Document [original]

Studies on stability and distribution of

polyunsaturated fatty acids in rat tissues

Studien zur Stabilität und zur Verteilung von polyungesättigten Fettsäuren

in Rattengewebe

vorgelegt von

MSc. Rokaia Ramadan Abdelsalam

aus El Minia, Ägypten

von der Fakultät III - Prozesswissenschaften

der Technischen Universität Berlin

zur Erlangung der akademischen Grades

Doktor der Naturwissenschaften

-Dr. rer. nat-

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender:

Prof. Dr. rer. nat. L. W. Kroh

Berichter : Prof. Dr. rer. nat. A. Hartwig

Berichter : PD Dr. J.-Th. Mörsel

Tag der wissenschaftlichen Aussprache: 19.12.2007

Berlin 2008

D 83

ACKNOWLEDGMENTS

I ACKNOWLEDGMENTS

I am deeply indebted to my supervisor PD.Dr.Thomas Mörsel for

suggesting this research study, continuous guidance, advices,

and encouragement through out the course of this work and

preparation of the dissertation and for his critical reading and

valuable revision of my dissertation. I was really pleased to be

one of his students and study in his lab and under his

supervision.

I would like to express my thanks to my supervisor Frau

Professor Dr. Andrea. Hartwig, for her guidance, encouragement

during my research and for critical reading my dissertation

My deep thanks and gratitude are for my lab mates, Herr Dr.

Mohamed F. Ramadan, Herr Martin Doert and Frau K.Wilcopolski

for their ultimate help, cooperation and advices during my

research experiments. Also, I would like to thank all the in

Institute of Food Chemistry and Technology.

My sincere thanks and gratitude are for my husband Dr. A.

Mustafa and my family throughout my studies and during these

years abroad. In spite of being away you were always present for

their advices and encouragement during my stay in Germany.

Finally, I would like to express my thanks and appreciation to the

Egyptian Embassy and Cultural Office in Germany, Herr Prof.

Galal Elgemaay for his ultimate help and to the Egyptian Ministry

of Higher Education and Scholarship Mission for providing me

with the fellowship to study for my Ph.D. in Germany.

SUMMARY

II SUMMARY

In the wider field of nutrition, intensive efforts are under way to determine

the effects of various nutrients on growth and development. The object of the

present study is to demonstrate the effects of different dietary levels of DHA and/

or GLA from different oils in the presence of constant amount of LCPs n-3 (EPA)

and AA as LCPs n-6 on the fatty acid patterns of brain, liver, plasma and their

phospholipids during experiment at two stage (the first one after four weeks and

the second after eight weeks). Rates with omega 6 diets were present various

metabolic fatty acids pathway, liver and plasma fatty acids were significantly

related after four weeks. Liver DHA was positively correlated with plasma DHA (r

= 0.81, P< 0.05) and liver AA was positively correlated with plasma AA (r = 0.86,

P< 0.05). Plasma and liver AA were not correlated with brain and kidney AA.

However, DHA in liver and plasma DHA were significantly associated with DHA

in brain (r = 0.81 and 0.92, P< 0.05), respectively. Brain DHA had not related with

the increase with DHA in diets. In all groups, there was trend for α-tocopherol to

decrease with time, especially between 4 week and 8 weeks of feeding with

significance difference between the low and high level of ω-3 as well as low ω-6

and high concentration. The extracted liver and brain oils from low ω-3 as or ω-6

fed rats had the strongest radical scavenging activity compared to high levels

with positive correlation between RSA and the levels of tocopherols in detected

oils. The storage test existed correlations between peroxide value (PV) and

conjugated dienes (CD) (P< 0.05) as well as 2-thiobarbituric acid-reactive

substances (TBARS) and headspace volatiles (hexanal and propanal) content

(P< 0.001) for most oils and extracted oils. Besides, it was found that a negative

correlation was demonstrated between the TBRAS formation and the total

phenolic contents. We can conclude that, high levels of DHA not contribute to

increase of DHA in brain, liver or plasma beside reduction of plasma AA by

adding EPA+ DHA to the diet with different amounts of GLA may be important to

use for infant fed formula with a family history of inflammatory condition such as

rheumatoid arthritis or AD diseases.

TABLE OF CONTENTS

III TABLE OF CONTENTS

Acknowledgements

Summary

Abbreviations

Zusammenfassung

1 Introduction

2 Review of the literature

2.1 Essential fatty acids and functions

2.2 Essential omega 3 and omega 6 fatty acids requirements

2.2.1 Pregnancy and lactation 8

2.2.2 Infancy and childhood 9

2.3 Essential omega-3 and omega-6 metabolism

2.3.1 Conversion of linoleic and α-linolenic acids up to eicosanoids 10

2.3.2 Competition between unsaturated fatty acid families 11

2.3.3 Omega 3, omega 6 and eicosanoids 12

2.3.4 Relationship between polyunsaturated fatty acids, lipid oxidation

and antioxidant

3 Material and Methods

3.1 Materials

3.2 Analytical Methods

3.2.1 Animals and experimental design 17

3.2.2 Diets and preparations 18

3.2.3 Lipid extraction and separation of phospholipids from the tissues,

plasma

3.2.4 Lipids transmethylation and gas chromatography 19

3.2.5 Determination of tocopherols by NP-HPLC 20

3.2.5.1 Apparatus and chromatographic conditions 20

3.2.5.2 Standard curves preparation

III

TABLE OF CONTENTS

3.2.6 Radical Scavenging Activity of total lipids from brain and liver against

DPPH radicals

3.3. Oxidative stability of oils and formulas during storage

3.3.1 Chromatographic purification 22

3.3.2 Preparation of the emulsions and samples 23

3.3.3 Determination of total phenolic compounds (TPCs) 23

3.3.4 Lipid classification by column chromatography (CC) 24

3.3.5 Peroxide, anisidine and conjugated diene values 24

3.3.6 Characterization of thiobarbituric acid reactive substrate and free

radical activity

3.3.7 GC-HS analysis of chosen secondary oxidation products 25

Results and Discussion

Part I Animal experiment

4.1 Effect of feeding on the weight and fatty acid composition

4.1.1 Impact of feeding on rat’s growth 27

4.1.2 Impact of feeding on liver weight and fatty acids 27

4.1.2.1 Omega-3 group 27

4.1.2.2 Omega-6 group 30

4.1.3 Impact of feeding on plasma fatty acids 32

4.1.3.1 Omega-3 group 32

4.1.3.2 Omega-6 group 34

4.1.4 Impact of feeding on brain weight and fatty acids 35

4.1.4.1 Omega-3 group 35

4.1.4.2 Omega-6 group 38

4.2 Effect of feeding on phospholipids fatty acid composition

4.2.1 liver phospholipids fatty acids profile 39

4.2.2 Plasma phospholipids fatty acids profile 40

4.2.3 Brain phospholipids fatty acids profile 41

TABLE OF CONTENTS

4.3 Effect of feeding on tocopherol levels

4.3.1 Impact of feeding on level of liver tocopherols 43

4.3.2 Impact of feeding on level of plasma tocopherols 43

4.3.4 Impact of feeding on level of brain tocopherols 44

4.4 Free Radical Activity in brain and liver extracted

Part II Oxidative stability of formula

5.5 Oxidation stability during oven test

5.5.1 Hydroperoxide formation

5.5.1.1 Peroxide and p-anisidine values 46

5.5.1.2 Ultraviolet absorptivity 47

5.5.2 Hydroperoxide of decomposition

5.5.2.1 Oxidative stability of bulk oils 49

5.5.2.2 Stability of extracted oils (EO) 49

5.5.2.3 Oxidative stability of oil fractions 50

5.5.2.4 Thiobarbituric acid reactive substances (TBARS) Assay 52

5.5.3 Effect of α-tocopherol in bulk oils and extracted oils

Conclusion

References

Tables and figures

ABBRIVIATIOS

VI ABBERVIATIONS USED

rachidonic acid

ADHD Attention hyperactivity disorder

AI Acceptable Intake

AMDR Acceptable Macronutrient Distribution Range

AV p-anisidine Value

CC Column Chromatography

CD Conjugated Diene

CHD Coronary heart disease

COX Cyclogenase

DHA Docosahexanoic acid

DGLA Dihomo-gamma-linolenic

DPPH 1, 1-diphenyl-2-picrylhydrazyl

EPA Eicosapentaenoic acid

FAME Fatty acids methyl esters

FAO Food and Agriculture Organization

FID Flame ionization detector

GC Gas Chromatography

GLA Gamma linolenic acid

GL Glycolipids

HS-GC Head space gas chromatography

IQ Intelligence quotient tests

α-LA Alpha-linolenic acid

LA Linoleic acid

LCPs Long-chain polyunsaturated fatty acids

LOX Lipoxygenase

MAD Malondialdehyde

MUSFA Monounsaturated fatty acids

ABBRIVIATIOS

VII

NL Neutral lipids

NP-HPLC Normal phase-high performance liquid

chromatography

PL Phospholipids

PUFAs Polyunsaturated fatty acids.

PV Peroxide value

RSA Radical scavenging activity

TBARS Thiobarbituric acid reactive substances

TEP 1, 1, 3, 3-tetraethoxypropane

TMSH N-trimethylsulfoniumhydroxide

TPCs Total phenolic compounds

ω-3 Omega 3

ω-6 Omega 6

WHO World Health Organization

ZUSAMMENFASSUNG

Studien zur Stabilität und zur Verteilung von

polyungesättigten Fettsäuren

in Rattengewebe

Einleitung

Muttermilch ist in ihrer Zusammensetzung ideal auf die biologischen

Bedürfnisse des Babys abgestimmt. Das Fett der Muttermilch und der

Nahrung sind der wichtigste Energieträger für den Säugling. Es liefert ca.

40 - 55 % der zugeführten Energie

Die wichtigsten Bestandteile der

Nahrungslipide

(Triglyceride, Phospholipide und Cholesterinester), nämlich die

langkettigen polyungesättigten Fettsäuren (LCPs) sind essenziell für das normale

Wachstum und die Entwicklung des Säuglings (3, 4). Die beiden Gruppen der

LCPs, die omega-3- (ω-3) und omega-6-Fettsäuren (ω-6) haben besonders

wichtige Funktionen: Docosahexaensäure (DHA; C22:6n-3) in Netzhaut und

Gehirn, und Arachidonsäure (AA; C20:4n-6) als Vorläufer der Eicosanoide, die

bei einer ganzen Reihe zellulärer Prozesse wichtig sind. Muttermilch wird als

beste Quelle für essentielle Fettsäuren betrachtet; neben anderen Fettsäuren

enthält sie die essentielle Linolsäure (LA; C18:2n-6) und alpha-Linolensäure (α-

LA; C18:3n-3), und die LCPs: Arachidonsäure (AA) und Docosahexaensäure

(DHA) (6-8).

Die Untersuchungen zeigen, dass das Neugeborene im Stande ist AA aus

LA und DHA aus alpha-LA zu synthetisieren. Jedoch ist der Betrag der LCPs,

besonders der an DHA, der erzeugt werden kann, infolge einer verminderten

Desaturasekapazität nicht ausreichend um den Entwicklungsanforderungen das

Säuglings gerecht zu werden, was eine exogene Versorgung mit LCPs während

der ersten Lebensmonate erfordert (6, 10).

Der Bedarf von Neugeborenen an

LCPs ist ein Forschungsschwerpunkt im letzten Jahrzehnt gewesen. Ein

Grund ist die Beobachtung, dass bei zu früh Geborenen die

ZUSAMMENFASSUNG

Blutplasmawerte an LCPs nach der Geburt in mit Frühchenmilch

gefütterten Säuglingen deutlich abnehmen verglichen mit fast

unveränderlichen Niveaus in mit Muttermilch genährten Frühchen (12).

Diese Studie wurde mit Fütterungsversuchen an Ratten durchgeführt

(Tabelle 1). Es wurden zwei Diätgruppen gebildet: eine omega-3-Gruppe

mit DHA (1, 2, 3%) und eine omega-6-Gruppe mit GLA (1, 3, 5%).

Ziel der

Studie war es (a) die Wirkung der zunehmenden DHA-Menge auf die

Fettsäurespektren in Gehirn, Leber und Blutplasma (Gesamt- und Phospholipide)

innerhalb der ersten acht Wochen zu studieren; und (b) die Wirkung von

diätetischer GLA in Gegenwart vom omega-3-Fettsäuren auf die

Fettsäuremustern (Eicosanoid-Vorgänger) zu bewerten. Der Tocopherolgehalt

Ausgehend von den Ergebnissen beider Gruppen wurden drei

verschiedene (Verhältnis %DHA zu %GLA; 1:1, 1:3, 3:0) pulverförmige

Babynahrungspräparate hergestellt (Öl-Emulsion/AIG93 Diät-Trocknung-

Lagerung) und deren Lagerfähigkeit bei 60°C überprüft.

Zusammenfassung

Die Gehalte von DHA (omega-3-Gruppe) in Plasma und Gehirn

korrelieren nach vier Entwicklungswochen miteinander (r = 0.82, P <0.05),

gleiches gilt für AA (r = 0.89, P <0.05). Nach acht Wochen ergeben sich im

Gehirn vergleichbare DHA-Werte, unabhängig von der Fütterungsmenge

(1-3%). In den omega-6-Fütterungsversuchen korrelieren Leber-DHA und

Plasma-DHA (r = 0.81, P <0.05), als auch Leber- und Plasma-AA (r = 0.86,

P <0.05). Leber- und Plasma-DHA korrelieren nach vier Wochen auch mit

Gehirn (r = 0.81, P <0.05) ,dies gilt aber nicht für AA. Als Grund wird die

Blockierung der ∆5-Desaturase durch die Konkurrenzreaktion EPA zu DHA

angenommen (Figure 1). Zusammenfassend lässt sich sagen, dass die

ZUSAMMENFASSUNG

Verhältnisse zwischen Nahrungsfetten und Körperfetten über den

Fettstoffwechsel sehr komplex sind. Die über die Fütterung verursachte

Verschiebung der Fettsäurezusammensetzung ist in der Leber am

größten. Hier findet der Stoffwechsel statt und über das Blut gelangen die

Fettsäuren ins Gehirn. Von dem DHA-Gehalt im Blutplasma kann man

direkt auf den Gehalt in den Geweben schließen. Die zur Entwicklung des

Gehirns, welche bei Ratten nach 28 Tagen beim Menschen nach dem 2.

Lebensjahr abgeschlossen ist, notwendige DHA wird bereits mit 1% im

Nahrungsfett ausreichend zugeführt. Die Verabreichung größerer Mengen

führt zu keinem weiteren Anstieg im Gehirn.

Im Gegensatz zu anderen Studien konnte gezeigt werden, dass die

Gabe von GLA, AA, EPA und DHA zu einer Senkung der Plasma-AA und

einer Steigerung der Gehirn-DHA führt. Diese Beobachtung könnte eine

neue Therapiemöglichkeit für Menschen eröffnen, die an Beschwerden

leiden, welche aus dem Arachidonsäure-Stoffwechsel (Eicosanoide)

herrühren, z.B. Neurodermitis und Multiple Sklerose.

Die untersuchten Proben enthalten α-Tocopherol und Spuren von γ-

Tocopherol. Innerhalb der ersten acht Entwicklungswochen nimmt die

Tocopherolgehalt stark ab, der Effekt wird durch steigende LCPs-

Verabreichung, sowohl in der omega-3- als auch in der omega-6- Gruppe

noch verstärkt (Tabelle 13). Dieses Ergebnis entspricht dem anderer

Studien an Ratten bzw. Mäusen (99, 100) die zeigten, dass höhere

Gehalte an omega-3-FA kleinere Tocopherolgehalte verursachen.

Zwischen der gemessenen antioxidativen Wirksamkeit (RSA) und der

Tocopherolkonzentration besteht ein direkter Zusammenhang.

Die Lagerstabilität der drei pulverförmigen Babynahrungspräparate

beträgt bei 60 °C unabhängig von der Formulierungsform (Öl/Pulver) etwa

ZUSAMMENFASSUNG

sechs Tage, dies entspricht etwa drei Monaten bei 20 °C. Die

unterschiedlichen Verhältnisse von DHA zu GLA haben keinen

signifikanten Einfluss. PV, AV und CD korrelieren miteinander, ebenso

TBARS und flüchtige Verbindungen. Die Aldehyde des Fettverderbs

korrespondieren mit den anderen Verderbsparametern und steigen nach

etwa 4-6 Tagen an. Der Verderb ist gleichermaßen über Propanal,

Pentanal, Hexanal und Nonanal verfolgbar. Der Zusatz von 150ppm

Tocopherol verzögert die Entstehung der Verderbsaldehyde signifikant

(Tabelle 15) und ermöglicht eine bessere Lagerstabilität der

Formulierungen.

INTRODUCTION

1 INTRODUCTION

Dietary lipids are the major energy source for infants and young

children. Lipid tissue accretion in growing infants is very high and consists

of 90% of all energy deposited in the body during the first six months of

infant’s life (1, 2). The most important functional components of dietary

lipids (triglycerides, phospholipids and cholesterol esters), practically long-

chain polyunsaturated fatty acids (LCPs) are essential to normal growth

and the infant development (3, 4). The two families of LCPs, the omega-3

(ω-3) and the omega-6 (ω-6) have specific functions: docosahexanoic acid

(DHA; C22:6n-3) in retina and brain, whereas arachidonic acid (AA;

C20:4n-6) is known to be a precursor of eicosanoids that are important for

a number of cellular processes.

AA and DHA are deposited in large amounts in the nonmyelin

membranes of the developing central nervous system. Adequate supplies

of ω-6 and ω-3 fatty acids during nervous system development are of

concern because of possible long-term changes in learning ability and

reduce visual function (5). Human milk is considered to be the best source

of fat and dietary essential fatty acids for infant feeding; as it provides a

complex mixture of fatty acids, including the essential polyunsaturated

linoleic acid (LA, C18:2n-6) and Alpha-linolenic acid (α-LA, C18:3n-3) and

LCPs (AA, DHA)(6-8).

Current evidence suggests that the newborn is able to synthesize

AA and DHA from LA and α-LA, respectively (9). However, the amount of

LCPs being produced, particularly of DHA, may not be sufficient to meet

the developmental requirements of the infant, because they have a limited

desaturation capacity and depends on an exogenous supply of LCPs

during the first months of life (6, 10). Moreover, a dietary lack of essential

INTRODUCTION

fatty acids and their derivatives is evident also in weaned children during

the second half of their first year of life (11).

Newborn requirements of the LCPs have been a major focus of

research for the past decade. The reason for this intense interest was

based on several observations such as: (a) plasma levels of LCPs

decrease markedly after birth in formula-fed preterm infants as compared

to almost constant levels in breast-fed infants of comparable gestational

age; (b) the level of LCPs in human milk is constant as a function of length

of lactation or geographical distribution (i.e., nutrition) and therefore to

what extend can human milk be the gold standard (12); and (c) studies that

evaluate the effect of formula supplementation with LCPs provide only

partial answers as to benefit of such supplementation to full-term and pre-

term infants (13). One important question of relevance to infant nutrition is

whether there are critical neurodevelopmental stages in which DHA is

required for optimal development (14). However, docosahexanoic acid and

arachidonic acid are commonly added to infant formula worldwide, but

dietary concentrations needed to obtain optimal tissue levels have not

been established.

On the light of the fore going observations the objectives of this

study were; (a) to study the effect of feeding various levels of DHA were (0,

2, 4, and 6 times those used in infant formulas) on rats growth ; brain, liver

and plasma fatty acids and phospholipids, (b) to evaluate the effect of

dietary gamma-linolenic acid (GLA, C18:3n-6) as an intermediate of AA

synthesis at three levels in the presence of DHA, EPA plus AA (eicosanoid

precursors) on the fatty acid patterns of tissues, plasma and their

phospholipids of rats tissues, (c) to report the relationship between mixed

oils and their antioxidant properties in vivo, (d) to report the effect of

mixed omega 3 and omega 6 feed oils on their radical scavenging activity

of brain and liver, (e) to assess oxidative stability of both of oils and

INTRODUCTION

formulas by monitoring oxidative products and (f) to examine the effect of

storage on the evolution as volatile secondary products of the oils and

fractions.

REVIEW OF LITERATURE

2 REVIEW OF LITERATURE

2.1 Essential fatty acids and functions

The 'essential' fatty acids were given their name when researchers

found that they were essential to normal growth in young children and

animals, whereas mammalian cells lack the ∆ -12 and ∆-15 desaturase

enzymes (found in most plants) for insertion of a double bond at the ω-6 or

ω-3 position, but able to synthesize (from non-fat precursors) saturated

fatty acids and unsaturated fatty acids of the ω-9 and ω-7 series (9, 15).

Thus, mammalian cells cannot synthesize ω-6 or ω-3 PUFAs de novo and

must obtain them from the diet. Omega 3 polyunsaturated fatty acids are

α-LA, EPA and DHA have 3, 5 or 6 double bonds in a carbon chain of 18,

20 or 22 carbon atoms, respectively. All double bonds are in the cis-

configuration. Linoleic acid and arachidonic acid are essential fatty acids

(5, 16). Omega 3 and omega 6 essential fatty acids are needed for the

membranes of all body cells as their role in health is wide reaching:

encompassing not only healthy heart and brain good function, but also

playing an important role in the normal function of the eyes, the nervous

system, the kidney, and the liver, in fact all body systems. Other functions

also include the contraction of muscles, blood clotting, and inflammatory

processes (6-8, 17).

2.2 Essential omega 3 and omega 6 fatty acids requirements

As macronutrients, fats are not assigned recommended daily

allowances, it have AI (Acceptable Intake) and AMDR (Acceptable

Macronutrient Distribution Range) instead of RDAs. The AI for ω-3 is 1.6

grams/day for men and 1.1 grams/day for women, while the AMDR is 0.6%

to 1.2% of total energy. The National Institutes of Health recently published

recommended daily intakes of fatty acids (18); specific recommendations

REVIEW OF LITERATURE

include 650 mg of EPA and DHA, (because both have much greater

physiological potency than α-LA for protection against coronary heart

disease), 2.22 g/day of α-LA and 4.44 g/day of LA. This means that the

ratio of ω-6 to ω-3 is 1.5:1.

2.2.1 Pregnancy and lactation

An adequate intake of DHA and EPA is particularly important during

pregnancy and lactation periods. During this time the mother must supply

all the baby's requirements of DHA and EPA because babies are unable to

synthesize these essential fatty acids themselves. DHA makes up 15 to

20% of the cerebral cortex and 30 to 60% of the retina, so it is absolutely

necessary for normal development of the fetus and baby as well (19, 20).

During pregnancy, the fetus completely depends on maternal sources of

DHA from lipid stores, maternal diet and nutritional supplements. During

fetal life, placenta selectively and substantially transports AA and DHA

from the mother to the fetus. During the third trimester of pregnancy, there

is an avid accretion of DHA in the liver, brain and retina of the fetus at a

rate of 4.13 g of EFA per week i.e. 0.59 g/day (18, 21). Pregnancy leads to

a progressive depletion of maternal plasma DHA, presumably due to the

increased supply of this critical nutrient to the developing fetal nervous

system (22, 23). According to WHO and FAO the pregnant woman should

take at least 2.6 g of ω-3 and 100-300 mg of DHA daily to meet the

requirements of her fetus (24). In countries where pregnant women

consume large amounts of fish, it seems to be having beneficial effects for

the mother: a slightly longer pregnancy (by 1-3 days), slightly larger birth

weight and also a possible decrease in the risk of pre-maturity (25-27). On

the other hand, there is some evidence that an insufficient intake of ω-3

fatty acids may increase the risk of premature birth and an abnormally low

REVIEW OF LITERATURE

birth weight. Also, there is emerging evidence that low levels of ω-3 are

associated with hyperactivity in children (28-34).

2.2.2 Infancy and Childhood

Once the baby is born, the mother needs a good supply of essential

omega 3 fatty acids; through eating more oil-rich fish to increase the

amount of DHA in the breast-milk infants, in order to improve the available

amounts available for brain, eyes and the nervous system development. It

is known that, infant and children are generally thought to require 1-2% of

total dietary energy as LA to prevent EFA deficiency, but pre-term infants

require at least 10% of this energy (18, 24, and 25). Babies who are born

pre-maturely and miss out on the umbilical DHA supply during the last

stage of development are born with low stores of DHA in the brain and

liver. This can lead to visual impairment and abnormal retina function.

Thus, pre-mature babies are given extra fatty acids in their feeds to avoid

this miss function of retina. As these fatty acids are needed for eyesight

and brain function, there is an evidence, which shows that a lack of DHA in

the pre-term infant can lead to neurological deficits, such as learning

disabilities, behavioral problems and perhaps lower scores on IQ

(intelligence quotient) tests (35, 36). The weaning foods and diets of

preschool children should contain DHA because metabolic conversion of

α-LA to DHA is limited to less than 0.2% in children. There is evidence to

suggest that, 40% of children with attention hyperactivity disorder (ADHD)

have significantly low levels of plasma ω-3 and DHA (37). Forethere, it is

recommended that weaning foods should be rich in DHA (fish oil, sea food

and dry fruits) or supplemented with nutritional supplements containing

DHA.

