Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1987_A6_7 V64.pdf [ 4.06MB ]
Metadata
JSON: 831-1.0097194.json
JSON-LD: 831-1.0097194-ld.json
RDF/XML (Pretty): 831-1.0097194-rdf.xml
RDF/JSON: 831-1.0097194-rdf.json
Turtle: 831-1.0097194-turtle.txt
N-Triples: 831-1.0097194-rdf-ntriples.txt
Original Record: 831-1.0097194-source.json
Full Text
831-1.0097194-fulltext.txt
Citation
831-1.0097194.ris

Full Text

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 B.Sc. The University of Alberta, 1978 Specialization Certificate in Microbiology, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (Department of Pathology) He accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1987 ( c ) Doris Elenore Volz, 1987 by DORIS ELENORE VOLZ MASTER OF SCIENCE in In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of rfYTHCUiQ G-The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ABSTRACT: The objective of this study was to establish conditions for the selective periodate oxidation of s i a l i c acid, and then use these conditions to develop a series of general methods for the simultaneous visualization of "neutral sugars" (ie. 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 selective conditions for the oxidation of s i a l i c acids demonstrated that oxidation for one hour at 4°C with 0.4 mM periodic acid in approximately 1M hydrochloric acid (PA*) oxidized a l l available s i a l i c acid residues of both the sialo 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 Schiff staining of either neutral macromolecules or vicinal diols located on the "neutral sugars" of sialo and sialosulphoglycoproteins, and did not result in the extraction of epithelial glycoproteins or in the de-O-acylation of side chain substituted s i a l i c acid residues. Therefore, PA* can be used as a specific reagent for the selective oxidation of s i a l i c acids. Studies of the mechanism of oxidation with PA* showed that the lack of PAS reactivity of "neutral sugars" was not due to the production of Schiff unreactive hemiacetals or hemialdals. It i s possible that the selective oxidation of s i a l i c acids with PA* results from an increase in the oxidation rate of s i a l i c acid residues together with a decrease in the oxidation rate of "neutral sugars". Based upon this method for the selective oxidation of s i a l i c acid residues (PA*), five new methods have been devised for the simultaneous visualization of "neutral sugars" and either s i a l i c acid and i t side chain 0-acyl i i 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, in 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 vicinal diols stain magenta. In the second method, the saponiflcatlon-selectlve periodate oxidation-thlonln Schiff-saponlflcatlon- borohvdrlde 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 in 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" in macromolecules which also contain s i a l i c acids can be achieved. i i i TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i i LIST OF ILLUSTRATIONS x LIST OF ABBREVIATIONS xiv ACKNOWLEDGEMENTS xvi INTRODUCTION 1 MATERIALS AND METHODS 15 I. Materials A. Tissues 15 B. Chemicals 15 I I . Histochemical Procedures 16 II 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. Method 4 (KOH/PA*/Bh/AB1.0/PAS) 24 E. Method 5 (KOH/PA»/Bh/PAS) 25 iv Table of Contents (cont'd.) PAGE RESULTS AND DISCUSSION 26 I. Establishment of Conditions for the Selective Periodate Oxidation of s i a l i c acid 26 I I . Investigation of 0 . 4 mM Periodate in 1M. HCl 3 2 A. Specificity 3 2 B. Mechanistic Studies of PA* Conditions 3 7 C. Discussion and Conclusions 4 3 I I I . Development of Methods Based on PA* 4 5 A. Results of Methods 1 - 5 4 5 B. Specificity of Methods 1 - 5 5 6 C. Discussion and Conclusions 6 1 GENERAL CONCLUSIONS 6 8 REFERENCES 6 9 ILLUSTRATION LEGENDS I-XIX 8 2 LIST OF PUBLICATIONS 1 0 1 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 selectivity of periodate oxidation with 33 0.4mM. periodic acid in 1M hydrochloric acid at 4°C for one hour V Predicted results of the application of the methods 45 outlined in Table I to tissue sites containing s i a l i c acids both with and without side chain 0-acyl substituents, "neutral sugar" vicinal diols, and O-sulphate ester v i L i s t of Tables (cont'd.) PAGE VI Methods for the verification of the specificity of 57 Methods 1 to 5 VII List of control methods which should accompany Methods 65 1 to 5 v i i 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 in selective detection of s i a l i c acid and the use of PAPS procedures in 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 Illustration of the 2,4-dinitrophenylhydrazine, 3,3'> 41 dimethoxybenzidene fluoroborate (DNPH-TDMBF) procedure v i i i L i s t of Figures (cont'd.) 7 The mechanism and expected staining patterns for Method 1, the selective perlodate oxidation-borohydride reduction-saponification-selective perlodate oxidation-thionin 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-saponiflcation-borohydride reduction-periodic acid-Schiff (KOH/PA*/T/KOH/Bh/PAS) technique 8 The mechanism and expected staining patterns for 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 for 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 specificity of PA* conditions for the 85 selective periodate oxidation of s i a l i c acids - PART A V Evidence for the specificity 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 87 "neutral sugars" x L i s t of Illustrations (cont'd.) PAGE VII The effect of PA* conditions on the oxidation of 88 "neutral sugars" in the presence of sialo- 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 Illustration of results obtained in mechanistic studies 92 of the selectivity of PA* XII Illustration of the selective perlodate oxidation- 93 borohydride reduction-saponification-selective perlodate oxidatlon-thionin Schiff-saponification-borohydride reduction-periodic acid-Schiff (PA*/Bh/KOH/PA*/T/KOH/Bh/PAS) technique (Method 1) x i L i s t of Illustrations (cont'd.) PAGE XIII Illustration of the saponification-selective periodate 94 oxidation-thionin Schiff-saponification-borohydride reduction-periodic acid-Schiff (KOH/PA«/T/KOH/Bh/PAS) technique (Method 2) XIV Illustration of the selective periodate oxidation- 95 thionin Schiff-borohydride reduction-periodic acid-Schiff -saponification (PA*/T/Bh/PAS/KOH) procedure (Method 3) XV Illustration of the saponification-selective periodate 96 oxidation-borohydride reduction-alcian blue pH 1.0-periodic acid-Schiff (K0H/PA*/Bh/AB1.0/PAS) procedure (Method 4) XVI Illustration of the saponification-selective periodate 97 oxidation-borohydride reduction-periodic acid-Schiff (KOH/PA«/Bh/PAS) procedure (Method 5) XVII Evidence that exchange of Schiff reagent does not occur 98 with Methods 1 and 2 xii 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 in Methods 1-3 x i i i 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 in 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 in minutes. xiv L i s t of Abbreviations (cont'd.) PA* = Periodate oxidation in 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 vici 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 li 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 in previous studies; Charles Ramey for advice on technical matters, and to Anne Bishop, Pat Bemoe, and a l l those who assisted in 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 x v i i - 1 -INTRODUCTION In order to understand the underlying mechanisms involved in 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 in that they allow for the identification 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 localization of molecules, and large quantities of samples are often required before precise identification and quantitation can be obtained. Further, for most procedures, only a few samples can be examined concurrently. In contrast, histochemical methods have the disadvantage in that small molecules are d i f f i -cult to identify as they tend to be removed during fixation 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, in that chemical events can be localized at the histological level, a characteristic unique to histo-chemistry. The sensitivity of histochemical methods i s such that a direct visual correlation can be obtained between structure and function such that individual cellular metabolic activities 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 foci 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 - 2 -as 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 3 0 , 0 0 0 5 um sections per colon. When epithelial cells 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 cells 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 in sensitivity of 1000 times. Histochemical techniques have been applied to the study of many diseased systems, in the diagnosis of certain leukemias and lymphomas, and for the determination of the site of origin of a variety of malignancies (Filipe and Lake, 1983). Histochemistry can increase the accuracy of histological diagnosis (Ehsanullah et a l . 