a l l y u n p r o d u c t i v e  or f i b r o s i s .  f o r the f i r s t  incidence  intestine.  , b u t whether these  changes a r e a  cause  o f the I n t e s t i n a l Glycoproteins  Viscous throughout  s e c r e t i o n s of the e p i t h e l i a l  the g a s t r o i n t e s t i n a l  as h i g h l y  hydrated  lubricate  t h e mucous membrane,  tract"'"''".  g e l s the apparent  These  cells  a r e found  secretions exist  f u n c t i o n s o f which a r e t o  t o p r o t e c t i t from m e c h a n i c a l  and  12 c h e m i c a l damage  and from  Chemically  are g l y c o p r o t e i n s .  they  carbohydrate teolysis  moiety  forms a p h y s i c a l  of the protein  carbohydrate glycoprotein, epithelial  moiety which  cells.  a t t a c k by enzymes a n d b a c t e r i a  core  I t i s speculated that the b a r r i e r which prevents  ( 3 ) . Thus,  may be e s s e n t i a l i n t u r n imparts  the i n t e g r i t y  f o r the s u r v i v a l protection  pro-  of the  of the  t o the mucosal  4  T h e r e have b e e n glycoproteins. fractionation Gottschalk of  3  s u r p r i s i n g l y few s t u d i e s  The p r o c e d u r e s e m p l o y e d o f these g l y c o p r o t e i n s  , Marshal  JL 3  starting material  have b e e n e m p l o y e d  origin  o r g a n s may  contain  make i t u n l i k e l y t h a t were i s o l a t e d connective  since  12 ' .  Three  types  f o r the e x t r a c t i o n of 11  epithelial  and  have b e e n r e v i e w e d by  t h e s e g l y c o p r o t e i n s : (a) m u c o s a l s c r a p i n g s 2324 2526 colon ' and (c) r e c t a l i r r i g a t e s ' . starting materials  intestinal  i n the i s o l a t i o n  14 , and S p i r o  , Horowitz  of  15-22 '  , (b) m i n c e d  The u s e o f s u c h  compounds o f p u r e l y  scrapings  and m i n c e d  t i s s u e contaminants  s u c h as t h e  15 16 20 22 glycosaminoglycans irrigates  could  stream b a c t e r i a I n many c a s e s ,  found  data  native  glycoproteins  Chemical  analyses  '  , while  of glycoproteins.  were n o t o b t a i n e d  i n the i s o l a t i o n  to a specific  '  the products of f a e c a l  and e n z y m a t i c d e g r a d a t i o n  i f not impossible  obtained  studies  be c o n t a m i n a t e d w i t h  p r o t e o l y s i s was u s e d difficult  i n some  procedure.  because  Thus, i t i s  t o r e l a t e any o f t h e s t r u c t u r a l glycoprotein. of the i n t e s t i n a l  glycoproteins  * i • 9,11,15-17,23 . , . . of s e v e r a l species revealed the presence of p r o t e i n , sialic  a c i d , fucose,  hexosamine  case o f sheep c o l o n i c mucin detailed  ^was  structural analysis.  was r e p o r t e d  i n the other  about the chemical glycoproteins  and g a l a c t o s e .  i n the  an a t t e m p t made t o p e r f o r m a  A n a l y t i c a l data  studies.  Only  Thus, very  of limited little  scope  i s known  and m a c r o m o l e c u l a r s t r u c t u r e o f t h e i n t e s t i n a l  o f man  and a n i m a l s .  3  Animal G l y c o p r o t e i n  Studies  15 Werner scrapings  s t u d i e d the  from g a s t r o - i n t e s t i n a l  p r o t e i n s were shown t o be mucins. lated one  On  the  rich  the  e x i s t e n c e o f two  I n o u e and  with  by  pronase.  contained two  the  and  tissues  o f washed  o f the  analytical  different  the  other  O-sulphate  ester.  groups: glycopeptides  rich  in sialic  postu-  acid.  glycopeptides scrapings  o b t a i n e d were a c i d i c  T h e y c o u l d be  and  various  intestinal glycoproteins,  d i g e s t i o n of p i g c o l o n i c mucosal fractions  Glyco-  d a t a , Werner  Yosizawa"^ examined the  A l l the  mucosal  pig.  a m a j o r component o f t h e  b a s i s of the  i n fucose,  obtained  composition  separated  glycosaminoglycans.  and into  The  glyco-  5 p e p t i d e s -(molecular weight fucose, glucosamine, The  heparin  and  the  fractions,  ) contained  galactose,  sialic  and  present  glycopeptides, contained  chondroitin sulphate.  glycosaminoglycans Nemoto and  X 10  galactosamine,  glycosaminoglycan  amounts t h a n  2-3  represented 17  Yosizawa  obtained  I t was  connective  acid  i n much heparin  Glycosaminoglycans  s c r a p i n g s o f dog  colon  smaller sulphate,  assumed t h a t tissue  s i m i l a r mixed  pronase d i g e s t s of mucosal s c r a p i n g s of r a b b i t intestine.  sulphate.  have a l s o been  the  contaminants.  fractions c o l o n and found  from small  i n mucosal  6  Kent e t on  the  water  al  have c a r r i e d o u t  soluble  glycoproteins  s h e e p c o l o n i c mucosa. found, with 2.15  acid-containing 2 X 10^. in  The  c o e f f i c i e n t s of  of  a rapid reduction and  from s c r a p i n g s  with  this  9.58,  6.1,  3.25 a  of  about  resulted  loss of  a partially  and  sialic  a molecular weight of  f r a c t i o n with papain  production  of  f r a c t i o n s were  i n v i s c o s i t y without'any  the  studies  m a j o r f r a c t i o n (9.58S) was  glycoprotein  Incubation  carbohydrate,  obtained  Four macromolecular  sedimentation  respectively.  detailed  dialysable  ,  degraded  5 glycoprotein ction be  of molecular weight of  (6.IS) was  d e g r a d e d by  1 X  s u b s t a n t i a l l y free of  papain  to  a 1.7S  10  .  The  sialic  second  a c i d , and  f r a c t i o n (molecular  fracould  weight  2  X  4 10  ).  The  mainly  remaining  two  nucleoproteins.In  labelled  fractions  (3.2S  and 2.15S) 2 7 28  subsequent s t u d i e s  9.58S g l y c o p r o t e i n  was  '  contained  an  isotope  i s o l a t e d from s c r a p i n g s  s h e e p c o l o n i c m u c o s a l t i s s u e w h i c h had  been i n c u b a t e d  Krebs-Ringer b u f f e r  f o r 4 hours  presence of  such m e t a b o l i t e s  D-(1- C)  as  35 threonme the  two  and  SO^  .  Neither  The  25%  sialic  protein,  the  glycoprotein,  acid  and  37%  N-acetylneuraminic  that  could  they  6. IS  the  72%  acid residues  of  implied  1 4  oxygen  glucose,  and  L-(U- C) 1 4  glycoprotein  f r a c t i o n s were l a b e l l e d u n d e r  13.7%  these residues  D-(2- C)  the  Chemical a n a l y s i s of  i t contained  sulphate.  and  in  2-  nucleoprptein  conditions. that  1 4  i n the  of  be  occupied  of  the  were made up  hydrolyzed terminal  by  these  9.58S g l y c o p r o t e i n  carbohydrate  acid.  nor  of The  and  4.6%  fraction, 6 3%  ester  representing  N-glycollylneuraminic  ease w i t h  d i l u t e acids  positions  showed  in a  or  which neuraminidase  "branched  7.  oligosaccharide  structure .  Sequential  v  "Smith  degradation"  35 of  the  S-labelled  glycoprotein  had a p e p t i d e  oligosaccharides galactose-amino aspartamido jacent  9.58S g l y c o p r o t e i n  (sialic  t o the peptide  extracted  acid-galactose-amino  this  an a r r a y o f sugar o r fucose  probably  t h r o u g h B-  Some o f t h e amino s u g a r r e s i d u e s chain  A glycoprotein was  "backbone" t o which  s u g a r ) were a t t a c h e d ,  linkages.  indicated that  were  ad-  sulphated.  containing  sulphate  from mucosal s c r a p i n g s  and s i a l i c  of r a t small  acid  intestine  19 with  isotonic saline  glycoprotein The  this be  was c a r b o h y d r a t e - r i c h  f r a c t i o n was r e s o l v e d  partially  mixture. sialic  i n t o two c o m p o n e n t s , w h i c h  h a d t h e same c h e m i c a l c o m p o s i t i o n  detected  sul-  glucosamine,  fucose,  i n a l l fractions.  they occupied  terminal  glycoprotein  galactose  strongly  by a c i d  that  Threonine, residues.  treatment o f the  galactosamine  t o the peptide through O - g l y c o s i d i c and t h r e o n i n e residues.  Both  hydrolysis  amino a c i d  from a l k a l i n e b o r o h y d r i d e suggested  and  labile.  positions.  a n d p r o l i n e made up 69% o f t h e t o t a l  Both  The amount o f  a c i d and f u c o s e were r a p i d l y r e l e a s e d that  could  as t h e p a r e n t  was 2.4%, 40% o f w h i c h was a c i d  r e s u l t s obtained  linked serine  and c o n t a i n e d  by S e p h a r o s e - 4 B c h r o m a t o g r a p h y .  Galactosamine,  suggesting serine  separated  a c i d were p r e s e n t  sulphate sialic  fractions.  On u l t r a c e n t r i f u g a t i o n and d i s c g e l e l e c t r o p h o r e s i s  subfractions  The  Chromatography o f t h i s s a l t - s o l u b l e  on a DEAE c e l l u l o s e c o l u m n g a v e t h r e e  major f r a c t i o n  phate.  .  linkages  residues  were  involving  Forstner et a l ^ i s o l a t e d u  weight small  g l y c o p r o t e i n (HMG) intestine.  in  rabbits  was  found  goblet cells was  and  i n the  high-molecular-  from mucosal  scrapings of r a t  A n t i b o d i e s a g a i n s t HMG  the  fluorescein-labelled  to s t a i n  cells  a  the  were  prepared  antisera  s u p r a n u c l e a r mucous  so  obtained  vesicles  i n t h e body o f t h e  s t o m a c h and  sublingual gland.  I t was  of  mucus-producing  concluded  a m a j o r component o f r a t s m a l l i n t e s t i n e  that  HMG  goblet-cell  21 mucus.  In a l a t e r  charged  macromolecule with  10  and  contained  10%  sialic  m a j o r and disc  acid, two  study  12% 6.6%  , HMG  was  found  t o be  a molecular weight  protein, fucose  23%  and  hexose,  less  than  and  by  analytical  of about  22.4% 1%  m i n o r components were i d e n t i f i e d  gel electrophoresis  a negatively  hexosamine,  sulphate. by  2 X  One  acrylamide  ultracentrifugation.  Human G l y c o p r o t e i n S t u d i e s  25 Roelf et a l Gas  chromatographic  revealed  , have e x a m i n e d human r e c t a l  analysis  the presence  of h y d r o l y s a t e s o f the  o f f u c o s e , mannosamine,  glucosamine,  N-acetyl-neuraminic  The  o f mannosamine was  presence  m e r i z a t i o n of glucosamine pyridine.  acid  and  thought  irrigates. irrigates  galactosamine,  N-acetyl-galactosamine.  to r e p r e s e n t the e p i -  as a c o n s e q u e n c e o f h e a t i n g w i t h  y  Two r e c t a l mucous p l u g s , with c y s t i c  fibrosis  o b t a i n e d from c h i l d r e n  o f the pancreas and r e l a t i v e l y uncon-  taminated by m a t e r i a l s such as  faeces and b l o o d , were  26 examined by Johansen  .  The samples were i n s o l u b l e  a wide v a r i e t y o f s o l v e n t s i n c l u d i n g w a t e r , urea,  formamide,  v/v),  0.1 M c y s t e i n e ,  They d i s s o l v e d  salt solutions  s l o w l y i n 0.1 M sodium h y d r o x i d e . fucose,  sialic  found i n e i t h e r  sample.  terminal p o s i t i o n ,  as  Sialic  a c i d and  Both samples  N-acetylglucosamine.  U r o n i c a c i d was  not  a c i d appeared t o occupy a  i t c o u l d be removed from the  c h a i n by v i b r i o c h o l e r a neuraminidase.  glycoproteins  (9:1  and m i n e r a l a c i d s .  sample c o n t a i n e d t r a c e s of mannose.  saccharide  and 9 M  saturated calcium c h l o r i d e , ethanol  contained g a l a c t o s e , One  3,6,  in  oligoBoth  were found to be r e s i s t a n t t o t r y p s i n , c h r y m o t r y p s i n ,  p e p s i n and lysozyme, but c o u l d be d i s p e r s e d  with papain,  23 Sky-Peck e t glycosaminoglycans colon.  al  have i s o l a t e d  glycopeptides  from p r o t e o l y t i c d i g e s t s of whole human  Human c o l o n s were minced and the crude  was i n c u b a t e d w i t h papain at  6 0 ° C for  the glycosaminoglycans  and a s u p e r n a t a n t .  Treatment of (CPC), to  pre-  p r e s e n t , y i e l d e d a CPC complex  Treatment of the m a t e r i a l  CPC complex w i t h t e s t i c u l a r  homogenate  24 h o u r s .  the incubate w i t h 1% c e t y l p y r i d i n i u m c h l o r i d e , cipitate  and  hyaluronidase  obtained  from the  f o l l o w e d by f r a c t i o n -  a t i o n of the d i g e s t by a combination of B i o g e l P30 and DEAE  1(  c e l l u l o s e chromatography y i e l d e d a major h e x o s e - r i c h f r a c t i o n free  of u r o n i c a c i d .  