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CHAPTER I

INTRODUCTION TO PHOSPHOLIPIDS

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1.1 DEFINITION AND CLASSIFICATION OF LIPIDS

Fatty acid and their derivatives and substances related

biosynthetically or functionally to this compounds, that are soluble in

organic solvents such as chloroform, ether, benzene, acetone and

insoluble in water are commonly known as lipids. Most of them form

molecules such as waxes, triglycerides, phospholipids, etc, whereas

other substances such as fat soluble vitamins, coenzymes, pigments,

terpenes, sterols and phenolics are also considered as lipids because

they are extracted with “fat” solvents1.

Lipids are mainly composed of carbon and hydrogen, elements

that confer a non-polar behavior, although they can also have polar

groups containing oxygen, nitrogen and phosphorous. The most

common functional chemical groups present in lipids are simple or

double carbon-carbon bonds, carboxylate esters, phosphate esters

andamides. Therefore, lipids are hydrophobic (usually with a polar

head connected to a non-polar structure), On the contrary,

amphipathic lipids tend to form surface mono-layers, bi-layers or

micelles in contact with water.

Lipids are found in all living beings, where they carry out a wide

range of functions due to their high chemical variability. They are

involved in forming biological membranes, energy storage, heat, water

or electric insulation, heat production and intracellular or intercellular

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signaling. Moreover, they act as hormones, pigments, vitamins,

enzymatic cofactors, electron transporters and detergents.

Lipids can be classified into two groups namely simple and

complex lipids based on chemical composition. Simple lipids contain

C, H and O and complex lipids contain one or more additional

elements, such as phosphorus, nitrogen or sulfur.

1.1.1 Simple lipids

Simple lipids can be suggested into structural types, which are

fatty acids (FA), waxes, triglycerides (TG) and sterols.

Fatty Acids: The general structure of fatty acids (Fatty Acids) is

made up of a long and straight aliphatic chain with a hydrophilic

carboxylate group attached to one end: CH3(CH2)nCOOH, although

some of them are dicarboxylic acylglycerols.

Acylglycerols: Acylglycerols (AGs) are also named glycerides or

neutral fats and they are glycerol esters of one, two or three fatty acids

– mono- (MAGs), di- (DAGs) and triacylglycerols (TAGs) respectively.

Waxes: Waxes are defined as the compounds formed by Fatty

Acids esterified to the alcohol group of fatty alcohols or other lipid

alcohols such as aminoalcohols, sterols, hydroxycarotenoids or

terpenols.

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Cyanolipids: Fatty Acids esterified to mono- or dihydroxynitrile

moieties are cyanolipids.

Terpenes: Terpenes are lipids constituted of a defined number of isoprene

units (2-methyl 1,3 butanodiene). Isoprene units may be linked in a head to

tail or in a head to head fashion and the resulting compounds can be

acyclic or cyclic and saturated or unsaturated. Many terpenes are

hydrocarbons, although some of them, designated terpenoids, contain

oxygen, alcohol (terpenols), aldehyde or ketone groups.

Steroids: Steroids are modified triterpenes derived from squalene.

Their nucleus is based on the saturated tetracyclic hydrocarbon 1, 2-

cyclopentanoperhydrophenanthrene or sterane, which can be modified

by C-C bond scissions, ring expansions or contractions,

dehydrogenation and substitutions.

1.1.2 Complex lipids

Complex lipids are frequently constituted by three or more

chemical identities (e.g. glycerol, fatty acids, sugar and other groups)

and they are usually amphiphatic.

Phenolic lipids: This heterogeneous group includes simple phenols and

polyphenols as well as their derivatives and can be classified into

coumarins, quinones and flavonoids, by far the largest group of phenolics.

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Glycolipids: Glycolipids are complex lipids containing a glycosidic

moiety and are major constituents of cell membranes in bacteria,

plants and animals, where they regulate cell interactions with other

cells or the environment. According to their structure, glycolipids

may be classified into the following groups:

Glycosides of fatty acids, lipid alcohols and steroids: These

compounds are made up of a glycosyl moiety (one or several

units) linked to one or more Fatty Acids, fatty alcohols or alkyl

chains.

Glycolipids based on glycerol (Glyceroglycolipids): These lipids consist

of a mono-, di- or oligosaccharide moiety linked glycosidically to the

hydroxyl group of glycerol, which may be acylated (or alkylated) with

one or two Fatty Acids.