1982a,b), has been used in the assessment of malignant transformation and plays a role in monitoring a patient's response to therapy (Filipe and Lake, 1983). In addition, histochemical methods have shown that there are changes in the epithelial 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; Filipe 1979, 1984; Filipe et_al., 1980; Franzin et a l . 1981, 1983a,b, 1984; Lev et a l . f 1985; Listinskl and Riddell, 1981; Nontero - 3 -and Segura, 1980; Reid et a l . T 1980, 1984c,d, 1985a,b; Rhatigan and Saffros, 1979; Spicer, 1965; Sunter et a l . . 1985). Most histochemical studies of the changes in epithelial 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 lectin 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 in 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 in the phenylhydrazine Schiff sequence, or i f the hydrazine can be coloured in 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 in 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 3 I NH/f-OH -|-OH CH2OH OH COOH OH 0=OCH, NH/iS:H0CHX900H ^H2OH / I _ Q OH Cec-C H 3 I NH f0H \ C O O H ^COCOC^yLo OH e. I-0-SO3H CH2OH OH OH OH g C H 3 \ OH OH CH,OH HNCOCH, - 6 -Fig. 2. Diagramatic representation of the mechanism(s) bv which the periodic  acld-phenvlhvdrazlne-Schiff (PAPSprocedure results in selective  detection of s i a l i c acids and the use of PAPS procedures in the  simultaneous visualization of s i a l i c acids and "neutral sugars" Periodate oxidation results in the oxidation of vicinal diols to aldehydes (a-b) which are then condensed with phenylhydrazine (b-c). Subsequent treat-ment 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 in selective Schiff staining of s i a l i c acids. If a coloured arylhydrazine i s substituted for phenylhydrazine or i f the hydrazine can be coloured in a subsequent reaction, then the simultaneous visualization of "neutral sugars" and s i a l i c acids can be obtained. - 7 -(a) C H P H OH ( b ) OH PA C O O H PA 0 = C - C H , C O O H O Ph CoC-CH3i 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 first oxidation must be carried out under reaction conditions in which a l l sialic acids oxidize without significant oxidation of other carbohydrate residues. The sialic acids are then either stained with Schiff or converted to Schiff unreactive alcohols with sodium borohydride. The second oxidation is then performed under conditions in which "neutral sugars" oxidize (Volz et al., 1987a,b). Previous attempts at developing "selective oxidation" conditions were based on the use of dilute periodic acid (Klessen, 1978; Roberts, 1977; Veh et al., 1979; Weber et a l . f 1975). Due to steric factors, the rate of oxidation of vicinal diols varies, with open chain diols (such as sialic 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 al., 1971). From this i t was concluded that i f limited quantities of periodate were used, only sialic acid diols would be oxidized. The periodic acid-Schiff (PAS) reaction could then be used to selectively detect sialic acids. It is d i f f i -cult, however, to estimate the amount of sialic 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 sialic 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 sialic acids and/or "neutral sugars". In preliminary studies, Volz et al. (1986) demonstrated that maximal PAS staining of sialic 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 in 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 - C 2 ) . 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 stains no Magenta Magenta 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 in l i v e r glycogen and the neutral glycoproteins of human stomach, oxidized significantly. However, when oxidation was performed with very dilute solutions of periodic acid in either 0.125N sulphuric acid (Illustration II) or 1M sodium chloride, the rate of oxidation of s i a l i c acid residues was increased significantly. 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 in 1M NaCl but was apparently decreased when 0.125 H 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 vicinal 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). If 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 in the s i a l i c acid oxidation rate may be such that the oxidation of other carbohydrate residues becomes significant. It becomes, - 12 -therefore, impossible to use very dilute periodic acid for the selective oxidation of s i a l i c acid residues. However, in 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. It appeared possible, therefore, that manipulation of the oxidation conditions could result in 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 in 0.125H sulphuric acid due to a lowering of the pH or to an increase in the ionic strength? 2. Would the use of 1M. sodium chloride or a lower pH improve the selectivity 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 in glycoproteins affect the rate of s i a l i c acid oxidation (possibly by altering the electrostatic f i e l d in 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 glycos-aminoglycans 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. Materials: A. 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 in 10% formalin calcium for at least 7 days. After fixation, specimens of rat colon and terminal ileum were prepared as a single "Swiss Roll" (Park et a l . , 1987a). The tissues were then processed through ethanol and xylene and embedded in 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 in an atmosphere of formalin vapour. B. Chemicals The product colour index number and supplier of staining dyes used in this study are as listed below: Color Index Dye Number Supplier Alcian Blue 8GX Pararosaniline hydrochloride Thionin 74240 42500 52000 Gurr Chemicals Fisher Scientific Company Fisher Scientific 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 in 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.) in each of the following solvents: d i s t i l l e d water, 1M. sodium chloride or IH hydrochloric acid. i i ) In studies of the mechanism of selective oxidation of s i a l i c acid, and in 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 in approximately 1M. hydrochloric acid at 4°C. The latter 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. i i ) Cool sections to 4°C and treat with a pre-cooled solution of 0.4mM periodic acid dissolved in approximately 1M_ hydrochloric acid. i i i ) Wash sections in running water for 10 minutes at 4°C. 7) Periodic Acid Schiff reaction (PAS). Sections were oxidized in W (w/v) aqueous periodic acid for one hour at room temperature, washed in 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 . f 1974). In this procedure, treatment with 1% (w/v) periodic acid for 2 hours at room temperature oxidizes vicinal 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 in K0H/PA3/Bh treated sections i s therefore confined to vicinal 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 3fV-dlmethoxybenzidene fluoroborate procedure (DNPH-TDMBF). In this procedure periodate derived aldehydes are blocked by treatment with a saturated solution of 2,4-- 19 -dinitrophenylhydrazine (DNPH) in 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) in 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 in 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 C7r C8, and C9, the selective  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 in a precooled solution of 0.4mM. periodic acid in approximately 1M HCl for 1 hour at 4°C. 3) Wash in running water for 10 minutes at 4°C. 4) Reduce with 0.1% (w/v) sodium borohydride in 1% (w/v) dibasic sodium phosphate (anhydrous) for 20 minutes at room temperature. 