Oil for Life to Balance omega-3 polyunsaturated fatty acids ... - Oil4Life

Oil for Life to
Balance omega-3
polyunsaturated fatty
acids in cell membrane
phospholipids improving
health condition and
wellness
Bruno Berra and
Angela Maria Rizzo
Institute of General Physiology and Biochemistry
“G. Esposito” University of Milan, Italy
Milano 2008

OIL FOR LIFE TO BALANCE OMEGA-3 POLYUNSATURATED
FATTY ACID IN CELL MEMBRANE PHOSPHOLIPIDS IMPROVING
HEALTH CONDITION AND WELLNESS
Bruno Berra and Angela Maria Rizzo
Institute of General Physiology and Biochemistry “G. Esposito” University of Milan, Italy
EXECUTIVE SUMMARY
Since the 1950’s there has been a rapid global shift towards a diet linked to the chronic
degenerative diseases such as Heart disease, Stroke, Cancer, Diabetes, Osteoporosis and
Alzheimer, the major causes of ill health and death. There are many ways in which the
modern diet triggers disease, but it appears likely that the most important mechanism is that
it creates a pro-inflammatory climate within the body, enhanced by smoking, our low-energy
lifestyle and obesity. Five out of six of 60-year-olds already have one or more of the chronic
degenerative diseases. Most of these people will not yet know that they have the disease -
because it has yet to become noticeable. Chronic degenerative diseases are slowly
progressing conditions, a pre-illness growing little by little every day for years or decades
until the clinical illness finally emerge, sometimes overnight.
Excessive and / or inappropriate oxidative and closely related inflammatory stressors are
profoundly involved in tissue damage, the ageing process and most of the chronic
degenerative diseases. Antioxidant and anti-inflammatory defence systems are required to
maintain health, but the modern diet has increased our exposure to oxidative and
inflammatory components, and simultaneously reduced the integrity and functionality of our
defences against these stressors. Our body needs to have a strong anti-inflammatory /
antioxidant defence system - in the same way as it needs a strong immune system. The main
defence against free radical attack are antioxidant enzymes and antioxidant nutrients. Free
radicals generated in cigarette smoke are known to deplete antioxidants, a mechanism by
which cigarette smoking will promote vascular disease. Many plant foods contain
antioxidants that survive cooking in a bio available form and are absorbed by the body. The
olive fruit, being an important part of the Mediterranean diet, is a plant that has the potential
of being a part of a strong antioxidant defences. Secoiridoids are a class of compounds found
only in virgin olive oil that are strong antioxidants. One important molecule in this family is
oleuropein, a bitter-tasting glycoside that is converted to p-hydroxyphenyl ethanol and 3,4dihydroxy-phenyl
ethanol. The mixture of these minor components works synergistic in their
antioxidant defence as in Oil4Life Balance. The described biophenols are absorbed by our
body and enter exclusively into lipoproteins containing cholesterol, protecting the
lipoproteins from oxidation.
The polyunsaturated fatty acid linoleic acid (omega-6) and α-linolenic acid (short chain
omega-3) from plant oils are essential to the human diet. Neither is synthesized
endogenously and the omega-3/omega-6 families cannot be interconverted. α-linolenic acid
may be convertred to the long chain marine omega-3 EPA and DHA in the body. However,
due to competition an omega-6 rich diet of plant oils may inhibit EPA and DHA production,
leading to depleted levels of EPA and DHA in the plasma, although the minimum levels to
prevent disease is obtained. Our early diet contained small, but approximately equal amounts
of omega-6 and omega-3 fatty acids, whereas the modern Western diet today contains an
excess of omega-6 due to high consumption of plant oils. This imbalance of omega-6 to
1

omega-3 has been associated with increased risks of cardiovascular disease,
neurodegenerative disorders and some cancers, including carcinoma of the breast. The
observed increased risk of breast cancer in Japanese women over the past four decades
correlates with an increased imbalance of dietary omega-6 to omega-3 ratio.
The membrane of our trillions of cells physically separates the intracellular components from
the extracellular environment. The movement of water, nutrients and waste across the
membrane can be either passive or active (energy required). The arrangement of hydrophilic
heads and hydrophobic tails in the lipid bilayer allow the cell to control the movement of
substances via transmembrane protein complexes such as pores and gates. The fatty acid
present in membrane lipids come from the diet. Phospholipids are main components of cell
membrane in our trillions of cells with a hydrophilic head and generally one saturated fatty
acid and one polyunsaturated fatty acid (omega-6 or omega-3) as the hydrophobic tails. The
length and the degree of unsaturation of fatty acids chains have a profound effect on
membranes fluidity. Unsaturated lipids create a kink, preventing the fatty acids from packing
tightly together, thus increasing the fluidity of the membrane and facilitating the exchanges
of substances. Human cells constantly receive signals from other cells and from the
environment, which they perceive, interpret and respond to with appropriate metabolic or
physiological changes.
An unbalanced ratio of mega-6 / omega-3 in cell membrane phospholipids may create a proinflammatory
climate within the cells. Inflammations are a key cause of pain. Phospholipase
A2 frees EPA (omega-3) and aracidonic acid (AA; omega-6) located in the 2 nd position of
the phospholipid molecule. The released AA and EPA are further oxidized by specific
enzyme to eicosanoids, signaling molecules called prostaglandins, leukotrienes,
thromboxane and prostacyclins. These hormone-like compounds are involved in tissue
damage, inflammation and pain. The enzymatic releases of arachidonic acid (AA - omega-6)
from membrane phospholipids enhance the synthesis of eicosanoids of the 2 and 4 series.
These eicosanoids are pro-inflammatory products that in turn create pain, which is
recognised by pain receptors and transmitted to the brain. On contrary the release of
eicosapentaenoic acid (EPA – omega-3) from membrane phospholipids enhance the
synthesis of eicosanoids of the 3 and 5 series being less inflammatory. The networks of
controls that depend upon eicosanoids are among the most complex in the human body. They
exert complex control over inflammation and immunity systems, and are messengers in the
central nervous system.
Menstrual pain is the most common gynecological complaint among female adolescents and
young women. The high intake of omega-6 fatty acids in the western diet is reflected in the
cell membrane phospholipids. Due to progesterone withdrawal before menstruation
arachidonic acid (omega-6) are released, and a cascade of eicosanoids is initiated in the
uterus. The inflammatory response mediated by these eicosanoids produces both cramps and
systemic symptoms such as nausea, vomiting, bloating and headaches, including ischemia,
pain and systemic symptoms of dysmenorrhoea. EPA and DHA, as in Oli4Life Balance,
compete with arachidonic acid for the production of eicosanoids. In the uterus, this
competitive interaction between omega-3 and omega-6 fatty acids may result in the
production of less potent eicosanoids and may lead to a reduction in the systemic symptoms
of dysmenorrhea.
The arachidonic acid metabolism is also altered in psoriasis. Proinflammatory leukotrienes
(LTB4) are markedly produced in the psoriatic lesions. Fish oil and omega-3 fatty acids are
2

known to decrease the production of LTB4, a metabolite of arachidonic acid produced by
activated neutrophils. Fish oil supplementation as in Oli4Life Balance offers an opportuinity
in management of psoriasis and inflammatory skin disorders with negligible side effects.
The immune system must be ready to mount acute inflammatory reactions to kill invading
micro-organisms, but it should not be so over-reactive as to create chronic inflammations
which damage the host. Asthma is a chronic inflammation of the airways, which in many
cases is considered to be due to an allergic reaction. The immune system learns to react to
the allergen and starts the chronic inflammatory process. The rate of asthma increases as
communities adopt western lifestyles and become urbanised. Diet is deeply implicated due to
reduced intake of inflammation-damping elements like the flavonoids, sterols and omega 3
fatty acids, and the simultaneous increase in intakes of pro-inflammatory compounds.
Potential therapeutic uses of prostaglandins include relief of asthma. A mixture of
eicosanoids of the 4 series (LTC4, LTD4 and LTE4) is a potent constrictor of the bronchial
airway musculature. They are also important regulators in many diseases involving
inflammatory or immediate hypersensitivity reactions, such as asthma.
The analysis of fatty acid composition of phospholipids in red blood cell (RBC) membranes
allows us to obtain relevant information about diet fatty acids deficiency, eicosanoids
biosynthesis and eventual metabolic abnormalies. However, this marker analysis require a
long lasting procedure. In our laboratory we have developed and tested a time saving and
reliable method to determine AA/EPA and omega-6/omega-3 ratios in whole blood. Our
method of AA/EPA ratio in whole blood demonstrate a good correlation with erythrocyte
phospholipids composition and a significant correlation with the AA/EPA value of RBC
membranes. This reliable biomarker method of dietary fat intake are at present being
commercialised by the Itogha group under the brand name of Test4Life. The AA/EPA ratio
may be considered a predictor of health status for risks of diseases such as cardiovascular
disease (CVD), diabetes, chronic inflammation and depression, as well as an index of wellbeing.
The Test4Life analysis can give information on cellular membrane fatty acid status
and potential error in eicosanoid biosynthesis. Test4Life is a reliable and low-time
consuming assays to establish a fatty acid balance for dietary advice and in the prevention
and control of chronic diseases.
The AA/EPA ratio found in healthy Italian subjects without omega-3 supplementation is
high and comparable to data reported for the Western population, indicating an unbalanced
omega-6/omega-3 fatty acid intake, leading to a subsequent imbalance in membrane
composition and the eicosanoid biosynthesis. We have also observed that patients with
allergic-, skin- and neurodegenerative diseases have higher values of the AA/EPA ratio than
other pathological subjects.
About 7% of children between the ages of 5-11 years have been diagnosed with Attention
Deficit Hyperactivity Disorder (ADHD). General consensus is that many genes are involved
in the transmission of the disorder. Diet may be an ethiological risk factor for ADHD.
Abnormalities in PUFA metabolism in red blood cell membranes has been reported in
children with ADHD, having lower levels of long chain omega-3 fatty acids (EPA+DHA) in
their blood due to lack of dietary intake in conjunction with a more rapid metabolism. Our
studies have highlighted a deficiency of the long chain omega-3 fatty acids in the membrane
phospholipids of patients affected with ADHD. The AA/EPA-ratio in phospholipids in blood
and in RBC membranes was elevated, indicating an increased upstream inflammatory
potential. In our study of 30 children with ADHD the diet were supplemented with 2.5 mg
3

10 kg/day of EPA+DHA 2:1 as in Oil4Life Balance. The supplementation of EPA and
DHA in relatively high doses compared to body weight, verified that an improved AA/EPA
balance in the cell membrane increased attention level and decrease both hyperactivity levels
and impulsiveness. There was a correlation between the dose of long chain omega-3 fatty
acids, the decrease of AA/EPA ratio and/or the entity of the clinical improvement. Our data
are in agreement with the results obtained by different Authors.
Depletion of omega-3 fatty acid levels in red blood cell membranes of depressed patients has
been reported. A significant positive relationship was observed between the severity of the
illness and the AA/EPA ratio in serum phospholipids and in erythrocyte membranes.
Preliminary results in our laboratory on depressed elderly patients demonstrated that
counteracting and balancing high levels of AA with EPA+DHA 2:1, as in Oil4Life Balance,
also decreased depression symptoms. Several authors have also reported lower
concentrations of erythrocyte essential fatty acids among schizophrenic patients as compared
with control. DHA is the major acid of neurological and retinal membranes. It makes up
more than 30% of the structural lipids of the neuron. Low levels of circulating DHA may be
a significant risk in the development of Alzheimer dementia. The inability to maintain a high
level of DHA may be due to a reduced capacity to synthesise DHA late in life, as the result
of a reduction in Δ-6-desaturase activity. Alterations in phospholipids, which are structural
components of all cell membranes in the brain, may induce changes in membrane fluidity
and, consequently, in various neurotransmitter systems that are believed to be related to the
pathophysiology of major depression.
4

CONTENTS
SUMMARY............................................................................................................................6
1.DEFINITION, NOMENCLATURE AND METABOLISM OF OMEGA-3
LCPUFA……………………………………………………………………………………13
2. DIETARY SOURCES .....................................................................................................14
3. CELL MEMBRANE COMPOSITION AND FUNCTION..........................................15
3.1 Lipids................................................................................................................................17
4. FUNCTION OF PUFA IN CELL MEMBRANE PHOSPHOLIPIDS......................... 19
4.1 Biosynthesis .................................................................................................................. 19
5. BIOMARKERS OF OMEGA-3 FATTY ACID NUTRITIONAL STATUS .............. 21
6.OMEGA-6/OMEGA-3 PUFA BALANCE AND CHRONIC DISEASES .................... 22
6.1. Smoking, hormonal contraception, pregnancy and premenstrual syndrome ............... 22
6.2. Coronary heart disease ................................................................................................. 23
6.3. Hypertension ................................................................................................................ 24
6.4 Diabetes........................................................................................................................ 24
6.5. Atopic eczema and psoriasis ........................................................................................ 24
6.6. Rheumatoid arthritis (RA) ........................................................................................... 25
6.7 Noreulogical diseases – alzheimer dementia, depression, attention deficit
hyperactivity disorder (ADHD) and schizophrenia ............................................................ 25
7. OLIVE OIL ....................................................................................................................... 26
7.1 Digestion and absorption of olive oil triglycerides ....................................................... 27
7.2 Use of Olive Oil for Secondary Prevention of Atherosclerosis .................................... 27
7.3 Biological and nutritional value of olive oil ................................................................. 28
7.4 Minor components of olive oil ...................................................................................... 28
7.5 Protective effects of secoiridoids, oleuropein and phenols in olive oil on protection
against LDL oxidation ........................................................................................................ 29
8. CLINICAL STUDIES AT THE UNIVERSITY OF MILAN – THE
DEVELOPMENT OF OIL4LIFE BALANCE: ................................................................. 30
8.1 The blood AA/EPA ratio. ............................................................................................. 30
8.2 Mood and brain wellness .............................................................................................. 31
8.3 ADHD in children and depression in elderly ................................................................ 33
9.CONCLUSIONS ................................................................................................................ 35
REFERENCES ...................................................................................................................... 36
5

SUMMARY
1. Olive oil - a strong antioxidant defence built into Oil4Life Balance
Excessive and / or inappropriate oxidative and closely related inflammatory stressors are
profoundly involved in tissue damage, the ageing process and most of the chronic
degenerative diseases such as Heart disease, Stroke, Cancer and Alzheimer. Antioxidant and
anti-inflammatory defence systems are required to maintain health, but the modern diet has
increased our exposure to oxidative and inflammatory components, and simultaneously
reduced the integrity and functionality of our defences against these stressors. Our body
needs to have a strong anti-inflammatory / antioxidant defence system - in the same way as it
needs a strong immune system. The main defence against free radical attack are antioxidant
enzymes and antioxidant nutrients. Free radicals generated in cigarette smoke are known to
deplete antioxidants, a mechanism by which cigarette smoking will promote vascular
disease. Many plant foods contain antioxidants that survive cooking in a bio available form
and are absorbed by the body. The olive fruit, being an important part of the Mediterranean
diet, is a plant that has the potential of being a part of a strong antioxidant defences.
The olive fruit contain a large amount of “vegetation water” with polyhydroxy compounds
and phenol derivatives present as glycoconjugates.These amphiphilic molecules are
distributed between the organic and aqueous phases as the oil is processed from the pericarp
of the olive fruit. In the oil phase these components protect virgin olive oil against oxidation.
Secoiridoids are a class of compounds found only in virgin olive oil that are strong
antioxidants. One important molecule in this family is oleuropein, a bitter-tasting glycoside.
Upon enzymatic hydrolysis oleuropein loses its saccharide component and becomes soluble
in the oil. Further hydrolysis converts it into p-hydroxyphenyl ethanol and 3,4-dihydroxyphenyl
ethanol. The mixture of these polar minor components works synergistic in their
antioxidant defence, and constitute the antioxidante defence built into a product developed
for the Itogha group, Oil4Life Balance. Olive oil has a very high digestibility in humans.
The described biphenols are absorbed and enter exclusively into lipoproteins containing
cholesterol, protecting the lipoproteins from oxidation. Oxidized lipoproteins, particularly
LDL cholesterol, play a fundamental role in the pathogenesis of arteriosclerosis leading to
heart attack and stroke. The oxysterol components of oxidized LDL cholesterol are known to
cause many harmful actions to cell life.
Except for the antioxidant properties only a few investigations have been made on the
nutritional functions of the minor components of olive oil. Olive oil contains many minor
components like hydrocarbons, fatty acid esters, monohydroxy and dihydroxy triterpenes,
sterols (β-sitosterol) and hydroxy triterpenic acids. These components shows bioactive
functions like hypocholesterolemic activity (β-sitosterol), healing and anti-inflammatory
activities (triterpenic acids), choleretic activity (caffeic and gallic acid) and anti catecholamin
O-methyl transferase activity.
2. Polyunsaturated fat are essential in the diet – omega-3 and omega-6
The polyunsaturated fatty acid linoleic acid (LA; 18:2, omega-6) and α-linolenic acid (ALA;
18:3, omega-3) are essential to the human diet. Neither is synthesized endogenously and the
omega-3/omega-6 families cannot be interconverted. The biosynthetic pathways of both the
omega-3 and the omega-6 families share an enzyme called Δ-6-desaturase. This enzyme has
a preference for AL, and is together with elongases responsible for the conversion of ALA to
6

eicosapentaenoic acid (EPA; C20:5, omega-3) and docosahexaenoic acid (DHA; C22:6,
omega-3). However, in the presence of high levels of plasma LA the preference may be
shifted towards the omega-6 pathway. Thus, an omega-6 rich diet of plant oils may lead to
depleted levels of EPA and DHA in the plasma, although the minimum levels to prevent
disease is obtained. Dermatitis is consistently the first sign of PUFA deficiency in both
animals and humans. The early diet contained small, but approximately equal amounts of
omega-6 and omega-3 fatty acids, whereas the modern Western diet today contains an excess
of omega-6. This imbalance of omega-6 to omega-3 has been associated with increased risks
of cardiovascular disease and some cancers, including carcinoma of the breast. The increased
risk of breast cancer in Japanese women over the past four decades correlates with an
increased imbalance of dietary omega-6 to omega-3 ratio.
The amount and type of dietary fats play a crucial and well-documented role on plasma lipid
concentration. In the Mediterranean countries the population have been adviced to improve
plasma lipid levels by shifting from a diet based on olive oil, rich in monounsaturated fat, to
a diet based on corn oil, rich in polyunsaturated fat (PUFA; omega-3 and omega-6). We have
studied the variations induced on plasma lipid levels by shifting back to the olive oil based
diet. Substitution of corn oil with olive oil does not cause hazards as far as haemostatic
functions, lipids and lipoprotein cholesterol are concerned, except for a mild elevation of
total cholesterol. In subjects using the olive oil diet we observed over a six-month period a
consistent decrease in LDL cholesterol and an increase in HDL cholesterol levels. We
concluded that it is not worthwhile to change compulsorily olive oil to corn oil or other
vegetable oils rich in PUFA.
3. The cell membrane – composition and function
The membrane of our trillions of cells physically separates the intracellular components from
the extracellular environment, provide shape to the cell, and help group cells together in the
formation of tissues. Special phospholipids are required for specific membrane structures
such as curved regions and junctions with adjacent membranes. The movement of water,
nutrients and waste across the membrane can be either passive or active (energy required).
The arrangement of hydrophilic heads and hydrophobic tails in the lipid bilayer allow the
cell to control the movement of substances via transmembrane protein complexes such as
pores and gates. Specific proteins embedded in the cell membrane act as molecular signals
that allow cells to communicate with each other and the environment. Proteins on the surface
of the cell membrane serve as "markers" that identify a cell to other cells. These markers
forms the basis of cell to cell interaction in the immune system.
The fatty acid present in membrane lipids come from the diet. The cell membrane contains
three classes of amphipathic lipids, phospholipids, glycolipids and cholesterol. Phospholipids
are normally the most abundant with a hydrophilic head and generally one saturated fatty
acid and one polyunsaturated fatty acid (omega-6 or omega-3) as the hydrophobic tails. The
length and the degree of unsaturation of fatty acids chains have a profound effect on
membranes fluidity. Unsaturated lipids create a kink, preventing the fatty acids from packing
tightly together, thus increasing the fluidity of the membrane facilitating the exchanges of
substances. Phospholipids made exclusively of saturated fatty acids will result in a very
“dense” membrane, which will not allow physiological exchanges. Cell cholesterol is
normally located between the hydrophobic tails providing stiffening and strengthening
effects that reduce the membranes fluidity.
7

