Carbohydrates

Carbohydrates

1. I. Neutral polysaccharides

   • glycogen
     • -simple polysaccharides -branched or straight chain of D-glucose units
     • where is it??
       • intracytoplasmic: 1.liver,cardiac and skeletal muscle 2.endometrial gland vaginal and cervical epithelium
     • fixation requirement
       • 1.fix at low temp 2. fix promptly -have to minimize streaming artefact
   • starch
     • -formed of two main constituents: amylose and  amylopectin -difficult to see under normal bright field microscopes, can see under polarized light: maltese cross birefringence -stain very strongly with PAS reaction.
   • cellulose
     • Undigested food particles in section of GI tract. Foreign bodies in skin.
   • chitin
     • -hyaline substance:in non-human tissues e.g. hydatid cysts of liver or lung,due to infestation by larvae of tape worm

2. II.Mucins

   • a defining structure;the presence of tandem repeats of amino acid sequence within the protein core -mucins are hexosamine-containing polysaccharides,covalently bound to various amount of proteins.
   • 1.Acid mucins
     • -contain carbohydrates with carboxylate or sulphonate  groups:ionized at physiologic pH,produce overall negative charge on these mucin molecules.
     • a.sulphated
       • Strongly sulphated connective tissue mucin
         • -react at low pH with cationic dye -usually PAS negative***
         • found in : endothelial cells, fibroblasts, chondrocytes, osteocytes, mast cells
         • types of sulphate
           • Chondroitin A
             • cartilage
           • Chondroitin B
             • -aorta -heart valves -dermis of skin
           • Chondroitin C
             • -cartilage  -umbilical cord -dermis of skin
           • Heparan/Heparin
             • (1)Heparan: aorta,cardiac connective tissue# (2)Heparin:mast cells
           • Keratan
             • -cornea -aging cartilage
       • strongly sulphated epithelial Mucin
         • -epithelial origin -react at low pH with cationic dyes -PAS +ve*** -present in bronchial sub-mucous gland
       • weak sulphated mucins
         • -widely distribution in tissue -react at slighly higher pH with cationic dyes
     • b.carboxylated
       • Sialomucins
         • -a mucin whose carbohydrate groups containing sialic acid -epithelial in origin
         • enzyme-liable
           • -it is digested by the enzyme sialidase=PAS positive -wide distribution -commonly as a mixture with other mucin
           • where: submandibular of salivary glands/goblet cells ofintestine.
         • enzyme-resistant
           • -resistant to denaturation by sialidase -PAS -VE -rare -found in mucosa of large intestine
       • sulphated mucins
       • Hyaluronic acid
         • -usually formed by fibroblasts -no sulpated radicals -important constituent of synovial fluid
         • -distinction by reaction with hyaluronidase and sialidase. -stains at similar pH with cationic dyes like sialomucins -found in umbilical cord,cardiac connective tissue and skin dermis
   • 2.Neutral mucins
     • -lack acidic groups and carry no net charge -epithelial in type
     • -consist of various hexosamines associated with free hexose groups. -abundant in brunner's gland and gastric lining cells
   • Fixation requirement
     • 1.aqueous fixations; formaline are satisfactory 2.mercuric chloride: enhance metachromatic staining 3.10% formal alcohol:for free hyaluronic acid

3. III. Glycoproteins

   • -protein with carbohydrate -found  in basement membrane, pituitary beta cells and amyloid -+PAS and protein reaction