5) Wash in running water for 10 minutes at room temperature. 6) Rinse in 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 in Table. Selective perlodate oxidation (PA*) = 0.4 mM periodic acid in approximately 1M hydrochloric acid for one hour at 4°C Borohydride reduction (Bh) = 0.1% sodium borohydride in 1% (anhydrous) dibasic sodium phosphate for 20 minutes at room temperature Saponification (KOH) = 0.5% potassium hydroxide in 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 in 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 2 3 Selective Periodate Oxidation (PA*) X Borohydride Reduction (Bh) X Saponification (KOH) X X -Selective Periodate Oxidation (PA*) 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 X X Saponification (KOH) X - 22 -7) Saponify with 0.5% (w/v) potassium hydroxide in 70% (v/v) ethanol for 15 minutes at room temperature. 8) Hash in running water for 10 minutes at room temperature. 9) Cool to 4°C and again oxidize in pre-cooled O.MmM. periodic acid dissolved in approximately 1M_ hydrochloric acid for 1 hour at 4°C. Wash in running water for 10 minutes at 4°C. Stain with freshly prepared thionin Schiff reagent for 2 hours at room temperature. Wash in running water for 10 minutes at room temperature. Rinse in 70% (v/v) ethanol. Saponify with 0.5% (w/v) potassium hydroxide in 70% (v/v) ethanol for 15 minutes at room temperature. Wash in running water for 10 minutes. Reduce with 0.1% (w/v) sodium borohydride in 1% (w/v) dibasic sodium phosphate (anhydrous) for 20 minutes at room temperature. Wash in running water for 10 minutes at room temperature. Oxidize in 1% (w/v) aqueous periodic acid for 1 hour at room temperature. Wash in running water for 10 minutes. Stain with freshly prepared pararosaniline Schiff reagent for 1 hour at room temperature. Wash in running water for 10 minutes. Dehydrate (through series of 95%, 100% alcohol), clear in 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 vicinal diols stain magenta. Mixtures of these components stain in various shades of purple. B. Method 2: Simultaneous visualization of "neutral sugars" and total  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 vicinal diols) stain magenta, and mixtures stain in 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 reduction- periodic 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 (in this order). - 24 -Staining results In this procedure sialic 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 in 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 in 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 saponification- selective 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 li 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 in tissues based upon the previous studies of Volz fit a l . (1986). To accomplish this, conditions were sought in 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 in 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 in 1M_ HCl and 1M. NaCl as solvents. These provided solutions similar in ionic strength, but differing in 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 in Table II (Page 27). These tissues were selected because they provided examples of neutral polysaccharides alone, sialoglyco-proteins, 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 in 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 in water for 2 minutes at 4°C and 4.0mM. perlodate in 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 celU ^ granules. Sublingual Acinar c e l l glycoprotein "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) Sialoglycoprotein containing side chain substituted s i a l i c acids (Park gt a l . , unpublished) Rat Colon /Epithelial (glycoprotelr Proximal (lower half of crypts) Distal 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) Human Colon Epithelial glycoproteins Sialosulphoglycoproteins containing side chain O-acylated s i a l i c acids (Filipe, 1979; Culling fit a l . , 1981) - 29 -was also obtained with 0.4mM periodate in 1M HCl for 1 hour at 4°C (Illustration Ib). This latter 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 III and Illustration 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. Finally, the rate of oxidation of s i a l i c acid residues was not affected by changes in the pH of the solvent used whether the tissue contained sulphate (rat colon) or was unsulphated (rat sublingual gland). 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 in the glycoprotein. In conclusion, i t appears that the oxidation rate of s i a l i c acid in 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 in the glycoprotein. - 30 -Table III 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 in 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+ t r 1+ t r 21 3+ 1+ 2+ t r 48 4+ 1+ 2+ t r - 32 -II. Investigation of oxidation with 0.4mM Periodic acid in approximately 1M HCl A. Specificity In the previous study i t was shown that treatment with 0.4mM periodic acid in 1M HCl at 4°C for one hour (PA*) resulted in the selective oxidation of a l l s i a l i c acids. To further investigate the selectivity of the PA* technique, a series of control experiments was performed to demonstrate that under conditions of PA*, a l l s i a l i c acids were oxidized, oxidation of "neutral sugars" was insignificant, and glycoproteins were not extracted or de-O-acylated. These experiments are summarized in Table IV (Page 33)-To study the oxidation of s i a l i c acid residues with the PA* technique, sections of rat ileum, colon, salivary glands, and human colon were treated with the PA/Bh/KOH sequence to confine vicinal diols to s i a l i c acid residues. The sections were then treated with each of 1% periodic acid for 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 for 1 hour at room temperature (PA/Bh/KOH/PA*/PA6o/S). No difference in the staining intensity of these three treatments could be detected (Illustration IV), suggesting that PA* oxidized a l l s i a l i c acid residues. In a second set of experiments, sections of human colon treated with the PA/Bh/KOH/PA*/S sequence yielded a strong (4+) Schiff reaction. However, when se r i a l sections were reduced with borohydride after the PA* step and then further oxidized with PA* (PA/Bh/KOH/PA*/Bh/PA*/S) or with 1% periodic acid for 1 hour at room temperature (PA/Bh/K0H/PA*/Bh/PA6Q/S), no Schiff staining was obtained. This indicated that when only s i a l i c acids were present, they are completely oxidized under PA* conditions (Illustration V). - 33 -Table IV Evidence for the selectivity 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 in these tissue sites i s summarized in 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 in 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 1.0, respectively (Culling 1974) 0 = no staining 4+ = strong staining Tissue Procedure and Results Interpretation Rat colon and sub-lingual gland and human colon Human colon PA/Bh/K0H/PA60/S = 4+ PA/Bh/KOH/PA*/S = 4+ PA/Bh/KOH/PA*/PA6o/S = 4+ PA/Bh/KOH/PA*/S = 4+ PA/Bh/KOH/PA*/Bh/PA*/S = 0 PA/Bh/K0H/PA*/Bh/PA6Q/S = 0 The PA/Bh/KOH sequence confines vicinal 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. Rat