EFFECfS OF DIETARY HEATED FATS AND CYCLIC FATTY ACID
MONOMERS ON RAT LIVER ENZYME ACTIVITY
BY
COURDJO LAMBONI
LICEN., Université du Bénin, Lomé-Togo, 1978
MAITR., Université de Dijon, Dijon-France, 1980
M.A.+, Université de Dijon, Dijon-France, 1981
oocr., Université de Djon, Dijon-France, 1983
.' .. '
-
NSE~:AFRICAIN ET MALGACHE
• ~~UR L'ENSEIGNEMENl SU~~~~~
\\ C. A. M. E. S. -
OU{g~~...... ,
THESIS
. Arr;vée 23 .OC1,·.
8'
\\ Emeglstré s~~.s n:t~o·'Lt6.:.:.~·
,---,----' ."
Subrnined in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Nutritional Sciences
in the Graduate College of the
University of Illinois at Urbana-Champaign, 1993
Urbana, Illinois

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UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
THE GRADUATE COLLEGE
AUGUST 16,1993
\\VE TIEREBY RECOMMEND THA.T THE THESIS KY
COURDJO LAMBONI
EFFECTS OF DIETARY HEATED FATS AND CYCLIC FATIY
ENTITLED
ACID MONOMERS ON RAT LNER ENZYME ACTNITY
BE :\\CCEPTED I:-.J PARTIAL Fl-LFILLME.KT OF THE RF~lUIREMENTS FOR
THE DEGREE OF
DOCTOR O~ PHILOSOPHY
_
~d~__
Director of Thesis Rescarc h
_ _ _Y,'Tb '0_ ~2A'),."--l
- - --'
Head of Dcpartment
jJ
Chai rl'c r$<)' L
t Rcquirerl for Joctor's rlcgrce but not for mastcr's.

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III
EFFEcrS OF DIETARYHEATED FATS ANDCYCLICFATTY ACID
MONOMERS ON RAT LNER ENZYME ACI1VITY
Courdjo Lamboni, Ph.D.
Division of Nutritional Sciences
University of minais at Urbana-Champaign. 1993
E.G. Perkins, Advisor
11le objective of mis research was to evaluale the effects of dietary heated Cats from a commercial
deep-fat frying operation and of cyelie Cauy acids (CFA) on rat livet enzyme activity. The fats were
panially hydrogenated soybean oil (PHSBO) used 4 days (4·MH) and 7 days (7-DII) for frying foodslllffs.
Balanced mets coowning these used fats were fed ta rats for 10 weeks. In a second experiment, rats were
pair-Ced diets containing graded doses of7-DH. In a thïrd. 0.15% CFA diet was (cd 10 animais. ln a fomth,
rats were pair-fed diet containing the '-OH treated (T-7DH) with magnesium silicate which removes sorne
polymers from lhe used oil. AIl four sets of experiments were conducted wilh control rats Ced non-heated
PHSBO (NB) dieL Ali diets were isocaloric with 15% faL
AnimaIs (cd 7-0H diet cithet ad libitum or by pair-feeding, showed significant increased contents
of cytoehromes P450 and bS as weil as increased activity of (EC 1.6.2.4) NADPH-cytoehrome P4SO
reductase when comparcd to controls. The 4-MH diet group sbowed the same trends Cor cytochromes
contents and reductase activilY in comparison LO controls. Similar resullS were obtained with CFA diet. The
activities oC (EC 2.3.1.21) carniline palmiloyllransfernse-I; (EC 1.1.1.42) isocillate dehydrogenase; were
significantly decreascd in each case either ad libitum or by pair·feeding in comparison ta controls.
Significantly depressed activity of (Ee 1.1.1.49) glucose 6-phosphate dehydrogenase was also noticed for
thcse animais wOOn compared ta NH. Similar results were recorded when fed CFA diet to rats. In addition,
liver and microsomal protein were significantly increased wOOn the used oils were fed ta rats either ad
libitum CI' by pair·Ceeding in comparison to NH. Enlarged and fauy livers were noted. Similar results were
obtained when Ced CFA diet. Livet glycogen wu signiflCaRt1y decreased when fed the used oils or CFA
diets in comparison to controls. However, when animais were fed T·7DH diet, the activities of the enzymes
were significantly improved.
Key words: rat; heated fats: cyclic fatty 8Cids: livet et\\Z)'ft\\e.

---

IV
Ta my Children:
Marie-Carmel Dambé & Théodore Damigou.
1have been away from YOll, but 1was always near YOll in
my daily thoughts throughoul the 4 years 1 spenl in the U.S.A.
We cenainly miss each other a lOI and 1 will he righl with
you. Nevertheless, 1want you to he proud of this work..

---

v
ACKNOWLEDGMENTS
1 would like to express my sinccre gratitude to my sponsors: Fulbright, for their
financial SUPPOlt during my English training year (Ohio University) and during my
graduate carecr at the University of Illinois, Urbana-Champaign, Illinois, U.S.A.
1 am also grateful to Dr. A.T. Gogué, Ph.D., vice rector of "Université du Bénin,
Lomé-Togo", former minister of "Plan et Aménagement du Territoire, Togo", for all you
did which belped me to achieve this degree.
1 would like to than!< my academic advisor Dr. Perkins for your fruitful advice and
for being present when 1 need you, and for providing me with all the materials and the
fmance needed for this projecL Il was my real pleasure to work with you.
Also, my appreciation goes ta Dr. Siedler for serving as chairman of my thesis
committee and for providing me with advice. 1 really appreciate how easy il was to
approach you personally and talked freely about my projecl.
1 am aIso gratefut ta the other committee members of this Ph.D. thesis: Dr. Artz
and Dr. Potter who helped me in understanding sorne of the aspects of this wark. 1 am
glad to have bad you in my committee.
SPECIAL TIIANKS TO:
John P. Jerrell, for his assistance in gas liquid chromatography for the analyses of
lipid and cyclic falty acid prafù.es. You are a nice guy John and il was my pleasure ta
work with you. 1 learned a lot from you about gas chrornatography and than!<s again.
Dr. T. Smith. for guiding me for the use of radioactive materials for the assay of
carnitine palmitoyltransferase-I activity.
F. Balmir. for reading the rrrst draft of this thesis and making corrections. You are
nice Frantzie and 1 am glad to have YOll as my best friend.
Dr. J. Robinson. for looking al this thesis and making suggestions about the fonn.
Linda Barenthin, the secretary (Nutritiona1 Sciences) for being so kind and helpful
in all the campus bureaucratie documents. Without you 1 would have been 10st in these
papers.
Dorothy Slavil<, the secretary (Burnsides) for taking care of ordering the reagents.
My laboratory mate Mike and Sean. You made the laboratory a goodenvironment
for work. 1 enjoyed your company.
Dr. S. Hill and T. Tiffany: 1 really enjoyed your company in this Burnsides
Research Laboratory before you graduated last year. 1 missed you a lot and you were both
Diee guys. It was my pleasure to work with you. l "follow" you too this year with my
graduation. Dr. Perkins is still the good rather you knew befote.

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vi
TABLE OF CONTENTS
CHAPTER 1. 1 NTR 0 D U CTI 0 N
1
CHAPTER ll. LIT E RAT URE RE VIE W
4
CHAPTER lll. B AC KG ROUND INFORMA TION AND
ENZYMES ASSAY SIGNIFICANCE
24
CHAPTER IV. MATERIALS AND METHODS
37
4.1) FATS
38
4.2) ANIMALS
38
4.2.1) AnimaiSpecies
38
4.2.2) Source
38
4.2.3) Housing
38
4.2.4) Group of Rats and Treatments
38
4.2.5) Diets
40
4.2.6) Feeding Pmeess
41
4.2.7) Growth
41
4.2.8) Length of the Experiments
42
4.3) PROCEDURES. EXTRACTIONS. AND ASSAyS
42
4.3.1) Procedures
42
4.3.2) Extractions
42
4.3.2.1) Microsornes Preparation
42
4.3.2.1.1) Preparation of Whole Homogenate
42
4.3.2.1.2) Preparation of Mitochondrial
Supernatant Fraction
42
4.3.2.1.3) Preparation ofWashed Microsomes
42
4.3.2.2) Preparation of Liver Mitochondria
43
4.3.2.3) Preparation of Liver Homogenate
43
4.3.2.4) Total Liver Lipid COnten!..
43
4.3.2.5) Isolation of Cyclic Fatty Acid Monomers
43
4.3.3) Assays
45
4.3.3.1) Cytochrome P450 Content
45
4.3.3.2) Cyrochrome b5 Content
45
4.3.3.3) NADPH-Cytochrome P450 Reduclase Activity
45
4.3.3.4) Camitine PalmilOy1transferase-1 Activity
45
4.3.3.5) Glucose 6-Phosphate Dehydrogenase Activity
46
4.3.3.6) Isocitrate Dehydrogenase Activity
46

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vii
4.3.3.7) Liver Glycogen Cootent
47
4.3.3.8) Alanine Aminottansferase (Glutamate Pyruvate
Transaminase GPT)
47
4.3.3.9) Protein Assay
48
4.3.3.10) Red Blond Cell Counts
48
4.3.3.11) Analysis of Oils Fed to Anima1s
49
4.3.3.11.1) lodine Value
49
4.3.3.11.2) Peroxide Valu•............................................. 49
4.3.3.11.3) Oxidative Stability Index
49
4.3.3.11.4) Free Fatty Acids
49
4.3.3.11.5) Color
49
4.3.3.11.6) Soap Value
50
4.3.3.11.7) Gel Permeation Chromatography
(GPC)
50
4.3.3.12) Fatty Acid Profiles
50
4.4) STATISTICALANALYSISOFDATA
51
CHAPTERV. RESULTS
52
5.1) ANALYSIS OF OILS FED TO ANIMALS
53
5.1.1) lodine Value (I.V.)
53
5.1.2) Peroxide Value (P.y.)
53
5.1.3) Oxidative Stability Index (O.S.I.)
53
5.1.4) Free Fatty Acids (FFA)
54
5.1.5) Color
54
5.1.6) SoapValue
54
5.1.7) Gel Permeation Chromatography (GPe)
54
5.1.8) Fatty Acid Composition
54
5.1.9) Quantitative Determination of Cyclic Fatty Acid
Monomers
55
5.2) FEED EFFICIENCY
56
5.3) WEIGHT GAIN
59
5.4) RED BLOOD CELL COUNTS
65
5.5) LIVER WEIGHT/BODY WEIGHT RATIO
66
5.6) PROTEIN CONTENT
70
5.6.1) Total Liver Protein
70
5.6.2) Microsomal Protein
75
5.7) LIVER LIPID CONTENT................................•......................................... 76

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viii
5.7.1) Total Lipid
76
5.7.2) Lipid Weight/Liver Weight Ratio
77
5.8) LIPIDIPR01EIN RATIO
78
5.9) LIVER GLYCOGEN CON1ENT
82
5.10) LIVER MICROSOMAL CYTOCHROMES CON1ENT
84
5.10.1) Cytochrome b5
84
5.10.2) Cytochrome P450
87
5.11) EN:lYMES ACTIVITY
88
5.11.1) Glutamate Pyruvate Transaminase
88
5.11.2) Microsomal NADPH-Cytochrome P450 Reductase
92
5.11.3) Carnitine Palmitoylttansferase-I (CPT-I)
96
5.1 lA) Isocitrate Dehydrogenase (ICDH)
100
5.11.5) Glucose 6-Phosphate Dehydrogenase (G 6-PDH)
I04
CHAPTERVI. DIS CV S SION 0 F RE S VL T S
109
SV M MAR Y
122
RE FER EN C E S
126
APPENDIX
137
Preparation of Whole Homogenate. POSl-mitochondriai Supernatant and
Microsomal Fractions
138
Preparntion ofPost-Mitochondriai Supematant
139
Preparation of Washed Microsomes
140
Detennination Of Cytochrome P450 COntenr..
142
Detennination Of Cytochrome b5 COnten!..
144
Determination OfNADPH-Cytochrome C (P450) Reductase Activity
148
Detennination Of Camitine Palmitoylttansferase 1 Activity
150
First Experiment:
Fany Acid Composition Of Liver Tissue (Weight %)
154
Second Experiment:
Fany Acid Composition Of Liver Tissue (Weight %)
155
Third Experiment:
Fany Acid Composition Of Liver Tissue (Weight %)
156
Founh Experiment:
Fany Acid Composition Of Liver Tissue (Weighl %)
157
Gel Permeation Chromatography Analysis OfOi!s Fed Ta Animals
158
Determination Of Cyclic Fany Acid Monomers in PHSBO Samples
159
Vita
160

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1
CHAPTERI
INTRODUCTION

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2
As societies develop, people's eating habits change. In the late twentieth century
in most d.eveloped counmes, fast food restaurants are papular. These restaurants seH for
example, French fries, merl chicken, fried fish etc. Such restaurants use a variety of oils
to deep fry f0005 and the ails are used over and over again. When fats or oils have been
subjected to deep frying, many new compounds 5uch as polymerie triglycerides (TG) and
oxidized TG derivatives are forrned. [1,2]. Snch compounds are absorbed iota the fricd
foods that are cateo. Oils sold in markets contain polyunsaturated fatty acids (PUFA) and
sorne of them, like safflower and sunflower oils, have high levels of unsaturation. Deep
fat frying causes destruction of unsaturation and foonation of decomposition products
containing carbonyl and hydroxyl groups [3]. This phenomenon is increased when fats
contain polyunsaturated fatty acids. Other products generated during deep fat frying are
cyc1ic fatty acid monomers originating from the intramolecular condensation of eighteen
carbon fatty acids.
In sorne cooking practices. a variety of oils are used to fry foods and the used oils
are usually kept for the next frying. Indeed, in sorne cases, any of a variety of fresh oils
are added to old oil rnerely to keep the oi1level high enough for the frying operation. For
example, in small restaurants that sell fried foods, a mixture of different kinds of oils are
used for frying foodstuffs and the oils are kept in use for up to two weeks.
The nature of the cornpounds formed during deep frying are dependent not only
upon the composition of the fats or oils which are used, but also on the frying conditions.
The temperature of frying must be taken into account as weil as the exposure of the fats
or oils to oxygen. In addition, the heating period and the frying procedure (continuous or
intermittent) influence oxidation [4,5]. Among the products generated during fat frying,
cyclic fatty acid monomers tend to cause nutritional toxicity [6,7] and celi membrane
damage [8]. It has also been noted that heated fats have other nutritional toxic effects on
rat morphology and physiology such as growth [8J,life span, and tissues (enlarged livers)
[4,6,9]'

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3
The mechanisms by which these compounds exert their adverse effects is not
completely understood. Andia & Street [10] have studied the effects of oxidized fats on
sorne enzyme activities snch as S-adenosylmethionine:phosphatidylethanolamine methyl
transferase. They reported an increased activity of the enzyme S-adenosylmethionine :
phosphatidylethanolamine methyl transferase. This may be a key regulatory enzyme for
the monooxygenase system, which is also affected. Yoshioka et al. [11] by studying the
impairments of metabolic fUfictions, induced by the toxicity of autoxidized safflower oils,
reported low activities of thiokinase and succinate dehydrogenase. They associated the
elevated carbonyl values of oxidized ails with ilieir taxie metabolic effects.
However, litùe is known about the effects of cyclic Catty acid monomers on liver
enzyme activity. Siess et al. [12] reponed an increased activity of NADPH-cytochrome
P4S0 reductase in female Wistar rats fed for four weeks a semi-synthetic diet containing
different quantities of cyelie monomers isolated from heated linseed oil.
The main objective of this study will he to elucidate the mechanism by which
dietary heated fats and cyclic fatty acid monomers fed to rats affect the activity of several
metabolic pathways by examining the following enzymes: glutamate pyruvate
transaminase; carnitine palmitoyltransferase-I; isocitrate dehydrogenase; glucose 6-
phosphate dehydrogenase; and NADPH-cytochrome P450 reductase.
In addition to the determination of these enzymes activity, the content of liver
protein, microsomal protein, liver glycogen, microsomal cytochrome P450, and liver lipid
will he determined. Furthermore, weight gain, feed efficiency, liver weight will be
recorded ta complete the study.