T h i s m a t e r i a l ' appeared t o be homogenous  by u l t r a c e n t r i f u g a t i o n and f r e e boundary e l e c t r o p h o r e s i s pH 8.4 of  and at pH 4 . 6 ,  hexose,  sialic  Chemical a n a l y s i s  r e v e a l e d the  a c i d , hexosamine and f u c o s e .  from the CPC p r e c i p i t a t i o n c o n t a i n e d hexose.  Galactose,  a c i d were p r e s e n t  mannose, hexosamine,  On f r a c t i o n a t i o n distinct  fucose  i n each o f these f r a c t i o n s .  c o u l d not be d e t e c t e d .  presence  The supernatant  on DEAE c e l l u l o s e chromatography i t y i e l d e d t h r e e fractions.  at  and s i a l i c  Hexuronic a c i d  In two of the f r a c t i o n s  hexosamine  was p r e s e n t o n l y as glucosamine w h i l e the t h i r d c o n t a i n e d o n l y galactosamine.  22 Korhonen and Makela  removed human c o l o n i c mucosa  at the l e v e l of the lamina m u s c u l a r i s mucosae. o b t a i n e d was d r i e d at  The t i s s u e  6 0 ° C f o r 24 h o u r s , homogenized, and then  heated f o r 20 minutes i n a b o i l i n g water b a t h .  Pronase d i -  g e s t i o n o f the heated homogenate was c a r r i e d out at  6 0 ° f o r 48  hours and the g l y c o p e p t i d e s o b t a i n e d were e x t r a c t e d w i t h chloroacetic acid.  E l e c t r o p h o r e s i s of the e x t r a c t  tri-  revealed  the presence o f four components, t h r e e o f which c o n t a i n e d s u l phate.  A l l these g l y c o p e p t i d e s  fucose,  hexosamine,  contained p r o t e i n ,  s i a l i c a c i d and u r o n i c a c i d .  a c i d was thought t o be p r e s e n t  hexose, The h e x u r o n i c  i n the c o l o n i c mucosa as  g l y c o p r o t e i n component and not as  a  a c o n n e c t i v e t i s s u e contaminant.  Kawaski e t a l ^ e x t r a c t e d g l y c o p r o t e i n s colon with contained  ethylene  galactose,  Threonine,  amino a c i d s . these  The m a j o r g l y c o p r o t e i n  a s much a s 80% c a r b o h y d r a t e ,  galactosamine, sulphate.  glycol.  fucose,  Immunofluorescent  similar  t o show t h a t t h e y  of  i n Ulcerative  ulcerative colitis  and h i s t o c h e m i c a l  of glycoproteins  attempt  of e p i t h e l i a l  histochemical  against  antigastric  glands.  Colitis  glycoproteins  have been d e m o n s t r a t e d by  methods.  i s an i m p o r t a n t  to elucidate their  were  i n "intestinalised  A d e t a i l e d knowledge  t h e normal and p a t h o l o g i c a l d i s t r i b u t i o n  types  antisera  towards normal g a s t r i c  Changes i n t h e p a t t e r n s  both chemical  a c i d and e s t e r  studies, using  Involvement o f G l y c o p r o t e i n s  associated with  obtained  composed o f g l u c o s a m i n e ,  sialic  to glycoproteins  mucosa" b u t were u n r e a c t i v e  The  human  s e r i n e and p r o l i n e were t h e p r e d o m i n a n t  g l y c o p r o t e i n s were s a i d  genically  from  biological  o f these  different  p r e r e q u i s i t e f o r any significance.  Although  methods c a n be p r e s u m e d t o show t h e p r e s e n c e and  location  o f some o f t h e s e  chemical  nature  glycoproteins i n a tissue,  o f such g l y c o p r o t e i n s  the exact  c a n n o t be d e t e r m i n e d  23  by  histochemistry  Histochemical The in  the colon  Studies normal p a t t e r n  of the e p i t h e l i a l  and r e c t u m o f man, as r e v e a l e d  by  glycoproteins histochemical  methods, has been d e s c r i b e d There i s general  agreement t h a t b o t h s u l p h a t e d  containing glycoproteins o f man b u t t h e r e distribution  by s e v e r a l i n v e s t i g a t o r s '  are present  and s i a l i c  i n t h e c o l o n i c mucosa  i s some d i f f e r e n c e i n o p i n i o n  o f these  acid  regarding the  m a t e r i a l s between t h e s u r f a c e  epithelium  36 and  t h e u p p e r and l o w e r h a l v e s  the  pattern  of the e p i t h e l i a l  of the crypts glycoproteins  .  Changes i n  are associated  7 8 with as  ulcerative colitis  t o the nature  a decrease in  o f these  i s no g e n e r a l  changes o t h e r  i n the quantity  one s t u d y  varied  ' , but there  agreement  than that there i s  of glycoprotein present.  the d i s t r i b u t i o n  of the c o l o n i c  Indeed  glycoproteins  from case t o case.  Chemical  Investigations  9 Soergel subjects  et a l  obtained  and f r o m p a t i e n t s w i t h  irrigation  o f the rectum w i t h  Chemical analyses  revealed  h e x o s a m i n e and s i a l i c n o r m a l and d i s e a s e d  p e r cent  tissue  higher  hypertonic  acid i n material  colons.  isolated  but the nitrogen  i n t h e samples  isolated  by  galactose,  from  both  composition content  from  Immunologic  showed t h a t g l y c o p r o t e i n s  normal  phosphate s o l u t i o n .  The c a r b o h y d r a t e  than from normal t i s s u e .  diffusion)  ulcerative colitis  the presence o f fucose,  appeared t o remain constant, 50  r e c t a l mucus f r o m  was  diseased  studies  f r o m n o r m a l human  (double rectal  mucosa contained a variable number of serum proteins, cularly  c< and  fl  globulin.  parti-  These serum proteins were  absent i n mucus from u l c e r a t i v e c o l i t i s patients.  The authors  postulated that these globulins might form an i n t e g r a l part of r e c t a l mucus and that highly viscous  °<^ - glycoproteins  might contribute to the physical and chemical properties of the mucus.  Teague et a l 1 ^ , i n a study using mucosal and mucosal scrapings,  reported the presence of two glyco-  proteins i n colonic mucus.  Both contained fucose,  N-acetyl-glucosamine, N-acetyl-galactosamine One however, contained mannose, whereas was a minor component.  biopsies  galactose,  and s i a l i c a c i d .  i n the other mannose  The amount of the mannose-rich  component was s i g n i f i c a n t l y increased i n u l c e r a t i v e  colitis.  The p o s s i b i l i t y that u l c e r a t i v e c o l i t i s i s an 5 37-42 auto-immune disorder has been extensively investigated ' 38 Broberger and Perlmann  found that the sera of 90% of  children and 40% of the adults with u l c e r a t i v e c o l i t i s contained an antibody that haemagglutinated with sheep red blood c e l l s pre-coated with a phenol extract of s t e r i l e human colon. Fluorescein l a b e l l e d gamma g l o b u l i n from such patients was absorbed p r e f e r e n t i a l l y onto the e p i t h e l i a l c e l l s of the crypts  of  the colon  sera  .  Absorption  was t h o u g h t t o be s p e c i f i c f o r  from u l c e r a t i v e c o l i t i s  involved  was i m m u n o l o g i c a l l y  substances. that  patients  sera  Further,  i n that  unrelated  fluorescent  f r o m some p a t i e n t s  the antigen  t o t h e ABH b l o o d  antibody  labelling  group  showed  with u l c e r a t i v e c o l i t i s  reacted 40  with the cytoplasm of t h e i r  own c o l o n i c e p i t h e l i a l  On c h e m i c a l a n a l y s i s , t h e p h e n o l e x t r a c t colon  was f o u n d t o c o n t a i n  fucose, sialic tive  xylose,  galactose,  a c i d and an a c i d h y d r o l y z a b l e  colitis  glycoproteins  human  mannose,  o f 3,6-dideoxyhexose,  lipid.  i s an autoimmune d i s e a s e , might p l a y  of sterile  glucose,  h e x o s a m i n e and t r a c e s  cells  Thus, i f u l c e r a -  i t i s possible  that  a central role i n the antigenic  44 stimulus C h e m i c a l changes i n c o l o n i c e p i t h e l i a l have been a s s o c i a t e d  with u l c e r a t i v e c o l i t i s .  c h a n g e s may be n o n - s p e c i f i c ^ ' ^ ' ^ , colonic epithelial  glycoproteins  with ulcerative c o l i t i s ing in  Although  i t s diagnosis.  i n normal s u b j e c t s  contribute  only  From t h e p r e s e n t  crude g l y c o p r o t e i n  a s was p o i n t e d  to a better  with g a s t r o i n t e s t i n a l secretions degradation.  literature,  extracts  o u t above, t h e y  these  a d e t a i l e d knowledge o f  o f t h e e t i o l o g y and p a t h o g e n e s i s o f t h e d i s e a s e  that and  could  glycoproteins  have b e e n  are probably  and t h o s e understandand may a i d  i t appears investigated, contaminated  and t h e p r o d u c t o f b a c t e r i a l  15.  The  Involvement o f G l y c o p r o t e i n s  Crohn's d i s e a s e small  and l a r g e  large  intestine i s often  logically  i n Crohn's  i s a chronic  intestine.  Diffuse  Disease  inflammation  of the  Crohn's d i s e a s e  of the  misdiagnosed  clinically,  and h i s t o p a t h o l o g i c a l l y a s u l c e r a t i v e  In  a chemical  analysis  radio. . 5 colitis.  of the i n t e s t i n a l w a l l i n  47 a case o f Crohn's d i s e a s e  o f the ileum,  that  the glycosaminoglycan  than  i n the healthy  collagen, the On  and t h e h e a l t h y  the glycosaminoglycans  were  i s o l a t e d from the  i s o l a t e d from g r a n u l a t i o n  was n o t f u r t h e r To  have been  implicated  chemical  i n nature, but i t  investigations  to reveal  differences  changes  i n Crohn's d i s e a s e .  study has been c a r r i e d o u t t o c o n f i r m  Crohn's d i s e a s e ,  i n gly-  investigated.  t h i s degree t h e r e f o r e ,  pathological  found  tissue of rats.  f r a c t i o n was t h o u g h t t o be g l y c o p r o t e i n  extensive  collagen  of the i n t e s t i n e .  f r a c t i o n w h i c h was s i m i l a r t o t h a t  cosaminoglycans  proteins  of t o t a l  i n t e s t i n e a l w a y s showed t h e p r e s e n c e o f an e x t r a ,  slow-moving,  and  parts  reported  i n the diseased  n e u t r a l - s a l t - s o l u b l e and a c i d - s o l u b l e  electrophoresis,  This  c o n t e n t was h i g h e r  t i s s u e , whereas t h e c o n t e n t s  same i n t h e d i s e a s e  diseased  Seppala e t a l  this finding.  No  i n glycoother Despite  the c l i n i c a l , r a d i o l o g i c a l  between u l c e r a t i v e c o l i t i s and  i t i s sometimes d i f f i c u l t  t o make a d i f f e r e n t -  5 ial  diagnosis  .  I t i s hoped  that  a d e t a i l e d knowledge o f t h e  chemical d i f f e r e n c e s , should proteins  i n these disease  differential  The  Present  diagnosis  this  fractionation  diseases.  proteins  were o b t a i n e d  particularly  epithelial  sources.  those resected  Epithelial  f o rulcerative  glycohuman colitis  disease.  studies,  r a t colon  m o d e l f o r e x p e r i m e n t s w i t h human t i s s u e .  fully  glyco-  f r o m b o t h n o r m a l and d i s e a s e d  In p r e l i m i n a r y  procedures  the i s o l a t i o n  and c h a r a c t e r i z a t i o n o f human c o l o n i c  from p u r e l y  Crohn's  o f t h e s e two  might a i d i n the  t h e s i s , the author describes  proteins  and  entities,  glyco  Investigation  In  colons  they e x i s t i n the c o l o n i c  was u s e d  I t was f o u n d  suitable f o r r a t colonic tissues could  applied  as a  t o a s t u d y o f human c o l o n i c t i s s u e .  that  be s u c c e s s  MATERIALS AND METHODS  I.  P r e p a r a t i o n of samples f o r GLC and C o l o r i m e t r i c A n a l y s i s .  In g e n e r a l , were drawn from a stock accurately  II.  samples f o r the v a r i o u s  analyses  aqueous s o l u t i o n of the g l y c o p r o t e i n o f  known c o n c e n t r a t i o n  (usually  2 mg g l y c o p r o t e i n per m l ) .  Gas L i q u i d Chromatographic A n a l y s i s .  Gas l i q u i d chromatography  (GLC) was c a r r i e d  on a Hewlett Packard model No. 7 610A h i g h e f f i c i e n c y gas  chromatography  f i t t e d w i t h d u a l hydrogen flame  d e t e c t o r s and "on column i n j e c t i o n " . gas.  The f o l l o w i n g columns were  (a) S.E.  8'  x 1/4"  out  d u a l columan  ionization  Helium was used as  carrier  used:  o.d.copper  52 on 80 - 100 mesh d i a t o p o r t  S,  columns c o n t a i n i n g 10% operated  isothermally  at  190°C. (b) sorb  103,  6'  operated  x 1/4"  o.d.  copper columns c o n t a i n i n g Chromo-  i s o t h e r m a l l y at  145°C.  