l i v e r and parotid gland PA*/S = 0 Oxidation with PA* produces no visible Schiff with neutral macromolecules. Rat colon KOH/PA3/Bh/PA6o/S = 4+ KOH/PA3/Bh/PA*/PA6o/S =4+ KOH/PA3/Bh/PA*/S = 0 Following removal of O-acyl esters with KOH, the PA3/BI1 sequence confines vicinal diols to "neutral sugars" in sialo- and sialosulphoglycoproteins. Absence of Schiff staining following PA* but strong staining after PAgg indicates PA* does not produce Schiff reactive sites from "neutral sugars". Rat colon and sub-lingual gland human colon K O H / P A * / S s 4+ K O H / P A * / B h / P A * / S = K0H/PA*/Bh/PA6Q/S 4+ PA* oxidizes a l l s i a l i c acids but does not produce Schiff positive sites with "neutral sugars". Rat colon and sub-lingual gland Rat colon and human colon 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+ PA* or 1 M HCl, 4°C, 1 hour does not extract 4+ epithelial glycoproteins. Rat colon PA/Bh/PAgQ/S = 0 and sub- PA/Bh/1M HCl, 4°C, lingual gland 1 h/PAgo/S=0 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 (Illustration Via) or the PAS positive granules of rat parotid gland. PAS reactivity was produced, however, when oxidation with O.iimM periodic acid in 1M. HCl at 1°C was performed for 24 hours (Illustration VIb) or when the routine PAS procedure was employed (Illustration Vic). These results Indicate that PA* does not produce significant Schiff staining of "neutral sugars". The oxidation of the "neutral sugars" of sialo- and sialosulphoglyco-proteins was investigated in sections of rat ileum-colon which were pretreated with the KOH/PAj/Bh sequence to confine vicinal 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 (Illustration 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 in 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 in the presence of the "neutral  sugars" of sialo- 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 in 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 possibility, PA/Bh/KOH treated sections of rat ileum-colon and salivary gland were incubated in 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 differ 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 (Illustration VIII). These data indicate that PA* does not extract epithelial 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 vicinal 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. Finally, 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 either 1M HCl for 1 hour at 4°C or with PA* (Illustration X). A minimal Schiff staining was obtained. However, when sections were incubated in PA* for 24 or 48 hours, strong Feulgen staining was observed. The Feulgen reaction, however, would not be expected to interfere with s i a l i c acid detection in the PA*/S procedure. B. Mechanistic Studies of PA* Conditions In the previous section i t was demonstrated that PA* did not form Schiff positive 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 either do not oxidize or do not produce a sufficient quantity of aldehydes to produce a visible Schiff reaction, was not determined. To investigate this, sections of rat l i v e r , ileum-colon and salivary gland complex were subjected to the KOH/PA^/Bh technique to confine v i c i n a l diols to "neutral sugars", and were then oxidized with one of PA* (K0H/PA3/Bh/PA*) (Fig. 5B), 1% periodic acid for 1 hour at room temperature (KOH/PA/Bh/PAgo) or both PA* and PAgn (K0H/PA/Bh/PA/PA60) (Fig. 5A). The sections were then treated with either Schiff reagent or with 2,4-dinitro-phenylhydrazine (DNPH) followed by tetrazotized 3,3'-dimethoxybenzidine fluoroborate (TDMBF) (Stoward, 1967b) (Fig. 5A and 5B). Sections exposed to the KOH/PA3/Bh/PA60 and KOH/PA3/Bh/PA*/PA6o were strongly Schiff-positive and yielded formazans on treatment with DNPH and TDMBF (Fig. 5A). However, sections exposed to the KOH/PA^/Bh/PA* sequence 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 (Illustration XIa & b). - 38 -Fig . 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 iver, 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. - 39 -Fig. 5A - 40 -- 41 -Fig. 6. Illustration of the 2f4-dlnltrophenvlhydrazlne 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-dlnltrophenyl-hydrazlne (DNPH) which reacts with the aldehydes to form phenylhydrazones (b-c). Next, treatment with tetrazotized 3,3'-dimethoxybenzidlne fluoroborate (TDMBF) (c-d) results in the formation of formazans (d). 42 H , O H PA CHbOH O DNPH C H 2 O H O i TDMBF C H 2 O H Ar-formazan formazan - 113 -C. Discussion and Conclusions In this study i t was shown that PA* (0.4 mM periodic acid in 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 visible staining of the neutral macromolecules In rat l i v e r and parotid glands, or of the "neutral sugar" residues of either sialo- or sialosulphoglycoproteins. In addition, the reagent did not extract epithelial glycoproteins, de-O-acylate the side chain of s i a l i c acid residues, or produce a significant Feulgen reaction. Therefore, oxidation with 0.4 mM periodic acid in 1M HCl at 4°C in 1 hour results in 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 in 1M HCl occurs has not been f u l l y established. These oxidation conditions did not produce Schiff-positive reactive sites 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. It i s probable, therefore, that oxidation of "neutral sugars" with PA* results in too few aldehyde groups to produce a visible 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 in 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 selectivity of the PA* technique i s probably a result of an increase in the oxidation rate of s i a l i c acids together with a decrease in 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 determina-tion of the types and relative proportions of the various sugar residues in normal tissues and i t would therefore be possible to detect changes in the types and ratios of these residues in 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 vicinal 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" in s i a l i c acid-containing tissues. The five histochemical procedures devised in this study are listed in 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 vicinal 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 C7f 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 f "neutral sugar" vicinal diols and O-sulphate ester a) Neutral sugar = hexose, 6-deoxyhexose and N-acetyl hexosamine residues b) If 9-0-acyl s i a l i c acids oxidize in the i n i t i a l PA* steps of methods 1 and 3, then they w i l l appear in 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 in Table I. Results of the application of Methods 1-5 on the staining patterns of the tissues listed here were predicted on the basis of the known composition of these tissues (Table II - Page 27). - 47 -Si a l i c Acid 0-acyl side chain substitution Method Procedure O-sulphate "Neutral Sugar" None b) C7 C8 and C9 ester vicinal diol a) 1 PA*/Bh/KOH/PA*/T/KOH/Bh/PAS 0 M O B 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; in 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 in 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" vicinal 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, Illustration 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 in Method 1 (Fig. 9, Illustration 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 reduction-periodic acid-Schlff (PA*/Bh/KOH/PA*/T/KOH/Bh/PAS) technique, and Method 2, the saponification-selective periodate oxidation-thionin Schiff-saponification-borohydride reduction-periodic acid Schiff (KOH/PA*/T/KOH/Bh/PAS) technique. In Method 1, in 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 in 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) in 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" vicinal 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 in 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 for 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 