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CHAPTER I
INTRODUCTION TO PHOSPHOLIPIDS
1
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
2
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.
3
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, 2cyclopentanoperhydrophenanthrene 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.
4
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
5
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 Oantigens 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-(1phospho)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,
6
phosphatidyl
inositol,
phosphatidyl
glycerol
andcardiolipin.
structural diversity within each type of phosphoglyceride
is
The
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 1,2diacyl-sn-glycero-3-phosphate. Phosphatidyl choline (Ptd Cho, PC) a
major component of soybean lecithin, is 1,2-diacyl-sn-glycero-3phosphocholine. 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-CO-R
CH-O-CO-R
O
II
I
CH2-O-P -O -X
O-
Fig. 1.1: General Structure of Phospholipids
7
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.
8
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
exception of
ingredient with the
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.
the highest portion of
Skim milk and milk serum have
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.
9
Table 1.1: Structures of Common Phospholipids
Class (commonly used abbreviation)
Substituent (-X)
Phosphatidic acid (PA)
-H
Name of the phospholipid
1,2- Diacyl -sn-glycero-3-phosphatidic acid
Phosphatidyl choline (PC)
+
-CH2-CH2-N(CH3)3
1,2- Diacyl-sn-glycero-3-phosphocholine
Phosphatidyl ethanolamine (PE)
+
-CH2-CH2-NH3
1,2-Diacyl-sn-glycero-3-phosphoethanolamine
Phosphatidyl serine (PS)
COO-CH2-CH +
NH3
1,2-Diacyl-sn-glycero-3-phosphoserine
Phosphatidyl inositol (PI)
HO HO
1,2-Diacyl-sn-glycero-3-phosphoinositol
OH
OH OH
Phosphatidyl glycerol (PG)
CH2
1,2-Diacyl-sn-glycero-
CHOH
3-phosphoglycerol
CH2-OH
Cardiolipin (CL)
Bis-glycero phosphate
O
CH2 O P O
HO _ CH
O
CH2-
CH2
CHOOC-R''
CH2-OOC-R'''
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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
the
fractionation and
starting
materials
of choice for further
purification processes to obtain phospholipid
lecithins
are
compositions suitable
for various industrial applications.
Soybean phospholipids are obtained from commercial soybean
lecithin. It is a complex mixture comprised of phospholipids,
11
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.
12
Table 1.2: Applications of Individual Phospholipids and Modified
Lecithins
Phospholipids / Modified
Applications
lecithins
Phosphatidyl choline
Provides free choline in the blood for the
manufacture of acetylcholine; regulates
digestive, cardiovascular and liver
functions
Enriched phosphatidyl
Pharmaceutical
choline
cosmetics.
Phosphatidyl
preparations,
choline: For the production of stable liposomes,
Phosphatidyl-ethanolamine
anti-spattering agent in margarine.
(80:20)
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
decline with age, memory enhancer.
Ethanol solubles of soybean Emulsifier in foods (e.g. chocolate)
lecithin
that
13
Hydrogenated lecithin
Exhibits
greater
reduced
used
oxidative
solubility,
in
less
lubrication
stability,
hygroscopic,
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
Baked goods
Function
Effect
Gluten
chemistry Improves
modifier;
emulsifier effect”
emulsification,
and stabilizer for fats; flavor
andshelf-life,
antioxidant;
wetting texture
agent
Pasta
products Inclusion
dried potatoes
“shortening
and
moisture
retention.
partner
for Improves
amylose;co-emulsifier
emulsification,
flavor, texture and shelf
14
for
mono
and life.
diglycerides;antioxidant
Wafers
Emulsifier;stabilizer;
Improves
emulsification,
antioxidant
flavor, texture and shelf
life.
Cream
like Emulsifier;
emulsions
stabilizer; Improves
antioxidant.
emulsification
and stabilizes emulsion;
Improves
emulsification
flavor, texture and shelf
life.
Candy
Emulsifier;
viscosity Aids mixture of sugar, fats
reducer; wetting agent; and
dispersant.
water
to
prevent
greasiness, graining and
streaking.