In the fluid membranes the components move around, are metabolized and subject to
metabolic turnover. The turnover of membrane components is especially important for the
cellular response to information from inside and outside the cell, like recognition, transfer,
amplification and signal transduction. These are processes that occur in or on the membrane
surface. Human cells constantly receive signals from other cells and from the environment,
which they perceive, interpret and respond to with appropriate metabolic or physiological
changes. Four phospholipids classes have been shown to participate in signal transduction.
These are phosphatidylinositols (PtdIns), which are the preferred substrate for liberating
arachidonic acid catalysed by phospholiphase A2, phosphatidylcholines, sphingomielin and
glycosylphosphatidyl-inositols.
4. Eicosanoids from omega-3 and omega-6 - the inflammatory climate within the body
The amounts and balance of omega-6 and omega-3 in a diet have a profound affect on the
body's eicosanoid controlled functions involved in cardiovascular diseases, triglyceride
consentration, blood preassure, arthritis, inflammation and oxidative/nitrosative conditions.
The eicosanoid biosynthesis begins when a cell is activated. Phospholipase A2 is released at
the cell membrane, where it frees the 20-carbon polyunsaturated fatty acids EPA (omega-3)
and aracidonic acid (AA; omega-6) located in the 2 nd position of the phospholipid molecule.
The released AA and EPA are further oxidized by specific enzyme to eicosanoids, signaling
molecules called prostaglandins (prostanoids), leukotrienes (LTs), thromboxane and
prostacyclins. Two families of enzymes catalyze the fatty acid oxygenation to produce the
eicosanoids, cyclooxygenases (COX) generating the prostanoids and lipoxygenases
generating the leukotrienes. Eicosanoids are not stored within cells, but are synthesized as
required.
The networks of controls that depend upon eicosanoids are among the most complex in the
human body. They exert complex control over inflammation and immunity systems, and are
messengers in the central nervous system. The eicosanoids of the 2 and 4 series from AA
(omega-6) are generally proinflammatory, while eicosanoids of the 3 and 5 series from EPA
(omega-3) are much less inflammatory. Eicosanoids of the 3 series (PG3 and TX3) formed
by oxidation of EPA inhibits the release of AA from phospholipids, and thus the formation
of the proinflammatory eicosanoids of the 2-series (PG2 and TX2). EPA has antiarrhythmic
effects and several antithrombotic actions, particularly inhibiting the synthesis of
thromboxane A2 (TX2). Thromboxanes and prostacyclins are antagonistic. Thromboxanes
are synthesized in blood platelets causing vasoconstriction and platelet aggregation, while
prostacyclins (PGI2) produced in the blood vessel walls is a potent inhibitor of platelet
aggregation. Thromboxanes of the 3-series (TXA3) are a wicker aggregator than
thromboxanes of the 2-series (TXA2), favouring longer clotting times. Fish oil retards the
growth of atherosclerotic plaque by reducing the amount of inflammatory interleukine 1
(IL1) and tumour necrosis factor (TNF), and by inhibiting both cellular growth factors and
migration of monocytes, which should aid in the amelioration of inflammation.
Potential therapeutic uses of prostaglandins include relief of asthma. A mixture of
leukotrienes of the 4 series (LTC4, LTD4 and LTE4) is a potent constrictor of the bronchial
air-way musculature. They are also important regulators in many diseases involving
inflammatory or immediate hypersensitivity reactions, such as asthma. Together with
leukotriene B4 (LTB4) these leukotrienes also cause vascular permeability and attraction and
activation of leukocytes.
8

Isoprostanes are prostaglandin-like compounds formed in vivo from free radical-catalyzed
peroxidation of primarily arachidonic acid (AA) without the action of cyclooxygenase
(COX) enzyme. These nonclassical eicosanoids possess potent biological activity as
inflammatory mediators that augment the perception of pain. These compounds are accurate
markers of lipid peroxidation in the body. The main isoprostanes (endocannabinoids,
endogenous cannabis-like substances) are small molecules that bind to a family of G-protein
coupled receptors that are densely distributed in areas of the brain related to motor control,
cognition, emotional responses, motivated behaviour and homeostasis. Outside of the brain,
the endocannabinoid system is one of the crucial modulators of the autonomic nervous
system, the immune system and microcirculation.
During pregnancy, there is a faster turnover of PUFA from fast storage that may modify the
profile of erythrocyte cell membrane fatty acids. Thus, it is suggested that omega-3 PUFA
intake during pregnancy should be increased in the last trimester. Menstrual pain or
dysmenorrhea is the most common gynecological complaint among female adolescents and
young women. The high intake of omega-6 fatty acids in the western diet is reflected in the
cell membrane phospholipids. Due to progesterone withdrawal before menstruation omega-6
fatty acids, in particular arachidonic acid, are released, and a cascade of prostaglandins and
leukotrienes is initiated in the uterus. The inflammatory response mediated by these
eicosanoids produces both cramps and systemic symptoms such as nausea, vomiting,
bloating and headaches, including ischemia, pain and systemic symptoms of dysmenorrhoea.
EPA and DHA compete with arachidonic acid for the production of prostaglandins and
leukotrienes through the incorporation into cell membrane phospholipids, and through
competition at the prostaglandin synthesis level. Long chain omega-3 can also inhibit
arachidonic acid formation at the level of the Δ-6-desaturase enzyme. In the uterus, this
competitive interaction between omega-3 and omega-6 fatty acids may result in the
production of less potent prostaglandins and leukotrienes and may lead to a reduction in the
systemic symptoms of dysmenorrhea.
The arachidonic acid metabolism is also altered in psoriasis. Proinflammatory leukotrienes
(LTB4) are markedly produced in the psoriatic lesions. Fish oil and omega-3 fatty acids are
known to decrease the production of LTB4, a metabolite of arachidonic acid produced by
activated neutrophils. Fish oil supplementation offers an opportuinity in management of
psoriasis and inflammatory skin disorders with negligible side effects. Clinical
improvements in tender joint scores and morning stiffness have also been reported with fish
oil supplementation, associated with a decreased production of pro-inflammatory
interleukine 1 (IL1) and LTB4.
5. Biomarker of dietary fat intake – the development of Test4Life
The turnover time for fatty acids (FA) in the adipose tissue is 1–3 years and unaffected by
temporary variations in diet. This make adipose tissue a good material to evaluate dietary FA
intake over extended periods of time. However, the FA composition of plasma lipids and the
membranes of platelets and erythrocytes provide a good assessment of the the dietary intake
of the long chain omega-3 fatty acids (EPA and DHA). It was shown that EPA and DHA
contents of adipose tissue aspirates and erythrocyte membranes have similar correlations
with fish consumption. The measurement of erythrocyte phospholipids fatty acid status
presents several advantages: (1) it is a reflexion spread over time of habitual dietary fat
intake in relation to the biological half life of erythrocytes, (2) the level of EPA can be used
as a specific marker for the intake of fish and fish oil, (3) phospholipids are a model of fatty
9

acid incorporated into cell membranes, (4) it gives an image of hepatic and extrahepatic fatty
acid metabolism, (5) erythrocyte phospholipids are in equilibrium with structural
phospholipids of tissues.
The analysis of fatty acid composition of phospholipids in red blood cell (RBC) membranes
allows us to obtain relevant information about diet fatty acids deficiency, eicosanoids
biosynthesis and eventual metabolic abnormalies. The determination of the fatty acid
composition of RBC membrane phospholipids is becoming a common procedure to evaluate
fat intake. The diagnostic value is well documented, particularly concerning cardiovascular
diseases. However this marker require a long lasting procedure. In our laboratory we have
developed and tested a time saving and reliable method to determine AA/EPA and omega-
6/omega-3 ratios in whole blood. The specific AA/EPA analysis was more significant than
the total ratio of omega-6/omega-3 fatty acids, even when the omega-6 fatty acids were
significantly decreased. Our method of AA/EPA ratio in whole blood, as a biomarker of fatty
acid intake, demonstrate a good correlation with erythrocyte phospholipids composition and
a significant correlation with the AA/EPA value of RBC membranes. This reliable biomarker
method of dietary fat intake are at present being commercialised by the Itogha group under
the brand name of Test4Life.
6. Test4Life – an index of well-being and predictor of health status
There are many ways in which the modern diet triggers disease, but it appears likely that the
most important mechanism is that it creates a pro-inflammatory climate within the body,
enhanced by smoking, our low-energy lifestyle and obesity. The major causes of ill health
and death now are the chronic degenerative diseases such as Heart disease, Stroke, Cancer,
Diabetes, Osteoporosis and Alzheimer diseases. Unbalanced PUFA status accompanied by
deficiency of antioxidants is observed in numerous of conditions linked to the chronic
degenerative diseases. Thus, early diagnosis of such impairment is of crucial importance to
prevention and control such diseases. Five out of six of 60-year-olds already have one or
more of the chronic degenerative diseases. Most of these people will not yet know that they
have the disease - because it has yet to become noticeable. Chronic degenerative diseases are
slowly progressing conditions, a pre-illness growing little by little every day for years or
decades until the clinical illness finally emerge, sometimes overnight. The AA/EPA ratio
may be considered a predictor of health status, as well as an index of well-being
The chronic degenerative diseases makes it difficult to choose healthy subjects for clinical
studies. Test4Life can give information on cellular membrane fatty acid status and potential
error in eicosanoid biosynthesis. Monitoring dietary fat intake as a biomarker of disease risk
by measuring erythrocyte fatty acids is becoming increasingly common in clinical nutrition.
It can also be used as a biomarker for risks of diseases such as CVD, diabetes, chronic
inflammation and depression. AA/EPA ratio in total blood may also be a reliable and lowtime
consuming assays for selection of healthy subjects for clinical studies, and to give
dietary advice on how to establish a fatty acids balance in the prevention and control of
chronic diseases.
Chronic diseases like coronary heart disease, hypertension, diabetes, cancer, inflammatory
and autoimmune disorders, atopic eczema, depression, schizophrenia, Alzheimer, dementia
and multiple sclerosis are frequently associated with abnormalities in polyunsaturated fatty
acid metabolism, and an unbalance in omega-6/omega-3 fatty acid ratio. Erythrocyte fatty
acid measurements can indicate fatty acid deficiencies or imbalances from the diet, but also
10

metabolic abnormalities (e.g. lack of Δ-6-desaturase) or peroxidation of membrane
phospholipids.
Red blood cell fatty acid analysis can give information on cellular membrane fatty acid status
and potential error in eicosanoid biosynthesis. Measuring erythrocyte fatty acids for
monitoring dietary fat intake, or as a biomarker of disease risk, is becoming increasingly
common in clinical nutrition. It can be used also as a biomarker for ascertain risks of
diseases such as CVD, diabetes, chronic inflammation, depression.
AA/EPA ratio in total blood is a reliable and low-time consuming assays to establish a fatty
acids balance for dietary advice and in the prevention and control of chronic diseases.
7. Clinical studies at the university of Milan – The development of Oil4Life Balance
The AA/EPA ratio found in healthy Italian subjects without omega-3 supplementation is
high and comparable to data reported for the Western population, indicating an unbalanced
omega-6/omega-3 fatty acid intake leading to a subsequent imbalance in membrane
composition and the eicosanoid biosynthesis. We have also observed that patients with
allergic, skin and neurodegenerative diseases have higher values of the AA/EPA ratio than
other pathological subjects.
Obesity and the control of body weight is influenced not only by the quality and quantity of
food intake, but also by hormonal, metabolic and genetic factors linking obesity to
cardiovascular, inflammatory and endocrine diseases. Diet with low carbohydrate content
and low glycemic index are believed to have beneficial effects on type 2 diabetes. In our
laboratory we have tested two diets with very different percentages of carbohydrate and
protein. The reduced carbohydrate and increased protein concentration in the second diet are
believed to have beneficial effects on type 2 diabetes, cardiovascular diseases and obesity. A
reduction of body fat was observed in subjects on the last diet. We also found that the
AA/EPA ratio, as measured by Test4Life, correlates with the changes in tryglicerides and
Trg/HDL ratio. The AA/EPA ratio was reduced by both diets, but most strongly by the diet
with a low carbohydrate content. By supplementing the diets with omega-3, the AA/EPA
ratio was greatly decreased. The relationship between AA/EPA and Trg/HDL, observed in
the above experiment, confirms the importance ascribed to the latter parameter in the
prevention of type 2 diabetes and cardiovascular disease. The omega-3 fatty acids have a
similar effect on lipid metabolism as a low glycemic index diet. In fact the long chain
omega-3 reduce triglycerides, have anti-inflammatory effects, reduce the insulin response to
glucose, and reduce the risk of cardiovascular diseases and cancer.
The free radicals are neutralized by a strong antioxidant defence system consisting of
enzymes and non-enzymatic antioxidants, including vitamin C (ascorbic acid). When free
radicals are generated they can attack polyunsaturated fatty acids in the cell membrane. This
leads to lipid peroxidation, which reduces the membrane fluidity, permeability and
excitability, and promote the formation of hydrocarbon gases and aldehydes such as MDA.
Plasma MDA is a marker of lipid oxidation. The plasma MDA levels in subjects on the two
diets above reported supplemented with omega-3 fatty acids were significantly reduced
compared to basal levels (with a greater decrease in the low carbohydrate diet subjects),
indicating that nutritional status has an important role in preventing lipid peroxidation by
increasing the plasma total antioxidant capacity.
11

Supplementation with omega-3 were linked to an increase of vigour and a decrease of
negative factors such as anger, anxiety and depression. These results confirm the influence of
omega-3 on the central nervous system. They are also in line with the suggested action of
these compounds on dementia, depression and mood disorders, in which they may act as
mood. The results suggest that some of these effects are due almost exclusively to diet, e.g
the reduction of body fat, while others, such as the mood state variations, are mainly due to
omega-3 supplementation and the strong antioxidant defence built into Oil4Life Balance.
About 7% of children between the ages of 5-11 years have been diagnosed with Attention
Deficit Hyperactivity Disorder (ADHD). It is one of most common neurodevelopmental
syndrome of childhood. The symptoms should present itself for at least six months before the
age of 7 years, accompanied by “clinically significant” impairment. The current consensus is
that no biochemical tests can reliably predict ADHD. However, a genetic feature of ADHD
is strongly suggested because the syndrome clusters in families, and two polymorphisms in
the dopamine transporter and receptor genes have been identified that seem to influence the
risk of ADHD. General consensus is that many other genes are probably involved in the
transmission of the disorder.
One hypothesis of the etiology of ADHD is concerned with the role of prostaglandins in the
dopaminergic synapses. According to this hypothesis, ADHD is caused or worsened by a
deficiency of Prostaglandin E1 (PGE1), which is again caused by the lack of the enzyme
Δ-6-desaturase. Also environmental toxicant (lead) might be an etiologic risk factor for
ADHD, as is cigarette smoking during pregnancy. Diet may also be an ethiological risk
factor for ADHD. Abnormalities in PUFA metabolism in red blood cell membranes has been
reported in children with ADHD. Children with ADHD have lower levels of long chain
omega-3 fatty acids (EPA+DHA) in their blood, probably due to lack of dietary intake in
conjunction with a more rapid metabolism. Our studies have highlighted a deficiency of the
long chain omega-3 fatty acids in the membrane phospholipids of patients affected with
ADHD. Furthermore, the ratio of AA/EPA in phospholipids both in blood and in RBC seems
to be elevated. The elevated AA/EPA ratio indicates an increased upstream inflammatory
potential. In our study of 30 children with ADHD the diet were supplemented with 2.5 mg
/10 kg/day of EPA+DHA 2:1 as in Oil4Life Balance. The supplementation of EPA and
DHA in relatively high doses compared to body weight, verified that an improved AA/EPA
balance in the cell membrane increased attention level and decrease both hyperactivity levels
and impulsiveness. There was a correlation between the dose of long chain omega-3 fatty
acids, the decrease of AA/EPA ratio and/or the entity of the clinical improvement (score).
Depletion of omega-3 fatty acid levels in red blood cell membranes of depressed patients has
been reported. A significant positive relationship was observed between the severity of the
illness and the ratio of arachidonic acid to eicosapentaenoic acid in serum phospholipids and
in erythrocyte membranes. Preliminary results in our laboratory on depressed elderly patients
demonstrated that counteracting and balancing high levels of AA with EPA+DHA 2:1, as in
Oil4Life Balance, also decreased depression symptoms. Several authors have also reported
lower concentrations of erythrocyte essential fatty acids among schizophrenic patients as
compared with control. DHA is the major acid of neurological and retinal membranes. It
makes up more than 30% of the structural lipids of the neuron. Low levels of circulating
DHA may be a significant risk in the development of Alzheimer dementia. The inability to
maintain a high level of DHA may be due to a reduced capacity to synthesise DHA late in
life, as the result of a reduction in Δ-6-desaturase activity. Alterations in phospholipids,
12

