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Development of methods for the simultaneous visualization of neutral sugars and either sialic acid and… Volz, Doris Elenore 1987

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DEVELOPMENT OF METHODS FOR THE SIMULTANEOUS VISUALIZATION OF NEUTRAL SUGARS AND EITHER SIALIC ACID AND ITS SIDE CHAIN O-ACYL VARIANTS OR O-SULPHATE ESTER BASED ON THE SELECTIVE PERIODATE OXIDATION OF SIALIC ACID by DORIS ELENORE VOLZ B.Sc. The University of Alberta, 1978 Specialization Certificate i n Microbiology, 1982  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology)  He accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1987 ( c ) Doris Elenore Volz, 1987  In  presenting  degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  thesis for by  this  his  or  and study.  representatives.  Department of  rfYTHCUiQ G-  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  that the  may be It  thesis for financial gain shall not  permission.  requirements  I further agree  scholarly purposes her  the  for  an  advanced  Library shall make it  that permission  for extensive  granted  head  is  by the  understood be  that  allowed without  of  my  copying  or  my written  ABSTRACT: The objective of t h i s study was to e s t a b l i s h conditions f o r the s e l e c t i v e periodate oxidation of s i a l i c a c i d , and then use these conditions to develop a s e r i e s of general methods f o r the simultaneous v i s u a l i z a t i o n of "neutral sugars" ( i e . hexose, 6-deoxyhexose and N-acetylhexosamine) and s i a l i c acid and i t s side chain O-acyl substituted variants, or O-sulphate ester. Investigations of s e l e c t i v e conditions f o r the oxidation of s i a l i c acids demonstrated that oxidation f o r one hour at 4°C with 0.4  mM periodic acid i n  approximately 1M hydrochloric acid (PA*) oxidized a l l a v a i l a b l e s i a l i c acid residues of both the s i a l o and sialosulphoglycoproteins of human and rat colon and the sialoglycoproteins of rat sublingual gland.  These conditions produced  no v i s i b l e S c h i f f s t a i n i n g of e i t h e r neutral macromolecules or v i c i n a l d i o l s located on the "neutral sugars" of s i a l o and sialosulphoglycoproteins, and did not r e s u l t i n the extraction of e p i t h e l i a l glycoproteins or i n the de-O-acylation  of side chain substituted s i a l i c acid residues.  Therefore,  PA*  can be used as a s p e c i f i c reagent f o r the s e l e c t i v e oxidation of s i a l i c acids.  Studies of the mechanism of oxidation with PA* showed that the lack of PAS  r e a c t i v i t y of "neutral sugars" was not due to the production of S c h i f f  unreactive hemiacetals or hemialdals.  I t i s possible that the s e l e c t i v e  oxidation of s i a l i c acids with PA* r e s u l t s from an increase i n the oxidation rate of s i a l i c acid residues together with a decrease i n the oxidation rate of "neutral sugars". Based upon t h i s method f o r the s e l e c t i v e oxidation of s i a l i c acid residues (PA*), f i v e new methods have been devised f o r the simultaneous v i s u a l i z a t i o n of "neutral sugars" and e i t h e r s i a l i c acid and i t side chain 0-acyl  ii  derivatives or O-sulphate ester. The f i r s t of these i s the selective periodate oxldatlon-borohvdrlde  reductlon-saponlflcatlon-selectlve periodate  oxidation-thlonln Schiff-saponlflcatlon-borohydrlde reduction-periodic acld-Schlff (PA»/Bh/KOH/PA»/T/KOH/Bh/PAS) technique, i n which s i a l i c acids with O-acyl substituents at C7, C8, or C9 (or which have two or three side chain O-acyl substituents) stain blue while "neutral sugars" with periodate sensitive v i c i n a l diols stain magenta. In the second method, the saponiflcatlon-selectlve periodate oxidation-thlonln Schiff-saponlflcatlonborohvdrlde reduction-periodic acld-Schlff fKOH/PA»/T/KOH/Bh/PAS) method a l l s i a l i c acids stain blue while "neutral sugars" stain magenta. In the third method, the selective periodate oxidation-thlonln Schiff-borohydrlde reduction-periodic acid-Schlff-saponification (PA»/T/Bh/PAS/KOH) method, s i a l i c acids without side chain substituents or which have an O-acyl substituent at C7 stain blue while "neutral sugars" stain magenta. In the fourth method, the saponiflcatlon-selectlve periodate  oxldatlon-borohvdride  reductlon-alclan blue PH 1.0-perlodic acid-Schlff (K0H/PA*/Bh/AB1.O/PAS) technique, O-sulphate esters stain aquamarine blue while "neutral sugars" stain magenta. In a l l of these techniques, mixtures of the components stain i n various shades of purple.  In the f i f t h and f i n a l method, the  saponiflcatlon-selectlve periodate oxldatlon-borohvdrlde  reduction-periodic  acld-Schlff (KOH/PA»/Bh/PAS) technique, selective identification of "neutral sugars" i n macromolecules which also contain s i a l i c acids can be achieved.  iii  TABLE OF CONTENTS  PAGE ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  viii  LIST OF ILLUSTRATIONS  x  LIST OF ABBREVIATIONS  xiv  ACKNOWLEDGEMENTS  xvi  INTRODUCTION  1  MATERIALS AND METHODS I.  II.  15  Materials A. Tissues  15  B. Chemicals  15  Histochemical Procedures  16  I I I . Methods based on the Selective Perlodate Oxidation of s i a l i c acid  19  A. Method 1 (PA*/Bh/KOH/PA*/T/KOH/Bh/PAS)  19  B. Method 2 (KOH/PA»/T/KOH/Bh/PAS)  23  C. Method 3 (PA»/T/Bh/PAS/KOH)  23  D.  24  Method 4 (KOH/PA*/Bh/AB1.0/PAS)  E. Method 5 (KOH/PA»/Bh/PAS)  iv  25  Table of Contents (cont'd.)  PAGE  RESULTS AND DISCUSSION I.  26  Establishment of Conditions for the Selective Periodate Oxidation of s i a l i c acid  II.  26  Investigation of 0 . 4 mM Periodate i n 1M. HCl  32  A. Specificity  32  B. Mechanistic Studies of PA* Conditions  37  C. Discussion and Conclusions  43  I I I . Development of Methods Based on PA*  45  A. Results of Methods 1 - 5  45  B. Specificity of Methods 1 - 5  56  C. Discussion and Conclusions  61  GENERAL CONCLUSIONS  68  REFERENCES  69  ILLUSTRATION LEGENDS I-XIX  82 101  LIST OF PUBLICATIONS  v  LIST OF TABLES  PAGE  I  Outline of methods for the simultaneous visualization  20  of "neutral sugars" and either s i a l i c acid and i t s side chain 0-acyl variants or O-sulphate ester  II  Location and histochemical characteristics of the  27  carbohydrate containing macromolecules studied during the investigation of methods for the selective oxidation of s i a l i c acids  III  The effect of pH on the oxidation of neutral macromolecules  30  IV  Evidence for the s e l e c t i v i t y of periodate oxidation with  33  0.4mM. periodic acid i n 1M hydrochloric acid at 4°C for one hour  V  Predicted results of the application of the methods outlined i n Table I to tissue sites containing s i a l i c acids both with and without side chain 0-acyl substituents, "neutral sugar" v i c i n a l d i o l s , and O-sulphate ester  vi  45  L i s t of Tables (cont'd.)  VI  PAGE  Methods for the verification of the s p e c i f i c i t y of  57  Methods 1 to 5  VII  L i s t of control methods which should accompany Methods 1 to 5  vii  65  LIST OF FIGURES  PAGE  1  Structure of sugar residues frequently encountered i n  4  colonic glycoproteins 2  Diagramatlc representation of the mechanism(s) by which  6  periodic acid-phenylhydrazine-Schiff (PAPS) procedures result i n selective detection of s i a l i c acid and the use of PAPS procedures i n the simultaneous visualization of s i a l i c acids and "neutral sugars"  3  A proposed selective oxidation mechanism for the  9  simultaneous visualization of "neutral sugars" and s i a l i c acid residues  4  Effect of the electrostatic f i e l d surrounding molecules  13  on periodate oxidation  5A & B  Flow diagram of the investigation into the mechanism by  38  which "neutral sugars" react following treatment with PA* and Schiff reagent  6  I l l u s t r a t i o n of the 2,4-dinitrophenylhydrazine,  3,3'>  dimethoxybenzidene fluoroborate (DNPH-TDMBF) procedure  viii  41  L i s t of Figures (cont'd.)  7  The mechanism and expected staining patterns f o r Method 1, the selective perlodate oxidation-borohydride reduction-saponification-selective perlodate oxidationthionin Schiff-saponiflcation-borohydride  reduction-  periodic acid-Schiff (PA*/Bh/KOH/PA«/T/KOH/Bh/PAS) technique, and Method 2, the saponification-selective perlodate oxidatlon-thionin Schiff-saponiflcationborohydride reduction-periodic  acid-Schiff  (KOH/PA*/T/KOH/Bh/PAS) technique  8  The mechanism and expected staining patterns f o r Method 3» the selective perlodate oxidation-thionin Schiff-borohydride  reduction-periodic  acid-Schiff-  saponification (PA*/T/Bh/PAS/KOH) technique  9  The mechanism and expected staining patterns f o r Method 4, the saponification-selective perlodate oxidation-borohydride reduction-alcian blue pH 1.0-periodic acid-Schiff (K0H/PA*/Bh/AB1.0/PAS) technique, and Method 5, the saponification-selective perlodate oxidation-borohydride  reduction-periodic  acid-Schiff (KOH/PA*/Bh/PAS) technique  ix  LIST OF ILLUSTRATIONS  PAGE  I  A) Sections of rat colon stained with the PA/Bh/K0H/PA2/S,  82  PA/Bh/KOH/PA*/S, and PA/Bh/KOH/PAgrj/S procedures B) Sections of rat l i v e r stained with the PA2/S, PA*/S and PA6Q/S procedures  II  Effect of pH on the rate of periodate oxidation of  83  s i a l i c acid residues  III  Effect of pH on the periodate oxidation of "neutral  84  sugars"  IV  Evidence for the s p e c i f i c i t y of PA* conditions for the  85  selective periodate oxidation of s i a l i c acids - PART A  V  Evidence for the s p e c i f i c i t y of PA* conditions for the  86  selective periodate oxidation of s i a l i c acids - PART B  VI  Effect of PA* conditions on the periodate oxidation of "neutral sugars"  x  87  L i s t of Illustrations (cont'd.)  VII  The effect of PA* conditions on the oxidation of  PAGE  88  "neutral sugars" i n the presence of s i a l o - and sialosulpho-containing glycoproteins  VIII  To determine i f glycoproteins are extracted when  89  treated with PA* conditions  IX  To determine whether PA* de-O-acetylates s i a l i c acids  90  X  To determine whether PA* conditions invoke a Feulgen  91  reaction  XI  I l l u s t r a t i o n of results obtained i n mechanistic studies  92  of the selectivity of PA*  XII  I l l u s t r a t i o n of the selective perlodate oxidationborohydride reduction-saponification-selective perlodate oxidatlon-thionin Schiff-saponificationborohydride reduction-periodic acid-Schiff (PA*/Bh/KOH/PA*/T/KOH/Bh/PAS) technique (Method 1)  xi  93  L i s t of Illustrations (cont'd.)  XIII  PAGE  I l l u s t r a t i o n of the saponification-selective periodate oxidation-thionin  94  Schiff-saponification-borohydride  reduction-periodic acid-Schiff (KOH/PA«/T/KOH/Bh/PAS) technique (Method 2)  XIV  I l l u s t r a t i o n of the selective periodate oxidationthionin Schiff-borohydride  reduction-periodic  95  acid-  Schiff-saponification (PA*/T/Bh/PAS/KOH) procedure (Method 3)  XV  I l l u s t r a t i o n of the saponification-selective periodate  96  oxidation-borohydride reduction-alcian blue pH 1.0periodic acid-Schiff (K0H/PA*/Bh/AB1.0/PAS) procedure (Method 4)  XVI  I l l u s t r a t i o n of the saponification-selective periodate oxidation-borohydride reduction-periodic  97  acid-Schiff  (KOH/PA«/Bh/PAS) procedure (Method 5)  XVII  Evidence that exchange of Schiff reagent does not occur with Methods 1 and 2  xii  98  L i s t of Illustrations (cont'd.)  XVIII  Evidence that the position of the saponification  (KOH)  step does not affect i t s a b i l i t y to remove non-specific thionin Schiff staining  XIX  Evidence that non-specific thionin Schiff staining does not occur i n Methods 1-3  xiii  LIST OF ABBREVIATIONS  Ac  =  Acetyl group ( C H 3 - C = 0)  AB1.0  =  Alcian blue 8GX at pH = 1.0  AB2.5  =  Alcian blue 8GX at pH = 2.5  Bh  =  Borohydride reduction  Co  = S i a l i c acids without side chain substituents  Cx  = S i a l i c acids with side chain substituents.  The numbers  7, 8 and 9 (in place of x) refer to which carbon of s i a l i c acid contains the acetyl group. DNPH-TDMBF  =  2,4 dinitrophenylhydrazine 3i3'-dimethoxybenzidene fluoroborate procedure. The product of this reaction i s a formazan (Fig. 6).  KOH  = Saponification. A solution of 0.5% potassium hydroxide (w/v) dissolved i n 70% ethanol (v/v) i s used to remove esters from sugar residues.  N/A  = Not applicable  "neutral sugars"  = Refers to hexose, 6-deoxyhexose and N-acetyl hexosamine sugars  %  -  Per cent  PA  = Oxidation with 40 mM periodic acid at room temperature; the subscripts 2, 3 60 refer to the time period of oxidation i n minutes.  xiv  L i s t of Abbreviations (cont'd.)  PA*  = Periodate oxidation i n 0.1 mM periodic acid dissolved in approximately  1M, HCl for 1 hr. at 40°C (selective  periodate oxidation) PA/Bh/KOH  = periodate oxidation-borohydride reduction-saponification technique used to confine oxidizable v i c i n a l diols to CT and C8 substituted s i a l i c acids.  In this procedure PA  was performed for 2 hours. PAPS  = Periodate oxidation-phenylhydrazine-Schiff procedure  S  = Pararosaniline Schiff reagent  SA  = S i a l i c acids  T  = Thionin Schiff reagent  xv  ACKNOWLEDGEMENTS  I would l i k e to thank my supervisor, Dr. P.E. Reid, for his support, enthusiasm, and guidance during the course of this project.  