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4
CHAPTER Il
LITERATURE
REVIEW

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5
It is now common [13-15,4,6] knowledge that rnany of the changes observed in
heated fats are the direct result of the incorporation of oxygen ioto the fats. Both
continuous or intennittent heating influence the fat in tenns of oxidation. Intennittent
heating [5] increases the degradation of fats, probably due to peroxides formation and
decomposition during the cooking and re-heating cycles. PUFAs that are contained in
those oils react with oxygen and generate free radicals (L") during frying and semi-stable
peroxides (LOOH) upon storage of the fat. The basis of the process is that the methylene
carbon between any two double bonds in PUFAs is susceptible to hydrogen abstraction
and the fonnation of free radical. As a consequence, oxygen can attach to the rnolecule
from which hydrogen has been abstracted fonning a lipid hydroperoxide free radicals.
The lipid hydroperoxide molecules decompose fonning aldehydes • such as hexanal and
many other aldehydes, ketones, etc. as weil as higher molecular weight products. These
are cyclic fany acids, dimers, trimers and polymers oftriglyceride. However. in deep-fat
frying sorne aldehydes further oxidize and decompose. Hydroperoxides fonnation. which
is a free radical process. involves three phases or steps that are: Initiation, Propagation,
and Tennination. These are described as follows:
1-) INITIATION STEP
It involves hydrogen ion abstraction. PUFAs are susceptible to hydrogen
ion abstraction because the double bond structure weakens the hydrogen bond [16] on the
carbon atom adjacent to the C=c.
LH (Lipid PUFA)
L' (Lipid Radical)
2-) PROPAGATION STEP
The propagation step is very fast in the presence of air [17]. The peroxy

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6
radicals formed react with unsaturated fats to fonu hydroperoxides, The free radical cycle
closes when a peroxy radical abstracts a bydrogen from another unsaturated fatty acid.
This reaction is the slowest and the rate-detennining.
Conjugated Diene
(Shiftofthe double bond)
LOO' (peroxy Radical or
Lipid Peroxyl Radical)
(Slow Reaction)
LOO' + LH - - - - - - - -....
~ LOOH + C
(HydroIX'lOxide)
Once formed, hydroperoxides readily decompose (especial1y in the presence of rnetals
sucb as rron) to give chain-initiating radicals. The reactions are the following:
LOO' + LH
------41...
~
LooH
+ L'
(Hydro~xide)
LOOH + Fe2+ - - - - -.........
-
Ln + OH- + Fe3+
(Alkoxyl)
LooH + Fe3+
.........
~
LOO' + H+ + Fe2+
-----41..._
L' + LOH
(Lipid Alcohol)
LOH + 'OH
3-) TERMINATION SIEP
Termination step DCcurs wben two radicals react and thus put an end to the
propagation. There is interaction of radicals producing non-initiating and non-
propagating species as follows:

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7
L' + L'
L' + LOO'
non-radical products.
LOO' + LOO'
Therefore, lipid peroxidation can be dermed as oxidarive deterioration of polyunsaturated
lipids. Also, it has been noted [18] that microsornes reduced hydroperoxides to a complex
range of products induding polymers suggesting that free radicals were formed. The
catalyst was identified as Cyt.P450. Moreover, polyunsaturated fany acid hydroperoxides
cause direct oxidation of various amino acids resulting in enzyme inactivation.
Furthermore, malondialdehyde, a decomposition product rnay polymerize membrane
proteins and phospholipids through Schiff base formation and form fluorescent
chromolipids and lipofuscin pigments. Perkins [4,6] showed that gross chemical changes
take place in a fat when it is heated, which will result in the formation of many new
chemical compounds containing cyclohexyl. cyclohexenyl, cyc1ohexadienyl, and
aromatic rings. (cyclic fatty acid monomers). Previous smdies [2,19-24] showed that
among the many cyclic monomers that have been isolated are monomers in which the
total number of carbons is 18. The most common typical cyclic monomer i5 that which 1-S
di-substituted with fatty acids and has the following general structure:
Where x+y=lO

---

8
These types of compounds arise from the intramolecular condensation of the eighteen
carbon polyunsaturated fatty acids. Meltzer et al.[25] quantitated the cyclic monomers
formed by thermal oxidation induced during deep-fat frying in order ta determine the
potential toxicity of commonly used vegetable oils. Heated soybean ails and partially
hydrogenated soybean oil had substantial chemical and physical alterations as indicated
by increased free fany acid (FFA) content of 5-10 fold and non-eluted materials which
reflect the presence of both polymerie and oxidative materials accounting for 18-21 %.
Furthermore. 0.3~0.6% of cyclic fany acid monomers as weIl as 0.4-0.9% of polar
materials were detected in the heated ails after concentration by low temperature
crystallization of hydrogenated samples to remove a major portion of the saturated
components which appeared in the gas chromatogram with the same retention time as
those in a cyclic monomer standard. These compounds were identified by gas
chromatography-mass spectrometry (GC-MS) as di-substituted C18 acids. The cyclic
monamer corresponding to linolenic acid (C18:3 n-3) was synthesized [26,27] as follows:
CH3-CH2-(CH=CH CH~3-(CH2h-COOH
~NaOH
Canjugated Linolenic Acid Isomers
~t
~~ ::::::::H
t (Œ2l.COOH
Where x+y=lO