which are structural components of all cell membranes in the brain, may induce changes in
membrane fluidity and, consequently, in various neurotransmitter systems that are believed
to be related to the pathophysiology of major depression.
1. DEFINITION, NOMENCLATURE AND METABOLISM OF OMEGA-3 LC-PUFA
Fatty acids (FA) typically have an even number of carbon atoms, in the range of 16–26. Fatty
acids with only single bonds between adjacent carbon atoms are referred to as 'saturated',
whereas those with at least one C=C double bond are called 'unsaturated'. The PUFA have
two or more double bonds and are named according to their position and to the total chain
length. For example, DHA (C22:6, n-3) is an omega-3 (n-3) fatty acid with 22 carbon atoms
and six double bonds. The term 'n-3' indicates that, counting from the methyl (CH3) end of
the molecule, the first double bond is located between the third and fourth carbons. As the
degree of unsaturation in fatty acids increases, the melting point decreases which confers to
PUFA the possibility to increase the fluidity of the membranes where they are inserted.
The DHA and EPA (C20:5, n-3) are synthesized from the n-3 precursor α-linolenic acid
(ALA; 18:3, n-3), whereas long chain n-6 PUFA such as arachidonic acid (AA, C20:4, n-6)
are synthesized from the precursor linoleic acid (LA; 18:2, n-6). The ALA and LA are
essential to the human diet because neither is synthesized endogenously by humans, and the
n-3/n-6 families cannot be interconverted. In theory, the ability to convert ALA to EPA and
DHA might indicate that humans have no need for an exogenous supply of these fatty acids.
However, there are two reasons why this is only partially true. First, the biosynthetic
pathways of both the n-3 and the n-6 families share an enzyme called δ-6-desaturase. This
enzyme, which is essential for the conversion of ALA to DHA and EPA, has a preference for
ALA but the presence of high levels of plasma LA (caused by high n-6 PUFA intakes) can
shift its actions towards the n-6 pathway (Budowski P., 1988). The result is the inhibition of
the pathway that converts ALA to EPA and DHA (Gerster H., 1998) with possible low
plasma levels of these fatty acids. Secondly, it has long been suspected that the conversion of
ALA to DHA is inefficient. Studies comparing supplementation using linseed oil rich of
ALA vs. fish oil rich in EPA + DHA have demonstrated that whereas linseed oil produces a
moderate increase in platelet EPA, fish oil produces a large rise in both platelet EPA and
DHA content (Sanders T.A. et al., 1983). The absence of EPA and DHA in the diet is
unlikely to lead to serious clinical deficiency but it is possible that people with enhanced
requirements could be disadvantaged by this type of diet.
A double enzymatic systems exist which made up continually the long chain omega-3 and
omega-6 polyunsaturated fatty acids which in turn will be incorporated in the new
phospholipids. Two types of enzymes will participate on the metabolism of these fatty acids,
elongase and desaturase. The first one allow to lengthen the essential fatty acid increasing
the number of carbon atoms. The desaturases allow to the body, depending on its needs, to
increase the number of double bonds.
The enzymatic potential of a person is depending on many circumstances: age, some
pathologies, erroneous diet; in this case the synthesis of long chain polyunsaturated fatty
acids became insufficient to satisfy the needs of membrane building. In this case a
supplementation of already prepared fatty acid must be performed to balance the FA
composition. The mentioned metabolic pathways are depicted in figure 2.
13

18:3 ω-3 ALA 18:2 ω-6 LA
Δ6-desaturase Δ6-desaturase
18:4 ω-3 18:3 ω-6 GLA
elongase elongase
20:4 ω-3 20:3 ω-6 DHGLA
Δ5-desaturase Δ5-desaturase
20:5 ω-3 EPA 20:4 ω-6 AA
elongase elongase
22:5 ω-3 DPA 22:4 ω-6
elongase elongase
24:5 ω-3 24:4 ω-6
Δ6-desaturase Δ6-desaturase
24:6 ω-3 24:5 ω-6
β-oxidation (-2C) β-oxidatione (-2C)
22:6 ω-3 DHA 22:5 ω-6
Figure 2: The actual pathways for n-6 and n-3 PUFA biosynthesis.
2. DIETARY SOURCES
To a considerable extent, the FA content of fish oils is determined by the food that is
available for the fish diet; this is influenced strongly by the geographic area in which the fish
live, the season of the year, and the fluctuations that occur from year to year. In consequence,
published data on the classes and levels of FAs in various species of fish vary widely, and
they still may not represent the true situation (Stansby M.F., 1986). Puustinen et al. (1985)
analyzed the total lipid content and the FA composition of 12 commonly eaten northern
European fish. They found that the range of total lipids amounts in different fish species was
greater than the variation of the percentage amount of EPA and DHA, which led them to
conclude that possible beneficial effects would be achieved by the intake of the fattier fish.
The United States Department of Agriculture published fat and FA values for 30 fish of
different species or location (Simopoulos A., 1991); 14 of these are listed in Table 1. The
total amount of fat in these fish ranges from 0.7 g/100 g in cod and haddock to 13.8 and 13.9
g/100 g in Greenland halibut and Pacific herring, respectively. The relative amounts of EPA
and DHA contained in fish oils vary considerably between species (Childs M.T. et al., 1990).
Moreover, some freshwater fish oils contain relatively low levels of EPA, but substantial
amounts of AA, compared with those present in marine fish oils; as a consequence their
14

consumption will have very different effects on the cell membrane FA profiles and
prostanoid production (Innis S.M. et al., 1995).
Table 1. The total lipid and omega-3 fatty content of 14 edible fish that are widely available
in the United States. From Simopouls A., 1991.
In addition to fish and fish oils, soybean and canola (low erucic acid rapeseed) oils may
provide a significant source of dietary n-3 FA in the form ALA (Hunter G.E., 1990), the
major FA in chloroplast lipids. In North American diets, the principal food sources of ALA
are salad, cooking oils and salad dressing products. The per capita intake in the United States
has been estimated to be 16–20 g/day for men and 12 g/day for women (Kim W.W. et al.,
1984).
The changes in the FA content of the typical human diet that have occurred over time since
the Paleolithic period were discussed by Simopoulos A. (1991). She pointed out that the
early diet contained small, but approximately equal, amounts of n-6 and n-3 FAs, whereas
the modern Western diet contains a relative excess of n-6 FA. It is this imbalance of n-6 FA
to n-3 FA that some investigators have associated with increased risks for both
cardiovascular disease and some cancers, including carcinoma of the breast. While this
aspect of nutritional cancer epidemiology will be discussed in detail below, one instructive
example may be mentioned at this point: the increased breast cancer risk in Japanese women,
which has taken place over the past four decades and which correlates with imbalance of
dietary n-3:n-6 FA ratio with a significant decrease of the ratio itself (Rose D.P., 1997a).
3.CELL MEMBRANE COMPOSITION AND FUNCTION
The cell membrane (Fig.3) is a semipermeable lipid bilayer found in all cells. It contains a
wide variety of biological molecules, primarily proteins and lipids, which are involved in a
vast array of cellular processes, and also serves as the attachment point for both the
intracellular cytoskeleton and, if present, the cell wall.
15

Figure 3: The cell membrane.
The cell membrane surrounds the cytoplasm of a cell and, in animal, physically separates the
intracellular components from the extracellular environment, thereby serving a function
similar to that of the skin. The cell membrane also plays a role in anchoring the cytoskeleton
to provide shape to the cell, and in attaching to the extracellular matrix to help group cells
together in the formation of tissues.
The barrier is selectively permeable and able to regulate the entry and the exit of material
from the cell, thus facilitating the transport of molecules needed for cell survival. The
movement of substances across the membrane can be either passive, occurring without the
input of cellular energy, or active, requiring the cell to spend energy in moving it. The
membrane also maintains the cell potential.
Specific proteins embedded in the cell membrane can act as molecular signals that allow
cells to communicate with each other. These receptors are found ubiquitously and their
function is to receive signals from both the environment and other cells. These signals are
transduced into a form that the cell can use to carry out a response. Other proteins on the
surface of the cell membrane serve as "markers" that identify a cell to other cells. The
interaction of these markers with their respective receptors forms the basis of cell-cell
interaction in the immune system.
The cell membrane consists of a thin layer of amphipathic lipids which spontaneously
arrange so that the hydrophobic "tail" regions are shielded from the surrounding polar fluid,
causing the more hydrophilic "head" regions to associate with the cytosolic and extracellular
faces of the resulting bilayer.
The arrangement of hydrophilic heads and hydrophobic tails in the lipid bilayer prevents
hydrophilic solutes from passively diffusing across the band of hydrophobic tail groups,
allowing the cell to control the movement of these substances via transmembrane protein
complexes such as pores and gates.
According to the fluid mosaic model of Singer and Nicolson, the biological membranes can
be considered as a two-dimensional liquid where all lipid and protein molecules diffuse more
or less freely. This picture may be valid in the space scale of 10 nm. However, the plasma
membranes contain different structures or domains that can be classified as (a) proteinprotein
complexes; (b) lipid rafts, (c) pickets and fences formed by the actin-based
cytoskeleton; and (d) large stable structures, such as synapses or desmosomes.
16

3.1 Lipids
Figure 4: Examples of lipids embedded in the cell membrane.
Examples of the major membrane phospholipids and glycolipids: phosphatidylcholine
(PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns),
phosphatidylserine (PtdSer) are presented in figure 4.
The cell membrane contains three classes of amphipathic lipids: phospholipids, glycolipids,
and colesterol. The relative composition of each depends upon the type of cell, but in the
majority of cases phospholipids are the most abundant. In RBC studies, 30% of the plasma
membrane was shown to be made up by lipids. The fatty chains in phospholipids and
glycolipids usually contain an even number of carbon atoms, typically between 14 and 24.
The 16- and 18-carbon fatty acids are the most common. Fatty acids may be saturated or
unsaturated, with the configuration of the double bonds nearly always cis. The length and the
degree of unsaturation of fatty acids chains have a profound effect on membranes fluidity as
unsaturated lipids create a kink, preventing the fatty acids from packing together as tightly,
thus decreasing the melting point (increasing the fluidity) of the membrane.
The morphology of phospholipids will be very different depending on the nature of the two
fatty acids included in the molecule. A phospholipids exclusively made of saturated fatty
acids will be very rigid and the assembly of such kind of phospholipids will result in a
membrane very “dense” which will not allow physiological exchanges. For these reasons
generally a phospholipids is constituted by one saturated fatty acid and by one
polyunsaturated fatty acid. In this case the membrane will be fluid and the exchanges will be
facilitated.
The fatty acid present in membrane phospholipids came from the diet; depending on the
chain length and of the degree of unsaturation of the fatty acids in the diet , e.g. a good
balance of n-6/n-3 fatty acids, membrane will result more or less fluid. In the case of
unbalanced ratio a certain number of pathologies can originate.
17

In animal cells cholesterol is normally found dispersed in varying degrees throughout cell
membranes, in the irregular spaces between the hydrophobic tails of the membrane lipids,
where it confers a stiffening and strengthening effect.
The major role of membranes is to maintain the structural integrity of cells and organelles
related also to their barrier function. As mentioned before , membranes are not rigid or
impermeable: they are fluid, and their components move around, are metabolized, and are
subject to metabolic turnover. The turnover of membrane components is especially important
for the cellular response to information from inside and outside the cell: recognition, transfer,
amplification and signal transduction processes occur in or on the membrane surface.
Cells of multicellular organisms constantly receive signals from other cells and from the
environment which they perceive, interpret and to which they respond with appropriate
metabolic or physiological changes. In the recent past it has been discovered that some
membrane-bound phospholipids play a vital role in signal transduction mechanisms of a
large number of receptors.
At present at least four phospholipid classes have been shown to participate in signal
transduction: phosphatidylinositols, phosphatidylcholines, sphingomielin and
glycosylphosphatidyl-inositols. Moreover, phospholipids regulate the fluid environment of
the membrane and also the activities of some enzyme. Particular phospholipids are required
for specific membrane structures, such as curved regions and junctions with adjacent
membranes.
Little variation on the percentage composition or in the molar ratio of the different classes of
phospholipids and glycolipids, modification of their fatty acid composition with balance or
imbalance of n-6/n-3 ratio, and changes in the amount of cholesterol cause variation not only
of the chemical and physical characteristics of the membrane, but also of the activity of
enzymes and/or ionic channels that are intrinsic protein of the cellular membrane.
As an example the phosphatidil inositol (PI) is phosphorylated to phosphatidil inositol-4phosphate
(PIP) and after to phosphatidil inositol-4,5-bis-phosphate (PIP2). The PIP2 is
hydrolyzed by a specific enzyme phospholipase C (PIP2 diesterase) to inositol triphosphate
(IP3) and diacilglycerol (DAG). The first one is responsible for the release of calcium whit
an increase of its intracellular concentration, the second one activates a specific protein
Kinase (protein kinase C, PKC). All these events determine a stimulation of the cellular
proliferation . It is important to consider that the regulation of the PtdIns turnover and the
regulation of the PIP2 effects is modulated from the lipid composition of the PIP2 itself ;
results from our laboratory indicate that an unbalance of the lipid pattern, probably due to an
altered activity of the Δ-6-desaturase - membrane bound enzyme - results in a longer PKC
activation that might causes a big abnormal cellular proliferation.
Moreover, the PtdIns constitute the preferential substrate for the phospholiphase A2 action,
that causes the liberation of arachidonic acid (the precursor of the 2 prostanoid family),with
the effects described below.
Besides the multiple and important functions of cholesterol in the body, in this context we
will discuss its role in the membrane equilibrium. At the level of cellular membranes the
cholesterol has a structural role; it penetrates in the interior of the double layer between
phospholipids molecules stabilizing the membranes and avoiding an excessive fluidity.
It must be pointed out that, when the membrane is rigid due to the presence of saturated fatty
acids, cholesterol is unable to penetrate between the phospholipids; it can return back to the
circulation being oxidized and favouring in this way the appearance of the atheromatic
plaque.
In conclusion membrane fluidity is greatly involved in cholesterol homeostasis.
18

4. FUNCTION OF PUFA IN CELL MEMBRANE PHOSPHOLIPIDS
Besides the structural role already described the long chain fatty acids incorporated in
membrane phospholipids has another important function, completely different.
After the release mediated by a specific phospholipase A2, AA and EPA are oxidized by
specific enzyme giving origin to cellular mediators, namely prostaglandins, leukotrienes,
thromboxane, prostacyclins.
However, several other classes can technically be termed eicosanoids, including the
hepoxilins, resolvins, isofurans, isoprostanes, lipoxins, epi-lipoxins, epoxyeicosatrienoic
acids (EETs) and endocannabinoids. LTs and prostanoids are sometimes termed 'classic
eicosanoids' (Van Dyke T.E. et al., 2003, Serhan C.N. et al., 2004, Anderle P. et al., 2004)
in contrast to the 'novel', 'eicosanoid-like' or 'nonclassic eicosanoids' (Evans A.R. et al., 2000,
O'Brien W.F. et al., 1993, Behrendt H. et al., 2001, Sarau H.M. et al., 1999) As already
reported above, eicosanoids are signaling molecules made by oxygenation of twenty-carbon
fatty acids. They exert complex control over many systems, mainly in inflammation or
immunity, and as messengers in the central nervous system. The networks of controls that
depend upon eicosanoids are among the most complex in the human body. Eicosanoids
derive from either omega-3 or omega-6. The n-6 eicosanoids are generally proinflammatory;
n-3's are much less so. The amounts and balance of fats in a diet will affect
the body's eicosanoid-controlled functions, with effects on cardiovascular disease,
triglycerides concentration, blood pressure, arthritis, inflammation and oxidative/nitrosative
conditions.
4.1 Biosynthesis
Two families of enzymes catalyze fatty acid oxygenation to produce the
eicosanoids:Cyclooxygenase, or COX, generates the prostanoids. Lipoxygenase, in several
forms, e.g. 5-lipoxygenase (5-LO) generates the leukotrienes. Eicosanoids are not stored
within cells, but are synthesized as required.
Eicosanoid biosynthesis begins when cell is activated by mechanical trauma, cytokines,
growth factors or other stimuli. (The stimulus may even be an eicosanoid from a neighboring
cell; the pathways are complex). Phospholipase is released at the cell membrane and travels
to the nuclear membrane. There, it frees 20-carbon fatty acids. This event appears to be the
rate-determining step for eicosanoid formation.
A schematic view of conversion of AA and EPA to prostanoids and leukotrienes through the
cyclo oxigenase and lipo oxigenase pathway are depicted in the following figure:
19

Figure 5: The three groups of eicosanoids and their biosynthetic origins. (PG, prostaglandin;
PGI, prostacyclin; TX, thromboxane; LT, leukotriene; LX, lipoxin; 1, cyclooxigenase; 2,
lipoxygenase pathway). The subscript denotes the total number of double bonds in the
molecules and the series to which the compound belongs.
Adapted by: Murray R.K. Granner D.K., Mayes P.A., Rodwell V.W. (2003). Harper’s
Illustrated Biochemistry. 23: 193.
Thromboxane are synthesized in platelets and upon release cause vasoconstriction and
platelet aggregation. Their synthesis is specifically inhibited by low-dose aspirin.
Prostacyclins (PGI2) are produced by blood vessel walls and are potent inhibitors of platelet
aggregation. Thus, thromboxanes and prostacyclins are antagonistic. PG3 and TX3 formed
from eicosapentaenoic acid inhibits the release of arachidonate from phospholipids and
deformation of PG2 and TX2. PGI3 is a potent antiaggregator of platelet as PGI2, but TXA3
is a wicker aggregator than TXA2, changing the balance and favouring longer clotting times.
Potential therapeutic uses of prostaglandins include prevention of conception induction of
labor at term, termination of pregnancy, prevention or alleviation of gastric ulcers, control of
inflammation and of blood pressure, and relief of asthma and nasal congestion.
Slow-reactive substance of anaphylaxis (SRS-A) is a mixture of leukotrienes C4, D4 and E4.
20