I would also l i k e  to thank my research committee, Drs. D.E. Brooks, W.L. Dunn and G.G.S. Dutton, for their support and suggestions; Mrs. Linda Truman and Barbara Meulchen for their technical assistance i n previous studies; Charles Ramey f o r advice on technical matters, and to Anne Bishop, Pat Bemoe, and a l l those who assisted i n the preparation of this manuscript. And especially to my fiance, Mike, without whose support this thesis would not have been possible.  xvi  One ought every day at least, to hear a l i t t l e song, read a good poem, see a fine picture and, i f i t were possible, to speak a few reasonable words.  Goethe from "Wilhelm Meister's Apprenticeship" Book V, Chapter 1  Intelligence i s quickness to apprehend as distinct from a b i l i t y which i s capacity to act wisely on the thing apprehended.  Whitehead from "Adventures of Ideas" pg. 135  xvii  - 1 -  INTRODUCTION In order to understand the underlying mechanisms involved i n a disease process, a knowledge of the normal molecular events within a c e l l or bodily system i s required.  There are two approaches by which such knowledge can be  obtained - either chemical, or histochemical, with each approach having both advantages and disadvantages.  Chemical procedures have the advantage i n that  they allow for the i d e n t i f i c a t i o n of small molecules and permit the determination of the structure of macromolecules.  In addition, chemical  methods tend to be both specific and quantitative, and the mechanism of the reaction used i s frequently understood. They do not, however, allow for the l o c a l i z a t i o n of molecules, and large quantities of samples are often required before precise identification and quantitation can be obtained.  Further, f o r  most procedures, only a few samples can be examined concurrently.  In contrast,  histochemical methods have the disadvantage i n that small molecules are d i f f i cult to identify as they tend to be removed during f i x a t i o n and processing, and structural, quantitative, and mechanistic studies are d i f f i c u l t to carry out. Histochemistry has the advantage, however, i n that chemical events can be localized at the histological l e v e l , a characteristic unique to histochemistry.  The s e n s i t i v i t y of histochemical methods i s such that a direct  v i s u a l correlation can be obtained between structure and function such that individual c e l l u l a r metabolic a c t i v i t i e s can be readily recognized.  In  addition, histochemical methods require a minimal amount of tissue, many sections can be studied simultaneously, and the functional heterogeneity at individual c e l l levels can be visualized.  For example, small f o c i of change  can be seen whereas such a change cannot be detected by chemical procedures (Reid et a l . . 1985a). Further, i t i s possible to perform retrospective studies  - 2as the tissues are preserved for long periods of time.  The sensitivity of  histochemical reactions can be illustrated with the following example: i f one considers the average rat colon to be approximately 15 cms long, there are 30,000  5 um sections per colon. When e p i t h e l i a l c e l l s are isolated from rat  colon, approximately 4 mg of purified glycoprotein i s obtained.  Such glyco-  protein contains approximately 15% by weight s i a l i c acid, i . e . 0.6 ng s i a l i c acid (Reid et a l . 1975, 1977). Therefore, there are 0.6 x 1000 x 1000 = 20 nanograms SA/section 30,000 If i t i s assumed that there are 100 epithelial c e l l s per transverse section, then 0.2 nanograms/cell of SA can be detected histochemlcally. Currently, 0.2 ug/tube of SA can be detected chemically. This represents a difference i n s e n s i t i v i t y of 1000 times. Histochemical techniques have been applied to the study of many diseased systems, i n the diagnosis of certain leukemias and lymphomas, and for the determination of the s i t e of origin of a variety of malignancies ( F i l i p e and Lake, 1983). Histochemistry can increase the accuracy of histological diagnosis (Ehsanullah et a l . 1982a,b), has been used i n the assessment of malignant transformation and plays a role i n monitoring a patient's response to therapy ( F i l i p e and Lake, 1983). In addition, histochemical methods have shown that there are changes i n the e p i t h e l i a l glycoproteins associated with various colonic diseases such as colonic cancer, ulcerative c o l i t i s , and Crohn's disease (Culling et a l .  T  1975, 1977, 1979, 1981; Fakan and Adamocova, 1981;  Fenger and F i l i p e , 1981; F i l i p e 1979, 1984; F i l i p e e t _ a l . , 1980; Franzin et a l . 1981, 1983a,b, 1984; Lev et a l .  f  1985; L i s t i n s k l and Riddell, 1981; Nontero  - 3 -  and Segura, 1980; Reid et a l . 1980, 1984c,d, 1985a,b; Rhatigan and Saffros, T  1979; Spicer, 1965; Sunter et a l . . 1985). Most histochemical studies of the changes i n e p i t h e l i a l glycoproteins associated with colonic disease have been based on methods for the detection of s i a l i c acids, 0-acyl s i a l i c acids and O-sulphate ester (Fig. 1). Studies of the "neutral sugars" of these glycoproteins (hexose, 6-deoxyhexose and N-acetyhexosamlne), however, have been confined to the use of appropriately labelled lectins (Iannoni et a l . , 1986; Boland et a l . , 1982a,b, 1984; Bresalier et a l . , 1984; Cooper 1980, 1982, 1983; Yonesawa et a l . . 1982, 1983; Schulte and Spicer, 1983). Although l e c t i n methods are specific, they most commonly detect non- reducing terminal sugar residues and have not been used to determine the relative proportions of anionic groups ( s i a l i c acids and sulphate) and "neutral sugars".  There existed, therefore, a need for general histochemical methods  for the detection of "neutral sugars" and the relative proportions of such sugars and either s i a l i c acids and 0-acyl s i a l i c acids or O-sulphate ester. The development of such methods i s the subject of this thesis. There are at least two theoretical approaches for the detection of "neutral sugars".  The f i r s t involves the use of the periodic acid-phenylhydrazine-  Schiff (PAPS) procedure (Spicer 1961) (Fig. 2).  In this technique, aldehydes  produced by the i n i t i a l perlodate oxidation are condensed with  phenylhydrazine.  Subsequent treatment with Schiff reagent reverses the blockage of s i a l i c acid monoaldehydes, but not that of "neutral sugar" dialdehydes, resulting i n selective staining of s i a l i c acids (Reid et a l . , 1984a,b). I f , therefore, a coloured arylhydrazine can be used i n the phenylhydrazine Schiff sequence, or i f the hydrazine can be coloured i n a subsequent reaction then "neutral sugars" and s i a l i c acids can be visualized simultaneously.  Such a procedure has been  - 4 -  Fig. 1. Structure of sugar residues frequently encountered i n colonic glycoproteins  Shown i s the structure of a) s i a l i c acid without side chain substituents; s i a l i c acid with side chain O-acyl substituents at (b) position C7, (c) position C8, and (d) position C9; (e) O-sulphate ester; ( f ) hexose; (g) 6-deoxyhexose; and (h) N-acetyl hexosamine.  - 5 -  0 = C-CH  I  3  NH/f-OH -|-OH CH OH  COOH  2  OH  OH  0=OCH,  Cec-C 3 H  I  iS: 0CHX900H H  NH/  ^H OH 2  /I_Q  OH  NH  f0H  \  C O O H  ^COCOC^yLo OH  CH OH 2  e.  I-0-SO3H  OH OH OH  CH,OH  g C  H  3  OH  \ OH  HNCOCH,  - 6-  Fig. 2.  Diagramatic representation of the mechanism(s) bv which the periodic acld-phenvlhvdrazlne-Schiff  (PAPSprocedure results i n selective  detection of s i a l i c acids and the use of PAPS procedures i n the simultaneous visualization of s i a l i c acids and "neutral sugars"  Periodate oxidation results i n the oxidation of v i c i n a l diols to aldehydes (a-b) which are then condensed with phenylhydrazine (b-c). Subsequent treatment with Schiff reagent reverses the blockage of s i a l i c acid monoaldehydes (large arrow), but not that of "neutral sugar" dialdehydes (small arrow), resulting i n selective Schiff staining of s i a l i c acids.  I f a coloured  arylhydrazine i s substituted for phenylhydrazine or i f the hydrazine can be coloured i n a subsequent reaction, then the simultaneous visualization of "neutral sugars" and s i a l i c acids can be obtained.  - 7-  (a)  CHPH COOH OH OH  PA  PA  (b)  0=C-CH, COOH O  Ph  CoC-CH  3i  NHyk>~vCOOH  OH  bis hydrazone  Azido derivative  Morpholene derivative  side chain binding  - 8 developed by Park eJt al.  (1987b) using the coloured hydrazine 2,4-dinitro-  phenylhydrazine. The second approach involves the use of "selective oxidation" in which two step-wise periodate oxidations are employed (Fig. 3).  The f i r s t oxidation must  be carried out under reaction conditions in which a l l s i a l i c acids oxidize without significant oxidation of other carbohydrate residues.  The s i a l i c acids  are then either stained with Schiff or converted to Schiff unreactive alcohols with sodium borohydride. The second oxidation i s then performed under conditions in which "neutral sugars" oxidize (Volz et a l . , 1987a,b). Previous attempts at developing "selective oxidation" conditions were based on the use of dilute periodic acid (Klessen, 1978; Roberts, 1977; Veh et a l . , 1979; Weber et a l . 1975). Due to steric factors, the rate of oxidation of f  vicinal diols varies, with open chain diols (such as s i a l i c acids) oxidizing at a faster rate than ring cis diols which in turn oxidize at a faster rate than ring trans diols (Culling and Reid, 1977; Suttajit, 1970; McLean et a l . , 1971). From this i t was concluded that i f limited quantities of periodate were used, only s i a l i c acid diols would be oxidized.  The periodic acid-Schiff (PAS)  reaction could then be used to selectively detect s i a l i c acids.  It i s d i f f i -  cult, however, to estimate the amount of s i a l i c acid in a given tissue section and therefore to determine the amount of periodate required for the oxidation. Further, the above studies did not establish whether or not a l l s i a l i c acids present in the tissue had oxidized under the conditions used, and no studies were made to determine what effect the pH or ionic strength of the periodate solution would have on the oxidation rate of s i a l i c acids and/or "neutral sugars". In preliminary studies, Volz et a l . (1986) demonstrated that maximal PAS staining of s i a l i c acid residues with minimal staining of other carbohydrate  - 9 -  Fig. 3«  A proposed selective oxidation mechanism for the simultaneous visualization of "neutral sugars" and s i a l i c acid residues  The f i r s t oxidation i s carried out under conditions i n which a l l s i a l i c acids oxidize without significant oxidation of other carbohydrate residues (PA*) (a-b).  The s i a l i c acids are then either stained by a process yielding covalent  bonds (b-ci) or converted to Schiff unreactive alcohols by reduction with sodium borohydride (b-CQ or  b-C2).  The second oxidation i s then performed  under conditions which oxidize "neutral sugars" (c-d). The resultant aldehydes are then stained with Schiff reagent (d-e).  - 10 -  stains Magenta  stains Magenta  no staining  - 11 residues could best be obtained by oxidation with either aqueous solutions of 40 mM periodic acid for 2 minutes at 4°C or with 4.0 mM periodic acid for 2 minutes at room temperature (Illustration IA). Specific PAS staining of s i a l i c acid residues could be obtained with other concentrations of periodic acid, but maximal staining of s i a l i c acids could not be obtained without significant staining of "neutral" carbohydrate residues.  In addition,  oxidation of s i a l i c acid residues with very dilute solutions of periodic acid (0.04 mM and 0.004 mM) was extremely slow, requiring 48-72 hours to reach completion. Under such conditions  neutral carbohydrate residues, such as  those present i n l i v e r glycogen and the neutral glycoproteins of human stomach, oxidized s i g n i f i c a n t l y .  However, when oxidation was performed with very dilute  solutions of periodic acid i n either 0.125N sulphuric acid ( I l l u s t r a t i o n II) or 1M sodium chloride, the rate of oxidation of s i a l i c acid residues was significantly.  increased  Parallel studies demonstrated that the oxidation of l i v e r  glycogen and stomach mucin was unaffected when the reaction was carried out with 0.04 mM periodic acid i n 1M NaCl but was apparently decreased when 0.125 sulphuric acid was used as the solvent (Illustration I I I ) . Scott and Harbinson (1968, 1969; and Scott and Dorling, 1969) showed that the slow oxidation rate of the 2-3 v i c i n a l diols of the uronlc acid residues of glycosaminoglycans was due to the electrostatic f i e l d surrounding these molecules which repels the periodate ion, thereby inhibiting or retarding oxidation, an effect that can be overcome by increasing the ionic strength of the oxidant solution (Fig. 4).  I f a similar repulsion effect occurs when  s i a l i c acids are oxidized under conditions of low periodic acid concentration, then the reduction i n the s i a l i c acid oxidation rate may be such that the oxidation of other carbohydrate residues becomes significant.  I t becomes,  H  - 12 therefore, impossible to use very dilute periodic acid for the selective oxidation of s i a l i c acid residues.  However, i n concentrated perlodate, the  dual effect of lowering the pH and increasing the ionic strength of the solution would be expected to suppress the ionization of the s i a l i c acid carboxyl groups leading to a faster and more selective oxidation of such residues.  