---

9
The complete isolation and purification of cyclîc fany acîd monomers were accomplîshed
by Rojo & Perk.îns [28,29] as weil as Sébédio et al.[30]. The general chart showing the
steps for the isolation of cyclic fany acid monomers is the fol1owing:
Linseed on
275°C/12hrsIN2
1 Saponification
,&Esterification
Methyl Esters
Column Chromatography
on Silicie Acid
Polar Fraction
Non-Polar Fraction
Urea Fractionation
16:0
Methylated CFA
18:0
(@ 99%)
~~~i} Isomers
18:3
'"
formation of numerous degradation products which W~Yf--'~-me-v-ne--\\s
properties [19-21]. Alexander [34] indicated that, during deep-fat frying the fat is often
kept for long periods of time at about 180°C while moisture and air are mixed. into the hot
ail. The fried foods absorb the heated fat as weil as any compounds in the oil and thus
become part of our diet. He then fed male weanlîng rats (l0 per group) four distinctly
different fats in a relatively short feeding period of 28 days in arder to detennine sorne of

---

10
the early effects of heated fat toxicity. These fats were corn oil, olive ail, low erucic acid
rapeseed ail, and lard. Heating conditions of the fats such as temperature, aeration, and
time were selected to produce a prcxiuct with sunHar toxicity to used fat samples ohtained
from commercial deep-fat frying operations. The fats were heated in stainless-steel
beakers for 72 hours at a controlled temperature of 180°C. Each day they were stirred
continuously for 12 hours with a mechanical stirrer and by hand every hour for the next
twelve hours ta ensure aeration and mixing. Analysis of the fats showed that the
saponification and free fatty acid values were essentially unchanged in the thermally
oxidized fats. But, the iodine values were reduced, and carbonyl values were greatly
inereased indicatîng the degree of degradation due ta oxygen attack. The relative
concentrations of the polyunsaturated fatty acids were reduced as the result of heating,
but the amount of the monoenes was not affected. AIso, the author found that all the
animals fed theffilally oxidized fats exhibited rough greasy haïr, possibly due to mild
seborrhea as weil as a noticeable deerease in grooming time. There was also evidence of
diarrhea and polyuria, concomitant with water consumption. There was very evident heart
injury due to fresh and oxidized low erucic aeid rapeseed oil and lard. Animais fed
oxidized low erucic acid rapeseed oil sustained the highest degree of injury to the heart.
Corn oil did not effeet the liver. Fresh low erucie acid rapeseed oil and lard produced
lesions. Thermal oxidation of olive ail, low erucic acid rapeseed oil, and lard significantly
increased liver damage for these three fats. Heated lard was most damaging to the lddney
followed by heated and fresh low erucic acid rapeseed oil and heated olive oil. Necrotic
fcci, manifested by focal granulomas, were numerous in
. ivers of.Ïil~s fed oxidized
,-':;:
- -- .-.... '\\
low erucic acid rapeseed oil or oxidized lard. They rep .~~t,J1t,ed/!~ inft~tory lesion
characterized by an invasion of histiocytes and lymphoA~es. Rà.ts~:~.t;J J'.
olive oil
had no distinct necrotic foci, but severe pyknosis of the\\h,ep':ltic ~' ..:'"cytoplasmic
,,_. ~ :.' " __ : ,.\\)'lllC
atrophy leading to cell death. The livers of these animais li
regeneration, as indicated by many mitotic hepatoeytes. These changes were not present

---

11
in control groups. Later, Alexander et al.[9] fed male rats laboratory heated corn oil
(HCO) and laboratory heated peanut oil (HPO) which resulted in elevated liver and
kidney weights. In addition, rats fed diets containing HeO or HPO demonstrated the
toxicity of thennally oxidized fats with the appearance of diarrhea, dennatitis, and hair
1055. Moreover, histological examinations revealed injury to the thymus for ail fat sample
tested except for fresh corn oil (FCO). The liver was damaged by HCO, HPO, and by
commercial pressure deep-fry peanut oil (PPO), while the testes and epididymides were
affected by HPO and PPO. They also emphasized that in the latter case, there was
complete cessation of spennatogenesis. However, ails prepared by this method in the
laboratory bear no resemblance to thase actually used in commercial practice. Therefore,
snch results are not applicable to reallife situations.
Gabriel et al.[B] intubated male weanling rats with 0.5 ml/day of distillable non-
urea adductable fractions (DNUA) from therrnally oxidized low-erucic acid rapeseed oil
and lard. The results showed that animaIs became irritable, disoriented AIso. loss of
weight was reported along with feed and water consumption which were decreased. In
addition, the rats exhibited diarrhea and seborrhea and the gross pathological exarnination
showed organ edema, retarded blood clotting time, flatulence, and gastric ulcers
extending into the outer muscular layer. Moreover, tissue damage as determined
histologically was extensive. cardiac lipids were increased significanùy compared to the
controls. The relative liver weights were higher compared to the controls.
Nolen et al.[35], however, using fats prepared by frying in a commercial
operation, indicated that such fats do not adversely affect rats even when they are fcd at
high levels for long time periods. The objective of their study was to determine whether
fats which had been exposed to the heat and aeration of actual frying differ significanùy
from fresh fats in their nutritional properties. Partially hydrogenated soybean oil,
cottonseed oil. and lard were used for frying under practical restaurant~type frying
conditions until they became unfit for funher use owing to excessive foaming during

---

12
frying. The used fats were fed to groups of 50 male and 50 female rats as 15% of the diet
for two years. They found that the uscd fats were slightly less ahsorbable than unheated
control fats. and gave correspondingly slower growth rates. They aIso reportcd that there
were no differences in dinical, metabolic, or pathological criteria between the two groups
to suggest that the used fats adversely affected the rats consuming them. Moreover. the
mortality among the heated-fats groups was not higher than among the control groups.
They aIso stated that when large doses of distillable non~urea-adductable fractions
concentrated from the used fats were administered by stomach tube to weanling rats, il
proved to he somewhat taxie. They concluded that, although heating of fats under actual
frying conditions caused the fonnation of substances which can he shawn to he taxie, the
level of such substances and the degree of their toxicity were so low as to have no
practical dietary significance. In another study, Nolen [36] fed two male and two female
dogs a semi-purified diet consisting of 15% of partially hydrogenated soybean oil that
had been used for deep.fat frying under commercial conditions until il reached the end of
its useful frying life. Their effects were compared to those of a commercial dog feed from
shortly after weaning until the dogs were 54 weeks old. He did not find any apparent
difference in the growth of female dogs fed a11 three diets (used partial1y hydrogenated
soybean oil, frying fat, and fresh fat control). The male dogs fed the diet with used fat
grew about the same as those fed the commercial dog feed, but both groups had reduced
growth compared to dogs fed the diet with fresh fat. He concluded that as in the rat
studies. this reduced rate of growth for males was attributed to the lower absorbability of
the used fat compared ta the fresh oil. Otherwise. histopathological and clinical
examinations showed mat aIl of me dogs fcd the three diets were in good health. In a 91
days study conducted by Miller & Long [37]. their main purpose was to assess the
potential toxicity of heated olestta/vegetable oil blends. Olestra was synthesized by
reacting sucrose with the methyl esters of fatty acids derived from edible oils in a solvent-
free system described by Rizzi & Taylor [38]. The synthesis produces a sucrose fany acid

---

13
ester with six, seven or eight Molecules of fatty acids with chain lengths defined. by the
source oil. They used refined, bleached and deodorlzed hydrogenated soya-bean oil to
make the olestra and used il in the olestra/vegetable blends. The samples tested were
prepared by heating under conditions simulating normal home use and conditions
consistent with a reasonable extreme of food-service practices. Unheated vegetable oil
and olestra/vegetable blends and vegetable oil heated und.er the same conditions as the
blends served as controls. Each of the 10 groups of animaIs consisted of 10 male and 10
female Sprague Dawley rats at 34 days of age acclimatized for 10 days before the star! of
the stndy. In addition to the control illet group, groups receiving unheated vegetable oil or
unheated olestra/vegetable blends served as contraIs. The diets were designed with 25%
casein vitamin free as the protein source. The heated blends were tested at 5% and 10%
(w/w) of the diet. The olestra/vegetable oil (35/65) was heated to 3800 P (@193°C) for 30
min. in a skillet ta simulate home pan-frying conditions. Filter paper soaked with distilled
water was added ta the skillet ta supply the moisture that would be present during actual
home use. Another sample of olestra/vegetable oil (75/25) was used to deep fry fresh-cut
potatoes under conditions simulating food-service deep-frying usage. The heating
conditions were 365°P (l85°C) for 7 days using 12 hours on-off cycles (84 hours of
frying in total). The frying kettles were topped up daily with fresh blend, as typically
done in food-service operations, ta compensate for absorption losses. Heated vegetable
oil controls were also prepared by bath frying methods. All samples were sealed under
nitrogen and stored at oop (32°C) until they were blended into the test diets. They found
that heating the vegetable oil and olestra/vegetable blends under the same conditions
resulted in similar increases in polymer and free fatty acid contents. which indicated
comparable stability of the blends and vegetable oil. The pan-fried 35/65
olestra/vegetable ail blend and pan~fried vegetable ail remained virtually unchanged
relative ta their respective unheated contraIs. The ten groups of animals fed the
olestra/vegetable blends. either heated or unheated, tended ta consume more feed than

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14
their corresponding vegetable oil contraIs which reflected the lower calorie density of the
diets containing olestra. Absolute or relative organ (ad.renal, heart, brain, liveT) weights
and organ-to-body-weight ratios did not show any statistical significant differences
between groups. for either males or females. In addition. survival. clinicat signs,
hematological parameters and histomorphology did not show any consistent statistically
significant differences between groups fed heated and unheated oleslra/vegetable blends
or between those groups and the appropriate vegetable oi! control groups. They finally
concluded that heated olestra was non-taxie and, in this respect, was not diffcrent from
unheated olestra or heated or unheated vegetable oil.
Although the adverse nutritional effcets observed when heated fats are [cd to
experimental animaIs can he attributed to the taxie compounds generated during the
heating process, a question can arise concems the handling of the diets containing heated
fats that are fed to animaIs when conducting experiments. Care should be taken to
minimize deterioration. Literature reported vitamin destruction due to oxidized fats.
Among these, Bames et al.[39] found that a biotin deficiency was due to oxidation of
biotin synthesized in the intestine when rancid fat was inc1uded in the diet. Also, Holman
[40] reponed the destruction of vitarnin A in the presence of oxidized fat, and before 10%
of linoleate carrier was oxidized, virtually aIl of the vitamin was gone. In addition,
Witting et al.[4l] stated that dietary riboflavin and pyridoxine levels were factors in the
nutritional behavior of thennally oxidized fats. Excess riboflavin seemed to be of special
value. Therefore, Alexander [42] suggested that it is possible that much of the adverse
effects observed in feeding studies where depressed growth has been seen may have
resulted from improper handling of the diets. Improper handling of the diet leads to
consequent oxidation reactions of the fats resulting in products that can cause subsequent
damage to other dietary nutrients. He then, conducted a four-week experiment on the
effoot of diet handling on nutritional studies with used frying fats fed to weanling rats at
levels of 15%. Fresh soybean oil was the fat in the control diet and the other fats, which

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15
had becn used to prepare food by a commercial-type deep-frying operation. were soybean
oil, partially hydrogenated soybean oil with iodine value 70, partially hydrogenated
soybean oil with iodine value 108. and cottonseed oil. A purified. diet was fcd ad libitum.
Each of the four used fats for the feeding study was prepared using a Toastmaster Fryer
that heated and thermostatically controlled the fats at 182°C. There were 10 fryings peT
day for 5 consecutive 8 hours days to give a total of 50 fryings during 40 hours of
heating. Each frying consisted of 1.5 lb of French-fry potatocs (6 ntin.) followed by 1.5 lb
of breaded shrimp (3 ntin.) and then 0.85 lb of onion rings (2 ntin.). Approximately 3 lb
of fresh fat was added to the fryer daily to maintain the charge. Experimental diets
prepared from these fats were then fcd to animals ad libitum for 4 weeks. Ali the
experimental fats were refrigerated until they were rnixed into the rations. Each of the
five fats was studied under (WQ different conditions of diet handling. In the first
condition. diets were prepared weekly. not refrigerated, and fed only twice a week. In the
second condition. diets were prepared and fed daily. In this case, enough basal diet,
minus fat. to last a week was mixed and refrigerated. Fat-soluble vitamins in soybean oil
as weil as the experimental fats were added to the basal diet during the daily preparation.
The fats for these diets were protected with 70 ppm of added antioxidant (BHA and
BHT). He then found thal used frying fat when fed to rats led to depressed growth and
lowered the efficiency of feed conversion unless special handling conditions were
employed to protect the ration against oxidation effects. Treatment of the dietary groups
in regard to preparation and handIing of the rations proved to be highly significant. That
is, as opposed to weekly mixing and twice weekly feeding along with the use of
antioxidants and refrigeration of the ingredients resulted in a much superior growth rate
and a higher efficiency of feed conversion. He thus concluded that since this very
significant response became apparent in less than four weeks, the importance of careful
handling to minimize secondary effect within the diet must be emphasized. The fresh
soybean oil control. and all of the used frying fats gave similar results. Researchers

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16
attempted to find out if by reducing oxidation of ails using antioxidants could decrease
the deleterious effects reported in the literature. Among them, Miller & Landes [43] fed
for eight weeks 4 weeks oid mate Sprague Dawley rats non-hydrogenated soybean oil
heated al 195°C. The oil was refined. deodorized and had no antioxidants added. This
feeding was applied to their frrst experiment. One batch of the oil was aerated by a stream
of compressed air for severa! months. In a second experiment. they [ed rats refined and
heated partially hydrogenated soybean oil that cantains BHA and BlIT as antioxidants.
While heating the oil, compressed air was bubbled through it for 100 hours. These were
compared to a control group. Each diet contained 40% of fresh or treated oil. In the frrst
experiment. all diets were fed ad libitum white in the second study rats were fed daily. In
this second experirnent. one group of rats was given the fresh oil diet and a second group
was fed the heated oil diet ad libitum. Each animal of a third group was given an amount
of the fresh oil diet equal to the average amount of the heated oil diet consumed by the