Glycolipids based on ceramides: They are known as glycosphingolipids

and they are based on a mono-, di- or oligosaccharide moiety linked to

the hydroxyl group of a ceramide backbone. The ceramide and the

glycosyl group(s), which can be neutral (unsubstitued) or acidic

(substituted with carboxyl, sulphate or phosphate group(s)), can have

further modifications.

Glycosides of lipoamino acids: Two groups of complex lipoamino acids

containing glycosyl moieties are known: (1) lipids having an amino

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acid with N-acyl and/or ester linkages and (2) lipids having a glycerol

and an amino acid with ether linkage.

Lipopolysaccharides: These complex compounds are the endotoxic O-

antigens found in outer membranes of Gram-negative bacteria. The

lipid part (Lipid A), responsible for the toxic activity of these bacteria

that results in septic shock, consists of a backbone of β- 1,6-(1-

phospho)glucosaminyl-(4-phospho)glucosamine. The 3-position of

glucosamine II establishes a glycosidic linkage with a long-chain

polysaccharide. The other hydroxyl and amine groups are substituted

with normal or hydroxy Fatty Acids.

Phospholipids: Phospholipids are complex lipids which contains

one or more phosphate groups. Phospholipids are amphipathic in

nature that is each molecule consists of a hydrophilic portion

and a hydrophobic portion thus tending to form lipid bilayers.

In fact, they are the major structural constituents of all

biological membranes, although they may be also involved in

other functions such as signal transduction. There are two

classes of phospholipids, those that have a glycerol backbone

and those that contain sphingosine. Phospholipids that contain

glycerol backbone are called as glycerophospholipids, which are

the most abundant class found in nature. The most abundant

types of naturally occurring glycerol phospholipids are phosphatidyl

choline, phosphatidyl ethanolamine, phosphatidyl serine,

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phosphatidyl inositol, phosphatidyl glycerol andcardiolipin. The

structural diversity within each type of phosphoglyceride is due

to the variability of the head group, variability of the chain

length and degree of saturation of the fatty acid ester

groups.

1.2 NOMENCLATURE OF PHOSPHOLIPIDS

The stereospecific nomenclature of glycerol phospholipids places

the phosphate at the sn-3 position (Table 1.1). The sn is an

abbreviation for stereospecific numbering.

Phosphatidyl (Ptd) - the radical of phosphatidic acid, is 1,2-

diacyl-sn-glycero-3-phosphate. Phosphatidyl choline (Ptd Cho, PC) a

major component of soybean lecithin, is 1,2-diacyl-sn-glycero-3-

phosphocholine. Initially PC was known as lecithin. Phosphatidyl

ethanolamine (Ptd Etn, PE), is 1,2-diacyl-sn-glycero-3-phospho

ethanolamine and it was previously called cephalin. Phosphatidyl

inositol (Ptd Ins, PI), is 1,2-diacyl-sn-glycero-3-phospho inositol (Fig.

1.1).

CH2-O-PIIO

O

-OI

CH2-O-CO-R

CH-O-CO-R

X-

-

Fig. 1.1: General Structure of Phospholipids

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Presently phospholipids are commercially available in several

formulations from natural and synthetic phospholipids. Many of

these products are defined according to the stage of the purification

process from which they are obtained and fall into three broad

categories like natural, refined and modified varying in their

constituents both quantitatively and qualitatively.

1.3 SOURCES OF PHOSPHOLIPIDS

Phospholipids are present in many natural sources like

human/animal tissues, plant sources and microbial sources.

1.3.1 Phospholipids in Human/Animal Tissues

Almost all body cells contain PLs. The common animal PLs are

made of sphingomyelin, PC, PE, PS, PI and other glycerol phosphatides

of complex fatty acid composition. These phospholipids occur

normally in cell membranes and lipid proteins, where they serve both

structural and functional purposes. Animal phospholipids are highly

valued for their desirable emulsifier and organoleptic properties. The

exact composition of human/animal phospholipids depends on the

source and the method of extraction and purification. The central

nervous system especially has high phospholipids content. The liver

is the site for their biosynthesis and the lipids of the mitochondria,

which are the regulators of cell metabolism and energy production in the

body, consist of up to 90% of PLs.

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1.3.2 Egg Phospholipids

The phospholipids in egg are mainly present in the yellow yolk

at least a portion of them is combined with protein and carbohydrates.