I t appeared possible, therefore, that manipulation of the oxidation  conditions could result i n a complete oxidation of s i a l i c acid residues without a significant oxidation of other carbohydrate residues. The objectives of this thesis were, therefore, to investigate the following questions: 1.  Is the increased oxidation rate i n 0.125H sulphuric acid due to a lowering of the pH or to an increase i n the ionic strength?  2.  Would the use of 1M. sodium chloride or a lower pH improve the s e l e c t i v i t y of the perlodate oxidation of s i a l i c acids, regardless of the periodic acid concentration used?  3.  Does the presence of sulphate esters i n glycoproteins affect the rate of s i a l i c acid oxidation (possibly by altering the electrostatic f i e l d i n the microenvironment of the s i a l i c acid molecule)?  i|.  Assuming that i t was possible to selectively oxidize a l l s i a l i c acids, then could such conditions be used to develop general methods for the detection of "neutral sugars" and the simultaneous visualization of these sugars and either s i a l i c acid and i t s side chain 0-acyl variants'*' or sulphate esters.  +  In the following account, s i a l i c acids without side chain 0-acyl substituents are referred to as CO and s i a l i c acids with 0-acyl substituents at positions 7, 8 and 9 are designated C7, C8 and C9 respectively. For histochemical purposes the C8 class of s i a l i c acids includes those with two- (C7C8, C7C9, C8C9) and three- (C7C8C9) side chain 0-acyl substituents.  - 13 -  Fig 4.  Effect of the electrostatic field surrounding molecules on periodate oxidation  Scott and Harbinson (1968,1969; Scott and Dorling, 1969) showed that the slow oxidation rate of the 2-3 vicinal diols of uronic acid residues of glycosaminoglycans was due to the electrostatic field surrounding these molecules which repels the periodate ion, thereby inhibiting or retarding oxidation (a). This effect can be overcome by increasing the ionic strength of the oxidant solution (b).  - 14 -  (a)  (b)  10."  10;  - 15 MATERIALS AND METHODS I. A.  Materials: Tissues Specimens of Spraque-Dawley rat l i v e r , salivary gland complex (including  the sublingual, submandibular, and parotid glands), and terminal ileum and colon obtained at autopsy immediately following death by ether anesthesia, and surgical specimens of human colon were fixed i n 10% formalin calcium for at least 7 days. After fixation, specimens of rat colon and terminal ileum were prepared as a single "Swiss R o l l " (Park et a l . , 1987a). The tissues were then processed through ethanol and xylene and embedded i n paraplast. Sections of 5 um thickness were cut with an E. Leitz Hetzlar Type 1212 model microtome, mounted on clean slides using chrome-alum gelatin as adhesive (Kiernan, 1981), and then treated overnight at 60°C i n an atmosphere of formalin vapour.  B.  Chemicals The product colour index number and supplier of staining dyes used i n this  study are as l i s t e d below:  Color Index Dye  Number  Supplier  Alcian Blue 8GX  74240  Gurr Chemicals  Pararosaniline hydrochloride  42500  Fisher S c i e n t i f i c Company  Thionin  52000  Fisher S c i e n t i f i c Company  - 16 I I . Histochemical Procedures 1) Sections were brought to water by treating successively with the following solutions for the times specified. xylene  5 min.  xylene  5 min.  100% alcohol  3 min.  100% alcohol  3 min.  95% alcohol  3 min.  70% alcohol  3 min.  water  rinse  2) Pararosanlllne (S) and thlonln (T) Schiff reagents were prepared by the method of Barger and DeLamater (1948).  3)  Sodium borohydrlde (Bh) reduction was performed with the procedure of L i l l i e and Pizzolato (1972).  4) Saponification (KOH) was carried out using 0.5% (w/v) potassium hydroxide dissolved i n 70% (v/v) ethanol for 15 minutes at room temperature (Culling et a l .  t  1974).  5) Periodate oxidation i)  In investigations of selective conditions for the oxidation of s i a l i c acids, periodate oxidation was performed for different time periods (some or a l l of 2,5,10,20 and 60 minutes and 1 and 2 hours) at either 4°C or room temperature using different concentrations of periodic  - 17 acid (0.004mM., 0.04mM_, 0.4mM., 4.0mM and 40.0mM.) i n each of the following solvents: d i s t i l l e d water, 1M. sodium chloride or IH hydrochloric acid.  ii)  In studies of the mechanism of selective oxidation of s i a l i c acid, and i n the development of methods for the simultaneous visualization of "neutral sugars" and either s i a l i c acid and i t s side chain O-acyl variants or O-sulphate ester, perlodate oxidation was performed with either 1% (w/v) aqueous periodic acid at room temperature, or with 0.4mM periodic acid i n approximately 1M. hydrochloric acid at 4°C. The l a t t e r was prepared by mixing equal volumes of stock solution of 0.8mM aqueous periodic acid and 2M. hydrochloric acid (HCl). The 2M. HCl was prepared by diluting concentrated HCl (1 volume) with d i s t i l l e d water (4 volumes). These stock solutions were stored at 4°C.  6)  Selective perlodate oxidation (PA») was performed as follows. i)  Bring sections to water.  ii)  Cool sections to 4°C and treat with a pre-cooled solution of 0.4mM periodic acid dissolved i n approximately 1M_ hydrochloric acid.  i i i ) Wash sections i n running water for 10 minutes at 4°C.  7)  Periodic Acid Schiff reaction (PAS). Sections were oxidized i n W (w/v) aqueous periodic acid for one hour at room temperature, washed i n running water for 10 minutes, and stained for 60 minutes with pararosaniline Schiff reagent. The staining intensity was visually assessed on the following  - 18 scale:  0 = no staining, t r = trace, 1 = weak, 2 = moderate, 3 = strong  and 4 = maximum.  8)  Periodic Acld-Borohvdrlde Reductlon-Saponlflcatlon (PA/Bh/KOH) method (Reid et a l .  T  1973; Culling et a l . 1974). In this procedure, treatment f  with 1% (w/v) periodic acid for 2 hours at room temperature oxidizes v i c i n a l diols to aldehydes which are then reduced with sodium borohydride (Bh) to primary alcohols. Following saponification, only s i a l i c acids with O-acyl substituents at C7, C8 or C9 (or which have 2 or 3 side chain O-acyl substituents) are PAS-positive.  9)  Saponlficatlon-Perlodlc acid oxldation-Borohvdrlde reduction sequence (K0H/PA3/Bh). In this procedure, acyl groups are removed by saponification.  Oxidation with 1% (w/v) periodic acid for 3 minutes  at room temperature (PA3) then converts a l l s i a l i c acids to the corresponding C7 aldehydes (Volz et a l . , 1986) which are then reduced to primary alcohols with sodium borohydride.  Positive PAS staining i n  K0H/PA3/Bh treated sections i s therefore confined to v i c i n a l diols on sugar residues other than s i a l i c acid (Volz gt a l . , 1986).  10) Alcian blue staining - was performed with alcian blue at either pH 1.0 or pH 2.5 as described by Culling (1974).  11) 2,4-Dlnltrophenylhvdrazlne - Tetrazotlzed 3 V-dlmethoxybenzidene f  fluoroborate procedure (DNPH-TDMBF). In this procedure periodate derived aldehydes are blocked by treatment with a saturated solution of 2,4-  - 19 -  dinitrophenylhydrazine (DNPH) i n 1M HCl at 4°C for 2 hours (Pearse 1968).  Formazans are then generated by treatment of the sections with  a freshly prepared solution of tetrazotized 3,3'-dimethoxybenzidene fluoroborate (TDMBF) i n 25% (v/v) aqueous pyridine for 3 minutes at room temperature (Stoward 1967a,b).  I I I . Methods based on selective oxidation of s i a l i c acid These methods are outlined i n Table I (Page 20).  A. Method 1. Simultaneous visualization of "neutral sugars" and s i a l i c acids with O-acyl substituents at positions C7 C8, and C9, the selective r  periodate oxidatlon-borohyride reductlon-saponlflcatlon-selectlve periodate oxidation-thlonln Schiff-saponlflcatlon-borohydride reduction-periodic acld-Schlff (PA»/Bh/KOH/PA»/T/KOH/Bh/PAS) procedure 1)  Bring sections to water.  2)  Cool sections to 4°C and oxidize i n a precooled solution of 0.4mM. periodic acid i n approximately 1M HCl for 1 hour at 4°C.  3)  Wash i n running water for 10 minutes at 4°C.  4)  Reduce with 0.1% (w/v) sodium borohydride i n 1% (w/v) dibasic sodium phosphate (anhydrous) for 20 minutes at room temperature.  5)  Wash i n running water for 10 minutes at room temperature.  6)  Rinse i n 70% (v/v) ethanol.  - 20 Table I  Outline of methods for the simultaneous visualization of "neutral sugars" and either s i a l i c acid and i t s side chain 0-acvl variants or 0-sulohate ester  x = step performed; - = step omitted a) Washing steps have not been included i n Table.  Selective perlodate oxidation (PA*) = 0.4 mM periodic acid i n approximately 1M hydrochloric acid for one hour at 4°C Borohydride reduction (Bh) = 0.1% sodium borohydride i n 1% (anhydrous) dibasic sodium phosphate for 20 minutes at room temperature Saponification (KOH) = 0.5% potassium hydroxide i n 70% ethanol 15 min at room temperature Thionin Schiff (T) = Thionin Schiff for 2 hr at room temperature Alcian blue pH 1.0 (AB 1.0)  = 0.3% Alcian blue 8GX i n 0.1 M hydrochloric  acid for 30 min. at room temperature (Culling  1974)  Periodic Acid - Schiff (PAS) = oxidation with 1% periodic acid for 1 hr at room temperature followed by pararosaniline Schiff for 1 hr at room temperature  - 21 -  Method( ) a  1  Selective Periodate Oxidation (PA*)  X  Borohydride Reduction  (Bh)  X  Saponification  (KOH)  Selective Periodate Oxidation (PA*)  2  X X  3  X X  X  Thionin Schiff  (T)  X  X  X  Saponification  (KOH)  X  X  -  Borohydride Reduction  (Bh)  X  X  X  Alcian Blue pH 1.0  (AB)  -  Periodic Acid Schiff  (PAS)  X  Saponification  (KOH)  X  X X  - 22 7)  Saponify with 0.5% (w/v) potassium hydroxide i n 70% (v/v) ethanol for 15 minutes at room temperature.  8)  Hash i n running water for 10 minutes at room temperature.  9)  Cool to 4°C and again oxidize i n pre-cooled O.MmM. periodic acid dissolved i n approximately 1M_ hydrochloric acid f o r 1 hour at 4°C. Wash i n running water for 10 minutes at 4°C. Stain with freshly prepared thionin Schiff reagent for 2 hours at room temperature. Wash i n running water for 10 minutes at room temperature. Rinse i n 70% (v/v) ethanol. Saponify with 0.5% (w/v) potassium hydroxide i n 70% (v/v) ethanol for 15 minutes at room temperature. Wash i n running water for 10 minutes. Reduce with 0.1% (w/v) sodium borohydride i n 1% (w/v) dibasic sodium phosphate (anhydrous) for 20 minutes at room temperature. Wash i n running water for 10 minutes at room temperature. Oxidize i n 1% (w/v) aqueous periodic acid for 1 hour at room temperature. Wash i n running water for 10 minutes. Stain with freshly prepared pararosaniline Schiff reagent for 1 hour at room temperature. Wash i n running water for 10 minutes. Dehydrate (through series of 95%, 100% alcohol), clear i n xylene and mount with permount.  - 23 Staining Results In this procedure, s i a l i c acids with 0-acyl substituents at positions C7, C8, or C9 (and s i a l i c acids containing two or three side chain O-acyl substituents) stain blue, while "neutral sugars" with oxidizable v i c i n a l diols stain magenta. Mixtures of these components stain i n various shades of purple.  B. Method 2: Simultaneous visualization of "neutral sugars" and t o t a l s i a l i c acid, the saponification-selective perlodate oxldatlon-thlonln Schiff-saponiflcation-borohydride reduction-periodic acid-Schiff (KOH/PA«/T/KOH/Bh/PAS) procedure 1)  Bring sections to water.  2)  Perform steps 6-22 of Method 1.  Staining Results In this procedure a l l s i a l i c acids stain blue, "neutral sugars" (with oxidizable v i c i n a l diols) stain magenta, and mixtures stain i n various shades of purple.  C. Method 3s  Simultaneous visualization of "neutral sugars" and s i a l i c acids  without side chain substituents or which have 0-acyl substituents at C7. the selective perlodate oxldatlon-thlonln Schiff-borohydride reductionperiodic acld-Schlff-saponlflcatlon fPA»/T/Bh/PAS/KOm procedure. 1)  Bring sections to water.  2)  Perform steps 9-12,  16-21, 13-15, and 22 of Method 1 ( i n this order).  - 24 Staining results In this procedure s i a l i c acids without O-acyl side chain substituents or with a substituent located at position C7 stain blue, "neutral sugars" (with oxidizable vicinal diols) stain magenta, and mixtures stain i n various shades of purple.  D.  Method 1 : Simultaneous visualization of "neutral sugars" and O-sulphate esters, the saponification-selective periodate oxldatlon-borohvdrlde reductlon-Alclan blue pH 1.0-perlodlc acld-Schlff (K0H/PA»/Bh/AB1.O/PAS) procedure. 1)  Bring sections to water.  2)  Perform steps 6-10 and 16-17 of Method 1.  3)  Stain with 0.3% (w/v) Alcian blue 8GX in 0.1M. HCl pH 1.0 for 30 minutes at room temperature.  4)  Rinse briefly with 0.1M. HCl pH 1.0.  5)  Wash i n running water for 10 minutes.  6)  Perform steps 18-22 of Method 1.  Staining results "Neutral sugars" (with oxidizable vicinal diols) stain magenta; O-sulphate esters stain aqua and mixtures stain in various shades of purple.  - 25 E. Method 5: Selective staining of "neutral sugars" the saponificationselective perlodate oxldatlon-borohvdrlde  reduction-periodic acld-Sohlff  (KOH/PA»/Bh/PAS) procedure. 1)  Bring section to water.  2)  Perform steps 6-10 and 16-22 of Method 1.  Staining results "Neutral sugars" stain magenta.  