animals of the second group for the previous 24 hOUTS. A fourth group of rats was given
ad libitum a diet prepared each day with the heated oil. AH diets but one contained 20%
casein. The casein content of one of the diets used in the second experiment was adjusted
so that the average proœin intake of the animaIs consuming it would equal that of the
group fed the fresh oil diet ad libitum. This diet was mixed daily. The amount of protein
to he added was based on feed and protein intake of the two groups of animals for the
previous 24 hours. They found that feed intake and growth of the animals were depressed
by the tteatrnents imposed on the dietary oils and increased with the severity of the oil
treatment. In the second experiment, the animais given the fresh oil diet in an amount
limited to that of rats fed the heated oil diet consumed their entire allotment of food
within a short period of rime. Animais fed a limited quantity of the fresh oil diet in
comparison to those that consumed an equal quantity of the heated oil diet had increased
rate of growth. This was evidence that poor performance of animaIs fed the damaged oil
cannot be attributed solely to decreased palatahility of the diet. When the protein content

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17
of the heated oil diet was increased sa that animaIs consumed the same amount of protein
as those fed the fresh oil diet ad libitum, feed efficiency was increased to that of the fresh
oil diet. Feed intake and growth were aIse improved but did not reach levels obtained
with the diet containing fresh oil. Livers of rats fed the heated oil had the same number of
celis as their pair-fed contraIs but the protein content per œli was significantly increased.
Thus. al equallevels of protein intake.less protein was used for somarie growth and more
was retained in the liver of animaIs fed the heated oil in comparison to rats given the
fresh oil. The addition of extra protein to the diet containing heated oil partially alleviated
the effects seen when the diet was fed with 20% protein. They concluded that the increase
of cellular protein in the liver of rats fed the heated oil diet suggested that an abnormal
ponion of the d.ietary protein was being retained. in the liver to cope with the metabolic
effects of the damaged fat. Possibly the protein is used. to synthesize hepatic enzymes
which metabolize the non-physiological portion of the dietary lipid to a form, or forms,
which can he expelled either through the lungs or the kidneys. They also reported
hepatomegaly in animaIs fed heated oils. In addition. their study found that both the
number of red. blood cells produced and the mean cell hemoglobin concentration were
depressed in animals given diets containing heated fats. Consequently, the hemoglobin
content and volume of cells in the whole blood were significantly reduced in these
animals in comparison to the animals fed the fresh oil diets.
Having reviewed the effects of heated fats under deep~fat frying operation. the
logical question one might ask concems the effects of heated fats under ordinary
conditions of kitehen use. Indeed, Simko et al.[44] conducted a study in which fats were
exposed to heating at the temperature of 170 ± SoC for a period of 120 minutes (except
one sample of butter heated for 30 minutes only). During heating. the fats were
continuously stirred and aerated. Guinea-pigs were then fed the heated fats: sunflower oil.
hydrogenated vegetable fat. lard. and butter for 10 weeks. Each experimental group
contained 8-12 animals and each guinea-pig received daily 1 ml of the respective fat by

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18
the peroraI route. The experimental animals were fed ad-libitum a semisynthetic ration.
At the end of the experimental period. histological examinations of the liver, the heart
with the thoracie part of the aorta. the stomach, kidneys. and testes were carried oul They
round histological changes in various parenchymatous organs. The changes were minute
in the guinea-pigs receiving fresh fats. whereas heated fats brought about substantially
more pronounced changes. In aIl animaIs. alterations of degenerative character
predominated. In sorne of the guinea-pigs fcd fresh sorts of fat, fauy infiltration of the
liver parenchymal cells in the fonn of smal1 fat draplets were noticed. In addition. the
extent of these changes was similar after the administration of all sorts of fresh fats,
except for the guinea-pigs receiving the fresh hydrogenated fat. They also found in five
animaIs from this group isolated foei of necrosis in the liver besides the fatty
degeneration. The fatty degeneration was of greater extent in the liver of animaIs fed with
heated fats. Guinea-pigs that received heated lard showed the least changes in the liver.
fatty infiltration of a Iow grade and a moderate infIltration of the ponobiliary spaces with
mononuclears. In aIl animaIs that had received butter heated for 30 minutes the
inflammatory infiltration of the ponobiliary spaces was more pronounced. After feeding
the butter heated for 120 minutes. the authors found a greater degree of fatty infiltration
of the liver and in four animaIs, there were inflammatory infiltrates appearing as
granulomatous focuses as weil as a hyperplasia of Kupffer's ceUs. Such focuses were also
found in the liver of four guinea-pigs that had received heated sunflower ail. The greatest
histological changes were found in the liver of animaIs that had received the heated
hydrogenated fat. The liver parenchyma of five animaIs from tbis group was infiltrated
with extensive necrotic foci with a surrounding giant-cell reaction (type of ceUs which are
ta he found around foreign bodies). Their results point to the possibility that harmful
products, peroxides and thermal polymers which have nutritional toxic effects, may
develop in ordinary conditions of ntchen processing of foodstuffs. Furthermore. in these
conditions close ta the usuaI cooking procedures there may occur changes in fats.

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19
identical with thase described by other authors using substantially longer thennal
processing.
These reports about heated fats have shown them to induce deleterious efrcets or
not to have adverse effcets on animaIs to which they are red. In view of the infonnation
provided in the literature about heated and oxidized fats, the question about the potential
toxicity of these fats remains a controversial issue. Scientists trierl to find out what kinds
of compounds are fonned. in fats during heating process throughout temperature cycles,
cooling and re-heating periods. Numerous studies [2,22,45,46] have becn reported in
which the amannt of taxie substances produced during deep-fat frying that cause adverse
biological effects were detennined.
Investigators continued to attempt to elucidate the effcets ingestion of these
derivatives might have on humans. Iwaoka & Perkins [31] fed maie rats different levels
of protein along with different leve1s (0.0075,0.0225, and 0.15%) of cyclic f.tty .cid
methyl esters (> 98% pure). The diets were stored under nitrogen in the cold between
feedings. Each group of rats was fed the diets for 6 weeks. They found that animaIs on
low protein diets (8 - 10% casein) exhibited decreased weight gains and feed
consumption with inereasing levels of cycUc esters in their diets after 6 weeks. Liver
enlargements, due to a significant (p< 0.01) accumulation of liver lipid, were noted in
animals receivîng 0.15% cyclic fatty aeid esters in their diets. Moreover, the lipid/liver
weight ratios showed a large difference for rats that consumed 0.15% cyclic fatty aeids in
their diets. The amount of lipid in the liver of animais eonsuming 0.15% eyclic fany acids
in their diets was slightly higher than the others. Funhermore, they showed that animals
exhibited differences in behavior and those fed 8% protein were cIearly more tense and
excitable than rats fed higher levels of protein. They stipul.ted th.t although sorne studies
[47-51J showed that cyelie fany acids in diets caused adverse physiological effects when
fed to rats, these diets contained only uncharacterlzed eoncentrated fractions fed at
rel.tively high levels rather than the pure cyclic f.tty .cids themselves. In another study,

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20
Perkins [6] adrrûnistered methyl 12-keto-octadec-9-enoate to rats via stomach tubes and
caHected the lymph for 48 hours. This keto ester is typical of those fanned during the
oxidation of fats. Many of the compounds formed in heated fats appeared ta be readily
but slowly absorbed. Analysis of the lymph indicated that 68% of the keto ester was
absorbed in 48 hours. To pursue the investigation, Perkins & Taubold [33] fed male
weanling Sprague Dawley rats methyl esters of non cyclic dimeric fatty acids for 6 to 8
weeks. Methyl esters of 14C-Iabeled dimeric fatty acids were prepared from uniformly
labeled methyl oleate according to the procedure described by Perkins & Iwaoka [27].
Rats were conditioned receiving the special diets for 8 weeks and then were immediately
placed in aH-glass metabolic cages for 48 hours after injection of the labeled sample. The
expired C02, urine and feces were recovered, and the radioactivity was determined by
liquid scintillation counting. The rats were sacrificed after the 48 hours period. Livers,
stomach, small and large intestines, epididymal fat, and peritoneal fat were quickly
removed for analyses. They found that rats fed diets containing less than 1% of the non
cyclic dimer methyl ester showed no significant differences in growth, and liver size from
those fed nutritionally adequate diets. Also, the fact tha,"-
in food consumption indicated that food. palatability r.-'-f; not a pro
o
CAM
metabolic studies indicated that an average of 85
tg t
E
'oaetivity was
\\,
found in the gastrointestinal tract and feces. In fact, [lJ,e Sl:Y!d~Ufi.~f1
.rIO/fin
ntS.,}
dimeric fatty acids in rats diets, paralleling a study condu
e
sieh & Perkins [52]
who found an average of 80% of the recovered radioactivity in urine and feces. They
stated that even though enlarged liveTS due accumulation of lipid have been reported as a
result of feeding heated or oxidized fats, the studies they conducted did not [lOd any
significant differences in liver weighVbody weight ratios. Furthennore, both studies with
cyclie dimers. bave indicated that dimeric fatty acids did not lead to enlarged or fatty
livers. They suggested limited absorption of dimerie fatty acids based on recovered

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21
radioactivity in the gastrointestinal ttact and feces. In conclusion, prefeeding dimer to rats
did not appear te influence the metabolism of cithet dimer or methyl oleatc.
In order to better understand how weB these cyclic compounds weIe metabolized
lwaoka & Perkins [7J studied the effeets of dietary eyelie fauy acid monomers on the
metabolism of unifonnly labeled cyclic fatty acids and on the rate of in vivo and in vitro
lipogenesis in rats. The unifonnly labeled cyclic monomers were synthesized from
unifonlÙy labeled linolenie acid aeeording to Perkins & lwaoka [27J. After purification,
the unifomùy labeled eyelie fauy acids obtained (as methyl esters) had a specifie aetivity
of about 500 mCi/g. The cyclic fatty acids wcre then dilutcd ioto corn oil to a specîfie
aetivity of 59 mCi/g when fed to experimental animals. Then, unifonlÙY labeled methyl
linoleate was di1uted with pure methyllinoleate to a specifie aetivity of 516 mCi/g and
further diluted in fresh corn oil to 49 mCi/g. They found that fatty aeids were partiaBy
oxidized to C02 and a panicn of the cyclic fatty acids. presumably the ring structure was
excreted in the urine. The unifonn1y labe1ed cyclic monomers used in the study were
synthesized from unifonnly labeled linolenic acid and were purified. The authors showed
that approximately 40% of the totaI radioactivity was found in the urine aiter 48 hours
with about 60% of that being excreted within 12 hours after ingestion. Also, they reported
decreased rates of lipogenesis in livers of animais fed 8% and 10% protein and higher
levels of cyclic fatty acids. These results suggested that the depression in hepatic
lipogenesis might he attributed to the action of cyclic fatty acids directly or indirectly on
fatty acid synthesis and the related supporting systems, i.e. reducing equivalent
production, (NADPH). In addition, the presence of eyelie faUy acids or long chain fauy
acids derived from the diet or CoA derivatives in the cytoplasm might aIso contribute to
the depression in enzyme activity.
Andia & Street [10] investigated functional changes associated with the
hepatomegaly commonly observed upon feeding rats thermaIly oxidized fats. They fed
rats purified diets in which the fat consisted of fresh corn oil, thennal1y oxidized oil, or

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22
the proportional arnount of non-urea-adduct fonning rnaterial from thennally oxidized ail
plus fresh oil. Increases in relative weights and the concentrations of microsomal protein
and endogenous malondialdehyde were observed when thennally oxidized oil or 000-
urea-adduct fraction plus fresh oil were fed rather than pure fresh oil with two types of
dietary protein, casein and soy. In addition. they found that bath the basal and DDT-
induced mixed function oxidase activities were higher in animais fcd thennally oxidized
oil and non-urea-adduct forming material plus fresh oil than thase given fresh oil. The
thermally oxidized oil also increased cytochrome P450 and the activity of S-
adenosylmethionine:phosphatidylethanolamine methyltransferase whereas the non-urea-
adduct forming material plus fresh ail did not. They finally conc1uded that oxidized fat
appeared 10 stimulate smooth endoplasmic reticulum proliferation and induce a complex
of microsomal enzymes.
As previously mentioned in the introduction, the objective of this study was to
examine the effects of dietary heated fats and cyclic fatty acid monomers on the activity
of several enzymes in rats.
Although previous research has provided scientists with infonnation regarding
abnormal enlarged livers which develop in rats fed heated fats, little is known conceming
the effects of cyclic fany acid monomers on the liver enzyme systems. Therefore, this
study will focus on determining why rats fed heated fats developed the adverse
physiological effects cited in the literature. Liver enzymes in four experimental groups of
rats will he compared ta a control group fed unheated partially hydrogenated soybean ail.
The heated ails will include partially hydrogenated soyhean ail (PHSBO) used for 4 days
of frying and PHSBO that has been used for 7 days of frying. Bath of the used ails were
obtained from a commercial deep-fat frying operation and had served ta fry fish, French
fries, etc. One of the experiments will be designed to evaluate pure cyclic fany acid
monorners. The last experiment will deal with the same batch of the partially

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23
hydrogenated soybean oil that has heen used for 7 days of frying but treated with an
active fù.ter aid.

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24
CHAPTER III
BACKGROUND INFORMATION AND
ENZYMES ASSAY SIGNIFICANCE

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25
It has been noted [12,53] that thermally oxidized oils, whole or fractionated into
cyclic fatty aeid monomers, when fed to rats lead to the increase of cytochrome P450
content (Cyt.P450) in the liver. For this reason, liver Cyt.P450 content and the activity of
the enzyme will be investigated since il perfonns a central mIe in the metabolism of
xenobiotics1 and various endogenous compounds. According ta Okita & Masters [54],
cytochromes P450 are a family of enzymes that metabolize a variety of lipophilic cyclic
compounds from endogenous or exogenous sources. When an -OH group is attached to a
xenobiotic compound by a methylene carbon or the carbon atom of a methyl group, it
makes it more polar and thus more soluble in the aqueous environment of the cell. These
enzymes catalyze hydroxylation of an aromatic ring to fonn a phenol, or are involved in