REVIEW OF LITERATURE

2.3 Essential omega 3 and omega 6 metabolism

2.3.1 Conversion of linoleic acid and linolenic acids up to

eicosanoid

The commonly consumed polyunsaturated fatty acids are LA and α-

LA. Once consumed, these fatty acids can be converted to the longer-

chain, more unsaturated derivatives. Thus, linoleic acid is converted via

GLA and dihomo-gamma-linolenic (DGLA; 20:3n-6) acids to AA. Likewise,

α-linolenic acid is converted to eicosapentaenoic acid (EPA; 20:5n-3)

(Figure 1). There is some controversy about the extent to which DHA can

be synthesized from EPA in humans (15-17, 38). There are two steps in

which dietary fatty acids can modulate eicosanoids biosynthesis from AA;

the first step is a desaturation and elongation and the second step in which

dietary fatty acids can modulate the biosynthesis of eicosanoids

(prostaglandins, leucotrienes and thromboxanes by cyclooxygenase and

lipoxygenase) is at the formations of endoperoxide intermediate (Figure 1).

EPA as ω-3 can be metabolized by both cyclooxygenase and 5-

lipoxygenase similarly to AA, leading to the formation of trienes

prostaglandins and the leukotriene 5 series. So, it acts as a competitive

inhibitor. Thus, increasing ω-3 in diets may reduce AA levels in tissue

lipids and may decrease the formation of eicosanoid derived from AA

through inhibiting the oxygenation of AA by cyclooxygenase. (15-17, 38-

39).

REVIEW OF LITERATURE

Figure 1: Metabolic pathway for conversion of the dietary essential fatty acids to

eicosanoids via the cyclooxygenase (COX) and Lipoxygenase (LOX) pathways.

2.3.2 Competition between unsaturated fatty acids families

The actions of the essential omega 3 and omega 6 fatty acids are best

characterized by their interactions; they

cannot be understood separately.

In the body, LA competes with α-LA, for ∆6-desaturase, and thereby

eventually inhibits formation of anti-inflammatory EPA (15, 16). In contrast,

GLA derives from LA and does not compete for ∆6-desaturase. GLA's

aturase

∆∆

∆6 Desaturase

∆5 Desaturase

oxidation

COX

LOX

COX

REVIEW OF LITERATURE

elongation product DGLA competes with 20:4n-3 for the ∆5-desaturase.

Arachidonic acid was derived from DGLA pathway. It sits at the head of the

"arachidonic acid cascade" more than twenty different signaling pathways

that control a bewildering array of bodily functions, but especially those

functions involving inflammation and the central nervous system (15, 40).

DGLA and EPA compete with AA for accessing to the cyclooxygenase and

lipoxygenase enzymes. So the presence of DGLA and EPA in tissues

lowers the output of AA's eicosanoids. For example, dietary GLA increases

tissue DGLA and lowers thromboxanes B

(16, 17, 41, and 42) .Likewise,

EPA inhibits the production of prostaglandins series-2 and thromboxanes

(40, 43).

This competition was recognized as important when it was found that

leukotrienes were similarly found to be important in immune inflammatory

system response, and therefore relevant to some diseases like arthritis

and asthma. These discoveries led to greater interest in finding ways to

control the synthesis of omega-6 eicosanoids. The simplest way would be

by consuming more ω-3 and fewer ω-6 fatty acids (17, 44).

2.3.3 Omega 3, omega 6 and eicosanoids

Eicosanoids are a family of very potent biological signaling molecules

that act as short-range messengers, affecting tissues near the cells that

produce them. In response to hormonal or phospholipase A2, which are

present in most types of mammalian cells, attacks membrane

phospholipids, releasing arachidonate from the middle carbon of glycerol.

Then arachidonate converted to prostaglandins, beginning with the

formation of prostaglandin H2 (PGH2), the immediate precursor of many

other prostaglandins and of thromboxanes (Figure. 1). The two reactions

that lead to PGH2 are catalyzed by a bifunctional enzyme, cyclooxygenase

(COX), also called prostaglandin H2 synthesis. In the first of the two steps,

REVIEW OF LITERATURE

the cyclooxygenase activity introduces molecular oxygen to convert

arachidonate to PGG2. The second step, catalyzed by the peroxidase

activity of COX, converts PGG2 to PGH2 (Figure. 1). Animal studies

showed that increased dietary ω-3 resulted in decreased AA in brain and

other tissues (15). Linolenic acid contributes to this by displacing LA from

the elongase and desaturase enzymes that produce AA (17). EPA inhibits

phospholipase A2's which releases AA from cell membrane. Other

mechanisms involving the transport of essential fatty acids may also play a

role (44, 45). Thus, eicosanoids are involved in reproductive function; in

the inflammation, fever, and pain associated with injury or disease; in the

formation of blood clots and the regulation of blood pressure; in gastric

acid secretion; and in a variety of other processes important in human

health or diseases (46-49).

2.3.4 Relationship between polyunsaturated fatty acids, lipid

oxidation and antioxidant

Vitamin E as an antioxidant prevents rancidity of fats in plant

sources and in the animal’s digestive tract, through inhibiting the oxidation

of PUFAs in tissue membranes, especially at the cellular level in the

membranes surround the cells, the sub-cellular particles, and the

ercythrocytes (50, 51). It also stabilizes the lipid parts of cells and protects

them from damage from toxic free radical formed from the oxidation of

PUFAs as well as being an end-product of the oxidation of α-tocopherol, as

the α-tocopherol quinone is the cofactor for the mitochondrial fatty acid

desaturation/elongation pathway (51-53). It has been suggested that the

severe neurological degeneration in patients with a genetic lack of α-

tocopherol transfer protein or abetalipoproteinemia is caused by failure of

synthesis of long-chain polyunsaturated fatty acids (50, 51).

REVIEW OF LITERATURE

Figure 2a

Radical chain reaction mechanism of lipid peroxidation.

Figure 2b: Reaction of tocopherol with lipid peroxides; the tocopheroxyl

radical can be reduced to tocopherol or undergo irreversible onward

oxidation to tocopherol quinone.

REVIEW OF LITERATURE

So, the primary function of vitamin E is to help protect the integrity of

cellular and intra-cellular structures. There is much controversy as to how

this function is carried out catalytically as a chain-breaking lipophilic

antioxidant in membranes and plasma lipoproteins? Because the

tocopheroxyl radical formed by reaction of α-tocopherol with the lipid

peroxide radical (Figure. 2) can be reduced to tocopherol in three main

ways by reaction with: (a) Ascorbate to yield the monodehydroascorbate

radical, this in turn can either be reduced to ascorbate or can undergo

dismutation to yield dehydroascorbate and ascorbate. There is an integral

membrane oxidoreductase that uses ascorbate as the preferred electron

donor, linked either directly to reduction of tocopheroxyl radical or via an

electron transport chain linked to the oxidation of NADH and succinate

involving ubiquinone in mitochondria (51, 54). (b) Other lipid-soluble

antioxidants in the membrane or lipoprotein, including ubiquinone, which is

present in large amounts in all membranes as part of an electron transport

chain, not just the mitochondrial inner membrane (53, 54, and 56-58). (c)

Glutathione is catalyzed by glutathione peroxidase, which is a specific

isoenzyme (selenoenzyme). Furthermore, selenium has a direct role in the

recycling of tocopherol (51, 55).

It is noteworthy that when fat is added to the diet it will destroy the

vitamin E in both of diet and digestive tract if rancidity occurs. For this

reason, the quantitative relationship between vitamin E and the amount

and kind of dietary fat is practical importance. The higher consumption of

PUFA fat related with higher vitamin requirement (50, 51, 54, and 58).

MATERIALS AND METHODS

3 MATERIALS AND METHODS

3.1 Materials

Oils were obtained from Henry Lamote GmbH, (Bremen, Germany).

AIN-93G was obtained from SSNIFF Spezialdiäten GmbH, Soest,

Germany. Vitamin E standard (α-, δ-, β-, γ-tocopherol) and Folin-Ciocalteu

reagent were obtained from Merck (Darmstadt, Germany). DPPH; 1, 1-

diphenyl-2-picrylhydrazyl (approximately 90%) was obtained from Sigma

(St. Louis, MO, Germany). p-Anisidine (4-Amino-anisol; 4-Methoxy-anilin)

and caffeic acid were from Fluka (Buchs, Swityerland). TMSH; N-

trimethylsulfoniumhydroxide (Macherey-Nagel, Düren, Germany). TEP; 1,

1, 3, 3-tetraethoxypropane from Sigma (approximately 97%, Steinheim,

Germany), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA) and

butylated hydroxyltoluene (BHT) were from Sigma (St. Louis, MO,

Germany). Propanal, pentanal, hexanal, octanal and neonanal were

purchased from Fluka (Steinheim, Germany). Toluene of HPLC grade was

used throughout the experiment. All other chemicals and solvents were

from the highest commercial grade and were used without further

purification.

3.2 Analytical Methods

3.2.1 Animals and experimental design

Long-Evans offspring pregnant rats were acquired at 10-12 day

gestation from the commercial supplier of Animal House. Only males were

used in this study to minimize possible effects of hormonal factors. Animals

were housed and cared there, and then kept in individual cages in a

thermo-neutral environment at room temperature (26 °C) and 60% relative

humidity with a cycle of 12 h light and 12 h dark. Each cage was equipped

with a water bottle with metal lid, a cup for administration attached to a

MATERIALS AND METHODS

stainless steel plate to avoid over throwing and spillage. Animals were

placed on a semi-synthetic diet, which was based on the AIN-93G (59). In

the first Phase, the newborn pups were selected on a random basis for

each experiment, with mean body weight with no significantly differences.

Each animal within a particular group was from a different litter and then

this litter was used as a basis for the number of animals. Rat pups were

dam-reared until 8 day of age. Dams were fed as described previously

(60). The artificially hand rearing system was used for pup feeding until

day 17 when rats were hand fed every 3-4 h. On the day 17-28 (after the

opening eye), artificial rat milk was fed in a 50 ml water bottle. In the

second Phase (from 4 weeks to 8 weeks), rats were fed a diet containing

the similar fatty compositions (Table 1, p 71).

3.2.2 Diets and preparations

The diets used were modified from AIN-93 G rodent diet (60). The

dietary oils were a mixture of hydrogenated coconut oil, flaxseed oil,

rapeseed oil, borage oil and marine oil. The oils were obtained from Henry

Lamote GmbH, (Bremen, Germany). Coriander oil was cold extracted by n-

hexane. AIN-93G was obtained from SSNIFF Spezialdiäten GmbH (Soest,

Germany). All formulas were prepared to provide about 15 % LA, 3 % α-

LA, 0.58% AA and 0.89% EPA of dietary fat in omega-3 and omega-6

groups, with the exception of deficient group (control only contained 15 %

LA and 3 % α-LA of dietary fat). The omega-3 group contained three levels

of docosahexanoic acid (DHA): 1%, 2% and 3 % DHA, while the omega-6

group contained 1%, 3% and 5% gamma-linolenic acid (GLA).

Compositions of dietary fat used oils in the experiment were determinate

by gas chromatography analysis.

MATERIALS AND METHODS

3.2.3 Lipid extraction and separation of phospholipids from

the tissues and plasma

At four time points, rats were killed and the liver and brain were

removed from the body and collected, washed three times in ice cold

saline, blotted dry and weighed. Lipids were extracted according to Folch

et al. 1957 (61). The organs were homogenized with twenty fold volume of

dichloromethane: methanol (2:1, v/v) containing 10 mg of buthylated

hydroxytoulene (BHT)/L as an antioxidant, which was then flushed with

nitrogen and left to be extracted overnight at 4 °C. The homogenate was

filtered and washed with additional 10 ml of solution mixed with 20 ml of

0.85% sodium chloride (v/v), shake and left to partition overnight at 4°C.

The lower organic phase was transferred and evaporated. Extracts were

combined and dried under a steam of nitrogen. Blood was collected,

transferred to a plastic tube containing EDTA, centrifuged at 3000 rpm at

4°C. An aliquot of the upper-phase (plasma) was transferred to another

tube. For lipid extraction from plasma 500 µL plasma was mixed with

ethanol containing 0.005% BHT (500 µL) and extracted with hexane (2 ml)

after manually shaking for 3 min (62). Phospholipids were obtained for

application on silica gel plates (thickness = 0.25 mm; Merk, Darmstadt,

Germany) activated at 120°C for two hours immediately before use. Plates

were developed with chloroform: methanol: acetic acid: water (25: 15: 4: 2,

by volume) (63), after visualization with 2, 7-dichlorofluorescein,

phospholipids band was isolated and transmethylated.

3.2.4 Lipids transmethylation and gas chromatography

A portion of total lipid extracts from each tissue was transmethylated

using methanol (1% H

in methanol) for three hour at 70°C (64). After

cooling the resulting fatty acid methyl esters (FAME) were extracted with n-

hexane, dried with anhydrous sodium sulfate and then concentrated to a

MATERIALS AND METHODS

small volume with a steam of nitrogen and transferred to micro vials for

gas chromatographic (GC) injection, whereas phospholipids were

transmethylated, phospholipids band was isolated and transesterified into

their methyl esters (FAME) using N-trimethylsulfoniumhydroxide (TMSH),

methyl esters were extracted twice into one ml hexane and the combined

extracts were dried with steam nitrogen.

The fatty acid methyl esters were then identified and quantified on a

Shimadzu GC-14A equipped with flame ionization detector (FID) and C-

R4AX chromatopac integrator (Kyoto, Japan).The samples were separated

on a 30m SP™ -2380 capillary column (Supelco, Bellefonte, PA, USA:

0.25 mm diameter, 0.2 µm film thickness) using helium at a flow rate of 0.6

ml/ min with a spilt ratio of 1:40. The chromatographic run parameters

included an oven starting temperature of 100°C that was increased by the

rate of 5°C/min to 175 °C and then were held for 10 min before increasing

to 220°C at 8°C/min, with a final hold of 10 min .T he injector and detector

temperatures were both constant at 250°C. Peaks were identified by

comparison of retention times with external standard mixture (Sigma, St.

Louis, MO, USA; 99% purity specific for GLC) on the same conditions.

3.2.5 Determination of tocopherols by NP-HPLC

3.2.5.1 Apparatus and chromatographic conditions

Separations were accomplished on a NP-HPLC with Lichrosorb Si

60, 5 µm (250 *4 mm, i.d.) analytical column from Knauer (Berlin,

Germany). Isooctane: acetyl acetate 96:4 (v/v) were used as a mobile

phase and delivered at a constant flow rate of 1.0 ml/ min with column

back-pressure of about 65–70 bar. The used solvent delivery module was

LC-9A from Shimadzu HPLC (Kyoto, Japan), while the chromatographic

system included a Model 87.00 variable wavelength monitor detector from

Knauer, Berlin, Germany. The solvent system selected, retention time, and

MATERIALS AND METHODS

UV detection of eluting components are shown in Table 2a. The (20 µL)

diluted oils solution (20-30 mg in 1ml) of the selected mobile phase) was

directly injected onto the HPLC column. Tocopherols were identified by

comparing their retention times with those of authentic standards.

3.2.5.2 Standard curves preparation

Standard solution of vitamin E (50 mg/L), were prepared in n-

hexane. The standard solutions were stable at least one month at 4º C in

an argon atmosphere. Fresh working standard solution was prepared daily

by approximate dilutions of standards in mobile phase solution. Twenty µL

were injected four times into the HPLC system and the peak areas were

determined to generate standard curve data.

Table 2a: Linearity in studied vitamin E by NP-HPLC at 295 nm.

Compound RT (min)* R

2**

Equation curve***

α-Tocopherol 9 0.9989 y = 10436 x - 141.48

ß-Tocopherol 13 0.9996 y = 11396 x - 5152.4

γ-Tocopherol 15 0.9967 y = 12124 x - 118.88

∆-Tocopherol 20 0.9935 y = 12224 x - 3532.6

* Rt, retention time

** Determination coefficient

*** X, concentration (µg/ml); y, peak area

Slope of standard curves (six concentrations levels) were obtained

by linear regression. Shimadzu C-R6A chromatopac integrator was used

for all quantization based on peak areas.

MATERIALS AND METHODS

3.2.6 Radical Scavenging Activity of total lipids from brain

and liver against DPPH radicals

The reduction of toluenic solution of DPPH was examined radically in

the presence of hydrogen donors in total lipids from brain and liver. Briefly,

freshly made DPPH radical at a concentration of 0.0001 M in toluene was

added into toluene solutions of total lipids from brain and liver to start the

reaction. For determination, ten mg (in 100 µL toluene) of total lipids from

brain or liver were mixed with 390 µL toluenic solution of DPPH radicals

and the mixture was vortexed for twenty seconds at ambient temperature.

The possible dilution in the cuvette was taken into account wherein the

final volume in all of the assays was 400 µL. The absorbance at 515 nm

was measured in 1-cm quartz cells using UV-260 visible recording

spectrophotometer (Shimadzu, Kyoto, Japan) against a blank of pure

toluene after the reaction was carried out at ambient temperature for 60

min (65). Radical DPPH scavenging activity was estimated from the

difference in absorbance with or without sample and expressed as percent

DPPH inhibiting. All tests were conducted in triplicates.

3.3. Oxidative stability of oils and formulas during storage

3.3.1 Chromatographic Purification.

Control oil (Table 1) was purified chromatographically to remove

tocopherols and other antioxidants and carotenoids by the following

procedure. A glass column was packed with a hexane slurry of 14 g of

high-purity Merck silica gel (grade 60, 230-400 mesh, activated overnight

at 120 °C), followed by a hexane slurry of 4 g of a ctivated carbon (100-200

mesh, Sigma Chemical Co., St. Louis, MO), and then was washed with

hexane. A 3.0 g of oil was passed through the column and eluted with 200

mL of hexane, followed by 100 mL of 10% ethyl ether in hexane (v/v). The

MATERIALS AND METHODS

chromatographed oil recovered in 98% yield was not significantly changed

in fatty acid composition. Another control was used containing 150 ppm α-

tocopherol.

3.3.2 Preparation of the emulsions and samples

Oil-in-water emulsions contained ten percent oil and one percent

lecithin and were prepared by a previously described procedure with a

slight modification (66) using a phosphate buffer (25 mM, pH 3.8), and

then were added to AIG 93G free fat diet, mixed for ten min as well as

freeze-dried, packed and sealed under vacuum condition for omega 3 and

omega 6 formula oils (1% DHA with1% GLA, 3% DHA and 3% GLA). All

samples were placed in the oven at 60ºC for storage study.

3.3.3 Determination of Total Phenolic Compounds (TPCs)

TPCs were quantified colorimetrically using Folin-Ciocalteu reagent.

Folin-Ciocalteu was diluted thirty fold in distilled water. Aliquots of oil (2 g)

were dissolved in five ml n-hexane and mixed with ten ml methanol

solution (80%) in glass tubes for two min in a vortex and then were

centrifuged at 3000 rpm for 10 min. The extracts were combined and

concentrated in vacuo at 30ºC until consistency was reached. After

purification with acetonitrile, 0.2 ml and one ml diluted Folin-Ciocalteu was

added, the flasks were shaken vigorously. After three min, 0.8 ml of 20%

were added and the mixtures were mixed thoroughly again. The

mixtures were allowed to stand for one hour protected from light. The

absorbance of the blue color produced was measured at 765 nm using a

UV-260 visible spectrophotometer (Shimadzu, Kyoto, Japan).The

concentration of the total phenolic compound for each extract was

expressed as caffeic acid in parts per million based on the calibration of

standard (six serial in triplicate).

MATERIALS AND METHODS

3.3.4 Lipid Classification by column chromatography (CC)

Oils and recovered oils in chloroform were separated into the

different classes neutral lipids (NL), glycolipids (GL) and phospholipids

(PL) by passing through a glass column (20 mm dia * 30 cm) packed with

a slurry of activated silicic acid (65- 230 mesh; Merck) in chloroform (20 %,

w/v) according to Rouser el al. (67). NL was eluted with three times the

column of chloroform, but GL and PL were eluted with five and four times

of acetone and methanol, respectively.

3.3.5 Peroxide, anisidine and conjugated diene values

Measuring progress of oxidation levels of the oils during storage

period was followed by determination of primary and secondary oxidation

products through changes in fat peroxide (AOCS Cd 8-53), whereas was

assayed by indirect titration with sodium thiosulfate, and the iodine

resulting from the oxidation of iodide by peroxides was measured. The oil

sample weight in the range of 0.2- 0.5 g was used. Also, a blank assay

was carried out. Peroxide values (PV) are expressed as mill moles (mmol)

of active oxygen per kg of oil; as well as p-anisidine (AOCS Cd 18-90) and

conjugated dienes and trienes were expressed as absorptitivies of the one

percent oils in isooctane at 232 and 270 nm. All tests were conducted in

triplicate and averaged. No significantly statistic difference (P> 0.05) was

found among the replicated experiments.

3.3.6 Characterization of thiobarbituric acid reactive substrate and

free radical activity

Compounds’ reacting with thiobarbituric acid (TBA) was determined

by the described Pegg’s method (68), with slight modification. Twenty µL

butylated hydroxyltoluene (BHT) (to prevent oxidation during the assay)

were added to 100-200 mg oil, two ml 10% (wt/v) trichloroacetic acid (TCA)

MATERIALS AND METHODS

in water and two ml 0.6% (wt/v) TBA in 0.25 M HCL. The tubes were

shaked and heated at 80ºC in a water bath for one hour, left to cool at

room temperature and centrifuged 3000 rpm for 20 min. Calibration curve:

0, 20, 40, 100, 150 or 200 µL 0.1 nM 1, 1, 3, 3-tetraethoxypropane (TEP),

an MDA precursor, was completed to 2ml with water. The procedure

described for samples was then applied. According to color intensity, as an

indicator of malondialdehyde (MDA) content, malondialdehyde was

measured at 532 nm, using a Shimadzu UV-260 (Kyoto, Japan)

spectrophotometer. Radical scavenging activity of oils and their fractions

were examined by reduction of DPPH in toluene. (See details 3.2.6).

3.3.7 GC-HS analysis of chosen secondary oxidation products

Oils and oils fractions (NL, GL and PL) were carried into special

headspace vials sealed with silicone rubber Teflon caps with a crimper.

Oils were heated at 60ºC for 17 min and the head gas phase was injected

for 60-90 seconds in HS-GC (a Hewlett–Packard 5890) with FID detector

and split/splitless injector. Chromatographic separation of butanal,

pentanal, hexanal, octanal and neonanal were carried out using a DB-1701

column (30 m length, 0.32 mm i.d., and 1µm film thickness; J & W

Scientific, Folsom, CA). The oven temperature was 40ºC for 10 min,

followed by temperature programming to 210 ºC at 4ºC/min and then was

held for 10 min. The FID temperature was 300ºC and the injection port was

held at 280ºC. Helium was used as the carrier gas. Peaks were identified

by comparison of retention times with external standard mixture (Fulka,

99% purity specific for GLC) on the same conditions. Results were

calculated in micro moles per kilogram of oil.

MATERIALS AND METHODS

Table 3b: Linearity in studied propanal, pentanal, hexanal and nonanal by

GC-HS.

Compound RT (min)* R

2**

Equation curve***

Propanal 4.9 0.994 y = 66233 x - 12490

Pentanal 7.7 0.998 y = 52444 x - 19676

Hexanal 11.3 0.998 y = 36956x + 2730

Nonanal 24.1 0.997 y = 2900.6 x + 848.12

* Rt, retention time

** Determination coefficient

*** X, concentration (µg/g); y, peak area

All rats fatty acids compositions are expressed as mean ± SD. Least

significance Difference was used to compare the different of means

between samples. Differences were analysis with the Correlation

coefficient and SPSS 12.0 for windows. Statistical were considered

significantly at a value of p < 0.05.

RESULTS

RESULTS AND DISCUSSION

Part I Animal experiment

4.1 Effect of feeding on the weight and fatty acid composition

4.1.1 Impact of feeding on rat’s growth

Experimental rats’ growth showed a slight increase in body weight of the

3% DHA group than another 1% DHA and 2% DHA groups at days 3, 6 until 56.

Body weights at day 56 were 178, 183 and 196 g for the 1% DHA, 2% DHA and

3% DHA omega 3 groups, respectively. On the other hand, there were no

significant differences in rat weight gains in omega-6 group (Figure 4, p 85).

Hence, it could be concluded that high levels of DHA in the omega-3 diet may

result in an increase in body weight of the experimental animals (69).