Peak areas were measured w i t h a Hewlett Packard model No. 3370B electronic integrator.  Analyses were c a r r i e d out by an i n t e r n a l  standard method u s i n g molar response laboratory  as d e s c r i b e d by Reid e t  al  f a c t o r s determined i n our  48  18.  A.  Neutral  Sugars  An  a c c u r a t e l y weighed  sample  (approximately  of each  f r e e z e - d r i e d m a t e r i a l was  hydrolyzed  tube at  1 0 0 ° C . f o r 36  according  to  50  hours  2  in a sealed t o the  mg) glass  procedure  49 of Lehnhardt analysed  as  and  Winzler  their  .  The  f r e e n e u t r a l sugars  trimethylsilyl  ethers  were  ( T M S - e t h e r s ) on  column  48 (a) by  t h e method o f  Reid  et a l  using  sorbitol  as  internal  standard. B.  0-acetyl  Groups  An lized xide The on  a c c u r a t e l y weighed  glycoproteins  (2 mg)  i n anhydrous methanol methyl acetate  column  III.  Normally, and  the these  one  calf  ml.)  at  4°C.  0.1  M  lyophi-  s o d i u m metho-  f o r 0.5  hours^.  t r a n s e s t e r i f i c a t i o n was the  internal  analysed  standard.  Analyses  various  components o f  the  glycoprotein  u n d e r a n a l y s i s were i n c l u d e d as c o n t r o l s .  were g a l a c t o s e ,  N-acetyl-glucosamine.  D-ribose,  (o.5  p r o d u c e d by  In most a s s a y s , than  treated with  (b) u s i n g b e n z e n e as  Colorimetric  other  was  sample o f e a c h o f t h e  thymus DNA  On and  fucose,  occassion, yeast  RNA  N-acetylneuraminic D-ribose, were a l s o  2-deoxyused.  acid  H e x o s e s were e s t i m a t e d  by  the p h e n o l - s u l p h u r i c  51 procedure (10:3  of Dubois  w/w)  as  the  using a mixture  standard.  o f g a l a c t o s e and  F u c o s e was  estimated  by  fucose  the  3 52 and S h e t t l e s ' . P r o t e i n was m e a s u r e d by 53 t h e method o f L o w r y w i t h H y l a n d c o n t r o l serum ( C a l i f o r n i a ,  method o f D i s c h e  U.S.A.) as  standard.  d u r e o f Chen by  54  and  P h o s p h o r u s was  DNA  by  Short.^. Hydrolysates  estimated  by  t h e method o f C e r i o t t i o f the  the  55  as  procemodified  glycoproteins prepared  49 and W i n z l e r were a n a l y z e d f o r 57 58 h e x o s a m i n e by m o d i f i c a t i o n of the procedure of W i n z l e r by  t h e method o f L e h n h a r d t  Total  and  bound s i a l i c  a c i d s were e s t i m a t e d  by  the  periodate  59 resorcinol a c i d s by IV.  procedures  t h e method o f  , and  free  sialic  Warren^".  Electrophoresis Studies  Cellulose on  of Jourdian e t a l  6"  x 1"  strips  using  Tris  barbital  Buffer,  a c e t a t e e l e c t r o p h o r e s i s was  of Sepraphore  G e l m a n ) , pH  III  (Gelman I n s t r u m e n t  sodium b a r b i t a l 8.8,  i = 0.1,  carried  buffer  out Co.)  (High R e s o l u t i o n  f o r 30 m i n u t e s a t 300  volts.  61 S t r i p s were s t a i n e d w i t h  alcian  blue  (pH  3.0).  Cellulose  a c e t a t e e l e c t r o p h o r e s i s o f r a t g l y c o p r o t e i n s a m p l e s was carried pyridine  out  i n 0.02  formate  M p y r i d i n e HCl  buffer,  pH  3.0.  buffer,  pH  5.5,  and  also i n 0.1  M  F i g u r e 1.  Flow diagram o f the method f o r the intact rat colonic e p i t h e l i a l  isolation  cells.  20b  WISTAR RAT (150-250 gm.)  1.  k i l l e d by e t h e r  2.  colon  anesthetia  resected  RAT COLON  Small p i e c e for histology  Main p o r t i o n  1.  everted  2.  i n f l a t e d with  3.  shaken w i t h  0.9% s a l i n e  0.01% Na EDTA 2  i n PBS  1 Cell Suspension  Colon Histology  Centrifuged 2000 rpm f o r 10 minutes  Isolated  Supernatant  Epithelia4 1. d i a l y z e d 2. 3.  (water) C e l l s  freeze-dried stored  Agarose g e l e l e c t r o p h o r e s i s commercial agarose g e l p l a t e s  was performed w i t h  ( A n a l y t i c a l Chemists I n c . )  1% agarose i n T r i s b a r b i t a l sodium b a r b i t a l b u f f e r , at  a constant v o l t a g e  of 90 v o l t s  f o r 30 m i n u t e s .  s t a i n e d w i t h a l c i a n b l u e , pH 3 . 0 .  containing  pH 8 . 8 , The p l a t e s were  Electrophoretic mobilities  were measured r e l a t i v e t o h e p a r i n . V . P r e l i m i n a r y Experiments on the I s o l a t i o n o f Rat C o l o n i c E p i t h e l i a l G l y c o p r o t e i n s (Figures A.  I s o l a t i o n of C o l o n i c E p i t h e l i a l Intestinal  1-3)  Cells  e p i t h e l i a l c e l l s were i s o l a t e d 62  procedure of C u l l i n g e t a l  (Figure 1 ) . W i s t a r r a t s  by the (150-250g)  were k i l l e d w i t h e t h e r and the colons were removed.  Epithelial  c e l l s were o b t a i n e d by shaking the e v e r t e d ,  colons  w i t h 0.01% EDTA i n PBS (phosphate-buffered Q2.g The at  KC1, 1.15g  s a l i n e ) ,. (8 g N a C l , '  N a 2 H P 0 4 and 0.2 g K H 2 P 0 4 i n 1 l i t r e of  c o l o n s were a l t e r n a t e l y  water).  shaken f o r 5 minutes and i n c u b a t e d  3 7 ° C f o r 5 minutes f o r a t o t a l p e r i o d of one h o u r , the shaking  medium b e i n g changed at 10 and 30 m i n u t e s . suspension was c e n t r i f u g e d at The  inflated  residue,  The pooled  cell  2000 rpm f o r 10 minutes at 4 ° C .  which c o n t a i n e d e p i t h e l i a l c e l l s ,  was washed twice  w i t h i c e - c o l d PBS and r e c o v e r e d by c e n t r i f u g a t i o n .  The  pooled supernatants were d i a l y s e d a g a i n s t d i s t i l l e d w a t e r , freeze  d r i e d and s t o r e d f o r f u t u r e Specimens  use.  (approximately 1/2  cm) were removed immediately  after  the c o l o n s had been e v e r t e d and at the c o n c l u s i o n o f  final  shaking p r o c e d u r e .  p a r a f f i n processed  These were f i x e d i n 10% f o r m o l c a l c i u m ,  and cut at 5 j i . S e c t i o n s were s t a i n e d w i t h  Hematoxylin and E o s i n (H & E) and w i t h p e r i o d i c 63 a l c i a n b l u e , pH 2.5 sections  the  .  acid/Schiff,  M i c r o s c o p i c examinations o f the  enabled the degree o f c e l l  stained  removal to be a s s e s s e d .  22a.  Figure  2.  Flow  d i a g r a m o f t h e method f o r t h e e x t r a c t i o n o f  glycoproteins  from r a t c o l o n i c e p i t h e l i a l  cells.  22b.  EPITHELIAL CELLS  1.  Suspended  i n 1 M NaCl  2.  Sonated  3.  C e n t r i f u g e d a t 2000rpm f o r 10 minutes  4.  Supernatant removed and r e s i d u e resuspended i n 1 M NaCl, steps  POOLED SUPERNATANT  2-4  repeated  RESIDUE  centrifuged at 20,000 g f o r 1 hour  1 RESIDUE  SUPERNATANT* c e n t r i f u g e d a t 105,000 g  (stored)  f o r 1 hour  RESIDUE (stored)  SUPERNATANT 1. d i a l y z e d a g a i n s t 1 M NaCl 2.  concentrated  CONCENTRATED SUPERNATANT  23a.  F i g u r e 3.  Flow diagram of the method f o r the f r a c t i o n a t i o n of Rat c o l o n i c e p i t h e l i a l  glycoproteins.  23b.  CONCENTRATED SUPERNATANT Gel chromatography on Biogel A15 M using 1 M NaCl solution as eluant AT.  FRACTION (Carbohydrate-rich)  A2 FRACTION (Protein-rich)  1. concentrate 2. dialyzed against 0.02 M pyridine-HCl buffer, pH 5.5 3. ion-exchange chromatography on DEAE c e l l u l o s e using 0.02 M pyridine-HCl buffer, tpH 5.5 as eluant and a NaCl gradient (0-1.5N A  FRACTION  B  FRACTION  LEAF 24 OMITTED IN PAGE NUMBERING.  B.  E x t r a c t i o n of C o l o n i c G l y c o p r o t e i n s  The procedure f o r the e x t r a c t i o n o f c o l o n i c g l y c o p r o t e i n s was a m o d i f i c a t i o n o f a method p r e v i o u s l y  des-  64 c r i b e d by Hakkinen saccharide  (figure  thelial cells periods  f o r the i s o l a t i o n of a c i d mucopoly2).  A suspension  i n i c e c o l d IM sodium c h l o r i d e was sonated  of 30 seconds w i t h a B i o s o n i k Sonator  Scientific)  a t s e t t i n g s of 1 and 80.  was then c e n t r i f u g e d  at  r e s i d u e was resuspended  (Bronswill  The c e l l  suspension  i n c o l d 1 M sodium c h l o r i d e T h i s procedure was  the q u a n t i t y of r e s i d u e  constant.  for  2000 rpm f o r 10 minutes and the  s o l u t i o n and again sonated. until  o f the c o l o n i c e p i -  repeated  o b t a i n e d by c e n t r i f u g a t i o n  The pooled supernatants were c e n t r i f u g e d  at  was  20,000  g f o r 1 hour and the r e s i d u e was washed w i t h 1 M sodium c h l o r i d e s o l u t i o n and r e c e n t r i f u g e d . o b t a i n e d were c e n t r i f u g e d  at  The combined s u p e r n a t a n t s  105,000 g f o r 1 hour and the  r e s i d u e was washed i n 1 M sodium c h l o r i d e and  recentrifuged.  The f i n a l pooled supernatants were d i a l y z e d a g a i n s t 1 M sodium c h l o r i d e s o l u t i o n f o r 24 hours at  4 ° C and c o n c e n t r a t e d to a  convenient volume by means of an Amicon u l t r a f i l t r a t i o n w i t h an XM 50 membrane, e x c l u s i o n l i m i t C.  apparatus  50^,000 M.W. .  F r a c t i o n a t i o n of Colonic Glycoproteins  The f r a c t i o n a t i o n of the c o n c e n t r a t e d supernatant  is  shown i n f i g u r e  3.  105,000 g  The c o n c e n t r a t e d  supernatant  was  a p p l i e d t o a c o l u m n o f B i o g e l A15M  RAD  Laboratories)  and  rich  fraction  0.02  M pyridine-HCl  eluted with  obtained  was  applied to  Whatman), and  was  5.5,  containing  various  I M NaCl.  concentrated  b u f f e r , pH  r e t e n t a t e was  (100-200 mesh)  5.5  at  and  4°C  eluted with  0.02  dialyzed  f o r 24  a convex NaCl g r a d i e n t  ( 0 - 1.5  f r a c t i o n s obtained  against  hours.  M pyridine-HCl  concentrated, d i a l y z e d against d i s t i l l e d by  carbohydrate-  a column o f D E A E - c e l l u l o s e  carbohydrate-rich  recovered  The  (BIO-  The  (DE22,  buffer, M).  were  pH  The  individually  water overnight  and  lyophilization.  In p r e l i m i n a r y e x p e r i m e n t s , the f r a c t i o n a t i o n p r o c e d u r e was  monitored  method o f D u b o i s e t a l and  for s i a l i c  a c i d by  f o r h e x o s e s by  51  the  , f o r p r o t e i n by  the  phenol-sulphuric  the  Lowry  procedure  d i p h e n y l a m i n e method o f W e r n e r  53  and  65  Odin  VI  A.  .  In  later  Isolation  experiments, the  sialic  a c i d a s s a y was  o f Human C o l o n i c E p i t h e l i a l  Glycoprotein :  Source o f Specimens A l l s p e c i m e n s were o b t a i n e d  department of the ported  t o the  Vancouver General  laboratory  in plastic  from the  Hospital. bags packed  omitted.  pathology T h e y were in ice.  transThe  l e n g t h o f time  after  s u r g e r y and b e t w e e n p r o c e s s i n g was  1 to 3 hours.  Cells  were o b t a i n e d f r o m t h e  specimens: obtained  from p a t i e n t s  29  t o 85,(  The  cells  sample. which  (a) t h e h i s t o l o g i c a l l y  average  53)  and  (b) The  entire  had b e e n s e l e c t e d  old  f e m a l e ) , one  and  two  and  38.year  and  cases o f Crohn's  B.  Isolation  on  colitis  (two  cells  the o u t s i d e .  each c o l o n  tube  specimens  (39 y e a r bowel  ileum  from the  (36 two  Cells  received  tubular  A cylindrical  the  ileum.  from the p a t h o l o g y  G e n e r a l H o s p i t a l had  original  and was  speci-  were p o o l e d as w e r e t h o s e f r o m  b e e n removed f r o m t h e s e r o s a l their  one  60 y e a r o l d  d i s e a s e of the large  The  as  cadaver  (c) S u r g i c a l  along the length of the c o l o n .  were s u t u r e d i n t o  treated  a 19 y e a r o l d m a l e  d i s e a s e of the t e r m i n a l  surgical  specimens  f o r carcinoma.  disease of the terminal  colitis  colon  r a n g i n g from  case of ischemic c o l i t i s  a t the Vancouver  longitudinally  surface  colectomy  o f Human C o l o n i c E p i t h e l i a l  The  o f 20  o f ages  as k i d n e y d o n o r .  o l d female p a t i e n t s ) .  