F. Control Methods When performing Methods 1-5, controls are required to ensure proper functioning of the method. These are as follows: 1) Sections of rat l i v e r and parotid gland treated with either the PA*/S or the PA*/Bh/PA*/S sequences should be unstained as they contain no s i a l i c acid, and "neutral sugars" should not oxidize under conditions of PA*. Sections of rat l i v e r and parotid glands should stain only magenta with Methods 1-5.  2) When treated with the PA/Bh/KOH/PA*/S sequence, sections of human colon containing 8-0-acyl s i a l i c acids should stain intensely while similar tissues exposed to the sequence PA/Bh/KOH/PA*/Bh/PA*/S should be unstained, an indication that a l l s i a l i c acid i s oxidized under conditions of PA*.  - 26 RESULTS AND DISCUSSION I.  Establishment of conditions for the selective oxidation of s i a l i c acid The f i r s t objective of this study was to develop a method for the selective  detection of s i a l i c acids i n tissues based upon the previous studies of Volz fit a l . (1986).  To accomplish t h i s , conditions were sought i n which s i a l i c  acids oxidized maximally but other carbohydrate residues did not oxidize significantly.  Two parameters were investigated simultaneously.  a) To determine whether the increased oxidation rate i n 0.125H sulphuric acid (Volz et a l . , 1986) was due to a lowering of the pH or was due to an increase in ionic strength, oxidations were performed i n 1M_ HCl and 1M. NaCl as solvents. These provided solutions similar i n ionic strength, but differing i n pH. b) To determine whether the oxidation rate of s i a l i c acid i s affected by the presence or absence of sulphate esters, comparative studies were carried out with the tissues shown i n Table I I (Page 27).  These tissues were selected  because they provided examples of neutral polysaccharides alone, sialoglycoproteins, and sialosulphoglycoproteins, and their histochemical staining patterns had been established. Untreated sections of rat l i v e r , and PA/Bh/KOH pretreated sections of rat salivary gland complex and rat colon were oxidized at 4°C with 40mM., 4.0mM., 0.4mM, 0.04mM. and 0.004mM periodic acid i n one of water, 1M_ sodium chloride, or 1M. hydrochloric acid for each of 2, 5 and 60 minutes and 24 and 48 hours, stained with Schiff reagent, and the intensity of staining assessed. The results of this experiment confirmed that 40mM. perlodate i n water for 2 minutes at 4°C and 4.0mM. perlodate i n water for 2 minutes at room temperature were selective for the oxidation of s i a l i c acids. Further, selective oxidation  J  - 27 -  Table U Location and histochemical characteristics of the carbohydrate containing macromolecules studied during the Investigation of methods for the selective oxidation of s i a l i c acids  - 28  -  Tissue  Macromolecule examined  Histochemical characteristics  Rat Liver  Glycogen  Neutral PAS reactive macromolecule  Rat Salivary Glands ^ Parotid / Acinar c e l U ^ granules.  Sublingual  Rat Colon  Acinar c e l l glycoprotein  /Epithelial (glycoprotelr  Proximal (lower half of crypts)  Sialoglycoprotein containing side chain substituted s i a l i c acids (Park gt a l . , unpublished)  Sialoglycoproteins containing side chain O-acylated s i a l i c acids but l i t t l e or no sulphate ester (Park et a l . , unpublished) Sialosulphoglycoproteins containing side chain O-acylated s i a l i c acids (Park et a l . , unpublished)  Distal  Human Colon  "Neutral" PAS reactive macromolecule (Munhoz, 1971: Schackleford and Klapper, 1962; Spicer and Duvenci, 1964; Simson gt a l . , 1973; Park e i a l . , published)  Epithelial glycoproteins  Sialosulphoglycoproteins containing side chain O-acylated s i a l i c acids ( F i l i p e , 1979; Culling fit a l . , 1981)  - 29 -  was also obtained with 0.4mM periodate i n 1M HCl for 1 hour at 4°C ( I l l u s t r a t i o n Ib). This l a t t e r set of conditions appeared to be the most selective since the staining of rat l i v e r and rat parotid gland (containing neutral carbohydrate molecules) was reduced under these conditions when compared to that obtained when either water or 1M NaCl were used as the solvent at the same periodate concentration (Table I I I and I l l u s t r a t i o n I I I ) . The different results obtained with 1M NaCl and 1M HCl, therefore, can be attributed to a lowering of pH rather than to an increase i n ionic strength. F i n a l l y , the rate of oxidation of s i a l i c acid residues was not affected by changes i n the pH of the solvent used whether the tissue contained (rat colon) or was unsulphated (rat sublingual gland).  sulphate  The rate of oxidation  of s i a l i c acid i s , therefore, independent of the presence or absence of O-sulphate ester i n the glycoprotein. In conclusion, i t appears that the oxidation rate of s i a l i c acid i n tissues i s dependent on the pH of the oxidation solution, the concentration of periodic acid used, and i s independent of the presence or absence of sulphate i n the glycoprotein.  - 30 -  Table I I I  The effect of pH on the oxidation of neutral macromolecules. The oxidation of neutral macromolecules with 0.4 mM. periodic acid (as demonstrated by the intensity of Schiff staining) i s greater when water (aq) or 1ft NaCl l s used as solvent i n place of 1M. HCl. The staining intensity was graded on the following scale: 0 = no staining, t r = trace, 1+ = weak, 2+ = moderate, 3+ = strong, 4+ = maximum.  - 31 -  TIME  Rat Liver  Rat Parotid Gland  (hours) IMNaCl  1M. HCl  IMNaCl  1M. HCl  1  2+  tr  1+  tr  21  3+  1+  2+  tr  48  4+  1+  2+  tr  - 32 I I . Investigation of oxidation with 0.4mM Periodic acid i n approximately 1M HCl  A.  Specificity In the previous study i t was shown that treatment with 0.4mM periodic acid  i n 1M HCl a t 4°C f o r one hour (PA*) resulted i n the s e l e c t i v e oxidation o f a l l s i a l i c acids. To further investigate the s e l e c t i v i t y o f the PA* technique, a s e r i e s o f control experiments was performed to demonstrate that under conditions o f PA*, a l l s i a l i c acids were oxidized, oxidation o f "neutral sugars" was i n s i g n i f i c a n t , and glycoproteins were not extracted or de-O-acylated.  These experiments are summarized i n Table IV (Page 33)-  To study the oxidation o f s i a l i c acid residues with the PA* technique, sections o f r a t ileum, colon, s a l i v a r y glands, and human colon were treated with the PA/Bh/KOH sequence t o confine v i c i n a l d i o l s to s i a l i c acid residues. The sections were then treated with each of 1% periodic acid f o r 1 hour at room temperature (PA/Bh/K0H/PA6n/S), PA* (PA/Bh/KOH/PA*/S) or with the PA* procedure followed by oxidation with 1% periodic acid f o r 1 hour at room temperature (PA/Bh/KOH/PA*/PA6o/S).  No difference i n the s t a i n i n g i n t e n s i t y  of these three treatments could be detected PA* oxidized a l l s i a l i c acid residues.  ( I l l u s t r a t i o n IV), suggesting  that  In a second s e t o f experiments,  sections of human colon treated with the PA/Bh/KOH/PA*/S sequence yielded a strong (4+) S c h i f f reaction.  However, when s e r i a l sections were reduced  with borohydride a f t e r the PA* step and then further oxidized with PA* (PA/Bh/KOH/PA*/Bh/PA*/S) o r with 1% periodic acid f o r 1 hour at room temperature (PA/Bh/K0H/PA*/Bh/PA6Q/S), no S c h i f f s t a i n i n g was obtained. This indicated that when only s i a l i c acids were present, they are completely oxidized under PA* conditions ( I l l u s t r a t i o n V).  - 33  -  Table IV  Evidence for the s e l e c t i v i t y of periodate oxidation with 0.4 mM periodic acid  in 1M hydrochloric acid at 4°C for one hour  The nature of the macromolecules studied i n these tissue sites i s summarized i n Table II (Page 27).  PA3  and  PA60  = oxidation with 1% (w/v) periodic acid at room temperature  for 3 and 60 minutes, respectively PA* = oxidation with 0.4 mM periodic acid i n approximately 1M hydrochloric acid at 4°C for one hour KOH = saponification Bh = borohydride reduction ( L i l l i e and Pizzolato 1972) S = treatment with pararosaniline Schiff AB 2.5 and AB 1.0 = staining with alcian blue 8GX at pH 2.5 and pH respectively (Culling 1974) 0 = no staining 4+ = strong staining  1.0,  Tissue  Procedure and Results  Interpretation  Rat colon and sublingual gland and human colon  PA/Bh/K0H/PA6 /S = 4+ PA/Bh/KOH/PA*/S = 4+ PA/Bh/KOH/PA*/PA6o/S = 4+  Human colon  PA/Bh/KOH/PA*/S = 4+ PA/Bh/KOH/PA*/Bh/PA*/S = 0 PA/Bh/K0H/PA*/Bh/PA6Q/S = 0  Rat l i v e r and parotid gland Rat colon  PA*/S = 0  Oxidation with PA* produces no v i s i b l e Schiff with neutral macromolecules.  KOH/PA /Bh/PA6o/S = 4+ KOH/PA /Bh/PA*/PA6o/S =4+ KOH/PA /Bh/PA*/S = 0  Following removal of O-acyl esters with KOH, the PA3/BI1 sequence confines v i c i n a l diols to "neutral sugars" i n s i a l o - and sialosulphoglycoproteins. Absence of Schiff staining following PA* but strong staining after PAgg indicates PA* does not produce Schiff reactive sites from "neutral sugars".  KOH/PA*/S s  PA* oxidizes a l l s i a l i c acids but does not produce Schiff positive sites with "neutral sugars".  0  The PA/Bh/KOH sequence confines v i c i n a l diols to s i a l i c acid residues. Subsequent oxidation with either PA50 or PA* gives Schiff staining of the same intensity and following borohydride reduction of PA* engendered aldehydes, PA* produces no further Schiff reactivity. Therefore, PA* oxidized a l l s i a l i c acids.  3  3  3  Rat colon and sublingual gland human colon Rat colon and sublingual gland  4+  KOH/PA*/Bh/PA*/S =  K0H/PA*/Bh/PA6Q/S  4+  AB 2.5 = 4+ PA/Bh/KOH/AB2.5 = 4+ PA/Bh/KOH/1M HCl, 4°C, 1  AB1.0 = 4+ K0H/PA+/Bh/AB1.0 = 4+ Rat colon PA/Bh/PAgQ/S = 0 and subPA/Bh/1M HCl, 4°C, lingual gland 1 h/PAgo/S=0 and human colon  4+  PA* or 1 M HCl, 4°C, 1 hour does not extract e p i t h e l i a l glycoproteins.  Rat colon and human colon  O-Acyl esters are stable to 1 M HCl at 4°C for 1 hour.  - 35  -  To study the effect of PA* on "neutral sugars", tissues containing macromolecules with only "neutral sugars", such as rat l i v e r and parotid gland, were subjected to PA* followed by Schiff. Oxidation under these conditions produced no significant staining of either l i v e r glycogen ( I l l u s t r a t i o n Via) or the PAS positive granules of rat parotid gland.  PAS reactivity was  produced, however, when oxidation with O.iimM periodic acid i n 1M. HCl at 1°C was performed for 24 hours (Illustration VIb) or when the routine PAS procedure was employed ( I l l u s t r a t i o n Vic). These results Indicate that PA* does not produce significant Schiff staining of "neutral sugars". The oxidation of the "neutral sugars" of s i a l o - and sialosulphoglycoproteins was investigated i n sections of rat ileum-colon which were pretreated with the KOH/PAj/Bh sequence to confine v i c i n a l diols to "neutral sugar" residues (Volz et a l . , 1986). Subsequent oxidation of these sections with PA* (K0H/PA3/Bh/PA*) produced no Schiff reactivity ( I l l u s t r a t i o n V i l a ) .  In  contrast, oxidation with either 1% periodic acid for 1 hour at room temperature (KOH/PA3/Bh/PA6o), or with PA* followed by 1% periodic acid for 1 hour at room temperature (KOH/PA3/Bh/PA*/PA6o)» resulted i n similar strong (4+)  PAS  staining, indicating that PAS-reactive "neutral sugar" residues are present and available for oxidation, but do not oxidize and then stain under conditions of PA*. To investigate the oxidation of s i a l i c acid i n the presence of the "neutral sugars" of s i a l o - and sulphosialoglycoproteins, sections of rat ileum-colon and salivary gland complex and human colon were f i r s t saponified to remove O-acyl esters.  Oxidation with PA* (KOH/PA*/S) then resulted i n strong (4+) Schiff  reactivity.  I f , however, saponification and PA* treatment were followed by  - 36  -  reduction with borohydride, further oxidation with PA* (KOH/PA*/Bh/PA*/S) followed by Schiff, produced no Schiff reactivity.  When, however, the second  oxidation step was carried out using 1% periodic acid for 1 hour at room temperature (KOH/PAVBh/PAgo)* a strong Schiff reaction resulted.  This  indicated that PA* oxidized a l l s i a l i c acids regardless of the presence of either "neutral sugars" or O-sulphate ester i n the glycoprotein. Next, a number of experiments were conducted to investigate any potential non-specific effects which may occur under PA* conditions.  The conditions of  PA* are acidic, therefore the extraction of glycoproteins from the tissues may occur. To eliminate this p o s s i b i l i t y , PA/Bh/KOH treated sections of rat ileum-colon and salivary gland were incubated i n PA* or 1M HCl at 4°C for 1 hour and were then stained with Alcian blue pH 2.5 (a stain which detects sulphate and carboxyl groups).  The Alcian blue pH 2.5 staining of these  sections did not d i f f e r from that of untreated control sections (Alcian blue pH 2.5).  Further, the Alcian blue pH 1.0 (stains sulphate groups) staining of  rat ileum-colon and human colon was unaffected by the K0H/PA*/Bh sequence ( I l l u s t r a t i o n VIII).  These data indicate that PA* does not extract e p i t h e l i a l  glycoproteins. To determine whether PA* de-O-acetylates s i a l i c acids, sections of rat ileum-colon and salivary gland complex and human colon were treated with the PA/Bh sequence to remove a l l oxidizable v i c i n a l diols.  Sections were then  incubated with 1M HCl for 1 hour at 4° C (Illustration IX). These sections were not PAS-reactive, indicating that such conditions do not de-O-acetylate s i a l i c acids.  