this method, the selective perlodate oxidation-thionin Schiff-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, Illustration XIV). Method 4: Simultaneous Visualization of A l l S i a l i c Acids and O-sulphate Esters In this method, the saponification-selective perlodate oxidation-borohydride 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. The O-sulphate esters are then stained aqua with Alcian blue at pH 1.0. The "neutral sugars" are then oxidized and stained magenta with the PAS procedure (Fig. 9, Illustration XV). Method 5: Method for the Selective Oxidation of "Neutral Sugars" In this method, the saponification-selective perlodate oxidation-borohydride 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 in the PAS procedure (Fig. 9, Illustration 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 C H p H T) 0 H CH2OH : H 2 O H O H C H 2 O H O O H ( a ) (b) ( c ) (d) ( e ) (f) (9) 0=C-CH3 N | - u t o H \ c O O H C H O \ C O O H l |T C C C - C H 3 ' N ^ H O ? ^ Q Q H O H . Q - C - C H 3 Bh H O T V C O O H 1 PA NVcH0f^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 reduction- periodic 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 vicinal diols, resulting in 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. This method therefore results in magenta staining of "neutral sugars". 55 :H 2 OH KOH H , O H PA 3 C H ? O H H 7 OH C ^ O H ( a ) (b) ( c ) (d) ( e ) (f) (9) 0 = C - C H O O H KOH C c C - C H 3 ^ l PA, 0 - c - C H 3 J 3 N ' t f £ ^ \ C O O H CcC-CH "•O AB 1.0 C O O H 571 ^ r - C H 3 jPA - 56 -B. Specificity of Methods 1-5 The specificity of these sequences was examined using the experiments outlined in 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 in 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 specificity of the KOH/Bh sequence used in 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 specificity of Methods 1 to 5. The nature of the macromolecules studied in these tissue sites in summarized in 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 in 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 Method Experimental Sequence Results To determine i f one Schiff reagent i s replaced by another PA*/Bh/KOH/PA*/T/KOH/Bh/ PA/Bh/S K0H/PA»/T/KOH/Bh/PA/Bh/S Thionin staining only 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 in 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, illu 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 Results To demonstrate that no non-specific staining with thionin Schiff occurs PA»/Bh/K0H/PA»/Bh/T/K0H7 Magenta Bh/PA/S staining only KOH/PA*/Bh/T/KOH/Bh/PA/S PA*/Bh/T/Bh/PA/S/KOH To determine i f 1 the resulting staining patterns in Methods 1-5 2 reflect those expected on the 3 basis of the known composition 4 of the various macromolecules 5 in the tissues (Table V) (Page 46) PA*/Bh/KOH/PA*/T/KOH/ Bh/PA/S KOH/PA*/Bh/T/KOH/Bh/PA/S PA*/T/Bh/PA/S/KOH K0H/PA*/Bh/AB1.0/PAS KOH/PA*/Bh/PA/S See Table V (Page 46) Interpretation No non-specific thionin Schiff staining occurs 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". - 60 -The KOH/Bh sequence could not be used in 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" in the subsequent PAS step. As a result, the saponification step was placed after the f i n a l pararosaniline Schiff treatment. The fea 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 in these control studies (Illustration XIX) ,indicating that non-specific staining with thionin Schiff had not occurred. Finally, in a l l five methods the f i n a l PAS procedure was performed under conditions which resulted in maximal staining of rat l i v e r glycogen and the neutral macromolecules of parotid gland acinar c e l l granules. The specificity 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 in the tissues (Table I I ) . As would be expected, l i v e r glycogen and the neutral macromolecules of the acinar cells of the rat parotid gland, which do not contain sialo-, 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 epithelial sialoglycoproteins of human colon and rat distal colon, which also contain "neutral sugars", stained in 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 in various shades of purple. The acinar cells 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 et_al . , 1987 unpublished), human colon (Culling et a l . f 1981) and the bases of the crypts of the rat proximal colon (Reid et a l . , 1973) stained in 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. Finally, 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 in various shades of purple with Method 3* C. Discussion and Conclusions These studies have resulted ln 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" in the presence of s i a l i c acid. The specificity of these procedures depends upon several factors. F i r s t l y , the selective perlodate oxidation procedure (PA*) used in 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 vicinal diols without producing a visible 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 in 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 in 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 in 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, in Methods 4 and 5, such oxidation would result in 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 in approximately 1M hydrochloric acid for 1 hour at 4°C. These conditions did not produce visible Schiff staining of either neutral macromolecules or the "neutral sugars" of sialo- 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* in 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 in the tissues examined in this study, and i f they oxidize under PA* conditions, then the observed thionin Schiff staining in 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> in 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. Acyl migration would not, however, affect the staining in Method 1. 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 in bovine submandibular glands using conditions which accounted for the slow oxidation of such acids. Secondly, the perlodate oxidation conditions employed in the f i n a l PAS step of a l l five methods must completely oxidize a l l "neutral sugar" vicinal diols and the Schiff reagent used should react completely with a l l aldehydes produced. If 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 in 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). It i s necessary, therefore, to - 64 -consider the possibility that an incomplete PAS reaction can occur. Furthermore, "neutral sugars" containing O-acyl esters upon potential vic i n a l diols w i l l not be detected in Method 3« Such residues have only been detected thus far i n one gastrointestinal mucin, the mucous cell s of the di 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 in these methods w i l l remove any O-acyl esters present before the PAS procedure i s carried out. Finally, as demonstrated in control experiments, the product of thionin Schiff reagent and s i a l i c acid monoaldehydes was sufficiently 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 in Table VII. - 65 -Table VII: List of Control Methods which should accompany Methods 1-5. x = step performed PA* = oxidation with 0.4 mM period acid in 1M hydrochloric acid for 1 hour at 4°C Bh = reduction with 0.1% (w/v) sodium borohydride in 1% dibasic sodium phosphate (anhydrous) for 15 minutes at room temperature KOH = saponification with 0.5% potassium hydroxide in 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. If Ia i s positive, then Ib should be carried out. If Ib i s also positive, then Methods 1-5 are not specific. - 66 -Tissue la) rat l i v e r rat parotid gland Procedure PA*/S Expected Result no staining Rationale These tissues do not contain s i a l i c acids and "neutral sugars" - should not oxidize under conditions of PA* b) rat l i v e r rat parotid gland PA*/Bh/PA*/S no staining any sugar residues oxidized in PA* should be reduced