4.1.2 Impact of feeding on liver weight and fatty acids composition

4.1.2.1 Omega-3 group

Liver fatty acids composition (Table 3, p 72), as affected by different

levels of GLA along with eight weeks, reflected more biological variability in

the liver. It has been reported that, liver cells are the main site for the

biosynthesis of LCPs from 18 carbon precursors, and for the formation of

lipoproteins that transport fatty acids in the plasma and uptake by most

other cells (70 and 71). LA was found to be higher (11.6%- 16.2%) than

that obtained in brain of all studied groups (0.8%- 1.53%) after 4 weeks

and 8 weeks. However, as dietary DHA increased, LA was increased after

8 weeks (11.6%, 14.4% and 16.2% in 1%, 2% and 3% DHA groups,

respectively) and AA decreased (10.5%, 9.88% and 9.72% in the same

order). These findings were supported by Laidlaw and Holub (72) and

Jensen et al. (73) that high levels of dietary DHA may inhibit ∆5- and ∆6-

desaturase activity and thereby decrease the formation of AA from LA. α-

LA was found to be 0.12% at birth , then significantly increased to 0.45%

RESULTS

and 0.52% after 4 and 8 weeks, respectively in control group, but this

amount was insufficient to meet the developmental requirements. This can

be emphasized by the low level of DHA (5.86%) at birth and after 4 and 8

weeks (4.93% and 6.03%, respectively). On the other hand, although α-LA

was not detected in all omega-3 groups after 4 weeks (may be due to the

complete conversion to another omega-3 metabolic product or DHA, it was

detected in decreased levels about 58.4, 66.7% and 33.3% in 1%, 2% and

3% DHA, respectively after 8 weeks compared to that obtained at birth

0.12%). However, it reflects high ∆6- and ∆5- desaturase activity to the

formation of C20:4n3, EPA and DHA from α-LA.

In regard to C18:4n3 and EPA which intermediate DHA synthesis,

the results illustrated that C18:4n3 was not detected in the profile of liver

fatty acids at birth and after 4 and 8 weeks in control group, may be due to

the formation of EPA from C18:4n3, particularly in the presence insufficient

amount of DGLA. It was reported that, DGLA competes with C20:4n3

(C18:4n3 elongation product) for the ∆5- desaturase and inhibit formation

of EPA, DHA and eicosanoids from EPA. In the other interpretation in the

body, LA ca competes with α-LA for ∆6- desaturase and thereby eventually

inhibits formation of C18:4n3, C20:4n3, EPA, DHA and eicosanoids (15

and 16). After 8 weeks, C18:4n3 was detected with insignificantly

differences between omega-3 groups (0.29%- 0.46%), which may be

attributed to the conversion of α-LA into C18:4n3.

EPA significantly showed lower level at birth (0.31%), compared to

those obtained after 4 weeks (1.49%- 5.52%) and (0.45%- 2.19%) after 8

weeks in all groups. After 8 weeks, 3%DHA group illustrated the highest

significantly EPA level (2.19%) and followed by 2%DHA group (1.39%),

1%DHA group (0.76%) and control group (0.45%). However, the

increasing of C18:4n3 and EPA compared to that obtained at birth can be

RESULTS

referred to the metabolism of α-LA, and this is in agreement with Sastry

(74), Lands (75) and Salem (76).

Liver DHA level was found to be 5.86% at birth and then significantly

increased to 6.03% in the control group and to 13.9%-14.8% in omega-3

groups after 8 weeks, with significantly differences between groups. The

data also showed that 3%DHA group had the highest DHA level (14.8%)

followed by 2%DHA group (14.3%) and 1%DHA group (13.9%). On the

other hand, the lowest DHA level and the increase in n-6 PUFA in the

control group reflects the low available supply of DHA to the nervous

system, but by contrast after 8 weeks, DHA was increased with increasing

LCPn-6 to 23.8%, 26.1% and 27.6% in 1%, 2% and 3% DHA, respectively

depending on the biological behavior in liver and dietary supplementation.

As previously mentioned, depending on inhibition of ∆6- and ∆5-

desaturase activity, the results showed that , as DHA level increased in the

diet, AA levels significantly decreased. Wherefore in 1%, 2% and 3% DHA

groups after 8 weeks, compared to that obtained after birth (19.8%).

In general, all groups showed very similar profile of liver fatty acids

after 8 weeks, which composed of 38.6%- 40.7% SFA, 12.4%- 22.7%

MUFA and 38.7%- 46.9% PUFA of total fatty acids; and 8.24%- 19.2% as

oleic acid which represents 53.1%- 84.6% of total MUFA. AA/ LA, DGLA/

AA and n-3/n-6 ratios reflect the change in enzymatic activities. The high

ratios of n-3/ n-6 after 8 weeks in omega-3 groups compared to that

obtained from control group, but with comparatively higher levels in

3%DHA group.

AA/ LA and DGLA/ AA ratios reflect the change in the ∆6- and ∆5-

desaturase activity in all groups. After 8 weeks, control group had higher

levels of AA/ LA and DGLA/ AA (1.23% and 423.3%, respectively)

compared to that obtained after 4 weeks. The higher ratios of AA/ LA and

DGLA/ AA can be due to EPA, which acts as competitive inhibitor that

RESULTS

decreases the formation of eicosanoids by inhibiting oxygenation of AA

(cyclooxygenase and lipoxygenase pathways). On the other hand, AA/ LA

and DGLA/ AA were decreased about 31.1% and 66.6%, respectively in

1%DHA group; 16.7% and 37.5%, respectively in 2%DHA group; and

25.9%- 24.6%, respectively in 3%DHA group after 8 weeks compared to

the ratios at 4 weeks, but with comparatively higher AA/ LA (0.9) and

DGLA/ AA (7.0) ratios after 8 weeks in 1%DHA group. This finding reflects

the effect of added EPA to diet that inhibits ∆5- desaturase activity and

thereby the formation of AA from DGLA (77). A DHA/EPA ratio reflects the

changes in the DHA status in all groups. After 8 weeks, the ratio was

increased by about zero, 191.7%, 81.8% and 273.5% compared to that

obtained after 4 weeks in control, 1%, 2% and 3% DHA groups,

respectively. The increase in DHA/EPA ratio reflects higher β-oxidation on

elongation EPA products to produce DHA. The average liver weight after

one week was 0.40 g with increasing to 8.00, 8.47 and 8.20 g in the 1%

DHA, 2% DHA and 3% DHA group after 8 weeks, respectively.

4.1.2.2 Omega-6 group

It has been reported that GLA reduces body fat content but not body

mass and facilitates fatty acid β-oxidation in the liver (78). In the present

study, the average liver weight after the first week was 0.40 g; whereas,

the relative weights of liver to total body weights were 4.4%, 4.6% and

4.1% for 1% GLA, 3% GLA and 5% GLA group after the fourth week,

respectively; with no significant difference at the end point.

Concerning liver fatty acids profile (Table 4, p 73), as affected by

different Levels of DHA during 8 weeks of age; the data revealed that, LA

acts as precursors to LCPsn-6, had higher levels in liver (11.6%- 13.8%)

than that obtained in brain of omega-3 and omega-6 groups at birth and

after 4 and 8 weeks. In respect to omega-3 and omega-6 groups, the

RESULTS

results showed that, 1%DHA and 1%GLA groups have the same LA level

(11.6%) after 8 weeks, but 3% and 5%GLA groups showed lower levels

(12.7% and 11.6%, respectively) compared to those obtained in 2% and

3%DHA groups (14.4% and 16.2%, in the same order). The reduction of

LA level in 3% and 5%GLA groups is due to the following, LA competes

with α-LA for ∆6- desaturase, and thereby evenly inhibits formation of EPA

(0.25% and 0.34%, respectively) than those obtained in 2% and 3%DHA

groups (1.39% and 2.19%, in the same order), this result is supported by

Chapkin (15); Calder and Field (16). With references to liver omega-6

groups, the result showed that, as dietary GLA increased up to 3%, LA and

AA increased, then decreased in 5%GLA group, but with higher AA levels

than in 1%GLA group. Wherefore, 3%GLA groups showed the highest LA

and AA levels (12.7% and 24.1%, respectively) and followed by 5%GLA

group (11.6% and 21.2% in the same order), and 1%GLA group (11.6%

and 15.6%, respectively). The higher formation of AA in 3%GLA and

5%GLA groups accompany with lower DHA levels after 8 weeks mean

that, DGLA compete with omega-3 fatty acids for the ∆5- desaturase to

produce AA, and thereby inhibition/ or production of lower DHA. On the

other hand, the lower formation of AA in 5%GLA group compared to that in

3%GLA group may be attributed to the presence of GLA, DGLA,

prostaglandin E1 and 15-hydroxy-eicosatrienoic acid or to the metabolic at

∆5- desaturase step mostly appeared to be of low enzymatic activity in the

synthesis of LCPs n-6 fatty acids (79). Moreover, AA/LA and DGLA/AA

ratios (provides a biochemical indexes of product-substrate relationship in

the LCPs n-6 pathway and ∆5-6 desaturase activity) has emphasized the

same results of AA in omega-6 groups, in which 3%GLA group showed the

highest AA/LA and DGLA/AA ratios (1.89% and 14.9%, in the same order)

and 1%GLA group (1.34% and 10.4%, respectively) after 8 weeks. The

results of EPA/AA support the foregoing findings, in which 3%GLA group

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the lowest EPA/AA ratio and followed by 5% and 1%GLA groups at the

end of experiment.

DHA/EPA ratio reflects the biochemical synthesis of DHA by β-

oxidation, in which 3%GLA group showed the highest ratio (21.7) and

followed by 5%GLA group (12.7), and 1%GLA (10.7) after 8 weeks. This

result showed that, 3%GLA group possesses the highest capacity of

converting the LCPn-3 especially DHA from its dietary precursors. The

higher ratio of n-3/n-6 in 1%GLA group (0.37) after 8 weeks reflects the

higher enzymatic activity, which reserves a normal brain behavior through

n-3 status (59). However, high dietary levels of GLA (3% and 5%) resulted

in higher levels of AA and lower EPA and DHA levels after 8 weeks.

Generally, omega-6 groups illustrated similar profile of liver fatty acids,

composed of 41.3%- 44.2% SFA, 15.3%- 20% MUFA, 39.5%- 45.7%

PUFA of total fatty acids and 9.05%- 11.9% oleic acid, which represents

19.8%- 30.1% of MUFA.

4.1.3 Impact of feeding on plasma fatty acids composition

4.1.3.1 Omega-3 group

Effect of supplement of different levels of DHA on the content of

plasma fatty acids, along 8 weeks and presented in tables 5 (p 74). It’s well

known that, after the biosynthesis of the fatty acids and the formation of

lipoproteins in liver cells, the latter transports the fatty acids through

plasma and uptake by most cells (70 and 71). Wherefore, the profile of

plasma fatty acids showed that, LA and α-LA, which acts as precursors for

LCPs n-6 and n-3 were found in higher levels (21.1%- 14.3% for the former

and 4.41%-1.56% for the latter) than those obtained in liver and brain of

omega-3 and omega-6 group, it can be seen that, as dietary DHA

increased up to 3%, LA levels increased and AA levels decreased, but with

higher LA(21.1%) and lower AA (4.37%) in DHA group means that,

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increasing omega-3 in the diet may reduce AA levels (15-17 and 38-39), in

which α-LA and C20:4n3 compete with LA and DGLA on ∆6- and 5-

desaturase, and thereby production of higher EPA (2.09%). The EPA

competes with the lower level of AA for accessing to the cyclooxygenase

and lipoxygenase to produce EPA’s eicosanoid, and thereby formation of

the lowest DHA (4.85%). Contrarily, 1%DHA group showed the lowest LA

and EPA levels and the highest α-LA, AA and DHA. This can be explained

on the basis that, the reduction of LA level is due to the competition of α-

LA for ∆6- desaturase, and thereby reduces the formation of EPA (1.04%)

and increases AA (8.93%) and α-LA (4.41%). Furthermore, the higher level

of DHA was to be accompanying with decreasing EPA. However, 2%DHA

group behaves between 1% and 3% DHA groups. The results of AA can

be emphasized by the results of AA/LA and AA/DGLA ratios, in which

3%DHA group had the lowest levels (0.21% and 5.14%, respectively),

followed by 2%DHA group (0.41% and 9.03%, in the same order) and

1%DHA group (0.62% and 16.54%, respectively) after 8 weeks.

The results of DHA can be explained by DHA/EPA ratios, which

reflect the biosynthesis of DHA from EPA in which 3%DHA group had the

lowest DHA level (2.32%) and followed by 2%DHA (3.52) and 1%DHA

group (6.68) after 8 weeks. This result showed that, 1%DHA group

possessed the highest capacity of converting the LCPs n-3 especially DHA

from its dietary precursors.

The results of EPA/AA ratios supported the foregoing results, in

which 3%DHA group had the highest ratio (0.48) and followed by 2%DHA

(0.22) and 1%DHA group (0.12) after 8 weeks. The higher ratio of n-3/n-6

in 1%DHA group (6.57) after 8 weeks reflects the higher enzymatic activity.

However, plasma omega-3 groups presented similar profile of fatty acids,

composed of 39.4%- 41.4% SFA, 20.2%- 23.2% MUFA, 37.2%-38.3%

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PUFA of total fatty acids after 8 weeks. Palmitic acid was detected (24.7%-

26.8%) as a major component (62.7%-66.5%) of SFA.

4.1.3.2 Omega-6 group

Tables 6 present the effect of different levels of GLA on plasma fatty

acids, along with eight weeks. As mentioned elsewhere, lipoproteins

transport fatty acids, in particular, LCPs by plasma to most cells (70 and

71). Wherefore, LA and α-LA, which consider as precursors for LCPs n-6

and n-3 were found to be in higher levels (ranged between 19.3% to 20.2%

for LA and 1.18% to 1.41% for α-LA) than those obtained in liver and brain

of omega-6 and control group.

In respect to plasma LA, as dietary GLA increased LA level in

significantly decreased, but with significantly increase in 5%GLA group

than that of 3%GLA group, after 8 weeks. This may be attributed to the

effect of intestinal up take of GLA (1.41%-1.3% in 3% and 5%GLA groups,

respectively) after 8 weeks. The results revealed that, there were

insignificant differences in DGLA level between all omega-6 groups after 8

weeks. Plasma AA, showed that 3% and 5% GLA groups had significantly

higher levels of AA (13.4%- 13.5%) than that obtained in 1%GLA group

(12.2%), but with significantly lower levels than obtained in control

group(24.2%) after 8 weeks. The highest level of AA in control group

accompany with the lowest EPA level (0.48%) and the highest DHA level

(4.5%), after 8 weeks reflect the competitive effect of DGLA with C20:4n3

for ∆5- desaturase, and thereby, the formation of AA. The significant higher

AA levels accompany with the highest EPA and the lowest DHA in 3%GLA

and 5%GLA groups compared to that obtained in 1%GLA after 4 weeks,

means that at higher dietary levels of GLA, DGLA compete with C20:4n3

for the ∆5- desaturase to produce higher AA. The highest EPA and the

lowest DHA, reflect the slower formation of DHA. On the other hand, the

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reduction formation of plasma AA in 1%GLA group than that in other

omega-6 groups, reflects the combination effect of EPA (1.18%), DHA

(3.05%), which lead to suppress the formation of AA and the inflammatory

mediator Leukotrienes in the plasma, because EPA blocks ∆5- desaturase

activity, the terminal enzymatic step in AA synthesis (77). These findings

were also reflected in the calculated AA/LA in which 1%GLA group had the

lowest ratio (0.6). Plasma DHA was found at significant higher

concentration in 1%GLA group than those of 3% and 5%GLA groups,

which could be supported by the highest DHA/EPA ratio (2.58) in 1%GLA

group after 8 weeks. The highest DHA level (3.05%), DHA/EPA (2.58) and

n-3/n-6 (0.21) in 1%GLA group, and the lowest AA level (12.2%) and by

AA/LA ratio (0.60) reflect the competition between the metabolic pathways

between w-3 and w-6 fatty acids (80-83). However, palmitic acid was found

as a major component on of SFA in all groups, with significant reverse

relationship between PUFA n-3 and PUFA n-6 at all experimental periods.

4.1.3 Impact of feeding on brain weight and fatty acids

4.1.3.1 Omega-3 group

The average brain weight of the 1

week was 0.75 g, increased to

1.42 g in 1% DHA and 1.65 for both of 2% and 3% DHA omega 3 groups

with brain to body weights ratios were 0.0079, 0.0086 and 0.0084 at the

end of the experiment.

In respect to brain fatty acids composition, as affected by different

levels of DHA during 8 weeks of age (Tables 7, p 76).α-LA levels

(C18:3n3) showed significant higher levels in all groups (0.41%- 0.52%)

after 8 weeks, compared to that obtained at birth (0.15%). The

comparatively higher levels of α-LA after 8 weeks can be attributed to the

exogenous supply of α-LA to the diets. On the other hand, the insignificant

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difference between α-LA level at birth and after 4 weeks (0.06%) means

that, the amount of α-LA almost completely used in the metabolic pathway.

The intermediate C18:4n3 and EPA (C 20:5n3) of DHA metabolite

were detected in the profile of brain fatty acids. Although C18:4n3 was not

detected at birth it showed significantly higher levels in all groups, with

comparatively higher levels in omega-3 groups after 4 and 8 weeks. The

higher levels of C18:4n3 was found to be 2.33, 2.42 and 2.21 fold in 1%,

2% and 3% DHA groups, respectively more than that obtained in control

group after 8 weeks. This can be attributed to the conversion of the

supplemented from α-LA to C18:4n3.

In regard to EPA, the results showed significantly lower level at birth

(0.24%) compared to those obtained after 4 and 8 weeks in all groups

(0.44%-0.49% and 0.45%- 1.78% respectively). After 8 weeks, it could be

noted that, 3% DHA group has the highest EPA (1.78%) followed by 2%

DHA groups (1.07%) and 1% DHA group (0.45%). However, control group

and 1%, 2% and 3%DHA showed higher levels of EPA by about 4.46,

1.88, 4.46 and 7.42, respectively fold more than that obtained at birth

(0.24%). Increasing C18:4n3 and EPA in all groups compared to that

obtained at birth refers to the metabolism of α-LA, and this is agreement

with Sastry (74), Lands (75) and Salem (76).

Brain DHA level was found to be 6.05% at birth, then significantly

increased to 6.55% in control group and to 13.9- 14.4% in all groups after

8 weeks, but with no significant differences between omega-3 groups. This

results are consistent with the observations of Pawlosky at al.(84), who

reported that, a human who consume a high omega-3 have a low rate of

conversion of DPA n-3 to DHA.

As expected, depending on inhibition of ∆5- and ∆6- desaturase

activity as DHA level increased in diet, it was found that AA (20:4n6)

significantly decreased after 4 and 8 weeks and by about 18.6%, 24.9%

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and 30.2% after 8 weeks in 1%, 2% and 3% DHA groups, respectively

compared to that obtained at birth (12.9%). However, rats in all groups

showed similar profile of fatty acids composition, which contains 44.6%-

54% SFA, 23.4%- 26.7% MUFA and 20.1%- 29.9% PUFA.

The higher ratio of n-3/n-6 in all groups, after 8 weeks compared to

those obtained after 4 weeks, with comparatively higher levels in omega-3

groups reflect the enzymatic activity, which reserve a normal brain

behavior through n-3 status (59).

AA/ LA and AA/ DGLA ratios illustrate the changes in AA status in all

groups. Control group showed higher levels of AA/ LA and AA/ DGLA after

8 weeks by about 26.9% and 593.8%, respectively than that obtained after

4 weeks. This higher ratios can be attributed to the higher level of EPA

(1.07%), which consider as competitive inhibitor that may decreases the

formation of eicosanoids through inhibiting oxygenation of AA by

cyclooxygenase (COX) and lipoxygenase (LOX) pathways (15-17 and 38-

39). On the other hand, in omega-3groups, AA/LA was decreased in 1%

and 2% DHA groups by about 13.8% and 0.33%, respectively, but it was

increased in 3% DHA group by about 6.88% after 8 weeks than that

obtained after 4 weeks. Furthermore, AA/ DGLA was decreased by about

35.5, 58.7 and 61.5% in 1%, 2% and 3% DHA groups, respectively after 8

weeks compared to that obtained after 4 weeks. This means that, addition

of EPA inhibits the activity of ∆5- desaturase and thereby, decreases the

formation of AA from DGLA (77 and 87).

The results of DHA/ EPA ratio, which reflects the changes in DHA

status, showed that, after 8 weeks DHA/ EPA was increased by about

14.7% in 1% DHA group, but decreased by about 50.4% and 72.5% in 2%

DHA and 3%DHA, respectively compared to those obtained after 4 weeks.

The decrease in DHA/ EPA ratios shows lower β-oxidation to produce DHA

and/ or modulation to eicosanoids synthesis from EPA by cyclooxygenase

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and lipoxygenase. On the other hand, the increase in DHA/ EPA reflects

higher β-oxidation to produce DHA.

4.1.3.2 Omega-6 group

The average brain weight after the 1

week was 0.75 g and after 28

days, the relative weights of brain to total body weights were 1.69%, 1.48%

and 1.55% for the 1% GLA, 3% GLA and 5% GLA group, respectively.

Hence, the effect of different levels of GLA on brain fatty acids

composition, along 8 weeks of age (Tables 8, p 77) showed marked effects

of supplementation on special fatty acids in brain. LA levels were

significantly decreased to about 0.92%- 1.42% and to 0.76%- 0.92% after

4 and 8 weeks, respectively in all groups, but with comparatively lower

levels in omega-6 groups (0.76%- 0.92%). In respect to brain DGLA, 5%

GLA group showed the significantly highest DGLA level (1.25%) after 8

weeks. Control group illustrated significant reduction in DGLA (80.5%),

after 8 weeks compared to that obtained after 4 weeks, but omega-6

groups showed significantly marked increase (24.2%, 27.4% and 43.2% in

1%, 3% and 5% GLA groups, respectively) after 8 weeks compared to

those obtained after 4 weeks. However, high dietary level of GLA (5%)

resulted in a slight significantly higher level of AA (11.2%) and lower DHA

level (12.9%) after 8 weeks.

AA/ LA and AA/ DGLA ratios reflect the changes in AA status in all

groups. In control group AA/LA and AA/ DGLA ratios were increased by

about 1.26 and 6.94, respectively fold more than that obtained after 4

weeks. This can be attributed to the higher levels of EPA (1.07%) after 8

weeks. On the other hand, while AA/LA ratio was decreasing by about

8.8% in 1%GLA, it increases by about 87.3% and 72.9% in 3%GLA and

5%GLA compared to that obtained after 4 weeks, respectively.

Furthermore, AA/ DGLA was decreased by about 32.4, 29.1 and 45.7% in

RESULTS

1%, 3% and 5%GLA groups, respectively compared to that obtained after

4 weeks. This can be attributed to the effect of EPA in metabolic pathway

to the formation of AA from DGLA (77, and 85-87).

The brain DHA level was found to be 6.05% at birth, which

significantly increased up to 8 weeks. Brain DHA level in 1%GLA group

was found to be 2.13, 1.07 and 1.08 fold more than that obtained in control

group, 3%GLA and 5%GLA groups, respectively after 8 weeks. DHA/ EPA

ratio reflects the changes in DHA status in all groups. After 8 weeks, DHA/

EPA was increased by about 93.2% in 1% GLA group, but it decreased by

about 44.2%, 49.4% and 35.7% in control group, 3% GLA and 5%GLA,

respectively compared to those obtained after 4 weeks.

In omega-6 group, rats showed similar profile of fatty acids

composition, which contains 44.1%- 54% SFA, 23.5%- 26.3% MUFA and

20.1%- 29.9% PUFA of total fatty acids; and 18%-20.1% as oleic acid

which represents 70%-76.5% of total MUFA. The higher ratio of n-3/n-6 in

all groups, after 8 weeks compared to those obtained after 4 weeks,

observe a normal brain behavior through n-3 status (59).

4.2 Effect of feeding on phospholipids fatty acids composition

4.2.1 Liver phospholipids fatty acids profile

In omega-3 group, they were a fall of AA and increase the level of

DHA in liver PL, whereas the level of ω-3 in cell very important But when

cell deficient in ω-3 fatty acid has a decrease in DHA and increase levels

of the end product of ω-6 metabolism, DPA within the sub-cellular

organelles and mitochondria seem to be more sensitive to a low dietary

supply as evidenced by the relative abundance of DHA and the changes

compositions of these organelles in response to dietary deprivation (77,

88, and 89). The evidence indicates that in early life α-LA precursors are

RESULTS

not sufficiently converted to DHA to allow the biochemical and function of

normal cycle (90). Summed of ω-3 FA after 4 week (Table 9, p 78)

increased gradually until 8 weeks. On the other hand, in omega-6 group,

there was a fall in the level of DHA in liver PL during the fourth week

(Table 10, p 79), whereas the decrease was similar in both 3%GLA and

5%GLA groups (about 17-18%). At the end of the experiment, all groups

showed decreases of about 1.36, 1.22 and 1.20 folds than the level at the

fourth week in the 1% GLA, 3% GLA and 5% GLA groups, respectively.

Moreover, rats liver PL had higher levels of AA at 28 day, after that the

levels were decreased in all groups till the end. It was found that the 5%

GLA group had the highest percentage of AA and DGLA at the end of the

experiment. AA was increased by about 25% and 11% than both of the

other groups and it had 2.69 and 2.35 folds more than group 1% GLA and

3% GLA of DGLA, respectively.

