LIPIDS Introduction Lipid is derived from the greek word lipos meaning fat.

Lipids were originally demonstrated in tissue sections for microscopy more than a century ago.Wide range of fat stains, sudan dyes, have been utilized for histology since 1896. Lipids may be defined as any one of a group of fats or fatlike substances characterized by their insolubility in water. These fats would include: true fatsesters of fatty acids and glycerol lipidsphospholipids, cerebrosides and waxes sterolscholesterol and ergosterol hydrocarbonssqualene and carotene. By definition an ester is a fragrant compound formed by the reaction of an acid and an alcohol or phenol without water. Lipids have a vital role in the normal cell membrane, myelin, hormones and secretions. The need to demonstrate lipids is important when they appear abnormal or fail to appear at all. Ideally histochemistry should be utilized with other techniques such as biochemistry and chromatography to demonstrate the exact nature of the lipid identified microscopically and then possibly quantified. Not all lipids resemble fats. For example, free cholesterol with its melting point of 144C is crystalline, and some of the phospholipids and gangliosides are even water-soluble. Loven's 1955 definition that lipids are 'actual or potential derivatives of free fatty acids and their metabolites' still holds true today. Definition Defined as naturally occuring fat like substances that are soluble in organic solvents but not in water .As per loverns 1955 definition lipids are actual or potential derivatives of free fatty acids and their metabolites.Lipids are defined as those naturally occuring substances which are insoluble in water but soluble in so called fat solvent (chloroform, benzene, petroleum, ether, acetone), which are related either actually or potentially to fatty acid esters and which are capable of being utilized by animal organism General chemistry of lipids

Classification 1. 2. Simple lipids Compound lipids Derived lipids Polar lipids Non polar lipids

Classification Polar lipids

Phospholipids Glycerol based Eg: Phophatidyl choline Phosphatidyl serine Phosphatidyl ethanolamine Plasmalogens Classification Non polar lipids Unconjugated lipids Eg 1. Fatty acids 2. Cholesterol esters eg sphingosine based eg: sphingomyelins

glycolipids eg: 1. Cerebrosides 2. Sulphatides 3. Ganglioside

1. Cholestryl esters 2. Mono, di & triglycerides 3. Waxes

Classification 1. Simple lipid esters of fatty acids and alcohols Eg: Neutral fats fats and oil in which alcohol is glycerol Ester waxes contains alcohol higher than glycerol 2. Compound lipids possess a phosphorus/nitrogenous base + fatty acid + alcohol Eg: Phospholipids contains a phosphoric acid radical. Eg: lecithin, cephalin, plasmals. Cerebrosides sphingosine + sugar + fatty acid Eg: Kerasine Phrenosine Nervosine/ nervone Classification Derived lipids are obtained from simple and compound lipid by hydrolysis. Eg: Fatty acids - are derived from triglycerides. Saturated fatty acids contains no double bond eg: stearic & palmitic acid Unsaturated fatty acids have one or double bonds . eg: oleic acid Alcohol of high molecular weight which are obtained from the hydrolysis of ester waxes Sterols cholesterol Straight chain alcohol Physical properties Triglycerides have specific gravity less than 1 They are solid fat or liquid oil , depends upon the room temperature and their MP.

MP depends on the chain length of the component fatty acids and their degree of unsaturation. Saturated fatty acids contain more than 8 C-atoms are solid. Introduction of double bond lowers the MP. Fixation Fixation is necessary so that lipids and the section themselves are able to withstand the potentially destructive or solvent effects of histochemical reagents. Fixatives used are: Osmium tetraoxide Chromic acid Formol calcium preferred for lipid histochemistry

Fixation Formaldehyde can chemically alter certain lipids lecithins and plasmalogens which are slowly degraded to their water- soluble derivatives. Formalin may oxidize to formic acid during prolong storage. Neutral lipids may crystallize during prolong immersion in this fixative and lose their original physical characteristics. Control sections The use of control sections is important in lipid demonstration methods to exclude interference from cross reacting non lipid compounds (negative control)& to verify lipidic nature of stained material As a positive control, bayliss high (1974) suggests the use of cryostat sections from a composite block comprising Rat or human brain (phospholipids) Fatty liver (triglycerides & fatty acids) Atheromatous artery or adrenal (free & esterified cholesterol). In negative control delipidized sections should be included along with normal sections

Lipids are extracted from control sections so that reaction product under consideration in untreated section can be attributed to lipid or to any interfering non lipid group Control sections Delipidisation: Lipids can be totally extracted with chloroform: methanol if 1% hcl is included in the solvent to release bound lipids. Addition of 4% water facilitates extraction of phospholipids Composition of lipid solvent: CHLOROFORM METHANOL DISTILLED WATER Conc. Hcl 66 ml 33 ml 4 ml 1 ml