addition of an oxygen atom across a carbon-carbon double bond to fonn an epoxide. The
epoxide may non-enzymatically decompose to an alcohol group [54]. Alcohols are further
oxidized to aldehydes by cytochrome P450 enzymes [54]. In addition, in mammalian
cells cytochromes P450 serve as tenninal electron acceptors and monooxygenases in
electron transport systems which are present either in the microsomes or in the inner
mitochondrial membrane. The Cyt.P450 protein contains a single iron protoporphyrin IX
prosthetic group and the resulting heme protein cantains binding sites for both an oxygen
molecule and the substrate. In order for the hydroxylation (monooxygenation) reaction
schematically written as the following
-----II.....
NADPH + Ir + O2 + SH
NADP+ + H20 + S-OH
Thîs reaction is referred to as a monooxygenation and
the enzyme as a DloDooxygeD8Se because only one of
the two oxygen atoms îs incorporated into the substrate (SH).
(NADP = Phosphorylated Nicotinamide Adenine Dinucleotide oxidized fonn)
1 Exogenous substmtes are often referred to as xeoobiotics, meaning "foreign ta Iife" and this tenn includes
drugs that are used therapeutically and non therapeutically chemicals used in the work place, industrial
byproducts that become environmental conlaminants, and food additives. From reference [54], P·995.

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26
ta QCcur. the heme iron must be reduced frorn the ferric (Fe3+) ta its ferrous (Fe2+) state
sa that oxygen may bind to the herne iron. A total of two electrons are required for the
monooxygenation reaction. The electrons are transferred to the Cyt.P450 molecule
individually. the tirst ta allow oxygen binding and the second ta c1eave the oxygen
molecule ta generate the active oxygen species for insertion into the reaction site of the
suhstrate as shown by the following diagram which is the sequence of reactions at
Cyt.P45û.
Product (S-OH)
Substrate (S)
P4S0-
~~
Fe
r~
3+-
P4So-Fe
3+
P4so-Fe
1
S
l'
~
)S ~
Pro-Fe\\..
2
s
~ P450-Fe +
r
l'
(
s
~
O2
The diagram shows lhe binding of substrate,
transfer of the fmt and the second electrons,
and binding of molecular oxygen [541'
Electrons are transferred frorn NADPH ta the cytochrorne P450. Pyridine nuc1eotides are
2e- donors. but Cyt.P45ü with its single herne prosthetic group can only accept le- at a
time. The problem is solved by the presence of NADPH-dependent flavoprotein reductase
which accepts 2e- from NADPH simultaneously but transfers the electrons individually ta
an intennediate iron-sulfur protein or directly ta Cyt.P450. NADPH-Cyt.P45ü reductase

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27
is the intermediate electron donor in the microsomes. The enzyme is bound by its
hydrophobie tail ta the membrane whereas Cyt.P450 is deeply embedded in the
membrane. In endoplasmic retieulum (microsome), NADPH donates electrons to a
flavoprotein called NADPH-Cyt.P450 reductase. This enzyme contains both flavin
adenine dinucleotide (FAO) and flavin mononucleotide (FMN) as prosthetic groups. It is
the only marrnnalian flavoprotein known to contain both FAD and FMN. The FAD serves
as the entry point for electrons frorn NADPH, and FMN serves as the exit point,
transferring electrons individually to Cyt.P450. But, in rnitochondria the difference
resides in the fact that a flavoprotein identified as NADPH-adrenodoxin reductase acts as
the electrons acceptor from NADPH.1t contains only FAD and is weakly associated with
ils membrane milieu, urùike ils flavoprotein counterpart in microsornal membranes or
Cyt.P450 molecules. The process of electron transfer from NADPH ta Cyt.P450 in
mitochondria is represented by the fol1owing diagram adapted from the same author:
Fe2+
NADPH
s+oz
Electrons are donated from NADPH 10 adrenodoxin reductase,
which reduces adrenodoxin, a non herne suIfur iron protein.
Adrenodoxin serves as an inlermediare between adrenodoxin
reductase and CytP450 because the reductase cannot direcùy
transfer either the fml or the second electron ID the herne
iron of Cyt.P4'0-
"P450 SCC" refeIS to side chain cleavage cytochrome P450
Therefore Cyt.P450 is involved in xenobiotic detoxification. A detennination of Cyt.P450
content and the activity of NADPH-cytochrome P450 reductase are bath Îndicated in this
research plan since cyclic fatty acid rnonorners have both 5 and 6 carbon rings in their

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28
structures and are foreign compounds ingested by rats from the given diets. Besides
Cyt.P45D. cytochrome b5 (Cyt.b5) is recognized as playing a role in drug oxidation [55)
and was shown to be an obligatory component of p-nitroanisole O~demethylationsystem.
It also was shown to interaet efficiently with Cyt.P450. donating one of the two electrons
required for the O-demethylation of p-nitroanisole catalyzed by the characteristic
Cyt.P45D. In addition. their results suggested that intact Cyt.b5 tightly binds to Cyt.P45D
at a malar ratio of 1 to 1 and that Cyt.P450 interacts with Cyt.bS. in such a way that the
second electron is transferred via Cyt.bS. exhibiting the maximal activity. Furthermore.
Okita & Masters [54] pointed out that in certain reactions catalyzed by the microsomal
P450, the transfer of the second electron may not be directly from NADPH-cytochrome
P450 reductase, but may occur from Cyt.bS. a small herne protein which is also present in
the microsomes. Cyt.bS is reduced either by NADPH-cytochrome P4S0 reductase or
another microsome-bound flavoprotein. NADH-cytochrome bS reductase, which is
specifie for NADH. Moreover, it is not known why certain reactions catalyzed by specifie
cytochromes P45D apparently require Cyt.b5 to transfer the second eJectron to Cyt.P450
for expression of maximal enzymatic activity. Okita & Masters [54] listed that NADH-
cytochrome bS reductase and Cyt.bS constitute the electron transfer system for NADH to
the iron·sulfur protein. desaturase, which catalyzes the formation of double bonds in fatty
acids. Therefore. in view of the information provided above, an investigation of Cyt.b5
content becomes necessary since heated fats contain substances (cyclic fatty acid
monomers) that are metabolized by the "mixed function oxidase system". Also. Cyt.bS is
involved in the expression of maximal enzymatic activity of Cyt.P45D.
Ingestion of oxidative lipids could have harmful effects on celllipid membrane
such as the 1055 of unsaturated fatty acids. By decreasing the amount of unsaturation,
membranes change their transition temperature. In fact. each of the double bonds of
PUFAs can have two geometrical isomers with the cis and the trans configuration. These
isomers exhibit different physical propenies. As the temperature of the molecule

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29
increases when it is heated, various degrees of disorganization iota a Molecule increase
and influence the physical stability of the PUFAs. This is evidenced in the me1ting points
which become modified. According to Stryer [56]. membrane lipids are amphipathic
Molecules Le. they have bath a hydrophilic and a hydrophobie moiety. They Conn
bimolecular sheets in aqueous media and are baniers to the flow of polar Molecules.
Hydrophobie interactions are the major drlving force for lipid bilayer fonnation. The
length and the degree of unsaturation of Catty aeid chains in membrane lipids have an
effeet on membrane fluidity. The Catty aeid chains of lipid Molecules in bilayer
membranes can cxist in an ordered rigid statc or in a relatively disordered fluid state. In
the ordered (rigid) statc, all the C-C bonds have a crans confonnation, whereas in the
disordered state, sorne are in the gauche (g) conformation (a 12ü-degree rotation of C-C
bonds in fatty acyl chains) which can be either g+ (clockwise rotation)
or g-
(counterclockwise rotation). The ordered state is favored by the presence of saturated
fatty acyl chains because their straight hydrocarbon chains interact favorably with each
other. The presence of cis double bonds produces a bend in the hydrocarbon chain. This
bend intetferes with a highly ordered packing of fatty acyl chains, and thus lower the
melting temperature. Therefore, membranes by changing their transition temperature
become less fluid and gellike. This state of membranes leads to the alteration of enzyme
activity. The barrier function of membranes is also lost in those condiùons.
When ingested. oxidaùve lipids can cause damage to DNA causing theîr cleavage
and cross-linkage between DNA molecules. 8uch linkages lead to cytotoxicity,
mutagenicity. membrane breakdown. and enzyme modification discussed above. ln
addition, when a lipid peroxidation process occurs in biological membranes
gross
alterations of structural organizations [57] and enzyme functions [58-60] May result.
Therefore, an investigation of transan'Ûnases is necessary in order to know if there is any
damage cause<! by the heate<! ails or cyclic fatty acid monamers when they are fe<! ta rats.
In fact, two transaminases of particular c1inical importance are glutamate oxaloacetate

---

30
transaminase (GOT) and glutamate pyruvate transaminase (GPT). Because these are
intracellular enzymes, their levels in blood are normally very low [59]. Any significant
tissue breakdown gives tise to high serum transaminase levels. For example, in
myocardial infarction, there is an increase in the serum level of GOT (heart muscle
contains high concentrations of this enzyme). On the other hand, GPT aIso called alanine
aminotransferase is an enzyme that indicates the level of liver cell damage. The higher
the activity of GPT in the blood, the more damaged are the liver tissue cells. It is clear
that when the liver tissue cells are destroyed, the organized membrane structure seen in
normalliver tissue and described above will no longer exist and therefore may impair
enzyme activity in liver cells. Thus, the investigation of this enzyme activity in blood in
necessary.
The next major enzyme activity to be considered is carnitine palmitoyItransferase
l (CPT-I) in rat liver mitochondria. This enzyme is located in the outer mitochondrial
membrane and plays an important role by catalyzing the transfer of long chain acyl
groups ta carnitine. Recall that the ~-oxidation of fany acids occurs in the mitochondrial
matrix. In order to be oxidized, fatty acids activated as acyl-CoA in the cytosol must
cross the mitochondrial membrane and thus. need the presence of carnitine as a carrier
molecule to do so. The structure of carnitine is as follows:
CH)
H
a
1
1
~
H3C - ~ - CH2 - C- CH2 - C
1
l
' 0 -
CH)
OH
1
Acyl group attached point
to camitine

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31
The acyl group of acyl-CoA is covalently attached to camitine at the cytoplasmic sid.e of
mitochondrial membrane by the point indicated by the arrow in camitine formula. The
transfer of the acyl group of acyl-CoA to camitine is catalyzed by the enzyme camitine
palmitoyltransferase~I (CPT-I) in the outer mitochondrial membrane. Recently, Schulz
[62] in his review article has discussed the transport of long chain fatty acids through
carnitine. He noticed that a significant modification was
made when a
carnitine:acylcarnitine translocase or exchange system was discovered in the inner
mitochondrial membrane. This translocase catalyzes the 1: 1 exchange of acylcarnitines
for carnitine or a slower unidirectional flux of carnitine across the inner mitochondrial
membrane. Thus, the pathway explaining the mitochondrial Catty acid oxidation is
schematically and sîmply represented as follows:
CYTOPLASM
ATP
Fatty Acids
Outer Mltochondrial
-----)2Tf:-~--~~--~-:-----~T ---------1
r
AMP + PPi
CoA-S
Acyl-carnitine
Acyl-CoA
__________:\\InnMer
b
em rane
---------------------- -~~~ ---------,1
MATRIX
1
1
Acyl-carnitine
Camitine
1
1
CoA-SH
Acetyl CoA
AS = Acyl-CoA Synthet.1se
CPT-II "" Camitine Palmitoyltransferase II
T "" Camitine:acylcamitine translocase

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32
CPT-I. the enzyme facing the cytosolic compartrnent is inhibited by malonyl-CoA
whereas CPT-II which is facing the matrix space is not affected by malonyl-CoA. In fact,
having the appropriate and non-deteriorated lipid in the diet is of capital importance as is
ilS Donnal metabolism. For example, Dannenberg & zakim [63] studied the effects of a
fat-free dîet on the amaunt and functional state of UDP-glucuronyltransferase in rat liver
microsomes. Measurements of the activity of the enzyme showed that the activity was
approximately 30% lower in untreated microsomes in response to the fat-free diet as
compared with the control diet. Immunoblotting with anti-UDP-glucuronosyltransferase
showed approximately 200% less enzyme in rats fed the fat-free diet. A kinetic method
for measuring total UDP-glucuronosyltransferase confmned the resuIt of the immunoblot.
Thus, the total amount of enzyme declined to a greater extent than enzyme activity.
Responses of the enzyme 10 activalion by palmitoyl-lysophosphatidylcholine or UDP-N-
acetyl-glucosamine suggested that rats fed the fat-free diet had a greater activity per
molecule of UDP-glucuronosyltransferase than did rats fed the control diet. The resull
explained the relatively small decline in enzyme activity as cornpared with enzyme
concentration in microsomes prepared from animals fed the fat-free diet. Fatty acid
analysis of microsornal lipids demonstrated that the fat-free diet was associated with
lower levels of arachidonic and linoleic acids and greater amounts of paluùtoleic, oleic
and cis-vaccenic acids. Therefore, it is obvious that when feeding rats fat that has been
used in deep frying, the metabolism of lipid he checked in order to understand the effects
that the used fat have on the fed rats in comparison with a control group. An investigation
ofCPT-I activity is necessary in titis research.
Wben fatty acyl-CoA are not degraded throngh the ~-oxidation pathway described
above for body energy need, they are used for lipid synthesis and deposit in adipose and
liver tissues. The excess of acetyl-CoA produced from ~-oxidation or carbohydrate
melabolism is used for fatty acids synthesis leading to lipid formation. Acetyl-CoA
carboxylase is the enzyme Ihat calalyzes the committed step in the process of fany acid

---

33
synthe sis and thus, the following reaction showing the conversion of acetyl-CoA to
malonyl-CoA becomes an essential control point in that synthesis.
-cxx::
o
Il
Acetyl-CoA Carboxylase
1
W
CH3 - C - SCoA
CH2 - C - SCoA
(Biotin)
Acetyl·CoA
Malonyl-CoA
AlP
(The reaction requires energy from ATP and uses
bicarbonate as source of CO2-)
In addition to the previous enzymes cited above (exception of acetyl-CoA
carboxylase) that are to be investigated, others also show their importance in this study
viz., isocitrate dehydrogenase and glucose 6-phosphate dehydrogenase activities.
Isocitrate dehydrogenase (ICDH) catalyzes the first of four oxidation-reduction
reactions which is also the first dehydrogenase reaction in the tricarboxylic acid cycle
(TCA cycle) located in the mîtochondrion. The reaction is schematically described as
follows:
coo-
coo·
CCXy
l
NAD+
NADH+H+
1
1
CH
COz
CH
CH2
2
2
1
~Mnz)
2+
1
H-C-COO·
... H-
_J.::;...._...,...~
C-COO·
H - ~ - H
1
Isocltro.te
Isocitrate
1
1
H- C- OH
DehydrogeMse
DehydrogeMse
C=O
C=O
1
1
1
exxr
coo-
coo-
Isocitra1e
Oxalosuecinate
a-Ketoglutarate
Isocitrate is convened to a-ketoglutarate in an oxidative decarboxylation reaction.
Oxalosuccinate, intennediate in this reaction, is an unstable p-keto acid. The flfSt of two

---

34
C02 in TCA cycle is generated as weIl as the firs! of the three NADH + H+. In
mitochondria of rnammalian tissues, ICDH requires NAD+ as the oxidized acceptar of
reducing equivalents. rCDH, catalyzing this reaction which i5 the second control point in
TCA cycle. is allosterically stimulated by ADP which enhances its affinity for substrates.
The binding of isocitrate, NAD+, Mn2+ or M g2+, and ADP i5 mutually cooperative. The
requirement of a divaient metai cation (Mn2+ or Mg2+) i5 necessary in the
d.ecarboxylation of the Pposition of the oxalosuccinate. The enzyme i5 inhibited by ATP
and NADH. Il is noteworthy to point out that mitochondria also possess an isocitrate
dehydrogenase that requires NADP+ as the oxidized coenzyme. Sinee the primary
metabolic fate of the acetyl group of acetyl-CoA produced in the various energy-