Egg yolk has about 70% PC, 24% PE, 4% Sphingomyelin, 1% PS, 1%

PI, lyso PC and lyso PE contribute the remaining 2% of the total

phospholipids3,4. Egg lecithin as a commercial ingredient with the

exception of some medical feeding program, is comparatively

expensive for the routine use in food5.

1.3.3 Milk Phospholipids

Milk has a phospholipid content of about 0.035% associated

with the fat by virtue of being part of a colloidal membrane, which

surrounds the fatty globule. Skim milk and milk serum have

the highest portion of polar lipids as percent of the total lipids,

while whole milk and cream have least of the polar lipids. PE

constitutes the largest component with PC and sphingomyelin

being present in about equal portions at a significantly lower

level5,6.

1.3.4 Brain Phospholipids

The brain is a rich source of phospholipids and, together

with the spinal cord, probably possesses the highest phospholipid

content of any of the organs.

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Table 1.1: Structures of Common Phospholipids

Class (commonly used abbreviation) Substituent (-X) Name of the phospholipid

Phosphatidic acid (PA) -H 1,2- Diacyl -sn-glycero-3-phosphatidic acid

Phosphatidyl choline (PC) -CH2-CH2-

+N(CH

3)3

1,2- Diacyl-sn-glycero-3-phosphocholine

Phosphatidyl ethanolamine (PE) -CH2-CH2-NH3

+

1,2-Diacyl-sn-glycero-3-phosphoethanolamine

Phosphatidyl serine (PS) -CH2-CH

COO-

NH3

+

1,2-Diacyl-sn-glycero-3-phosphoserine

Phosphatidyl inositol (PI) HO

OH

OH

HO

OH

1,2-Diacyl-sn-glycero-3-phosphoinositol

Phosphatidyl glycerol (PG) CH2

CHOH

CH2-OH

1,2-Diacyl-sn-glycero-

3-phosphoglycerol

Cardiolipin (CL)

CHOOC-R''

CH2-OOC-R'''

-_

CH2O

O

O

CH2

CH

CH2-

HO

PO

Bis-glycero phosphate

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There are many and different types of phospholipids

present in the central nervous system.

1.3.5 Phospholipids in Liver, Kidney, Muscles and other Tissues

Organ meats such as liver, kidney and muscles are major

source of dietary phospholipids. In blood PC is quantitatively the most

important phospholipid. Total blood contains about 0.2 to 0.3%

of phospholipids.

1.3.6 Plant Sources of Phospholipids

Vegetable materials usually contain only small amounts of

phospholipids, ranging from 0.3 to 2.5 wt%. The major phospholipids

present in plant sources are PC, PE and PI. The plant sources of

phospholipids are soybean7, rapeseed8, sunflower9, cottonseed and

peanut10, ricebran11, palm, coriander, carrot12, palash, janglibadam,

papaya13, olive, barley, cucurbit14, corn15, karanza16, castor bean17,

cocoa18, neem19, sesame20, khakan21, pear, quince22, tobacco23.

Phospholipids are removed as by-product during the degumming

process of vegetable oil refining. Crude vegetable oil lecithins are

the starting materials of choice for further fractionation and

purification processes to obtain phospholipid compositions suitable

for various industrial applications.

Soybean phospholipids are obtained from commercial soybean

lecithin. It is a complex mixture comprised of phospholipids,

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triglycerides with minor amounts of other substituents, i.e.

phytoglycolipids, phytosterols, tocopherols and fatty acids. The world’s

first industrial processing of soybean and production of lecithin was

carried out in Harmburg and the driving force behind this

development was Herman Bollmann (1880-1934). Soybean lecithin

mainly used because of its availability and excellent properties,

especially emulsifying behavior, color and taste.

Other lecithins like rice bran11, corn15, rapeseed8, sunflower9,

cottonseed and peanut10a are also good phospholipid sources and

some of these lecithins are being exploited for commercial

applications.

1.3.7 Microbial Sources of Phospholipids

Microorganisms also contain phospholipids and these entities

are of interest for clinical research. The diversity of lipid types is

enormous, and all the major phospholipids of plants and animals

have been recovered from at least one microorganism10b,c.

1.4 APPLICATIONS OF PHOSPHOLIPIDS

Phospholipids are being used for several applications as a

mixture or as an individual component. Some of the applications

are listed in Table 1.2. PLs play very important role in several

products and some representative examples are given in Table 1.3.