F i n a l l y , to determine whether conditions of PA* invoke a Feulgen  reaction, sections of rat l i v e r , ileum-colon, salivary gland complex and human  - 37 colon were incubated with e i t h e r 1M HCl f o r 1 hour at 4°C or with PA* ( I l l u s t r a t i o n X). A minimal S c h i f f staining was obtained.  However, when  sections were incubated i n PA* f o r 24 or 48 hours, strong Feulgen s t a i n i n g was observed.  The Feulgen reaction, however, would not be expected to i n t e r f e r e  with s i a l i c acid detection i n the PA*/S procedure.  B. Mechanistic Studies of PA* Conditions In the previous section i t was demonstrated that PA* d i d not form S c h i f f p o s i t i v e products with "neutral sugars".  However, whether "neutral sugars"  oxidize i n PA* and form Schiff-negative products such as hemialdals or hemiacetals, or whether they e i t h e r do not oxidize or do not produce a s u f f i c i e n t quantity of aldehydes to produce a v i s i b l e S c h i f f reaction, was not determined.  To investigate t h i s , sections of r a t l i v e r , ileum-colon and  s a l i v a r y gland complex were subjected to the KOH/PA^/Bh technique to confine v i c i n a l d i o l s to "neutral sugars", and were then oxidized with one of PA* (K0H/PA3/Bh/PA*) ( F i g . 5B),  1% periodic acid f o r 1 hour a t room  (KOH/PA/Bh/PAgo) or both PA* and PAgn (K0H/PA/Bh/PA/PA6 ) 0  temperature  ( F i g . 5A).  The sections were then treated with either S c h i f f reagent or with 2,4-dinitrophenylhydrazine (DNPH) followed by tetrazotized 3,3'-dimethoxybenzidine 1967b) ( F i g . 5A and 5B).  fluoroborate (TDMBF) (Stoward,  Sections exposed to  the KOH/PA /Bh/PA6 and KOH/PA /Bh/PA*/PA6o were strongly S c h i f f 3  0  3  p o s i t i v e and yielded formazans on treatment with DNPH and TDMBF ( F i g . 5A). However, sections exposed to the KOH/PA^/Bh/PA* sequence were S c h i f f unreactive and showed only traces of formazan with the DNPH/TDMBF sequence ( F i g . 5B). These data imply that PA* does not produce hemiacetals or hemialdals ( I l l u s t r a t i o n XIa & b ) .  - 38 -  F i g . 5A and 5B. Flow diagram of the investigation into the mechanism by which "neutral sugars" react following treatment with PA* and Schiff reagent  Sections of rat l i v e r , ileum-colon and salivary glands were subjected to the K0H/PA3/Bh technique to confine vicinal diols to "neutral sugars", and were oxidized with either 1% periodic acid for 1 hour at room temperature, or both PA* and PA6Q (Fig. 5A) or PA* (Fig. 5B).  Sections were then treated with  either Schiff reagent or 2,4-dinitrophenylhydrazine (DNPH) followed by tetrazotized 3,3-dimethoxybenzidine fluoroborate (TDMBF) (Stoward, 1967b). Sections treated with PA or the PA*/PA combination were strongly Schiff positive and yielded formazans on treatment with DNPH and TDMBF (Fig. 5A). Sections treated with PA*, however, were Schiff-unreactive and showed only traces of formazan with the DNPH/TDMBF sequence (Fig. 5B). These data imply that PA* does not produce hemiacetals or hemialdals.  -  Fig. 5A  39 -  - 40 -  - 41 -  Fig. 6. I l l u s t r a t i o n of the 2 4-dlnltrophenvlhydrazlne f  3,V-dlmethoxvbenzidlne  fluoroborate (DNPH-TDMBF) procedure  Sections of glycoprotein containing tissues (such as rat colon) are oxidized to produce aldehydes (a-b). Sections are then treated with 2,4-dlnltrophenylhydrazlne (DNPH) which reacts with the aldehydes to form phenylhydrazones (b-c).  Next, treatment with tetrazotized 3,3'-dimethoxybenzidlne fluoroborate  (TDMBF) (c-d) results i n the formation of formazans (d).  42  H,OH  PA CHbOH  DNPH CH OH 2  O  i TDMBF CH OH 2  O  Arformazan formazan  - 113 C.  Discussion and Conclusions In this study i t was shown that PA* (0.4 mM periodic acid i n 1M. HCl at  4°C for 1 hour) completely oxidizes the side chain t r i o l of a l l s i a l i c acid residues.  Under such conditions there was no v i s i b l e staining of the neutral  macromolecules In rat l i v e r and parotid glands, or of the "neutral sugar" residues of either s i a l o - or sialosulphoglycoproteins.  In addition, the  reagent did not extract e p i t h e l i a l glycoproteins, de-O-acylate the side chain of s i a l i c acid residues, or produce a significant Feulgen reaction. oxidation with 0.4  Therefore,  mM periodic acid i n 1M HCl at 4°C i n 1 hour results i n the  selective visualization of total s i a l i c acids and can be used to identify s i a l i c acids with PAS techniques. The mechanism by which the selective oxidation of s i a l i c acid residues with 0.4  mM periodic acid i n 1M HCl occurs has not been f u l l y established.  These oxidation conditions did not produce Schiff-positive reactive s i t e s with "neutral sugars". This does not appear to be due to the formation of Schiff unreactive hemiacetals or hemialdals (Guthrie, 1961; Stoward, 1967a,b,c) since such derivatives would be expected to form bis 2,4-dinitrophenylhydrazones and therefore produce formazans on subsequent treatment with tetrazotized  3»3'-  dimethoxybenzidine fluoroborate (Mester, 1955, 1958; Chittenden & Guthrie, 1963; Stoward, 1967a). Further, extended oxidation of "neutral sugars" with PA* produced Schiff reactivity.  I t i s probable, therefore, that oxidation of  "neutral sugars" with PA* results i n too few aldehyde groups to produce a v i s i b l e reaction product with Schiff reagent. Volz et a l . (1986) suggested that the increased rate of oxidation observed when s i a l i c acid residues are oxidized with very dilute periodic acid i n 0.125  N sulphuric acid was due to the suppression of the ionization of the  - 44 carboxyl groups of the s i a l i c acid residues.  Therefore, the s e l e c t i v i t y of  the PA* technique i s probably a result of an increase i n the oxidation rate of s i a l i c acids together with a decrease i n the oxidation rate of "neutral sugars". The selective periodate oxidation method for s i a l i c acids (PA*/S), described here, provides a rapid, technically simple substitute for the PAPS reactions, a method currently used to selectively visualize s i a l i c acids.  - 45 I I I . Development of Methods Based on PA* Selectivity Having established conditions for the selective oxidation of s i a l i c acids which do not significantly oxidize other carbohydrate residues, the potential existed for the development of methods for the simultaneous visualization of "neutral sugars" and either s i a l i c acid and i t s side chain 0-acyl variants or O-sulphate ester. The development of such methods would permit the determination of the types and relative proportions of the various sugar residues i n normal tissues and i t would therefore be possible to detect changes i n the types and ratios of these residues i n diseased tissues. The f i n a l objective of this study was, therefore, to develop methods for (1) the simultaneous visualization of "neutral sugars" with perlodate sensitive v i c i n a l diols (hexose, 6-deoxyhexose and N-acetyl hexosamine) and either s i a l i c acids and their 0-acyl side chain variants or O-sulphate ester and ( i i ) the selective identification of "neutral sugars" i n s i a l i c acid-containing tissues. The five histochemical procedures devised i n this study are l i s t e d i n Table I. Table V shows the results expected when these methods are applied to tissues composed of structural elements including "neutral sugars" with perlodate oxidizable v i c i n a l diols, s i a l i c acids without side chain 0-acyl substituents or with an 0-acyl substituent at C7, C8, or C9 (or which have either two or three side chain 0-acyl substituents) and O-sulphate esters.  A.  Results of Methods 1-5  Method 1:  Method for the Simultaneous Visualization of S i a l i c Acids with  0-acyl substituents at positions C7  f  C8. and C9 and "Neutral Sugars"  Method 1, the selective perlodate oxidation-borohydride  reductlon-  saponification-thionin Schiff-saponiflcation-borohydride reduction-periodic  - 46 Table V Predicted results of the application of the methods outlined In Table 1 to tissue sites containing s i a l i c acids both with and without side chain O-acvl substituents "neutral sugar" v i c i n a l diols and O-sulphate ester f  a) Neutral sugar = hexose, 6-deoxyhexose and N-acetyl hexosamine residues b) I f 9-0-acyl s i a l i c acids oxidize i n the i n i t i a l PA* steps of methods 1 and 3, then they w i l l appear i n the CO class of s i a l i c acids. c) For histochemical purposes 8-0-acyl s i a l i c acids include s i a l i c acids with two -(C7C8, C7C9, C8C9) or three - (C7C8C9) O-acyl substituents.  0 = no staining; M = magenta; B = blue; A = aqua. The term aqua i s used to distinguish the aquamarine shade imparted by Alcian blue from the grape blue produced following staining with thionin Schiff. Other abbreviations used are as i n Table I.  Results of the application of Methods 1-5 on the staining patterns of the tissues l i s t e d here were predicted on the basis of the known composition of these tissues (Table II - Page 27).  - 47 -  S i a l i c Acid 0-acyl side chain substitution Method  Procedure  O-sulphate ester  "Neutral Sugar" v i c i n a l d i o l a)  O  None b)  C7  B  C8 and C9  1  PA*/Bh/KOH/PA*/T/KOH/Bh/PAS  0  M  B  2  KOH/PA*/T/KOH/Bh/PAS  0  M  B  B  B  3  PA«/T/Bh/PAS/KOH  0  M  B  B  0  4  K0H/PA*/Bh/AB1.0/PAS  A  M  0  0  0  5  KOH/PA*/Bh/PAS  0  M  0  0  0  - H8 acid-Schiff technique; i n the i n i t i a l PA* oxidation step of this procedure, s i a l i c acids without side chain O-acyl substituents or with an O-acyl substituent at C7 are selectively oxidized to aldehydes. The O-acyl groups of s i a l i c acids with side chain O-acyl substituents at C7, C8 and C9 are then removed (saponification step), rendering them available for oxidation i n the second PA* step. The aldehydes produced are then stained blue with thionin Schiff reagent.  In the subsequent steps of the sequence, non-specific thionin  staining i s removed by saponification (KOH), the sections are reduced with sodium borohydride, and then "neutral sugar" v i c i n a l diols are oxidized and stained magenta with the periodic acid Schiff technique.  In this method,  s i a l i c acids with side chains O-acyl substituents at C7, C8 and C9 stain blue, and "neutral sugar" residues stain magenta (Fig. 9, I l l u s t r a t i o n XII).  Method 2: Method for the Simultaneous Visualization of Total S i a l i c Acid Residues and "Neutral Sugars" In this method, the saponification-selective periodate oxidation-thlonln Schiff-saponification-borohydride reduction-periodic acid-Schiff technique, the i n i t i a l saponification step removes O-acyl esters from a l l s i a l i c acids. These residues are then oxidized with PA* and subsequently stained blue with thionin Schiff reagent. The "neutral sugars" are then stained magenta with the same KOH/Bh/PAS technique as used i n Method 1 (Fig. 9, I l l u s t r a t i o n XIII).  - 49  -  Fig. 7: The mechanism and expected staining patterns for Method 1, the selective periodate oxidation-borohydride  reduction-saponification selective  periodate oxidatlon-thionin Schiff-saponification-borohydride reductionperiodic acid-Schlff (PA*/Bh/KOH/PA*/T/KOH/Bh/PAS) technique, and Method 2, the saponification-selective periodate oxidation-thionin Schiff-saponificationborohydride reduction-periodic acid Schiff (KOH/PA*/T/KOH/Bh/PAS) technique. In Method 1, i n the i n i t i a l PA* step (a-b), s i a l i c acids without side chain O-acyl substituents or with an O-acyl substituent at C7 are oxidized to aldehydes and reduced to Schiff unreactive primary alcohols with borohydride. They are not, therefore, identified i n this procedure. Saponification (c-d) then removes acyl groups including those located on s i a l i c acids at position C7, C8, or C9 (or which had 2 or 3 side chain substituents) which are then available for oxidation (d-e) i n the second PA* step (d-e) and are stained with thionin Schiff (e-f).  Non-specific thionin Schiff staining i s then removed by  saponification (f-g), tissues are reduced with borohydride (g-h), and then "neutral sugar" v i c i n a l diols are oxidized with PA (h-i) and stained magenta with Schiff reagent ( i - j ) .  In Method 2, the i n i t i a l saponification step (c-d)  results i n the oxidation of a l l s i a l i c acids (d-e) which were then stained with thionin Schiff (e-f) following the i n i t i a l PA* step (d-e).  Non-specific  staining of thionin i s then removed with KOH (f-g), tissues are reduced with borohydride (g-h) and "neutral sugars" are then oxidized with PA (h-i) and stained magenta with pararosaniline Schiff ( i - j ) .  -  50 -  - 51 Method 3: Method f o r the Simultaneous Visualization of S i a l i c Acids without Side Chain Substituents or with O-Acyl Substituents at C7 and "Neutral Sugar" Residues In t h i s method, the selective perlodate oxidation-thionin S c h i f f borohydride reduction-periodic acid-Schiff-saponification technique, s i a l i c acids without side chain substituents or with 0-acyl substituents at C7 stain blue with the selective perlodate oxidation-thionin Schiff sequence. Following borohydride reduction, "neutral sugars" are stained magenta with the PAS method and non-specific thionin staining i s then removed by saponification (Fig. 8, I l l u s t r a t i o n XIV).  Method 4:  Simultaneous Visualization of A l l S i a l i c Acids and O-sulphate Esters  In t h i s method, the saponification-selective perlodate oxidationborohydride reduction-Alcian blue pH 1.0-periodic acid-Schiff technique, the i n i t i a l KOH/PA*/Bh sequence renders a l l s i a l i c acids perlodate unreactive. 