This mixture is a potent constrictor of the bronchial air-way musculature. These leukotrienes
together with leukotriene B4, also cause vascular permeability and attraction and activation
of leukocytes and are important regulators in many diseases involving inflammatory or
immediate hypersensitivity reactions, such as asthma.
Leukotrienes are vasoactive and 5-lypoxigenase as been found in artherial walls.
Evidence supports a role for lipoxins in vasoactive and immunoregulatory function. (Murray
R.K. et al., 2003). Resolvin was discovered in vivo during the resolution phase of
inflammation in exudates from inflamed tissues in a mouse model. The main task of resolvin
E1 appears to be to serve as a counterregulator to proinflammatory mediators and turn down
acute inflammatory processes before too much damage is done to normal tissue (Arita M. et
al., 2005).
Isofurans are nonclassic eicosanoids formed nonenzymatically by free radical mediated
peroxidation of arachidonic acid. The isofurans are similar to the isoprostanes and are
formed under similar conditions, but contain a substituted tetrahydrofuran ring. The
concentration of oxygen affects this process; at elevated oxygen concentrations, the
formation of isofurans is favored whereas the formation of isoprostanes is disfavored
(Roberts L.J. et al., 2004). The isoprostanes are prostaglandin-like compounds formed in
vivo from the free radical-catalyzed peroxidation of essential fatty acids (primarily
arachidonic acid) without the direct action of cyclooxygenase (COX) enzyme. These
nonclassical eicosanoids possess potent biological activity as inflammatory mediators that
augment the perception of pain. These compounds are accurate markers of lipid peroxidation
in both animal and human models of oxidative stress (Evans A,R. et al., 2000). The
endogenous cannabinoid system is an ubiquitous lipid signalling system that appeared early
in evolution and which has important regulatory functions throughout the body in all
vertebrates. The main endocannabinoids (endogenous cannabis-like substances) are small
molecules derived from arachidonic acid, anandamide (arachidonoylethanolamide) and 2arachidonoylglycerol.
They bind to a family of G-protein-coupled receptors, of which the
cannabinoid CB1 receptor is densely distributed in areas of the brain related to motor
control, cognition, emotional responses, motivated behaviour and homeostasis. Outside the
brain, the endocannabinoid system is one of the crucial modulators of the autonomic nervous
system, the immune system and microcirculation (Rodriguez de Fonseca F. et al., 2005).
5. BIOMARKERS OF OMEGA-3 FATTY ACID NUTRITIONAL STATUS
The association of dietary fats with diseases risk or outcome could be determined from
epidemiological studies and/or from food frequency questionnaires; however the FA
composition of the plasma lipids and the membranes of platelets and erythrocytes provides a
better assessment of the the dietary intake of the long-chain n-3 FAs (Bjerve K.S. et al.,
1993; Andersen L.F. et al., 1996; Innis S.M. et al., 1988, Brown A.J. et al., 1991). Other
investigators have utilized adipose tissue obtained by percutaneous biopsy, for which the
turnover time for FAs has been estimated to be 1–3 years; however it must be pointed out
that this procedure is not only invasive but also time consuming (Field C.J. et al., 1984).
Marckmann P. et al. (1995) found adipose tissue DHA and docosapentaenoic acid (C22:5,
DPA) levels to be much higher than that of EPA, although the food record estimates of
dietary EPA and DHA intakes were both considerably higher than that of DPA. The apparent
discrepancy may have resulted from the elongation of EPA to the less metabolically active
DPA. In this study, the DHA content of adipose tissue provided a better marker of fish and
marine n-3 FA intake than did EPA, a result that was also obtained by Tjønneland A. et al.
(1993).
Two other studies provided a comparison of data from food-frequency questionnaires, diet
records, and subcutaneous adipose tissue FA content in American postmenopausal women
21

and men, respectively (London S.J. et al., 1991, Hunter D. et al., 1992). Both showed
significant correlations between the percentage of n-3 FAs in adipose tissue, and the
percentage of n-3 FAs in the diet, as determined by a food-frequency questionnaire. In the
study of American males, dietary intakes were also assessed by 7-day diet records, which are
generally regarded as more accurate. However, this method of evaluation gave a similar level
of correlation to that obtained with the semi-quantitative food-frequency questionnaire,
suggesting that the less complex method provides an equally reliable assessment. In neither
study were separated the data collected for EPA and DHA and so, there were no results upon
which to judge the relative importance of the two n-3 FAs as markers of n-3 FA intake. One
point deserves a critical consideration: the evaluation of n-3 FA intake used in this and other
epidemiological studies do not distinguish between the types of fish consumed, which, given
the wide variations in n-3 content of fish, may reduce the sensitivity of the obtained results.
Although the analysis of adipose tissues is necessary to evaluate dietary FA intake over
extended periods of time and is unaffected by temporary variations in diet, their acquisition
is not amenable to the large-scale sampling required for many epidemiological studies, and
the presence of large amounts of nonessential FAs can obscure the relatively low levels of n-
3 FAs. With this in mind, it is encouraging that a carefully executed comparative study by
Godley P.A. et al. (1996) found that the EPA and DHA contents of adipose tissue aspirates
and erythrocyte membranes had similar correlations with fish consumption, as estimated by a
food-frequency questionnaire.
PUFA status in Human can be assessed on several blood lipid classes. Erythrocyte
phospholipids fatty acid status presents several advantages: (1) it is a reflexion spread over
time of habitual dietary fat intake in relation to the biological half life of erythrocytes
(Romon M. et al., 1995); (2) the level of EPA can be used as a specific marker for the intake
of fish and fish oil ( Brown A.J. et al., 1990); (3) phospholipids are a model of fatty acid
incorporation into a cellular membrane; (4) it gives an image of hepatic and extrahepatic
fatty acid metabolism; (5) erythrocyte phospholipids are in equilibrium with structural
phospholipids of tissues; (6) it has been correlated reasonably well with the food- frequency
questionnaire (ffq) for the dietary intakes of polyunsaturated fatty acids (Parra M.S. et al.,
2002). However the ffp is time consuming and tedious and can lack accuracy in comparison
with the precision of erythrocyte fatty acid measurements.
Our studies on the AA/EPA ratio determined on whole-blood as a biomarker of fatty acid
intake or supplementation, demonstrate, as also described below, its good correlation with
erythrocyte phospholipids composition and its reliability.
6.OMEGA-6/OMEGA-3 PUFA BALANCE AND CHRONIC DISEASES
Unbalance PUFA status is observed in numerous conditions and especially in diseases
chronic and/or degenerative accompanied by deficiency of the antioxidant system. Thus, the
early diagnosis of such impairment is of crucial importance, since it is recognized that the
dietary PUFA balance could help in the prevention and the control of such diseases.
6.1. Smoking, hormonal contraception, pregnancy and premenstrual syndrome
Free radicals generated in cigarette smoke are known to deplete antioxidants and may result
in increased lipid peroxidation which leads to decreased concentrations of long chain PUFA.
This may be due to an inhibitory effect of tobacco, or its metabolites on erythrocyte fatty
acid metabolism as their PUFA content was decreased. The negative effect of cigarette
smoking on PUFA stores may be one further mechanism by which cigarette smoking
promotes vascular disease (Pawlosky R. et al., 1999).
A potential adverse effect of hormonal contraception on erythrocyte fatty acid status has
been observed by increasing some saturated fatty acids such as C16:0 and decreasing other
22

unsaturated fatty acids such as EPA ( Berry C. et al., 2001). PUFA play an important role in
the brain and during vascular development. Also the normal course of pregnancy, can be
affected as well as fetal growth retardation ( Matorras R. et al., 1999) and preeclampsia
(Wang Y. et al., 1991, Sattar N. et al., 1998). During pregnancy, there is a faster turnover of
PUFA from fast storage that may modify the profile of erythrocyte cell membrane fatty acids
(Parra M.S. et al., 2002). A significant decrease in the proportion of n-3 PUFA from the first
to the third trimester has been noted (Matorras R. et al., 2001). Thus, it is suggested that n-3
PUFA intake during pregnancy should be increased in the last trimester.
Menstrual pain or dysmenorrhea is the most common gynecological complaint among
female adolescents and young women. The majority of dysmenorrhea has a physiologic
cause, with occasional psychological components. The high intake of n-6 fatty acids in the
western diet results in a predominance of these fatty acids in the cell membrane
phospholipids. After the onset of progesterone withdrawal before menstruation, these n-6
fatty acids, particularly arachidonic acid, are released, and a cascade of prostaglandins and
leukotrienes is initiated in the uterus (Harel Z. et al., 1996). The inflammatory response,
which is mediated by these eicosanoids produces both cramps and systemic symptoms such
as nausea, vomiting, bloating and headaches. The prostaglandins E2 and F2α, cycloxygenase
metabolites of arachidonic acid, cause especially potent vasoconstriction and myometrial
contractions, which lead to ischemia, pain and systemic symptoms of dysmenorrhoea.
Several double blind, placebo controlled trial studies have demonstrated that dietary
supplementation with n-3 fatty acids has a beneficial effect on symptoms of dysmenorrhea
(Deutch B.,1995, Drevon C.A., 1992).
EPA and DHA compete with arachidonic acid for the production of prostaglandins and
leukotrienes through the incorporation into cell membrane phospholipids and through
competition at the prostaglandin synthesis level. PUFA n-3 can also inhibit arachidonic acid
formation at the level of the Δ6 desaturase enzyme (Deutch B., 1995). In the uterus, this
competitive interaction between n-3 and n-6 fatty acids may result in the production of less
potent prostaglandins and leukotrienes and may lead to a reduction in the systemic symptoms
of dysmenorrhea (Harel Z. et al., 1996).
6.2. Coronary heart disease
Altered fatty acid metabolism has been reported in patients with angiographically
documented coronary disease (Sigel E.N. et al., 1994). Carotid intima-media thickness has
been associated significantly and positively with saturated fatty acids and inversely with n-3
PUFA composition in both plasma phospholipids and cholesterol esters (Ma J. et al., 1997).
The n-3 fatty acids of fish and fish oil have great potential for the prevention and treatment
of patients with coronary artery disease. One of the most important effects of n-3 EPA and
DHA is their capacity to inhibit ventricular fibrillation and consequent cardiac arrest; for
these reasons their use in primary and secondary prevention for CVD is now suggested.
(Simopoulos A.P., 1999, Albert C.M., 2002). EPA has antiarrhythmic effects and several
antithrombotic actions, particularly inhibiting the synthesis of thromboxane A2. Fish oil
retards the growth of the atherosclerotic plaque by reducing the amount of pro-inflammatory
interleukine 1 (IL1) and tumour necrosis factor (TNF) and by inhibiting both cellular growth
factors and migration of monocytes. Moreover the n-3 fatty acids promote the synthesis of
nitric acid oxide in the endothelium. Finally experiments in humans demonstrate a
hypolipemic effect of fish oil, especially lowering plasma triglyceride (Weber T. et al., 2000
Scientists at the university of Tromsø have previously shown that a mixture of minor components
from the olive fruit and the protective nutrient omega-3 from fish oil work synergetic to reduce
inflammations leading to heart attack and stroke in the process of atherosclerosis (Østerud and
Elvevoll, 2007).
23

6.3. Hypertension
Different mechanisms appear to be involved in hypertension. Changes were reported in
eicosanoid metabolism, viscosity, loss of sodium, increase in potassium in cells and decrease
in intracellular calcium (Williams R. et al., 1990). In clinical studies, alfa-linolenic acid
contributed to lowering blood pressure (Berry E.M. et al., 1986). In a population- based
intervention trial it has been reported that a relationship may exist between n-3 fatty acid
concentration in plasma phospholipids and blood pressure. There was a lower blood pressure
at the baseline in subjects who habitually consume large quantities of fish, suggesting that
supplementation with fish oils would be important from the primary prevention standpoint
(Bonaa K.H et al., 1990).
6.4 Diabetes
Type 2 diabetes is a multigenic, multifactorial disorder. There is an interaction with genetic
predisposition, diet and exercise in the development of this disease. Type 2 diabetes is
characterised by hyperglycemia, insulin resistance and vascular complications. In animal
studies, the increased content of polyunsaturated fatty acids in the cell membrane enhances
the insulin receptor number and binding while saturated fat decreases binding and transport
(Field C.J. et al., 1990). Limited clinical studies are suggestive of a similar effect in human
(Harel Z. et al., 1996).
In diabetic patients the concentrations in erythrocytes of linoleic acid metabolites [alfalinolenic
acid garuma linolenic acid (GLA 18:3n-6) and arachidonic acid] are consistently
below normal (Horrobin D.F., 1993). The reason is that in diabetes the Δ6 and Δ5 desaturase
enzyme activities are greately impaired (Horrobin D.F., 1993). Decreased content of long
chain polyunsaturated fatty acids, in particular arachidonic acid, and the total percentage of
C20–C22 polyunsaturates of the n-6 family is associated with decreased insulin sensitivity as
reported in an old paper by Pelikanova (Pelikanova T. et al., 1989).
In a recent clinical study on erythrocyte membranes, n-6 polyunsaturated fatty acids were
positively correlated to insulin sensitivity while saturated fatty acids appear to have the
opposite effect (Clifton P.M. et al., 1998).
Another study suggests that hyperinsulinemia and insulin resistance are inversely associated
to the amount of 20 and 22 LC PUFA both of the n-6 and n-3 families in muscle cell
membrane phospholipids in patients with coronary heart disease and in normal volunteers
(Borkman M. et al., 1993).
6.5. Atopic eczema and psoriasis
Atopic eczema is an inherited form of dermatitis that almost always develops initially during
the first year of life. It remits and relapses throughout life, often showing considerable
improvement around the time of puberty. Patients with atopic eczema have an abnormal
immune function, with high concentrations of immunoglobulin E (IgE) and an elevated ratio
of T-helper to T-suppressor lymphocytes (Bordoni A. et al., 1987). Dermatitis is consistently
the first sign of PUFA deficiency in both animals and humans. Adult patients with atopic
eczema were compared with normal individuals as to the fatty acid composition of plasma
phospholipids with the following results: (Manku M.S. et al., 1984) Linoleic acid
concentration was slightly above normal while its metabolites GLA, dihomo-gamma-
linolenic acid (DGLA 20:3n-6) and arachidonic acid were below normal. Reduced LA
metabolites has also been found in children with atopic eczema, in cord blood of babies at
risk for atopic eczema, (Hibbeln J.R. et al., 1995) in triglycerides of adipose tissue and in the
breast milk of people with atopic eczema and at last in the red blood cells of patients with
eczema (Oliewiecki S. et al., 1990). This suggests that either Δ6 desaturase activity is
somewhat reduced in atopic eczema, or that the consumption of the metabolites is excessive
and could not be compensated due to the rate- limiting enzyme activity of the desaturase. By-
24

passing the Δ6 desaturase step by giving evening primrose oil rich in GLA led to a partial
normalisation of fatty acid phospholipid composition and to an increase in the formation of
prostaglandin E1, thus producing clinical improvement (Bordoni A. et al., 1987). Also in
psoriasis, arachidonic acid metabolism is altered. Proinflammatory leukotrienes (leukotriene
B4 LTB4) are markedly produced in the psoriatic lesions. The addition of fish oil to the
standard treatment produces further improvement with the decrease in LTB4 (Oliewiecki S.
et al., 1990). This approach provides an alternative or adjunct protocol for the management
of psoriasis and inflammatory skin disorders with negligible side effects (Ziboh V.A., 1991).
6.6 Rheumatoid arthritis (RA)
Investigations have examined the effects of dietary fatty acid supplementation in a variety of
autoimmune diseases. Effects of both n-6 and n-3 fatty acids on rheumatoid arthritis has been
reported (Kremer J.M., 1996, Simopoulos A.P., 2002). In a well designed study,
investigators treated patients with RA with 1.4 g of GLA daily over a period of 24 weeks.
They observed a clinically important reduction in both the tender and swollen joint counts of
36 and 38% of the patients respectively, while the placebo group showed no improvement in
these parameters (Leventhal L.J., 1993). It has previously been demonstrated that GLA can
inhibit interleukin- 2 (IL-2) production and may reduce the activation of T lymphocytes
(Santoli D. et al., 1990).
The effects of a dietary fish oil supplement on active rheumatoid arthritis has also been
shown in a double-blind study. Clinical improvements in tender joint scores and morning
stiffness have been reported with the fish oil (Guesens P. et al., 1994). In addition, fish oil
supplements are associated with a decreased production of IL-1 and LTB4 which should aid
in the amelioration of inflammation.
6.7 Noreulogical diseases – alzheimer dementia, depression, attention deficit
hyperactivity disorder (ADHD) and schizophrenia
Docosahexaenoic acid (DHA) is the major acid of neurological and retinal membranes. It
makes up more than 30% of the structural lipids of the neuron (Wainwright P., 1992). In a
retrospective study including 1188 elderly American subjects, the authors suggested that low
levels of circulating DHA may be a significant risk in the development of Alzheimer
dementia. The inability to maintain a high level of DHA may be due to a reduced capacity to
synthesise DHA late in life as the result of a reduction in Δ6 desaturase activity (Kyle D.J. et
al., 1999). Abnormalities in fatty acid composition may play a role also in psychiatric
disorders, including depression. Alterations in phospholipids which are structural
components of all cell membranes in the brain may induce changes in membrane fluidity
and, consequently, in various neurotransmitter systems, which are thought to be related to the
pathophysiology of major depression (Hibbeln J.R. et al., 1995). Depletion of n-3 fatty acid
levels in red blood cell membranes of depressed patients has been reported (Peet M. et al.,
1998). A significant positive relationship has also been noted between the severity of the
illness and the ratio of arachidonic acid to eicosapentaenoic acid in serum phospholipids and
in erythrocyte membranes (Adams P.B. et al., 1995).
In children the ADHD syndrome has many etiologies and in many cases is likely to be
inherited.
It includes deficits in sustained attention, in impulse control, and in the regulation of the
activity level to situational demands. These children are usually described as hyperactive
from their early pre-school years. Biochemical research indicates an aberrant metabolism of
dopaminergic transmitters in the central nervous system (Castellanos F.X., 1997). One
hypothesis of the etiology of ADHD is concerned with the role of prostaglandins in the
dopaminergic synapses. Prostaglandin E1 (PGE1) is considered to have a modulating
25

function in the dopaminergic synapses, influencing the release of transmitters and their
functions. As above reported PGE1 is synthesised from linoleic acid in several steps. The
first step, from LA to gamma-linoleic acid (GLA), is catalysed by the enzyme Δ6
desaturase. According to this hypothesis, ADHD is caused or worsened by a deficiency of
PGE1 which is again caused by the lack of the enzyme. Abnormalities in PUFA metabolism
in red blood cell membranes has been also reported in children with ADHA (Stevens L.J. et
al., 1995).
Several hypotheses have been advanced stating that genetic disturbances in phospholipids
and prostaglandin metabolism may contribute to the etiology and severity of schizophrenia
(Fenton W.S. et al., 2000). The observation that abnormalities in n-3 PUFA may play a
critical role has been supported by data of three types of essential fatty acid aberrations
among schizophrenic patients (Assies J. et al., 2001, Peet M. et al., 2001). First, several
authors have reported lower concentrations in erythrocyte essential fatty acids among
schizophrenic patients as compared with control subjects (Assies J. et al., 2001, Peet M. et
al., 1995). A second set of findings has correlated lower erythrocyte essential fatty acid
concentrations with greater severity of negative symptoms
(Yao J.K. et al., 1994, Gen A.I. et al., 1994). Third, findings of bimodal distributions of
arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid concentrations among
schizophrenic patients have raised the possibility that distinct subgroups of schizophrenia
could be identified based on abnormalities in fatty acid composition (Peet M. et al., 1994,
Norrish A. et al., 1999). For further information, see chapter 8.3 (page 33).
7. OLIVE OIL
Olive oil is still one of the most important fats in the diet for a large proportion of the
Mediterranean population. Its average chemical composition is 50% water, 22% oil, 19.1%
sugar, 5.8% cellulose 1.6% protein and 1.5% ash. 96-98% of the oil is located in the pericarp
of the fruit and includes lipids (triglycerides, fatty acids, phospholipids and non-glyceride
components) and water-soluble molecules present in the olive itself. Oleic acid is clearly the
main fatty acid. It accounts for 56-82% of the total: the second most abundant acid is
palmitic (from 8-18%) followed by linoleic (4-18%) and stearic (2-4%). Very little polyunsaturated
fatty acids are present. The fatty acids are distributed in olive oil triglycerides
according to the 1.3 random, 2 random rule observed for the majority of vegetable oils.
Normally the 2 position is occupated by unsaturated fatty acids while in the 1,3 position
saturated fatty acids with different chain length are found.
As in other vegetable oils, olive oil contains several substances referred to as “minor
components”, being essential components of the Itogha-products Oil4Life Cardio and
Oil4Life balance Some of them, however are specific to olive oil, or are present in higher
amounts then other vegetable oils. Minor components include:
� hydrocarbons. The polyunsaturated triterpenic hydrocarbon squalene, is the main
component of this fraction. Worthy of note is the content of squalene in virgin olive oil
(1500-7000 ppm) as compared to other vegetable oils (50-150 ppm);
� fatty acid esters. Esters of n-aliphatic alcohols, sterols and triterpenic alcohols are present
in the nonglyceride fraction. It is worth noting that in this fraction linoleic acid is abundant
(about 25% of the total fatty acids).
� monohydroxy and dihydroxy triterpenes. Several other triterpenic alcohols are present
in olive oil, some of them as esterified compounds. The more important, also because of
their potential biological significance, are cycloartenol, α- and β-amyrin, 24methylencycloartenol
and erythrodiol;
26