two  had  from  case o f Crohn's  cases of u l c e r a t i v e  tissues  colon  one  cases o f Crohn's  and  parts  were p o o l e d and  cases of u l c e r a t i v e  female p a t i e n t s ) ,  department  sexes  undergoing  from t h e s e specimens  mens f r o m two  into  of both  normal  following  from  a l l been c u t  After  surface,  the  the c o l o n s  form w i t h the  b a l l o o n , was  then i n f l a t e d  fatty  mucosal  inserted  with isotonic  saline.  Figure of  4.  Flow diagram o f the procedure  colonic epithelial  cholera  glycoproteins  neuraminidase.  Analyses  f o r the d i g e s t i o n  o f r a t and human u s i n g  fortotal  (PRT) s i a l i c  studies vibrio  a c i d s by  59 the on  periodate samples  r e s o r c i n o l method o f J o u r d i a n  of the retentate  and t h e f i l t r a t e  untreated  o r (2) t r e a t e d w i t h  erature.  Samples  the  retentate  for  glycosidically  (both  0.1 N KOH  t h e KOH t r e a t e d  and t h e f i l t r a t e  were  bound s i a l i c  et a l  performed  w h i c h were e i t h e r (1)  f o r 1 h o u r a t room  temp-  and non-KOH t r e a t e d )  further analyzed  acids  were  of  respectively  (PRB) b y t h e p e r i o d a t e  59 resorcinol (TB)  method o f J o u r d i a n  et a l  and f o r f r e e s i a l i c 60 b y t h e t h i o b a r b i t u r i c a c i d method o f W a r r e n  acids  283:  SAMPLE  incubated with v i b r i o a t 37 C f o r 65 h o u r s Suction  filtered  cholera  through a d i a l y s i s membrane  RETENTATE  FILTRATE  divided into 2 portions and one was t r e a t e d w i t h _j KOH  divided into 2 portions and one was t r e a t e d w i t h KOH  1  KOH treated  non-KOH treated  KOH treated  non-KOH treated  1.  TB  1.  TB  1.  PRT  1.  PRT  2.  PRT  2.  PRT  2.  PRB  2.  PRB  Colonic by  epitehlial  shaking with  tion rat  0.02% EDTA i n PBS.  p r o c e d u r e s were colons  cell  C.  c e l l s were removed f r o m e a c h  identical  (see f i g u r e  r e m o v a l , was  Extraction  2).  The s h a k i n g  specimen and  incuba-  t o t h o s e employed w i t h t h e  H i s t o l o g i c examinations,  performed  as d e s c r i b e d  and F r a c t i o n a t i o n  on page 21  o f Human C o l o n i c  to monitor •  Epithelial  Glycoproteins  The described  VII.  with  p r o c e d u r e s e m p l o y e d were s i m i l a r t o t h o s e r a t colons  Saponification  ( s e e p a g e s 22 t o 2 4 ) .  Studies  A known w e i g h t o f e a c h (o.2  mg/mg) was  2 hours  treated  with  a t room t e m p e r a t u r e .  freeze  dried  glycoprotein  0.1 N. p o t a s s i u m h y d r o x i d e f o r Aliquots  (0.2 t o 0.5 ml) were  w i t h d r a w n a t 0, 0.5,  1 and 2 h o u r s and a f t e r n e u t r a l i z a t i o n  with  quantity  the appropriate  analyzed  for sialic  o f IN s u l p h u r i c  a c i d by t h e p e r i o d a t e  acid  were  r e s o r c i n o l method  59 of Jourdian  VIII.  et a l  Digestion  Studies  T h e s e were c a r r i e d o u t as shown accurately  weighed  glycoprotein  was  sample  dissolved  (5 t o 12 mg)  i n figure  o f each  4.  freeze  i n 0.05 M s o d i u m a c e t a t e  An  dried  buffer,  3(  pH  5.5  (Containing 2 X  chloride) units  / ml  further and  incubated  incubate  100  exclusion  )  % sodium a z i d e  with  Vibrio  was  continued  f o r 24  incubate  through  of approximately  d i a l y s a t e was  %  calcium  was  then  a dialysis  100,000 MW..  water. Analyses  (100  hours a t 37°C.  o b t a i n e d were f r e e z e d r i e d  known v o l u m e of- d i s t i l l e d  1 X 10  The  sac w i t h The  and  A  added  f o r a f u r t h e r 41 h o u r s .  vacuum f i l t e r e d  limit  and  c h o l e r a neuraminidase  (Behringwerke)  u n i t s o f enzyme p e r ml  i n c u b a t i o n was  incubate  and  and  10  an  retentate  redissolved in a  for total  (PRT)  sialic 59  a c i d s by  the  p e r f o r m e d on were e i t h e r  p e r i o d a t e r e s o r c i n o l method o f J o u r d i a n samples o f the (1) u n t r e a t e d  r e t e n t a t e and  or  (2)  1 h o u r a t room t e m p e r a t u r e . non-KOH t r e a t e d )  of the  analyzed  respectively  (PRB)  the  and  by  for free  sialic  treated with  Samples  r e t e n t a t e and  filtrate 0.1N  ( b o t h t h e KOH the  for glycosidically  periodate  the  filtrate bound  KOH  (TB)  by  the  for  Immunodiffusion  sialic  thiobarbituric  acids 59 et a l acid  Studies.  T h e s e were c a r r i e d Ouchterlony  plates  (Hyland,  out  on  commercially  California,  U.S.A.).  and  were f u r t h e r  method o f Warren**^. IX.  were  which  treated  r e s o r c i n o l method o f J o u r d i a n  acids  et a l  available  F i g u r e 5.  Photomicrographs of s e c t i o n s  specimen r e s e c t e d CA) b e f o r e  f o r carcinoma of the c o l o n .  and (B) a f t e r  phosphate b u f f e r e d  of human colon o b t a i n e d from  treatment w i t h 0,02%  saline.  The t i s s u e was taken (0.54 mM) EDTA i n  In t h i s c a s e , complete removal of  e p i t h e l i a l mucus s e c r e t i n g c e l l s  i n (Bl)  and (B2) was seen and there  was no apparent damage t o the basement membrane.  Sections  (Al) and  (Bl) were s t a i n e d w i t h a l c i a n b l u e - PAS technique and s e c t i o n s and (B2) were s t a i n e d by H & E .  (A2)  31b.  32.  X.  Preparation  of Antisera  A modification followed  i n the preparation  epithelial  glycoprotein.  ( f r a c t i o n A) was of  10 mg  m i x t u r e was  f e m a l e New of  then  rabbit. was  T h r e e weeks  Laboratories).  30 m i n u t e s .  concentration  later,  of rat glycoprotein  and P e r t u s i s V a c c i n e Two weeks  a booster  dose  After a furi n j e c t i o n of  i n 1.4 m l o f a  (Connaught M e d i c a l  a f t e r the l a s t  glass.  f o o t pads o f a 3 l b .  a subcutaneous  was drawn f r o m t h e m a r g i n a l v e i n  obtained  glycoprotein  i n j e c t e d subcutaneously. given  was  an e q u a l v o l u m e o f c o m p l e t e  i n j e c t e d i n t o the four  o f 6.6 mg  m i x t u r e o f PBS  dried  i n PBS, pH 7.3, a t a  2 weeks, t h e r a b b i t was  blood  of freeze  66  rat colonic  (DIFCO Lab.) b y s q u i r t i n g i n t o a s h o t  Zealand  a suspension  was  7.7 mg  suspended  t h e same s t r e n g t h  ther  of antisera against  p e r ml and was m i x e d w i t h  Freunds adjuvant The  o f t h e method o f Kopp e t a l  injection,  Research  5 ml o f  o f the r a b b i t e a r .  by c e n t r i f u g i n g t h e c l o t t e d b l o o d  1:1  Serum  a t 3000 rpm f o r  RESULTS  AND  DISCUSSION  1. I s o l a t i o n  of E p i t h e l i a l  Complete c e l l experiments with  (0.27 mM)  were removed d u r i n g epithelial cells cell  cell  was washed  (figure  o f the c e l l s  crypts  proved  Extraction  The sonated  procedure  t h e u s e o f 0.02%  twice with  ml o f c e l l s was  complete 5), while  EDTA  i n the o t h e r s ,  of Colonic E p i t h e l i a l  isolated  cells  were  packed  per r a t  was cells.  removal o f c e l l s  t o be t h e most d i f f i c u l t  and t h e  a p p l i e d to specimens  (0.54 mM)  were r e m o v e d . The c e l l s  The  PBS  The a v e r a g e  removal o f the e p i t h e l i a l  o f the specimens,  accomplished  2.  30 m i n u t e s o f s h a k i n g .  1.16 + 0.24  isolation  to achieve  shaken  i n PBS. The m a j o r i t y o f t h e c e l l s  the f i r s t  volume o b t a i n e d was  necessary  %  EDTA  in a l l rat  r a t c o l o n s were  were r e c o v e r e d by c e n t r i f u g a t i o n .  human c o l o n ,  40%  accomplished  and e v e r t e d  suspension  c o l o n . When t h i s of  r e m o v a l was  when i n f l a t e d  0.01%  Cells  In  was  from  10 t o 90  a t the base o f the t o remove.  Glycoproteins  suspended  i n an i c e - w a t e r b a t h . N o r m a l l y ,  i n 1 M NaCl,  complete  and  rupture of a l l  the c o l o n i c  epithelial  cells  required  5 t o 6 periods o f sonation  of  30 s e c o n d s  by  the occurrence o f a constant residue after  d u r a t i o n each.  2000 rpm f o r 10 m i n u t e s . residue  appeared  membrane  (exclusion  t h e g l y c o p r o t e i n s was u s u a l l y  The  efficiency  Pestle  cells  OMNI-MIXER,  centrifugation at  microscope,  membrane  this  fragments.  limit  50,000 M.W.).  g r e a t e r than  used  The  obtained  o f i t s v o l u m e b y Amicon  homogenization  were h o m o g e n i z e d w i t h  Recovery  90% ( s e e page 37 ) .  o f the sonation procedure  p a r e d w i t h two o t h e r c u r r e n t l y volumes o f packed  t o be c o m p l e t e  a t 105,000 g a n d t h e s u p e r n a t a n t  concentrated, t o one-tenth  ultrafiltration of  Under t h e l i g h t  t o be composed o f c e l l  s o n a t e was c e n t r i f u g e d was u s u a l l y  S o n a t i o n was j u d g e d  was comm e t h o d s . Known  (a) a  motor-driven  (b) a p r e s s u r e h o m o g e n i z e r a n d (c) s o n a t i o n .  Each  homogenate o b t a i n e d was c e n t r i f u g e d  rpm,  20,000 g a n d 105,000 g .  3 r e s i d u e s from each  procedure  successively  T h e 105,000  supernatant  a t 2000 and t h e  were a n a l y z e d f o r p r o t e i n b y t h e  53 Lowry p r o c e d u r e  a n d f o r h e x o s e by t h e p h e n o l - s u l p h u r i c  51 method o f D u b o i s  etal  .  The r e s u l t s  s o n a t i o n g a v e a more c o m p l e t e r i c h m a t e r i a l s from in  a much s h o r t e r  tion  the c e l l s ,  time.  obtained indicated  extraction and t h a t  Furthermore,  o f the carbohydrate this  quantity  from  the sonation procedure  o f DNA.  c o u l d be a c c o m p l i s h e d  spectrophotometric estima-  o f t h e DNA i n t h e 105,000 g s u p e r n a t a n t s  supernatant  that  indicated  that the  contained the l e a s t  TABLE 1.  Percentage  concentration  o f the  r e c o v e r y o f hexose and s i a l i c 105,000  acid  after  g supernatant o f i n t e s t i n a l e p i t h e l i a l  g l y c o p r o t e i n by the v a r i o u s procedures  Method  Hexose r e c o v e r y (%)  Sephadex G-25  Amicon  5  67.33  83.8  4  85.81  33.0  1  fibre  (BIO-RAD)  Note:  67.3  ultra-  filtration  Hollow  S i a l i c acid r e c o v e r y (%)  0  1  (a) T h e number i n t h e b r a c k e t r e p r e s e n t s t h e number o f e x p e r i -  ments done t o a r r i v e (b) The d e g r e e  a t t h e above  averages.  o f c o n c e n t r a t i o n i n each  c a s e was a b o u t  8 t o 10 t i m e s .  51 (c)  Hexose was d e t e r m i n e d  and  sialic  and  Odin  by t h e p h e n o l - s u l p h u r i c p r o c e d u r e  a c i d was e s t i m a t e d b y t h e d i p h e n y l a m i n e  o f Dubois  method o f Werner  65 (3)  was  The e x c l u s i o n  10,000  (PM10).  limit  o f t h e membrane u s e d  i n Amicon  ultra-filtration  Figure  6.  Monitoring of the fractionation  g l y c o p r o t e i n s by c e l l u l o s e s t r i p s were s t a i n e d w i t h II,  fraction  B;  acetate electrophoresis  alcian  105,000g s u p e r n a t a n t ; VI, fractions  of r a t colonic  blue  a t pH 3.  I I I ,fraction  Al.;  A & B combined.  epithelial  a t pH 8.8.  I, herapin IV, f r a c t i o n  A l l  standard; A;  V,  0)  37.  During was  necessary  preparation  to concentrate  occassions. tration  the  I t was  For  this  concentration (coarse),  s o l u t i o n s on  caused  reason,  the  three  dialysis.  The  results  i n Table  1,  smallest  l o s s o f g l y c o p r o t e i n as  was  by  a c i d and  total  rinsing  85%  t i o n ) , has  (a)  and  hexose. of  the  By  of t h i s  been c l a i m e d  concentrating  biological  t o be  glyco-  the  A  recovered,  the  and  led to  the  recovery  f u r t h e r 5 to i n some  pores  t o be  shown  average  most s e l e c t i v e  materials  G-25  recovery  solution.  size-limiting  of  fibre  study,  t h i s method, the  1 M NaCl  membranes w i t h  concen-  Sephadex  starting material.  