1.0.  The O-sulphate esters are then stained aqua with Alcian blue at pH  The "neutral sugars" are then oxidized and stained magenta with the PAS  procedure (Fig. 9, I l l u s t r a t i o n XV).  Method 5: Method f o r the Selective Oxidation of "Neutral Sugars" In t h i s method, the saponification-selective perlodate oxidationborohydride reduction-periodic acid-Schiff technique, a l l s i a l i c acid residues are rendered Schiff unreactive by the K0H/PA*/Bh sequence. "Neutral sugars" are then stained magenta i n the PAS procedure (Fig. 9, I l l u s t r a t i o n XVI).  - 52 -  Fig. 8: The mechanism and expected staining patterns for Method 3, the selective periodate oxldatlon-thlonin Schiff-borohydride reduction-periodic acid-Schiff-saponification (PA»/T/Bh/PAS/KOH) technique.  S i a l i c acids without side chain substituents or with O-acyl substituents at C7 stain blue with the selective periodate oxidation-thionin Schiff sequence (a-c).  Following borohydride reduction (c-d), "neutral sugars" are oxidized  with PA (d-e), stained magenta with pararosaniline Schiff (e-f), and then non-specific thionin staining i s removed by saponification (f-g).  53 CHpH  0=C-CH  3  N|-utoH\cOOH  (a)  C H O \ C O O H  (b) T) CH OH 0  H  l  |T  2  CCC-CH3' N  ^ H O ? ^ Q Q H  (c) O H : H  2  O H  Q- -CH C  (d)  .  Bh  3  H O T V C O O H  1 PA N  (e)  O H  C H  2  O H O H  O  (f)  (9)  VcH0f^00H -O  - 54 -  Fig. 9 : The mechanism and staining patterns expected for Method 4 , the saponification-selective perlodate oxidation-borohydride reduction-Alclan blue pH 1.0-periodic acld-Schlff (K0B7PA*/Bh/AB1.O/PAS) technique, and Method 5. the saponification-selective perlodate oxidation-borohydride reductionperiodic acld-Schlff (KOH/PA*/Bh/PAS) technique.  In Method 4 , the i n i t i a l saponification step (a-b) removes 0-acyl groups from v i c i n a l d i o l s , resulting i n oxidation of a l l tissue s i a l i c acids with PA* (b-c).  These s i a l i c acids are then reduced to Schiff unreactive primary  alcohols with borohydride (c-d).  Sulphate esters are stained aqua with Alcian  blue pH 1.0 (d-e) and "neutral sugars" are then oxidized with 1% periodic acid (e-f) and stained magenta with Schiff (f-g).  "Neutral sugars", therefore,  stain magenta and sulphate esters stain aqua. Method 5 i s similar to Method 4 except that the Alcian blue pH 1.0 step (d-e) i s omitted. therefore results i n magenta staining of "neutral sugars".  This method  55 :H OH  0=C-CH  2  OOH  (a) KOH  KOH H,OH  CcC-CH  3  (b) PA CH OH 3  ^  0- -CH J  ?  c  N  (c)  PA,  l  3  3  'tf£^\COOH  CcC-CH  H OH 7  (d)  "•O  AB 1.0 COOH  (e)  57  1  ^r-CH  (f) C^OH  (9)  3  jPA  - 56 B.  Specificity of Methods 1-5 The s p e c i f i c i t y of these sequences was examined using the experiments  outlined i n Table VI. The order of application of thionin and pararosaniline Schiff reagents (thionin f i r s t , pararosaniline second) in Methods 1,2, and 3 was based upon past studies of double Schiff procedures (Van Duijn, 1956; Culling et a l . , 1976; Reid et a l . , 1984c) and was necessary to prevent the replacement of one reagent by the other.  In contrast to previous investigations (Reid et a l . ,  1984c), i t was found i n i n i t i a l studies that i t was not necessary to prepare thionin Schiff reagent by the method of Van Duijn (1956) as the faster method of Barger and DeLamater (1948) was satisfactory.  In addition, use of this  reagent diminished the time required for maximum staining with thionin Schiff from 4 hours (Reid et a l . . 1984c) to only 2 hours. A KOH/Bh sequence was used following the thionin Schiff step of Methods 1 and 2 (Tables I and V).  In previous studies (Reid et a l . , 1984c), i t was shown  that the saponification step was required to remove non-specific  thionin  staining of nuclei as well as for the de-O-acylation of s i a l i c acids, and that the borohydride reduction step was necessary to prevent anomalous pararosaniline Schiff staining.  In this study a number of controls were  carried out to establish the s p e c i f i c i t y of the KOH/Bh sequence used i n Methods 1-3.  When a borohydride reduction step was inserted between the f i n a l periodic  acid and Schiff steps of Methods 1 and 2, (PA*/Bh/KOH/PA*/T/KOH/Bh/ PA/Bh/S or KOH/PA*/T/KOH/Bh/PA/Bh/S) to prevent staining with pararosaniline Schiff, only blue staining (thionin) was obtained (Illustration XVII).  This indicated that  there was no exchange of Schiff reagents in these procedures.  - 57 Table VI: Methods for the verification of the s p e c i f i c i t y of Methods 1 to 5.  The nature of the macromolecules studied i n these tissue sites i n summarized i n Table II (Page 27).  P A 3 and PAgo = oxidation with 1% (w/v) periodic acid at room temperature for 3 and 60 minutes, respectively PA* = oxidation with 0.4 mM periodic acid i n approximately 1M. hydrochloric acid at 4°C for one hour KOH = saponification Bh = borohydride reduction ( L i l l i e and Pizzolato, 1972) S = treatment with pararosaniline Schiff AB 2.5 and AB 1.0 = staining with Alcian blue at pH 2.5 and pH 1.0, respectively (Culling, 1974) 0 = no staining 4+ = strong staining  - 58 -  Purpose  To determine i f one Schiff reagent i s replaced by another  Method  Experimental Sequence  PA*/Bh/KOH/PA*/T/KOH/Bh/ PA/Bh/S  Results  Thionin staining only  K0H/PA»/T/KOH/Bh/PA/Bh/S  PA»/T/Bh/PA/Bh/S/KOH  Thionin staining only  Interpretation  Insertion of the borohydride sequence step between the f i n a l periodic acid and Schiff steps of Methods 1 and 2 w i l l prevent staining with Schiff. Since no pink (only blue) staining was observed, no exchange of Schiff reagents occurs i n these procedures. When a borohydride reduction step was placed between the PAgg and Schiff steps, thereby preventing staining with pararosaniline Schiff, only thionin staining occurred, i l l u s t r a t i n g that no exchange of Schiff reagents occurs, and that moving the position of the KOH step does not affect staining results.  - 59 -  Purpose  Method  Experimental Sequence PA»/Bh/K0H/PA»/Bh/T/K0H7 Bh/PA/S  To demonstrate that no nonspecific staining with thionin Schiff occurs  KOH/PA*/Bh/T/KOH/Bh/PA/S  Results  Interpretation  Magenta staining only  No non-specific thionin Schiff staining occurs  See Table V (Page 46)  Staining patterns obtained with these methods were as predicted on the basis of the known composition of these tissues. These methods represent, therefore, valid methods for the detection of s i a l i c acid and i t s 0-acyl substituents and O-sulphate esters and "neutral sugars".  PA*/Bh/T/Bh/PA/S/KOH  To determine i f the resulting staining patterns in Methods 1-5 reflect those expected on the basis of the known composition of the various macromolecules in the tissues (Table V) (Page 46)  1  PA*/Bh/KOH/PA*/T/KOH/ Bh/PA/S  2  KOH/PA*/Bh/T/KOH/Bh/PA/S  3  PA*/T/Bh/PA/S/KOH  4  K0H/PA*/Bh/AB1.0/PAS  5  KOH/PA*/Bh/PA/S  - 60 The KOH/Bh sequence could not be used i n Method 3 (PA*/T/Bh/PAS/KOH), however, since the saponification (KOH) step would de-esterlfy 7- and 9-0-acyl s i a l i c acids which would then be identified as "neutral sugars" i n the subsequent PAS step.  As a result, the saponification step was placed after the  f i n a l pararosaniline Schiff treatment.  The f e a s i b i l i t y of moving the placement  of the KOH step was ascertained by demonstrating that, when a borohydride reduction step was placed between the PA6o and Schiff steps of Method 3 (PA*/T/Bh/PA6o/Bh/S/KOH/), only thionin Schiff staining was obtained. To demonstrate that there was no non-specific staining with thionin Schiff (Methods 1, 2 and 3), a borohydride reduction step was placed between the periodate oxidation and thionin Schiff steps. Only magenta staining was obtained i n these control studies (Illustration XIX) ,indicating that non-specific staining with thionin Schiff had not occurred. F i n a l l y , i n a l l five methods the f i n a l PAS procedure was performed under conditions which resulted i n maximal staining of rat l i v e r glycogen and the neutral macromolecules of parotid gland acinar c e l l granules. The s p e c i f i c i t y of the methods was also investigated by comparing the staining patterns obtained when they were applied to sections of rat l i v e r , salivary gland complexes and colon and human colon to that predicted on the basis of the known composition of the various macromolecules i n the tissues (Table I I ) .  As would be expected, l i v e r glycogen and the neutral  macromolecules of the acinar c e l l s of the rat parotid gland, which do not contain s i a l o - , sialosulpho-, or sulphated glycoproteins, stained only magenta with a l l methods. With Methods 4 ("neutral sugars" vs sulphate) and 5 ("neutral sugars" only), the rat sublingual gland, terminal ileum and crypt bases of the proximal colon of the rat, which contain only sialoglycoproteins  -  61  -  and "neutral sugars" but no sulphate, stained magenta. In contrast, the heavily sulphated e p i t h e l i a l sialoglycoproteins of human colon and rat d i s t a l colon, which also contain "neutral sugars", stained i n various shades of purple with Method 4 but magenta with Method 5. In Method 2, the glycoproteins of the rat ileum-colon and sublingual glands and human colon, which contain varying proportions of s i a l i c acids and "neutral sugars", stained i n various shades of purple.  The acinar c e l l s of rat  submandibular glands, however [which contain only traces of s i a l i c acid as shown by staining with the high iron diamine-Alcian blue pH 2.5 technique (Spicer, 1965)], stain only magenta with Method 2. In Method 1 (C7, C8 and C9 SA vs "neutral sugars"), tissues containing large quantities of these s i a l i c acids such as the rat sublingual gland (Park e t _ a l . , 1987 unpublished), human colon (Culling et a l . 1981) and the bases of f  the crypts of the rat proximal colon (Reid et a l . , 1973) stained i n varying shades of purple.  Tissues which contain less C7, C8, and C9 O-acyl substituted  s i a l i c acids, such as the upper halves of the crypts of rat proximal colon (Reid et a l . , 1973) stained with a redder hue, while tissues containing l i t t l e or no C7» C8 and C9 substituted s i a l i c acids, such as rat terminal ileum, stained magenta. F i n a l l y , sites containing significant quantities of s i a l i c acids without side chain substituents such as rat sublingual gland and rat terminal ileum and colon, stained i n various shades of purple with Method 3*  C. Discussion and Conclusions These studies have resulted l n the development of methods for the selective oxidation of s i a l i c acid, the simultaneous visualization of "neutral sugars" and either s i a l i c acid and i t s side chain O-acyl variants or O-sulphate ester,  - 62 and a general method for the identification of "neutral sugars" i n the presence of s i a l i c acid. The s p e c i f i c i t y of these procedures depends upon several factors.  F i r s t l y , the selective perlodate oxidation procedure (PA*) used i n  the i n i t i a l steps of Methods 2-5 (Table I) and for the f i r s t two oxidation steps of Method 1 (Table I ) , must oxidize a l l available s i a l i c acid v i c i n a l diols without producing a v i s i b l e Schiff reaction by the oxidation of "neutral sugars".  In Methods 2-5, the incomplete oxidation of s i a l i c acid residues w i l l  result i n their identification as "neutral sugars".  In Method I, those s i a l i c  acids f a i l i n g to oxidize i n the i n i t i a l PA* step, w i l l subsequently be Identified as 7-, 8- or 9-0-acyl substituted s i a l i c acids, and those that do not oxidize i n the second oxidation step w i l l be identified as "neutral sugars".  In addition, i f a significant oxidation of "neutral sugars" occurs  during the i n i t i a l oxidation steps of Methods 2 and 3, and during the f i r s t two oxidation steps of Method 1, these sugars would subsequently be identified as s i a l i c acids.  Further, i n Methods 4 and 5, such oxidation would result i n an  underestimate of the quantity of "neutral sugars" present. However, control studies carried out during the investigation of the PA* method demonstrated that the complete oxidation of s i a l i c acids without side chain 0-acyl substituents can be achieved using 0.4 mM periodic acid i n approximately 1M hydrochloric acid for 1 hour at 4°C. These conditions did not produce v i s i b l e Schiff staining of either neutral macromolecules or the "neutral sugars" of s i a l o - and sialosulphoglycoproteins, therefore the i n i t i a l perlodate oxidation steps of Methods 2, 4 and 5 can be considered specific for s i a l i c acids. The interpretation of the results obtained following the oxidation of 0-acyl side chain substituted s i a l i c acids with PA* i n Methods 1 and 3 i s  - 63 more complicated.  -  Chemical studies (Haverkamp et a l . , 1975; Schauer, 1982;  Shukla and Schauer, 1982; Diaz and Varki, 1985) have shown that 9-0-acyl s i a l i c acids oxidize at a much slower rate than s i a l i c acids without side chain 0-acyl substituents.  However, i f 9-0-acyl s i a l i c acids are present i n the tissues  examined i n this study, and i f they oxidize under PA* conditions, then the observed thionin Schiff staining i n Method 1 would be due to s i a l i c acids with an 0-acyl substituent at positions C7 or C8 only.  