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Table 1.2: Applications of Individual Phospholipids and Modified

Lecithins

Phospholipids / Modified

lecithins

Applications

Phosphatidyl choline Provides free choline in the blood for the

manufacture of acetylcholine; regulates

digestive, cardiovascular and liver

functions

Enriched phosphatidyl

choline

Pharmaceutical preparations,

cosmetics.

Phosphatidyl choline:

Phosphatidyl-ethanolamine

(80:20)

For the production of stable liposomes,

anti-spattering agent in margarine.

Phosphatidic acid Provides less absorption of oil into raw

materials, retains the intrinsic flavor of

raw materials.

Phosphatidyl serine Essential to the functioning of all body

cell, supports brain functions that

decline with age, memory enhancer.

Ethanol solubles of soybean

lecithin

Emulsifier in foods (e.g. chocolate)

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Hydrogenated lecithin Exhibits greater oxidative stability,

reduced solubility, less hygroscopic,

used in lubrication oil additives,

emulsions for intravenous injections in

liposomes.

Hydroxylated lecithin Suitable for oil in water emulsions

Acetylated lecithin Improved fluid properties, water

dispersibility andeffective oil in water

emulsions.

Lyso lecithin Effective for oil in water emulsions,

stable at high temperatures, low pH

and high salt concentrations

Table 1.3: Uses and Functions of Phospholipids in Various Products

Product Function Effect

Baked goods Gluten chemistry

modifier; emulsifier

and stabilizer for fats;

antioxidant; wetting

agent

Improves “shortening

effect” emulsification,

flavor andshelf-life,

texture and moisture

retention.

Pasta products

dried potatoes

Inclusion partner for

amylose;co-emulsifier

Improves emulsification,

flavor, texture and shelf

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for mono and

diglycerides;antioxidant

life.

Wafers Emulsifier;stabilizer;

antioxidant

Improves emulsification,

flavor, texture and shelf

life.

Cream like

emulsions

Emulsifier; stabilizer;

antioxidant.

Improves emulsification

and stabilizes emulsion;

Improves emulsification

flavor, texture and shelf

life.

Candy Emulsifier; viscosity

reducer; wetting agent;

dispersant.

Aids mixture of sugar, fats

and water to prevent

greasiness, graining and

streaking.

Margarine

shortening

Emulsifier; antioxidant Decreases pattering,

Improves emulsification,

flavor, texture and shelf-

life.

Meat (sliced

bacon)

Releasing agent;

antioxidant.

Improves separation of

refrigerated slices shelf

life.

Instant foods,

whole milk

Instantizer; emulsifier;

antioxidant; wetting

Speeds and improves

reconstitution,

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powder agent; dispersant;

nutritional supplement.

emulsification, flavor

texture and shelf life.

Dietic food Emulsifier; antioxidant.

Wetting and

strengthening agent;

nutritional supplement.

Improves emulsification,

flavor texture and shelf

life.

Pan coating Release and lubricate Improves appearance

and shelf life; prevents

surface greasiness.

Pharmaceuticals Emulsifiers; carrier;

antioxidant; donates

choline and linoleic

acid.

Improves emulsification,

dispersion and shelf- life.

Cosmetics Emulsifier and foam

stabilizer; emollient;

dispersing agent;

resorceable refatting

agent with depth

effects; wetting agent;

antioxidant; donates

choline, inositol and

linoleic acid. Vitamin

source; pantothenic

Improves dispersion of

various components

(pigments and shelf- life)

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acid, thiamine, folic

acid, riboflavin,

pyridoxine, biotin and

niacin.

Dog food Releasing agent;

emulsifier; antioxidant

wetting and

strengthening agent;

nutritional supplement.

Aids blending of fats and

water andclean release

from equipment and can;

improve animal coat

glossiness.

Calf and cow

milk

Emulsifier; antioxidant

wetting and

strengthing agent;

nutritional supplement.

Improves feed utilization

and nutrition

Poultry feed Releasing agent;

antioxidant; emulsifier;

wetting and

strengthening agent

nutritional supplement.

Improves emulsification

and dispersion; feed

utilization and nutrition

prevents lodging of food in

poultry beak and resulting

necrosis.

Live stock feed Emulsifier; antioxidant

wetting and

strengthening agent;

nutritional supplement;

Improves emulsification

and dispersion, food

utilization and nutrition.