t h e membrane w i t h  a  (c) h o l l o w  j u d g e d by  g l y c o p r o t e i n c o u l d be  which u t i l i z e s  three  losses of  i n d i c a t e d t h a t Amicon u l t r a f i l t r a t i o n  more t h a n  of the  least  c u r r e n t l y u s e d methods  (b) Amicon u l t r a f i l t r a t i o n  sialic  at  smallest  were i n v e s t i g a t e d : n a m e l y ,  concentration  of  glycoproteins, i t  d e s i r a b l e t h e r e f o r e , t o choose  procedure which  protein.  of the  the  This  10%  cases, procedure  (ultrafiltramethod  for  least injurious  67 to protein solutes in  a l l of the  used 3.  A.  in this  .  Thus, Amicon u l t r a f i l t r a t i o n  concentration  and  most o f  Preliminary  Model E x p e r i m e n t s w i t h  Electrophoretic at  dialysis  employed  processes  work.  F r a c t i o n a t i o n of Colonic E p i t h e l i a l  natant  the  was  pH  8.8  examination  i n d i c a t e d the  Glycoproteins  Rat  of  Glycoproteins  the  presence of  105,000 g two  super-  components  Figure  7.  Agarose A15M  g concentrated  column chromatography of the 105,000  supernatant  e x t r a c t e d from the  isolated  c o l o n i c e p i t h e l i a l c e l l s of a human sample r e s e c t e d f o r c a r cinoma of the c o l o n . were e l u t e d with  Column s i z e 83X  1 M NaCl.  Hexose  (0)  2.5  cm..  and  were determined by the procedures of Dubois respectively.  A carbohydrate-rich  r i c h f r a c t i o n A2 were  obtained.  Fractions  protein 51  and  f r a c t i o n A l and  (•)  Lowry  53  a protein-  38b  which s t a i n e d w i t h a l c i a n b l u e at pH 3 . 0 .  The m o b i l i t y of  the f a s t e r moving band was s i m i l a r to t h a t of the h e p a r i n standard and was approximately twice as slower moving band (see  figure  fast  as t h a t of  the  6).  F r a c t i o n a t i o n o f t h i s supernatant by g e l chromatography on B i o g e l A15M u s i n g 1 M NaCl as  eluant  y i e l d e d a c a r b o h y d r a t e - r i c h f r a c t i o n A l and a c a r b o h y d r a t e poor but p r o t e i n - r i c h f r a c t i o n A2. revealed that  An immunodiffusion study  f r a c t i o n A2 r e a c t e d w i t h a n t i r a t plasma p r o t e i n  prepared i n r a b b i t s  while fraction A l didnot.  f r a c t i o n A2 c o n t a i n e d plasma p r o t e i n s weight s u b s t a n c e s .  Presumably,  and o t h e r low m o l e c u l a r  F r a c t i o n A l was c o n c e n t r a t e d , d i a l y z e d  a g a i n s t 0.02 M p y r i d i n e H C l b u f f e r ,  pH 5 . 5 ,  and was then  f r a c t i o n a t e d by i o n exchange chromatography on DEAE c e l l u l o s e u s i n g 0.02 M p y r i d i n e H C l b u f f e r , s a l t gradient  pH 5 . 5 ,  (0 t o 1.5 M NaCl) as e l u a n t .  c o n t a i n i n g a convex Two c a r b o h y d r a t e -  r i c h peaks were o b t a i n e d which were d e s i g n a t e d and B a c c o r d i n g t o t h e i r o r d e r o f e l u t i o n . electrophoresis  showed t h a t  fractions A  C e l l u l o s e acetate  f r a c t i o n B corresponded t o  the  f a s t moving band i n the 105,000 g supernatant w h i l e f r a c t i o n A r e p r e s e n t e d the slower moving band  (figure 6 ) .  This  fractiona-  t i o n procedure was repeated on two new samples of the 105,000 g supernatant  of r a t  colonic e p i t h e l i a l c e l l s ,  r e s u l t s were o b t a i n e d .  and s i m i l a r  40a.  Figure of  8,  DEAE c e l l u l o s e  the carbohydrate-rich  A15M column  of the colon.  o f human  f r o m a sample  Column s i z e  83  chromatography from Agarose  colonic cell resected  sap  (105,000g  f o r carcinoma  X 2.5 cm.. F r a c t i o n s were  0.02 M p y r i d i n e HC1 b u f f e r , pH 5.5, c o n t a i n i n g  a convex NaCl g r a d i e n t obtained  f r a c t i o n A l obtained  chromatography  supernatant) obtained  eluted with  ion^-exchange c o l u m n  (0-1.5 M).  ( f r a c t i o n s A and B ) .  procedure o f D u b o i s ^  1  Two d i s t i n c t  Hexose  peaks  was e s t i m a t e d  and p r o t e i n m e a s u r e d  were  by t h e  by t h e method o f  53 Lowry  .  gradient.  (0) , h e x o s e ;  (•) , p r o t e i n and (••.•• ) t h e s a l t  40b.  4:  TABLE 2.  Agarose g e l e l e c t r o p h o r e t i c m o b i l i t i e s of the sample  o b t a i n e d from the v a r i o u s  stages i n the f r a c t i o n a t i o n of c o l o n i c  e p i t h e l i a l glycoproteins extracted  from normal and d i s e a s e d  human c o l o n s .  SAMPLES Normal (Cancer)  Heparin  Normal  Ischemic Crohn 1 s Crohn 1 s (colon) (ileum) Colitis  Ulce ativi Coli  1.0  1.0  1.0  1.0  1.0  1.0  0.29  0.34  0.35  0.22  0.20  0.34  0.79  0.82  0.79  0.89  0.83  0.87  Fraction A l (carbohydrate-rich)  0.29  0.35  0.36  0.19  0.25  0.37  0.74  0.78  0.87  0.91  0.88  0.85  F r a c t i o n A2  1.0  1.011  0.982  1.05  1.033  1.01  F i r s t DEAE f r a c t i o n  0.35  0.35  0.35  0.20  0.24  0.37  0.82  0.84  0.87  0.87  0.85  0 to 0.9  0 to 0.91  0 to 0.58  105,000g  Supernatant  ( f r a c t i o n A) Second DEAE f r a c t i o n ( f r a c t i o n B)  0.84  4  C a l f thymus DNA c o n t r o l (streak)  0 to 0.88  Note: (1)  t r a c e s of a l c i a n blue s t a i n i n g  (2)  and  (3) ,very f a i n t  m o b i l i t i e s of 0.45, (4)very of  faint  0.34 was  0.41  and 0.3  0 to 0.89  with  r e s p e c t i v e l y were found.  t r a c e of a l c i a n blue p o s i t i v e m a t e r i a l w i t h m o b i l i t y observed.  Figure the  9.  I l l u s t r a t i o n of a t y p i c a l agarose g e l e l e c t r o p h o r e s i s p a t t e r n  various fractions  human sample. isolated colon.  i n the  fractionation  In t h i s case, the  colonic  used as the  w i t h commercial agarose p l a t e  The  7,  105,000g supernatant was  pH  8.8,  standard.  fractions  e x t r a c t e d from  B combined.  a the  the  performed  agarose i n T r i s b a r b i t a l  at a constant v o l t a g e of 90 v o l t s f o r 30  f r a c t i o n A l , 4,  A and  E l e c t r o p h o r e s i s was  c o n t a i n i n g 1%  p l a t e s were s t a i n e d w i t h A l c i a n  supernatant; 3,  105,000g supernatant of  e p i t h e l i a l c e l l s of a sample r e s e c t e d f o r carcinoma of  Heparin was  barbital buffer,  of the  of  b l u e , pH  3.  1, h e p a r i n ; 2,  f r a c t i o n A2;  5,  f r a c t i o n A;  6,  sodium  minutes.  105,000g fraction  B;  VE  direction of migration  starting  t  n  PI  2  3  II  point  1  4  5  6  7  8  B.  Experiments on Human C o l o n i c E p i t h e l i a l  S i m i l a r procedures  Cells  to those developed on the  rat  model were a p p l i e d t o the e x t r a c t i o n of g l y c o p r o t e i n s from human c o l o n i c e p i t h e l i a l c e l l s . g supernatant  F r a c t i o n a t i o n of the 105,000  o f the 1 M NaCl e x t r a c t  of these c e l l s  on B i o g e l  A15M u s i n g 1 M NaCl as e l u a n t y i e l d e d a c a r b o h y d r a t e - r i c h f r a c t i o n A l and a p r o t e i n - r i c h f r a c t i o n A2. ( f i g u r e 7 )  Fraction  A l was d i a l y z e d a g a i n s t 0.02 M p y r i d i n e HC1, pH 5 . 5 ,  and was  f r a c t i o n a t e d by i o n exchange chromatography on DEAE  cellulose  u s i n g 0.02 M p y r i d i n e H C l , pH 5 . 5 ,  c o n t a i n i n g a convex NaCl  gradient  Two a p p a r e n t l y homogenous  fractions  ( 0 - 1.5  M ) as e l u a n t .  (fractions  A and B) were o b t a i n e d from each normal  and d i s e a s e d sample  ( f i g u r e 8 ) except i n one case o f C r o h n ' s  d i s e a s e of the t e r m i n a l iteum where m u l t i p l e peaks were observed i n F r a c t i o n B.  The r e s u l t s in table  2.  of agarose g e l e l e c t r o p h o r e s i s  A l l the 105,000 g supernatants  show two a l c i a n b l u e s t a i n i n g bands  (figure  are shown  were found t o 9 ).  One of  these  had a m o b i l i t y s i m i l a r to t h a t o f h e p a r i n w h i l e the o t h e r migrated at a much slower r a t e .  F r a c t i o n A l , o b t a i n e d by B i o g e l  A15M f r a c t i o n a t i o n , was found t o c o n t a i n both a l c i a n b l u e p o s i t i v e bands.  DEAE c e l l u l o s e  the two bands i n t o f r a c t i o n s to have a m o b i l i t y  chromatography of A l s e p a r a t e d  A and B.  F r a c t i o n B was found  s i m i l a r to h e p a r i n and approximately  twice t h a t of F r a c t i o n A .  A l l fractions  B showed s i m i l a r  4  TABLE 3.  Chemical Analyses o f f r a c t i o n s B. ( A l l v a l u e s are expressed i n ug/mg o f f r e e z e  dried  sample).  Protein  Phosphorus  DNA  Hexose  GLC Hexose fucose  Sialic acid  84  74.0  1080  245  0  16  NORMAL (Cancer)  122.5  67.4  1030  235  0  15  NORMAL  123.5  67.9  1090  250  0  17  ULCERATIVE COLITIS  11.9  76.8  1090  250  0  19  CROHN S (Colon)  16.6  77.9  1180  251  CROHN S (Ileum)  22.4  66.8  920  19  131.8  47.3  690  35  RAT  1  16  1  ISCHEMIC COLITIS  CALF THYMUS DNA (CONTROL)  18.3  66.8  1000  210  -  11  RNA (CONTROL)  27.4  76.8  0  340  -  2  Chemical a n a l y s e s were performed by the f o l l o w i n g procedures:hexose 51 53 by the method o f Dubois , p r o t e i n by the procedure o f Lowry , 54 phosphorus by method o f Chen , DNA by the i n d o l e procedure o f C e r i o t t i ^ as m o d i f i e d by S h o r t ^ and s i a l i c a c i d by the p e r i o d a t e 59 r e s o r c i n o l procedure o f J o u r d i a n  .  Other than the r a t and the  two n u c l e i c a c i d c o n t r o l s , a l l the samples colons.  were o b t a i n e d from human  4  TABLE 4.  Molar r a t i o of sugars i n c o l o n i c e p i t h e l i a l g l y c o proteins  ( f r a c t i o n A)  Molar R a t i o s o f Sugar Fucose  Galactose  Hexosamine  Sialic acid  RAT  1  3.35  2.2  1.45  NORMAL (Cancer)  1  3 .15  3.7  2.7  NORMAL  1  2.9  2.6  1.7  ULCERATIVE COLITIS  1  2.1  2.1  1.5  CROHN'S  (colon)  1  1.35  1.2  0.32  CROHN'S  (ileum)  1  5.3  2.1  3.73  1  3.4  3.35  3.3  ISCHEMIC COLITIS  N o t e : G l y c o p r o t e i n samples were t r e a t e d w i t h Dowex 50H at  i n 0.01 HCl  1 0 0 ° C f o r 36 t o 50 hours and the H y d r o l y s a t e s were analysed  for  fucose and g a l a c t o s e by GLC and f o r hexosamine by a m o d i f i c a t i o n ^of  a procedure o f W i n z l e r ^ .  the t o t a l s i a l i c of  the f u l l y  S i a l i c a c i d values  represented  a c i d o b t a i n e d from a p e r i o d a t e - r e s o r c i n o l  s a p o n i f i e d g l y c o p r o t e i n samples.  assay  TABLE 5.  Chemical a n a l y s e s of the c o l o n i c e p i t h e l i a l g l y c o proteins  (fraction A).  ug per mg of f r e e z e  Protein  RAT  A l l values  are expressed i n  d r i e d sample.  Hexose  Phosphorus  Fucose  84.3  230  0.2  60.1  58.5  170  0  41.3  NORMAL  65.0  195  0.2  57.6  ULCERATIVE COLITIS  82.8  140  0.2  41.2  CROHN'S  (Colon)  67.9  105  0.2  46.2  CROHN'S  (Ileum)  116.0  -  0.1  NORMAL  (Cancer)  ISCHEMIC COLITIS  53.7  —  0.1  Chemical a n a l y s e s were performed by the f o l l o w i n g p r o c e d u r e s : 53 p r o t e i n by the procedure of Lowry , hexose by the p h e n o l 51 s u l p h u r i c procedure of Dubois Chen ^ 4 and fucose (-)  indicates  , phosphorus by the method o f  by the procedure of Dische and  t h a t the a n a l y s i s  was not performed.  Shettles4'"*  -  -  F i g u r e 10. GLC o f the t r i m e t h y l s i l y l e t h e r s o f the n e u t r a l sugars o f c o l o n i c e p i t h e l i a l g l y c o p r o t e i n . The sample i l l u s t r a t e d i n t h i s t y p i c a l chromatogram was  o b t a i n e d from the h i s t o l o g i c a l l y normal p a r t o f s u r g i c a l  specimens  carcinoma  o b t a i n e d from p a t i e n t s undergoing colectomy. Only fucose and  g a l a c t o s e were d e t e c t e d and each o f the two sugars had r e s o l v e d i n t o three peaks  (1) ,  (2) and  (3) .  electrophoretic mobilities. the two samples o b t a i n e d disease  4.  In the case of f r a c t i o n s  from specimens  resected for Crohn's  moved much slower than f r a c t i o n s  from o t h e r  isolated  contaminated by b l o o d .  