In Method 3> i n contrast,  the staining of s i a l i c acid would be due to unsubstituted s i a l i c acids or s i a l i c acids with a single 0-acyl substituent at position C7.  Also, i f 0-acyl  migration (Cheresh and Reisfeld, 1984; Schauer, 1982; Varkl and Diaz,  1984;  Diaz and Varki, 1985) from position C7 to position C8 or C9 occurs during the i n i t i a l oxidation step of Method 3, then 7-0-acyl s i a l i c acids would not be identified. Method 1.  Acyl migration would not, however, affect the staining i n In studies performed on gastrointestinal mucins (Reid e i a l . ,  1977,  1978), 9-0-acyl s i a l i c acids were not detected; however, they were demonstrated in bovine submandibular gland (Reid et a l . , 1978). Veh et a l . (1979) have detected 9-0-acyl s i a l i c acids i n bovine submandibular glands using conditions which accounted for the slow oxidation of such acids. Secondly, the perlodate oxidation conditions employed i n the f i n a l PAS step of a l l five methods must completely oxidize a l l "neutral sugar" v i c i n a l diols and the Schiff reagent used should react completely with a l l aldehydes produced.  I f either of these reactions i s incomplete, then the quantity of  "neutral sugars" present w i l l be underestimated. The conditions chosen for the f i n a l PAS reaction result i n maximal staining of neutral macromolecules.  The  intensity of the PAS reaction i s , however, dependent upon the structure of the macromolecule oxidized (Reid & Culling, 1980). I t i s necessary, therefore, to  - 64 -  consider the p o s s i b i l i t y that an incomplete PAS reaction can occur. Furthermore, "neutral sugars" containing O-acyl esters upon potential v i c i n a l d i o l s w i l l not be detected i n Method 3«  Such residues have only been detected  thus f a r i n one gastrointestinal mucin, the mucous c e l l s of the d i s t a l rat colon (Park et a l . , 1987, unpublished).  O-acetyl "neutral sugars" w i l l not,  however, complicate the interpretation of staining patterns seen i n Methods 1, 2, 4 and 5, since the saponification step i n these methods w i l l remove any O-acyl esters present before the PAS procedure i s carried out. F i n a l l y , as demonstrated i n control experiments, the product of thionin Schiff reagent and s i a l i c acid monoaldehydes was s u f f i c i e n t l y stable to survive the steps following i t , and therefore anomalous pararosaniline Schiff staining of s i a l i c acid residues did not occur. A l i s t of control experiments which should be done concurrently with Methods 1-5 i s shown i n Table VII.  - 65 -  Table VII: L i s t of Control Methods which should accompany Methods 1-5.  x = step performed  PA* = oxidation with 0.4 mM period acid i n 1M hydrochloric acid f o r 1 hour at 4°C Bh = reduction with 0.1% (w/v) sodium borohydride i n 1% dibasic sodium phosphate (anhydrous) for 15 minutes at room temperature KOH = saponification with 0.5% potassium hydroxide i n 70% ethanol for 15 minutes at room temperature PA = oxidation with 1% periodic acid for 2 hours at room temperature S = pararosaniline Schiff for 1 hour at room temperature T = thionin Schiff reagents for 2 hours at 4°C  Note:  In most cases i t i s only necessary to perform control step Ia of step  Ia and b, and not both steps Ia and Ib. I f Ia i s positive, then Ib should be carried out.  I f Ib i s also positive, then Methods 1-5 are not s p e c i f i c .  - 66 -  Tissue la) r a t l i v e r rat parotid gland  b) rat l i v e r rat parotid gland  II) r a t l i v e r rat parotid gland  I l i a ) human colon  Procedure  Expected Result  Rationale  PA*/S  no staining  These tissues do not contain s i a l i c acids and "neutral sugars" - should not oxidize under conditions of PA*  PA*/Bh/PA*/S  no staining  any sugar residues oxidized i n PA* should be reduced by borohydride and therefore be unavailable f o r subsequent oxidation (PA* second) and staining with S c h i f f reagent  Method 1 PA*/Bh/KOH/PA*/T/ KOH/Bh/PAS Method 2 KOH/PA*/T/KOH/Bh/PAS Method 3 PA*/T/Bh/PAS/KOH Method 4 K0H/PA*/Bh/AB1.O/PAS Method 5 KOH/PA*/Bh/PAS  Magenta  These tissues do not contain s i a l i c acids and therefore should not be oxidized by PA*, but only by PA, resulting i n magenta staining  PA/Bh/KOH/PA*/S  Magenta Magenta Magenta Magenta  intensely magenta  A l l unsubstituted s i a l i c acids and s i a l i c acid substituted at C7 w i l l be oxidized and reduced by the PA/Bh sequence. C8 substituted s i a l i c acids w i l l then be de-O-acylated by KOH, then oxidized by PA* and stained magenta with S c h i f f reagent  - 67 -  b) human colon  PA/Bh/KOH/PA*/Bh /PA*/S  no staining  A l l s i a l i c acids and "neutral sugars" should be oxidized by either the PA or PA* step, and then reduced by the Bh step following each of these oxidations. No s i a l o - sugars should remain, therefore, to be oxidized i n the second PA* step, resulting i n lack of staining i n tissues treated i n this manner.  - 68 -  GENERAL CONCLUSION: This study was comprised of three parts.  The f i r s t involved the  development of selective conditions for the oxidation of s i a l i c acids, and therefore a selective method for the detection of s i a l i c acids with the PA /S #  technique.  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Histochemical evidence for  l i p o i d a l material i n secretory granules of rat salivary glands. Histochem. J . 5,  239-54.  Spicer, S.S.  (1961)  The use of cationic reagents i n histochemical  differentiation of mucopolysaccharides.  Spicer, S.S. and Duvenci, J . ( 1 9 6 4 )  Am. J. C l i n . Path.  36_, 3 9 3 - 4 0 9 .  Histochemical characteristics of  mucopolysaccharides l n salivary and exorbital lacrimal glands. Anat. Rec. 142,  333-58.  -  79  -  Spicer, S.S. (1965) Diamine methods for differentiating mucosubstances histochemically.  J. Histochem. Cytochem. 13*. 211-34.  Stoward, P.J. (1967a) Histochemical studies of the formazan reaction  I. A  theoretical review of some of the factors that can affect the conversion of perlodate oxidized mucosubstances into formazans. J. Roy. Micr. Soc. 87, 393-406.  Stoward, P.J. (1967b)  Histochemical studies of the formazan reaction.  I I . The conversion of perlodate reactive mucosubstances into diphenyl and phenyl 1-4'-diazo-3,3'-dimethoxybiphenyl formazans and related derivatives. J. Roy Micr. Soc. 8J, 407-35.  Stoward, P.J. (1967c) Studies in fluorescence histochemistry  I I I . The  demonstration with hydrazide of the aldehydes present in perlodate oxidized mucosubstances.  J. Roy. Mic. Soc. 81, 247-57.  Sunter, J.P., Higgs, M.J. and Cowan, W.K. (1985) Mucosal abnormalities at the anastomosis site in patients who have had intestinal resection for colonic cancer.  J. Clin Path. 38, 385-9.  Suttajit, M. (1970) Modification of s i a l i c acid in glycoproteins by perlodate oxidation followed by borohydride reduction. Structural and biological studies.  Ph.D. thesis, State University of New York at Buffalo, NY, USA.  - 80 Van Duijn, P. (1956)  A histochemical s p e c i f i c thionine-S02 reagent and i t s  use i n a bi-colour method f o r deoxyribonucleic acid and periodic a c i d - S c h i f f p o s i t i v e substances.  J . Histochem. Cytochem. 4, 55-63.  Varki, A. and Diaz, S. (1984)  The release and p u r i f i c a t i o n of s i a l i c acids  from glycoconjugates: methods to minimize the loss and migration of 0-acetyl groups.  Anal. Biochem. 131, 236-47.  Veh, R.W., C o r f i e l d , A.P., Schauer, R. and Andrews, K.H. (1979) The bovine submandibular gland. Int.  I I . Histochemical and biochemical r e s u l t s .  Symp. Glycoconjugates, pp. 652-53-  Proc. 5th  Thieme: S t u t t g a r t .  Volz, D., Reid, P.E., Park, CM., Owen, D.A., Dunn, H.L. and Ramey, C H . (1986)  Can "mild" periodate oxidation be used f o r the s p e c i f i c histochemical  i d e n t i f i c a t i o n of s i a l i c acid residues?  Histochem. J . 18, 579-82.  Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, H.L. (1987a)  A new  method f o r the s e l e c t i v e periodate oxidation of t o t a l tissue s i a l i c acids. Histochem. J . i n press.  Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, H.L. (1987b) Histochemical procedures f o r the simultaneous v i s u a l i z a t i o n of "neutral sugars" and e i t h e r s i a l i c acid and i t s side chain O-acyl variants or O-sulphate ester I. Methods based upon the s e l e c t i v e periodate oxidation o f s i a l i c acids. Histochem. J . i n press.  - 81 -  Weber, P., Harrison, F.W. and Hof, L. (1975) The histochemical application of danzylhydrazine as a fluorescent labelling agent for s i a l i c acid i n c e l l u l a r glycoproteins.  Histochemie M5, 217-7.  Yonezawa, S., Nakamura, T., Tanaka, S. and Sato, E. (1982) Glycoconjugate with Ulex europaeus agglutinin-1 binding sites i n normal mucosa. Adenoma and carcinoma of the human large bowel. J . Nat. Can. Inst. 6.9., 777-83.  Yonezawa, S., Nakamura, T., Tanaka, S., Marata, K., Nishi, M. and Sato, E. (1983) Binding of Ulex europaeus agglutinin-1 i n polyposis c o l i .  Comparative  study with solitary adenoma i n the sigmoid colon and rectum. J . Nat. Can. Inst. I I , 17-24.  - 82 -  I l l u s t r a t i o n I: A.  Sections of rat colon stained with the PA/Bh/K0H/PA /S (Slide A l ) . 2  PA/Bh/KOH/PA*/S (Slide A l i ) and PA/Bh/KOH/PAfip/S (Slide A l i i ) procedures.  The intensity of the Schiff staining of the colonic  goblet c e l l s i s similar regardless of the oxidation technique used, indicating that PA* oxidizes a l l s i a l i c acid d i o l s .  B.  Sections of rat l i v e r stained with the PA2/S, PA*/S and procedures.  PA6Q/S  There i s l i t t l e or no staining of glycogen with the  PA2/S and PA*/S techniques (Slides B i and B i i ) , but strong staining following treatment with the PAgp/S (Slide B i l l ) .  This indicates  that glycogen does not stain significantly with the PA2/S and PA*/S methods. Note the s l i g h t l y greater intensity obtained with the PA2/S technique, indicating the greater s p e c i f i c i t y of the PA*/S.  -  83  -  I l l u s t r a t i o n I I : Effect of pH on the rate of perlodate oxidation of s i a l i c acid residues.  PA/Bh/KOH treated sections of human colon were oxidized i n  0.004 mM periodic acid dissolved i n water (Slide Ha) or 0.125M sulphuric acid (Slide l i b ) for 24 hours, and then stained with Schiff reagent. Note that the intensity of staining i n acid solvent (Slide l i b ) i s greater than that obtained when water was used as solvent.  - 84 -  I l l u s t r a t i o n I I I : Effect of pH on the perlodate oxidation of "neutral sugars".  Sections of rat l i v e r were oxidized i n 0.4mM periodic acid at 4°C  using either water (Slide I l i a ) or 0.125M sulphuric acid (Slide I l l b ) as solvent, for 24 hours, then stained with Schiff.  Note:  The oxidation of "neutral sugars" i s depressed i n acid conditions.  Oxidation i n 1M NaCl (not shown) resulted i n a staining pattern similar to that observed when water l s used as a solvent.  - 85 -  I l l u s t r a t i o n IV: Evidence for the s p e c i f i c i t y of PA* conditions f o r the selective periodate oxidation of s i a l i c acids - Part A. Sections of rat colon were treated with the PA/Bh/KOH/PA6o/S (Slide IVa), PA/Bh/KPH/PA*/S (Slide IVb) and the PA/Bh/KOH/PA*/PA/S (Slide IVc) sequence. Note that no difference in the staining intensity of these treatments i s evident, suggesting that PA* oxidized a l l s i a l i c acid residues.  - 86  -  I l l u s t r a t i o n V: Evidence for the s p e c i f i c i t y of PA* conditions for the selective periodate oxidation of s i a l i c acids - Part B. Sections of human colon were treated with the (a) PA/Bh/KOH/PA*/S (Slide Va), (b) PA/Bh/KOH/ PA*/Bh/S (Slide Vb) and (c) PA/Bh/KOH/PA*/Bh/PA6o/S (Slide Vc) sequences. Treatment with sequence (a) resulted i n strong (4+) Schiff staining of the tissue.  However, when s e r i a l sections were reduced with borohydride after the  PA* step and then further oxidized with PA* (sequence b - Slide Vb) or reduced with borohydride and further oxidized with PA50 (sequence c - Slide Vc), no Schiff staining was obtained.  This indicated that when only s i a l i c acids were  present, they are completely oxidized by PA* conditions.  - 87  -  I l l u s t r a t i o n VI: Effect of PA* conditions on the perlodate oxidation of "neutral sugars". Sections of rat l i v e r were treated with (a) PA*/S, (b) perlodate oxidation with 0.4 mM periodic acid i n 1M HCl at 4°C f o r 24 hours, followed by Schiff, and (c) the routine PAS procedure.  Oxidation  with (a) PA*/S produced no significant staining of l i v e r glycogen (Slide Via). PAS r e a c t i v i t y of glycogen did occur s l i g h t l y i n (b) and intensely with (c). These results indicate that PA* conditions do not result i n any significant staining of "neutral sugars". Note the Feulgen staining of nuclei i n (b) (See also I l l u s t r a t i o n X).  Such staining i s insignificant i n (a) or (c). Similar  results were obtained with rat parotid gland.  - 88 -  I l l u s t r a t i o n VII: Effect of PA* conditions on the oxidation of "neutral sugars" l n the presence of s i a l o - and slalosulpho-contalning glycoproteins.  Sections  of rat colon were pre-treated with the K0H/PA3/Bh sequence to confine v i c i n a l diols to "neutral sugar" residues.  Sections were then treated with (a) PA*  (KOH/PA /Bh/PA*), (b) PA6Q (KOH/PA /Bh/PA6o) or (c) PAVPA60 3  3  (KOH/PA /Bh/PA*/PA) and stained with Schiff reagent. No Schiff reactivity 3  was produced with (a); however, strong (4+) staining was obtained with (b) and (c).  