� sterols: β-sitosterol (24-ethyl-�5-cholestene-3β-ol) is the main component of this fraction.
Other sterols, stigmasterol and campesterol, are also present;
� hydroxy triterpenic acids: oleanolic, maslinic, ursolic and its deoxy and 2α-hydroxy
derivatives are well represented; due to their chemical structures, these compounds should
be good candidates for biological and nutritional investigations; up to now little research
has been done on this interesting subject;
� olives contain a large amount of water, the so-called “vegetation water”: which is
squeezed out together with the oil and is usually removed by centrifugation. Vegetation
water contains sugars, nitrogen derivatives, organic acids, pectin, salts, polyhydroxy
compounds and phenol derivatives. These compounds are mainly present as
glycoconjugates; one of them, oleoeuropeine, is typical of olive oil and is the precursor of
bioactive phenols (see below). Some of these molecules, which are of amphiphilic nature,
are distributed between the organic and aqueous phases as the oil is processed. Those
retained in the oil phase are important because they favourably protect the stability of
virgin oil against oxidation. From the vegetation water, the following components were
extracted and identified: β(4-hydroxyphenyl) ethanol, β(3.4 dihydroxy-phenyl)ethanol and
o-hydroxyphenol.
7.1 Digestion and absorption of olive oil triglycerides
Fat digestibility depends upon the chain length and the structure and distribution of fatty
acids in the triglyceride molecule. Triglycerides with lower melting points are digested and
absorbed more rapidly; the rate of hydrolysis is hindered by the presence of saturated fatty
acids and fostered by the unsaturated ones. The presence of oleic acid in the 2 position of
many 2-monoglycerides results in a better stabilization of emulsion which can penetrate
more easily into the intestinal mucosa (Viola P. et al., 1975). As a consequence, olive oil is
better hydrolyzed than some other dietary fats. Fatty acids and the 2-monoglycerides are
absorbed from the intestinal mucosa cells by free diffusion, due to the linkage of fatty acids
to a fatty acid binding protein, which allows their intracellular transport to the endoplasmic
reticulum where they are activated and reesterified by a specific synthase. The activity of this
enzyme is induced by oleic acid. These results together warrant the assumption of very high
digestibility of olive oil both in laboratory animals and in humans (Berra B. et al., 1996).
7.2 Use of Olive Oil for Secondary Prevention of Atherosclerosis
The amount and type of dietary fats play a crucial and well-documented role on plasma lipid
concentration (Ahrens E.H. et al., 1957, Keys A. et al., 1957). In particular, diets with highly
polyunsaturated fats have been suggested for the prevention and treatment of atherosclerosis.
WHO recommended considering usual nutritional habits, which should not be changed
abruptly (Organization Mondiale de la Santè, 1982). This aspect is particularly important in
Mediterranean countries, where the population was induced to move from a diet based on
olive oil to a new one in which corn oil represented the main fat. Polyunsaturated fatty acids
(PUFA) abundant in corn oil as well as other vegetable fats were found, on the basis of
epidemiological studies, to decrease plasma cholesterol and low density lipoproteins
(Vergroesen A.J., 1975). The mechanism whereby PUFA administration results in lowering
plasma lipid levels is still questionable, despite the many experiments made on this topic
(Connor W.E. et al., 1982). Moreover, nowadays linoleic acid seems to be atherogenic if
assumed in large amounts (Tobarek M. et al., 2002); diets rich in polyunsaturated fats may
entail other harmful effects, such as formation of cholesterol gallstones (Grundy S. M.,
1975), increased vitamin E requirements and promotion of obesity.
For these discrepancies and according to the WHO recommendations, we evaluated the
variations induced on plasma lipid levels by changing the dietary fat composition from corn
27

oil to olive oil in arteriopathic patients (Zoppi S. et al., 1985). In particular, our aim was to
investigate the use of olive oil in a standard hypolipidemic diet suitable for the secondary
prevention of atherosclerosis. Our study indicates that the substitution of corn oil with olive
oil does not cause hazards as far as haemostatic functions, lipids and lipoprotein cholesterol
are concerned, except for a mild elevation of total cholesterol.
In the patients on the olive oil diet we observed a consistent decrease in LDL sterols and an
increase in HDL cholesterol levels over the six-month trial. Our results were confirmed very
recently by the new guideline issued by the Harvard Medical School (Willet W.C., 2001)
where the consumption of high amounts of monounsaturated fatty acids was recommended.
It can be concluded that it is not worthwhile to change compulsorily olive oil to corn oil or
other vegetable oils rich in PUFA. This conclusion applies not only to patients with vascular
diseases but also to healthy subjects within the population at large (see also Elvevoll and
Østerud, 2007, and Oil4Life Cardio).
7.3 Biological and nutritional value of olive oil
Studies on the biological and nutritional value of olive oil have been focused mainly on the
triglyceride fraction. In this respect, oleic acid has been shown to favour bile secretion
(Argon M.C. et al., 1975) and has been used as a “pharmacological” agent in
gastroenteropathic patients (Bucko A. et al., 1975). Moreover extensive investigations were
made on the nutritional and health values of olive oil in general, and oleic acid in particular.
However, many of these studies were of too short duration and disputed by other researchers.
7.4 Minor components of olive oil
On the contrary very few investigations have been made on the nutritional functions of the
so-called minor components of olive oil, which can be summarized as follows (Berra B. et
al., 1996): hypocholesterolemic activity of β-sitosterol; healing and anti-inflammatory
activities of triterpenic acids; choleretic activity of caffeic and gallic acid; anti-COMT
(catecholamin O-methyl transferase) and protection against lipid peroxidation of the phenolic
fraction (see also below). Effects on digestive function by cycloarterenol (abundant in olive
oil) have been extensively studied by Zambotti and his group (Berra B. et al., 1996).
Pancreatic lipase is activated by 2-phenylethanol, which is present in many vegetable oils,
and by a mixture of triterpenic acids (oleanolic and maslinic acids) which are peculiar to
olive oil. They increase the Vmax without changing the Km of the enzyme, suggesting that
the role of these substances resides in the stabilization of the substrate emulsion.
We have studied the influence of cycloartenol on cholesterol absorption because of the
similarities between this molecule and �-sitosterol. First we demonstrated that in rats
cycloartenol is absorbed and is stored mainly in the liver (Zambotti V. et al., 1975).
Subsequently, the influence of absorbed cycloartenol on cholesterol metabolism was
investigated (Zambotti V. et al., 1978). The results indicate that the amount of circulating
cholesterol is lower in animals which received cycloartenol than in untreated animals; at the
same time the excretion of bile acids was significantly increased in the treated rats.
Interestingly, the formation of bile salts takes place in liver where we found an accumulation
of the administered cycloartenol. Finally it was shown (Zambotti V. et al., 1978) that 2phenylethanol
in combination with triterpenic acids inhibits cholesterolesterase in a
noncompetitive way. Cholesterol esters are thus hydrolyzed at a slow rate and their
absorption is decreased. These results indicate that olive oil is active in preventing
hypercholesterolemia, justifying on a biochemical basis the epidemiological results of many
authors.
These investigations have shed some light on the physiological role of these minor
components and opened new perspectives from a biological and nutritional point of view.
28

Unfortunately, these results were neglected and disregarded for many years; only recently
the importance of the minor components of virgin olive oil was raised because of its
antioxidant properties (Berra B., 1998, Owen R.W. et al., 2000).
7.5 Protective effects of secoiridoids, oleuropein and phenols in olive oil on protection
against LDL oxidation
Secoiridoids are a class of compounds found only in virgin olive oil. In view of their
physico-chemical characteristics, it is believed that they may be the chief causes of the
antioxidant activity ascribed to the polar minor components of olive oil. One molecule that is
particularly well represented in this family is oleuropein (Berra B. et al., 1989, Cortesi N. et
al., 1995) a bitter-tasting glycoside. Upon enzymatic hydrolysis, oleuropein loses its
saccharide component and becomes soluble in the oil. Further hydrolysis converts it into phydroxyphenyl
ethanol and 3,4-dihydroxy-phenyl ethanol. We have carried out research on
the possible protection provided by olive oil phenols against LDL oxidative processes.
Recent studies have in fact shown that oxidized lipoproteins (particularly LDLs) play an
important if not fundamental role in the pathogenesis of arteriosclerosis. Oxidised LDLs
contain oxysterols to which have been attributed many actions harmful to cell life (Guardiola
F. et al., 1996). This theory is supported by the discovery of the presence of peroxidised
LDL in the atheroma (Carpenter K.L. et al., 1995) and by proof of the anti-arteriosclerotic
effect of antioxidants (Visioli F. et al., 1995, Wiseman S.A. et al., 1996).
The study in an in vitro model of human LDL indicates that both tyrosol and an extract
containing polar minor components of virgin olive oil inhibit oxysterol formation and
peroxidation of the protein component of the LDLs.
The mixture of polar minor components is more active than the single ingredients on their
own, presumably because of a synergistic effect of the different molecules, all of which have
antioxidant activity. The substances present in virgin olive oil exert a protective effect at a
lower concentration than do other natural antioxidants such as vitamin E (Berra B. et al.,
1995, Caruso D. et al., 1999).
Further studies in humans indicate that these biophenols are absorbed, metabolized and
excreted as glucurone-derivatives (Bonanome A. et al., 2000, Visioli F. et al., 2000). Worthy
of note is the evidence that they are found only in lipoproteins containing cholesterol.
To conclude, it can be asserted that virgin olive oil displays a marked stability to oxidation
due to its good monounsaturates/polyunsaturates ratio and to the presence of various natural
antioxidants. It is readily digested and absorbed and it has proved to work very well in
cholesterolaemia control. For all these reasons, virgin olive oil has distinctive nutritional
properties, as has been proven in epidemiological and biochemical studies, that make it an
ideal fat for the population in general and not just for the peoples of the Mediterranean area
where it has been consumed since time immemorial. The clinical studies also mentioned in
this article support nutritional research in an “extreme model” capable of highlighting the
mechanisms whereby the components in the oil itself act. At the same time they strive to
identify a useful element in the oil that could be incorporated into a nutraceutical or a
functional food. In fact, virgin olive oil should not be considered a drug but a food
exclusively, albeit one that it is distinctive and healthy as used in the Itogha products
Oil4Life Cardio and Oil4Life Balance.
29

8. CLINICAL STUDIES AT THE UNIVERSITY OF MILAN – THE
DEVELOPMENT OF OIL4LIFE BALANCE
8.1 The blood AA/EPA ratio.
As above indicated LCPUFAs, in particular those of n-3 family, play important and positive
roles in maintaining normal physiological conditions and a correct health status. Some
chronic pathology is often associated to a defective metabolism of these fatty acids.
The analysis of the fatty acid composition in phospholipids of RBC membranes allows
obtaining relevant information about diet fatty acids deficiency, eicosanoids biosynthesis and
eventual metabolic anomalies (e.g. Δ6-desaturase deficiency). For this reason, in clinical
practice, the determination of RBC fatty acid composition is becoming a common procedure
to evaluate fat intake; it is also used as biomarker for different pathologies (Sands S.A. et
al., 2005, Harnis W.S. et al., 2004).
The diagnostic value of the fatty acid composition of RBC membrane phospholipids is well
documented, particularly concerning cardiovascular diseases (Pappit S.D. et al., 2005,
Nishizawa H. et al., 2006, Fujioka S. et al., 2006). However this marker requires a long
lasting procedure.
In our laboratory we tested a time saving and reliable method to determine AA/EPA and n-
6/n -3 ratios on whole blood.
In our observational study, when the AA/EPA ratios were correlated with age of healthy
Italian subjects (Fig. 5), a statistically significant decrease was observed in subjects over 40
yrs, resulting from an increase of EPA simultaneously with a decrease of AA. Less
significant differences were observed with the n-6/n -3 ratio even if the total content of n-6
decreases significantly.
35
30
25
20
15
10
5
0
0-20 21-40 41-60 over 60 Age
§§
* *
AA/EPA
omega-/6/omega-3
Previous studies on Canadian population have shown only a higher amount of n-3 in elderly
subjects (Dewailly E. et al., 2002) probably due to an increased consumption of seafood. As
a working hypothesis, we attributed the lower amount of circulating AA to a reduction of �6
desaturase activity; our hypothesis is supported by observation on prostaglandin synthesis
(Hornych A. et al., 2002). However these data need to be confirmed by further larger studies.
The AA/EPA ratio found in healthy Italian subjects without supplementation is high and
comparable to the reported data for the Western population indicating an unbalance n-6/n -3
fatty acid intake with a subsequent changes in membrane composition (Simopoulos A.P.,
2002, Urquiaga I. et al., 2004). In subject that declare to use a supplement the ratio indicates
a physiological balance(Fig. 6).
§§
§
30

20
16
12
8
4
0
Healthy without
omega-3
Healthy with
omega-3
**
Pathological
without omega-3
AA/EPA
omega-6/omega-3
** **
**
##
## ##
##
Pathological with
omega-3
Patients with allergic, skin and neurodegenerative diseases had higher values of AA/EPA
ratio as compared to other pathological subjects, with an unbalance of fatty acid composition
Finally our results show that a significant correlation exists between the values in the whole
blood and the value in RBC membranes demonstrating the reliability of our method (Fig. 7).
AA/EPA in whole blood
49
42
35
28
21
14
7
0
R2 = 0,8675
0 20 40 60 80 100
AA/EPA in RBC membrane phospholipids
The data obtained suggest that particular attention have to be paid in clinical trials
considering AA/EPA and n-6/n-3 ratios as a biomarker due to the differences found
according to age.
8.2 Mood and brain wellness (Fontani et al 2005)
Following a diet without immoderate food intake is commonly considered a valid way to
reduce health risks and improve the quality of life. In addition, diets with or without certain
components are necessary in some pathological situations or are used to maintain particular
physical conditions (De Lopregeril M. et al., 1999). With increasing knowledge over the
years, several diets have been proposed to face specific problems. One current problem is
obesity and the control of body weight, which is influenced not only by the quality and
quantity of food intake but also by hormonal, metabolic and genetic factors that link obesity
to cardiovascular, inflammatory and endocrine diseases.
Therefore, the beneficial effects of a controlled diet also in healthy subjects who have never
followed any particular and specific nutritional regiment can be expected. Such beneficial
31

effects were found also in our experiments, involving healthy subjects performing daily
physical activities. However, it is interesting to note how different dietary approaches can
have different effects on biological parameters and on the well-being of a subject. In our
case, the two diets had very different percentages of carbohydrate and protein: 55%
carbohydrate and 15% protein in the N diet vs 40% carbohydrate and 30% protein in the Z
diet. The fat percentage was equal and accounted for 30% of the total calories. The reduced
carbohydrate and increased protein concentration are believed to have beneficial effects on
type 2 diabetes, cardiovascular diseases and obesity, thus reducing the risk of cardiovascular
diseases (Barvata D.M. et al., 2003, Deprès J.P. et al., 1996, Pereira M.A. et al., 2004);
however this opinion has been criticized by authors who fear the risk of ketone bodies
accumulation in low-carbohydrate diets (Baravata D.M. et al., 2003). Other authors consider
this diet of little use in endurance athletes because of its low carbohydrate content and point
out the relative lack of scientific evidence in favour of the proposed diets (Baravata D.M. et
al., 2003, Cheuvront S.N., 1999, Cheuvront N.S., 2003). Nevertheless, recent papers have
provided some supporting evidence for the use of the hypoglycaemic diet. For example, a
post-prandial reduction of glycaemia has been found in patients with type 2 diabetes
following this type of diet, with no effects on kidney function (Gannon M.C. et al., 2003).
This diet also results in the reduction of hunger, insulin resistance and in lower body weight,
along with an increased adipose tissue metabolism and a better glycemic control (Layman
D.K. et al., 2003, Layman D.K. et al., 2003, Sears B., 2002, Dumesnil J.G. et al., 2001,
Sears B., 1995, Sears B., 2002, Pereira M.A. et al., 2004). Increased blood levels of GH and
IGF-1 have also been described in subjects on low-carbohydrate diets and this effect is
accompanied by a lower triglyceride level (Nuttal F.Q. et al., 2003). On the other hand, it has
been reported that a high dietary glycemic load from refined carbohydrates increases the risk
of coronary heart diseases (Liu S. et al., 2002, Ludwig D.S. 2002, Lamarche B. et al., 1998).
The n-3 fatty acids have similar effects on lipid metabolism of a low glycemic index diet. In
fact they reduce triglycerides, have anti-inflammatory effects, reduce the insulin response to
glucose, and reduce the risk of cardiovascular diseases and cancer (Donaldson M.S., 2004,
Saldeen P. et al., 2004, Jho D.H. et al., 2004, Larsson S.C. et al., 2004, Lopez Mata P. et al.,
2003, Nordoy A., 2002, Leitzmann M.F. et al., 2004, Holness M.J. et al., 2003, Jayasooriya
A.P. et al., 2004). The effects of n-3 fatty acids are improved by aerobic exercise (Thomas
T.R. et al., 2000, Warner J.G. et al., 1989), which is also advisable for subjects whit diet
involving an high protein intake (Lemon P.W.R., 2000).
The results we obtained indicate that the diet can induce variations in some parameters like
cholesterol, tryglicerides and Trg/HDL, but the low glycemic diet has a greater effect and its
efficacy is increased by n-3 supplementation. An important example of the changes we found
is the AA/EPA ratio; is influenced more strongly by the low glycemic diet and is greatly
decreased by the addition of n-3.
The AA/EPA ratio is considered a predictor of health status, as well as an index of wellbeing
(Sears B., 2002, Mathers C.D. et al., 2001). The relationship between AA/EPA and
Trg/HDL observed in the above experiment confirms the importance ascribed to the latter
parameter in the prevention of type 2 diabetes and cardiovascular disease (Boizel R. et al.,
2000). Moreover, there are similar relationships between AA/EPA and the homocysteine and
insulin levels. These relationships are on line with the dietary recommendations for patients
with type 2 diabetes (Neff L.M., 2003, Steyn N.P. et al., 2004) and for the control of body
weight (Johnston C.S. et al., 2002, Markovich T.P. et al., 1998). The latter aspect is
particularly evident in the reported experiment, as there was a reduction of body fat in
subjects on the Z diet. These subjects (like all the others tested) were healthy individuals
without any apparent pathological condition and regularly performing physical activity. The
diet used with n-3 supplementation seems to increase what can be appointed as well being.
32