from some human samples were  In view of t h i s ,  f r a c t i o n a t i o n procedures  described  samples of whole human b l o o d .  none o f . t h e  glycoproteins  the s o n a t i o n  Blood components had a s i m i l a r (see  above).  examined below appeared  from c o n t a m i n a t i o n w i t h b l o o d  and  above were c a r r i e d out on  e l u t e d volume to the i n c l u d e d second peak  to  Thus,  arise  proteins.  Analyses of F r a c t i o n s B  The r e s u l t s o b t a i n e d from an a n a l y s i s of B are  shown i n t a b l e 3,  i n which i s  purposes s i m i l a r a n a l y s e s o f c a l f All  sources.  Blood Contamination  The c e l l s  5.  A,  these f r a c t i o n s  absorbed  g r a t e on e l e c t r o p h o r e s i s fucose,  included for  g a l a c t o s e or hexosamine.  comparative  thymus DNA and y e a s t RNA.  strongly  at pH 3.0  fractions  at  260nui, f a i l e d  to m i -  and c o n t a i n e d no d e t e c t a b l e Based upon t h e i r h i g h 56  phosphorus  content,  the r e s u l t s o f the Indole assay  s i m i l a r i t y of the a n a l y t i c a l fractions  B are p r o b a b l y DNA.  r e s u l t s to c a l f  and the  thymus DNA,  48b.  The  small  fractions B using due  to  the  interference  The  r e s u l t of  unlikely  that  RNA  low  DNA  was  p r o b a b l y due  was  to  present by  the  the  i n any  was  sample w i t h  presence of  found  low  indicates these  the  2%  v a l u e was  standard,  calf  insufficient  d a t a i n t h i s s t u d y t o draw any  significance  of  these d i f f e r e n c e s  6.  of  Fractions  The fractions tained Gas  are  fucose,  exhaustive  associated  table  2).  analysis.  12% for  colitis,  s i m i l a r to the  dialysis.  the  was that protein  There  conclusion  i n Lowry p r o t e i n  is  on  the  values.  A  r e s u l t s obtained shown i n t a b l e s  from a n a l y s e s o f  4 and  5.  A l l the  hexose, hexosamine, p r o t e i n  chromatographic  (see  thymus DNA,  b e e n removed by  The  alcian  Lowry p r o t e i n  very  i t is  It is  faint  for ulcerative  1 to  DNA  colitis  electrophoresis)  t h i s sample  of  fractions.  i n a l l human s a m p l e s e x c e p t  protein  probably  that  Ischaemic  o f w h i c h had  Analyses  is  quantities  Lowry p r o t e i n  where o n l y  reference  in  a contaminant.  from c o l o n s r e s e c t e d  This  i n the  of  found  assay  large  (indole)  (in agarose g e l  Crohn's d i s e a s e ,  found  test  i n t e r e s t i s the  obtained  detected.  acid  t h i s contamination produced  Lowry p r o t e i n  and  DNA  s e c o n d DEAE f r a c t i o n o f  Of  three  sialic  periodate-resorcinal  the  b l u e p o s i t i v e band w i t h the  of  i n t h i s a s s a y by  values given  probable that  quantity  analysis  of  the  and  hydrolyzates  these fractions  sialic  con-  acid.  indicated  that  F i g u r e 1 1.  I l l u s t r a t i o n o f a t y p i c a l s a p o n i f i c a t i o n time curve i n which  a sample of human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n , o b t a i n e d from a s u r g i c a l specimen r e s e c t e d  f o r carcinoma of the c o l o n , was t r e a t e d with 0.5% KOH.  The amount of t o t a l s i a l i c by the p e r i o d a t e  a c i d present was estimated 59  r e s o r c i n o l method of J o u r d i a n  appeared t o be completed at  0.5  hours.  .  at 0 . 5 ,  1 and 2 hour  Saponifications  300  galactose ratio  of  was the  significant  7.  the  only  sugars  hexose p r e s e n t  i n e a c h sample  q u a n t i t i e s of  Saponification  The  molar  4.  In-  detected.  Studies  same c o l o n i c e p i t h e l i a l a c i d by  10).  i s shown i n t a b l e  p h o s p h o r u s were  When u n t r e a t e d  sialic  (figure  the  and  KOH  treated  glycoprotein  periodate  samples o f  were t e s t e d  for  r e s o r c i n a l procedure  the total  (PRT)  of  59 Jourdian t o be that  et  al  greater  the i n the  some s i a l i c  amount o f fully  acids  sialic  acid detected  saponified  s t a t e were r e n d e r e d d e t e c t a b l e  tions.  I t has  the  r e a c t i o n of  p r o d u c e d by acid or  been r e p o r t e d  f o r the  side  C8  of  estimation  the  the  '  side  b o n d b e t w e e n C7  the  sialic  t r e a t m e n t w i t h KOH)  of  This sialic  can  are  will  and  C8  total'sialic  be  groups will  used  alkalie-labile  must be  condi?-  of  the  group, sialic  a t e i t h e r C7  oxidation,  leaving the  and  of  removal  ( f o r example, acids  reactive  discrepancy and  i n the fully  glycoprotein.  present  upon  clevage  unsaponified  same c o l o n i c e p i t h e l i a l  the  the  sialic  the  in  aldehyde  Consequently,  r e n d e r the  i n the  prevent  chemically  to explain  an  present  periodate  t o PRT.  PRT  a c i d depends  a t p o s i t i o n C7  therefore  by  implied  periodate-resorcinol  Substituents  acid detected  samples o f t h e which  .  acid unreactive  these substituent  PRT.  of  the  found  under a l k a l i n e  r e s o r c i n a l reagent with  chain  the  of  that  periodate oxidation, 59 68  chain  This  w h i c h were u n d e t e c t a b l e by  natural  procedure  sample.  was  a t C7  and  to amount  saponified Substituents  or  C8  of  5.  TABLE 6.  Percentage acids  of s i d e c h a i n s u b s t i t u t i o n  i n the  sialic  of c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s .  Source of Sample  Percentage  side chain  substitution in  sialic  acids  NORMAL (Cancer)  49.2  NORMAL  25.0  ULCERATIVE COLITIS  55.4  CROHN'S  (Colon)  41.0  CROHN'S  (Ileum)  53.1  ISCHEMIC COLITIS  61.0  Note: C o l o n i c e p i t h e l i a l g l y c o p r o t e i n s were s a p o n i f i e d i n 0.5%KOH f o r 120 m i n u t e s . Samples of the f u l l y s a p o n i f i e d and the unsaponifiec g l y c o p r o t e i n were t e s t e d f o r t o t a l s i a l i c a c i d by the p e r i o d a t e r e s o r c i n o l procedure of J o u r d i a n (59). The percentage of s i d e c h a i n s u b s t i t u t i o n i n s i a l i c a c i d i s g i v e n by the f o l l o w i n g e x p r e s s i o i c  Percentage o f side chain substitution  =  100 X  where PRT= p e r i o d a t e  PRT  , (sap,  , x sample) PRT  -  (sap.  PRT  (unsap.  . x sample)  sample)  r e s o r c i n o l estimate of t o t a l  sialic  acid.  some of the s i a l i c Furthermore,  a c i d s i d e chains of t h i s g l y c o p r o t e i n .  analysis  (GLC) of the methyl a c e t a t e produced  by treatment of the g l y c o p r o t e i n w i t h sodium methox i d e i n methanol i n d i c a t e d t h a t a l k a l i n e - l a b i l e 0 - a c e t y l  groups  were p r e s e n t i n these g l y c o p r o t e i n s .  In o r d e r t o o b t a i n a t r u e e s t i m a t e o f sialic is  acids  necessary  treatment  present  the  i n the g l y c o p r o t e i n s under s t u d y ,  to remove a l l the s u b s t i t u e n t  (saponification)  before  groups by a l k a l i n e  the PRT t e s t i s  applied.  To f i n d the o p t i m a l time f o r the a l k a l i n e treatment each c o l o n i c e p i t h e l i a l was s u b j e c t e d time s t u d y . figure  to a  was complete between 30 and 60 m i n u t e s . minutes was chosen as  procedure,  saponification  A t y p i c a l s a p o n i f i c a t i o n time curve i s  11. F o r the g l y c o p r o t e i n s s t u d i e d ,  it  shown i n  saponification  As a r e s u l t ,  the optimum time f o r a l l  60  subsequent  s a p o n i f i c a t i o n procedure.  Thus, f o r a p a r t i c u l a r g l y c o p r o t e i n ,  the t o t a l amount o f s i a l i c  a c i d present  v a l u e of a f u l l y of the f u l l y  s a p o n i f i e d sample.  is  e s t i m a t e d by the PRT  The d i f f e r e n c e  s a p o n i f i e d and the u n s a p o n i f i e d g l y c o p r o t e i n  measure o f the amount o f s i a l i c a c i d s u b s t i t u t e d or C8.  i n PRT v a l u e s  C o n s e q u e n t l y , the percentage  of s i a l i c  at e i t h e r C7  a c i d having  s i d e - c h a i n s u b s t i t u t i o n at C7 o r C8 can be c a l c u l a t e d . results  isa  The  o b t a i n e d w i t h the v a r i o u s c o l o n i c g l y c o p r o t e i n s under  study are t a b u l a t e d an i n c r e a s e  i n table  i n the percentage  6.  As compared w i t h the n o r m a l s ,  of s i d e c h a i n s u b s t i t u t i o n  s i a l i c a c i d s was found i n a l l the d i s e a s e d c o l o n s .  of  However,  more normal colons must be a n a l y s e d . b e f o r e  c o n c l u s i o n s can  be drawn.  8.  D i g e s t i o n Study of V i b r i o C h o l e r a Neuraminidase  Digest  In an independent experiment c a r r i e d out i n our l a b o r a t o r y ,  a sample of r a t  colonic e p i t h e l i a l glyco-  p r o t e i n was i n c u b a t e d w i t h v i b r i o c h o l e r a neuraminidase 48 h o u r s .  The amount of f r e e  sialic  acid released  for  by the  enzyme was estimated by the t h i o b a r b i t u r i c a c i d procedure of Warren^.  The r e s u l t s  total sialic  revealed that  acid present  l e s s than 30% of  i n the g l y c o p r o t e i n was  the  released.  F a i l u r e of the enzyme t o b r i n g about t o t a l r e l e a s e of  the  sialic  to  a c i d from the g l y c o p r o t e i n c o u l d be a t t r i b u t e d  the presence  of s u b s t i t u e n t  at C4 of the s i a l i c s i a l i c acids  groups  a c i d , for i t  substituted  is  such as 0 - a c e t y l  groups  generally believed  at C4 are r e s i s t a n t t o v i b r i o  c h o l e r a neuraminidase d i g e s t i o n ^ . then removal of these s u b s t i t u e n t s  If  t h i s were  should i n c r e a s e  true, the sus*-  c e p t i b i l i t y of the g l y c o p r o t e i n t o enzyme d i g e s t i o n . when a sample of a f u l l y  that  saponified rat  Indeed,  colonic e p i t h e l i a l  g l y c o p r o t e i n was t r e a t e d w i t h v i b r i o c h o l e r a neuraminidase for as  53 h o u r s , about 95% of the s i a l i c free  sialic  substituted  acid.  a c i d s were  released  Thus, i t was concluded t h a t s i a l i c  at C4 were p r e s e n t  acids  in rat colonic glycoproteins  and t h a t s a p o n i f i c a t i o n was necessary  t o b r i n g about  total  l i b e r a t i o n of N - a c e t y l n e u r a m i n i c a c i d by n e u r a m i n i d a s e .  S u b s t i t u t i o n o f the s i a l i c a c i d s by a l k a l i n e - l a b i l e groups i s not however c o n f i n e d  s o l e l y t o p o s i t i o n C4 .  As  was d i s c u s s e d  i n the s e c t i o n on s a p o n i f i c a t i o n , the c o l o n i c  glycoproteins  under study have some s i a l i c a c i d s w i t h  0-acetyl  groups a t p o s i t i o n C7 and or C8 of the polyhydroxy s i d e  chain.  The d i g e s t i o n study o u t l i n e d i n f i g u r e 4 was designed t o permit a p a r t i a l e s t i m a t i o n  of the percentage  s u b s t i t u t i o n o c c u r r i n g a t each o f p o s i t i o n s C4, on the s i a l i c a c i d r e s i d u e .  C7 and C8  A f t e r e x h a u s t i v e d i g e s t i o n of the  g l y c o p r o t e i n w i t h v i b r i o c h o l e r a neuraminidase, the  incubate  was vaccuum f i l t e r e d through a membrane w i t h an e x c l u s i o n l i m i t of approximately 100,000 M.W.,  S i a l i c a c i d r e l e a s e d by  the enzyme would pass through the membrane and be found as f r e e s i a l i c a c i d i n the f i l t r a t e . at  As s i a l i c  acids  substituted  C4 are r e s i s t a n t t o v i b r i o c h o l e r a neuraminidase d i g e s t i o n ,  these f r e e s i a l i c a c i d s would not be expected t o c o n t a i n any s u b s t i t u t e n t s a t C4.  