generating catabolic pathways of most cells is its complete oxidation in the TCA cycle,
the investigation of ICDH activity becomes necessary in tbis research where heated fats
will be fed to rats.
Besides rCDH reaction described above, the last enzyme activity to be carefully
examined is that of glucose 6-phosphate dehydrogenase(G 6-P DH). In fact, this enzyme
is the one catalyzing the frrst reaction of the pentose phosphate pathway. It starts with the
dehydrogenation of glucose 6-phosphate at C-l position described as follows:
H - P
~
H-~-OH
NADP+
NADPH + H+
0
1
1
~ )
H - f - OH
1
HO-C-H
0
_--':::-..<O._ _..,..
~ HO - C- H
0
Dehydrogellllse
H -
=:J
Glucose 6-Phosphale
1
T- OH
H - f
OH
1
H-C
H-c
1
1
H -C-OPO,Z·
H -C-OPO,z·
1
1
H
H
Glucose 6-phosphate
6-Phosphoglucono-&.1actone

---

35
This enzyme was the fiTst round to he specifie for NADP+. The product 6-
phospoglucono-é-lactone generated is an intramolecular ester between the C-l carboxyl
group
and the C-5 hydroxyl group. This lactone fonned is unstable and hydrolyzes
spontaneously. But, a specifie lactonase, 6-phosphoglucolactonase causes a more rapid
ring opening and ensures that the reaction goes to completion and thus, leads to the
formation of 6-phosphogluconatc. The pentose phosphate pathway is particularly
important in animal cells and functions side by side with glycolysis and TCA cycle
pathways in crder to produce reducing power in the fonn of NADPH and pentose
intennediates. Thus, the metabolic significance of this pentose phosphate pathway is not
to obtain energy from the oxidation of glucose in animal tissues. Starting with glucose 6-
phosphate, there is neither ATP generated nor any required.. It is important here to point
out that the fundamental distinction between NADH and NADPH in most biochemical
reactions is that NADH is oxidized by the respiration chain to produce ATP, whereas
NADPH serves as a hydrogen and electron donor in reductive biosyntheses. The pentose
phosphate pathway is active in tissues where biosynthesis of fatty acids and steroids are
of major imponance, such as in adipose tissue, liver, adrenal conex, mammary glands,
but in other ceUs, such as skeletal muscle cells, this pathway is vinually nonexistent. G6-
P DH is the key regulatory enzyme of the pentose phosphate pathway and
NADP+/NADPH ratio is to he taken ioto account. Therefore, the as say of G 6-P DR
activity in this study has its significance in this study. Moreover, it is important to recall
that NADPH is necessary in the detoxification process where it serves as a donor of
electrons ta the P450 mixed function oxidase system described carlier in this writing.
In arder ta give an overall point of view of this study where heated fats will he fed
to rats, additional assays will be performed as follows: dietary intake; weight gain; liver
weight; liver glycogen content.
The glycogen content of liver will he determined in rats fed heated fats or cyclic
fatty acid monomers in order to know if its content is maintained at the same level as that

---

36
in the control group considered as reference in this study. AIso, carbohydrates are sources
of energy for the body. Protein content of liver will be detennined since it is involved in
enzyme synthesis and must be adequate to facilitate body metabolism. Aiso ît is involved
in the expression of results in terrns of enzyme activity.
Both protein content of microsomes and lipid content of liver will he detennined
to ascertain how much fat was accumulated in livers of animais fed heated or cyeUe fatty
acid monomers in comparison with the control group.

---

37
CHAPTER N
MATERIALS AND METHüDS

---

38
4.1) FATS
Fats used in this research are:
1- Non-heated partially hydrogenated soybean oil (PHSBO).
2- PHSBO used 4 days for frying foodstuffs.
3- PHSBO use<! 7 days for frying foodstuffs.
Bath of the used cils (4 days and 7 days) were obtained from a commercial deep-fat
frying operation and had served 10 fry fish, French fries, etc.
4.2) ANIMALS
4.2.1) Animal Species
Male weanling rats of Sprague Dawley strain weighing about 60g when received
were used in four sets of experiments.
4.2.2) Source
Animals were purchased from Harlan Sprague Dawley Ine., Indianapolis. IN.
4.2.3) Housing
The rats were individually housed and randomly distributed in steel wire mesh
cages in the Animal Care Facility of the Plant and Animal Biotechnology Laboratory
building. The randomization was carried out by use of a random number table [64].
Twelve hOUT light and clark cycles were set from 8:00 a.m. to 8:00 p.m.. The relative
humidity (40 - 70%), temperature (@ 73'F), and ventilation of the animals'room were
centra11y controlled by a computer system. Animais were switched te clean cages every
other week.
4.2.4) Group ofRats and Treatments
4.2.4.1) First Set of Experiment
Animais were randomly divided into 4 groups as follows:
1- Group of rats fed 15% ofPHSBO (non-heated oil) or simply designated
NB
n =10

---

39
2- Group of rats fed 15% of the 4 days frying PHSBO (mildly heated
PHSBO) or simply designated 4-MH
n = 10
3- Group of rats fed 15% of the 7 days frying PHSBO (discarded heated
PHSBO) or simply designated 7-DH
n = II
4.2.4.2) Second Set of Experiment
Animais were randonùy divided iota 4 groups in this second set of experiment as
follows:
1- Group of rats fed 15% ofPHSBO or NH
n =10
2- Group of rats fed 5% of 7-DH and 10% of PHSBO or simply named 5%
7-DH.
n = 10
3- Group ofrats fed 10% of7-DH and 5% of PHSBO or designated 10%
7-DH
n = 10
4- Group of rats fed 15% of7-DH or simply designated 15% 7-DH or
7-DH
n = 10
4.2.4.3) Third Experiment
1- Group of rats fed 15% ofPHSBO or simply named NH........n = 10
2- Group ofrats fedO.15% ofa mixture ofcyc1ic fauy acid methyl esters'
dlssolved in 14.85% of PHSBO or simply called CFA
n =11
4.2.4.4) Fourth Experiment
1- Group of raIS fed 15% of PHSBO or simply designated NH
n=1O
2- Group of rats [cd 15% of the 7-DH oil treated with 10% magnesium silicate or
simply designated T-7DH.
n=10
A synthetic magnesium silicate removes from the used oil sorne of the polymers, free
fany acids and other compounds generated during the heating process of the oil. (See
Appendix for gel permeation chromatograms).
2 Cyclic fatty acid methyl esters (>98% pure) was kindly provided by Jean Louis Sébédio, IN.R.A.,
Station de Recherches sur la Qualité des Aliments de l'Homme, 17. rue Sully 21034 Dijon Cédex, France.

---

40
4.2.5) Diets
The designed groups in each set of experiment were switched to their respective
experimental diets after one week of feeding them the regular chow. AH diets were
weekly prepared or as needed in 1.5 kg batches and the stock diets in capped plastic
containers were stored under nitrogen in a 4°C cold room. The preparation and mixing of
the diets were accomplished in the diet room using the facility provided for researchers in
the Animal Care Facility of the Plant and Animal Biotechnology Laboratory building.
The nitrogen tank was installed in that room. The composition of the diets given to
animals are as fol1ows:
4.2.5.1) First & Tbird Sets of Experiments
Table 1: Diets Composition & Ingredients
NH
4-MB
7-DH
: CFA
Casein*
(%)
15
15
15
15
Dextrose anhydrous (% )
65
65
65
65
Vitamin mixture·
(%)
1
1
1
1
1\\.1ineral mixture'"
(%)
4
4
4
4
Fat PHSBO (NH)
(%)
15
0
0
14.85
Fat4-MH
(%)
0
15
0
0
Fat7-DH
(%)
0
0
15
0
Cyclic fatty acids
(%)
0
0
0
0.15
Animals were provided with food ad libitum and had free access to water.
4.2.5.2) Second & Fourth Sets of Experiments

---

41
Table 2: Diets Composition in Pair-Feeding Experiments
NH
5%7DH 10%7DH 15% 7DH T-7DH
Casein*
(%)
15
15
15
15
15
Dextrose anhydrous (%)
60
60
60
60
60
Cellulose .*
(%)
5
5
5
5
5
Vitamin mixture'"
(%)
1
1
1
1
1
Mineral mixture'"
(%)
4
4
4
4
4
Fat PHSBO (NB)
(%)
15
10
5
0
0
Fat 7-DH
(%)
0
5
10
15
0
Fat T-7DH
(%)
0
0
0
0
15
(* From Harlan Teklad. P.O. Box 44220. Madison, WI 537444220 U.SA.)
(** From Sa/ka· Floc. James Rjver Corporation, Berlin-Gorham Group, 650 Main Street, Berlin.
New Hampshire 03570 U.SA).
Animals in this set of experiment were fed different dilution of the 7 days frying PHSBO
in a paired feeding protocol. However, the rats had free access to wateT.
4.2.6) Feeding Process
In the four sets of experirnents, feed were given to animais in glass feed jars that
were weighed with food hefore introducing it in their cages, and the next morning with
the remaining food was weighed in order to determine their feed consumption. The
remaining feed in jars was simply discarded while the dirty feed jars were sent ta be
cleaned for the next day feeding.
4.2.7) Growth
AnimaIs were weighed when received and their body weight was determined. each
morning when the lights came on, until the end of the experiments.

---

42
4.2.8) Length of the Experiments
Rats were fcd the different diets described above for 10 weeks in cach set of
experiment.
4.3) PROCEDURES, EXTRACTIONS, AND ASSAYS
4.3.1) Procedures
After 10 weeks on the experimental diels. a total number of 6 rats peT day
randomly chosen from cach of the different groups were killed by Guillotine.
Blooo and liver were taken from cach animal. The blood was collected under
heparin and ilien centrifuged to ohtain plasma. The plasma was immediately frozen by
use of liquid nitragen and slored al -?O°C for future analyses. The liver was quickly
removed and weighed. Two gram portions were introduced into the appropriate cooled
buffer (kept in iee) for subsequent enzyme analysis. The rest of the liver was frozen by
use of liquid nitragen and then, stored al -?O°C for future analyses.
4.3.2) Extractions
4.3.2.1) Microsomes Preparation
43.2.1.1) Preparation ofWhole Homogenate
Microsomes were obtained according to the method described by Lake [65] after
homogenizing the liver in a 0.05M Tris-HCl buffer, pH 7.4 containing 1.15% KCI. (See
Appendix for details).
43.2.1.2) Preparation of Mitochondrial
Supernarant Fraction
The mitochondrial supematant fraction was prepared through the procedure of
Lake [65] after a series of centrifugation. (See Appendix for details).
43.2.1.3) Preparation ofWashed Microsomes
Washed microsomes were obtained according to the procedure described by Lake
[65] by resuspending the obtained pellets in a 1.15% KCl solution containing \\0 mM of

---

43
ethylenediamine tetraacetic acid (EDTA) and centrifugation al 105,000 x g for 60 min.
(See Appendix for details).
4.3.2.2) Preparation of Liver Mitochondria
The extraction of liver mitochondria was accomplished by following the method
"C" described by McGarry et al.[66] which yields minimal contamination from other sub
cellular fractions. (See Appendix for details).
4.3.2.3) Preparation of Liver Homogenate
About 2g of fresh liveT tissue were homogenized for 2 min. in (@ 0.04 ml/mg wet
weight) physiological saline solution containing 0.66 mM EDTA according to Lôhr &
Waller [67] by using an ice-cold Potter-Elvehjem homogenizer. The homogenate was
then centrifuged. for 20 min. al 31,000 x g between O-1.5°C. The clear homogenate
31,000 x g supernatant fluid was then collected for enzyme assay and determination ofits
protein concentration. The detennination of the protein concentration of this supernatant
willlead to the expression of the aetivity of the enzyme assayed in it as specifie aetivity.
4.3.2.4) Total Liver Lipid Content
The extraction of liver lipid was accomplished according ta Folch et 01.[68]. The
totallipid content of the liver was obtained by gravimetrie method after evaporation of
the solvent with a rotary evaporator whieh is described by Lamboni [69]. It eonsists of
keeping the rounded boiling flask eontaining the total lipid previously obtained after
solvent evaporation in a dessicator which is covered with aluminum foil and maintained
under vacuum for 24 hours before weighing it to get the amount of the tota1lipid. This is
done by subtracting the weight previously obtained from the emptied boiling flask
4.3.2.5) Isolation of Cyclic Fatty Acid Monomers
The quantitative determination of eyclie fatty aeid monomers from the 4 and 7
days frying PHSBO was done according ta the procedure described by Roja & Perkins
[28]. A CPSIL 88 column (60 m x 0.25 mm x 0.2 Ilm) was used for the analysis of the
cyc1ic fauy acid monomer profiles in a 5890 Hewlett Paekard gas ehromatograph.

---

44
Hydrogen was used as camer gas and was set al 24 psi. The conditions were set as
follows: 160°C (0), ZOC/min, 220°C. The procedure cited above is schematically
represented by Diagram J.
Diagram J: Steps of Isolation and Quantitation ofCyclic Patty Acid
Monomers
PHSBO
]
[ (Non-llealed or HeaIed)
Add Triheptadecanoin
(As Internai Standard)
Preparation of FAME
(Saponification/BF, MeOI!)
1
Check Recovery
~
inGLC
Hydrogenation
(5-10 p~, PtO" 2 hours
Add Phenanthrene
(As Internai Standanl)
Urea Fractionation
(5g Urea, 21 ml MeOH, Heat. Dark 18 hours
,
t
( Non-Urea Adduct 1
(Urea AdduCl )
1
1
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ J
Y t
Extraction CFA
GLC Analysis
..
(CH
(Capillary Column: CPSn.. 88)
2Clz}

---

45
4.3.3) Assays
4.3.3.1) Cytoehrome P4S0 Content
The determination of Cyt.P450 content was done according to the procedure
described by Lake [65] using the microsomal suspension prepared carlier and described
above. (See Appendix for detai!s). The peak al 450 nro specifie to Cyt.P450 was reeorded
with a Beckman Model 26 spectrophOlometer (UV/visible) upgraded with temperature
controller and coupled with a Beckman Model 24 recorder. The Cyt.P450 content was
expressed in tenns of nanomales per mg microsomal protein.
4.3.3.2) Cytoehrome bS Content
Cytochrome bS was also determined according te the method. described by Lake
[65] in the microsomal suspension. (See Appendix for details). The peak al 424 nm due
te Cyt.b5 was recorded with the same instruments described above. Cyt.b5 was expressed
in terms of nanomales peT mg microsomal protein.
4.3.3.3) NADPH-Cytoehrome P4S0 Reduetase
Aetivity (EC 1.6.2.4)
The activity the microsomal flavoprotein enzyme EC 1.6.2.4 was detennined in
the microsomal suspension with cytochrome C as an anificial electron acceptor according
to Lake (65J. The initial rate of the activity of the enzyme was obtained from the same
Beckman apparatus described above. The reductase activity was expressed in tenns of
nanomoles of oxidized (fenic) Cyt.P450 converted to reduced (ferrous) form of the
cytochrome per minute per mg microsomal protein.
4.3.3.4) Carnitine Palmitoyltransferase-I Aetivity
(EC 2.3.1.21)
Carnitine palmitoyltransferase-I activity was assayed in the mitochondrial-KCI
suspension using D,L(methyl-14C] carnitine3 according to the modified "assay II''
J It was purchased from ICN Bîomcdicals, Ine., Radiochcmieals, Irvine, CA (V.S.A.) and had a specifie
acûvity of 6 mCi/nmol. and a concentration of 30.4 ~Cilmg. Ils Chemical formula is:
CIC*CH3)3 NCH2CH(OH)CH2COOH with a molecular weight of 197.7.

---

46
described in McGarry et a1.[66,70]. (See Appendix for details). The counting of the
labeled palmitoylcarnitine formed was perfonned using a Beckman Model LS 3801
muhi-sample. multi~channel beta spectt'ophotometer for quantifying radioactive beta
emiuers. The transferase EC 2.3.1.21 activity was expressed in tenns of nanomale
palmitoylcarnitine formed per minute per mg protein.
4.3.3.5) Glucose 6-Phosphate Dehydrogenase
Activity (EC 1.1.1.49)
The activity of D-glucose 6-phosphate:NADP-oxidoreductase, EC 1.1.1.49, was
determined in the homogenate 31,000 x g supematant fluid previously obtained by using
Sigma Kit, procedure # 345-UV. The rate of fonnation of NADPH is proportion al to the
G 6-P DH activity and was measured spectrophotometrically as an increase in absorbance
al 340 nm. The production of a second malar equivalent of NADPH by the liver tissue 6-
phosphogluconate dehydrogenase according to the foUowing reaction:
NADP+
NADPH+H+
"'= /
6-phosphogluconate --''''''-=---l''~ Ribulose 5-phosphate
6-Phosphoglucona/e
Dehydrogenase
was prevented by use of maleimide (C4H3NOz) (12 mmoVl) which is an inhibitor of 6-
phosphogluconate dehydragenase. The temperature of the reaction mixture was
maintained at 30°C in a Beckman Model 26 spectrophotometer (U.Y/visible) npgraded
with temperature contraUer. Glucose 6-phosphate dehyclrogenase activity was expressed
in terms of its specifie activity (!lInol of NADPH fonned/min/mg pratein).
4.3.3.6) Isocitrate Dehydrogenase Activity