by boro-hydride and therefore be unavailable for subsequent oxidation (PA* second) and staining with Schiff reagent II) rat l i v e r rat parotid gland Method 1 Magenta PA*/Bh/KOH/PA*/T/ KOH/Bh/PAS Method 2 Magenta KOH/PA*/T/KOH/Bh/PAS Method 3 Magenta PA*/T/Bh/PAS/KOH Method 4 Magenta K0H/PA*/Bh/AB1.O/PAS Method 5 Magenta KOH/PA*/Bh/PAS 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 in magenta staining I l i a ) human colon PA/Bh/KOH/PA*/S intensely A l l unsubstituted magenta 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 Schiff reagent - 67 -b) human PA/Bh/KOH/PA*/Bh no staining colon /PA*/S 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 sialo- sugars should remain, therefore, to be oxidized in the second PA* step, resulting in lack of staining in tissues treated in 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. The second involved an investigation of the mechanism of this selectivity, and the third involved the application of the selective periodate oxidation of s i a l i c acids to the development of general methods for the detection of "neutral sugars" and for the simultaneous visualization of s i a l i c acids and i t s O-acyl side chain substituents and O-sulphate ester, and "neutral sugars". These methods provide new techniques for the examination of epithelial glycoproteins in normal and diseased tissues. - 69 -REFERENCES Barger, J.D. and DeLamater, E.D. (1948) The use of thionyl chloride in the preparation of Schiff reagent. Science 108, 121-2. Boland, C.R., Montgomery, C.K. and Kim, Y.S. (1982a) Alteration in human colonic mucin occurring with cellular differentiation and malignant transformation. Proc. Nat. Acad. Sci. 19, 2051-5. Boland, C.R., Montgomery, C.K. and Kim, Y.S. (1982b) A cancer associated mucin. Alteration in benign colonic polyps. Gastroenterology 82, 664-72. Boland, C.R., Lance, P., Lewin, B., Riddell, R.H. and Kim, Y.S. (1984) Abnormal goblet c e l l glycoconjugates in rectal biopsies associated with an increased risk of neoplasia i n patients with ulcerative c o l i t i s : early results of a prospective study. Gut 25, 1364-71. Bresaller, R.S., Boland, R. and Kim, Y.S. (1984) Characteristics of colorectal carcinoma cells with high metastatic potential. Gastroenterology 87., 115-22. Cheresh, D.A. and Reisfeld, R.A. (1984) 0-acylation of disialo ganglioside G D 3 by human melanoma cells creates an unique determinant. Science 225, 844-6. Chittenden, G.J.F. and Guthrie, R.G. (1963) Nitrogen-containing carbohydrate derivatives. Part I I I . Some methyl 3-arylazo-4,6-0-benzylidine-3-deoxy -D-glucosides. J. Chem. Soc. 3658-65. - 70 -Cooper, H.S., Cox, J., Patchefsky, A.S. (1980) Immunohistologic study of blood group substances In polyps of the distal colon. Expression of a fetal antigen. Am. J. Clin. Path. 13, 345-50. Cooper, H.S. (1982) Peanut lectin binding sites in large bowel carcinoma. Lab. Invest. Ml, 383-9. Cooper, H.S. and Reuter, V.E. (1983) Peanut lectin binding sites in polyps of the colon and rectum. Adenomas, hyperplastic polyps, and adenomas with in situ carcinoma. Lab. Invest. 4_9_, 655-61. Culling, C.F.A. (1974) Handbook of Histochemical and Histopathological Technique. 3rd Edn. London Butterworth. Culling, C.F.A., Reid, P.E., Dunn, W.l. and Clay, M.G. (1974) The histochemical demonstration of O-acylated s i a l i c acids in gastrointestinal mucins. Their association with the potassium hydroxide-periodic acid-Schiff effect. J. Histochem. Cytochem. 22, 826-31. Culling, C.F.A., Reid, P.E., Burton, J.D. and Dunn, H.L. (1975) A histo-chemical method of differentiating lower gastrointestinal tract mucin from other mucins in primary or metastatic tumors. J. Clin. Path. 28_, 656-8. Culling, C.F.A., Reid, P.E. and Dunn, W.L. (1976) A new histochemical method of use in the interpretation and diagnosis of adenocarcinoma and villous lesions in the large Intestine. J. Clin. Path. 30, 1056-62. - 71 -Culling, C.F.A. (1977) The apparent failure of sodium borohydride reduction to block further PAS reactivity in rat epithelial mucins. Histochem. J. 9, 781-5. Culling, C.F.A., Reid, P.E. and Dunn, H.L. (1979) A histochemical comparison of the O-acylated s i a l i c acids of the epithelial mucins in ulcerative c o l i t i s , Crohn's disease and normal controls. J. Clin. Path. 32, 1272-7-Culling, C.F.A., Reid, P.E., Dunn, H.L. and Freeman, H.J. (1981) The relevance of the histochemistry of colonic mucins based upon their PAS reactivity. Histochem. J. 13, 899-903. Diaz, S. and Varki, A. (1985) Metabolic labelling of s i a l i c acids in tissue culture c e l l lines: methods to identify substituted and modified radioactive neuraminic acids. Anal. Biochem. 150, 32-46. Ehsanullah, M., F i l i p e , M.I. and Gazzard, B. (1982a) Morphological and mucous secretion c r i t e r i a for differential diagnosis of solitary ulcer syndrome and non-specific proctitis. J. Clin. Path. 35, 26-30. Ensanullah, M., F i l i p e , M.E. and Gazzard B. (1982b) Mucin secretion in inflammatory bowel disease: correlation with disease activity and dysplasia. Gut 23, 485-9 F i l i p e , M.I. and Lake, B.D. (1983) Histochemistry in Pathology. London: Churchill Livingstone Press. - 72 -Fakan, F. and Adamocova, J. (1981) Diagnostic value of the PBT/KOH/PAS method for identification of primary cancer from metastases. Acta. Histochem. 6J£, 176-80. Fenger, C. and Fi l i p e , M.I. (1981) Mucin histochemistry of the anal canal epithelium. Studies of normal anal mucosa and mucosa adjacent to carcinoma. Histochem. J. J 3 , 921-30. F i l i p e , M.I. (1979) Mucins in the human gastrointestinal epithelium: a review. Invest. Cell Path. 2, 195-216. F i l i p e , M.I. (1984) Transitional Mucosa. Histopathology 8_, 707. F i l i p e , M.I., Mughal, S. and Bussey, H.J. (1980) Pattern of mucus secretion in the colonic epithelium in familial polyposis c o l i . Invest. Cell Path. 3 , 329-43. Franzin, G., Scapa, A., Dina, R. and Novelli, P. (1981) 'Transitional' and hyperplastic-metaplastic mucosa occurring in a solitary ulcer of the rectum. Histopathology 55, 27-33-Franzin, G., Grigioni, W.F., Dina, R., Scarpa, A. and Zamboni, G. (1983a) Mucin secretion and morphological changes of the mucosa in non-neoplastic diseases of the colon. Histopathology 7_, 707-18. - 73 -Franzin, G., Zamboni, G., Dina. R., Scarpa, A. and Fratton, A (1983b) Juvenile and inflammatory polyps of the colon - a histological histochemical study. Histopathology I, 719-28. Franzin, G., Dina, R., Zamboni, G., Iannucci, A., Scarpa, A. and Novell!, P. (1984) Hypoplastic (metaplastic) polyps of the colon. Am. J. Surg. Path. 5, 687-98. Guthrie, R.D. (1961) The "Dialdehydes" from the periodate oxidation of carbohydrates. Adv. Carbohydr. Chem. 16, 105-58. Haverkamp, J., Schauer, R., Wember, M., Kamberling, J.F. and Vliegenthart, J.P.F. (1975) Synthesis of 9-O-acyl and 4,9-di-O-acetylneuraminic acid methylglycoside. Z. Physiol. Chem. 3_6, 1575-83. Iannoni,C, Marcheggiano, A., Pallone, F., r i e r i , G., Gallucci, M., Di Silverio, F. and C a p r l l l i , R. (1986) Abnormal patterns of colorectal mucin secretion after urinary diversion of different types: histochemical and lectin binding studies. Hum. Path. J J , 834-40. Kiernan, J.A. (1981) Histological and histochemical methods: theory and practice pp. 35. Toronto: Pergamon Press. Klessen, C. (1978) Demonstration lines a l k a l i PAS effektes bei Verwendung. Von Perjodsaure in nierdriger Kenzentration. Histochemistry, 56, 299-305. - 74 -Lev, R., Lance, P. and Comava, P. (1985) Histochemical and morphological studies of mucosa bordering rectosigmoid carcinoma. Comparison with normal, diseased and malignant epithelium. Hum. Path. 16, 151-61. L i l l i e , R.D. and Pizzolato, P. (1985) Histochemical use of borohydrides as aldehyde blocking reagents. Stain Technol. 