The condition of well-being could also have been influenced by oxidative effects. Free
radicals and ROS are continuously produced by cells, and the free radicals are neutralized by
an elaborate antioxidant defence system consisting of enzymes and non-enzymatic
antioxidants including vitamin C (ascorbic acid). Our results show that the N and Z diets
reduced the oxidative stress of healthy subjects: there was a strong reduction of MDA, a
plasma marker of oxidative damage to lipids, and considerably higher concentrations of
vitamin C. MDA has been used in several experiments as a measure of oxidative stress, e.g.
that due to exercise. When free radicals are generated, they can attack polyunsaturated fatty
acids in the cell membrane. This leads to lipid peroxidation, which reduces the membrane
fluidity, permeability and excitability, producing hydrocarbon gases and aldehydes such as
MDA. Plasma MDA is a marker of lipid oxidation (Gokhan M. et al., 2003, Urso M.L. et al.,
2003) and its measurement may provide a further indication of oxidative injury in vivo.
Quantification of MDA by HPLC is recommended because of its high analytical sensitivity
and specificity, especially in the study of lipid peroxidation in human subjects (Karatas F. et
al., 2002). Our study shows that the plasma MDA levels in subjects on the N and Z diets
supplemented with n-3 fatty acids were significantly reduced with respect to basal levels
(with a greater decrease in the Z diet subjects), indicating that nutritional status has an
important role in preventing lipid peroxidation by increasing the plasma total antioxidant
capacity. For the above experiment, it must be underlined that olive oil (used as placebo)
contains antioxidant components and thus cannot be considered inactive. Therefore, some of
the antioxidant effects observed in the related groups may have been due to the olive oil as
indicated above (see paragraph 7.5).
Another important result of this experiment is the modulation of mood state, as revealed by
the POMS questionnaire. Supplementation with n-3 seems to be linked to an increase of
vigour and a decrease of negative factors such as anger, anxiety and depression. The ratio
between vigour and the mean of the negative factors (i- POMS) increased after n-3
supplementation and there was a negative relationship between i-POMS and AA/EPA. This
correlation was only present in subjects on the Z diet. These results confirm the influence of
n-3 on the central nervous system (Neuringer M. et al., 1994, Heude B. et al., 2003 ),
probably involving neuronal excitability (Rybach R., 2001). They are also in line with the
suggested action of these compounds on dementia, depression and mood disorders, in which
they may act as mood stabilisers (Silvers K.M. et al., 2002, Heude B. et al., 2003, Rybach
R., 2001, Haag M., 2003, Mishoulon D. et al., 2000, Rogers P.J., 2001 ).
On the whole, the results of this experiment in healthy subjects provide confirmation of the
effects of diet and micronutrients described in pathological conditions. They highlight the
value of dietary rules in improving health and add new evidence concerning the effects of the
diet with low carbohydrate content and with low glycemic index food. The results suggest
that some of these effects are due almost exclusively to diet, e.g the reduction of body fat,
while others, such as the mood state variations, are mainly due to n-3 supplementation.
8.3 ADHD in children and depression in elderly (Germano et al, 2007)
Attention-deficit/hyperactivity disorder (ADHD) is one of most common
neurodevelopmental syndrome of childhood.
Recent reviews estimate ADHD prevalence between 2%-18% (Goldman L.S. et al., 1998;
Elia J. et al., 1999; Brown R .T. et al., 2001). The 2002 National Health Survey of the CDC
indicates that 7% of children between the ages of 5-11 have been diagnosed with ADHD
(Dey A.N. et al., 2002).
The diagnosis of the syndrome is complicated by the frequent occurrence of comorbid
conditions such as learning disability, behavior, and anxiety disorder.
33

In the 1994 Diagnostic and Statistical Manual for Mental Disorders (DSM-IV) three specific
subtypes of ADHD are identified:
1. ADHD, Combined Type: if the following criteria, i.e. (a) “often fails to give close
attention in tasks or play activities” and (b) “often fidgets with hands or feet or
squirms in seat” were together for the past six months.
2. ADHD, Predominately Inattentive Type: if criterion (a) is met but criterion (b) is not
met during the past six months.
3. ADHD, Predominately Hyperactive-Impulsive Type: if criterion (b) is met but
criterion (a) is not met for the past six months.
Therefore the symptoms must have been present for at least six months, and accompanied by
“clinically significant” impairment (American Psychiatric Association, 1994). DSM-IV
requires that symptoms and impairment should be present before the age of 7 years.
Measurement of catecholamines or metabolites in plasma and urine of ADHD patients has
been implemented in order to investigate the possibility to have a laboratory test, but with
mixed success. (Halperin J.M. et al., 1993; Pliszka S.R. et al., 1994). The current consensus
(as stated in DSM-IV) is that no biochemical tests reliably predict ADHD. Therefore, teacher
and parent rating scales or interviews about the children’s behaviour continue to be the most
important diagnostic procedures available.
Three main areas i.e. neuroimaging studies, genetic studies, and other etiologic studies were
investigated to find evidence suggesting a biologic basis for ADHD.
Some findings generally converge on “dysfunction and deregulation of cerebellarstriatal/adrenergic-prefrontal
circuitry” (Castellanos F.X., 2001), and abnormal right
prefrontal anatomy and function have been found in several studies.
A genetic feature of ADHD is strongly suggested because the syndrome clusters in families
(Thapar A. et al., 2005). Thanks to studies at molecular level, two polymorphisms in the
dopamine transporter and receptor genes that seem to influence the risk of ADHD have been
identified (Swanson J. et al., 2001). The genetic links involving these two polymorphisms
have been replicated many times but the general consensus is that many other genes are
probably involved in the transmission of the disorder (Swanson J. et al., 2001).
A suggestive evidence that an environmental toxicant might be an etiologic risk factor for
ADHD is for lead. The literature on this element is important because it creates a paradigm
for understanding how an environmental agent might increase the risk of ADHD: e.g.
cigarette smoking during pregnancy has been reported to increase the risk (Millberger S. et
al., 1996).
Diet too seems to be an etiological factor for ADHD; it is known that children with ADHD
have lower levels of long-chain omega-3 fatty acids in their blood (Stevens L.J. et al., 1995;
Burgess J.R. et al., 2000). This is thought to be due to lack of dietary intake in conjunction
with a more rapid metabolism (Ross B.M. et al., 2003).
DHA is the most prevalent fatty acid in cerebral grey matter phospholipids and constitutes 45
% to 65 % of fatty acids in the nervous tissues (Hamilton L. et al., 2000) and in brain is
involved in the regulation process of cognitive function (Stillwell W. et al., 2003).
Some studies have highlighted a deficiency of LCPUFAs in the membrane phospholipids in
the patients affected with ADHD (Burgess J.R. et al., 2000; Stevens L.J. et al., 1995).
Furthermore, the rate of arachidonic acid to eicosapentaenoic acid in blood and red blood
cell membrane phospholipids seems to be elevated. The ratio AA/EPA is indicative of
increased upstream inflammatory potential. However the data reported in the literature are
34

controversial mainly for the gaps in the used protocols (e.g. the lack of adequate control, the
used dosage, the type of supplement, the age of onset) (Richardson A.J., 2004 a, Richardson
A.J., 2004 b).
Moreover some studies reported no benefit at all from supplementation with DHA alone
(Voight R.G. et al., 2001; Hirayama S. et al., 2004).
The results from our study, led on 30 children with ADHD with a diet supplemented with
2.5 mg /10 Kg/day of EPA+DHA (2:1), allowed us to verify that:
1. the supplementation with EPA and DHA in quiet high doses, related to the patient
weight, might result in attention level improvement and/or a decrease of the
hyperactivity levels/impulsiveness;
2. in patients presenting an high ratio AA/EPA the supplementation with EPA and DHA
determine in blood and in cell membrane phospholipids a new balance of the rates of
these fatty acids;
3. there is a correlation between the dose of n-3 LCPUFAs, the decrease of the
AA/EPA ratio and/or the entity of the clinical improvement (score);
4. the change in the AA/EPA ratio due to the increased intake of n-3 LCPUFAs is
accompanied in RBC membranes by a change in the cholesterol amount and in the
phospholipids profile.
Preliminary results on a depressed elderly patients, demonstrated that n-3 supplementation
which counteract and balance high levels of n-6 LC PUFA is able to decrease depression
symptoms. (Berra B. et al., 2007).
9.CONCLUSIONS
Fatty acids carry out many functions necessary for normal physiological health. Chronic
diseases are frequently associated with abnormalities in polyunsaturated fatty acid
metabolism, and unbalance in n-6/n -3 fatty acid ratio e. g. coronary heart disease,
hypertension, diabetes, cancer, inflammatory and autoimmune disorders, atopic eczema,
depression, schizophrenia, Alzheimer dementia, multiple sclerosis. Numerous studies in
recent years, indicate that n-3 PUFA supplementation results in important health benefits.
Erythrocyte fatty acid measurements can indicate fatty acid deficiencies or imbalances from
the diet, but also metabolic abnormalities (e.g. lack of Δ-6 desaturase) or peroxidation of
membrane phospholipids. Red blood cell fatty acid analysis can give information on cellular
membrane fatty acid status and potential error in eicosanoid biosynthesis. Measuring
erythrocyte fatty acids for monitoring dietary fat intake or as a biomarker of disease risk is
becoming increasingly common in clinical nutrition; it can be used also as a biomarker for
ascertain risks of diseases such as CVD, diabetes, chronic inflammation, depression.
AA/EPA ratio in total blood was found as a reliable and low-time consuming assays for
dietary advice to establish a fatty acids balance in the prevention and control of chronic
diseases.
35

REFERENCES
� Adams P.B., Sinclair A.J., Lawson .S, Sanigorski A. (1995) Arachidonic acid to
eicosapentaenoic acid ratio in blood correlate positively with clinical symptoms of
depression. In: abstract book 2nd International Congress of the International Society
Study Fatty acids and Lipids, Bethesda, 1995.
� Ahrens E.H., Hirsh J., Insull W. (1957) The influence of dietary fats on serum lipid
levels in man. Lancet 1: 943.
� Albert CM. (2002) Blood levels of long chain n-3 acids and the risk of sudden death.
N. Engl. J. Med. 15: 1113-1118.
� American Psychiatric Association. 1994. Diagnostic and Statistical manual of
Mental Disorders, fourth ed., American Psychiatryc Press, Washington, DC.
� Anderle P., Farmer P., Berger A., Roberts M.A. (2004). "Nutrigenomic approach
to understanding the mechanisms by which dietary long-chain fatty acids induce gene
signals and control mechanisms involved in carcinogenesis". Nutrition (Burbank, Los
Angeles County, Calif.) 20 (1): 103–8.
� Andersen L.F., Solvoll, K. and Drevon, C.A. (1996). Very-long-chain n-3 fatty
acids as biomarkers for intake of fish and n-3 fatty acid concentrates. Am. J. Clin.
Nutr. 64: 305–311.
� Argon M.C., Lopez M.A., Murillo A. (1975) Proc. Second Int. Congress on the
Biological Value of Olive Oil, 365-372.
� Arita M., Bianchini F, Aliberti J. (2005). Stereochemical assignment, antiinflammatory
properties, and receptor for the omega-3 lipid mediator resolving E1. J.
Exp.Med. 201: 713–722.
� Assies J., Lieverse R., Vreken P., Wanders R.J., Dingemans P.M., Linszen D.H.
(2001) Significantly reduced docosahexaenoic and docosapentaenoic acid
concentrations in erythrocyte membranes from schizophrenic patients compared with
a carefully matched control group. Biol. Psychiatry 6: 510-522.
� Bates D., Cartlidge N., French J.M., Jackson M.J., Nightingale S., Sinclair H.M.
(1989) A double-blind controlled trial of long chain n-3 polyunsaturated fatty acids in
the treatment of multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 52: 18-22.
� Behrendt H., Kasche A., Ebner von Eschenbach C., Risse U., Huss-Marp J.,
Ring J. (2001). "Secretion of proinflammatory eicosanoid-like substances precedes
allergen release from pollen grains in the initiation of allergic sensitization". Int.
Arch. Allergy Immunol. 124 (1-3): 121–5.
� Beluzzi A., Brignola C., Campieri M., Pera A., Boschi S., Miglioli M. (1996)
Effect of an enteric coated fish oil preparation on relapses in Crohn's disease. N. Eng.
J. Med. 334: 1557-1560.
� Berra B. (1998) Biochemical and nutritional aspects of the minor components of
olive oil. Olivae (english edition) 73:29-30.
� Berra B. Cortesi N., Rapelli S. (1989) Actes du Congres Int. Chevreul pour l’étude
des corps gras. 590-595.
� Berra B. Rapelli S. (1996) Nutritional and biochemical aspects of olive oil. Proc.
World Conference on Emerging Technologies in the Fats and Oil Industries, A. R.
Baldwin (ed.), AOCS Publ., 308-311.
� Berra B., Caruso D., Cortesi N., Fedeli E., Rasetti M.F., Galli G. (1995)
Antioxidant properties of minor polar components of olive oil in the oxidative
processes of cholesterols in human LDL. Riv. Ital. Sostanze Grasse 72, 285-288.
36

� Berra B., Rondanelli M., Montorfano G., Adorni L., Negroni M., Corsetto P.,
Berselli P. Rizzo A.M. Oral presentation: Geriatric depression syndrome is
ameliorated by integration with LC Omega-3 fatty acids, Proceedings 5 th Euro Fed
Lipid Congress. Gothenburg, 16-19 Sept. 2007, p. 81.
� Berry C., Montgomery C., Sattar N., Norrie J., Weaver L.T. (2001) Fatty acid
status of women of reproductive age. Eur. J. Clin. Nut.r 55: 518-524.
� Berry E.M., Hirsch J. (1986) Does dietary linoleic acid influence blood pressure?
Am. J. Clin. Nutr. 44: 336-340.
� Bjerve K.S., Brubakk, A.M., Fougner, K.J., Johnsen, H. and Midthjell, K.
(1993). Omega-3 fatty acids: essential fatty acids with important biological effects,
and serum phospholipid fatty acids as markers of dietary ω3-fatty acid intake. Am. J.
Clin. Nutr. 57 suppl: 801S–806S.
� Boizel R., Benhamou P.Y., Lardy B., Laporte F., Foulon T., Halimi S. (2000)
Ratio of triglycerides to HDL cholesterol is an indicator of LDL particle size in
patients with type 2 diabetes and normal HDL cholesterol levels. Diabetes Care 23:
1679– 85.
� Bonaa K.H., Bjerve K.S., Straum B., Gram I.T., Thelle D. (1990) Effect of
eicosapentaenoic and docosahexaenoic acids on blood pressure in hypertension. A
population-based intervention trial from the tromso study. N. Engl. J. Med. 322: 795-
801.
� Bonanome A., Pagnan A., Caruso D. et al. (2000) Evidence of postprandial
absorbtion of olive oil phenols in humans. Nutr. Metab. Cardiovasc. Dis. 10, 111-
120.
� Bordoni A., Biagi P., Masi P. (1987) Evening primrose oil (Efanol) in the treatment
of children with atopic eczema. Drugs Exp. Clin. Res. 14: 291-297.
� Borkman M., Storlien L.H., Pan D.A., Jenkins A.B., Chisholm D.J., Campbell
L.V. (1993) The relation between insulin sensitivity and the fatty acid composition of
skeletal-muscle phospholipids. N. Engl. J. Med. 328: 238-244.
� Bougnoux P., Germain E., Lavillonière F., Cognalult S., Jourdan M.L., Chajes
V., Lhuillery C. (1999) Polyunsaturated fatty acids and breast cancer. Lipids 34:
S99.
� Bravata D.M., Sanders L., Huang J., Krumholz H.M., Olkin I., Bravata D.A.M.
(2003) Efficacy and safety of low-carbohydrate diets. JAMA 289: 1837– 50.
� Brown A.J, Robert D.C.K. (1990) Erythrocyte EPA as a marker for intake of fish
and fish oil. Eur. J. Clin. Nutr. 44: 487-488.
� Brown A.J., Pang E., Roberts, D.C.K. (1991). Persistent changes in the fatty acid
composition of erythrocyte membranes after moderate intake of n-3 polyunsaturated
fatty acids: study design implications. Am. J. Clin. Nutr. 54: 668–673.
� Brown R.T., Freeman W.S., Perrin J.M., Stein M.T., Amler R.W., Feldman
H.M., Pierce K.,Wolraich M.L. (2001) Prevalence and assessment of attention
deficit/hyperactivity disorder in primary care settings. Pediatrics 107:143.
� Bucko A, Ginter E. (1975) Proc. Second Int. Congress on the Biological Value of
Olive Oil, 353-363.
� Budowski P. (1988). Omega 3 fatty acids in health and disease. World Rev. Nutr.
Diet. 57: 214– 274.
� Burgess J.R., Stevens L., Zhang W., Peck L. (2000) Long-chain polyunsaturated
fatty acids in children with attention-deficit hyperactivity disorder. Am. J. Clin. Nutr.
71(suppl):327S-330S.
37