4.2.2 Plasma phospholipids fatty acids profile

The fatty acids profiles of plasma phospholipids followed closely the

degree of supplementation in omega-3 group and illustrate the competitive

interactions between fatty acid (ω-3 compared with ω-6) metabolic

pathways (77, 82 and 91). For example, in all groups supplement with a

constant amount of EPA and AA, fatty acid concentration of AA decreased

linearly with increasing amounts of DHA supplements. In our results, DHA

recorded more fold decreases incorporation from plasma into the rat brain

as well as a reduction in recycling via deacylation/reacylation reactions.

The overall rate process of DHA uptake from the circulation into the

brain/retina appears slow and may be rate-limiting for DHA repletion. In

both of Figures 5 and 6, changes of the plasma phospholipids were shown

during the experiment especially after 28 days and 56 days of feeding.

RESULTS

All the omega 6 groups showed similar and significant group mean

reduction in AA concentrations in plasma phospholipids with

supplementation. After 8 weeks, the reduction in the 5%GLA group was

about 25% and 44% in both of the 1% GLA and 3% GLA groups,

respectively coming with the first four weeks. The multiple factors that

likely account for an actual reduction in AA concentrations include

competition by EPA and DGLA for esterification into cellular phospholipids

and the attenuating effect of n-3 fatty acids on ∆5-desaturase, which

affects the conversion of DGLA to AA (82, 83, 92, and 93). Addition of EPA

and DHA to formula increased the proportion of LCPs n-3 in plasma

phospholipids but did not prevent n-6 depletion (82, 88).

4.2.3 Brain phospholipids fatty acids profile

Rat brain phospholipids contained only a small amount of α-LA and

these results are in agreement with the literature (77, 88). Recycling

(deacylation and reacyclation) of PUFAs (AA and DHA) in brain

phospholipids is a going and active process, but unlike α-LA they are much

susceptible to β-oxidation and thus are conserved in the phospholipids by

the recycling. Brain phospholipids AA decrease and DHA increase with

decrease in 18:4n3 and EPA. At the end of experiment, the proportion of

DHA increased in 1%DHA and 2%DHA group (~79% and ~74%,

receptively) with similar decrease of EPA (Table 11, p 80). On the other

hand, in the omega-6 group, there was a parallel in the n-3 and n-6 fatty

acids in PL compared with the brain FA. Between the 4 and 8 weeks, there

was found a fall in the level of AA about 26-29% in all groups, related to

reverse relationship with DGLA. The proportion of DHA was decreased in

1% GLA group and 3% GLA (~ 13% and ~9%, respectively) at the 28 day

and (19% and ~20%, receptively) at the end of experiment comparing to

the level at the fourth week (Table 12, p 81). These data are in agree with

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that of Wainwright (94) who noted that feeding with oil containing high

level of GLA decreased the level of DHA in brain PL by competition

between the n-6 fatty acid GLA and the n-3 fatty acid DHA. Thus, a long-

sanding question in essential fatty acid research has been whether LCPs

are accumulated in tissues via metabolism of 18-carbon precursors

normally abundant in the diet or rather are incorporated directly from

preformed dietary LCPs. Many previous studies had demonstrated that

various pathway, either metabolic or involving preformed LCP

intermediated, are operative for various tissues. What has been total

lacking in any approach that could assess the quantitative contributions of

the various known pathways for LCP accumulation? One key aspect of this

problem is the source of brain DHA because maintenance of its level is

critical for optimal neutral function. The author chose to hold LA and α-LA

of the diets constant and change the levels of DHA and GLA at two

experiments. Rates with omega 3 diets were present were significantly

related after four weeks. Control rat showed decreased level of DHA

during 4 weeks. Brain DHA was positively correlated with plasma DHA (r =

0.82, P< 0.05) and Brain AA was positively correlated with plasma AA (r =

0.89, P< 0.05). Brain DHA had not related with the increase with DHA in

diets. We suggested to plasma DHA may be used as a marker of brain

DHA status. On the other side, rates with omega 6 diets were present

various metabolic fatty acids pathway, liver and plasma fatty acids were

significantly related after four weeks, which liver cells are the main site for

the biosynthesis of LPCs from 18 carbon precursors, and for the formation

of lipoproteins that transport fatty acids in the plasma and uptake by most

other cells. Liver DHA was positively correlated with plasma DHA (r = 0.81,

P< 0.05) and liver AA was positively correlated with plasma AA (r = 0.86,

P< 0.05). Plasma and liver AA were not correlated with brain AA. However,

DHA in liver and plasma DHA were significantly associated with DHA in

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brain (r = 0.81 and 0.92, P< 0.05) and kidney (r = 0.98 and P< 0.05),

respectively.

4.3. Effect of feeding on tocopherol levels

It is important to detect antioxidant status, which is very important

when consumed LCPs in infant fed formulas. Free radical especially

superoxide (O

2•

) and other reactive species such as H

, are continuously

produced in vivo. Superoxide in particular is produced by leakage from the

electron transport chains within the mitochondria system. Risk of oxidative

damage for premature infant is very important because they are born with

immature antioxidant status.

4.3.1 Impact of feeding on level of liver tocopherols

Liver is the major storage organ of α-tocopherol (95). Table 13

showed that, liver α-tocopherol was decreased by about 10.7-36.6% and

3-6% and in the omega-3 and omega-6 groups, receptively after 4 weeks

compared with the level after 2 weeks. Furthermore, liver α- tocopherol in

the 1% DHA group showed 1.58 fold more than the liver in the 3% DHA

group after 8 weeks of ages. However it was be found that, 5% GLA group

had lesser liver α- tocopherol (40.8%) compared with that obtained with

1%GLA after 8 weeks of age. Our results are in agreement with those of

other studies which have shown that diets induced high n-3 FA had oils

lower α- tocopherol content in the rats liver (96) or mice(97).

4.3.2 Impact of feeding on level of plasma tocopherols

The α-tocopherol level in plasma rapidly raised to about 0.52–11.1

mg/L in omega-3 group after 4 weeks, while in omega-6 group was

increased to about 4.1- 8.82 mg/L at the same period (Table 13, p 81).

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Continuous decrease in plasma α- tocopherol was noted after 6 weeks in

both groups and with some differences after 8 weeks. This can be due to

interaction of omega-3 or omega-6 oils and tocopherol at the gut level, or

to enhanced post absorptive utilization of tocopherol compared to rats fed

low level and high level of omega oils.

4.3.3 Impact of feeding on level of brain tocopherols

Concentration of α-tocopherol in rat’s brain was found to be 12.22

µg/ total brain at birth. Table 13 showed that, α-tocopherol significantly

decrease after 4 weeks and up to 8 weeks in all omega-3 and omega-6

groups. The consumption of α-tocopherol was lead to %- loss about 29-

40% and 31-38% in the omega-3 and omega-6 groups, respectively after 4

weeks and loss about 87%- 89% in each after 8 weeks. It was found that,

the change in brain α–tocopherol being

much smaller than that in plasma

and other tissues, including

liver (98-100). Furthermore, the

high content of

PUFA and, more specifically, DHA in rat brain fed omega-3 diet may

have

enhanced the consumption of antioxidants. Gamma-tocopherol was found

as very small amount in all tissues and plasma. Summary of this section,

all omega-3 and omega-6 groups, there was trend for α- tocopherol to

decrease with time, especially between 4 week and 8 weeks of feeding

with significance difference between the low and high level of ω-3 as well

as low ω-6 and high concentration.

4.4 Free Radical Activity in brain and liver extracted

Pervious studies on radical scavenging activity (RSA) have used

different solvents to dissolve the free radicals in different crude oils (91),

but were not detected before in brain and liver oil extraction. Hence, the

results here were difficult to be compared because the reactions were

done under different conditions. Characteristics of the antioxidant-free

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radical reactions in the present study were evaluated using total lipids by

spectrophotometric method with the stable DPPH radical. This method has

been reported as a simple method for evaluation of the radical scavenging

activity of a given substance, absorbance at 515 nm decreases as a result

of a color change from violet to yellow as the radical is scavenged by

antioxidants through donation of hydrogen to form the stable DPPH-H. So,

more rapidly of the absorbance decreases, the more potent the antioxidant

activity of the compounds was in terms of hydrogen donating ability (101).

Total DPPH radical scavenging capacities of fractions were

measured and compared in the two groups with different levels of omega-3

and omega-6. The liver and brain fractions from rat which fed 3% DHA had

a much weaker radical scavenging activity compared to 2% DHA and 1%

DHA fed rats firstly at all period of age studied. After 4 weeks of the

experiment and at a reaction time of 1 min, the liver and brain extract from

rat 1% DHA and 1% GLA exhibited the strongest activity, and followed by

the another omega-3 and omega-6 groups as shown in Figure 8a and 8b.

On the same per weight basis at 4 weeks and after 1 h of incubation,

26.4% of DPPH radicals were quenched by brain extract from rats fed the

1% DHA, while brain extract from the 2% DHA and 3% DHA were able to

quench 33.6% and 53.5%, respectively. Free radicals and radical-

mediated oxidation play roles in many aging-related health problems

including cancers and heart diseases. To better understand the beneficial

effects of natural antioxidant in oils, which used in processing infant

formula or food and differ in their reactions with free radicals, depending on

biological actions, more experiments should be done. In our present study,

positive correlation was found between RSA and the levels of tocopherols

in brain and liver extracted.

RESULTS

5.5 Oxidation stability during oven test

In this part of work, the objectives were to determine the oxidative

stability of the oils and their formulas for 12 days at 60°C, whereas the

times of storage was roughly comparable more to 6 months at room

temperature (20°C), when the general concept of 2 times the oxidation

reaction rate increment with 10°C increment (Q

= 2) was applied. On the

other hand, oil stability is determined under accelerated conditions (60°C

or more) for several reasons (i) ambient conditions demand an excessively

long period, (ii) ensure that the starting lipid does not contain high levels of

oxidation products (e.g. transition metals hexanal, free fatty acids and lipid

hydroperoxides) and (iii) at high temperatures can contribute to

decomposition of hydroperoxides, decomposition or volatility of

antioxidants, and decrease of oxygen solubility rapidly.

5.5.1 Hydroperoxide formation

5.5.1.1 Peroxide and p-anisidine values

Peroxide value is used to quantify the hydroperoixdes formed;

however these are intermediate products in the formation of carbonyl and

hydroxyl-compound (102). The oils were compared at three different of

LCPs (1% GLA containing 1% DHA, 3% GLA and 3% DHA) and oils

extracted from manufactured formulas containing the same fatty acids

composition. The changes of PV for oils and extracted oils from formulas

are illustrated in (Figure 9, p 88). PV remained with small changed at a

very low level (7.1 mmol/kg oil) over 192h in EO 1% GLA (Figure 9),

whereas the peroxides accumulated in EO 3% DHA to a high level after

eight days of storage and then decreased, as a result of hydroperoixdes

Part II Oxidative stability of formula

RESULTS

decomposition (102). With respect to control oils (Figure 9), PV sharply

increase up to 96h and then decreased throughout the rest of the storage

period, whereas PV was gradual increased by increasing content of LCPs;

suggested more oxidative problems with these group contained 3%DHA

compared to the others. To better elucidate degradation of the fat, a p-

anisidine assay was used to determine the aldehyde content of all oils

(Figure 10, p 89). Like peroxides, p-anisidine values were increased.

Thereafter, p-anisidine values were decreased, but to a lesser extent than

PV. Therefore, p-anisidine values, like peroxide values, proved also

unreliable as indicators of the degree of rancidity for fats exposed to

extreme oxidative challenge (103).

5.5.2 Ultraviolet absorptivity

LCPs oxidation can be analyzed by the absorptive increase in the

UV spectrum (104). During the storage time, lipids that have dienes and

polienes showed a changed in the double bonds positions, due to

isomeration and conjugation in the molecule (105). Dienes and trienes

formation on the oxidation initial steps and they show intense absorption at

233 and 270 nm, respectively (Figure 12, 13, 14 and 15, p 91-94).

Absorptive at 233 nm increased gradually with the increase in time, due to

the formation of conjugated dienes (CD) (Figure 12) during 288h. The CD

from both oils contained 3% DHA and neutral lipid were increased sharply

and peaked after 144 h. The sharp increase in the CD might be accounted

for by the formation of more and more hydroperoxides as primary products

of oxidation according to structure of non-polar lipid, whereas it is the

major component of lipid. On the other hand, control with α-tocopherol was

more stable than the all samples. After 192 h of storage, the CD values

were decreased, possibly due to the breakdown of unstable

hydroperoxides. On the another side, CD of EO containing 1% GLA as

RESULTS

well as netural lipid had low change during the storage, from 0 to 288 h,

indicating a good oxidative stability. Meanwhile, the changes in conjugated

tienes (CT) were less marked than the changes in CD during the storage

period. So, increases absorptivity at 270 nm after 6 days of heating being

as a result of rancid off-flavor compounds formation (aldehydes and

ketones). Parallel was observed between trends for changes in CD, PV

and AV.

The polar fractions extracted from formulas containing 1% emulsifier

had better OS compared to polar bulk oils fractions at the same induction

periods (Figure 14 and 15). It is seem to be due to the refining, bleaching

and deodorization process in which the phospholipids were almost

completely removed. The incomplete removal of phospholipid, promote the

initial breakdown of the primary antioxidants. Thus, any subsequent

storage, due to the lower level of antioxidant, will show a decreased

stability and a pro-oxidant effect of the PL (106). By comparison,

phospholipids gained of emulsifiers may be acting by quenching the free

radicals arising from PUFAs oxidation and thus improving the OS (104).

Therefore, it could be said that polar fractions added in bulk oils or oils

used in food industry were mainly responsible for their stabilities (105).

Ultraviolet scans between 220 and 320 nm after 12 days storage at 60

were shown in Figure 11.

5.5.2 Hydroperoxide decomposition

The primary oxidation products for unsaturated fatty acids are the

hydroperoxides, highly reactive compounds that decompose rapidly,

yielding a complex mixture of non-volatile and volatile compounds such as

hydrocarbons, aldehydes and ketones. Aldehydes are practically important

in stability of infant foods including DHA, which related to flavor alteration

and quality product (107).We were investigated the decomposition of

RESULTS

hydroperoxide as propanal, pentanal, hexanal and nonanal of oils

prepared from the same amount of LA, α-LA, AA and EPA, but only

different was the amount of DHA and/or GLA with 150 ppm of α-

tocopherol. Calibration curves were drawn for the four aldehydes. The

curves were followed linear relationship with highly significant correlation

coefficients (R

better than 0.9 and equal 0.99 for a large number of

compounds) (Figure 3b, p 84, Table 3b, p 26).

5.5.2.1 Oxidative stability of bulk oils

Pentanal and hexanal were originated from n-6 fatty acids (LA and

AA), while the propanal and nonanal were originated from n-3 and n-9 fatty

acid, respectively. In the control oil, the initial rate of hydroperoxides

decomposition increased sharply after storage period of 96 h. The low

propanal, hexanal, pentanal and nonanal values in 1% GLA oil containing

1% DHA agreed with the corresponding PV obtained for this oil (Figure 9).

With respect to the high content of n-3 fatty acids in 3%DHA oil was higher

propanal (33%), lower hexanal (49%) and pentanal (53%), after 192 h than

those obtained from 3% GLA oil (Figure 16, p 95-102). The lower hexanal

content without any off-flavor may be due to the addition of coriander oil.

However, these differences in relative stability may be due to not only to

fatty acids composition but also to interaction of α-tocopherols as

antioxidant (108).

5.5.2.2 Stability of extracted oils (EO)

Oil-in-water emulsions were prepared with oil 10 wt% of the same

LCPs containing DHA and/or GLA described above and 1% soy lecithin as

emulsifier in phosphate buffer at pH 3.8. The oxidative stability (OS) of

these extracted oils were significantly higher during the first 96 h than that

of the corresponding oils (Figure 16); and this may be resulted from many

RESULTS

factors (i) scavenging free radicals by amino acids of protein (109); (ii)

lecithin may be increasing the contact between the antioxidant and the

oxidizing fat (103) and/or; (iii) effecting of PC on the decomposition of

hydroperoxides by catalyze a non-radical pathway (104). Propanal

analysis was found to be the highest oil after 192 h in 3% DHA oil, while

the oppositely effect was observed in pentanal formation (Figure 16). On

the basis of hexanal formation, hexanal did not increase until 96 h of

storage period, and after 192 h the level was decreased. Decrease of

hexanal could be as a result of finished fatty acids oxidation, although this

assertion can not be categorical, because the differences could also be

ascribed to the manufacturing process

5.5.2.3 Oxidative stability of oil fractions

Bulks oils were sequentially fractionated with chloroform to recover

NL; followed by acetone to recover GL then methanol to recover PL.

Numerous studies have been focused on the oxidative stability of emulsion

containing lecithin. The OS of NL, GL and PL neither is so far nor reported

in the published literature. Hence, we may have for the first time definitively

established the OS of oil fractions. The importance of studying oil stability

fractions is reflected in the utilization of each fraction in the industry. As

expected in oils, NL (constituted mainly of triacylglycerols) was the major

fraction followed by GL and PL, respectively (Table 14, p 81).

RESULTS

Propanal and hexanal by GC-HS: chromatography of glycolipids of oils (A

)

1%GLA, (B) 3%GLA,

3%GLAand (F) 3%DHA after 96h at 60 ºC. Peaks numbered correspond to 1,

propanal; 2, pentanal; 3, hexanal; and 4, nonanal.

The ratio of saturated to polyunsaturated fatty acids (S/P) in the oil

fractions is summarized in Table 14. The fractions had a rather similar S/P

pattern wherein the ratio increased with the increase of the polarity of the

oil fraction. It was worthy to mention that the S/P ratio recorded the highest

level in the polar fractions (GL and PL).This may be explanation; the major

propanal content in polar fractions. On the basis of hexanal formation, in

the control oil, the rate of hexanal formation increased sharply after 96 h

(Figure 16), but a different order of stability was observed after 96 to 192 h

of storage period, detected at the higher level in polar lipids than the

neutral lipid fraction with increasing in the 3% GLA (Figure 16).Extended

storage, without daylight, results in high concentrations of propanal and

hexanal in polar fractions.

RESULTS

5.5.2.4 Thiobarbituric acid reactive substances (TBARS) Assay

A portion of the lipid hydroperoxides that form in the early stages of

lipid oxidation are broken down to form the low molecular weight volatile

compound (secondary products) that impart rancidity. Various low

molecular weight aldehydes, alkanals and nonvolatile precursors of these

substances react with 2-thiobarbituric acid (TBA), resulting in chromogens

termed 2-thiobarbituric acid-reactive substances (TBARS) that can be

determined spectrometric at 532 nm. TBARS values of oils and extracted

oils (EO) as well as the control are shown in Figure 17. During the test;

statistical evaluations (p< 0.05) showed that PUFAs significantly affected

the TBARS values. After the first 2 days of incubation, the lowest values

were observed for oil containing the lowest level of PUFAs. As the

accelerated storage period was extend up to 72 h, TBARS values of 3%

DHA oil increased gradually (Figure17, p 103).

TBARS of this oil was considerably higher than those of the EO 3%

DHA over the entire storage period. As explained earlier, removal of

antioxidant during process is responsible for the compromised stability of

the modified product. Meanwhile, TBARS values of the EO 1% DHA, as

such, remained nearly closed with control during the entire storage period,

indicated its good stability under oven conditions at 60 ºC. The general

increase in TBARS values of oils during storage time may be due to the

breakdown of lipid hydroperoxides and the production of secondary

products (110). Correlations were existed between peroxide value (PV)

and p-anisidine value (AV) (P <0.05) as well as 2-thiobarbituric acid-

reactive substances (TBARS) and headspace volatiles (propanal) content

(P<0.001) for most oils and extracted oils. Besides, it was found that a

negative correlation was demonstrated between the TBRAS formation and

total phenolic contents.

RESULTS

For each of the time periods studies, we measured the ratio of EPA

and DHA to palmitic acid. These values were used as markers of the lipid

oxidation progress. We would expect to decreases this ratio with an

increase in oxidation of EPA and DHA.

5.5.3 Effect of α-tocopherol in bulk oils and extracted oils (EO)

Lipid oxidation is the most critical parameter affecting in the shelf life

of the newer food products, which LCPs lipids have been incorporated.

The many different fatty acids hydroperoxide positional and geometrical

isomers that are formed during the oxidation of the ω-3 and ω-6 PUFAs up

on storage rise to a complex mixture of secondary oxidation products.

Therefore, we was investigated the effect of the conditions of (O/W

emulsion), pH 3.8, 150 ppm α-tocopherol and temperatures 50°C on the

oxidative stability in ω-3 and ω-6 enriched formulas and compared with the

same composition of bulk oils in a storage period (12 days) at 60°C.

Hydoperoxide formation and decomposition as well as antioxidant activity

were quantified for oils and finished products in storage. Quality testing of

several indicators of degradation in new oils enriched in omegas is

essential to enhancing quality, stability, and nutritional value of these foods

especially infant formulas.

The tocopherols are the most important natural antioxidants in fats

and oils. To understand the mechanism of α-tocopherol as an antioxidant

compound, it is well known that lipid oxidation started by the abstraction of

a hydrogen atom from an allylic or bis- allylic position of an USFA (RH) to

generate an alkyl radical , which combines with molecular oxygen at

diffusion- controlled rate to produce a lipid peroxyl radical (ROO

•

). The

ROO

•

, having longer lifetimes (ca. 7s) than R

•

(ca.

-8

), propagate the

oxidation reaction by selectively abstracting hydrogens from RH to form

RESULTS

lipid hydroperoxides (ROOH) and another R

•

Unsaturated fatty acids have

different susceptibilities to hydrogen abstraction, depending on the

dissociation energies of labile C-H bonds within the molecules. C-H bonds

adjacent to a double bond or at a tertiary carbon atom are weaker and

easier to break. For the FA series

–CH

-(CH= CH- CH

)n- CH

(n = 1-3), the

relative oxidation rates increase in the order 1: 40: 80, respectively. It was

established that the fatty acids oxidizability is much lower for MUSF

substrates (e.g. oleic acid) than mainly PUFAs. Thus the oxidize ability of

FA mixture is mainly depended on the number of allylic methlenes therein.

Thus, the α-tocopherol affected not only the overall formation of volatile

secondary oxidation products, but also the composition of this group of

oxidation products (109). In our study, the control oil without antioxidant

oxidised very rapidly and all the treatments related to hydroperoxide

formation and decomposition. The inhibition of hydroperoxide

decomposition by α-tocopherol between the control and sample containing

α-tocopherol increased with storage time (Table 15, p 82). With 150 ppm of

α-tocopherol, hexanal and propanal increased after 96 h. The inhibition

was increased after 144h with decreased in α-tocopherol amount; no

hexanal detected after this time. Mixture oil with 1% GLA in the presence

of 150 ppm had a good scavenging peroxyl radicals activity compared with

others but all oil mixtures had a very low total polyphenols. However; polar

lipids are found in high levels in EO, strong RSA of these components can

be expected as well as synergistic activity with α-tocopherol. On the other

hand, addition of lecithin containing phosphatidylcholine and

phosphatidyelethanolamine apparentely all facilitate hydrogen or electron

donation to tocopherols by the amine group. Hence, PL could be extending

the effectiveness of tocopherols by delaying the irreversible oxidation to

tocopheryl quinone, thereby delaying the oxidation. As detected, we

conclusion that these oils in stable up to 144 hour at 60ºC. Additional

RESULTS

studies are necessary to show the antioxidant activity between different

levels of tocopherols and PL, metal ions and poly phenols plus the effect of

LCPs n-3 on the oxidative and flavour stability of triglycerides remains

however an open and important question.

CONCLUSION

5 CONCLUSIONS

Each of the two experiment design contributes some evidence that

is relevant to possible causal linkages between tissues and plasma

concentrations of DHA and eicosanoid precussour. The relationship

between dietary fat and structural fats are very complex. The diet-

induced shifts in the fatty acid composition were greatest in liver, followed

by brain and then plasma. Plasma DHA can be used as proxies for

tissues DHA. Accretion liver of LCPs n-6 is more rapid than the LCPs n-3

series, rendering the latter more dependent upon dietary

supplementation than the former, while brain accretion is reversible,

depending on the metabolic pathway.

It would be expected that human infant who received vegetable oil-

based formulas which the essential fatty acids supplied by corn oil,

sunflower or safflower oil would exhibit a marked decline in brain DHA in

the first months of life, similar to that observed for the n-3 Def rats in the

present study.

It could be recommended that higher levels of DHA not contribute

to increase of DHA in brain, liver or plasma. Whereas addition 1% DHA

to formula had significant effect in an important time period during four

weeks, wherein this period is encompasses the majority of rat brain.

In the present study, adding a source of EPA+ DHA to the diet

concomitant with different amounts of GLA reduced mean AA

concentration in plasma and increased concentration of EPA. However,

DGLA increased when the ratio of EPA+DHA to GLA was 1: 0.5, 1: 1.5 or

1: 2.5. That is may be important to use for infant fed formula with a family

history of inflammatory or AD diseases.