42, 13-6. Listinski, CM. and Riddell, R.H. (1981) Patterns of mucin secretion in neoplastic and non-neoplastic disease of the colon. Human Path. 12, 923-9. McLean, R.L., Sultajit, M., Beidler, J. and Winzler, R.J. (1971) N-acetyl neuraminic acid analogues. I. Preparation of the 7 carbon and 8 carbon compounds. J. Biol. Chem. 246, 803-9. Mester, L. (1955) The formazan reaction in proving the structure of perlodate oxidized polysaccharides. J. Am. Chem. 22, 5432-3. Mester, L. (1958) The formazan reaction in carbohydrate research. Adv. Carbohydr. Chem. 13, 103-67. Montero, C. and Segura, D.I. (1980) Retrospective histochemical study of mucosubstances in adenocarcinomas of the gastrointestinal tract. Histopathology 4_, 281-91. Munhoz, C.O.G. (1971) Histochemical classification of acini and ducts of parotid glands from artiodactyles, carnivores and rodents. Acta. Histochem. 32, 302-17. - 75 -Park, CM., Reid, P.E., Walker, D.C. and MacPherson, B.R. (1987a). A simple, practical 'Swiss r o l l ' method of preparing tissues for paraffin and methacrylate embedding. J. Microsc. Jj£5, 115-20. 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 0-acyl variants or O-sulphate ester. I I . Methods based upon the periodic acid-phenylhydrazine-Schlff reaction. Histochem. J. 13 in press. Pearse, A.G.E. (1968) Histochemistry: Theoretical and Applied. Vol. 1, 3rd Ed. p. 706. London: Churchill Livingstone. Reid, P.E., Culling, CF.A. and Dunn, W.L. (1973) Saponification induced increase in the periodic acid Schiff reaction in the gastrointestinal tract: mechanism and distribution of the reactive substance. J. Histochem. Cytochem. 21, 473-83. Reid, P.E., Culling, C.F.A., Ramey, C.W., Dunn, W.L. and Clay, M.G. (1977) A simple method for the determination of the 0-acetyl substitution pattern of the s i a l i c acids of colonic epithelial glycoprotein. Can. J. Biochem. 55, 493-503. Reid, P.E., Culling, CF.A. and Dunn, W.L. (1978) A histochemical method for the demonstration of 9-0-acyl s i a l i c acids. An investigation of bovine submaxillary mucin and intestinal mucins. J. Histochem. Cytochem. 2j>, 187-92. / - 76 -Reid, P.E. and Cu l l i n g , C.F.A. (1980) The periodate oxidation of carbohydrates i n r e l a t i o n to the PAS reaction. J . Histotechnol. 3, 82-90. Reid, P.E., Dunn, W.L., Ramey, CW., Coret, E., Trueman, L. and Clay, M.G. (1984a) Histochemical I d e n t i f i c a t i o n of side chain substituted O-acylated s i a l i c acids: the PAT/KOH/Bh/PAS and the PAPT/KPH/Bh/PAS procedures. Histochem. J . 16, 623-39. Reid, P.E., Dunn, W.L., Ramey, C.W., Coret, E., Trueman, L. and Clay, M.G. (1984b) Histochemical studies of the mechanism of the periodic aci d -phenylhydrazine-Schiff (PAPS) procedure. Histochem. J. 16, 641-9. Reid, P.E., Cu l l i n g , C.F.A., Dunn, W.L., Ramey, CW. and Clay, M.G. (1984c) Chemical and histochemical studies of normal and diseased gastrointestinal t r a c t . I. A comparison between h i s t o l o g i c a l l y normal colon, colonic tumors, ulcerative c o l i t i s and d i v e r t i c u l a r disease of the colon. Histochem. J . 16, 235-51. Reid, P.E., Cu l l i n g , C.F.A., Dunn, W.L., Ramey, CW. and Clay, M.G. (1984d) Chemical and histochemical studies of normal and diseased gastrointestinal t r a c t . I I . A comparison between h i s t o l o g i c a l l y normal small intestine and Crohn's disease of the small intestine. Histochem. J. 16, 253-64. - 77 -Reid, P.E., Owen, D.A., Dunn, W.L., Ramey, C.W., Lazosky, D.A. and Clay, N.G. (1985a) Chemical and histochemical studies of normal and diseased gastro-intestinal tract. I I I . Changes in the histochemical and chemical properties of the epithelial glycoproteins in the mucosa close to colonic tumours. Histochem. J. H, 171-82. Reid, P.E., Owen, D.A., Ramey, C.H., Dunn, W.L., Jones, E.A., Lazosky, D.A., Allen, E. (nee Atkins), Park, CM. and Clay, N.G. (1985b) Chemical and histochemical studies of normal and diseased gastrointestinal tract. V. A differential diagnostic method for the histochemical classification of glycoproteins. Histochem. J. U, 891-903. Rhatigan, R.M. and Saffros, R.O. (1979) Mucosal hyperplasia in colonic diverticula. Histopathology 3 , 153-60. Roberts, CP. (1977) Histochemical detection of s i a l i c acid residues using perlodate oxiation. Histochem. J. 2 , 97-102. Schauer, R. (1982) Chemistry metabolism and biological functions of s i a l i c acid. Adv. Carb. Chem. Biochem. 4J), 131-234. Shukla, A.K. and Schauer R. (1982) Fluorometric determination of unsubstituted and 9 (8)-0-acylated s i a l i c acids in erythrocyte membrane. Hoppe-Seyler's Z. Physiol Chem. 3i3, 255-262. - 78 -Schulte, B.A. and Spicer, S.A. ( 1983 ) Light microscopic histochemical detection of sugar residues in secretory glycoproteins of rodent and human tracheal glands with lectin-horseradish peroxidase conjugates and the galactose oxldase-Schiff sequence. J. Histochem. Cytochem. 31, 3 9 1 - 4 0 3 . Scott, J.E. and Harbinson, R.J. ( 1968 ) Periodate oxidation of acid polysaccharides. Inhibition by the electrostatic f i e l d of the substrate. Histochemie J l , 215-20. Scott, J.E. and Darling, J. (1969) Periodate oxidation of acid polysaccharides I I I . A PAS method for chondroitin sulphates and other glycosamlno-glycuronans. Histochemie 12, 2 9 5 - 3 0 1 . Shackleford, J.M. and Klapper, C.E. ( 1962 ) Structure and carbohydrate histochemistry of mammalian salivary glands. Am. J . Anat. 1 1 1 , 25 -47. Simson, J.A.V., Hail, B.J. and Spicer, S.S. ( 1973 ) Histochemical evidence for lipoidal material in secretory granules of rat salivary glands. Histochem. J. 5 , 2 3 9 - 5 4 . Spicer, S.S. ( 1961 ) The use of cationic reagents in histochemical differentiation of mucopolysaccharides. Am. J. Clin. Path. 36_, 3 9 3 - 4 0 9 . Spicer, S.S. and Duvenci, J. ( 1964 ) Histochemical characteristics of mucopolysaccharides ln salivary and exorbital lacrimal glands. Anat. Rec. 142, 3 3 3 - 5 8 . - 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 sialic 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 specific thionine-S02 reagent and i t s use in a bi-colour method for deoxyribonucleic acid and periodic acid-Schiff positive substances. J. Histochem. Cytochem. 4, 55-63. Varki, A. and Diaz, S. (1984) The release and purification 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., Corfield, A.P., Schauer, R. and Andrews, K.H. (1979) The bovine submandibular gland. II. Histochemical and biochemical results. Proc. 5th Int. Symp. Glycoconjugates, pp. 652-53- Thieme: Stuttgart. Volz, D., Reid, P.E., Park, CM., Owen, D.A., Dunn, H.L. and Ramey, CH. (1986) Can "mild" periodate oxidation be used for the specific histochemical identification of s i a l i c acid residues? Histochem. J. 18, 579-82. Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, H.L. (1987a) A new method for the selective periodate oxidation of total tissue s i a l i c acids. Histochem. J. in press. Volz, D., Reid, P.E., Park, CM. Owen, D.A. and Dunn, H.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. in 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 in cellular glycoproteins. Histochemie M5, 217-7. Yonezawa, S., Nakamura, T., Tanaka, S. and Sato, E. (1982) Glycoconjugate with Ulex europaeus agglutinin-1 binding sites in 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 in polyposis c o l i . Comparative study with solitary adenoma in the sigmoid colon and rectum. J. Nat. Can. Inst. I I , 17-24. - 82 -Illustration I: A. Sections of rat colon stained with the PA/Bh/K0H/PA2/S (Slide Al).  