(EC 1.1.1.42)

---

47
The activity of isocitrate:NADP-oxidoreductase. decarboxylating. EC 1.1.1.42
was determined in the liver by the use of Sigma Kit, procedure #153-UV. Even though
ICDH is widely distributed in body tissue, the enzyme is primarily of liver origin. The
activity of the enzyme was measured in terms of the increase in absorbance al 340 nrn
that accurs when NADP is reduced to NADPH. The activity is proportional to the
concentration of NADPH formed. The temperature of the reaction mixture was
maintained al 37°C in the same Beckman apparatus described above. The activity of
ICDH was expressed in terms of its specifie activity (JlIDO! of NADPH fonnedlmin/mg
protein).
4.3.3.7) Liver Glycogen Content
The content of liver glycogen was determined according to the rnethod described
by Siu Lo [71] and modified by Lamboni [69]. The absorbance of the colorimetrie
reaction was recorded at 490 nm with the aid of the Beckman apparatus described above.
4.3.3.8) Alanine Aminotransferase
(Glutlmate Pyruvate Transaminase GPT) (EC 2.6.1.2)
The activity of L-alanine:et-ketoglutarate aminoo-ansferase, EC 2.6.1.2, was
determined in plasma and in liver tissue homogenate by use of Sigma Kit. procedure
#505. The enzyme catalyzes the transfer of et-amino group from alanine to (X-
ketoglutarate yielding glutamate and pyruvate according to the following reaction:
COlY
coer
CCXY
COO-
l
1
1
1
H,N'- C-H
+
o=c
GPT
.. c=o + H,N'- C-H
1
1
1
1
CH,
CH2
Of,
Of2
1
1
CH2
Of2
1
1
COO-
COO-
L~Alanîne
a·Ketoglut.arate
Pyruvate
Glutamate

---

48
The principle of this assay resides in the fact that pyruvate produced in the above reaction
reacts with 2,4-dinitrophenylhydrazine to fonn 2,4-dinitrophenylhydrazone of pyruvate.
The color intensity of the resulting phenylhydrazone is proponional to the transaminase
activity. The absorbance of the color is then measured al a wavelength of 505 nm. The
temperature of the reaction mixture was kept al 37°C in the same Beckman apparatus
upgraded with temperature contraller. GPT was expressed in tenns of international unit
CU). One international unit (U) of an enzyme is defined as that amannt of enzyme that
will convert 1 !lIDol of substrate per minute under the specified conditions of the
procedure.
4.3.3.9) Prolein Assay
Protein determination in liver tissue, microsome suspension, and homogenate
31,000 x g supernatant fluid was done according to Lowry et al. [72]. The absorbance
values were recorded at 750 nm with the aid of the Beckman apparatus described above.
4.3.3.10) Red Blood Cell Counls
This measurement was done in the whole blood by use of Coulter Counter Model
ZBL The dilution was accomplished by taking 20 ~l of whole blood and diluted in 10 ml
of Isoton-ll. Then, 100 J.ll of the previous dilution were diluted again into la ml of Isotan-
il for counting. FinaUy, these lead to a 1/50,000 dilution.
4.3.3.11) Analysis of Oils Fed 10 Animais
43.3.]].1) lodine Value
The iodine value (LV.) of oils (PHSBO, 4 days frying PHSBO, 7 days frying
PHSBO) fed to rais was measure<! by use of Wijs method of A.O.C.S Official Method Tg
1a-64 [73]. LV is a measure of the degree of un saturation of a fat or ail. Il is defined as
the number of grams of iodine absorbed by 1()() grams of fat sample (% iodine absorhed).
43.3.]].2) Peroxide Value
Peroxide value (P.V.) of oils fed 10 animais was delennined by use of A.O.C.S.
Method Cd 8-53 modified by Oil-Dri Corporation of America [74]. P.V. is a measure of

---

49
the amount of peroxides or hydroperoxides fonned. when oxygen is added to unsaturated
fatty acids. It has becn performed using the semi-automated 702 SM Titrino apparatus
(Brikman Q Metrohm; Swiss). This modified method uses iso-Octane as an alternative
solvent for peroxide value instead of chlorofonn. In addition, while the proportions
remain the same i.e. acetic acid:iso-Octane (3:2), the volume of the mixture has becn
changed to 50 où. It is expressed in milli-equivalents of peroxide peT kilogram of fat.
4.3.3.11.3) Oxidative Stability [ndex
Oxidative stability index (O.S.I.) of ails was determined using an automated
apparatus which function is based on conductometric principles. 0.5.1 measures the
resistance rime of an ail or fat ta oxidation.
4.3.3.11.4) Free Fatty Acids
Free fatty acids (FFA) content of ails fcd to rats was analyzed according to
A.D.C.S Dfficial Method Ca 5a-40 [75].
4.3.3.115) Color
Il was measured in the ails fcd to rats according to A.O.C.S Official
Method Cc 13b-45 [76]. Tintometer A.D.C.S made was used to read the color of oils that
have becn fcd to rats during the 10 weeks experiment. The color of an oil reflects ilS
quality and the highest quality oil approaches a water-white color like [77].
4.3.3.11.6) Soap Value
It has been assayed according 10 A.D.C.S Tittimetric Method Cc 17-79 [78] using
the semi-aulomated 702 SM Titrino apparatus. The soap value of a fat of oil is indicative
of the alkalinity of the fat or oil. Il is calculated as sodium aleate.
4.3.3.11.7) Gel Permeation Chromatography (GPC)
Il is a mechanical sarting of Molecules which is based on the size of the Molecules
that are in a given sample solution. The principle is hased on the faet that the smallest
components in the solution quickly migrate iota the parons packing gel in the columns
and are retained while the higher molecular weight components are flIS! excluded. The

---

50
gel must he compatible with the mobile phase. This assay was perfonned in the ails fcd te
rats by using a HPLC Pump Waters SOI, a differential refractometer Waters 410, and a
series of size exclusion columns (2 columns of Ultrastyragel SOOA, 30 cm, Part #10571
Waters; 2 columns # HIOXH0066 with G1000 HXL, 30 cm, Supelco, Inc).
Tetrahydrofuran was used mobile phase and the rate was 0.7 mVrnin.. while the pressure
was 1.5 x 103 psi.
4.3.3.12) Fatty Acid Profiles
Fany aeid methyl esters (FAMES) were prepared from oils fed to animais as well
as that extnlcted from liver tissue according to A.O.C.S Officia! Method Ce- 2-66 [79].
Triheptadecanoin was used as internai standard for quantîtation. FAMES were then
analyzed by gas liquid chromatography (OLe) under the following descriptions:
1-) Analysis of oils [cd 10 rats
GLC 5890A Gas Chromatograph Hewlett Packard
28 psi-H2,
Column DB-Wax 40 m xO.18 mm ID x 0.30)lm film thickness
Conditions: 150°C (1), 4°C/min., 245°C
2-) LiVet tissue FAMES
GLC 5730A Gas Chromatograph Hewlett Packard
15 psi-H2, Split 32: l,
Column Omegawax 250 (Fused silica capillary column)
30 m x 0.25 mm ID x 0.25 )lm film thickness (+ 5 m guard
column)
Conditions: 100°C (0), 4°C/min., 220°C (8)
For the second and fourth experiments the characteristics are the fol1owing:
GLC 5890 Series il Gas Chromatograph Hewlett Packard

---

51
12 psi-H2, Split 82:1,
Column Omegawax 250 (Fused siliea eapillary eo!umn)
30 m x 0.25 mm ID x 0.25 j.lm film thiekness (+ 5 m guard
eolumn)
Conditions: 120°C (0), 4°Clmin., 230°C (3)
4.4) STATISTICAL ANALYSIS OF DATA
Data were analyzed by analysis of variance (ANOYA) for a completely
randomized design using StatView statistical software package [80]. When significant
(p< 0.05) F test were detected. pairwise comparisons of mean among groups were
performed by Fisher's proteeted least signifieant difference (PLSD) [81].

---

52
CHAPTERV
RESULTS

---

53
5.1) ANALYSIS OF OILS FED TO ANlMALS
5.1.1) lodine Value (l.V.)
Iodine value measure<! in the non·heated PHSBO, 4 days and 7 days use<! PHSBO
are presented in Table 3. The results showed a decreased LV. from non·heated PHSBO
(100.43) ta 7 days used ail (94.18).
Table 3: Selected Parameters/or ails Analyses
Non-Beated
Four Days Frying
Seven Days Frying
PHSBO
PHSBO
PHSBO
(NB)
(4--MH)
(7·DH)
Iodine Value
100.43
98.77
94.18
Free Fatty Acids
0.002
1.39
6.28
(%)
Color
27red
>27.9 red
.
& 79 yellow
& >79 yellow
Soap Value
.
21
83
(ppm)
O.sJ
9.90
3.09
1.69
(hows)
Peroxide Value
2.0
7.86
6.99
(meq/k.g of fat)
5.1.2) Peroxide Value (P.v.)
The results obtained from the measure of peroxide value are in Table 3. P.V. is
the lowest in non-heated PHSBO (2.0 meq./lcg) while it is increased in 4 and 7 days used
ails.
5.1.3) Oxidative Stability 1ndex (O.S.1.)
The results of oxidative stability index measured. frorn the different kinds of ails
fed te rats are reported in Table 3. The value obtained for non-heated oil (9.90 hours) was

---

54
decreased in the four days used PHSBO sample (3.09 hours). A further decrease was
observed in the seven days used oil with 1.69 hours.
5.1.4) Free Fatty Acids (FFA)
The results shawn in Table 3 indicate that the seven days used oil contains the
highest percentage of FFA when compared to the non-heated PHSBO with almost no
FFA. The four days frying PHSBO showed only 1.39% offree fatty acltls after heating.
5.1.5) Color
The results of the color of oils fed 10 rats are presented in Table 3. When
compared to the control oil, the seven days used oil was too dark and was over the scale
(>27.9 red & >78 yellow). The color of the four days used PHSBO fit into the range 27
rcd and 79 yellow showing that the oil was clearer than the seven days used oil. The noo-
heated oil did fiat show any red or yellow value.
5.1.6) Soap Value
The soap values of the 4 and 7 days used oils are reported in Table 3. The results
showed that the alkalinity of the 7 days frying oil was greater than the four days used
PHSBO.
5.1.7) Gel Permeation Chromatography (GPC)
The analysis of the oils fed to rats showed that the non-heated PHSBO contained
100% TGs and no polymers. In contrast. the 4 days used oil contained about 3% of
polymers, 96% of TGs and 1% of FFA while the 7 days used oil had more polymer (@
6%) and FFA (@ 5%) but less TG (@ 70%). ln addition, the 7 days used oil had about
19% DGs. The treated used oil with 10% magnesium silicate showed less polymer
content with about 4%. (Sec Chromatograms in Appendix).
5.1.8) Fatty Acid Composition
Pany acid profiles determined from Ge analysis are compiled in Table 4. These
data show that the percentage of C18:3n.3 and C18:3n-6 are the lowest in the sample of the
seven days foodstuffs frying wlth partially hydrogenated soybean oil. The saturated

---

55
components of C16:0 and C18:0 increased in the seven days used oil ta 11.14% and
6.84%, respectîvely.
Table 4: Selected Fatty Acid Profiles OfOUs Fed to Animais
Farty Acid
Non-Heated
Four Days Frying
Seven Days F'11ng
Composition
PHSBO (NH)
PHSBO
(4-MH)
PHSBO
(7- H)
(Fames)
(FAMES in %)
(FAMES in %)
(FAMES in %)
Myris/ie Add
0.11
0.22
DAO
(CI4:0)
Palmitic Add
10.47
10.65
11.14
(Cl6:0)
Paimitoleic Add
0.10
0.20
0.31
(C l6:ln.,)
Slearie Acid
6.75
5.73
6.84
(C":O)
26.43
Oleic Acid
27.56
26.79
(C IS:ln.•)
Linoleic Acid
28.24
26.13
23.01
(C":2n.')
1.55
Linolenic Add
1.85
1.28
(C":'n.')
D.!1
5.02
3.81
Non-Elu/ab/e
O/herPeaks
24.81
24.07
26.42
5.1.9) Quantitative Determination ofCyclic Fatty Acid
Monomers
The values of cyelie fatly aeid monomers obtained from the four and the seven
days used PHSBO were 0.11% and 0.16%, respectively. (See Appendix for the
comparison of chromatograms showing Ûle used ails content of cyelie compounds). In the
non-heated PHSBO, peaks for the cyelie compounds showed up but the total value was
very low when compared to mose for 4 and 7 days llSed ails.

---

56
5.2) FEED EFFICIENCY
5.2.1) First Experîment
Feed efficiency of rats fed non~heated PHSBO diet (NH group), four days frying
PHSBO diet (4-MH group), or seven days frying PHSBO diet (7-DH group) show non
significant differences among groups as reported in Figure 1. The 7-DH diet seems to
increase the appetite of rats which are fed the diet.
Figure 1: Feed Efficiency OfRats Fed NH, 4-MH & 7-DH Diets
0.25 -r--------------__.
0.2
~
v
c
tU
0.15
r31 NH
·C
ia
tI;l
lm 4-MH
"0
0.1
ll,I
EJ 7-DH
tU
~
0.05
Non Significant
0=10
0=10
0=11
Group or Rats
5.2.2) Second Experîment
In this second experirnent where animais were pair-fed, the results appeared not to
be significantly different among groups as evidenced by Figure 2.

---

57
Figure 2: Feed Efficiency Of Rats Fed NH & Graded doses Of7-DH Diets
0.25 .......- - - - - - - - - - - - - - - - - - .
0..
NH
~ 5% 7-DH
&":3
lO%7-DH
El 15%7-DH
Non Significant
0=10
Group of Rats
5.2.3) Third Experiment
In comparison with the control group of rats, feed efficiency of those fed 0.15%
pure cyelie fatty acid monomers in the diet did not show a significant difference even
though the latter group did eat more food and had a slighùy higher effieiency. The results
are presented in Figure 3.

---

58
Fi&Ure 3: Feed Efficiency Of Rats Fed NH & CFA Diets
0.25
••••••••••
••••••••••
••••••••••
0.2
••••••••••
••••••••••
••••••••••
~
••••••••••
••••••••••
"
c
0.15
••••••••••
•
'ü
••••••••••
0::
••••••••••
~
••••••••••
'"
••••••••••
...
0.1
•
••••••••••
•
••••••••••
..
••••••••••
~
gj
CFA
••••••••••
••••••••••
••••••••••
0.05
••••••••••
Non Significant
••••••••••
••••••••••
••••••••••
••••••••••
0
D=10
0=11
Group of Rats
5.2.4) Fourth Experiment
The results of feed efficiency of rats fed T-7DH diet in this pair-feeding
experiment are shown in Figure 4 in comparison to the control group of rats fed NH diet.
There was no significant difference between the two groups. In addition. no significant
difference was noticed when the T-7DH group of rats was compared to that [cd the 000-
treated used oil diet.

---

59
Figure 4: Feed Efficiency OfRats Fed NH. 7-DH & T-7DH Diets
0 . 2 5 . . . , - - - - - - - - - - - - - - ,
0.2
~
...
[] NH
Cl
0.15
."
41
.;::;
t::
....
~ 15%7-DH
~
"c:l
0.1
tsJ T·7DH
41
41
t.ro
0.05
Non Significant
0=10
Group or Rats
5.3) WEIGHT GAIN
5.3.1) First Experiment
The results of weight gain of rats fed 4-MH and 7-DH diets did not show any
significant difference among groups in comparison with the control group fed NB diet.
(Table 5). In addition to Table 5, Figure 5 reponing the datiy weight of NB and 7-DH
diet groups c1early shows that 7-DH group grew less when compared to the control group
of rats. The weight gain of animaIs in 7-DH group was similar to controls until 35 days of
diet feeding at which point they dec1ined in weight gain. In addition to their slower
growth. animals fed the 7-DH diet lost hair and showed signs of dennatitis (Figure 6).

---

60
Table 5: Weight Gain OfRats Fed NH, 4-MH & 7-DH Diets
/
NB
(Mean ± STDEY)
WeightGain
273.24 ±25.81
266.60 ± 17.77NS
257.23 ± 18.04NS
(in Grams)
(n=lO)
(n=10)
(n=ll)
NS = Non Significant
Fi@re 5: Average Daily Weight Of Rats F ed NH & 7-DH Diets
400 "T""-------------...,
Switch to 7-DH Diet
(Day Zero ofExperimental Diet)
300
NH
.5:
200
••----••-'V- ••••••••
7.DH
100
O+--,.....--r-__-r----r-__-,.....--r.....
-10
0
10
20
30
40
50
60
70
Day of Diet Feediog

---

61
Figure 6: Picture Of Rats F ed Different Diets
The rat with the red mark on the tail was fed 15% 7-DH diet
and displays hair loss on its back

---

62
5.3.2) Second Experiment
In this pair-feeding experiment, the weight gain of rats fed graded doses of the 7-
DH diet did not show any significant difference among groups, when compared to the
control group of rats fed NH diet. Results are to be seen in Table 6. Even though animais
were pair~fed. the hair loss was observed in sorne animals in groups fed 10% and 15% of
7-DH diets. The hair loss was greater in 4 rats fed 15% of 7-DH die!. See Figure 6.
Table 6: Weight Gain OfRats Fed NH & Graded Doses Of7-DH Diets
Nll
5% 7-Dll
10% 7-Dll
15% 7-Dll
(Mean ± STDEV & n=10)
WeightGain
NS
NS
267.43± 25.41
265.53 ±2I.OI
264.46 ± 19.66
267.39 ± 15.17NS
(in Grams)
NS = Non Significant
5.3.3) Third Experiment
When rats are fed 0.15% pure cyelie fany acid monomers in the diet, their weight
gain was not significantly different frOID the control group of rats fed NB diet. Results are
shown in Table 7. In addition, 3 animais in the group fed cyelie compounds exhibited a
considerable hair 10ss as compared to the others in the same group. Also. these rats
developed severe dermatitis. See Figure 7 showing hair loss and dermatitis.

---

63
Figure 7: Picture Of Rat Fed CFA Diet
This rat which was fed cyclic fatty acid monomers (CFA)
exhibits hair loss and dermatitis on ilS back legs and abdomen

---

64
Table 7: Weight Gain Of rats Fed NH & CFA Diets
NH
CFA
(Mean ± STDEV)
WeightOain
273.24 ± 25.81
270.07 ± 25.18NS
(in Granl:~)
(n=10)
(n=11)
NS = Non Signiflcant
5.3.4) Fourth Experiment
The results obtained from the pair-feeding experiment when rats are fcd T-7DH