Extraction of lipid The criteria for extraction of lipids and naturally occurring fat- like substances is their solubility in fat solvents and insolubility in water. These methods are used either for the removal of lipids or to demonstrate their differential solubility in such solvents. Methods: Nile blue sulphate technique. Bromine sudan black B method Identification of lipid Solubility Examination by polarised light Reduction by osmium tetroxide Demonstration of fat soluble dyes Other staining & histochemical method

 Cains nile blue method for differentiating neutral fat & fatty acid Bakers acid hematein for phospholipids Fischlers method for fatty acid Schultz method for cholesterol & cholesterol esters Examination by polarized light By examining the sections with polarised light, it can be determined whether the substances is isotropic or anisotropic Three types of refractility may be seen: Isotropic (mono refringent): neutral fat, fatty acid and cholesterol esters in any state which prevents formation of liquid crystals Anisotropic (bi refringent): an appearance which may be found in any lipid in a crystalline state. Examination by polarized light Maltese cross birefrigent: polarised light can be used to distinguish between glycerol esters and cholesteryl esters. Both esters stain identically with sudan black but cholesteryl esters in fresh tissue display a distinct type of double refraction called maltese cross birefringent / conic focal anisotropism. Reason: this is due to liquid crystalline structure of the esters and this physical state is usually due to admixture of phospholipid and influenced by temperature, fixation and presence of water. Reduction by osmium tetroxide Osmium tetroxide oxidizes the ch=ch bond and reduced to a black substance probably osmium dioxide. Soluble in polar and non polar solvents and serves to stain both hydrophilic and hydrophobic bonds. Results: unsaturated lipids black Demonstration with fat soluble dyes 1. Sudan iii or iv Solution sudan iii or iv in 70% alcohol

 Results : Lipids orange red Nuclei blue 2. Oil red o-Solution: 1. Oil red o solution oil red o Absolute isopropyl alcohol 3. Sudan iv or fettrot in propylene glycol. 4. colloidal suspensions of sudan dyes in gelatine. Demonstration of fat soluble dyes Sudan black b Solution: Bromine water Sodium metabisulphite Sudan black b Counterstain alums brazilin or mayers carmalum * If bromination is omitted, then free cholesterol is not stained and phospholipid are less strongly stained. Result Lipids appear in different shades of deep gray, dark blue and black Oil red O in dextrin Place slides directly into filtered 0.5% oil red O in dextrin. Stain for 20 mins Rinse with running water Counterstain with hematoxylin for 20-30 sec. Rinse in water Coverslip with aqueous mounting media.

Results Fat Nuclei brilliant red blue

Application of lipid histochemistry in pathology Nervous system The myelin sheath is particularly rich in lipids, being composed of compacted cell membrane, a lamellar structure of cholesterol and phospholipids. Primary demyelination occurs when the myelin sheath is damaged by infectious agents, toxins or allergic influences and in some cases the toxins remain intact eg: multiple sclerosis. Dysmyelination implies a failure to myelinate properly during development. Application of lipid histochemistry in pathology Both demyelination and dysmyelination can be demonstrated in paraffin sections by conventional histological methods for myelin. Frozen sections are preferred because The most important feature of demyelination is the transformation of normal myelin, with its distinctive hydrophilic properties due to its polar phospholipids into fatty droplets comprising cholesterol esters within macrophages, with an affinity for sudan dyes. Application of lipid histochemistry in pathology Methods to demonstrate myelin 1. Luxol fast blue 2. Dichromate acid hematein oil red o provide a good color contrast between the blue myelin and red stained esters in lipophages. 3. Osmium tetroxide - naphthylamine technique. 4. Sudan black b normal myelin appear grey rather than the intense blue color of the degenerating myelin lipids. Application of lipid histochemistry in pathology

 The CNS is also the target for parkinsons disease and the characteristic lewy bodies have been shown to include sphingomyelin. Demonstrated by: Sodium hydroxide acid hematein procedure. Sudan black b viewed in polarised light to show the characteristic red birefringence attributable to sphingomyelin. Application of lipid histochemistry in pathology Metabolic disorders affecting the nervous system 1. Neurolipidoses: in which the lipids accumulate chiefly within neurons. Eg: battens disease 2. Leucodystrophies: in which the lipids accumulate in the cells of the mononuclear phagocyte series in the white matter of the brain. Eg: metachromatic leucodystrophy (sulphatide) and krabbes leucodystrophy (galactocerebroside). 3. Visceral lipidoses: where lipid storage is found predominantly in the mononuclear phagocyte system. Application of lipid histochemistry in pathology Cardiovascular system Cholesterol deposition in the human arterial wall leading to atherosclerosis can be demonstrated by oil red o. Lipids can also accumulate in the heart muscle of patient with cardiomyopathies induced by viral infections, alcohol abuse and toxins. In this case lipids in question are triglycerides demonstrated by Fat stains Calcium lipase method. Application of lipid histochemistry in pathology 3. Skeletal muscle Oil red o is routinely used to detect an excess of neutral fats in muscle biopsies. Sudan black may also be used.