CONCLUSION

Reduction of plasma AA in the second stage of our experiment

between 4 weeks and 8 weeks, is very important factor for patients with

CD or inflammatory condition, such as rheumatoid arthritis, are needed for

the development of additional treatment strategies of these patients.

Direct evaluation of antioxidants from tissues and plasma

contributed information to the antioxidant status of an individual. This

maybe useful for evaluating the risk for degenerative diseases and radical

scavenging activity could be a good marker for lipid peroxidation.

The OS of NL, GL and PL is neither so far nor reported in the

published literature. Hence, we may have for the first time definitively

established the OS of oil fractions. The importance of studying oil stability

fractions is reflected in the utilization of each fraction in the industry

especially infant rich PUFAs formulas processing.

The results of the present investigation indicated that level of LCPs,

initial PV and the level of polar lipids were significantly affect in the RSA of

the oils and EO. Beside, α-tocopherol was affected by the overall formation

of volatile secondary oxidation products.

In the light of these results, further studies will be required to:

1. Clarify the effects of long terms administration of DHA oil on lipid

metabolism and peroxidation.

2. Molecular and enzymatic studies for consumption a mixtures of

DHA, EPA plus GLA in some infant or adult inflammatory history

diseases.

3. Antioxidant activity between different levels of tocopherols and PL,

metal ions and polyphenols plus the effect of LCPs n-3 on the

oxidative and flavour stability of triglycerides remains an open and

important question.

REFERENCES

7 REFERENCES

1. Koletzko B (1997) Lipid supply for infants with special needs. Eur.J. Med.

Res. 21: 69–73.

2. Koletzko B (1999) Response to and range of acceptable fat intakes in

infants and children. Eur. J. Clin. Nutr. 53:78–83.

3. Giovannini M, Agostoni C, Salari PC (1991) The role of lipids in nutrition

during the first months of life. J. Int. Med.Res. 19:351–362.

4. Clandinin MT (1999): Brain development and assessing the supply of

polyunsaturated fatty acid. Lipids. 34:131–137.

5. Innis SM (1994) The 1993 Borden Award Lecture. Fatty acid requirements

of the newborn. Can. J. Physiol. Pharmacol. 72:1483–1492.

6. Gibson RA, Makrides M (1998) The role of long chain polyunsaturated fatty

acids (LC-PUFA) in neonatal nutrition. Acta Paediatr. 87:1017– 1022.

7. Uauy R, Mena P, Velenzuela A (1999) Essential fatty acids as

determinants of lipid requirements in infants, children and adults. Eur.

J. Clin. Nutr. 53:66–77.

8. Gibson RA, Neumann MA, Makrides M (1996) Effect of dietary

docosahexaenoic acid on brain composition and neural function in term

infants. Lipids. 1996, 31, 177–181.

9. Koletzko B, Demmelmair H, Hartl W, Kindermann A, Koletzko S,

Sauerwald T, Szitanyi P (1998) The use of stable isotope Techniques

for nutritional and metabolic research in Paediatrics. Early Hum. Dev.

53:77–97.

10. Kohn G, Sawatzki G, Biervliet JP, Rosseneu M (1994) Diet and the

essential fatty acids status of term infants. Acta Paediatr. Suppl.

402:69–74.

REFERENCES

11. Giovannini M, Agostoni C, Riva E (1994) Fat needs of term infants and fat

content of milk formulae. Acta Pediatric Suppl. 402:59–62.

12. Hamosh M, Salam N (1998) Long chain polyunsaturated fatty acids. Biol.

Neonate. 74:106-120.

13. Hamosh M (1998) Long chain polyunsaturated fatty acids- who needs

them? Biochem. Soc. Trans. 26:96-103.

14. Dobbing J (1990) vulnerable period in developing brain. In: Brain

Behaviour and Iron in the infant diet. Dobbing (Ed), Springer Verlag

(London). pp.1-17.

15. Chapkin RS (2000) Reappraisal of the essential fatty acids: Fatty acids in

foods and their health implications.

th edition

, Marcel Dekker, Inc. New

York, Basel. pp. 557- 568.

16. Calder PC, Field CJ (2002) Nutrition and Immune Function, CAB

International .pp. 67-101.

17. David NL, Michael MC (2005) Principles of Biochemistry. 4th edition, pp.

632-655.

18. Food and Nutrition Broad. Dietary Reference intake for Energy,

Carbohydrate, Fiber, Fat, Fatty acids, Cholesterol, Protein and Amino

acids. Washington, DC: The National Academies Press, 770. 2005.

19. Simopoulos A (1991) Omega-3 fatty acids in health and disease and in

growth and development. American Journal of Clinical Nutrition.

54:438-463.

20. Uauy DR, Valenzuela A (1996) Marine oils: the health benefits of n-3 fatty

acids. Nutrition Reviews. 54:S102-S108.

REFERENCES

21. Clandinin MT, Chappel JE, Leong S, Heim T, Swyer PR, Chance GW

(1980) Intrauterine fatty acid accretion in human brain : implications for

fatty acid requirements. Early Hum Dev. 4:121-129.

22. Uauy R, Mena P, Rojas C (2000) Essential fatty acids in early life:

Structural and functional role. Proceedings of the Nutrition Society.

59:3-15.

23. Smuts CM, Huang M, Mundy D (2003) A randomized trial of

decosahexaenoic acid supplementation during the third trimester of

pregnancy. Obstet Gynecol .101:469-479.

24. Simopoulos AP, Leaf A, Salem M (2000) Workshop on the essentiality of

and recommended dietary intakes of omega-6 and omega-3 fatty acids.

Prostaglandins Leukot Essent Fatty Acids. 63:119-121.

25. Olsen SF (1993) Consumption of marine n-3 fatty acids during pregnancy

as a possible determinant of birth weight. A review of current

epidemiological evidence. Epidemiologic Reviews. 15:399-413.

26. Hornstra G (2000) Essential Fatty acids in mothers and their neonates.

American Journal of Clinical Nutrition. 71:1262-1269

27. Wijendran V, Bendel RB, Couch SC, Philipson EH, Cheruku S, Lammi-

Keefe CJ (2000) Fetal Erythrocyte phospholipid polyunsaturated fatty

acids are altered in pregnancy complicated with gestational diabetes

mellitus. Lipids. 35:927-931.

28. Jensen CL, Maude M, Andreson RE, Heird WC (2000) Effect of

docosahexaenoic acid supplementation of lactating women on the fatty

acid composition of breast milk lipids and maternal and infant plasma

phospholipids. American Journal of Clinical Nutrition. 71:292S-299S.

REFERENCES

29. Makrides M, Gibson RA (2000) Long-chain polyunsaturated fatty acid

requirements during pregnancy and lactation. American Journal of

Clinical Nutrition. 71:307S-311S.

30. Connor WE, Lowensohn R, Hatcher L (1996) Increased docosahexaenoic

acid levels in human newborn infants by administration of sardines and

fish oil during pregnancy. Lipids. 31:183-187.

31. Cunnane SC, Francescutti V, Brenna JT, Crawford MA (2000) Breast-fed

infant achieve a higher rate of brain and whole body

docosahexaenoate accumulation than formula-fed infants not

consuming dietary docosahexaenoate. Lipids. 35:105-111.

32. Carlson SE (1999) Long-chain polyunsaturated fatty acids and

development of human infants. Acta Paediatr Suppl. 430:72-77.

33. Mitchell EA, Aman MG, Turbot SH, Manku M (1987) Clinical

characteristics and serum essential fatty acid levels in hyperactive

children. Clin Pediatr (Phila). 26:406-411.

34. Stevens LJ, Zentall JL, Abate ML, Watkins BA, Lipp SR, Burgess JR

(1995) Essential fatty acid metabolism in boys with attention-deficit

hyperactivity disorder. American Journal of Clinical Nutrition. 62:761-

768.

35. Innis SM, Adamkin DH, Hall RT (2002) Docosahexanoeic acid and

arachidonic acid enhance growth with no adverse effects in preterm

infants fed formula. Journal of Pediatrics. 10:547-554.

36. Birch EE, Garfield S, Hoffman DR, Uauy R (1992) Dietary essential fatty

acid supply and visual acuity development. Investigative

Ophthalmology and Visual Science. 33:3242-3253.

REFERENCES

37. Burgess JR, Stevens L, Zhang W, Peck L (2000) Long-chain

polyunsaturated fatty acids in children with attention deficit hyperactivity

disorder. Am J Clin Nutr . 71:327-330.

38. Shils ME, Olson JA, Shike M, Ross AC (1999) Modern Nutrition in Health

and Disease. 9th Edition, Williams & Wilkins, Inc., pp. 61-75.

39. Chow CK (2000) Fatty acids in foods and their health implications.

2th

Edition, Marcel Dekker, Inc. New York, Basel, pp.585- 595.

40. Stillwell W, Wassall SR (2005) Problems with essential Fatty acids: time

for a new Paradigm? Prog. Retinal Eye Res. 24:87-138.

41. Guivernau, M, Meza N, Barja P, Roman O (1994) Clinical and

experimental study on the long term affect of dietary GLA on plasma

fatty acids, paletet aggregation, thromoboxane formation, and

prostacyclin production. Prostaglandins Leukot Essent Fatty Acids. 51:

311-316.

42. Karlstad MD, DeMichele SJ, Leathem WD, Peterson MB (1993) Effect of

intravenous lipid emulsions enriched with gamma-linolenic acid on

plasma n-6 fatty acids and prostaglandin biosynthesis after burn and

exotoxin injury in rats. Am. J. Respir. Crit. Care Med. 21:1740-1749.

43. Calder PC (2006) fighting internal fires with fish fats: omega 3s for asthma

and more. Vital Choices. Issue 90, volume 3.

44. Nelson GJ (2000) Effects of dietary fatty acids on lipid metabolism: Fatty

acids in foods and their health implications.

2th Edition, Marcel Dekker,

Inc. New York, Basel, pp. 481- 516.

45. Horrobin DF, Bennett CN (1999b) New gene targets related to

schizophrenia and other psychiatric disorders: enzymes, binding

proteins and transport proteins involved in phospholipid and fatty acid

metabolism. Prostaglandins Leukot Essent Fatty Acids. 60:141–167.

REFERENCES

46. KP Su, SY Huang, Chiu CC, Shen WW (2003) Omega-3 fatty acids in

major depressive disorder. A preliminary double-blind, placebo-

controlled? European Neuropsychopharmacology. 13:267-271.

47. Conquer JA, Tierney MC, Zecevic J, Bettger WJ, R. H. Fisher RH (2000)

Fatty acid analysis of blood plasma of patients with Alzheimer's

disease, other types of dementia, and cognitive impairment. Lipids.

35:1305–1312.

48. Monsonego A, Maron R, Zota V., Selkoe DJ, Weiner HL (2001) Immune

hyporesponsiveness to amyloid beta-peptide in amyloid precursor

protein transgenic mice: implications for the pathogenesis and

treatment of Alzheimer's disease. Proc Natl Acad Sci USA. 98:10273-

10278.

49. Moriarty DM, Blackshaw AJ, Talbot PR, Griffiths HL (1999) Memory

dysfunction in multiple sclerosis corresponds to juxtacortical lesion load

on fast fluid-attenuated inversion-recovery MR images. Am J

Neuroradiol. 20:1956–1962.

50. Cohen M, Robinson RS, Bhagavan HN (2000) Antioxidant in dietary lipids:

Fatty acids in foods and their health implications.

2 th Edition, Marcel

Dekker, Inc. New York, Basel, pp. 439-450.

51. Bender DA (2003) Nutritional Biochemistry of the Vitamins. Cambridge

University Press, New York, pp 109-130.

52. Asplund K (2002) Antioxidant vitamins in the prevention of cardiovascular

disease: a systematic review. Journal of Internal Medicine. 251:372–

392.

53. Brigelius-Flohe R, Traber MG (1999) Vitamin E: function and metabolism.

FASEB Journal. 13:1145–1155.

54. Herrera E, Barbas C (2001) Vitamin E: action, metabolism and

perspectives. Journal of Physiology and Biochemistry. 57:43–56.

REFERENCES

55. Behne D, Kyriakopoulos A (2001) Mammalian selenium containing

proteins. Annual Reviews of Nutrition. 21:453–473.

56. Kamal-Eldin A, Appelqvist LA (1996) The chemistry and antioxidant

properties of tocopherols and tocotrienols. Lipids. 31:671–701.

57. Traber MG, Arai H (1999) Molecular mechanisms of vitamin E transport.

Annual Reviews of Nutrition. 19:343–55.

58. Bermond P (1990) Biological effects of food antioxidants: Food

antioxidants. Elsevier Applied Science, Clearance Center, Inc. London

and New York, pp. 204-211.

59. Lefkowity W, Lim S, Salem N (2005) Where does the developing brain

obtain it is docosahexanoic acid? Relative contributions of dietary

alpha-linolenic acid, docosahexanoic acid,and body stores in the

developing rat. Pediatric Research. 57:157-165.

60. Reeves PG, Neilsen FH, Fahey GC (1993) Committee report on the AIN-

93 purified rodent animal and diet. J. Nutr. 123:1939-1951.

61. Folch AC, Lees M, Sloane-Stanley GM (1957) A simple method for

isolation and purification of total lipids from animal tissues. J. Biol.

Chem. 226:497-509.

62. Kamal-Eldin A, Jan F, Alexander R, Bengt V (2001) Effect of dietary

Phenolic Compounds on Tocopherol, Cholesterol and fatty acids in

Rats .Lipids. 4:427-435.

63. Parker F, Peterson NF (1965) Quantitative analysis of phospholipids and

phospholipid fatty acids from silica gel thin-layer chromatograms.

J.Lipid Res. 6:455–460.

64. Makrides MM, Neumann RW, Byard KS, Gibson RA (1994) Fatty acid

composition of brain, retina and erythrocytes in breast and formula fed

infants. Am. J. Clin. Nuti. 60:189-194.

REFERENCES

65. Sang S, Lapsley K, Jeong WS, Lanchane PA, Ho CT, Rosen RT (2002)

Antioxidative phenolic compounds isolated from almond skins (Prunus

amygdalus Batsch). J. Agric. Food Chem. 50:2459-2463.

66. Huang SW, Satue-Gracia MT, Frankel EN, German JB (1999) Effect of

lactoferrin on oxidative stability of emulsions and liposomes. J. Agric.

Food Chem. 47:1356-1361.

67. Rouser G, Kritchevsky D, Simon G, Nelson GJ (1967) Quantative analysis

of brain and spinach leaf lipids employing silicic acid column

chromatography and acetone elution of glycolipids. Lipids. 2:37-42.

68. Pegg RB (2002) Spectrophotometric measurement of secondary lipid

oxidation products. In Current Protocols in Food Analytical chemistry;

Wrolstad, R. Ed.; Wiley: New York, pp D2.4.1- D2.4.18.

69. Kozak W, Soszynski D, Rudolph K, Conn CA, Kluger M J (1997) Dietary

n-3 fatty acids differentially affect sickness behavior in mice during local

and systemic inflammation. Am. J. Physiol. 272:R1298–R1307.

70. Das UN (2006) Essential fatty acids: biochemistry, physiology and

pathology. Biotechnology Journal.1:420-439.

71. Moriguchi T, Salem NJ (2003) Recovery of brain docosahexaenoate leads

to recovery of spatial task performance. J. Neurochem.87:297–309.

72. Laidlaw M, Holub B (2003) Effects of supplementation with fish oil-derived

n-3 fatty acids and gamma-linolenic acid on circulating plasma lipids

and fatty acid profiles in woman. The American of Clinical Nutrition.

77:37-42.

73. Jensen C, Chen H, Fraley K, Anderson RE, Heird W (1996). Biochemical

Effects of Dietary Linoleic/ alpha-LinolenIenic acid Ratio in Term Infant.

Lipids. 31:107-113.

REFERENCES

74. Sastry PS (1985) Lipids of nervous tissue: composition and metabolism.

Prog. Lipid Res. 24:69–176.

75. Lands WEM, Crawford GC (1976) Enzymes of membrane phospholipids

metabolism, In: The Enzymes of Biological Membranes (Marinosi, A.,

Ed.), Plenum, New York. pp. 3–85.

76. Greiner RS, Catalan JN, Salem NJ (2003) Docosapentanoic acid does

completely replace DHA in n3 FA deficient rats during early

development. Lipids. 38:431-435.

77. Barham JB, Edens MB, Fronteh AN, Johnson MM, Chilton FH (2000)

Addition of eicosapentaenoic acid to γ-linolenic acid-supplemented

diets prevents serum arschidonic acid accumulation in humans.J. Nutr.

130:1925–1931.

78. Anita Royneberg (2005) The impact of dietary sources of marine

polyunsaturated fatty acids on the fatty acid composition of rat brain,

liver and red blood cells. Master thesis for the degree in experimental

and human physiology. 47-63.

79. Ziboh VA, Fletcher MP (1992) Dose-response effects of dietary gamma-

linolenic acid-enriched oils on human polymorphonuclear-neutrophil

biosynthesis of leukotriene B4. Am. J. Clin. Nutr. 55:39–45.

80. Luthria DL, Sprecher H (1995) Metabolism of deuterium-labeled linoleic,

6, 9, 12 Octadecatrienoic, 8, 11, 14-eicosatrienoic, and arachidonic

acids in the rat. Journal of Lipid Research. 36:1897-1904.

81. Fan Y, Chapkin R, (1998) Importance of dietary -linolenic acid in human

health and nutrition. Journal of Nutrition. 128:1411–1414.

82. Rubin D, Laposata M (1992). Cellular interactions between n-6 and n-3

acids: a mass analysis of fatty acid elongation/desaturation, distribution

among complex lipids and conversion to eicosanoids. J. Lipid Res.

33:1431–1440.

REFERENCES

83. Graham J, Franks S, Bonney RC (1994) In vivo and in vitro effects of

gamma-linolenic acid and eicosapentaenoic acid on prostaglandin

production and arachidonic acid uptake by human endometrium.

Prostaglandins Leukot Essent Fatty Acids. 50:321–329.

84. Pawlosky RS, Hibbeln JR, Lin Y, Good son S, Riggs P, Sebring N,

Salem NJ (2003) Effect of beef and fish-based diets on the Kinetics of

n-3 fatty acid metabolism in human subjects. Am. J. Clin. Nutr. 77:565-

572.

85. Blok WL, Katan MB (1996) Modulation of inflammation and cytokine

production by dietary (n-3) fatty acids. J. Nutr. 126:1515–1533.

86. Mantzioris E, Cleland LG, Gibson RA, Neumann MA, Demasi M, James

MJ (2000) Biochemical effects of a diet containing foods enriched with

n-3 fatty acids. Am. J. Clin. Nutr. 72:42–48.

87. Chapkin RS, Somers SD, Erickson KL (1988) Dietary manipulation of

macrophage phospholipid classes: selective increase of dihomo

gamma linolenic acid. Lipids. 23:766–670.

88. DeMar JCJ, Ma K, Chang L, Bell JM, Rapoport SI (2005) Alpha-Linolenic

acid does not contribute appreciably to docosahexaenoic acid within

brain phospholipids of adult rats fed a diet enriched in

docosahexaenoic acid. Journal of Neurochemistry. 94:1063–1076.

89. Uauy R, Castillo C (2003) Liquid requirements of infants: implications for

nutrient composition of fortified complementary foods. Journal of

Nutrition. 133:2962S-2972S.

90. Uauy R, Mena P, Salem NJR (2000) Long chain polyunsaturated fatty

acid formation in neonates: effect of gestational ages and intrauterine

growth. Pediatr. Res. 47:127-135.

91. Boehm G, Borte M, Böhles HL, Müller H, Kohn G, Moro G (1996)

Docosahexanoic and arachidonic acid content of serum and red blood

REFERENCES

cell membrane phospholipids or preterm infants fed breast milk

standard formula or formula supplemented with n-3 and n-6 long-chain

polyunsaturated fatty acids. Eur.J. Pediatric. 155:410-416.

92. Sharrett AR, Sorlie PD, Chambless LE (1999) Relative importance of

various risk factors for asymptomatic carotid atherosclerosis versus

coronary heart disease incidence—the Atherosclerosis Risk in

Communities Study. Am. J. Epidemiol. 149:843–852.

93. Simon JA, Hodgkins ML, Browner WS, Neuhaus JM, Bernert JT, Hulley

SB (1995) Serum fatty acids and the risk of coronary heart disease.

Am. J. Epidemiol.142:469–476.

94. Wainwright PE, Huang YS, Coscina DV, Levesque S, Cutcheon DMc

(1994) Brain and behavioral effects of dietary n-3 de-ficiency in mice: a

three generational study. Dev. Psychobiol. 27:467–487.

95. Donzel AJ, Guenot L, Maupoil V, Rochette L, Rocquelin G (1993) Rat

Vitamin E Status and Heart Lipid Peroxidation: Effect of Dietary α-

Linolenic Acid and Marine n-3 Fatty Acids. Lipids. 28:651-655.

96. Chautam M, Calaf R, Honardi J, Charbonnier M, Andre M, Portugal H,

Pauli A-M, Lafont H, Nalbone G (1990).J. Lipid Res. 31:2201-2208.

97. Meydani SN, Shapiro AC, Meydani M, Macauley JB, Blumberg JB (1987).

Lipids. 22:345-350.

98. Amusquivar E, Rupérez FJ, Barbas C, Herrera E (2000) Low Arachidonic

Acid Rather than -Tocopherol Is Responsible for the Delayed

Postnatal Development in Offspring of Rats Fed Fish Oil Instead of

Olive Oil during Pregnancy and Lactation. Journal of

Nutrition.130:2855-2865.

99. Martin A, Janigian D, Shukitt-Hale B, Prior RL, Joseph JA (1999) Effect of

vitamin E intake on levels of vitamins E and C in the central nervous

REFERENCES

system and peripheral tissues: Implications for health

recommendations. Brain Res. 845:50-59.

100. Vatassery GT, Brin MF, Fahn S, Kayden HJ, Traber MG (1988) Effect of

high doses of dietary vitamin E on the concentration of vitamin E in

several brain regions, plasma, liver and adipose tissue of rats. J.

Neurochem. 51:621-623.

101. Martin TS, Kikuzaki H, Hisamoto M, Nakatani N (2000) Constituents of

Amomum tsao-ko and their radical scavenging and antioxidant

activities. J. Amer. Oil Chem. Soc. 77:667-673

102. Oliveira JTG, Regitano-D’Arce MAB (2004) Determination economical

TBHQ doses for corn oil stability. Cienc. Tecnol.Aliment. 24: 413-418.

103. Boyd, L.C (2001) Application of natural antioxidant in stabilizing

polyunsaturated fatty acids in a model systems and foods. In Omega-3

Fatty Acids, Chemistry, Nutrition and Healthy Effects; Finle JW, Shahidi

F., Eds; American Chemical Society: Washington, DC, pp 258-279.

104. Kolanowski W, Jaworska D and Weissbrodt J (2007) Importance of the

instrumental and sensory analysis in the assessment of oxidative

deterioration of omega 3 Long chain polyunsaturated fatty acid rich

foods. Journal of the Science of Food and Agriculture. 87: 181-191.

105. Shaihidi F, hamam F (2004) Synthesis of structured lipids via hydrolysis

of docosahexanoic acid single cell oil with capric acid. Agricultural and

Food Chemistry.52: 2900-2906.

106. Fomuso, LB, Corredig M, Akoh,CC (2002) Effect of emulsifier on

oxidation properties of fish oil-based structured lipid emulsions.

Agricultural and Food Chemistry.50: 2957-2961.

107. Llatas GG, Lagarada MJ, Romero F, Abellan P and Farre R (2006) A

headspace solid-phase microextraction method of use in monitoring

REFERENCES

hexanal, pentane during storage: Application to liquid infant foods and

powdered infant formulas. Food Chemistry. 101: 1078-1086.

108. Cortinas L, Galobart J, Barroeta Ana, Baucells MD and Grashorn MA

(2003) Change in α-tocopherol contents, lipid oxidation and fatty acid

profile in egg enriched with Linolenic acid or very Long-chain omega 3

polyunsaturated fatty acids after different processing methods. J. of the

Science of Food and Agricultural.83: 820-829.

109. Hu M, Clements DJ and Decker EA (2003) Impact of whey protein

emulsifiers on the oxidative stability of salmon oil-in-water emulsions. J.

Agricultural and Food Chemistry.51: 1435-1439.

110. Makinen M, Eldin AK, Lampi AM and Hopia A (2001) Tocopherols as

inhibitors of isomerization and decomposition of cis, trans methyl

linoleate hydroperoxides .Eur.J.Lipid Sci. Technol. 103:286-291.