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releasing agent; anti-

dusting agent.

Insecticides Emulsifier; dispersant;

stabilizer.

Disperses and stabilizers

pesticides and surfactants

in water.

Inks Emulsifiers;

dispersant; stabilizer;

grinding aid.

Improves pigment

solubility and flow

properties; stabilizes

dispersion.

Magnetic tape Emulsifier; dispersing

agent; antioxidant.

Improves dispersion and

shelf life.

Lacquers, paints

and other

coatings

Hydrophilic emulsifying

and wetting agent;

dispersing agent,

stabilizer; antioxidant.

Improves dispersion of

pigments, stability and

shelf- life.

Leather Softening agent, oil

penetrant

Improves the process of

fat liquoring.

Plastics Emulsifier; dispersing

agent; releasing agent.

Improves dispersion of

pigments and mold

release.

Rubber Emulsifier; dispersing

agent; releasing agent;

antioxidant.

Improves dispersion of

pigments mold release

and shelf –life.

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Among the PLs, PC is the most ubiquitous as it plays a vital role

in liver and cell functions and is an alternative to choline chloride and

choline bitartarate which are commonly used in vitamin and

nutritional supplements.

1.5 SEPERATION AND ANALYSIS OF PHOSPHOLIPIDS

1.5.1 Separation of Phospholipids

The major portion of tissue lipids are bound to proteins and

carbohydrates. Solvents such as chloroform, ether or benzene are

generally used in combination with methanol or ethanol. Various

methods for extraction of lipids are reported in literature24-27. The

most extensively used extraction procedure was reported by Folch et

al28 in which the tissue or seeds were extracted with chloroform:

methanol (2:1, vol/vol) solvent mixture. The Bligh and Dyer29 method

was also widely used for extraction of lipids, in which the tissue or

seeds were extracted with solvent mixture of chloroform : methanol :

water (1:20:8, vol/vol/vol). But some plant tissues contain active

enzymes, which are not inactivated either by chloroform or methanol

andreadily cause breakdown of phospholipids. In this case the method

of Kates30 was used in which the enzymes were deactivated by freezing

the seeds with liquid nitrogen and washing with 2-propanol.

Mostly the phospholipids from oil seeds and oils were isolated

by extraction of source material by Folch et al28 method followed by

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acetone precipitation of the extract31 or by extracting acetone defatted

materials with chloroform: methanol32. Phospholipids were further

purified by silicic acid column chromatography33. Commercially the

phospholipids were isolated from oils by degumming with steam34 or

weak boric acid35 or with sodium chloride solution36 or with acetic

anhydride37.

1.5.2 Analysis of Phospholipids

The quantitative and qualitative analysis of total phospholipids

is carried out by several methods namely, solvent fractionation,

counter-current distribution, paper chromatography, thin layer

chromatography, column chromatography, high-performance liquid

chromatography, proton nuclear magnetic resonance spectroscopy,

mass spectra and gas chromatography. The total phospholipids were

fractionated into alcohol soluble (PC rich fraction) and alcohol

insolubles (PE rich fraction), based on the solubility of phospholipids

in solvents38,39. Later Scholfield et al40 used counter current

distribution technique to soybean and corn phospholipids using

hexane and 95% methanol solvents. The composition was found to be

29% lecithin, 31% cephalin and 40% PI. However these classical

techniques are labourious and require large amounts of the sample.

Hence these have been substituted by modern chromatographic

methods. Paper chromatographic technique was used to study the

qualitative identification of phospholipids41. This technique was

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significantly improved by use of modified papers such as acetylated,

formaldehyde treated, impregnated with phosphate and alumina. The

most commonly used method consists silicic acid impregnated paper,

which has been used by several authors to anlayse phospholipid

classes42-44. Thin-layer chromatography (TLC) technique is extensively

used in area of phospholipid research. The various forms of TLC like

qualitative, quantitative and preparative methods have been used to

isolate and to determine the composition of the individual

phospholipid classes from phospholipid mixture. The adsorbent used

were alumina, hydroxylaptite, cellulose, polyamide, silicic acid. The

commonly used adsorbent was silica with 15 % calcium sulphate as

binder to separate phospholipid classes45,46. Skipski et al47 achieved

the separation of PE and PS using silica gel-H (without binder) plates

which were prepared in 1 mm aqueous sodium carbonate solution.