On the other  hand, s i a l i c a c i d s  found  i n the r e t e n t a t e would s t i l l be bound t o the g l y c o p r o t e i n and would t h e r e f o r e be s u b s t i t u t e d a t C4. amount o f f r e e s i a l i c p r e s e n t  Q u a n t i t a t i v e l y , the  i n the f i l t r a t e  as e s t i m a t e d  by the t h i o b a r b i t u r i c a c i d t e s t o f Warren f o r f r e e acid  (TB) would r e p r e s e n t  the amount o f s i a l i c a c i d  f r e e o f s u b s t i t u t e n t s a t C4. s i a l i c acid containing the p e r i o d a t e  residues  On the o t h e r hand, the amount o f  s u b s t i t u t e n t s a t C4 would be g i v e n by  resorcinol test  on the r e t e n t a t e .  sialic  (PRB) f o r bound s i a l i c  acid  The sum o f the f r e e and bound s i a l i c  acids  TABLE 7.  Substitution patterns epithelial  of s i a l i c acids  in colonic  glycoproteins.  % o f s u b s t i t u t i o n o c c u r r i n g at ^  C  + C  7°  NORMAL (Cancer)  34.0  16.3  18.6  NORMAL  58.1  19.8  16 .8  5.3  ULCERATIVE COLITIS  19 .0  12.0  23.4  45 .6  CROHN'S (Colon)  38.3  9.2  22.8  29.7  ISCHEMIC COLITIS  17.2  10.6  21.7  50.4  Note; for  .  4  analysis.  8  31.1  sample f o r C r o h n ' s d i s e a s e of the ileum was not  this  r C  positions  available  56  would give a t o t a l amount o f s i a l i c a c i d s p r e s e n t .  Hence,  the percentage o f C4 s u b s t i t u t i o n o c c u r r i n g i n the s i a l i c acid residue  o f a g l y c o p r o t e i n can be c a l c u l a t e d .  Saponified filtrate  and u n s a p o n i f i e d  and r e t e n t a t e were analyzed  samples o f both the  fortotal  sialic 59  a c i d by the p e r i o d a t e - r e s o r c i n o l procedure o f J o u r d i a n The  eta l  degree o f s u b s t i t u t i o n a t C7 o r C8 o f the f i l t r a t e and  the r e t e n t a t e was then c a l c u l a t e d s e c t i o n s on s a p o n i f i c a t i o n ) . s i a l i c acids were found.  unsubstituted  ( f o r d i s c u s s i o n see p r e v i o u s  F o r the f i l t r a t e  a t C 4 ) , two types o f s i a l i c  These were e s t i m a t e d as f o l l o w s :  f o r the non-KOH t r e a t e d sample would r e p r e s e n t unsubstituted  sialic  acids  (containing  (unsubstituted  (b) the d i f f e r e n c e i n PRT v a l u e s  (a) the PRT v a l u e the amount o f  a t C4, C7 and C8)  o f the KOH t r e a t e d and non-KOH  t r e a t e d samples would i n d i c a t e the amount o f s i a l i c s t i t u t e d a t C7 and o r C8.  acids  acids  sub-  In the r e t e n t a t e , two types o f s i a l i c  a c i d s s u b s t i t u t e d a t C4 were found and e s t i m a t e d as f o l l o w s : (a) the PRT value  o f the non-KOH  treated retentate  sample i n d i c a t e s  the amount o f s i a l i c a c i d s w i t h no s u b s t i t u t i o n s a t C7 o r C8. (b) the d i f f e r e n c e i n PRT v a l u e s  o f the KOH t r e a t e d and non-KOH  t r e a t e d samples would i n d i c a t e the amount o f s i a l i c s t i t u t e d a t C4, C7 and o r C8.  acids  sub-  5:  With the procedures employed i n t h i s study, i s not possible to d i f f e r e n t i a t e and C8.  between substitutents  it at C7  Neither can the procedures be u t i l i z e d to estimate  substitutents  at C9.  The results  of t h i s digestion study,  performed on human colonic e p i t h e l i a l glycoproteins obtained from normal and diseased colons, are as shown i n table  7.  The s i a l i c acid substitution of colonic glycoproteins obtained from diseased colons appears to be d i f f e r e n t from the c o n t r o l s . Since the results  are based on single specimens the  results  must be interpreted with a large degree of caution.  9.  GLC Analysis of 0-acetyl Groups  The number of 0-acetyl groups present i n the colonic e p i t h e l i a l glycoprotein was estimated by GLC as the methyl acetate after  t r a n s - e s t e r i f i c a t i o n with 0.1 M sodium  methoxide i n anhydrous methanol.  0-acetyl groups were found  in a l l the samples but the r e s u l t s were inconsistent, and i t was not possible to obtain quantitative  data.  GENERAL DISCUSSION  Fractions A  F r a c t i o n s A g l y c o p r o t e i n s appear  to be homogeneous  by DEAE chromatography, agarose g e l e l e c t r o p h o r e s i s and  c e l l u l o s e acetate electrophoresis  analysis  at pH 8 . 8 .  at pH 8.8  Chemical  o f these g l y c o p r o t e i n s r e v e a l the presence  g a l a c t o s e , hexosamine, phosphorus.  sialic  of  fucose,  a c i d , p r o t e i n and l i t t l e o r no  Q u a l i t a t i v e l y , they resemble  glycoproteins  . , . , . . . 70 . . 11,16,17,24 i s o l a t e d from g a s t r i c j u i c e , c o l o n i c mucosa ' 11,17,19,21  • 4. 4.. i n t e s t i n e mucosa  .  . .  9  and r e c t a l mucus  a p p a r e n t l y uncontaminated w i t h n u c l e i c a c i d . p r o t e i n s were e x t r a c t e d  free  t i s s u e components and m a t e r i a l s Further,  , , and they  are  S i n c e the g l y c o -  from washed e p i t h e l i a l c e l l  they should b e ' r e a s o n a b l y  n  preparations  of contaminants such as  connective  a r i s i n g from the f a e c a l  stream.  s i n c e p r o t e o l y t i c d i g e s t i o n was a v o i d e d , and r e l a t i v e l y  m i l d f r a c t i o n a t i o n t e c h n i q u e s were employed, i t t h a t an undegraded g l y c o p r o t e i n was o b t a i n e d . be no o t h e r r e p o r t i n the l i t e r a t u r e  is  possible  There appears  to  of the i s o l a t i o n and c h a r a c t e r "  i z a t i o n of c o l o n i c g l y c o p r o t e i n s from a p u r e l y e p i t h e l i a l The data p r e s e n t e d i n t a b l e  4 shows:  (a)  that  source. there  was a d i f f e r e n c e  i n the molar r a t i o of the sugars o f the g l y c o -  protein isolated  from the normal and d i s e a s e d human c o l o n s ,  (b)  t h a t i n each o f the g l y c o p r o t e i n s t u d i e d , the four types o f s i a l i c a c i d s found, C9  substituted s i a l i c acids  different  (that i s : ( i ) u n s u b s t i t u t e d o r ( i i ) s i a l i c acids substituted at  C4 and o r C9, ( i i i ) s i a l i c a c i d s s u b s t i t u t e d a t C7 and o r C8, (iv)  s i a l i c a c i d s s u b s t i t u t e d a t C4 and a t C7 and o r C8) were  present  i n d i f f e r e n t q u a n t i t i e s (see t a b l e 7 ) .  I t i s tempting  t o conclude t h a t these d i f f e r e n c e s are r e l a t e d t o changes associated with  the d i s e a s e processes  i n s u f f i c i e n t data a v a i l a b l e t o support  but there i s c l e a r l y such a c o n c l u s i o n .  Under normal c o n d i t i o n s , v a r i a t i o n s o f the chemical compositions o f the c o l o n i c g l y c o p r o t e i n s might occur f o l l o w i n g manner;  i n the  (a) along the l e n g t h o f the c o l o n , (b)  from the bottom t o the t o p o f the c r y p t s o f L i e b e r k u h n .  Thus,  the d i f f e r e n c e s found i n t h i s study c o u l d be a t t r i b u t e d t o the d i f f e r e n t anatomical mens were o b t a i n e d in  r e g i o n o f the c o l o n from which the s p e c i -  r a t h e r than t o t h e d i s e a s e p r o c e s s .  some experiments n o t a l l the c e l l s were removed, w i t h  at the base o f the c r y p t s the most d i f f i c u l t has  Also,  been r e p o r t e d  t o remove.  those It  t h a t c o l o n i c g o b l e t c e l l s mature as they 71  migrate from the bottom t o the t o p o f the c r y p t s  .  Failure-  t o remove the c e l l s i n the lower h a l f o f the c r y p t s i n some experiments may t h e r f o r e have e x c l u d e d the m a j o r i t y o f young cells.  In a d d i t i o n to the above, m i c r o h e t e r o g e n e i t y can occur i n the normal b i o s y n t h e s i s proteins.  Despite extensive  of c o l o n i c e p i t h e l i a l g l y c o -  i n v e s t i g a t i o n on the  biosynthesis  3  o f i n t e s t i n a l g l y c o p r o t e i n s , the d e t a i l e d mechanism remains unknown. In g e n e r a l , g l y c o p r o t e i n s are s y n t h e s i z e d i n c o l o n i c 3 4 epithelial chain i s  cells  by the f o l l o w i n g mechanism '  .  The p e p t i d e  s y n t h e s i z e d i n the rough endoplasmic r e c t i c u l u m  a c c o r d i n g the t o  Orthodox RNA template t h e o r y .  added i n a s t e p - w i s e sequence  as monosaccharide u n i t s .  enzyme attachment o f the f i r s t  are The  sugar v i a a sugar n u c l e o t i d e  t r a n s f e r a s e proceeds w h i l e the p e p t i d e the polyribosomes o r s h o r t l y a f t e r remainder o f the o l i g o s a c c h a r i d e  is  still  attached  their separation.  chain i s  to  The  completed i n t h e  smooth membranes and i n the g o l g i a p p a r a t u s . t r a n s f e r a s e s are s p e c i f i c  Sugars  The g l y c o s y l  f o r both the a c c e p t o r  and the donor.  The o r d e r o f the sugar sequence depends on the k i n e t i c sequence of the t r a n s f e r a s e s  involved.  F o r example,  sialy-  t r a n s f e r a s e s r e q u i r e a h i g h m o l e c u l a r weight a c c e p t o r and t h i s explains  the t e r m i n a l p o s i t i o n o f s i a l i c  saccharide  chain.  a c i d i n an o l i g o -  In s p i t e of the non-randomness i n the s y n -  t h e s i s of the o l i g o s a c c h a r i d e of the t r a n s f e r a s e s ,  c h a i n as w e l l as the  t h i s mechanism of s y n t h e s i s ,  specificity u n l i k e the  RNA-directed s y n t h e s i s  of p r o t e i n s , may i n t r o d u c e m i c r o h e t e r o -  g e n e i t y as a r e s u l t  (a)  of  the p r o d u c t i o n o f incomplete o l i g o -  saccharide  c h a i n s and  (b)  the o c c u r r e n c e of i n c o r r e c t mono-  saccharide  sequence.  The r e s u l t s  i n t h i s work may t h e r e f o r e  r e p r e s e n t m i c r o h e t e r o g e n e i t y o f one o r both o f these  types.  TABLE 8.  Molar r a t i o s  of sugar found i n c o l o n i c e p i t h e l i a l  g l y c o p r o t e i n s expressed as  FUCOSE  percentages.  GALACTOSE  HEXOSAMINE  SIALIC ACID  9.5  29.9  35.0  25.6  NORMAL  12 .2  35.4  31.7  20.7  ULCERATIVE COLITIS  14.9  31.3  31.3  22.4  CROHN'S  (Colon)  25.8  34.8  31.0  8.3  CROHN'S  (Ileum)  8.2  43.7  17.3  30.6  9.1  30.8  30.3  29.9  NORMAL (Cancer)  ISCHEMIC COLITIS  Note: at  G l y c o p r o t e i n samples were t r e a t e d w i t h Dowex 5 0 H T i n 0.01 N HCl  80^C. f o r 36 t o 50 hours and the h y d r o l y s a t e s were analysed  for 57 fucose and g a l a c t o s e by GLC and f o r hexosamine by a m o d i f i c a t i o n 58 of a procedure of W i n z l e r . S i a l i c a c i d s were e s t i m a t e d by the 59 p e r i o d a t e r e s o r c i n o l procedure of J o u r d i a n for t o t a l s i a l i c acid.  The and  f o l l o w i n g d i s c u s s i o n i s very t e n t a t i v e t h e r e f o r e ,  i t i s r e a l i z e d t h a t the d i f f e r e n c e s d i s c u s s e d may  be  a  r e s u l t o f normal v a r i a t i o n s and be e n t i r e l y u n r e l a t e d t o disease process. chemical  Under p a t h o l o g i c a l c o n d i t i o n s , changes i n the  composition  of the c o l o n i c e p i t h e l i a l  found i n t h i s study might be general  c l a s s e s of changes  a s c r i b e d t o one  glycoproteins  o r both o f  (a) a l t e r a t i o n s i n the  two  normal  b i o s y n t h e s i s o f the g l y c o p r o t e i n s at d i f f e r e n t stages s y n t h e s i s and,  the  of  the  (b) a l t e r a t i o n s i n the normal d e g r a d a t i o n  of  the g l y c o p r o t e i n a f t e r s y n t h e s i s i s complete.  In t h i s study, of C4 s u b s t i t u t e d s i a l i c l a t e d from a l l d i s e a s e d believed that s i a l i c  a general a c i d s was  samples  i n c r e a s e i n the  proportion  found i n g l y c o p r o t e i n s  ( t a b l e 7).  I t i s now  iso-  generally  a c i d s s u b s t i t u t e d at C4 are r e s i s t a n t 79  t o v i b r i o c h o l e r a neuramindase d i g e s t i o n environment, constant a c i d s might occur  In a  pathologxcal  removal of n o n - C 4 - s u b s t i t u t e d  sialic  r e s u l t i n g i n the presence of a h i g h  of C4-substituted s i a l i c these c o u l d be c a l l e d hand, the  .  