TABLES AND FIGURES

Table 1:

Composition of the two experimental groups and deficient group diets

Omega-3 group

Omega-6 group

Ingredient

(g/ 100 g diet)

1% DHA

2% DHA

3% DHA

1% GLA

3% GLA

5% GLA

Deficient

group

Skim milk

powder 20 20 20 20 20 20 20

Carbohydrate 60 60 60 60 60 60 60

Sucrose 10 10 10 10 10 10 10

Malto-dextrin 15 15 15 15 15 15 15

Cornstarch 15 15 15 15 15 15 15

Dextrose 20 20 20 20 20 20 20

Fat 10 10 10 10 10 10 10

Hydrogenated

coconut oil

7.12

7.1

7.12 6.95

6.87

8.1

Mixed oils

2.88

2.91

3.00

2.87

3.05

3.23

1.9

Additives: 10 10 10 10 10 10 10

Cellulose 5 5 5 5 5 5 5

Salts 3.5 3.5 3.5 3.5 3.5 3.5 3.5

Vitamins 1 1 1 1 1 1 1

Choline

chloride 0.25 0.25 0.25 0.25 0.25 0.25 0.25

L-Cystine 0.25 0.25 0.25 0.25 0.25 0.25 0.25

THBQ* 0.002 0.002 0.002 0.002 0.002 0.002 0.002

Fatty acids composition

SFA 69.8 70.9 71.2 69.8 68.7 66.3 75.5

MFA 10.2 9.8 9.2 10.2 9.5 8.9 14.2

C18:2n-6 15.24 15.2 15.53 15.19 15.25 15.51 15.96

C18:3n-3 3.01 2.98 3.11 2.98 3.01 3.04 3.2

C18:3n-6 0.099 0.098 0.105 0.99 3.01 4.98 nd

C20:4n-6 0.58 0.55 0.53 0.58 0.59 0.57 nd

C22:5n-3 0.89 0.89 0.85 0.89 0.87 0.88 nd

C22:6n-3 0.99 2.01 3.1 0.99 1.01 1.11 nd

n-6 15.92 15.85 16.17 16.76 18.85 21.06 15.96

n-3 4.89 5.88 7.06 4.86 4.89 5.03 3.2

n-6/n-3 3.26 2.70 2.29 3.45 3.85 4.19 4.99

18:2/18:3 5.06 5.10 4.99 5.10 5.07 5.10 4.99

* THBQ, t-butylhydroquinone; SFA, total saturated fatty acids; MFA, total

monosaturated fatty acids; nd, not detected

TABLES AND FIGURES

Table 3: Liver fatty acids composition (%) of different levels of DHA during eight weeks

% DHA Control 1% DHA 2% DHA 3% DHA

Weeks At birth

4 8 4 8 4 8 4 8

C14:0 0.57± 0.00 0.25 ± 0.00 0.28 ± 0.00 0.40 ± 0.01 0.64 ± 0.02 0.63 ± 0.05 0.69 ± 0.05 0.46 ± 0.01 0.43 ± 0.05

C16:0 21.7 ± 0.21 25.2 ± 0.21 25.6 ± 0.11 20.4 ± 0.25 22.4 ± 0.74 21.2 ± 0.45 23.3 ± 0.55 21.3 ± 0.35 23.6 ± 0.23

C16:1n-7 1.06 ± 0.00 0.23 ± 0.00 0.28 ± 0.02 1.86 ± 0.12 3.91 ± 0.09 1.83 ± 0.17 3.31 ± 0.60 1.40 ± 0.14 1.31 ± 0.07

C18:0 19.9 ± 0.17 11.3 ± 0.05 12.5 ± 0.09 16.5 ± 0.36 16.1 ± 0.26 15.6 ± 0.35 15.4 ± 0.27 16.0 ± 0.45 15.6 ± 0.23

C18:1n-9 10.8 ± 0.08 19.5 ± 0.10 19.2 ± 0.12 12.0 ± 0.25 10.9 ± 0.14 13.1 ± 0.27 8.24 ± 0.11 10.2 ± 0.15 9.21 ± 0.12

C18:1n-7 2.02 ± 0.04 3.20 ± 0.05 3.10 ± 0.03 2.01 ± 0.04 4.59 ± 0.07 2.52 ± 0.09 3.89 ± 0.02 2.01 ± 0.07 1.31 ± 0.04

C18:2n-6 11.9 ± 0.12 12.3 ± 0.16 13.8 ± 0.13 13.3 ± 0.06 11.6 ± 0.11 15.3 ± 0.21 14.4 ± 0.45 14.0 ± 0.23 16.2 ± 0.14

C18:3n-6 0.61 ± 0.02 0.06 ± 0.00 0.04 ± 0.00 0.43 ± 0.00 0.21 ± 0.00 0.25 ± 0.00 0.22 ± 0.05 0.19 ± 0.02 0.18 ± 0.00

C20:0 0.16 ± 0.00 nd nd 0.44 ± 0.01 0.19 ± 0.01 0.92 ± 0.05 0.12 ± 0.00 0.59 ± 0.01 0.33 ± 0.00

C18.3n-3 0.12 ± 0.00 0.45 ± 0.04 0.52 ± 0.01 nd 0.05 ± 0.00 nd 0.04 ± 0.00 nd 0.08 ± 0.00

C18:4n-3 nd nd nd 0.23 ± 0.02 0.29 ± 0.00 0.35 ± 0.00 0.41 ± 0.03 0.86 ± 0.04 0.46 ± 0.01

C20:3n-6 0.91 ± 0.05 0.06 ± 0.00 0.04 ± 0.00 0.84 ± 0.03 1.50 ± 0.03 1.21 ± 0.14 1.58 ± 0.05 1.28 ± 0.07 1.46 ± 0.04

C22:0 0.31 ± 0.00 nd nd nd 0.19 ± 0.00 0.73 ± 0.05 nd 0.24 ± 0.00 0.04 ± 0.00

C20:4n-6 19.8 ± 0.23 14.1 ± 0.12 16.9 ± 0.07 17.6 ± 0.15 10.5 ± 0.20 12.1 ± 0.39 9.88 ± 0.38 11.3 ± 0.24 9.72 ± 0.11

C22:1n-9 nd nd nd 0.13 ± 0.00 nd 0.10 ± 0.00 nd 0.29 ± 0.00 0.27 ± 0.00

C20:5n-3 0.31 ± 0.02 0.49 ± 0.02 0.45 ± 0.03 1.5 ± 0.05 0.76 ± 0.01 1.75 ± 0.16 1.39 ± 0.09 5.52 ± 0.04 2.19 ± 0.02

C24:0 2.33 ± 0.02 3.68 ± 0.01 0.24 ± 0.00 0.66 ± 0.00 0.64 ± 0.06 0.39 ± 0.02 1.13 ± 0.04 0.53 ± 0.00 0.65 ± 0.01

C24:1 nd 2.45 ± 0.01 0.12 ± 0.00 0.21 ± 0.00

0.21 ± 0.00

0.15 ± 0.00

0.09 ± 0.00 0.42 ± 0.01 0.32 ± 0.00

C22:5n-3 1.49 ± 0.09 1.63 ± 0.02 0.75 ± 0.02 1.77 ± 0.12 1.39 ± 0.16 1.86 ± 0.09 1.60 ± 0.10 2.97 ± 0.12 1.82 ± 0.14

C22:6n-3 5.86 ± 0.11 4.93 ± 0.04 6.03 ± 0.05 9.41 ± 0.28 13.9 ± 0.52 9.91 ± 0.89 14.3 ± 0.33 9.98 ± 0.24 14.8 ± 0.19

∑ SFA

42.7 ± 0.35 40.43 ± 0.35 38.6 ± 0.28

38.4

± 0.41

40.2

± 0.67

39.5

± 0.55

40.6

± 0.64

39.1

± 0.48

40.7

± 0.41

∑ MUFA

14.2 ± 0.12 25.38 ± 0.15 22.7 ± 0.12

16.2

± 0.21

19.6

± 0.12

19.6

± 0.32

15.5

± 0.23

14.3

± 0.28

12.4

± 0.14

∑ PUFA

43.1 ± 0.42 34.02 ± 0.35 38.6 ± 0.29

45.1

± 0.33

40.5

± 0.49

42.7

± 0.95

43.8

± 0.45

46.1

± 0.35

46.9

± 0.25

n-3/n-6 0.29 0.28 0.25

0.4 0.69 0.48 0.68 0.72 0.7

AA/LA 1.66 1.15 1.23

1.32 0.91 0.79 0.69 0.81 0.6

AA/DGLA 21.79 235 423.25

20.95 7 10 6.25 8.83 6.66

EPA/AA 0.12 0.03 0.03

0.09 0.07 0.14 0.14 0.49 0.23

DHA/EPA 2.52 10.06 13.4

6.27 18.29 5.66 10.29 1.81 6.76

PI**

164.70 122.63 138.73 182.47 182.87 169.4 191.97 196.72 203.13

*Each parameter is present as the mean form three rat ± SD; SFA:

Total saturated fatty acids; MUFA: total

monounsaturated fatty acids; PUFA: to

tal polyunsaturated fatty

acids; LA: linoleic acid; DGLA: dihomogamma

-linolenic acid; AA: arachidonic acid; EPA: eicosapentanoic acid and DHA: docosahexanoic acid. nd, not detected

PI: Peroxidizability index (sum of percentages of individual fatty acids х number of active methylenes).

TABLES AND FIGURES

Table

Liver

fatty acids composition (%) of different levels of GLA during eight weeks

% GLA Control 1% GLA 3% GLA 5% GLA

Weeks At birth

4 8 4 8 4 8 4 8

C14:0 0.57± 0.00 0.25 ± 0.00 0.28 ± 0.00 0.40 ± 0.01 0.64 ± 0.02 0.40 ± 0.05 0.42 ± 0.00 0.49 ± 0.01 0.51 ± 0.05

C16:0 21.7 ± 0.21 25.2 ± 0.21 25.6 ± 0.11 20.4 ± 0.18 22.5 ± 0.23 19.3 ± 0.32 22.0 ± 0.11 19.3 ± 0.21 23.6 ± 0.35

C16:1n-7 1.06 ± 0.00 0.23 ± 0.00 0.28 ± 0.02 1.86 ± 0.00 3.50 ± 0.06 1.35 ± 0.05 1.40 ± 0.02 1.64 ± 0.04 2.11 ± 0.11

C18:0 19.9 ± 0.17 11.3 ± 0.05 12.5 ± 0.09 16.5 ± 0.12 16.1 ± 0.18 17.6 ± 0.29 18.2 ± 0.14 17.2 ± 0.16 18.9 ± 0.21

C18:1n-9 10.8 ± 0.08 19.5 ± 0.10 19.2 ± 0.12 12.0 ± 0.08 11.9± 0.21 9.20 ± 0.18 9.05 ± 0.11 9.82 ± 0.18 10.77 ± 0.14

C18:1n-7 2.02 ± 0.04 3.20 ± 0.05 3.10 ± 0.03 1.97 ± 0.02 4.59 ± 0.04 2.10 ± 0.04 2.10 ± 0.02 1.78 ± 0.12 2.13 ± 0.01

C18:2n-6 11.9 ± 0.12 12.3 ± 0.16 13.8 ± 0.13 13.3 ± 0.09 11.6 ± 0.07 12.3 ± 0.10 12.7 ± 0.16 12.5 ± 0.14 11.6 ± 0.18

C18:3n-6 0.61 ± 0.02 0.06 ± 0.00 0.04 ± 0.00 0.43 ± 0.02 0.21 ± 0.00 0.98 ± 0.04 0.31 ± 0.01 1.10 ± 0.00 0.40 ± 0.01

C20:0 0.16 ± 0.00 nd nd 0.44 ± 0.01 0.19 ± 0.00 0.17 ± 0.00 0.12 ± 0.00 0.37 ± 0.01 0.18 ± 0.00

C18.3n-3 0.12 ± 0.00 0.45 ± 0.04 0.52 ± 0.01 0.03 ± 0.00 0.05 ± 0.00 0.07 ± 0.00 0.06 ± 0.00 0.03 ± 0.00 0.05 ± 0.00

C18:4n-3 nd nd nd 0.23 ± 0.00 0.29 ± 0.04 0.30 ± 0.01 0.23 ± 0.03 0.47 ± 0.01 0.27 ± 0.00

C20:3n-6 0.91 ± 0.05 0.06 ± 0.00 0.04 ± 0.00 0.84 ± 0.03 1.5 ± 0.11 1.10 ± 0.03 1.47 ± 0.08 1.17 ± 0.01 1.42 ± 0.07

C22:0 0.31 ± 0.00 nd nd nd 0.19 ± 0.02 nd 0.15 ± 0.00 nd 0.16 ± 0.00

C20:4n-6 19.8 ± 0.23 14.1 ± 0.12 16.9 ± 0.07 18.1 ± 0.23 15.6 ± 0.32 25.9 ± 0.45 24.1 ± 0.32 24.9 ± 0.25 9.72 ± 0.11

C22:1n-9 nd nd nd 0.13 ± 0.00 nd nd nd 0.07 ± 0.00 0.27 ± 0.00

C20:5n-3 0.31 ± 0.02 0.49 ± 0.02 0.45 ± 0.03 1.50 ± 0.01 0.76 ± 0.04 0.28 ± 0.00 0.25 ± 0.04 0.45 ± 0.01 2.19 ± 0.02

C24:0 2.33 ± 0.02 3.68 ± 0.01 0.24 ± 0.00 0.66 ± 0.01 0.64 ± 0.06 0.90 ± 0.12 0.52 ± 0.11 1.39 ± 0.04 0.85 ± 0.04

C24:1 nd 2.45 ± 0.01 0.12 ± 0.00 nd nd 0.30 ± 0.01 0.36 ± 0.04 0.03 ± 0.00 0.26 ± 0.02

C22:5n-3 1.49 ± 0.09 1.63 ± 0.02 0.75 ± 0.02 1.77 ± 0.04 1.39 ± 0.04 1.72 ± 0.01 1.14 ± 0.09 1.35 ± 0.04 0.93 ± 0.07

C22:6n-3 5.86 ± 0.11 4.93 ± 0.04 6.03 ± 0.05 9.41 ± 0.13 8.13 ± 0.24 5.96 ± 0.11 5.43 ± 0.17 5.59 ± 0.08 4.33 ± 0.21

SFA 42.7 ± 0.35 40.43 ± 0.35 38.6 ± 0.28 38.4 ± 0.28 40.3 ± 0.41 38.3 ± 0.61 41.4 ± 0.35 38.8 ± 0.37 44.2 ± 0.54

MUFA 14.2 ± 0.12 25.38 ± 0.15 22.7 ± 0.12 16.0 ± 0.07 20.0 ± 0.28 13.0 ± 0.21 12.9 ± 0.15 13.6 ± 0.35 15.3 ± 0.24

PUFA 43.1 ± 0.42 34.02 ± 0.35 38.6 ± 0.29 45.6 ± 0.35 39.5 ± 0.74 48.6 ± 0.58 45.7 ± 0.68 47.6 ± 0.55 40.5 ± 0.68

n-3/n-6 0.29 0.28 0.25

0.4 0.37 0.21 0.18 0.20 0.17

AA/LA 1.66 1.15 1.23

1.36 1.34 2.11 1.89 2.00 1.82

AA/DGLA 21.79 235 423.25

21.6 10.4 23.5 16.4 21.3 14.9

EPA/AA 0.12 0.03 0.03

0.08 0.05 0.01 0.01 0.02 0.02

DHA/EPA 2.52 10.06 13.4

6.27 10.70 21.29 21.72 12.42 12.72

PI 164.70 122.63 138.73 184.6 157.1 181.4 165.6 174.6 143.7

** Each parameter given is the mean from three rat’s ± SD. For abbreviations see table 3.

TABLES AND FIGURES

Table

Plasma

fatty acids composition (%) of different levels of DHA during eight weeks

% DHA Control 1% DHA 2% DHA 3% DHA

Weeks At birth

4 8 4 8 4 8 4 8

C14:0 1.41± 0.00 0.43 ± 0.00 0.38 ± 0.00

1.05 ± 0.03 1.36 ± 0.09 1.72 ± 0.02 1.92 ± 0.01 1.88 ± 0.02 1.56 ± 0.02

C16:0 28.7 ± 0.10 30.1 ± 0.14 31.1 ± 0.11

23.8 ± 0.15 26.3 ± 0.19 25.2 ± 0.25 26.8 ± 0.36 23.9 ± 0.16 24.7 ± 0.17

C16:1n-7 1.64 ± 0.00 0.52 ± 0.00 0.58 ± 0.02

0.53 ± 0.01 1.58 ± 0.02 1.74 ± 0.08 1.77 ± 0.02 1.32 ± 0.05 1.84 ± 0.04

C18:0 8.38 ± 0.07 12.5 ± 0.06 12.7 ± 0.09

12.1 ± 0.09 12.4 ± 0.06 8.91 ± 0.14 10.1 ± 0.15 10.3 ± 0.28 11.8 ± 0.19

C18:1n-9 17.5 ± 0.08 14.1 ± 0.11 15.2 ± 0.12

15.4 ± 0.14 16.4 ± 0.17 18.4 ± 0.21 18.8 ± 0.25 19.0 ± 0.06 19.3 ± 0.26

C18:1n-7 1.62 ± 0.04 nd nd

1.94 ± 0.04 1.91 ± 0.09 1.16 ± 0.05 1.45 ± 0.06 1.69 ± 0.07 1.78 ± 0.09

C18:2n-6 21.1 ± 0.11 16.4 ± 0.16 9.70 ± 0.13

14.5 ± 0.28 14.3 ± 0.19 18.0 ± 0.24 17.1 ± 0.19 21.9 ± 0.29 21.1 ± 0.19

C18:3n-6 1.23 ± 0.02 0.11 ± 0.00 0.06 ± 0.00

0.20 ± 0.00 0.66 ± 0.00 0.39 ± 0.04 0.76 ± 0.04 0.27 ± 0.01 0.88 ± 0.03

C20:0 0.42 ± 0.00 nd nd

0.32 ± 0.00 0.43 ± 0.03 0.34 ± 0.01 0.47 ± 0.01 0.35 ± 0.08 0.42 ± 0.02

C18.3n-3 1.26 ± 0.00 0.64 ± 0.04 0.54 ± 0.01

6.05 ± 0.11 4.41 ± 0.09 2.67 ± 0.09 2.64 ± 0.06 2.78 ± 0.07 1.56 ± 0.01

C18:4n-3 nd 0.28 ± 0.00 nd

1.69 ± 0.09 1.02 ± 0.11 1.56 ± 0.06 1.36 ± 0.05 1.59 ± 0.03 1.14 ± 0.04

C20:3n-6 0.73 ± 0.05 0.03 ± 0.00 0.02 ± 0.00

0.54 ± 0.00 0.54 ± 0.04 0.98 ± 0.04 0.78 ± 0.03 0.59 ± 0.02 0.85 ± 0.02

C22:0 0.63 ± 0.01 nd nd

0.36 ± 0.00 0.31 ± 0.01 0.35 ± 0.01 0.34 ± 0.01 0.27 ± 0.01 0.35 ± 0.01

C20:4n-6 8.89 ± 0.09 18.1 ± 0.12 24.2 ± 0.07

14.3 ± 0.35 8.93 ± 0.28 10.7 ± 0.35 7.04 ± 0.38 7.16 ± 0.29 4.37 ± 0.19

C22:1n-9 nd nd nd

0.38 ± 0.01 0.30 ± 0.02 0.63 ± 0.05 0.45 ± 0.04 0.21 ± 0.02 0.22 ± 0.01

C20:5n-3 3.08 ± 0.02 2.5 ± 0.02 0.48 ± 0.03

1.63 ± 0.04 1.04 ± 0.01 2.15 ± 0.07 1.57 ± 0.15 2.58 ± 0.05 2.09 ± 0.08

C24:0 0.24 ± 0.02 0.18 ± 0.01 0.21 ± 0.00

0.53 ± 0.01 0.63 ± 0.08 0.64 ± 0.01 0.67 ± 0.03 0.66 ± 0.08 0.59 ± 0.07

C24:1 nd nd nd nd

C22:5n-3 0.61 ± 0.09 0.60 ± 0.02 0.15 ± 0.02 0.85 ± 0.02 0.47 ± 0.01 0.79 ± 0.03 0.42 ± 0.01 0.71 ± 0.04 0.45 ± 0.08

C22:6n-3 2.02 ± 0.11 3.50 ± 0.04 4.50 ± 0.05 3.26 ± 0.18 6.95 ± 0.48 3.58 ± 0.36 5.53 ± 0.29 2.77 ± 0.13 4.85 ± 0.19

SFA 39.5 ± 0.18 43.2 ± 0.18 44.4 ± 0.18 38.2 ± 0.18 41.4 ± 0.28 37.2 ± 0.35 40.3 ± 0.45 37.4 ± 0.35 39.4 ± 0.28

MUFA 23.8 ± 0.11 14.6 ± 0.12 15.8 ± 0.09 18.3 ± 0.13 20.2 ± 0.21 21.9 ± 0.28 22.5 ± 0.28 22.2 ± 0.18 23.1 ± 0.29

PUFA 36.3 ± 0.25 42.1 ± 0.28 39.8 ± 0.23 43.9 ± 0.58 38.3 ± 0.94 40.9 ± 0.95 37.2 ± 0.65 40.4 ± 0.55 37.3 ± 0.59

n-3/n-6 0.14 0.22 0.17 0.46 0.57 0.36 0.45 0.35 0.37

AA/LA 0.42 1.11 2.49 0.99 0.62 0.60 0.41 0.33 0.21

AA/DGLA 12.18 603.33 1210.0 26.56 16.54 10.99 9.03 12.14 5.14

EPA/AA 0.05 0.14 0.02 0.11 0.12 0.20 0.22 0.36 0.48

DHA/EPA 4.70 1.40 9.38 2.00 6.68 1.67 3.52 1.07 2.32

PI 86.10 138.40 148.39 133.62 130.48 122.23 115.80 106.64 104.34

** Each parameter given is the mean from three rat’s ± SD. For abbreviations see table 3.

TABLES AND FIGURES

Table

Plasma

fatty acids composition (%) of different levels of GLA during eight weeks

% GLA Control 1% GLA 3% GLA 5% GLA

Weeks At birth 4 8 4 8 4 8 4

C14:0 1.41± 0.00 0.43 ± 0.00 0.38 ± 0.00 1.05 ± 0.03 1.26 ± 0.09 1.22 ± 0.02 1.28 ± 0.01 1.18 ± 0.02

1.24 ± 0.02

C16:0 28.7 ± 0.10 30.1 ± 0.14 31.1 ± 0.11 24.5 ± 0.18 25.6 ± 0.24 24.1 ± 0.23 26.7 ± 0.29 23.2 ± 0.23

27.0 ± 0.35

C16:1n-7 1.64 ± 0.00 0.52 ± 0.00 0.58 ± 0.02 1.53 ± 0.01 2.95 ± 0.01 1.01 ± 0.01 1.90 ± 0.07 1.91 ± 0.04

2.69 ± 0.02

C18:0 8.38 ± 0.07 12.5 ± 0.06 12.7 ± 0.09 10.1 ± 0.11 11.0 ± 0.14 11.2 ± 0.14 11.5 ± 0.24 11.7 ± 0.18

11.5 ± 0.17

C18:1n-9 17.5 ± 0.08 14.1 ± 0.11 15.2 ± 0.12 15.6 ± 0.12 14.4 ± 0.21 13.2 ± 0.08 14.4 ± 0.29 13.0 ± 0.12

15.0 ± 0.21

C18:1n-7 1.62 ± 0.04 nd nd 1.94 ± 0.04 2.91 ± 0.05 1.85 ± 0.04 2.55 ± 0.05 1.76 ± 0.01

2.35 ± 0.04

C18:2n-6 21.1 ± 0.11 16.4 ± 0.16 9.70 ± 0.13 14.3 ± 0.21 20.2 ± 0.21 15.5 ± 0.09 19.3 ± 0.32 16.5 ± 0.18

19.5 ± 0.32

C18:3n-6 1.23 ± 0.02 0.11 ± 0.00 0.06 ± 0.00 0.25 ± 0.00 0.23 ± 0.00 1.62 ± 0.04 1.40 ± 0.04 1.70 ± 0.04

1.30 ± 0.07

C20:0 0.42 ± 0.00 nd nd 0.32 ± 0.01 0.43 ± 0.01 0.32 ± 0.00 0.14 ± 0.00 0.33 ± 0.00

0.20 ± 0.00

C18.3n-3 1.26 ± 0.00 0.64 ± 0.04 0.54 ± 0.01 6.05 ± 0.17 1.41 ± 0.04 6.51 ± 0.21 1.18 ± 0.11 6.91 ± 0.15

1.19 ± 0.02

C18:4n-3 nd 0.28 ± 0.00 nd 1.89 ± 0.03 1.02 ± 0.02 0.43 ± 0.02 0.99 ± 0.08 0.90 ± 0.01

0.81 ± 0.01

C20:3n-6 0.73 ± 0.05 0.03 ± 0.00 0.02 ± 0.00 0.54 ± 0.02 0.84 ± 0.01 0.46 ± 0.04 1.01 ± 0.01 0.37 ± 0.04

0.89 ± 0.03

C22:0 0.63 ± 0.01 nd nd 0.16 ± 0.00 0.11 ± 0.00 0.43 ± 0.00 0.10 ± 0.00 0.17 ± 0.00