The complex mixture of phospholipids was efficiently separated by

TLC technique 48-50. The preparative TLC was used to separate

individual phospholipids in large quantities51. Spanner52 reviewed

some well tried systems for different phospholipids with their Rf

values. The quantitative TLC method was used by several workers53-55

to determine the phospholipid composition. TLC coupled with

densitometric estimation of phospholipids was also used to study the

phospholipid composition56,57. TLC of complex lipids has been

reviewed by skipski et al58 and Rouser59. Okumura et al 60 developed

reusable TLC rod with a sintered silica gel layer in collaboration with

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Iatron laboratories of Japan based on the flame ionization detection

principle. Ackman et al 61 comprehensively reviewed the applications

of this technique for lipid analysis.

The column chromatography has been used extensively in the

analysis of phospholipids. Several adsorbents were used for

separation of phospholipids such as alumina, silica acid, magnesium

silicate (florisil), diethylamino ethyl (DEAE) cellulose, triethylamino

ethyl (TEAE) cellulose and hydroxylapatite. Sweeley62 and Rouser63

reviewed the applications of various methods for anlaysis of complex

lipids. Another most promising chromatographic technique is High

Performance Liquid Chromatography (HPLC) to determine

phospholipid composition. Hurst and martin64 reported the

phospholipid composition of PC, PE, PI and PS of soy lecithin by this

method and several studies were reported using this technique. High

performance liquid chromatography coupled with an evaporative light

scattering detector (HPLC/ELSD) provides a universal separation and

detection method for phospholipids65-68. Methods include normal

phase and reversed phase techniques utilizing several solid phase

species.

Nuclear Magnetic Resonance spectroscopy for phosphorus is

quickly becoming the definitive method for quantitation of

phospholipid mixtures. 1H and 13C NMR are available for molecular

characterization of unknown lipid compounds. Electrospray mass

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spectrometry (ESMS)69,70 is a low fragmentation technique whereby

molecular weight information can be obtained from a small sample

either in positive or negative mode. Infusion of phospholipid solutions

into the electrospray interface can identify the molecular species for

each headgroup present. Quantitation can be accomplished with

HPLC separation prior to mass spectral detection. Use of modern

capillary columns with a choice of several stationary phases, coupled

with flame ionization detection71, provides % composition of fatty acids

present in a sample either as a total or as esterified fatty acids from

phospholipids or glycerides. Data from this method can be used to

calculate the ratio of unsaturated vs. saturated fatty acids present in

a sample, replacing the old iodine number wet chemistry assay. The

spray reagents72,73 used for the identification of PLs are Dragendroff for

PC, ninhydrin for PE identification and Ammonium molybdate-

perchloric acid reagent for identification of PLs.

1.6 MAJOR CLASSES OF PHOSPHOLIPIDS

1.6.1 Phosphatidyl choline

Phosphatidyl choline (PC) is the major component of lecithin. It

is also a source for choline in the synthesis of acetylcholine in

cholinergic neurons. PC is one of the primal class of substances

ubiquitous among life fonns74. PC is the predominant phospholipid of

all cell membranes and of the circulating blood lipoproteins. It is the

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main functional constituent of the natural surfactants and the body's

foremost reservoir of choline, an essential nutrient75. PC is a normal

constituent of the bile that facilitates fat emulsification, absorption

and transport and is recycled via entero-hepatic circulation. Lecithin

preparations enriched in PC at or above 30 percent by weight are

considered PC concentrates.

PC is usually the most abundant PL in animal and plants, often

amounting to almost 50% of the total and as such it is obviously the

key building block of membrane bilayers. In particular, it makes up a

very high proportion of the outer leaflet of the plasma membrane. PC

is also the principal PL circulating in plasma, where it is an integral

component of the lipoproteins, especially the HDL. On the other hand,

it is less often found in bacterial membranes, perhaps 10% of species.

It is a neutral or zwitterionic PL over a pH range from strongly acid to

strongly alkaline.

In most other species, it would be expected that the structure of

the PC in the same metabolically active tissue would be somewhat

similar. On the other hand, the PC in some organs contains relatively

high proportions of disaturated molecular species. For example, it is

well known that lung PC in all animal species studied to date contains

a high proportion (50% or more) of dipalmitoylphosphatidyl choline76.

It appears that this is the main surface-active component, providing

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alveolar stability by decreasing the surface tension at the alveolar

surface to a very low level.