This indicates that the PAS reactive "neutral sugar" residues present are  available for oxidation, and stain with the PAS. The absence of PA*/S staining indicates that PA* does not produce significant "neutral sugar" staining.  - 89 -  I l l u s t r a t i o n VIII: To determine i f glycoproteins are extracted when treated with PA* conditions.  Sections of rat colon were treated with (c) K0H/PA/Bh/AB1.0 (Slide VIII 1) or (b) AB1.0 (Slide VIII i i ) .  No difference i n the staining patterns occurred  between (a) and (b).  These data indicate that PA* does not extract e p i t h e l i a l glycoproteins.  - 90 -  I l l u s t r a t i o n IX: To determine I f PA* de-O-acetylates s i a l i c acids.  Sections of  rat colon were treated with the PA/Bh sequence to remove a l l oxidizable v i c i n a l d i o l s , incubated with 1M HCl for 1 hour at 4°C, and then treated with the technique (PA/Bh/1M HCl 1 hr at *»°C/PAS) (Slide IX).  PAS  These sections were not  PAS reactive, but sections treated with the sequence PA/Bh/1M HCl 1 hr at i|°C KOH/PAS (not shown) stained intensely, indicating that such conditions do not de-O-acetylate s i a l i c acids.  -  91  -  I l l u s t r a t i o n X: To determine I f PA* conditions Invoke a Feulgen reaction. Sections of rat colon were incubated with either 1 M HCl for 1 hour at 4°C (Slide X) or with PA* (not shown). Minimal Schiff staining of nuclei was obtained.  However, when sections were incubated i n PA* for 24 hours, a  strong Feulgen staining resulted i n addition to mucin staining.  - 92 -  I l l u s t r a t i o n XI.  I l l u s t r a t i o n of results obtained i n mechanistic studies of  the s e l e c t i v i t y of PA*.  Sections of r a t colon were treated with the  KOH/PAg/Bh sequence to confine v i c i n a l d i o l s to "neutral sugar" residues. Sections were then oxidized with (a) PA* (KOH/PA /Bh/PA*) (Slide XIa), 3  (b) PAgo (KOH/PA /Bh/PA6o) (Slide Xlb), or (c) PA*PAg (KOH/PA / 3  0  3  Bh/PA*/PA6o) (not shown) and stained with S c h i f f (not shown) or DNPH-TDMBF ( S l i d e s XIa and b). Sections exposed to (b) or (c) (not shown) were strongly S c h i f f p o s i t i v e and yielded formazans on treatment with DNPH and TDMBF (Slide XIa).  Sections exposed to (a), however, were S c h i f f unreactive and showed only  traces o f formazan with the DNPH/TDMBF sequence (Slide X l b ) . that PA* does not produce hemiacetals or hemialdals.  These data imply  - 93 -  I l l u s t r a t i o n XII. Illustration of the selective periodate oxldatlonborohvdrlde reductlon-saponlficatlon-selective periodate oxldatlon-thionln Schiff-saponlfication-borohvdrlde reduction-periodic acld-Schlff (PA»/Bh/KOH/PA»/T/KOH/Bh/PAS) technique (Method 1). Sections of rat l i v e r (Slide X l l a ) , salivary gland complex (Slide X l l b ) , proximal and d i s t a l colon (Slide XIIc and X l l d respectively) and human colon (Slide Xlle) were treated with the PA*/Bh/KOH/PA*/T/KOH/Bh/PAS technique.  In this method s i a l i c acids  with O-acyl substituents at C7, C8 and C9 stain blue, "neutral sugars" stain magenta, and mixtures stain i n varying shades of purple.  Note that rat l i v e r  (Slide X l l a ) , rat submandibular gland (Slide Xllb) and the neutral macromolecules of the acinar c e l l s of rat parotid gland (not shown) which either do not contain sialoglycoproteins, or only contain traces, stain magenta, while rat sublingual gland (Slide X l l b ) , human colon (Slide Xlle) and the bases of the crypts of the rat proximal colon (Slide XIIc), which contain large quantities of C7, C8 and C9 s i a l i c acids, stain i n various shades of purple. Note that the upper crypts of rat proximal colon, which do not contain much s i a l i c acid, stain magenta.  - 94 -  I l l u s t r a t i o n XIII.  I l l u s t r a t i o n of the saponification-selective  periodate  oxidation-thlonln Schiff-saponiflcatlon-borohvdride reduction-periodic acid-Schiff (K0n7PA»/T/K0H/Bh/PAS) method (Method 2).  Sections of rat l i v e r ,  salivary gland complex, colon and human colon were treated with the KOH/PA*/T/KOH/Bh/PAS method. In t h i s method a l l s i a l i c acids stain blue, "neutral sugars" stain magenta, and mixtures stain In varying shades of purple.  Tissues that contain no s i a l i c acids or only traces of such residues  such as rat l i v e r (staining results similar to X l l a ) , submandibular gland (Slide XIIla) and parotid gland (not shown) stain magenta. Tissues which contain varying proportions of s i a l i c acids and "neutral sugars" such as rat colon (Slide XHIb), sublingual gland (Slide XHIa) and human colon (Slide X I l i e ) , stain i n varying shades of purple.  -  I l l u s t r a t i o n XIV.  95  -  I l l u s t r a t i o n o f the s e l e c t i v e perlodate  t h i o n i n Schiff-borohydride  oxidation-  reduction-periodic a c l d - S c h l f f - s a p o n i f i c a t i o n  (PA*/T/Bh/PAS/KOH) technique (Method 3).  Sections of r a t l i v e r , s a l i v a r y gland  complex, r a t colon and human colon were treated with PA*/T/Bh/PAS/KOH.  In t h i s  method s i a l i c acids without side chain substituents or with side chain substituents at p o s i t i o n C7 s t a i n blue, "neutral sugars" s t a i n magenta, and mixtures s t a i n i n varying shades of purple.  Tissues containing no s i a l i c acids  or only traces o f t h i s residue such as r a t l i v e r (staining s i m i l a r to that seen i n S l i d e X l l a ) , submandibular gland (Slide XlVa) and parotid gland ( S l i d e XlVb), s t a i n magenta, while tissues containing large quantities of s i a l i c a c i d without side chain substituents such as r a t sublingual gland (Slide XlVa) and rat d i s t a l colon (Slides XIVc and XlVd respectively) s t a i n i n varying shades of purple.  Tissues with l i t t l e unsubstituted s i a l i c acid s t a i n magenta (rat  proximal colon (Slide XIVc) and r a t d i s t a l colon ( S l i d e XlVd)).  - 96 -  I l l u s t r a t i o n XV. Illustration of the saponification-selective perlodate oxidation-borohydride reductlon-Alclan blue pH 1.0-perlodlc acid-Schiff (K0H/PA»/Bh/AB1.0/PAS) procedure (Method i n . Sections of rat l i v e r , salivary gland complex, rat colon and human colon were treated with the K0H/PA*/Bh/AB1.C7PAS technique.  In this method, sulphate esters stain aqua  and "neutral sugars" stain magenta. Tissues which contain only sialoglycoproteins such as the rat sublingual gland (Slide XVa) and crypt bases of the proximal colon (Slide XVb) and "neutral sugars" ( l i v e r (see X l l a ) , submandibular gland (Slide XVa) and parotid gland (not shown)) but no sulphate, stain magenta. Heavily sulphated epithelial sialoglycoproteins, as are found in human colon (not shown) and rat d i s t a l colon (Slide XVc), which also contain "neutral sugars", stain i n varying shades of purple.  -  97  -  I l l u s t r a t i o n XVI. Illustration of the saponlfication-seleotlve periodate oxidation-borohydride reduction-periodic acid-Schlff (KOH/PA»/Bh/PAS) procedure (Method 5).  Sections of rat l i v e r , salivary gland complex, rat colon and human  colon were treated with the KOH/PA*/Bh/PAS procedure.  In this method "neutral  sugars" alone stain magenta. Since a l l tissues used contain "neutral sugars", they stain i n varying intensities of magenta. Shown are rat submandibular and sublingual gland (Slide XVIa), rat proximal (Slide XVIb) and d i s t a l (Slide XVIc) colon and human colon (Slide XVId).  - 98 -  I l l u s t r a t i o n XVII. Evidence that exchange of Schiff reagents does not occur with Methods 1 and 2, Sections of human colon and rat salivary gland complex (containing submandibular and sublingual glands) were treated with a variation of Methods 1 and 2 i n which a borohydride reduction step was placed between the f i n a l periodic acid and Schiff steps of method 1 (a) PA*/Bh/KOH/PA*/T/ KOH/Bh/PA/Bh/S and Method 2 (b) KOH/PA*/T/KOH/Bh/PA/Bh/S to prevent staining with pararosaniline Schiff.  Only blue (thionin) staining was obtained  i l l u s t r a t i n g that no exchange of Schiff reagent occurs i n these procedures. A i s i l l u s t r a t e d with human colon (Slide XVIIa) and (b) with rat submandibular and sublingual glands (Slide XVIIb).  -  I l l u s t r a t i o n XVIII.  99 -  Evidence that the position of the  saponification  (KOH)/borohydrlde sequence does not affect i t s a b i l i t y to remove non-specific thionin Schiff staining. non-specific  The KOH/Bh sequence l s necessary for the removal of  thionin Schiff staining and to prevent anomalous pararosaniline  Schiff staining (Reid et a l . , 1984b), and i s usually placed immediately following the thionin Schiff step (see Methods 1 and 2).  This placement i s  not possible i n Method 3 (PA*/T/Bh/PAS/KOH) since the KOH step would de-esterify 7- and 9-0-acyl s i a l i c acids which would then be identified as "neutral sugars".  As a result, the KOH step was placed following  the  pararosaniline Schiff treatment. To demonstrate that moving the position of the KOH step does not affect i t s a b i l i t y to remove non-specific  thionin Schiff  staining, sections of rat colon were treated with a variation of Method 3 i n which a borohydride step was placed between the PA6Q and Schiff steps of Method 3 (PA*/T/Bh/PA6o/Bh/S/KOH) (Slide XVIII).  Only thionin Schiff  staining was obtained, i l l u s t r a t i n g the KOH step i s s t i l l functional i n i t s new  position.  - 100 -  I l l u s t r a t i o n XIX. Evidence that non-specific thionin Schiff staining does not occur i n Methods 1-3. Sections of human colon were treated with a variation of Methods 1, 2 and 3, i n which a borohydride reduction step was placed between the periodate oxidation and thionin Schiff steps - Method 1 (PA*/Bh/K0H/PA*/Bh/T/K0H/Bh/PA/S - see Slide XIX); Method 2 (K0H/PA*/Bh/TV KOH/Bh/PA/S - not shown); and Method 3 (PA*/Bh/T/Bh/PA6o/S/KOH - not shown). Only magenta staining was obtained, indicating that non-specific thionin Schiff staining had not occurred.  - 101 L i s t of Publications: 1.  Volz, D.E., Reid, P.E., Park, CM., Owen, D.A., Dunn, W.L. and Ramey, CW. (1986) Can 'mild' periodate oxidation be used for the specific histochemical identification of s i a l i c acid residues? Histochem. J . 18, 579-582.  2.  Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, W.L. (1987a) A new method for the selective periodate oxidation of t o t a l tissue s i a l i c acids. Histochem. J . i n press.  3.  Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, W.L. (1987b) Histochemical procedures for the simultaneous visualization of "neutral sugars" and either s i a l i c acid and i t s side chain O-acyl variants or O-sulphate ester I. Methods based upon the selective periodate oxidation of s i a l i c acids. Histochem. J. i n press.  4. Park, CM., Reid, P.E., Owen, D.A., Dunn, W.L. and Volz, D.E. (1987b) Histochemical procedures for the simultaneous visualization of neutral sugars and either s i a l i c acid and i t s O-acyl variants or O-sulphate ester. I I . Methods based upon the periodic acid-phenylhydrazine-Schiff reaction. Histochem. J. 19. i n press. 5.  Reid, P.E., Volz, D., Park, CM., Owen, D.A. and Dunn, W.L. (1987) Methods for the identification of side chain O-acyl substituted s i a l i c acids and for the simultaneous visualization of s i a l i c acid, i t s O-acyl variants and O-sulphate ester. Histochem. J. 19, i n press.  6. Park, CM., Reid, P.E., Owen, D.A., Volz, D. and Dunn, W.L. (1987) Light microscopic histochemical studies of e p i t h e l i a l c e l l glycoproteins in normal rat colon. Submitted to Histochem. J .  List  of Publications:  1.  Volz, D.E., Reid, P.E., Park, CM., Owen, D . A . , Dunn, W.L. and Ramey, CW. (1986) Can 'mild' periodate oxidation be used f o r the s p e c i f i c histochemical I d e n t i f i c a t i o n of s i a l i c acid residues? Histochem. J . J_8, 579-582.  2.  Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, W.L. (1987a) A new method f o r the s e l e c t i v e periodate oxidation of t o t a l tissue s i a l i c acids. Histochem. J . i n press.  3.  Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, W.L. (1987b) Histochemical procedures f o r the simultaneous v i s u a l i z a t i o n of "neutral sugars" and e i t h e r s i a l i c acid and i t s side chain O-acyl variants or O-sulphate ester I. Methods based upon the s e l e c t i v e periodate oxidation of s i a l i c acids. Histochem. J . i n press.  4.  Park, CM., Reid, P.E., Owen, D . A . , Dunn, W.L. and Volz, D.E. (1987b) Histochemical procedures f o r the simultaneous v i s u a l i z a t i o n of neutral sugars and e i t h e r s i a l i c acid and i t s O-acyl variants or O-sulphate ester. I I . Methods based upon the periodic acid-phenylhydrazine-Schiff reaction. Histochem. J . 1_9_ i n press.  5.  Reid, P.E., Volz, D., Park, CM., Owen, D.A. and Dunn, W.L. (1987) Methods f o r the i d e n t i f i c a t i o n of side chain O-acyl substituted s i a l i c acids and f o r the simultaneous v i s u a l i z a t i o n of s i a l i c a c i d , i t s O-acyl variants and O-sulphate ester. Histochem. J . 19, i n press.  6.  Park, CM., Reid, P.E., Owen, D . A . , Volz, D. and Dunn, W.L. (1987) Light microscopic histochemical studies of e p i t h e l i a l c e l l glycoproteins i n normal rat colon. Submitted to Histochem. J .  

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