0.21 ± 0.00

C20:4n-6 8.89 ± 0.09 18.1 ± 0.12 24.2 ± 0.07 14.3 ± 0.07 12.2 ± 0.21 13.6 ± 0.21 13.4 ± 0.35 13.8 ± 0.21

13.5 ± 0.17

C22:1n-9 nd nd nd 0.08 ± 0.00 0.03 ± 0.00 0.20 ± 0.00 0.14 ± 0.00 0.24 ± 0.00

0.12 ± 0.00

C20:5n-3 3.08 ± 0.02 2.5 ± 0.02 0.48 ± 0.03 1.63 ± 0.02 1.18 ± 0.02 5.28 ± 0.11 2.14 ± 0.07 3.07 ± 0.02

2.01 ± 0.09

C24:0 0.24 ± 0.02 0.18 ± 0.01 0.21 ± 0.00 1.01 ± 0.02 0.63 ± 0.01 0.85 ± 0.04 0.60 ± 0.02 0.85 ± 0.11

0.78 ± 0.04

C24:1 nd nd nd nd nd nd nd nd

C22:5n-3 0.61 ± 0.09 0.60 ± 0.02 0.15 ± 0.02 0.55 ± 0.04 0.47 ± 0.00 0.43 ± 0.01 0.25 ± 0.01 0.61 ± 0.04

0.35 ± 0.00

C22:6n-3 2.02 ± 0.11 3.50 ± 0.04 4.50 ± 0.05 4.06 ± 0.10 3.05 ± 0.04 1.64 ± 0.42 1.04 ± 0.01 1.82 ± 0.05

1.01 ± 0.08

SFA 39.5 ± 0.18 43.2 ± 0.18 44.4 ± 0.18 37.2 ± 0.16 39.0 ± 0.40 38.1 ± 0.12 40.4 ± 0.52 37.4 ± 0.16

40.9 ± 0.54

MUFA 23.8 ± 0.11 14.6 ± 0.12 15.8 ± 0.09 19.2 ± 0.48 20.3 ± 0.27 16.2 ± 0.51 18.9 ± 0.39 16.9 ± 0.19

18.75 ± 0.23

PUFA 36.3 ± 0.25 42.1 ± 0.28 39.8 ± 0.23 43.5 ± 0.18 40.6 ± 0.54 45.5 ± 0.23 40.7 ± 0.94 45.6 ± 0.37

40.4 ± 0.74

n-3/n-6 0.14 0.22 0.17 0.48 0.21 0.46 0.16 0.41

0.15

AA/LA 0.42 1.11 2.49 1.00 0.60 0.88 0.70 0.83

0.70

AA/DGLA 12.18 603.33 1210.0 26.39 14.52 29.70 13.28 37.19

15.21

EPA/AA 0.05 0.14 0.02 0.11 0.10 0.39 0.16 0.22

0.15

DHA/EPA 4.70 1.40 9.38 2.49 2.58 0.31 0.49 0.59

0.40

PI 86.10 138.40 148.39 138.5 112.8 136.8 107.2 130.2

104.7

** Each parameter given is the mean from three rat’s ± SD. For abbreviations see table 3.

TABLES AND FIGURES

Tab

Brain

fatty acids composition (%) of different levels of DHA during eight weeks

% DHA

Control

1% DHA 2% DHA 3% DHA

Weeks At birth 4 8 4 8 4 8 4 8

C14:0 0.87 ± 0.00 1.40 ± 0.00 1.60 ± 0.01 0.24 ± 0.00 0.41 ± 0.02 0.21 ± 0.01 0.34 ± 0.02 0.52 ± 0.03 0.4 ± 0.01

C16:0 25.3 ± 0.11 26.8 ± 0.12 26.4 ± 0.14 21.2 ± 0.15 21.6 ± 0.12 21.8 ± 0.11 22.4 ± 0.16 23.1 ± 0.15 22.1± 0.15

C16:1n-7 1.46 ± 0.01 1.15 ± 0.01 1.24 ± 0.03 0.47 ± 0.01 0.47 ± 0.03 0.45 ± 0.00 0.41 ± 0.01 0.6 ± 0.05 0.4 ± 0.02

C18:0 18.8 ± 0.12 21.7 ± 0.05 22.1 ± 0.08 20.2 ± 0.31 21.5 ± 0.25 21.7 ± 0.14 22.1 ± 0.18 21.7 ± 0.12 20.9 ± 0.15

C18:1n-9 19.5 ± 0.04 18.0 ± 0.07 18.4 ± 0.04 19.5 ± 0.25 19.1 ± 0.08 18.4 ± 0.23 18.0 ± 0.35 18.7 ± 0.28 18.2 ± 0.23

C18:1n-7 2.82 ± 0.01 4.98 ± 0.03 3.50 ± 0.01 4.26 ± 0.03 4.01 ± 0.01 4.4 ± 0.1 4.01 ± 0.01 4.3 ± 0.15 4.01 ± 0.02

C18:2n-6 8.51 ± 0.02 1.42 ± 0.00 1.53 ± 0.02 0.94 ± 0.01 0.97 ± 0.05 0.93 ± 0.02 0.8 ± 0.02 1.12 ± 0.05 0.95 ± 0.02

C18:3n-6 0.15 ± 0.00 1.04 ± 0.00 0.11 ± 0.00 0.07 ± 0.00 nd 0.05 ± 0.00 0.1 ± 0.01 0.85 ± 0.04 0.11 ± 0.01

C20:0 0.15 ± 0.00 0.12 ± 0.00 0.30 ± 0.00 0.07 ± 0.00 nd 0.03 ± 0.00 nd 0.16 ± 0.00 nd

C18.3n-3 0.15 ± 0.00 0.06 ± 0.00 0.41 ± 0.00 0.49 ± 0.02 0.52 ± 0.00 0.5 ± 0.01 0.41 ± 0.02 0.46 ± 0.01 0.44 ± 0.01

C18:4n-3 nd 0.75 ± 0.01 0.78 ± 0.01 1.61 ± 0.03 1.82 ± 0.01 1.36 ± 0.02 1.89 ± 0.05 1.74 ± 0.21 1.72 ± 0.04

C20:3n-6 1.01 ± 0.01 1.28 ± 0.04 0.25 ± 0.00 0.69 ± 0.01 0.91 ± 0.06 0.53 ± 0.00 1.1 ± 0.02 0.65 ± 0.02 1.53 ± 0.05

C22:0 0.14 ± 0.00 0.42 ± 0.00 0.45 ± 0.01 0.47 ± 0.02 0.52 ± 0.01 0.55 ± 0.00 0.5 ± 0.01 0.48 ± 0.02 0.69 ± 0.02

C20:4n-6 12.9 ± 0.15 9.74 ± 0.08 13.2 ± 0.13 11.8 ± 0.15 10.5 ± 0.23 11.3 ± 0.14 9.69 ± 0.24 9.93 ± 0.25 9.01 ± 0.21

C22:1n-9 nd 0.12 ± 0.00 0.05 ± 0.00 0.4 ± 0.01 0.19 ± 0.01 0.31 ± 0.00 0.29 ± 0.01 0.35 ± 0.01 0.71 ± 0.02

C20:5n-3 0.24 ± 0.00 0.49 ± 0.00 1.07 ± 0.03 0.49 ± 0.01 0.45 ± 0.00 0.49 ± 0.03 1.07 ± 0.04 0.44 ± 0.01 1.78 ± 0.06

C24:0 1.68 ± 0.01 3.56 ± 0.02 1.45 ± 0.03 2.82 ± 0.02 1.85 ± 0.04 2.42 ± 0.01 1.55 ± 0.03 1.65 ± 0.02 1.71 ± 0.03

C24:1 nd 1.46 ± 0.01 0.35 ± 0.00 0.59 ± 0.00 0.96 ± 0.01 0.96 ± 0.01 0.63 ± 0.01 0.24 ± 0.01 0.6 ± 0.01

C22:5n-3 0.23 ± 0.00 0.25 ± 0.00 0.23 ± 0.00 0.32 ± 0.02 0.25 ± 0.00 0.3 ± 0.00 0.25 ± 0.02 0.43 ± 0.01 0.65 ± 0.02

C22:6n-3 6.05 ± 0.05 5.10 ± 0.07 6.55 ± 0.03 13.2 ± 0.11 13.9 ± 0.08 13.3 ± 0.11 14.4 ± 0.41 12.6 ± 0.53 14.0 ± 0.24

∑ SFA

45.3 ± 0.22 54.0 ± 0.18 52.3 ± 0.21 45.0 ± 0.23 45.9 ± 0.19 46.7 ± 0.16 46.9 ± 0.19 47.3 ± 0.11 45.8 ± 0.12

∑ MUFA

24.0 ± 0.04 25.7 ± 0.09 23.5 ± 0.03 25.2 ± 0.18 24.7 ± 0.23 24.5 ± 0.23 23.4 ± 0.34 24.2 ± 0.12 23.9 ± 0.15

∑ PUFA

30.7 ± 0.19 20.1 ± 0.10 24.1 ± 0.21 29.6 ± 0.14 29.3 ± 0.28 28.8 ± 0.12 29.7 ± 0.48 28.4 ± 0.38 29.9 ± 0.31

n-3/n-6 0.36 0.49 0.60 1.19 1.37 1.25 1.54 1.27 1.58

AA/LA 1.52 6.86 8.63 12.55 10.82 12.15 12.11 8.87 9.48

AA/DGLA 12.77 7.61 52.80 17.10 11.54 21.32 8.81 15.28 5.89

EPA/AA 0.13 0.05 0.08 0.04 0.04 0.04 0.11 0.04 0.20

DHA/EPA 3.60 10.41 6.12 26.94 30.89 27.14 13.46 28.64 7.87

123.19 94.02 119.78 168.17 169.13 165.48 174.04 159.66 173.89

** Each parameter given is the mean from three rat’s ± SD. For abbreviations see table 3.

TABLES AND FIGURES

Tab

Brain

fatty acids composition (%) of different levels of GLA during eight weeks

% GLA

Control

1% GLA 3% GLA 5% GLA

Weeks

At birth 4 8 4 8 4 8 4 8

C14:0 0.87 ± 0.00 1.40 ± 0.00 1.60 ± 0.01 0.24 ± 0.00 0.41 ± 0.02 0.32 ± 0.01 0.13 ± 0.02 0.30 ± 0.03

0.13 ± 0.01

C16:0 25.3 ± 0.11 26.8 ± 0.12 26.4 ± 0.14 21.2 ± 0.32 20.3 ± 0.24 21.1 ± 0.23 21.2 ± 0.18 21.4 ± 0.12

20.1 ± 0.32

C16:1n-7 1.46 ± 0.01 1.15 ± 0.01 1.24 ± 0.03 0.47 ± 0.01 0.47 ± 0.01 0.61 ± 0.01 0.45 ± 0.05 0.46 ± 0.01

0.38 ± 0.01

C18:0 18.8 ± 0.12 21.7 ± 0.05 22.1 ± 0.08 20.2 ± 0.12 21.3 ± 0.21 21.4 ± 0.29 21.7 ± 0.24 19.9 ± 0.14

21.9 ± 0.21

C18:1n-9 19.5 ± 0.04 18.0 ± 0.07 18.4 ± 0.04 19.5 ± 0.11 20.1 ± 0.14 18.9 ± 0.24 19.4 ± 0.32 19.2 ± 0.12

18.5 ± 0.25

C18:1n-7 2.82 ± 0.01 4.98 ± 0.03 3.50 ± 0.01 4.33 ± 0.09 4.58 ± 0.09 4.29 ± 0.11 4.58 ± 0.17 4.32 ± 0.09

4.08 ± 0.17

C18:2n-6 8.51 ± 0.02 1.42 ± 0.00 1.53 ± 0.02 0.94 ± 0.02 0.92 ± 0.05 1.35 ± 0.02 0.79 ± 0.04 1.37 ± 0.03

0.76 ± 0.04

C18:3n-6 0.15 ± 0.00 1.04 ± 0.00 0.11 ± 0.00 0.13 ± 0.00 0.20 ± 0.00 0.24 ± 0.00 0.02 ± 0.00 0.48 ± 0.00

0.02 ± 0.00

C20:0 0.15 ± 0.00 0.12 ± 0.00 0.30 ± 0.00 nd nd 0.02 ± 0.00 nd 0.02 ± 0.00

C18.3n-3 0.15 ± 0.00 0.06 ± 0.00 0.41 ± 0.00 0.49 ± 0.01 0.52 ± 0.00 0.39 ± 0.00 0.45 ± 0.02 0.56 ± 0.01

0.54 ± 0.01

C18:4n-3 nd 0.75 ± 0.01 0.78 ± 0.01 1.61 ± 0.04 2.00 ± 0.02 1.41 ± 0.07 1.98 ± 0.06 1.73 ± 0.09

1.78 ± 0.11

C20:3n-6 1.01 ± 0.01 1.28 ± 0.04 0.25 ± 0.00 0.69 ± 0.01 0.91 ± 0.01 0.53 ± 0.01 0.73 ± 0.04 0.71 ± 0.04

1.25 ± 0.07

C22:0 0.14 ± 0.00 0.42 ± 0.00 0.45 ± 0.01 0.47 ± 0.00 0.52 ± 0.00 0.39 ± 0.00 0.39 ± 0.00 0.52 ± 0.00

0.38 ± 0.00

C20:4n-6 12.9 ± 0.15 9.74 ± 0.08 13.2 ± 0.13 11.8 ± 0.14 10.5 ± 0.21 11.1 ± 0.23 10.9 ± 0.21 11.7 ± 0.11

11.2 ± 0.25

C22:1n-9 nd 0.12 ± 0.00 0.05 ± 0.00 nd nd 0.16 ± 0.00 0.28 ± 0.00 0.21 ± 0.00

0.29 ± 0.00

C20:5n-3 0.24 ± 0.00 0.49 ± 0.00 1.07 ± 0.03 0.31 ± 0.02 0.30 ± 0.00 0.28 ± 0.00 0.56 ± 0.04 0.63 ± 0.02

1.03 ± 0.06

C24:0 1.68 ± 0.01 3.56 ± 0.02 1.45 ± 0.03 2.82 ± 0.12 1.65 ± 0.08 3.35 ± 0.01 2.43 ± 0.02 3.34 ± 0.08

2.88 ± 0.07

C24:1 nd 1.46 ± 0.01 0.35 ± 0.00 0.59 ± 0.01 0.96 ± 0.04 0.66 ± 0.07 0.61 ± 0.01 0.73 ± 0.01

1.64 ± 0.03

C22:5n-3 0.23 ± 0.00 0.25 ± 0.00 0.23 ± 0.00 0.32 ± 0.01 0.25 ± 0.01 0.30 ± 0.01 0.21 ± 0.00 0.18 ± 0.00

0.25 ± 0.00

C22:6n-3 6.05 ± 0.05 5.10 ± 0.07 6.55 ± 0.03 13.4 ± 0.21 13.9 ± 0.23 12.9 ± 0.28 13.1 ± 0.21 12.3 ± 0.18

12.9 ± 0.23

∑ SFA

45.3 ± 0.22 54.0 ± 0.18 52.3 ± 0.21 45.0 ± 0.45 29.6 ± 0.51 46.8 ± 0.28 45.7 ± 0.58 45.5 ± 0.45

46.4 ± 0.62

∑ MUFA

24.0 ± 0.04 25.7 ± 0.09 23.5 ± 0.03 25.1 ± 0.25 17.0 ± 0.24 24.6 ± 0.52 25.3 ± 0.32 24.9 ± 0.29

24.8 ± 0.35

∑ PUFA

30.7 ± 0.19 20.1 ± 0.10 24.1 ± 0.21 29.9 ± 0.17 11.6 ± 0.23 28.6 ± 0.29 28.7 ± 0.24 29.6 ± 0.18

29.7 ± 0.32

n-3/n-6 0.36 0.49 0.6 1.21 1.36 1.16 1.31 1.08 1.25

AA/LA 1.52 6.86 8.63 12.5 11.4 8.25 13.8 8.5 14.7

AA/DGLA 12.77 7.61 52.8 17.0 11.5 21.0 14.9 16.4 8.9

EPA/AA 0.13 0.05 0.08 0.04 0.029 0.03 0.05 0.05 0.09

DHA/EPA 3.60 10.41 6.12 27.43 46.5 46.32 23.34 19.52 12.55

123.19 94.02 119.78 170 169.8 161.7 164.5 162.3 167.9

** Each parameter given is the mean from three rat’s ± SD. For abbreviations see table 3.

TABLES AND FIGURES

Table 9: Fatty acids compositions (%) of liver phospholipids after 4 and 8 weeks

% DHA

1% DHA 2 % DHA 3% DHA

Weeks

4 8 4 8 4 8

C14:0

1.51 ± 0.02 1.53 ± 0.12 1.22 ± 0.10 1.26 ± 0.02 0.58 ± 0.03 0.68 ± 0.02

C16:0

34.0 ± 0.23 34.0 ± 0.49 33.3 ± 0.32 33.1 ± 0.35 32.2 ± 0.39 32.1 ± 0.65

C16:1n-7

1.52 ± 0.11 1.79 ± 0.12 1.63 ± 0.11 1.65 ± 0.14 0.88 ± 0.08 0.97 ± 0.08

C18:0

24.1 ± 0.24 25.6 ± 0.35 24.6 ± 0.32 25.7 ± 0.28 25.2 ± 0.31 25.7 ± 0.24

C18:1n-9

13.2 ± 0.15 13.3 ± 0.12 13.7 ± 0.15 12.7 ± 0.18 11.3 ± 0.12 11.1 ± 0.21

C18:1n-7

2.75 ± 0.12 2.93 ± 0.09 2.90 ± 0.05 3.94 ± 0.06 1.68 ± 0.08 1.72 ± 0.09

C18:2n-6

5.96 ± 0.54 5.13 ± 0.32 6.90 ± 0.35 6.94 ± 0.27 11.2 ± 0.35 12.3 ± 0.32

C18:3n-6

0.96 ± 0.15 0.60 ± 0.11 0.58 ± 0.02 0.51 ± 0.00 0.26 ± 0.00 0.21 ± 0.11

C20:0

0.18 ± 0.00 0.16 ± 0.00 nd nd nd nd

C18.3n-3

1.89 ± 0.12 0.62 ± 0.08 1.90 ± 0.03 0.92 ± 0.03 0.82 ± 0.11 0.45 ± 0.12

C18:4n-3

0.49 ± 0.12 0.33 ± 0.00 0.42 ± 0.02 0.26 ± 0.02 1.11 ± 0.01 0.80 ± 0.13

C20:1n-9

0.62 ± 0.11 0.55 ± 0.00 0.43 ± 0.03 0.46 ± 0.01 1.10 ± 0.09 0.96 ± 0.21

C22:0

0.58 ± 0.02 0.65 ± 0.01 0.88 ± 0.12 0.72 ± 0.09 0.50 ± 0.02 0.40 ± 0.04

C20:4n-6

7.02 ± 0.28 4.27 ± 0.24 6.10 ± 0.15 2.97 ± 0.21 5.81 ± 0.56 2.94 ± 0.19

C22:1n-9

0.35 ± 0.02 0.10 ± 0.00 0.50 ± 0.03 0.55 ± 0.01 0.12 ± 0.00 0.40 ± 0.03

C20:5n-3

0.71 ± 0.04 0.50 ± 0.02 0.73 ± 0.11 0.15 ± 0.00 1.76 ± 0.05 1.02 ± 0.04

C24:0

1.23 ± 0.13 1.13 ± 0.01 1.03 ± 0.03 1.14 ± 0.03 0.66 ± 0.03 0.68 ± 0.08

C22:5n-

0.22 ± 0.00 0.18 ± 0.00 0.21 ± 0.01 0.06 ± 0.00 1.67 ± 0.04 0.39 ± 0.03

C22:6n-3

2.62 ± 0.14 6.54 ± 0.31 2.69 ± 0.21 6.95 ± 0.32 2.89 ± 0.21 6.98 ± 0.54

SFA

61.6 ± 0.45 63.1 ± 0.65 61.0 ± 0.65 61.9 ± 0.63 59.1 ± 0.69 59.6 ± 0.75

MUFA

17.8 ± 0.32 18.1 ± 0.21 18.7 ± 0.21 18.9 ± 0.32 13.9 ± 0.25 14.2 ± 0.27

PUFA

20.5 ± 0.65 18.7 ± 0.60 19.9 ± 0.52 19.2 ± 0.71 26.6 ± 0.87 26.1 ± 0.75

n-3/n-6

0.41 0.77 0.42 0.77 0.45 0.59

AA/LA

1.18 0.83 0.88 0.43 0.52 0.24

AA/DGLA

11.32 7.76 14.19 6.46 5.28 3.06

EPA/AA

0.10 0.12 0.12 0.05 0.30 0.35

DHA/EPA

3.69 13.08 3.68 46.33 1.64 6.84

69.93 83.92 66.43 80.97 87.29 95.15

* Each parameter given is the mean from three rat’s ± SD. For abbreviations see table 3.

TABLES AND FIGURES

Table 10: Fatty acids composition (%) of liver phospholipids after 4 and 8 weeks*

% GLA

1% GLA 3% GLA 5% GLA

Weeks

4 8 4 8 4 8

C14:0

1.56 ± 0.08 1.62 ± 0.04 0.49 ± 0.01 0.82 ± 0.03 0.57 ± 0.01 0.64 ± 0.03

C16:0

22.4 ± 0.17 26.4 ± 0.21 26.8 ± 0.29 28.0 ± 0.32 24.4 ± 0.38 27.7 ± 0.39

C16:1n-7

1.93 ± 0.11 1.66 ± 0.03 1.25 ± 0.03 2.08 ± 0.08 1.23 ± 0.04 0.99 ± 0.04

C18:0

24.0 ± 0.32 25.6 ± 0.21 23.0 ± 0.17 25.2 ± 0.28 24.9 ± 0.28 25.8 ± 0.38

C18:1n-9+n-7

15.1 ± 0.27 16.9 ± 0.24 11.2 ± 0.21 13.3 ± 0.15 12.3 ± 0.21 13.2 ± 0.28

C18:2n-6

6.15 ± 0.12 5.05 ± 0.11 7.92 ± 0.15 6.59 ± 0.21 8.74 ± 0.15 7.28 ± 0.19

C18:3n-6

0.99 ± 0.05 0.64 ± 0.07 0.58 ± 0.02 0.27 ± 0.00 0.85 ± 0.11 0.19 ± 0.00

C20:0

0.68 ± 0.01 0.16 ± 0.00 0.26 ± 0.00 0.75 ± 0.01 0.16 ± 0.00 0.19 ± 0.00

C18.3n-3

1.10 ± 0.06 0.65 ± 0.05 0.96 ± 0.04 0.99 ± 0.05 0.82 ± 0.08 0.82 ± 0.05

C18:4n-3

0.51 ± 0.01 0.35 ± 0.03 0.30 ± 0.00 0.30 ± 0.00 0.30 ± 0.00 0.42 ± 0.04

C20:3n-6

0.65 ± 0.02 0.58 ± 0.01 0.76 ± 0.06 0.65 ± 0.01 0.89 ± 0.03 1.54 ± 0.07

C22:0

1.48 ± 0.06 1.41 ± 0.07 0.55 ± 0.01 1.59 ± 0.09 0.39 ± 0.01 0.81 ± 0.06

C20:4n-6

12.1 ± 0.34 10.5 ± 0.11 16.8 ± 0.19 12.4 ± 0.32 16.9 ± 0.21 13.9 ± 0.18

C22:1n-9

0.37 ± 0.00 0.10 ± 0.00 nd nd nd nd

C20:5n-3

0.74 ± 0.07 0.53 ± 0.02 0.99 ± 0.08 0.62 ± 0.01 0.88 ± 0.08 0.19 ± 0.00

C24:0

0.66 ± 0.03 0.93 ± 0.07 0.74 ± 0.04 0.63 ± 0.03 0.68 ± 0.04 0.68 ± 0.07

C22:5n-3

0.98 ± 0.03 0.23 ± 0.00 0.82 ± 0.05 0.59 ± 0.02 0.63 ± 0.02 0.62 ± 0.01

C22:6n-3

8.51 ± 0.11 6.25 ± 0.21 6.53 ± 0.09 5.35 ± 0.15 6.02 ± 0.21 4.99 ± 0.11

SFA

50.9 ± 0.55 56.2 ± 0.51 51.9 ± 0.46 57.0 ± 0.68 51.1 ± 0.67 55.9 ± 0.45

MUFA

17.4 ± 0.37 18.7 ± 0.26 12.4 ± 0.23 15.3 ± 0.23 13.5 ± 0.25 14.2 ± 0.32

PUFA

11.8 ± 0.27 8.01 ± 0.32 9.61 ± 0.25 7.85 ± 0.24 8.65 ± 0.32 7.03 ± 0.22

n-3/n-6

0.60 0.48 0.37 0.39 0.32 0.31

AA/LA

1.97 2.08 2.12 1.88 1.93 1.91

AA/DGLA

18.62 18.10 22.11 19.08 18.99 9.03

EPA/AA

0.06 0.05 0.06 0.05 0.05 0.01

DHA/EPA

11.50 11.79 6.60 8.63 6.84 26.26

140.91 107.22 144.33 111.65 140.21 114.79

* Each parameter given is the mean from three rat’s ± SD. For abbreviations see Table 3.