a c i d s i n the g l y c o p r o t e i n p o o l .  "residual" glycoproteins.  increase i n C4-substituted s i a l i c  be the r e s u l t of the p r o d u c t i o n the e p i t h e l i a l c e l l s .  proportion  On  the  a c i d s may  of "adaptive"  other  also,  glycoproteins i n  That i s , the g l y c o p r o t e i n s  synthesized  are capable of s u r v i v i n g the p a t h o l o g i c a l environment. d e t a i l e d k i n e t i c study p r o t e i n i n normal and  Thus,  A  on the b i o s y n t h e s i s of i n t e s t i n a l i n p a t h o l o g i c a l c o n d i t i o n s would  i n f o r m a t i o n on whether or not such " r e s i d u a l " o r  glyco-  provide  "adaptive"  g l y c o p r o t e i n s are i n v o l v e d i n these d i s e a s e  processes.  When the molar r a t i o s of the sugars of the (fucose, g a l a c t o s e , hexosamine and as percentage v a l u e s galactose  and  colon.  ( t a b l e 8) i t was  found t h a t the amount of  from the d i f f e r e n t d i s e a s e d  i n the  from d i s e a s e  to disease.  the  fucose were  Of p a r t i c u l a r i n t e r e s t  the low percentage of s i a l i c a c i d s found i n the  p r o t e i n obtained  glyco-  t i s s u e s of  However, the amount of s i a l i c a c i d s and  found t o vary was  s i a l i c acid) were expressed  hexosamines were q u i t e c o n s t a n t  proteins obtained  glycoproteins  from Crohn's d i s e a s e of the l a r g e  glycointestine.  T h i s low percentage v a l u e does not e x p l a i n the slower m o b i l i t y of the g l y c o p r o t e i n on agarose g e l e l e c t r o p h o r e s i s s i n c e the g l y c o p r o t e i n o b t a i n e d t e r m i n a l ileum which contained  (table 2),  from Crohn's d i s e a s e  of  the  t h r e e t o f o u r times more  sialic  a c i d , e x h i b i t e d a s i m i l a r slow e l e c t r o p h o r e t i c m o b i l i t y . low m o b i l i t y however, was from u l c e r a t i v e c o l i t i s  not found i n g l y c o p r o t e i n s (table 2).  The  and  Crohn's d i s e a s e of the  obtained  above f i n d i n g , i f  confirmed, might a i d i n the d i f f e r e n t i a l colitis  diagnosis of u l c e r a t i v e  colon.  I t i s i n t e r e s t i n g t o note t h a t the percentage of s u b s t i t u t e d s i a l i c a c i d dropped from 58% 35%  i n glycoproteins  from p a t i e n t s w i t h colitis  and  ischaemic  colitis.  non-  i n the normal to about  from p a t i e n t s w i t h Crohn's d i s e a s e  carcinoma and  This  t o about 18% This reduction  and  i n both u l c e r a t i v e is  counterbalanced  by a c o r r e s p o n d i n g i n c r e a s e acids  substituted  the percentage  i n the percentages of  at C4 o r at C7 and o r C8.  sialic  In g e n e r a l ,  of s i d e c h a i n s u b s t i t u t i o n was  elevated  i n a l l of the d i s e a s e s s t u d i e d and s u p r i s i n g l y i n t h e t i s s u e s resected  for carcinoma.  In view o f the h i g h r i s k of  mali-  72 gnant change a s s o c i a t e d w i t h u l c e r a t i v e c o l i t i s i n c r e a s i n g number o f r e p o r t s Crohn's disease ^ , t h i s  and the  of carcinoma c o m p l i c a t i n g  finding,  i f c o n f i r m e d , might c o n t r i -  bute t o an u n d e r s t a n d i n g of the e t i o l o g y and  pathogenesis  of these d i s e a s e s . Teague e t a l ^ r e p o r t e d  the presence o f two  g l y c o p r o t e i n s i n human c o l o n i c mucosa. from Sepharose 2B, was mannose f r e e ;  One o f t h e s e ,  the o t h e r which was of  m o l e c u l a r w e i g h t , and which c o n t a i n e d s i g n i f i c a n t o f mannose. protein,  S i n c e mucosal s c r a p i n g s  quantities  were the source of g l y c o -  contamination o f the p r e p a r a t i o n s  i s probable.  excluded  by serum p r o t e i n s  These would be expected t o appear  i n the i n c l u d e d  f r a c t i o n o b t a i n e d from Sepharose 2B and c o n t r i b u t e mannose this fraction.  It  is  surprising therefore  to  t h a t serum p r o t e i n s  c o u l d not be d e t e c t e d by immunological methods i n e i t h e r of the Sepharose f r a c t i o n s  o b t a i n e d by Teague e t a l " ^ .  mannose were found t o be p r e s e n t  i n the g l y c o p r o t e i n s  Traces of obtained  26  from "mucous p l u g s " by Johansen , and i n the g l y c o p e p t i d e s i s o l a t e d by p r o t e o l y t i c d i g e s t i o n of minced c o l o n by Sky-Peck 23  et al  .  testinal  glycc  However, the presence of mannose in intestinal glyco11,16,17,19-21,2  p r o t e i n s has not been r e p o r t e d by o t h e r w o r k e r s .  The  a n a l y t i c a l methods employed i n t h i s  g a t i o n w i l l enable the d e t e c t i o n of a q u a n t i t y as low as 2% by weight o f the g a l a c t o s e approximately 0.2%  detected  i s o l a t e d i n t h i s study but c o n t a i n i n g g l y c o p r o t e i n was weight  'A2  of the  (that i s ,  glycoprotein). glycoproteins  i t i s p o s s i b l e t h a t a mannose present  f r a c t i o n ' obtained  fractionation.  i n any  of mannose  present,  of the dry weight of the  Mannose c o u l d not be  investi-  i n the  from the  low  molecular  agarose  A15M  T h i s f r a c t i o n p r o b a b l y corresponds t o  the  i n c l u d e d Sepharose 2B f r a c t i o n i n v e s t i g a t e d by Teague e t al ^ 1  and  i t i s t h e r e f o r e o f i n t e r e s t t h a t the  t e s t e d i n t h i s work c o n t a i n e d  'A2*  fractions  serum p r o t e i n s .  F u r t h e r Work Proposed  As was  mentioned p r e v i o u s l y  (see page 59)  /  colonic  e p i t h e l i a l c e l l s p r o g r e s s through a m a t u r a t i o n c y c l e as  they  71 migrate from the bottom t o the top of the c r y p t s to assess whether there  .  In  order  i s a s i g n i f i c a n t v a r i a t i o n i n the  chemical composition o f the g l y c o p r o t e i n s  obtained  from c o l o n i c  c e l l s a t d i f f e r e n t phases of t h e i r m a t u r a t i o n c y c l e , e p i t h e l i a l c e l l s at d i f f e r e n t l e v e l s o f the c r y p t should Removal o f c e l l s  from d i f f e r e n t l e v e l s o f the c r y p t s i n r a t 73  s m a l l i n t e s t i n e had  been r e p o r t e d  by Weimer  i s a l s o n e c e s s a r y to i n v e s t i g a t e any p r o t e i n s along  be i s o l a t e d .  .  Furthermore, i t  v a r i a t i o n s i n the  the whole l e n g t h of the  colon.  glyco-  Once t h e p o s i t i o n a l  variations  purified  g l y c o p r o t e i n s s h o u l d be o b t a i n e d  diseased  colons.  out  to test  Chemical  analyses  the r e p r o d u c i b i l i t y  each type  o f amino a c i d  from normal and  o f these  s h o u l d be  of the present  I n a d d i t i o n , t h e amount o f O - a c e t y l of  i f any a r e known,  present  groups and t h e i n these  carried data.  percentage  glycoproteins  s h o u l d b e a s c e r t a i n e d and t h e s t r u c t u r e o f t h e o l i g o s a c c h a r i d e units  should  be e l u c i d a t e d .  g a t i o n s would permit proteins present  The r e s u l t s  from these  investi-  a much b e t t e r c o m p a r i s o n o f t h e g l y c o -  i n the normal andin  the various p a t h o l o g i c a l  conditions.  Once p u r i f i e d  g l y c o p r o t e i n s from d e l i n e a t e d areas o f  each normal and d i s e a s e d these  s h o u l d be u s e d  tract,  e p i t h e l i u m have been  i n the preparation of antisera.  a n t i s e r a would a l l o w cross r e a c t i v i t y )  colonic  o f these  glycoproteins i n the g a s t r o i n t e s t i n a l facilitate  whether o r n o t t h e a n t i g e n i c determinants are disease  the investigation o f present  i n the glyco-  specified.  In view o f t h e d i f f i c u l t i e s  experienced  i n .obtaining  human s a m p l e s , m i n i a t u r i z a t i o n o f t h e f r a c t i o n a t i o n procedures small  would enable  samples  formol-calcium tissues  such  i n v e s t i g a t i o n s t o be c a r r i e d  as b i o p s y  fixed,  are readily  Such  an a s s e s s m e n t o f t h e d i s t r i b u t i o n ( o r  and i n a d d i t i o n , would  proteins  obtained  specimens.  paraffin  available,  processed  and  chemical  o u t on  Also, since blocks of tissues of diseased  the s u c c e s s f u l a p p l i c a t i o n  of  such  micro techniques allow  an  to the  investigation  extensive investigation  on  of these  readily  tissues  available  would material.  BIBLIOGRAPHY  1.  S p i r o , R.G.: New E n g l . J . Med. 281, 991  (1969).  2.  S p i r o , R.G.: New E n g l . J . Med. 281, 1001 (1969).  3.  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Digestion  (1958).  APPENDIX  Since the c o m p l e t i o n of the e x p e r i m e n t a l work r e p o r t e d i n t h i s thesis,  an independent study c a r r i e d out i n t h i s  has found t h a t . t h e substituted  at  laboratory  e s t i m a t e of the q u a n t i t y of s i a l i c and or Cg i s  acids  in error.  In the PRT a s s a y , the sample under t e s t i s  first  treated  periodic acid.  at  and Cg are  susceptible  S i a l i c acids  to periodate  c a r b o n y l groups  at C ^ .  r e s o r c i n o l reagent maxima a t  630 m . .  unsubstituted  o x i d a t i o n r e s u l t i n g i n the formation o f These c a r b o n y l groups  r e a c t w i t h the  forming a chromogen, w i t h an a b s o r p t i o n S i a l i c acids  r e s i s t a n t to periodate  substituted  at C^ and o r C g are  o x i d a t i o n and i t was assumed  t h a t they were u n r e a c t i v e towards the r e s o r c i n o l  therefore,  reagent.Recently  however, i t has been shown t h a t the r e s o r c i n o l reagent the  and o r Cg s u b s t i t u t e d  reaction gives  s i a l i c acids  a s i g n i f i c a n t O . D . r e a d i n g at  T h u s , the O . D . r e a d i n g at  a t r u e e s t i m a t i o n o f C^ u n s u b s t i t u t e d  acids  630 m  sialic  unsubstituted  is  no l o n g e r  acids  and v a l u e s  f o r KOH t r e a t e d  need not be c o r r e c t e d as none of the s i a l i c substitutents  This  630 m^j , thereby  r e p o r t e d i n t a b l e s 6 and 7. need t o be r e c a l c u l a t e d . be noted however, t h a t the PRT v a l u e s  reacts with  (PRS r e a c t i o n ) .  r a i s i n g the PRT r e a d i n g a t t r i b u t e d t o s i a l i c a t C_ and C 0  with  acids  at C^ and would a l l p a r t i c i p a t e  It  should  samples  contain  i n the PRT r e a c t i o n .  The e x t i n c t i o n c o e f f i c i e n t of the PRS r e a c t i o n i s  0.279 8 times  that in  o f t h e PRT a s s a y .  tables  In r e c a l c u l a t i n g  6 and 7, t h e f o l l o w i n g  P R T  equator  (observed,  the data was  as r e p o r t e d  employed.  non-KOH)  -  0  -  2  7  9  8  P R T  (KOH)  (actual) 0 .7202  The  recalculating  comparison that  data  a r e as shown i n t a b l e s  of the o r i g i n a l  the o r i g i n a l  and r e v i s e d  interpretation  A a n d B.  calculations  of the data  still  A  suggests holds  true.  TABLE A.  Percentage s i d e c h a i n  of c o l o n i c e p i t h e l i a l  Source o f Sample  s u b s t i t u t i o n i n the s i a l i c  acids  glycoproteins.  Percentage s i d e  chain  substitution i n s i a l i c acids  NORMAL (Cancer)  65.9  NORMAL  34.9  ULCERATIVE COLITIS  80.0  CROHN'S (Colon)  44.0  CROHN'S  73.4  (Ileum)  ISCHEMIC COLITIS  84.7  TABLE B.  Substitution patterns  epithelial  of s i a l i c  acids  in colonic  glycoproteins.  % of s u b s t i t u t i o n o c c u r i n g at  positions  C  C7orC8  C4  C4+C7orC8  NORMAL (Cancer)  27.7  22.6  6.5  43.2  NORMAL  50.4  27.5  14.7  7.4  ULCERATIVE COLITIS  14.3  16.7  5.7  63.3  CROHN'S  34.7  12.8  11.3  31.2  13.1  14.7  2.1  70.0  (Colon)  ISCHEMIC COLITIS  

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