� Byers T., Gieseker K.(1997) Issues in the design and interpretation of studies of
fatty acids and cancer in humans. Am. J. Clin. Nutr. 66S: 1541-1547.
� Caroll K. (1997) The role of dietary in breast cancer. Curr. Opin. Lipidol. 8: 53-56.
� Carpenter K.L., Taylor S.E., van der Veen C., Williamson B.K., Ballantine J.A.
(1995) Lipids and oxidised lipids in human atherosclerotic lesions at different stages
of development. Biochim.Biophys Acta 1256, 141-150.
� Caruso D., Berra B., Giavarini F, Cortesi N., Fedeli E., Galli G. (1999) Effect of
virgin olive oil phenolic compounds on in vitro oxidation of human low density
lipoproteins. Nutr.Metab.Cardiovasc.Dis. 9, 102-107.
� Castellanos F.X. (1997) Toward a pathophysiology of attention-deficit/hyperactivity
disorder. Clin. Pediatr. 36: 381-393.
� Castellanos F.X. (2001) Neuroimaging studies of ADHD. In:Solanto MV. Et al.,
eds. Stimulant drugs and ADHD: basic and clinical neuroscience. Oxford. Oxford
University Press, 243-258.
� Cheuvront N.S. (2003) The zone diet phenomenon: a closer look at the science
behind the claims. J. Am. Coll. Nutr. 22: 9– 17.
� Cheuvront S.N. (1999) The zone diet and athletic performance. Sports Med. 27:
213– 28.
� Childs M.T., King I.B. and Knopp R.H. (1990). Divergent lipoprotein responses to
fish oils with various ratios of eicosapentaenoic acid and docosahexaenoic acid. Am.
J. Clin. Nutr. 52: 632–639.
� Christophe A., Robberecht E. (2001) Directed modification instead of
normalization of fatty acid patterns in cystic fibrosis: an emerging concept. Curr.
Opin. Clin. Nutr. Metab. Care 4: 111-113.
� Clifton P.M., Nestel P.J. (1998) Relationship between plasma insulin and
erythrocyte fatty composition. Prostaglandins Leukot. Essent. Fat. Acids. 59: 191-
194.
� Connor W.E., Connor S.L. (1982) The dietary treatment of hyperlipidemia. Med.
Clin. North. Am. 66, 485.
� Cortesi N., Azzolini M., Fedeli E. (1995) Determination of minor polar components
in virgin olive oil. Riv. Ital. Sostanze Grasse 72, 333-337.
� Cunnane S., Ho S., Dore-Duffy P., Elis K., Horrobin D. (1989) Essential fatty
acids and lipid profiles in plasma and erythrocytes in patients with multiple sclerosis.
Am. J. Clin. Nutr. 50: 801-806.
� Das U.N. (1999) Reversal of tumor cell drug resistance by essential fatty acids.
Lipids (Suppl) 34: 103.
� De Lorgeril M., Salen P., Martin J.L., Monjaud I., Delaye J., Mamelle N. (1999)
Mediterranean diet, traditional risk factors, and the rate of cardiovascular
complications after myocardial infarction. Circulation 99: 779– 85.
� Desprès J.P., Lamarche B., Mauriège P., Cantin B., Dagenasi G.R., Moorjani S.
et al. (1996) Hyperinsulinemia as an independent risk factor for ischemic heart
disease. N. Engl. J. Med. 334: 952– 7.
� Deutch B. (1995) Menstrual pain in Danish women correlated with low n-3
polyunsaturated fatty acid intake. Eur. J. Clin. Nutr. 49: 568-516.
� Dewailly E., Blanchet C., Gingras S., Lemieux S., Holub B.J. (2002)
Cardiovascular disease risk factors and n-3 fatty acid status in the adult population of
James Bay Cree. Am. J. Clin. Nutr. 76: 85-92.
38

� Dey A.N., Shiller J.S., Tai D.A. (2002) Summary Health Statistics for U.S.
Children: National Health Interview Survey,. National Center for Health Statistics.
Vital and Health Stat. 10: 221.
� Donaldson M.S. (2004) Nutrition and cancer: a review of the evidence for an anticancer
diet. J. Nutr. 3: 19.
� Drevon C.A. (1992) Marine oils and their effects. Nutr. Rev. 50: 38-45.
� Dumesnil J.G., Turgeon J., Tremblay A., Poirier P., Gilbert M., Gagnon L. et al.
(2001) Effect of a low-glycaemic index-low-fat-high protein diet on the atherogenic
metabolic risk profile of abdominally obese men. B.r J. Nutrition. 86: 557– 68.
� Elia J., Ambrosiani P.J., Rappoport J.L. (1999) Treatment of attention-deficithyperactivitydisorder
New Engl. J. Med. 11: 780-788.
� Evans A.R., Junger H., Southall M.D. (2000). "Isoprostanes, novel eicosanoids that
produce nociception and sensitize rat sensory neurons". J. Pharmacol. Exp. Ther. 293
(3): 912–20.
� Evans A.R., Junger H., Southall M.D. (2000). "Isoprostanes, novel eicosanoids that
produce nociception and sensitize rat sensory neurons". J. Pharmacol. Exp. Ther. 293
(3): 912–20.
� Fenton W.S., Hibbelin J.R., Knable M. (2000) Essential fatty acids, lipid
membrane abnormalities, and the diagnosis and treatment of schizophrenia. Biol.
Psychiatry 47: 8-21.
� Field C.J., Clandinin M.T. (1984). Modulation of adipose tissue fat composition by
diet: a review. Nutr. Res. 4: 743–755.
� Field C.J., Ryan E.A., Thomson A.B., Clandinin M.T. (1990) Diet fat composition
alters membrane phospholipids composition, insulin binding, and glucose
metabolism in adipocytes from control and diabetic animals. J. Biol. Chem . 265:
11143-11150.
� Fontani G., Corradeschi A., Felici A., Slfatti F., Burgarini R., Fiaschi AI.,
Cerretani D., Montorfano G., Rizzo AM., Berra B. (2005). Blood profiles, body
fat and mood state in healthy subjects on different diets supplemented with omega-3
polyunsaturated fatty acids.Eur. J. Clin Invest. 35, 499-507.
� Fujioka S., Hamazaki K., Itomura M., Huan M., Nishizawa H., Sawazaki S. et
al. (2006) The effects of eicosapentaenoic acid-fortified food on inflammatory
markers in healthy subjects- A randomized, placebo-controlled, double-blind study.
J. Nutr. Sci. Vitaminol. 52 (4): 261-265.
� Gannon M.C., Nuttall F.Q., Saeed A., Jordan K., Hoover H. (2003) An increase
in dietary protein improves the blood glucose response in persons with type 2
diabetes. Am. J. Clin. Nutr. 78: 734– 41.
� Gen A.I., Glen E.M., Horrobin D.F., Vaddali K.S., Spellman M., Morse-Fisher
N. (1994) A red cell membrane abnormality in a subgroup of schizophrenic patients :
evidence for two diseases. Schizoph. Res. 12: 53-61.
� Germano M ; Meleleo D.; Montorfano G.; Adorni L.; Negroni M.; Berra B.;
Rizzo A M. Plasma, red blood cells phospholipids and clinical evaluation after long
chain omega-3 supplementation in children with attention deficit hyperactivity
disorder (ADHD)( 2007).Nutritional Neuroscience,
� Gerster H. (1998). Can adults adequately convert alpha-linolenic acid (18:3 n-3) to
eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3)? Int. J. Vitam.
Nutr. Res. 68: 159– 173.
� Godley P.A., Campbell M.K., Miller C., Gallagher P., Martinson F.E., Mohler
J.L., Sandler, R.S. (1996). Correlation between biomarkers of omega-3 fatty acid
39

consumption and questionnaire data in African American and Caucasian United
States males with and without prostatic carcinoma. Cancer Epidemiol. Biomarkers
Prev. 5: 115–119.
� Gokhan M., Atukeren P., Alturfan A.A., Gulyasar X.X., Kaya M., Gumustas
M.K. (2003) Lipid peroxidation, erythrocyte superoxide-dismutase activity and trace
metals in young male footballers. Yonsei Med. J. 44: 979– 86.
� Goldman L.S., Genel M., Bezman R.J., Slanetz P.J.(1998) Diagnosis and
treatment of attention-deficit/hyperactivity-disorder in children and adolescents.
JAMA 279: 1100-1107.
� Grønn M., Gørbitz C., Christensen E., Levorsen A., Ose, L., Hagve T.A.,
Christophersen B.O. (1991). Dietary n-6 fatty acids inhibit the incorporation of
dietary n-3 fatty acids in thrombocyte and serum phospholipids in humans: a
controlled dietetic study. Scand. J. Clin. Lab. Invest. 51: 255–263.
� Grundy, S.M. (1975) Effects of polyunsaturated fats on lipid metabolism in patients
with hypertriglyceridemia. J. Clin. Invest. 55:269.
� Guardiola F., Codony R., Addis P. B., Rafecas M., Boatella J. (1996) Biological
effects of oxysterols: current status. Food Chem.Toxicol. 34, 193-211.
� Guesens P., Wouters C., Nijs J., Jiang Y., De Queker J. (1994) Long-term effect
of n-3 fatty acid supplementation in active rheumatoid arthritis. A 12 month double
blind controlled study. Arth. Rhum. 37: 824-829.
� Haag M. (2003) Essential fatty acids and the brain. Can. J. Psychiat. 48: 195– 203.
� Halperin J.M., Newcorn J.H., Schwartz S.T., et al. (1993) Plasma catecholamine
metabolite levels in ADHD boys with and without reading disabilities. J. Clin. Child.
Psycol. 22: 219-225.
� Hamilton L., Greiner R., Salem N. Jr., et al. (2000) N-3 fatty acid deficency
decrease phosphatylserine accumulation selectively in neuronal tissues. Lipids 35:
863-869.
� Harel Z., Biro F.M., Kottenhahn R.K., Rosenthal S. (1996) Supplementation with
omega-3 polyunsaturated fatty acids in the management of dysmenorrhea in
adolescents. Am. J. Obstet. Gynecol. 174: 1335-1338.
� Harris W.S., Von Schacky C. (2004) RBC EPA+DHA: a new risk factor for sudden
cardiac death? Prev. Med. 39: 212-220.
� Heude B., Ducimetiere P., Berr C. (2003) Cognitive decline and fatty acid
composition of erythrocyte mebranes-The EVA study. Am. Clinic. Nutr. 77: 803– 8.
� Hibbeln J.R., Salem N. (1995) Dietary polyunsaturated fatty acids and depression:
when cholesterol does not satisfy. Am. J. Clin. Nutr. 62: 1-9.
� Hirayama S., Hamazaki T., Terasawa K. (2004) Effect of docosahexaenoic
acidcontaining food administration on symptoms of attention-deficit/hyperactivity
disorder:a placebo-controlled double-blind study. Eur. J. Clin. Nutr. 58: 467-473.
� Holness M.J., Greenwood G.K., Smith N.D., Sugden M.C. (2003) Diabetogenic
impact of long-chain omega-3 fatty acids on pancreatic beta-cell function and the
regulation of endogenous glucose production. Endocrinology 144: 3858– 68.
� Hornych A., Oravec S., Girault F., Forette B., Horrobin DF. (2002) The effect of
gammalinolenic acid on plasma and membrane lipids and renal prostaglandin
synthesis in older subjects. Bratisl. Lek. Listy 103 (3): 101-107.
� Horrobin D.F. (1993) Fatty acid metabolism in health and disease: the role of 6desaturase.
Am. J. Clin. Nutr. 57S: 732S-736S.
� Hubard V.S., Dunn G.D. (1980) Fatty acid composition of erythrocyte
phospholipids from patients with cystic fibrosis. Clin. Chim. Acta 102: 115-118.
40

� Hunter D. Rimm E.B., Sacks F.M., Stampfer M.J., Colditz G.A., Litin L.B.,
Willett W.C. (1992). Comparison of measures of fatty acid intake by subcutaneous
fat aspirate, food frequency questionnaire, and diet records in a free-living population
of US men. Am. J. Epidemiol. 135: 418–427.
� Hunter J.E. (1990). n-3 fatty acids from vegetable oils. Am. J. Clin. Nutr. 51: 809–
814.
� Innis S.M., Kuhnlein H.V., Kinloch D. (1988). The composition of red cell
membrane phospholipids in Canadian Inuit consuming a diet high in marine
mammals. Lipids 23: 1064–1068.
� Innis S.M., Rioux F.M., Auestad N., Ackman R.G. (1995). Marine and freshwater
fish oil varying in arachidonic, eicosapentaenoic and docosahexaenoic aids differ in
their effects on organ lipids and fatty acids in growing rats. J. Nutr. 125: 2286–2293.
� Jayasooriya A.P., Weisinger R.S., Weisinger H.S., Mathai M., Puskas L.,
Kitajika K. et al. (2004) Dietary omega-3 fatty acid supply influences mechanisms
controlling body weight and glucose metabolism. Asia Pac .J Clin. Nutr. S13: 51.
� Jho D.H., Cole S.M., Lee E.M., Espat N.J. (2004) Role of Omega-3 fatty acid
supplementation in inflammation and malignancy. Integr. Cancer. Ther. 3: 98– 111.
� Johnston C.S., Day C.S., Swan P.D. (2002) Postprandial thermogenesis is increased
100% on a high-protein, low-fat diet versus a high-carbohydrate, low-fat diet in
healthy, young women. J. Am. Coll. Nutr. 21: 55– 61.
� Karatas F., Karatepe M., Baysar A. (2002) Determination of free malondialdehyde
in human serum by high-performance liquid chromatography. Anal Biochem. 311:
76– 9.
� Keys A., Anderson J. T., Grande F. (1957) Serum Cholesterol response to dietay
fat. Lancet 1: 787.
� Kim W.W., Kelsay J.L., Judd, J.T., Marshall M.W., Mertz W., Prather E.S.
(1984). Evaluation of long-term dietary intakes of adults consuming self-selected
diets. Am. J. Clin. Nutr. 40: 1327–1332.
� Kobayashi M., Sasaki S., Hamada G., Tsugane S. (1999) Serum n-3 fatty acids,
fish consumption and cancer mortality in six Japanese population in Japan and Brasil.
Jpn. J. Cancer. Res. 90: 914-921.
� Kremer J.M. (1996) Effects of modulation of inflammatory and immune parameters
in patients with rheumatic and inflammatory disease receiving dietary
supplementation of n-3 and n-6 fatty acids. Lipids 31: S243-S247.
� Kyle D.J., Shaeter E., Patton G., Beiser A. (1999) Low serum docosahexaenoic
acid is a significant risk for Alzheimer's dementia. Lipids 34: S245.
� Lamarche B., Tchernof A., Mauriège P., Cantin B., Dagenais G.R., Lupien P.J.
et al. (1998) Fasting insulin and apolipoprotein B levels and low-density lipoprotein
particle size as risk factors for ischemic heart disease. JAMA 279: 1955– 61.
� Larsson S.C., Kumlin M., Sundberg M.I., Wolk A. (2004) Dietary long-chain n-3
fatty acids for the prevention of cancer: a review of potential mechanisms. Am. J.
Clin. Nutr. 79: 935– 45.
� Layman D.K., Boileau R.A., Erickson D.J., Painter J., Shiue H., Sather C. et al.
(2003) A reduced ratio of dietary carbohydrate to protein improves body composition
and blood lipid profiles during weight loss in adult women. J. Nutr. 133: 411– 7.
� Layman D.K., Shiue H., Sather C., Erickson D.J., Baum J. (2003) Increased
dietary protein modifies glucose and insulin homeostasis in adult women during
weight loss. J. Nutr. 133: 405– 10
41

� Leitzmann M.F., Stampfer M.J., Michaud D.S., Augustsson K., Colditz G.C.,
Willet W.C. et al. (2004) Dietary intake of n-3 and n-6 fatty acid and the risk of
prostate cancer. Am. J. Clin. Nutr. 80: 204– 16.
� Lemon P.W.R. (2000) Beyond the zone: Protein needs of active individuals. J. Am.
Coll. Nutr. 19: 513– 21.
� Leventhal L.J., Boyce E.G., Zurier R.B. (1993) Treatment of rheumatoid arthritis
with gammalinolenic acid. Ann. Intern. Med. 119: 867-873.
� Liu S., Willet W.C. (2002) Dietary glycemic load and atherothrombotic risk. Cur.r
AtherosclerRe.p 4: 454– 61.
� London S.J., Sacks F.M., Caesar J., Stampfer M.J., Siguel E., Willett W.C.
(1991). Fatty acid composition of subcutaneous adipose tissue and diet in
postmenopausal US women. Am. J. Clin. Nutr. 54: 340–345.
� Lòpez Mata P., Ortega R.M. (2003) Omega-3 fatty acids in the prevention and
control of cardiovascular disease. Eur. J. Clin. Nutr. 57: 22– 5.
� Ludwig D.S. (2002) Physiological mechanisms relating to obesity, diabetes, and
cardiovascular disease. JAMA 287: 2414– 23.
� Ma J, Folsom R, Lewis L, Eckfeldt H. (1997) Relation of plasma phospholipids and
cholesterol ester fatty acid composition to carotid artery intima-media thickness: the
atherosclerosis risk in communities (ARIC) study. Am. J. Clin. Nutr. 65: 551-559.
� Manku M.S., Horrobin D.F., Morse N.L. (1984) Essential fatty acids in the plasma
phospholipids of patients with atopic eczema. Br. J. Dermatol. 110: 643-648.
� Marckmann P., Lassen A., Haraldsdóttir J., Sandström B. (1995). Biomarkers of
habitual fish intake in adipose tissue. Am. J. Clin. Nutr. 62: . 956–959.
� Markovic T.P., Campbell L.V., Balasubramanian S., Jenkins A.B., Fleury A.C.,
Simons L.A. et al. (1998) Beneficial effect on average lipid levels from energy
restriction and fat loss in obese individuals with or without type 2 diabetes. Diabetes
Care 21: 695– 700.
� Mathers C.D., Sadana R., Salomon J.A., Murray C.J.L., Lopez A.D. (2001)
Healthy life expectancy in 191 countries 1999. Lancet 357: 1685– 91.
� Matorras R., Perteagudo L., Sanjurjo P., Ruiz J. (1999) The intake of long chain
n-3 PUFA during pregnancy and their levels in the mother influence the new-born
levels. Eur. J. Obstet. Gynecol. Reprod. Biol. 83: 179-189.
� Matorras R., Ruiz J., Perteagudo L., Barbazan M.J, Diaz A., Velladoid A.,
Sanjurjo P. (2001) Longitudinal study of fatty acids in plasma and erythrocyte
phospholipids during pregnancy. J. Perinat. Med. 29: 293-297.
� McKarney C., Everard M., N’Diaye T. (2007) Omega-3 fatty acids (from fish oils)
for cystic fibrosis. Cochrane Database Syst. Rew. 17; (4):CD002201.
� Millberger S., Biederman J., Faraone S.V., Chen L., Jones J. (1996) Is maternal
smoking during pregnancy a risk factor for attention deficit hyperactivity disorder in
children? Am. J. Psychiatry 153: 1138-1142.
� Miller A., Berrino F., Hill M., Pietnen P., Piboli E., Wahrendorf J. (1990) Diet in
the aetiology of cancer: a review. Eur. J. Cancer 30: 207-220.
� Mischoulon D., Fava M. (2000) Docosahexanoic acid and n-3 fatty acids in
depression. Psychiat. Clin. North Am. 23: 785– 94.
� Murray R.K. Granner D.K., Mayes P.A., Rodwell V.W. (2003). Harper’s
Illustrated Biochemistry. 23: 195-96.
� Neff L.M. (2003) Evidence-based dietary recommendations for patients with type 2
diabetes mellitus. Nutr. Clin. Care 6: 51– 61.
42