1.6.2 Phosphatidyl ethanolamine

1,2-Diacyl-sn-glycero-3-phospahtidyl ethanolamine (PE) is

usually the second most abundant PL in animal and plant lipids and

it is frequently the main lipid component of microbial membranes77.

As such, it is obviously a key building block of membrane bilayers. It

is a neutral or zwitterionic phospholipid (at least in the pH range 2 to

7)78. In mammalian and plant tissues PE occurs in lesser amounts

than PC where as in bacteria, it is the principal PL present79.

Unusual PE analogues containing a carbon-phosphorus bond

instead of the classical carbon-oxygen-phosphorus bond are described

in marine invertebrates and protozoa. These phosphonolipids, often

termed PE, are extremely resistant to acid hydrolysis.

Although PE is sometimes equated with PC in biological

systems, there are significant differences in the chemistry and

physical properties of these lipids and they have different functions in

biochemical processes. Both are key components of membrane

bilayers. However, PE has a smaller head group and it can hydrogen

bond through its ionizable amine group. In bilayers, it can undergo

distinctive physical transitions. Much of the evidence for the unique

properties of PE come from studies of the biochemistry of E.coli, where

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this lipid is a major component of the membranes. There is evidence

that PE acts as a 'chaperone' during the assembly of membrane

proteins to guide the folding path for the proteins and to aid in the

transition from the cytoplasmic to the membrane environment 80.

1.6.3 Phosphatidyl inositol

Phosphatidy inositol (PI) is an important lipid, both as a key

membrane constituent and as a participant in essential metabolic

processes in all plants and animals and in some bacteria

(actinomycetes), both directly and via a number of metabolites. It is an

acidic (anionic) PL that in essence consists of a phosphatidic acid

backbone, linked via the phosphate group to inositol

(hexahydroxycyclohexane). In most organisms, the stereochemical

form of the last is myo-D-inositol (with one axial hydroxyl in position 2

with the remainder equatorial), although other forms (scyllo- and

chiro-) have been found on occasion in plants.

PI is especially abundant in brain tissue, where it can amount

to 10% of the PL and it is present in all tissues and cell types. There is

usually less of it than of PC, PE and PS. In animal tissues, PI is the

primary source of the arachidonic acid required for biosynthesis of

eicosanoids, including prostaglandins, via the action of the enzyme

phospholipase A2, which releases the fatty acids from position sn-2.

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In addition to functioning as negatively charged building blocks

of membranes, the inositol phospholipids appear to have crucial roles

in interfacial binding of proteins and in the regulation of proteins at

the cell interface. As phosphoinositides are polyanionic, they can be

very effective in non-specific electrostatic interactions with proteins.

1.7 STRUCTURED PHOSPHOLIPIDS

The aim to alter the existing fatty acids in the natural

phospholipids is to improve the properties of phospholipids or to meet

particularly functional requirements. Phospholipids obtained after

such modifications are known as structured phospholipids. Highly

unsaturated fatty acid containing phospholipids are currently

receiving attention because of their novel physiological functions.

Yazawa et al81 reported that decrease in the weight of adipose tissue

among the major organs (perirenal adipose tissue, paraepididymal

adipose tissue;) after the administration of the eicosapentaenoic acid

containing PL (EPA-PL) suggests a specific effect of this novel chemical

form of EPA. Suzuki et al82., reported that docosahexaenoicacid (DHA)-

containing PC isolated from rainbow trout embryos, induces

differentiation of murine undifferentiated tumour cells. Kohno et al.83

observed that the rate of retinoic acid-induced differentiation of HL-60

human leukemia was accelerated by HUFA-PC. 5-Lipoxygenase is

known to catalyze the first step in leukotriene production. Matsumo et

al84 showed that DHA-PC can inhibit this enzyme. This study also

27

indicated that sn-1 18:1/sn-2 DHA-PC is the most potent inhibitor of

5-lipoxygenase. The effect of n-3 fatty acids on the metabolism of

prostaglandins and on serum lipid, cholesterol, phospholipids,

triglycerides, platelet aggregation as well as on immune regulation,

have been demonstrated. The role of n-3 fatty acids in health

promotion and disease prevention, especially in the treatment of

cardiovascular disease is under extensive investigations85-90. It is

believed that PC with EPA and DHA at the second position could more

easily be digested by the body and might be of value in nutritional and

medical applications91. For example, PLs with enriched DHA at the

second position have potential medical applications, such as in

promoting cell differentiation in leukemia, enhancing survivals of

tumor bearing mice and preventing cerebral apoplexy92-95.

