6 - Carbohydrates and Lipids

Functions and classification of mucins

• Storage, lubricants, cell adhesion, structure, protection against microbial invasion, cancer diagnostics

• Simple - purely CHO, mono, di and polysaccharides

• Glycoproteins - CHO and protein or lipid molecules e.g. glycolipids, proteoglycans, mucins, glycosaminoglycans

Mono and polysaccharides

• Monosaccharides - glucose, fructose, galactose, water soluble, lost during fixation, difficult to stain

• Polysaccharides - glycogen (repeating glucose), storage form of sugar found in liver and muscle, only polysaccharide that can be demonstrated histochemically, glycogen storage disorder results in deposits in liver

• Seminomas - testicular tumour that results in excess glycogen deposition

Proteoglycans and glycosaminoglycans

• Proteoglycans - found in connective tissue, 95% CHO component (glycosaminoglycans)

• Glycosaminoglycans - composed of repeating disaccharide units e.g. hyaluronic acid, heparin sulphate, dermatan sulphate

Mucins

• Glycoproteins with large CHO component, central protein core found in epithelial cells

• Natural mucins - found in brunners glands in duodenum, no charge, stain with periodic acid schiff (pink) not alcian blue

• Acid mucins - negative charge, can be simple (carboxyl group) or complex (sulphuric acid group), can be strongly or weakly sulphated, found in GI (goblet cells) and respiratory tracts, alcian blue positive, excess in epithelial malignancies

Clinical significance of mucins

• Mucins provide lubrication, membrane bound ones assist in cell adhesion, may be present in malignancy

• Excess production in adenocarcinomas - breast, lung, ovary, colon

• Excess in lung disease - asthma, bronchitis, COPD, CF

• Mucin subtypes can be used as diagnostic markers

Other glycoproteins

• Other CHO-protein conjugates are not easily categorised as they vary in composition, structure and function

• Expressed throughout tissues and cells in body e.g. CD markers involved in lymphocytic cell adhesion CD4/8

Periodic acid schiff technique for detection of carbohydrates

• Reacts with glycogen, starch, mucin, basement membrane, reticulin, fungi

• Used to distinguish between types of glycogen storage diseases, classify tumours of epithelial origin, assess basement membrane thickness (glomerulonephritis), identify fungi in tissue (candida), infection

• Method - oxidation in 1% periodic acid to produce free aldehyde groups, addition of schiff reagent made from basic fuschin, forms a pink compounds in presence of the aldehyde groups

Diastase control

• Helps differenciate glycogen, one slide with, one slide without

• Glycogen is the only CHO that is diastase sensitive (will be destroyed)

• If slide is clear after PAS staining the initial structure was glycogen

Alcian blue stain for mucins

• Dye contains copper based ring linked to functional groups, binds to acid mucins and proteoglycans at a pH of 2.5, can be reduced to 1.5 for subtypes of mucins

• May be combines with PAS to distinguish between natural and acid mucins, AB first PAS second

• Southgates mucicarmine - another carbohydrate stain (yellow)

Classification of lipids

• Saturated and unsaturated fatty acids

• Cholesterol, triglycerides

• Phospholipids, sphingolipids, glycolipids

Properties of lipids

• Soluble in organic solvents, removed during paraffin processing

• Fixation - formalin may be detrimental to some, frozen sections used to avoid solvent

• Histochemistry - determined by hydrophobic/phillic nature and reactive groups e.g. sugars or phosphates

• Lysochrome dyes - lipid soluble dyes e.g. sudab black B, oil red O

• Modified PAS stain can detect the carbohydrate portion of glycolipid

• Implicated in cardiovascular disease, muscle and liver disease, tumours, metabolic disorders