TABLES AND FIGURES

Table 11: Fatty acids compositions (%) of brain phospholipids after 4 and 8 weeks

% DHA 1% DHA 2% DHA 3% DHA

Weeks 4 8 4 8 4 8

C14:0 0.93 ± 0.02 1.04 ± 0.06 0.62 ± 0.03 0.64 ± 0.05 0.67 ± 0.04 0.62 ± 0.09

C16:0 37.5 ± 0.25 37.6 ± 0.32 37.0 ± 0.28 37.2 ± 0.35 33.7 ± 0.39 33.8 ± 0.21

C16:1n-7 1.20 ± 0.02 1.44 ± 0.05 1.09 ± 0.06 1.26 ± 0.02 0.82 ± 0.02 1.37 ± 0.05

C18:0 17.3 ± 0.15 17.6 ± 0.21 17.2 ± 0.18 17.3 ± 0.16 18.3 ± 0.21 18.6 ± 0.24

C18:1n-9 19.5 ± 0.18 19.8 ± 0.23 20.4 ± 0.31 20.6 ± 0.24 20.4 ± 0.25 20.9 ± 0.21

C18:1n-7 5.47 ± 0.06 5.77 ± 0.11 4.72 ± 0.08 4.83 ± 0.09 5.60 ± 0.14 4.78 ± 0.11

C18:2n-6 0.76 ± 0.02 0.72 ± 0.05 0.95 ± 0.05 1.56 ± 0.02 0.95 ± 0.07 2.16 ± 0.09

C18:3n-6 0.44 ± 0.01 0.57 ± 0.02 0.30 ± 0.00 0.68 ± 0.03 0.28 ± 0.02 0.79 ± 0.07

C20:0 nd nd 0.36 ± 0.00 0.41 ± 0.08 0.43 ± 0.04 0.41 ± 0.04

C18.3n-3 1.36 ± 0.06 0.84 ± 0.09 1.74 ± 0.02 0.31 ± 0.00 1.88 ± 0.06 0.91 ± 0.02

C18:4n-3 2.07 ± 0.08 1.53 ± 0.07 1.72 ± 0.03 1.42 ± 0.05 2.67 ± 0.04 2.13 ± 0.11

C20:3n-6 0.20 ± 0.00 0.24 ± 0.00 0.33 ± 0.05 0.55 ± 0.02 0.36 ± 0.01 0.54 ± 0.03

C22:0 0.41 ± 0.04 0.18 ± 0.05 0.41 ± 0.02 0.26 ± 0.01 0.60 ± 0.01 0.28 ± 0.04

C20:4n-6 6.28 ± 0.19 3.92 ± 0.21 5.90 ± 0.09 3.79 ± 0.11 5.82 ± 0.18 3.70 ± 0.10

C22:1n-9 0.48 ± 0.01 0.24 ± 0.02 0.13 ± 0.00 0.43 ± 0.03 0.08 ± 0.00 0.06 ± 0.00

C20:5n-3 0.40 ± 0.02 0.22 ± 0.01 0.56 ± 0.02 0.22 ± 0.00 0.58 ± 0.00 0.28 ± 0.00

C24:0 1.40 ± 0.04 1.41 ± 0.02 1.68 ± 0.11 1.72 ± 0.12 1.61 ± 0.08 1.56 ± 0.09

C22:5n- 0.15 ± 0.02 0.10 ± 0.00 0.32 ± 0.00 0.22 ± 0.03 0.21 ± 0.00 0.12 ± 0.00

C22:6n-3 3.78 ± 0.14 6.76 ± 0.28 3.98 ± 0.19 6.91 ± 0.21 4.78 ± 0.32 6.81 ± 0.21

SFA 57.5 ± 0.37 57.8 ± 0.48 57.3 ± 0.41 57.5 ± 0.42 55.3 ± 0.58 55.3 ± 0.41

MUFA 26.7 ± 0.18 27.3 ± 0.35 26.4 ± 0.34 26.8 ± 0.28 26.9 ± 0.38 27.1 ± 0.28

PUFA 15.4 ± 0.30 14.9 ± 0.41 15.8 ± 0.28 15.7 ± 0.25 17.5 ± 0.45 17.4 ± 0.32

n-3/n-6 1.01 1.73 1.11 1.38 1.37 1.43

AA/LA 8.26 5.44 6.21 2.43 6.13 1.71

AA/DGLA 31.40 16.33 17.88 6.89 16.17 6.85

EPA/AA 0.06 0.06 0.09 0.06 0.10 0.08

DHA/EPA 9.45 30.73 7.11 31.41 8.24 24.32

PI 72.37 82.50 73.95 84.07 83.60 87.52

* Each parameter given is the mean from three rat’s ± SD. For abbreviations see Table 3.

TABLES AND FIGURES

Tab

Fatty acids co

mposition (%) of Brain

phospholipids after 4 and 8 weeks

% GLA 1% GLA 3% GLA 5% GLA

Weeks 4 8 4 8 4 8

C14:0 0.95 ± 0.02 1.07 ± 0.05 0.71 ± 0.02 0.98 ± 0.08 0.45 ± 0.02 0.54 ± 0.02

C16:0 30.4 ± 0.23 32.5 ± 0.32 32.5 ± 0.21 32.9 ± 0.35 30.9 ± 0.32 32.0 ± 0.38

C16:1n-7 1.47 ± 0.02 1.23 ± 0.04 1.28 ± 0.04 1.15 ± 0.09 0.59 ± 0.00 0.53 ± 0.02

C18:0 18.8 ± 0.25 19.1 ± 0.19 18.2 ± 0.21 17.5 ± 0.11 20.1 ± 0.09 22.5 ± 0.21

C18:1n-9+n-7 25.2 ± 0.32 25.3 ± 0.21 24.0 ± 0.30 24.0 ± 0.24 24.1 ± 0.12 24.1 ± 0.25

C18:2n-6 0.78 ± 0.05 0.73 ± 0.01 2.83 ± 0.11 3.31 ± 0.13 2.55 ± 0.06 0.95 ± 0.01

C18:3n-6 0.46 ± 0.01 0.59 ± 0.03 0.58 ± 0.07 0.91 ± 0.07 0.48 ± 0.02 0.99 ± 0.01

C20:0 0.40 ± 0.02 0.62 ± 0.01 0.44 ± 0.05 0.44 ± 0.01 0.57 ± 0.03 0.63 ± 0.02

C18.3n-3 0.87 ± 0.08 1.39 ± 0.05 1.23 ± 0.04 2.27 ± 0.12 0.59 ± 0.03 0.89 ± 0.01

C18:4n-3 1.57 ± 0.04 2.11 ± 0.11 1.18 ± 0.03 1.31 ± 0.10 1.70 ± 0.01 2.04 ± 0.03

C20:3n-6 0.21 ± 0.00 0.25 ± 0.00 0.22 ± 0.00 0.87 ± 0.02 0.48 ± 0.01 0.36 ± 0.01

C22:0 0.83 ± 0.05 0.95 ± 0.02 0.50 ± 0.01 0.85 ± 0.01 0.57 ± 0.04 0.79 ± 0.01

C20:4n-6 7.98 ± 0.15 5.64 ± 0.11 8.23 ± 0.21 5.98 ± 0.09 8.45 ± 0.11 6.25 ± 0.11

C22:1n-9 0.49 ± 0.02 0.27 ± 0.01 0.12 ± 0.00 0.23 ± 0.00 0.50 ± 0.02 0.33 ± 0.00

C20:5n-3 0.41 ± 0.00 0.43 ± 0.02 0.23 ± 0.00 0.47 ± 0.01 0.60 ± 0.01 0.79 ± 0.01

C24:0 1.41 ± 0.11 1.44 ± 0.03 1.32 ± 0.01 1.59 ± 0.18 1.57 ± 0.11 1.61 ± 0.03

C22:5n-3 0.10 ± 0.00 0.16 ± 0.00 0.11 ± 0.00 0.14 ± 0.00 0.19 ± 0.00 0.13 ± 0.00

C22:6n-3 7.62 ± 0.15 6.18 ± 0.11 6.36 ± 0.21 5.12 ± 0.14 5.57 ± 0.24 4.61 ± 0.08

SFA 52.8 ± 0.48 55.7 ± 0.54 53.6 ± 0.47 54.2 ± 0.49 54.2 ± 0.44 58.1 ± 0.60

MUFA 27.2 ± 0.33 26.8 ± 0.25 25.4 ± 0.33 25.4 ± 0.32 25.1 ± 0.13 24.9 ± 0.26

PUFA 20.0 ± 0.48 17.5 ± 0.35 20.9 ± 0.54 20.4 ± 0.61 20.6 ± 0.41 17.0 ± 0.25

n-3/n-6 1.12 1.42 0.77 0.84 0.72 0.99

AA/LA 10.2 7.73 2.91 1.81 3.31 6.58

AA/DGLA 38.00 22.56 37.41 6.87 17.60 17.36

EPA/AA 0.05 0.08 0.03 0.08 0.07 0.13

DHA/EPA 18.59 14.37 27.65 10.89 9.28 5.84

PI 106.76 89.84 98.09 85.82 96.18 81.61

* Each parameter given is the mean from three rat’s ± SD. For abbreviations see Table 3.

TABLES AND FIGURES

Table 13

Concentration of alpha tocopherols at all time points in

tissues and plasma in omega 3 group and omega 6 groups

Groups

Tissues

Weeks

Formulas

2 4 6 8

1 DHA

78.62 48.57 18.57 9.88

2 DHA

69.21 41.46 13.58 8.18

Brain

µg

/total brain

3 DHA

55.34 39.04 8.44 6.11

1 DHA

89.97 80.29 77.9 24.68

2 DHA

77.26 65.74 57.26 18.99

Liver

µg

/total liver

3 DHA

65.67 41.61 19.57 15.53

1 DHA

5.34 11.1 4.30 5.66

2 DHA

7.48 8.52 5.70 5.74

Omega-3

group

Plasma

/ L Plasma

3 DHA

8.90 8.62 4.78 3.00

1 gamma

72.51 47.57 18.97 7.88

3 gamma

65.91 45.46 16.24 6.94

Brain

µg

/total brain

5 gamma

53.12 32.54 16.82 6.84

1 gamma

80.29 77.9 24.68 21.13

3 gamma

77.15 68.41 21.13 16.6

Liver

µg

/total liver

5 gamma

62.52 58.77 18.04 12.5

1 gamma

3.10 5.34 4.30 5.66

3 gamma

1.98 8.82 6.58 5.68

Omega-6

group

Plasma

/L plasma

5 gamma

1.76 4.08 2.50 2.48

Table 14: Percent of fatty acids classification of oils and extracted oils (EO)

Oils EO

Oils component

Oils fractions

1%GLA

3%GLA

3%DHA

1%GLA

3%GLA

3%DHA

NL 90.1 91.0 93.2 81.1 80.2 79.9

GL 6.9 6.7 4.9 8.1 8.6 9.5

PL 2.9 1.5 1.8 10.2 11.0 10.3

S/ P* 0.652 0.523 0.498 0.751 0.526 0.485

Total phenolic** 4 5 10 5 7 11

* S/ P ratio saturated to unsaturated fatty acids in phospholipids.

**Total phenolic excess as caffeic acid (ppm).

TABLES AND FIGURES

Table

Inhibition of formation of propanal and hexanal by tocopherol in bulk

oils and extracted oils (EO) and their fractions (Percent Mean Inhibition

SD)

Propanal Hexanal

sample day 4 day 6 day 4 day 6

control oil 0.0 0.0 0.0 0.0

1% GLA 33.5 41.7 62.5 66.7

3% GLA 3.1 18.8 41.7 33.3

3% DHA -2.6 6.1 45.8 66.7

EO control 0.0 0,0 0.0 ND

EO 1%GLA 22.7 79,9 44.4 ND

EO 3% GLA -0.3 75,8 22.2 ND

EO 3% DHA -30.9 63,8 33.3 ND

control oil 0.0 0.0 0.0 0.0

1% GLA 37.2 50.8 91.1 92.7

3% GLA -1.0 31.8 73.2 78.2

3% DHA 12.6 20.5 ND ND

EO control 0.0 0.0 0.0 0.0

EO 1% GLA 6.2 60.1 16.3 98.1

EO 3% GLA -10.4 54.3 -44.9 96.3

EO 3% DHA -71.3 25.9 -4.1 96.3

Polar lipid day 4 day 8 day 4 day 8

control oil 0.0 0.0 0.0 0.0

1% GLA 59.8 75,4 38.7 90.1

3% GLA 45.9 74,5 -79.9 76.7

3% DHA 33.5 51,7 96.2 93.5

EO control 0,0 0,0 0,0 0,0

EO 1%GLA 47,8 70.8 40.7 77.0

EO 3% GLA 43,3 60.4 -75.2 41.0

EO 3% DHA 0,1 51.9 97.4 90.1

% inhibition = (C-S)/S*100; C=hexanal or propanal formation in control and S =

hexanal or propanal formation in sample. Negative represent prooxidant activity;

SD standard deviations; the initial inhibition of hexanal formation was not

calculated because hexanal formation did not increase until after 4 days of

oxidation.

TABLES AND FIGURES

Figure 3a: Tocopherols Standard, 1: α-Tocopherol, 2: β-Tocopherol, 3: ｰ-

Tocopherol

, 4: ∆-Tocopherol detection with NP-HPLC at 295 nm and used

isooctane: acetylacetate 96:4 (v/v) as a mobile phase.

Figure 3b: Chosen volatile aldehyde standards by GC-

HS.Peaks numbered

correspond to 1, propanal; 2, penatanl; 3, hexanal; 4, octanal and 5

nonanal

TABLES AND FIGURES

100

150

200

250

0 20 40 60

Days

Gram

1DHA

2DHA

3DHA

100

150

200

0 20 40 60

Days

Gram

1gamma

3gamma

5gamma

Figure 4: Changes in rat weight gains during 56 days.

TABLES AND FIGURES

% Fatty acids

LA ALA AA EPA DHA

Figure 5:

Changes in the fatty acids in plasma

phospholipids after 4 weeks

1%DHA

2%DHA

3%DHA

% Fatty acids

LA ALA AA EPA DHA

Figure 6:

Changes in the fatty acids in plasma

phospholipids after 8 weeks

1%DHA

2%DHA

3%DHA

0.2

0.4

0.6

0.8

ratio of n-6/n-3

4 8

weeks

Figure 7: Ratio of n-6/n-3 during 4 and 8 weeks in brain

1DHA

2DHA

3DHA

TABLES AND FIGURES

Figure 8a: Effect of brain total lipid on DPPH

100

120

1% DHA 2% DHA 3% DHA

% Remaining DPPH

% Inhibition of

DPPH radical at 4

weeks after 1 min

% Inhibition of

DPPH radical at 4

weeks after 60 min

% Inhibition of

DPPH radical at 8

weeks after 1 min

% Inhibition of

DPPH radical at 8

weeks after 60 min

Figure 8b: Effect of brain total lipid on DPPH

100

1% GLA 3% GLA 5% GLA

% Remaining DPPH

% Inhibition of DPPH

radical at 4 weeks

after 1 min

% Inhibition of DPPH

radical at 4 weeks

after 60 min

% Inhibition of DPPH

radical at 8 weeks

after 1 min

% Inhibition of DPPH

radical at 8 weeks

after 60 min

TABLES AND FIGURES

0 48 96 144 192 240 288

Hours

PV (mmol/ kg oil)

Control oil

1%GLA

3% GLA

3% DHA

0 48 96 144 192 240 288

Hours

PV (mmol/ kg oil)

Control oil

EO 1%GLA

EO 3% GLA

EO 3% DHA

Figure 9: Changes in peroxide levels of bulk (A) and extracted (B) oils d

uring

storage test

TABLES AND FIGURES

0 48 96 144 192 240 288

Hours

Cont rol oil

1% GLA

3% GLA

3% DHA

120

0 48 96 144 192 240 288

Hours

Cont rol oil

EO 1%GLA

EO 3%GLA

EO 3%DHA

Figure 10: Changes in p-anisidine values of bulk (A) and extracted (B) oils

during storage test

TABLES AND FIGURES

(I) Neutral lipids

(II) Glycolipids

(III) Phosopholipids

Figure 11

: Ultraviolet scans between 220 and 320 nm after 12 days storage at 60

ºC, (A), Oils: 1, 1%GLA; 2, 3%GLA; 3, 3%DHA; (B), EO: 4, EO 1%GLA; 5

, EO

3%GLA; 6, EO 3%DHA.

TABLES AND FIGURES

0.1

0.2

0.3

0.4

0.5

0.6

0 48 96 144 192 240 288

Hours

Absorptivity at 233

Control oil Control oil with Toc. 1% GLA

3% GLA 3% DHA

0.08

0.16

0.24

0.32

0.4

0.48

0 48 96 144 192 240 288

Hours

Absorptivity at 233 nm

0.2

0.4

0.6

0.8

1.2

0 48 96 144 192 240 288

Hours

Absorptivity at 233 nm

0.5

1.5

2.5

0 48 96 144 192 240 288

Hours

Absorptivity at 233 nm

Figure 12: Absorpitivity at 233 nm of oils (A), Neutral lipids (B), Glycolipids (C) and phospholipds (D) during storage

period at 60 ºC. Error bars show the variations of the three determinations in terms of standard deviation.

TABLES AND FIGURES

0.08

0.16

0.24

0.32

0 48 96 144 192 240 288

Hours

Absorptivity at 270 nm

Cont rol oil wit h Toc. 1%GLA 3%GLA

3%DHA

0.08

0.16

0.24

0.32

0 48 96 144 192 240 288

Hours

Absorptivity at 270 nm

0.08

0.16

0.24

0.32

0.4

0 48 96 144 192 240 288

Hours

Absorptivity at 270 nm

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 48 96 144 192 240 288

Hours

Absorptivity at 270 nm

Figure 13: Absorpitivity at 270 nm of oils (A), Neutral lipids (B), Glycolipids (C) and phospholipds (D) during storage

period at 60 ºC. Error bars show the variations of the three determinations in terms of standard deviation.

TABLES AND FIGURES

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 48 96 144 192 240 288

Hours

Absorptivity at 233

Cont rol oil EO 1%GLA

EO 3%GLA EO 3%DHA

Cont rol oil with Toc.

0.1

0.2

0.3

0.4

0.5

0 48 96 144 192 240 288

Hours

Absorptivity at 233 nm

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 48 96 144 192 240 288

Hours

Absorptivity at 233 nm

0.2

0.4

0.6

0.8

1.2

1.4

1.6

0 48 96 144 192 240 288

Hours

Absorptivity at 233 nm

Figure 14: Absorpitivity at 233 nm of extracted oils (EO); (A), Neutral lipids (B), Glycolipids (C) and phospholipds (D) during

storage period at 60 ºC. Error bars shows the variations of the three determinations in terms of standard deviation.

TABLES AND FIGURES

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 48 96 144 192 240 288

Hours

Absorptivity at 270 nm

Cont rol oil wit h Toc. EO 1%GLA

EO 3%GLA EO 3%DHA

0.05

0.1

0.15

0.2

0.25

0 48 96 144 192 240 288

Hours

Absorptivity at 270 nm

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 48 96 144 192 240 288

Hours

Absorptivity at 270 nm

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 48 96 144 192 240 288

Absorptivity at 270 nm

Hours

Figure 15: Absorpitivity at 270 nm of extracted oils (EO); (A), Neutral lipids (B), Glycolipids (C) and phospholipds (D)

during storage period at 60 ºC. EO, extracted oil from formulas .Error bars shows the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

0 48 96 144 192

Hours

Propanal( µM/kg oil)

Control oil

1% GLA

3% GLA

3% DHA

0 48 96 144 192

Hours

Propanal( µM/kg NL)

Control oil

1% GLA

3% GLA

3% DHA

120

160

200

240

280

0 48 96 144 192

Hours

Propanal( µM/kg PL)

Control oil

1% GLA

3% GLA

3% DHA

Figure 16a: Propanal content in (A), Oils; (B), Neutral lipids and polar lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

0 48 96 144 192

Hours

Propanal( µM/kg oil)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

0 48 96 144 192

Hours

Propanal( µM/kg NL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

100

125

150

175

200

0 48 96 144 192

Hours

Propanal( µM/kg PL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

Figure 16b:

Propanal content in (A), Extracted oils; (B)

, Neutral lipids and polar

lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

0 48 96 144 192

Hours

Hexanal( µM/kg oil)

Control oil

1% GLA

3% GLA

3% DHA

0 48 96 144

Hours

Hexanal( µM/kg NL)

Control oil

1% GLA

3% GLA

3% DHA

0 48 96 144 192

Hours

Hexanal( µM/kg PL)

Control oil

1% GLA

3% GLA

3% DHA

Figure 16c: Hexanal

content in (A), Oils; (B), Neutral lipids and polar lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

0 48 96 144 192

Hours

Hexanal( µM/kg oil)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

0 48 96 144

Hours

Hexanal( µM/kg NL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

0 48 96 144 192

Hours

Hexanal( µM/kg PL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

Figure 16d:

Hexanal content in (A), Extracted oils; (B)

, Neutral lipids and polar

lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

0 48 96 144

Hours

Pentanal( µM/kg oil)

Control oil

1% GLA

3% GLA

3% DHA

0 48 96 144 192

Hours

Pentanal( µM/kg NL)

Control oil

1% GLA

3% GLA

3% DHA

100

0 48 96 144 192

Hours

Pentanal( µM/kg PL)

Control oil

1% GLA

3% GLA

3% DHA

Figure 16e: Pentanal content in (A), Oils; (B), Neutral lipids and polar lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

100

0 48 96 144 192

Hours

Pentanal( µM/kg oil)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

0 48 96 144 192

Hours

Pentanal( µM/kg NL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

0 48 96 14

Hours

Pentanal( µM/kg PL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

Figure 16f: Hexanal content in (A), Extracted oils; (B)

, Neutral lipids and polar

lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

101

0 48 96 144 192 240 288

Hours

Nonanal( µM/kg oil)

Control oil

1% GLA

3% GLA

3% DHA

0 48 96 144 192 240 288

Hours

Nonanal( µM/kg NL)

Control oil

1% GLA

3% GLA

3% DHA

0 48 96 144 192

Hours

Nonanal( µM/kg PL)

Control oil

1% GLA

3% GLA

3% DHA

Figure 16g: Pentanal content in (A), Oils; (B), Neutral lipids and polar lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

102

0 48 96 144 192

Hours

Nonanal( µM/kg oil)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

0 48 96 144 192

Hours

Nonanal( µM/kg NL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

100

0 48 96 144 192

Hours

Nonanal( µM/kg PL)

EO Control

EO 1% GLA

EO 3% GLA

EO 3% DHA

Figure 16h:

Nonanal content in (A), Extracted oils; (B)

, Neutral lipids and polar

lipids (C)

during storage period at 60 ºC. Error bars show the variations of the three

determinations in terms of standard deviation.

TABLES AND FIGURES

103

0 48 96 144 192

Hours

mM MAD /kg oil

Cont rol oil wit h Toc.

1% GLA

3% GLA

3% DHA

0 48 96 144 192

Hours

mM MAD/ kg oil

Cont rol oil wit h Toc.

EO 1%GLA

EO 3%GLA

EO 3%DHA

Figure 17: Malondialdehyde contents in (A) bulk and (B) extracted oils

during storage period.

Curriculum Vitae

Rokaia Ramadan Abdelsalam, she was born in Minia,

Egypt, on the 1

of July, 1974. She is married since 2000.

She graduated (B.SC.) in Agricultural Science from the

University of Minia, Egypt, in June 1995. She received the

M.SC. degree (May, 2001) in Agricultural Science (Food

Science from the Faculty of Agricultural, University of

Minia, Egypt. From December 1995 to June 2001, she

worked as a Demonstrator and taught Food Science and

Technology at the Faculty of Agricultural, University of

Minia, Egypt. From June 2001 till now she is an Assistant

Lecturer at the Food Science and technology Department,

Faculty of Agricultural, University of Minia, Egypt. In March

2002 she started her Ph.D at Food Science and Technology

Institute, Technical University of Berlin, Germany.

LIST OF PUPLICATION RELATED TO THE STUDY

1. Abdelsalam, R. and Mörsel T. 2007. Docosahexanoic acid does not raise

LC-PUFA n-3 in rat brain, liver and plasma. (Submitted for publication).

2. Abdelsalam, R. and Mörsel T. 2007. Effect of long time feeding with different

levels of gamma-linolenic acid with a constant amount of docosahexanoic acid

and eicosapentanoic acid on the developing and lipids profile of rat. (under

puplications).

3. Abdelsalam, R. and Mörsel T. 2007. A Rapid Method for Determination of

Vitamin e and Radical Scavening Activity on Plasma and Tissues. (submitted

for publication).