4. IV. Glycolipids

   • -lipid and carbohydrates +PAS and lipid reaction

5. Demonstration^^^

   • 1.Chemical methods
     • Periodic acid-schiff reagent
       • -can stain carbohydrate: polysaccharides,mucopolysaccharides,glycoproteins and glycolipids
       • -using picric-acid as oxidizing agent -PAS:basic fuchsin and sulfurous acid
       • the role of sulfurous acid : decolorize the dye of reagent. staining principal: the single bonds of schiff's reagent reform in a quinoid ring by addition of aldehyde.
       • (additional step)Sulphite rinse
         • applied in Feulgen reaction(DNA staining): since schiff reagent would behave as a basic dye and stain the tissue,cause false positive, diluted sulphurous acid is used to wash off Schiff's reagent before washing the tissue in water. 
       • The target tissue can show PAS positive
         • Basic criterias: 1.must contain the 1-2 glycol grouping,an equivaent amino or alkylamino grouping or the oxidation product CHOH-CO
         • 2.must not diffuse during fixation 3.give an oxidation product:does not diffuse
         • -must be presented in sufficient concentration to produce detectable colour.
       • possible results
         • 1.acid mucins can weak positive(need to contain sufficient sialic acid) or negative
         • 2.Prolonged oxidation time:connective tissue mucin(PAS positve),except keratan sulphate
   • 2. Cationic(basic) dye
     • a. Alcian blue
       • features: -positively charged dye(basic dye) so it can forming electrostatic bonds with tissue polyanions(including sulphate and carboxyl groups) -stain sulphated and carboxylated acid mucin blue -glycogen and neutral mucin are negative
       • (varying the pH of the alcian blue solution: can further characterize the subtypes of acid mucins)
       • at the condition of pH 1: sulphated mucin can ionize at pH1:can stained by alcian blue-pH1; but carboxylated mucin cannot ionize at this pH, can only strongly stained by alcian blue-pH 2.5
       • principal
         • all acid mucins can ionize at pH 2.5,produce anionic groups: pH 2.5-alcian blue can stains all acid mucins
       • Important factor:pH
         • pH 0.5
           • only stain strongly sulphated mucin
         • pH1.0
           • stain both weak and strong sulphated acid mucin
         • pH 2.5
           • stain all acid mucins
     • b. metachromasia
       • the phenomenon is described as the ability of a dye to change from orthochromatic or usual color to a metachromatic color. -it is the result of polymerization
       • capable of staining tissues with colours different from it self
       • common dyes
         • 1.Methylene blue 2.Methyl violet 3.Crystal violet 4.Toluidine blue  5.Azure A 6.Thionine 
       • target tissues
         • 1. cartilage 2. mucins 3. Mast cell granules
         • sulphatd mucin
           • it shows metachromasia below pH  (except carboxylated mucin)
       • factor can alter the result
         • 1.alc dehydration 2.mountant used 3.storage
         • 4. enhancement: uranyl nitrate- can increase brightness, mordant-differentiator and alc fastness
   • 3.Traditional staining
     • a. Best's Carmine
       • -selective for glycogen -weakly stain fibrin and neutral mucin
       • principal
         • 1. form hydrogen bond between OH group on the glycogen and H atoms on the carmine acid
       • important agents involved
         • 1.K(2)CO3 and KCl inhibit electrostatic bonds between negatively charged carmine and basic protein in tissue
         • 2.NH3 act as solventfor carminic acid and increase pH: reduce electrostatic linkage 
     • b. Southgate's mucicarmine
       • a chelate complex formed between cationic aluminum ions and carminic acid
       • principal
         • the aluminum cations confer overall positive charge to the large carmine complex
       • results
         • electrostatic attraction to the anionic group of acid mucins: 1. tissue sites that contain plenty of neutral mucin:little or no staining 2.the site of GI tract that acid mucin:stain strongly
       • other applications
         • stain capsule of the fungus, crytococcus neoformans.
   • 4.Silver method
     • gomori's methenamine silver method
   • 5.immunohistochemistry
     • use specific recognition of carbohydrate recognition
   • 6.Affinity cytochemistry
     • -a technique to detect distribution of  specific cell-surface receptors.
   • enzyme used to enhance staining
     • -to increase specificity by treating the section with enzyme to remove non target carbohydrate
     • Amylase
       • -catalyses the hydrolysis of glucosidic linkage of glycogen
     • Diatase
       • contain both alpha and beta amylase: beta amylase is usually employed to digest glycogen
     • Hyaluronidase
       • -attacks the glycosidic linkage of some proteoglycans such as Hyaluronic acid.
     • sialidase
       • -digest sialic mucin:except those are enzyme resistant -loss of alcian blue staining -give positive PAS