1.7.1 Enzymatic and Chemical Methods for the Preparation of

Structural Phospholipids

The molecular structure of PL can be changed by either

enzymatic or chemical means. The aim of all these process is to obtain

tailormade PLs. The interest in new PLs and PL analogues results from

their potential use in different fields of application96 for example as

biodegradable surfactants, as carriers of drugs or genes or as

biologically active compounds in medicine and agriculture. The

synthesis of new PLs and PL analogues97 using both enzymatic and

chemical methods had gained importance. In recent years, enzymatic

28

catalysis particularly with lipases and phospholipises98 has gained

increasing importance to replace chemical methods or to permit

synthesis of compounds which have not been accessible by chemical

means.

Best way for the partial synthesis of PLs is enzymatic

modifications. Different enzymes are employed to tailor PLs with

defined fatty acid composition at the sn-1 and sn-2 positions. Using

enzymatic acyl exchange it would be possible to acquire PLs for

specific application requirements in food, pharmaceutical and

cosmetics by altering the technical or physiological properties of the

natural compounds. Most work in this direction focuses on the

incorporation of saturated fatty acids (including both medium and

long chain)or polyunsaturated fatty acids into PLs. Lipase catalyzed

enzymatic acidolysis reaction between soy PLs and CLA and

phospholipase D catalyzed transposphotidylation reaction between

PLs and sterols were used to synthesize structured PLs with modified

fatty acid (CLA) and head group (sterol). Compared to chemical

methods, enzymatic modifications of PLs have few advantages like

selectivity or specificity of enzyme is one of the most important

properties of enzymes that makes the modification of PLs simple and

easy (Fig 1.2). With possible and available enzymes, the manipulation

of PL structure can be complicated but versatile.

29

CH2-O-PIIO

O

-OI

CH2-O-CO-R

CH-O-CO-R

X-

PLA1

PLA2

PLC PLD

Fig 1.2: Enzymatic Hydrolysis of Phospholipids

There are various ways to chemically modify PL molecules, but

only few of them are commercialized. The reason is that none of the

resulting products have food grade status except products like

hydroxylated and acetylated lecithins. However, a substantial

development and application work has been reported on the

chemically modified PLs for application in pharmaceutical and

cosmetic products. The major problem in PL synthesis is to construct

the chiral structure and keep the configuration in the further chemical

processing. The general procedure for the synthesis of ether and

ester glycerophospholipids includes preparation of stereospecific acyl

or ether-substituted glycerol backbone and further phosphorylation of

glycerol derivatives. Sterospecific glycerol derivatives could be

synthesized via ring opening reaction using (S)-glycidol,(R)-glycidol

tosylates and (R)-isopropylidene-rac-glycero, etc as starting materials

99-101. The resulting glycerol derivatives were then phosphorylated with

the phosphorylating agents such as (2-bromoethyl)

phosphochloridate, 2-chloro-2-oxo-1,3,2-dioxoaphospholane and N,N-

30

diisopropylmethylphosphoramidic chloride102,103. Using the above

routes or similar methods as well the methods of phosphate

chemistry, a variety of optically active PLs and analogues with similar

structures, such as esters, ether (PAF), thioether, thioester and amide

PLs can be synthesized.

Chemical and physical properties of PLs depend on their

molecular structure. To meet different industial application

requirements, hydrolysis, hydroxylation, acetylation and

hydrogenation have been applied to the chemical modifications of

commercial lecithin to generate lyso-PLs, hydroxylated PLs, acylated

PE, hydrogenated PLs and other PLs104,105. Alternately, PLs can also

be prepared by utilizing natural PLs as precursors106. However the

glycerol derivatives or sphingosines obtained by chemical or enzymatic

cleavage are usually structural or stereo mixtures that are difficult to

be isolated and purified. The advantage of semi-synthesis is its low

cost due to its naturally available source of precursors and fewer

reaction steps. Synthetic routes for PL synthesis been reviewed by

Gunston et al107. The work reported in the present study involves

preparation of novel PLs such as platelet activating factor (PAF) PC

and PE analogues, PE-N-amino acid derivatives and hydroxyl fatty

acid containing PLs. The synthesis of these PLs were carried out by

total and partial synthesis employing enzymatic and chemical

approaches.

31

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