� Neuringer M, Reisbick S., Janowsky. (1994) The role of n-3 fatty acids in visual
and cognitive development: current evidence and methods of assessment. J. Pediatr.
125: 39– 47.
� Nishizawa H., Hamazaki K., Hamazaki T., Fujioka S., Sawazaki S. (2006) The
relationship between tissue RBC n-3 fatty acids and pulse wave velocity. In Vivo 20
(2): 307-310.
� Nordoy A. (2002) Statins and omega-3 fatty acids in the treatment of dyslipidemia
and coronary heart desease. Minerva Med. 93: 357– 63.
� Norrish A., Skeaff C., Arribas G., Jackson R. (1999) Prostate cancer risk and
consumption of fish oils: dietary biomarker-based case-control study. Br. J. Cancer
81: 1238-1242.
� Nuttal F.Q., Gannon M.C., Saeed A., Jordan K., Hoover H. (2003) The metabolic
response of subjects with type 2 diabetes to a high-protein, weight-maintenance diet.
J. Clin. Endocrinol. Metab. 88: 3577– 83.
� O'Brien W.F., Krammer J., O'Leary T.D., Mastrogiannis D.S. (1993). "The
effect of acetaminophen on prostacyclin production in pregnant women". Am. J.
Obstet. Gynecol. 168 (4): 1164–9.
� Oliewiecki S., Burton J.L., Elles K., Horrobin D.F. (1990) Levels of essential and
other fatty acids in plasma and red cell phospholipid from normal controls and
patients with atopic eczema. Acta Derm. Venoral. 71: 224-228.
� Owen R.W., Giacosa A., Hull W.E., Haubner R., Wurtele G., Spiegelhalder B.,
Bartsch H. (2000) Olive-oil consumption and health: the possible role of
antioxidants. Lancet Oncol. 1:107-112.
� Pariza M. (1997) Animal studies: summary, gaps, and future research. Am. J. Clin.
Nutr. 66S: 1539-1540.
� Parra M.S., Schnaas L., Meydani M., Perroni E. (2002) Erythrocyte cell
membrane phospholipids levels compared against reported dietary intakes of
polyunsaturated fatty acids in pregnant Mexican women. Public Health Nutrition 5:
931-937.
� Pawlosky R., Hibben J., Wegher B., Sebring N., Salem J. (1999) The effects of
cigarette smoking on the metabolism of essential fatty acids. Lipids 34: S287.
� Peet M., Brind J., Ramchand C.N., Shah S., Vankar K.G. (2001) Two doubleblind
placebo-controlled pilot studies of eicosapentaenoic acid in the treatment of
schizophrenia. Schizoph. Res. 49: 243-251.
� Peet M., Laugharne J., Rangarajan N., Horrobin D., Reynolds G. (1995)
Depleted red cell membrane essential fatty acids in drug-treated schizophrenic
patients. J. Psychiatr. Res. 29: 227-232.
� Peet M., Laugharne J.D., Horrobin D.F., Reynolds G.P. (1994) Arachidonic acid:
a common link in the biology of schizophrenia? Arch. Gen. Psychiatry. 51: 665-666.
� Peet M., Murphy B., Shay J., Horrobin D. (1998) Depletion of omega-3 fatty acid
levels of depressive patients. Biol. Psychiatry 43: 315-319.
� Pelikanova T., Kohout M., Valek J., Base J., Kazdova L. (1989) Insulin secretion
and insulin action related to the serum phospholipid fatty acid pattern in healthy men.
Metabolism 38: 188-192.
� Pereira M.A., Swain J., Goldfine A.B., Rifai N., Ludwing D.S. (2004) Effects of a
low-glycemic load diet on resting energy expenditure and heart disease risk factor
during weight loss. JAMA 292: n 20.
� Pliszka S.R., Maas J.W., Javors M.A., Rogeness G.A., Baker J. (1994) Urinary
catecholamines in attention-deficit hyperactivity disorder with and without comorbid
43

anxiety. Journal of the American Academy of Child & Adolescent Psychiatry
33(8):1165-73.
� Poppitt S.D., Kilmartin P., Butler P., Keogh G.F. (2005) Assessment of
erythrocyte phospholipid fatty acid composition as a biomarker for dietary MUFA,
PUFA or saturated fatty acid intake in a controlled cross-over intervention trial.
Lipids. Health. Dis. 4: 30.
� Prevention des cardiopathies coronariennes (1982) Rapport d’un Comité
de’experts de l’OMS Organization Mondiale de la Santé, Série de Rapport techniques
678. Organization Mondiale de la Santé, Geneve.
� Puustinen T., Punnonen K., Uotila P. (1985). The fatty acid composition of 12
North-European fish species. Acta Med. Scand. 218: 59–62.
� Richardson A.J. (2004 a) Clinical trials of fatty acid treatment in ADHD, dyslexia,
dyspraxia and the autistic spectrum. Prostaglandins, leukotrienes and essential fatty
acids 70: 383-390.
� Richardson A.J. (2004 b) Long-chain polyunsaturated fatty acids in childhood
developmental and psychiatric disorders. Lipids 39: 1215-1222.
� Roberts L.J., Fessel, J.P. (2004). "The biochemistry of the isoprostane,
neuroprostane, and isofuran pathways of lipid peroxidation.". Chem. Phys. Lipids 128
((1-2)): 173-86.
� Rodriguez de Fonseca R., Del Arco I., Javier Bermundez-Silva F., Bilbao A.,
Cippitelli A., Navarro M. (2005). The Endocannabinoid system: physiology and
pharmacology. Alcohol and Alcoholism 40(1):2-14.
� Rogers P.J. (2001) A healthy body, a healthy mind: long-term impact of diet on
mood and cognitive function. Proc. Nutr. Soc. 60: 135– 43.
� Rogiers V., Crockaert R., Vis H.L. (1980) Altered phospholipid composition and
changed fatty acid pattern of the various phospholipid fractions of red cell
membranes of cystic fibrosis children with pancreatic insufficiency. Clin. Chim. Acta
105: 105-115.
� Romon M., Nuttens M.C., Theret N., Delbart C., Lecerf J.M., Fruchart J.C.,
Salomez J.L. (1995). Comparison between fat intake assessed by a 3-day food record
and phospholipid fatty acid composition of red blood cells. Metab Clin Exp 44: 1139-
1145.
� Rose D.P. (1997). Dietary fat, fatty acids and breast cancer. Breast Cancer (Japan) 4:
7–16 a.
� Rose D.P.(1997) Dietary fatty acids and cancer. Am. J. Nutr. 66S: 998-1003.
� Ross B.M., McKenzie I., Glen I., Bennett C.P. (2003) Increased levels of ethane, a
non-invasive marker of n-3 fatty acid oxidation, in breath of children with attention
deficit hyperactivity disorder. Nutr. Neurosci .6(5): 277-281.
� Ryback R. (2001) Bioelectrical modulators and the cell membrane in psychiatric
medicine. Psycopharmacol. Bull. 35: 5– 44.
� Saldeen P., Saldeen T. (2004) Women and Omega-3 fatty acids. Obstet. Gynecol.
Surv. 59: 720– 30.
� Sanders T.A. & Roshanai F. (1983). The influence of different types of omega 3
polyunsaturated fatty acids on blood lipids and platelet function in healthy volunteers.
Clin. Sci. 64: 91– 99.
� Sands S.A., Reid K.J., Windsor S.L., Harris W.S. (2005) The impact of age, body
mass index and fish intake on the EPA and DHA content of human erythrocytes.
Lipids 40: 343-347.
44

� Santoli D., Philips P.D., Zurier R.B. (1990) Suppression of interleukine-2
dependant human T cell growth by prostaglandin E and their precursor fatty acids. J.
Clin. Invest. 85: 424-432.
� Sarau H.M., Foley J.J., Schmidt D.B. (1999). "In vitro and in vivo pharmacological
characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with
anti-inflammatory activity". Prostaglandins Leukot. Essent. Fatty Acids 61 (1): 55–
64.
� Sattar N., Berry C., Greer I. (1998) Essential fatty acids in relation to pregnancy
complications and fetal development. B.r J. Obstet. Gynecol. 105: 1248-1255.
� Sears B. (1995) The Zone. New York, NY: Regan Book; 1995. 27
� Sears B. (2002) The anti-Aging Zone. New York, NY: Regan Book 2002.
� Sears B. (2002) The Omega Rx Zone. New York, NY: Regan Book; 2002.
� Serhan C.N., Gotlinger K., Hong S., Arita M. (2004). "Resolvins, docosatrienes,
and neuroprotectins, novel omega-3-derived mediators, and their aspirin-triggered
endogenous epimers: an overview of their protective roles in catabasis".
Prostaglandins Other Lipid Mediat. 73 (3-4): 155–72.
� Sigel E.N., Lerlan R.H.(1994) Altered fatty acids metabolism in patients with
angiographically documented coronary artery disease. Metabolism 43: 982-993.
� Silvers K.M., Scott K.M.(2002) Fish consumption and self-reported physical and
mental health status. Public Healt Nutr. 5: 427– 31.
� Simopoulos A. (1991). Omega-3 fatty acids in health and disease and in growth and
development. Am. J. Clin. Nutr. 54: 438–463.
� Simopoulos A.P. (1999) Essential fatty acids in health and chronic disease. Am. J.
Clin. Nutr. 70S: 560S-569S.
� Simopoulos A.P. (2002) Omega-3 fatty acids in inflammation and auto-immune
disease. J. Am. Coll. Nutr. 21: 495-505.
� Simopoulos A.P. (2002) The importance of the ratio of omega-6/omega-3 essential
fatty acids. Biomed. Pharmacother. 56: 365-379.
� Stansby M.E. (1986). Fatty acids in fish. In: Simopoulos, A.P., Kifer, R.R. and
Martin, R.E. Editors, 1986. Health Effects of Polyunsaturated Fatty Acids in
Seafoods ( Academic Press, New York, pp. 389–401.
� Stenson W.F., Cort D., Beeken W., Rodgers J., Burakoff R. (1991) Trial of fish
oil supplemented diet in ulcerative colitis. Word Rev. Nutr. Diet 66: 533.
� Stevens L.J., Zentall S.S., Deck J.L., Abate M.L., Watkins B.A., Lipp S.R.,
Burgess J.R.. (1995) Essential fatty acid metabolism in boys with attention-deficit
hyperactivity disorder. Am. J.Clin. Nutr. 62:761-768.
� Stevens L.J., Zentall S.S., Deck J.L., Abate M.L., Watkins BA., Lipp S.R.,
Burges J.R.. (1995) Essential fatty acid metabolism in boys with attention-deficit
hyperactivity disorder. Am. J. Clin. Nutr. 62: 761-768.
� Steyn N.P., Mann J., Benett P.H., Temple N., Zimmet P., Tuomilehto J. et al.
(2004) Diet, nutrition and the prevention of type 2 diabetes. Public Health Nutr. 7:
147– 65.
� Stillwell W., Wassal S.R. (2003) Docosahexaenoic acid: membrane properties of a
unique fatty acid. Chem. Phys. Lipids 126: 1-27.
� supplementation in children with attention-deficit/hyperactivity disorder. J. Pediatr.
139: 189-196
� Swanson J., Deutsh C., Cantwell D., et al. (2001) Genes and attention-deficit
hyperactivity disorder. Clinical neuroscience research 1: 207-216.
45

� Thapar A., O’Donovan M., Owen M.J. (2005) The genetics of attention deficit
hyperactivity disorder. [Review]. Hum. Mol. Genet .14 Spec No. 2:R275-82.
� Thomas T.R., Fischer B.A., Kist W.B., Horner K.E., Cox R.H. (2000) Effects of
exercise and n-3 fatty acids on postprandial lipemia. J. Appl. Physiol. 88: 2199– 204.
� Tjønneland A., Overvad K., Thorling E., Ewertz, M. (1993). Adipose tissue fatty
acids as biomarkers of dietary exposure in Danish men and women. Am J Clin Nutr
57: 629–633.
� Tobarek M., Lee Y.W., Garrido R., Kaiser S., Henninh B. (2002) Unsaturated
fatty acids selectively induce an inflammatory environment in human endothelial
cells. Am. J. Clin. Nutr. 75:119-125.
� Urquiaga I., Guasch V., Marshall G., San Martin A., Castillo O., Rozowski J. et
al. (2004) Effect of mediterranean and occidental diets, and red wine, on plasma fatty
acids in humans. An intervention study. Biol. Res. 37 (2): 253-261.
� Urso M.L., Clarkson P.M. (2003) Oxidative stress, exercise and antioxidant
supplementation. Toxicology 189: 41– 54.
� Van Dyke T.E., Serhan CN. (2003). Resolution of inflammation: a new paradigm
for the pathogenesis of periodontal diseases. J. Dent. Res. 82 (2): 82–90.
� Vergroesen A.J. (1975). Blood and arterial wall in atherogenesis and artherial
thrombosis. J.G.A.J. Mantlast, R.J.J. Hermus & F. van der Haar (Eds.). Influence of
dietary fatty acids on blood lipids. Brill: Leiden.
� Viola P., Audisio M. (1975) Proc. Second. Int. Congress on the Biological Value of
Olive Oil, 115-127.
� Visioli F., Bellomo G., Montedoro G.F., Galli C. (1995) Low density lipoprotein
oxidation inhibited in vitro by olive oil constituents. Atheroscleoris 117, 25-32.
� Visioli F., Galli C., Bornet F., Mattei R., Patelli R., Galli G., Caruso D. (2000).
Olive oil phenolics are dose-dependently absorbed in humans. FEBS Letters 468,
159-160.
� Voigt R.G., Llorente A.M., Jensen C.L., Fraley J.K., Berretta M.C., Heird W.C.
(2001) A randomized, double-blind, placebo-controlled trial of docosahexaenoic acid
� Wainwright P. (1992) Do essential fatty acids play a role in brain and behavioural
development? Neurosci Biobehav. Rev. 16: 193-205.
� Wang Y., Kay H., Killian A.P. (1991) Decreased levels of polyunsaturated fatty
acids in preeclampsia. Am. J. Obstet. Gynecol. 164: 812-819.
� Warner J.G., Ullrich I.H., Albrink M.J., Yeater R.A. (1989) Combined effects of
aerobic exercise and omega-3 fatty acids in hyperlipidemic persons. Med. Sci. Sports.
Exerc. 21: 498– 505.
� Weber P., Raederstorff D. (2000) Triglyceride-lowering affect of omega 3 LC
polyunsaturated fatty acids. A review. Nutr. Metab. Cardiovasc. Dis. 10: 28-37.
� Willet W.C. (1994) Diet and health: what should we eat? Science 264: 532-537.
� Willet W.C. (Ed) (2001) Eat, Drink and Be Healthy, Simon and Schuster Publ.
� Williams R., Hunt S.C., Hasstedt S.J. (1990) Hypertension: genetics and nutrition.
In: Simopoulous AP, Childs B (Eds), Genetic variation and nutrition. World Rev.
Nutr. Diet 63: 116-130.
� Wiseman S.A., Malthot N.N.J., de Fouw N. J., Tijburg L.B.M. (1996) Dietary
non-tocopherol antioxidants present in extravirgin olive oil increase the resistance of
low density lipoproteins to oxidations in rabbits. Atherosclerosis 120, 15-23.
� Yao J.K., Van Kammen D.P., Gurkis J. (1994) Red blood cell membrane dynamics
in schizophrenia. Correlation of fatty acid abnormalities with clinical measures.
Schizophr. Res. 13: 227-232.
46

� Zambotti V, Masserini M, Fedeli E. (1978) Atti Simposium su “Il contenuto
ottimale di acido linoleico nella dieta”, 63-66.
� Zambotti V, Masserini M, Fedeli E. (1978) Atti Simposium su “Il contenuto
ottimale di acido linoleico nella dieta”, 53-61.
� Zambotti V., Preti A., Berra B. (1975) Proc. Second Int. Congress on the
Biological Value of Olive Oil 399-407.
� Ziboh V.A. (1991) n-3 polyunsaturated fatty acids constituents of fish oil and the
management if skin inflammatory and scaly disorders. World Rev. Nutr. Diet 66:
425-435.
� Zoppi S., Vergani C., Giorgetti P., Rapelli S., Berra B. (1985) Effectiveness and
reliability of medium term treatment with a diet rich in olive oil of patients with
vascular diseases. Acta Vitaminol. Enzymol. 7:3-8.
� Østerud B. And Elvevoll, E. (2007) OliVita Scientific Report, OliVita AS.
47

www.itogha.com
www.oil4life.uk.co
Adding life
to your years