Margarine
Emulsifier; antioxidant
shortening
Decreases
pattering,
Improves
emulsification,
flavor, texture and shelflife.
Meat
bacon)
(sliced Releasing
antioxidant.
agent; Improves
separation of
refrigerated
slices
shelf
life.
Instant
whole
foods, Instantizer; emulsifier; Speeds
milk antioxidant;
and
wetting reconstitution,
improves
15
powder
agent;
dispersant; emulsification,
flavor
nutritional supplement. texture and shelf life.
Dietic food
Emulsifier; antioxidant. Improves
Wetting
emulsification,
and flavor texture and shelf
strengthening
agent; life.
nutritional supplement.
Pan coating
Release and lubricate
Improves
appearance
and shelf life; prevents
surface greasiness.
Pharmaceuticals Emulsifiers;
carrier; Improves
antioxidant;
choline
emulsification,
donates dispersion and shelf- life.
and
linoleic
acid.
Cosmetics
Emulsifier
and foam Improves
stabilizer;
emollient; various
dispersing
refatting
with
depth
effects; wetting agent;
antioxidant;
choline,
donates
inositol
and
linoleic acid. Vitamin
source;
pantothenic
of
components
agent; (pigments and shelf- life)
resorceable
agent
dispersion
16
acid,
thiamine,
acid,
folic
riboflavin,
pyridoxine, biotin and
niacin.
Dog food
Releasing
agent; Aids blending of fats and
emulsifier; antioxidant water
wetting
strengthening
andclean
release
and from equipment and can;
agent; improve
animal
coat
nutritional supplement. glossiness.
Calf
and
cow Emulsifier; antioxidant Improves feed utilization
milk
wetting
strengthing
and and nutrition
agent;
nutritional supplement.
Poultry feed
Releasing
agent; Improves
antioxidant; emulsifier; and
wetting
strengthening
emulsification
dispersion;
and utilization
and
feed
nutrition
agent prevents lodging of food in
nutritional supplement. poultry beak and resulting
necrosis.
Live stock feed
Emulsifier; antioxidant Improves
wetting
strengthening
and and
emulsification
dispersion,
food
agent; utilization and nutrition.
nutritional supplement;
17
releasing agent; antidusting agent.
Insecticides
Emulsifier; dispersant; Disperses
stabilizer.
and stabilizers
pesticides and surfactants
in water.
Inks
Emulsifiers;
Improves
dispersant;
pigment
stabilizer; solubility
grinding aid.
and
properties;
flow
stabilizes
dispersion.
Magnetic tape
Emulsifier;
dispersing Improves dispersion and
agent; antioxidant.
shelf life.
Lacquers, paints Hydrophilic emulsifying Improves
dispersion
and
stability
coatings
other and
wetting
dispersing
agent; pigments,
of
and
agent, shelf- life.
stabilizer; antioxidant.
Leather
Softening
agent,
oil Improves the process of
penetrant
Plastics
Emulsifier;
fat liquoring.
dispersing Improves
agent; releasing agent.
pigments
dispersion
and
of
mold
release.
Rubber
Emulsifier;
dispersing Improves
agent; releasing agent; pigments
antioxidant.
dispersion
mold
and shelf –life.
of
release
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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
19
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,
chromatography, column chromatography,
thin
layer
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
20
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
21
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
phospholipid
composition.
Hurst
and
(HPLC)
martin64
to
determine
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
phospholipid mixtures. 1H and
13C
method
for
quantitation
of
NMR are available for molecular
characterization of unknown lipid compounds. Electrospray mass
22
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 molybdateperchloric 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
23
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
24
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
25
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.
26
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.
unsaturated
fatty
acid
containing
phospholipids
are
Highly
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
PLA1
CH2-O-CO-R
PLA2
CH-O-CO-R
O
II
I
CH2-O-P -O - X
O
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
The resulting glycerol derivatives were then phosphorylated with
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.
requirements,
To
hydrolysis,
meet
different
hydroxylation,
industial
application
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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