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 cells i s similar regardless of the oxidation technique used, indicating that PA* oxidizes a l l s i a l i c acid diols. B. Sections of rat l i v e r stained with the PA2/S, PA*/S and PA6Q/S procedures. There i s l i t t l e or no staining of glycogen with the PA2/S and PA*/S techniques (Slides Bi 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 slightly greater intensity obtained with the PA2/S technique, indicating the greater specificity of the PA*/S. - 83 -Illustration 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 in 0.004 mM periodic acid dissolved in 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 in acid solvent (Slide l i b ) i s greater than that obtained when water was used as solvent. - 84 -Illustration I I I : Effect of pH on the perlodate oxidation of "neutral  sugars". Sections of rat li v e r were oxidized in 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 in acid conditions. Oxidation in 1M NaCl (not shown) resulted in a staining pattern similar to that observed when water l s used as a solvent. - 85 -Illustration IV: Evidence for the specificity of PA* conditions for 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 -Illustration V: Evidence for the specificity 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 in strong (4+) Schiff staining of the tissue. However, when serial 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 -Illustration 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 in 1M HCl at 4°C for 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 reactivity of glycogen did occur slightly in (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 in (b) (See also Illustration X). Such staining i s insignificant in (a) or (c). Similar results were obtained with rat parotid gland. - 88 -Illustration VII: Effect of PA* conditions on the oxidation of "neutral sugars"  ln the presence of sialo- and slalosulpho-contalning glycoproteins. Sections of rat colon were pre-treated with the K0H/PA3/Bh sequence to confine vicinal diols to "neutral sugar" residues. Sections were then treated with (a) PA* (KOH/PA3/Bh/PA*), (b) PA6Q (KOH/PA3/Bh/PA6o) or (c) PAVPA60 (KOH/PA3/Bh/PA*/PA) and stained with Schiff reagent. No Schiff reactivity 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 -Illustration 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 in the staining patterns occurred between (a) and (b). These data indicate that PA* does not extract epithelial glycoproteins. - 90 -Illustration IX: To determine If 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 vicinal diols, incubated with 1M HCl for 1 hour at 4°C, and then treated with the PAS technique (PA/Bh/1M HCl 1 hr at *»°C/PAS) (Slide IX). 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 -Illustration X: To determine If PA* conditions Invoke a Feulgen reaction. Sections of rat colon were incubated with either 1M 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 in PA* for 24 hours, a strong Feulgen staining resulted in addition to mucin staining. - 92 -Illustration XI. Illustration of results obtained in mechanistic studies of the selectivity of PA*. Sections of rat colon were treated with the KOH/PAg/Bh sequence to confine vicinal diols to "neutral sugar" residues. Sections were then oxidized with (a) PA* (KOH/PA3/Bh/PA*) (Slide XIa), (b) PAgo (KOH/PA3/Bh/PA6o) (Slide Xlb), or (c) PA*PAg0 (KOH/PA3/ Bh/PA*/PA6o) (not shown) and stained with Schiff (not shown) or DNPH-TDMBF (Slides XIa and b). Sections exposed to (b) or (c) (not shown) were strongly Schiff positive and yielded formazans on treatment with DNPH and TDMBF (Slide XIa). Sections exposed to (a), however, were Schiff unreactive and showed only traces of formazan with the DNPH/TDMBF sequence (Slide Xlb). These data imply that PA* does not produce hemiacetals or hemialdals. - 93 -Illustration XII. Illustration of the selective periodate oxldatlon- borohvdrlde 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 Xllb), proximal and distal colon (Slide XIIc and Xlld 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 in 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 macro-molecules of the acinar cells of rat parotid gland (not shown) which either do not contain sialoglycoproteins, or only contain traces, stain magenta, while rat sublingual gland (Slide Xllb), 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 in 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 -Illustration XIII. Illustration 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 this 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. - 95 -Illustration XIV. Illustration of the selective perlodate oxidation- thionin Schiff-borohydride reduction-periodic acld-Schlff-saponification  (PA*/T/Bh/PAS/KOH) technique (Method 3). Sections of rat l i v e r , salivary gland complex, rat colon and human colon were treated with PA*/T/Bh/PAS/KOH. In this method s i a l i c acids without side chain substituents or with side chain substituents at position C7 stain blue, "neutral sugars" stain magenta, and mixtures stain in varying shades of purple. Tissues containing no s i a l i c acids or only traces of this residue such as rat l i v e r (staining similar to that seen i n Slide Xl l a ) , submandibular gland (Slide XlVa) and parotid gland (Slide XlVb), stain magenta, while tissues containing large quantities of s i a l i c acid without side chain substituents such as rat sublingual gland (Slide XlVa) and rat d i s t a l colon (Slides XIVc and XlVd respectively) stain in varying shades of purple. Tissues with l i t t l e unsubstituted s i a l i c acid stain magenta (rat proximal colon (Slide XIVc) and rat dist a l colon (Slide XlVd)). - 96 -Illustration 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 sialoglyco-proteins such as the rat sublingual gland (Slide XVa) and crypt bases of the proximal colon (Slide XVb) and "neutral sugars" (liver (see X l l a ) , sub-mandibular 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 distal colon (Slide XVc), which also contain "neutral sugars", stain in varying shades of purple. - 9 7 -Illustration 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 in varying intensities of magenta. Shown are rat submandibular and sublingual gland (Slide XVIa), rat proximal (Slide XVIb) and di s t a l (Slide XVIc) colon and human colon (Slide XVId). - 98 -Illustration 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 in 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 il l u s t r a t i n g that no exchange of Schiff reagent occurs in these procedures. A i s illustrated with human colon (Slide XVIIa) and (b) with rat submandibular and sublingual glands (Slide XVIIb). - 99 -Illustration XVIII. 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. The KOH/Bh sequence l s necessary for the removal of non-specific 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 in 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 in 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, illustrating the KOH step i s s t i l l functional in i t s new position. - 100 -Illustration XIX. Evidence that non-specific thionin Schiff staining does  not occur in Methods 1-3. Sections of human colon were treated with a variation of Methods 1, 2 and 3, in 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 -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 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 total tissue s i a l i c acids. Histochem. J. in 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. in 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. in 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, in press. 6. Park, CM., Reid, P.E., Owen, D.A., Volz, D. and Dunn, W.L. (1987) Light microscopic histochemical studies of epithelial c e l l glycoproteins in normal rat colon. Submitted to Histochem. J . 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. J_8, 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 total tissue s i a l i c acids. Histochem. J. in 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. in 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. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction. Histochem. J. 1_9_ in 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, in press. 6. Park, CM., Reid, P.E., Owen, D .A. , Volz, D. and Dunn, W.L. (1987) Light microscopic histochemical studies of epit h e l i a l c e l l glycoproteins in normal rat colon. Submitted to Histochem. J . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0097194/manifest

Comment

Related Items