diet showed non significant difference when compared to a control group of rats fed NH
diet. There was no significant difference between the treated used oil and the non-treated
usect ail diets when fed to rats. Table 8 has the report. In this group the haïr 10ss was not
accentuated.
Table 8: Weight Gain OfRats FedNH. 7·DH & T·7DH Diets
NH
15% 7-DH
T-7DH
(Mean ± SIDEV & n= 10)
WeightGain
267.43±25.41
NS
267.39 ± 15.17
267.91 ± 25.50NS
(in Grams)
NS = Non Significant

---

65
5.4) RED BLOOO CELL COUNTS
No data was recorded for the frrst and third experiments.
5.4.1) Second Experiment
Red blood cell counts have been done in the pair-feeding experiment and the
results are represented by Figure 8. When 15% of the 7-DH diet was fed to rats. they
exhibited a significantly lower number (p<O.OS) of red blood ceUs in comparison to the
control group of rats and to the others fed graded doses of the same used ail. The same
level of significance (p<O.OS) was observed when 10% of the 7-DH diet was fed ta rats in
comparison to the control group. Feeding rats with 5% of the 7-DH ruet did not show any
significant difference in comparison with those fed the NH diet.
Figure 8: Red Blood Cell Counts O/Rats Fed NH & Graded Doses Of7-DH
Diets
(n=10 & Mean ± SEM)
10
l'''l
COI:]
•
,.
e 7.5
c~o
a
[][][]
••• .".."..;.;
-.
CCO
•••
...........-..
=
'(:.:!l,.!l,.!
lm NH
.~
[]C[]
••• ...."'."..".
=
[][][]
•••
..........,...
.......". • .1'
[][][]
•••
..........
El
If . . . . " , , . . , , . . . .
~ 5%7-DH
5
[]CC
•••
..........
.~ • ."fa". ...
ccc
•••
. '. . . .:'1.11
.5
.,JI.J'• ., ....
I:JI:1C
......
~ 10% 7·DH
BCC
•••
Il'\\.......''..
..
...
~.~.~
:!:
••
.~.,
• "'."'• .1.,1
CC
••• .,...........
'il
• ,J .........."
tll:ll:l
ccc
•••
III'........
..
...
U
~.~.~
~ 15% 7-DH
ccc
•••
............
....
......... ...
2.5
..
••• .". ".,.
....
"CI
~.~.~
CCtl
•••
..........
."...."..{'
CI::
CCC
••• ...........
.",.J'."'."
occ
••• ....,....
I:ltltl
••• '!:.:':"~:"!
.-"..".,,1 ......
c[]
••• ..........
.".."..".."
...........
• p< 0.05
0
Gr~orRa~
_

---

66
5.4.2) Fourth Experiment
In this pair-feeding experiment where T-7DH diet was [cd to rats, no significant
difference of red blood ecH counls was observed when compared to the control group of
rats as evidenced by Figure 9. The slight increased red ceUs number observed in the
group of rats fed the T-7DH diet was not significantly different from the counts recorded
for those fcd the non-treated used oil.
Figure 9: Red Blood Cell Counts OfRats Fed NH , 7-DH & T-7DH Diets
(n=lO & Mean ± SEM)
10
•
NS
7.5
••••
••••
••••
••••
ê"lNH
••••
5
••••
UU
I!l 15% 7·DH
••••
••••
o T-7DH
tt::
2.5
••••
nn
• p< 0.05
o
!liS =Non Significant
Group or Rats
5.5) LIVER WEIGHT/BODY WEIGHT RAno
5.5.J) First Experiment
Even though an increase was ohserved in the ratio of liveT weight/body weight
when rats are fed 7-DH diet, the difference was not significant when compared to the

---

67
control group fcd NH diet. Aiso. no significant difference was noticed when animaIs are
fcd 4-MH met. The liver weight/body weight ratio for rats fed the 4-:MH diet was not
significantly different from that of animaIs fed the 7-DH diet The results are presented in
Table 9.
Table 9: Several Hepatic Parameters 0IRats FedNH, 4-MH & 7-DH diets
NH
4·MH
7·DH
(Mean ± SEM)
% (Lirer Wt.l
Body Wt.) Ratio
3.21 ± 0.04
3.20 ± 0.05
3.25 ± 0.06
(n=lO)
(n=10)
(n=ll)
Liver
Prote in
235.89 ± 22.46
242.53 ± 21.05
479.13 ± 16.12"·
(mg/g)
(n=lO)
(n=lO)
(n=lI)
...
Microsomal
30.39 ± UO
30.71 ± 0.66
60.40 ± 1.23
Protein
(mg/8)
(n=8)
(n=lO)
(n=ll)
Liver
Lipid
57.73 ± 1.58
59.28 ± 0.90
70.80 ± 1.27"·
(mg/g)
(n=lO)
(n=10)
(n=ll)
•••
% (Lipid Wt.l
5.77 ± 0.16
5.93 ± 0.09
7.08 ± 0.13
Liver Wt.) Ratio
(n=10)
(n=10)
(n=lI)
LiveT Glycogen
10.65 ± 0.66
9.05 ± 0.43
6.77 ± DAO·"
(mg/g)
(n=10)
(n=10)
(n=ll)
Li.,er Glycogen
126.28 ± 10.08
104.14 ± 6.16+
76.76 ± 5.64"·
(mg/liver wet weighl)
(n=10)
(n=lO)
(n=ll)
+ p< 0.05
Wt.= weight
••• IX 0.0001
5.5.2) Second Experiment
In the pair-feeding experiment, the results showed significant differences (p<
0.05) in the ratios of liver weight/body weight when animaIs are fed 10% or 15% of 7-

---

68
DR diets as evideneed by the data in Table 10. Rats fed 5% of 7-DR diet did not show
any significant difference when compared to the control group fed NH diet.
Table 10: Several Hepatic Parameters Of Rats Fed NH & Graded Doses
Of 7-DH Diets
NH
5% 7·DH
10% 7·DH
15% 7-DH
(Mean ± SEM & n::::lO)
% (Li ver WU
Body Wt.)
Ratio
2.75 ± 0.05
2.84 ± 0.07
2.94 ± 0.07+
2.95 ± 0.02+
Liver
Protein
392.65 ± 7.44
398.93 ± 7.92
462.45 ± 14.90·
593.22 ± 17.50'"
(mgjg)
Microsomal
30.51 ± 1.01
30.55 ± 1.00
42.84 ± 1.8S"·
56.47 ± 1.55"""
Prottin (mg/g)
Liver
Lipid
63.88 ± 1.69
65.78 ± 1.40
71.72 ± 1.27-
79.66 ± 1.86"""
(mgjg)
% (Lipid WU
6.39±O.17
6.58 ± 0.14
7.17 ± 0.13-
7.96 ± 0.18"""
Liver Wt.)
Ratio
Liver Glycogen
11.03 ± 0.78
9.55 ± 0.29+
7.00 ± Q.41"""
6.32 ± 0.34"""
(mgjg)
Liver Glycogen
107.89 ± 7.33
94.19 ± 5.02
72.13 ± 5.39"·
65.44 ± 2.84"·
(mg/livet wet weight)
+ p<O.OS
Wt.= weight
• p< 0.01
••• p< 0.0001
5.5.3) Third Experiment
Table 11 displays the results obtained from the liver weight/body weight ratio
when animais are fed 0.15% CFA diels. Although rats fect eyelie fatty aeid monomers

---

69
showed an increased ratio, the difference observed was not significantly different from
the control group of animals fed NH diet.
Table Il: Several Hepatic Parameters Of Rats Fed NH & CFA Diets
NH
CFA
(Mean±SEM)
% (UverWtJ
3.21±0.04
3.23± 0.04
Body Wt.) Ratio
(n=lO)
(n=lO)
Liver Protein
235.89 ± 22.46
460.39± 15 22'"
(mg/g)
(n=lO)
(n=l1)
Microsomal
30.39 ± 1.10
63.15 ± 1.34'"
Pro/ein (mg/g)
(n=8)
(n=l1)
Liver Lipid
57.73± 1.58
70.17± UXr-
(mg/g)
(n=lO)
(n=l1)
% (LipidWtJ
5.77±O.16
7.02±0.IO'"
Liver Wt.) Ratio
(n=lO)
(n=l1)
u
Liver Glycogut.
1O.65±0.66
6.75±O.64 •
(mg/g)
(n=lO)
(n=l1)
..
Liver Glycogen
126.28 ± 10.08
80.34 ± 7.62
(mg/liver wet weight)
(n=lO)
(n=l1)
... p< O.lXH
Wt.= weight
... p< 0.0001
5.5.4) Fourth Experiment
When rats was fed diet containing the 7-DH oil treated with magnesium silicate
(T-7DH). the resuIts showed in Table 12 indicate an incTease of the ratio of liver
weight/body weight but it was not significantly dUfcrent from the control group in this
pair-feeding experiment. When the T-7DH group was compared to thase fed diet

---

70
containing the 7-DH ail, the decreased liver weight/body weight ratio noticed in the
treated used oil group of rats was not significantly different from the non-treated used oil
group of animals. However, the 15% 7-DH diet group was significantly different from
control animais (p< 0.05).
Table 12: Several Repalie Parameters OfRats FedNR, 7-DR & T-7DR Diets
NH
15% 7·DH
15% T·7DH
(Mean ± SEM & n=10)
% (Li ver WU
2.75 ± 0.05
2.95 ± 0.02+
2.89 ± O.û6NS
Body Wl.) Ratio
Liver
Protein
u
392.65 ± 7.44
593.22 ± 17.50·
487.09 ± 25.84"
(mg/g)
Microsomal
30.51 ± LOI
56.47 ± 17.50'"
30.60 ± 1.32NS
Protein
(mg/g)
Liver
Lipid
63.88 ± 1.69
79.66 ± 1.86"·
72.86 ± 2.07"
(mg/g)
% (Lipid WU
6.39 ± 0.17
7.96 ± 0.18'"
7.28 ± 0.21"
LiveT Wr.) Ratio
Liver Glycogen
11.03 ± 0.78
6.32 ± 0.34"·
8.81 ± 0.56"
(mg/g)
Liver Glycogen
107.89 ± 7.33
65.44 ± 2.84"·
86.732 ± 6.678+
(mg/liver wet weight)
... p< 0.05
Wt.= weight
• p< 0.01
. . . p< 0.0001
.. p<OJXn
NS = Non Significant
5.6) PROTEIN CONTENT
5.6.1) Total Liver Protein
5.6.1.1) First Experiment

---

71
In comparison 10 the control group of rats, the group of animaIs fed 7-DH diet
showed a tremendous amount of liver protein as evidenced by the highly significant
difference (p< 0.0001) (Table 12) and shown in Figure 10. The 4-MH d.iet d.id not show
any significant difference in liver protein content. The liver protein content expressed in
mg/g wet liver tissue (Table 9) showed the same trend with a highly significant increase
(p< 0.0001) when animals are fed 7-DH diet. The 4-MH d.iet did not show any significant
difference in liver protein content when compared 10 the control group of rats. However,
the 4-1vIH diet group of rats showed a significantly less increase (p< 0.(001) from the
greates t increase of liver protein noticed when animals were fed the 7-DH diet
Fi~ure 10: Total Lipid & Protein Contents in Liver OfRats Fed NH, 4-MH
& 7-DH Diets
(Mean ± SEM)
6000
•••
5000
0.. NH(Lipid)
ea
4000
~ NH (Protein)
e
.e
(SJ
...
4-MH (Lipid)
Cl
QI
...
• 4·MH(Protein)
Cl
~
U
~ 7-DH (Lipid)
~ 7-DH (Protein)
•• pc: 0.001
••• p< 0.0001
0=10
0=10
0=11
Group or Rats

---

72
5.6.1.2) Second Experiment
In the pair-feeding experiment, the group of rats fed 10% of 7-DH diet showed a
significant increase (p< 0.01) in liver protein content as compared to the control group.
The difference observed in liver protein content when animaIs are fed 15% of 7-DH diet
was a highly significant increase (p< 0.0001) when compared to the control group of
animaIs fed NH diet. These results are presented in Figure Il. When animaIs are fed 5%
of 7-DH diet, no significant difference was observed. When the resu1ts are expressed in
mg protein/g (Table 10) liver, the ratios followed the same trend with levels of
significance of p< 0.01 for rats fed 10% of 7-DH diet and p< 0.0001 when animaIs are
fed 15% of7-DH diet.
Fi&\\lre Il: Total Lipid & Protein Contents in Liver OfRats Fed NH & Graded
Doses Of7-DH Diets
(n=10 & Mean ± SEM)
7000-
•••
T
~ NH (lipid)
6000-
~:~:~:~:~:~:~
fir?
~
• NH(PrOlein)
••
••
5000 -
.~
m 5% 7·DH (lipid)
E!
.........
.9
4000-
" " "
-
...
...
...
...
...
...
~ 5% 7-DH (Protein)
'" " "
Cl
!~!~~(
-
"...""
...
...
Qol
!,,?:,-;:,
"..."..."...
Cl
...
...
...
f;]
- 3000- ~~!:.~,
" " "
10% 7-DH (lipid)
Q
~~-t~~
"..."..."...
u
-::,?:.{.::
"..."..."...
" " "
~ 10% 7·DH (Proleîn)
2000- -:.::,?,{:,
...
...
...
?:,-::,{:,
"..."..."...
-::.!:,~:.
"..."..."...
~'--::.{~
'"..."..."...
53 15% 7-DH (Lipid)
1000 - !:.~:.(:.
" " "
.........
"'."'.".
.......
"..." "
......
-:o\\.-:",~:,
"...""
...
...
.........
CI
..
15% 7·DH (Proleîn)
-:o\\.~~
" ""
0
" '" "
• p< 0.05
•• p< 0.01
••• p< 0.0001
Group of Rats

---

73
5.6.1.3) Third Experiment
Figure 12 shows a highly significant increase (p< 0.0001) in liver protein content
for rats fed 0.15% CFA diet when compared to a control group of rats fed non-heated
partially hydrogenated soybean oil diet. The highly significant increase (p< 0.0001) of
liver protein observed in animais fed cyc1ic fatty acid monomers diet in Figure 12 is also
seen when the results are expressed in mg/g wet tissue (Table Il) as compared ta the
control animais.
Fi&ure 12: Total Lipid & Protein Contents in Liver OfRats F ed NH & CFA
Diets
(Mean ± SEM)
6000
•••
"
" ,;"~" "
" .;",;'"
5000
, " ' "
,; " ~ ~ ,; "
"""
" .; ~ ,; ; ;
" , ' , " ,
, , ; , ; ~ ,
""''"
,; , , ,. " ,
, " , ;
CIO
4000
'""",;-
" " "
~ NH(Lipid)
E
" " " ~ " ,
"""
"," , ,. ./ ,
" " " '"
.S!
D NH (Protein)
- 3000
=
~
~ CFA (Lipid)
-=QU 2000
[3
CFA (Protein)
1000
. . . p< 0.0001
0
D=10
8=11
Group of Rats

---

74
5.6.1.4) Fourth Experiment
When the T-7DH diet was fcd to rats in a pair-feeding experiment, their liveTs
showed a significant increase in protein content to p< 0.01 when compared to a control
group of rats fed NH diet (Figare 13). When rats fed the treated used oil diet were
compared to thase fcd the non-treated used oil diet, the fonner group showed a decreased
liveT protein (p< 0.05). When expressed in mg/g WC! liver tissue, the protein content of
liveT also showed a significant increase (p< 0.01) in comparison with the control group of
rats fed NH diet. (Table 12).
Fi~ 13: Total Lipid & Protein Contents in Liver Of Rats Fed NH, 7-DH &
T-7DH Diets
13 NH(Lipid)
l1li NH (Prolein)
m 15% 7-DH (Lipid)
m 15% 7-DH (Protein)
!il T-7DH(Lipid)
~ T·7DH (Protein)
• p< 0.05
.. p< 0.01
••• p< 0.0001
Group or Rais

---

75
1
1
5.6.2) Microsomal Protein
1
5.6.2.1) First Experiment
Livers of rats fed 7-DH diet (Table 9) have shown a highly significant increase
1
(p< D.DOOI) for microsomal protein (MP) in comparison with the control rats. When
1
animals were fcd 4-l\\1H diet no significant differences were observed when compared te
the control group. AIso, results displayed in Table 9 show that MP in 7-DH diet group
1
was double of that obtained for the control group of rats. In addition, the increased level
1
of microsomal protein noticed for the group of rats fcd the ?-DH diet was significantly
different (p< 0.00(1) from that observed for animais fed the 4-MH diet.
5.6.2.2) Second Experiment
After 10 weeks of pair-feeding rats with 15% of 7-DH diet, microsomal protein
content has significantly increased (p< 0.0001) when compared to a control group of rats
fed NH diet. The group fed 10% of 7-DH die' showed the same trend with a significant
increase al p< 0.0001. Feeding rats with 5% of ?-DH diet did not show any signifieant
difference when compared to the control group. (Table 10).
5.6.2.3) Thire! Experiment
When rats are fcd 0.15% CFA diet, their microsomal protein content increased
significantly (p< 0.0001) when cornpared to the control rats fed NH diet. The increase
was double the value of that of the control rats as evidenced by the data presented in
Table Il.
5.6.2.4) Fourth Experiment
ln the pair-feeding experiment when T-7DH diet was fed to rats, their microsomal
protein content did not show any significant difference as compared to the control rats fed
NH diet. (Table 12). However, when the group fed the treated used oi! diet was compared
to the group of rats fed the non-treated used oil, the decreased level of the microsornal _ _

---

76
protein observed for the fonner group was significant1y different from the latter group
(p< 0.0(1).
5.7) LIVER LIPID CONTENT
5.7.1) Tolal Lipid
5.7.1.1) FirstExperiment
Liver lipid content of rats fed 7-DH diet is significantly increased (p< 0.001) as
compared to the control animaIs. When rats were fed 4-MH diet, no significant
differences were ohserved when compared to the control group as shown in Figure 10.
When results are expressed in mg/g (Table 9). the 7-DH group of rats showed a highly
significant increase (p< 0.0001) in lipid content in comparison to the control animaIs. The
increased content of liver lipid observed for the group of rats [ed the 7-DH diet was
significantly different (p< 0.0001) from that noticed for animaIs fed the 4-MH diet. In
addition.livers of rats fed 7-DH diet presented in aspect sorne fatty infiltration of the liver
cells as small fat droplets.
5.7.1.2) Second Experiment
In the pair-feeding experiment, the level oflipid in livers of rats fed 15% of7-DH
diet showed to be highly increased (p< 0.0001) when compared ta the control animais fed
NH diet. Feeding 10% of 7-DH diet ta rats showed a significant increase (p< 0.05) of
liver lipid content in comparison with the control animals. AnimaIs fed 5% of the llsed ail
did not show any significant increase of liver lipid content as presented in Figure 11.
Table 10 displays the results expressed in mg/g and shows that Ûle 15% of 7-DH diet
group of rats had a highly significant increase (p< 0.0001) of liver lipid content when
compared ta Ûle control group. When animais are fed.lO% of7-DH diet in comparison ta
the control group, the significant increase observed in liver lipid content was at p< 0.01.
Rats fed 5% of 7-DH diet did not show any significant difference when they are
compared ta the control animals.

---

77
5.7.1.3) ThirdExperiment
Figure 12 c1early shows the highly significant increase (p<O.OOOI) of liver lipid
content when animaIs are fed 0.15% of a mixture of cyclic fatty acid monomers in the
diet as il is compared to a control group of rats fed 15% of a non~heated partially
hydrogenated soybean oil diet. Lipid content of liver expressed in mg/g (Table 11)
showed a highly significant increase (p< 0.0001) when rats are fed the 0.15% ŒA diet in
comparison with the control group. In addition, small fat droplet infiltrated in liver cens
showed up and were extensive.
5.7.1.4) Fourth Experiment
The slight increase (p< 0.05) of liver lipid content observed wben animals are fed
T-7DH diet in a pair-feeding experiment is to be seen in Figure 13 as it is compared to a
control group of rats fed NB diet. Results expressed in mg/g we! tissue (Table 12)
showed a significant increase (p< 0.(01) ofliver lipid content when animaIs are fed 15%
of the T-7DH diet in comparison to the control rats. The decreased liver lipid content
observed for the group of rats fed the T~7DH diet was not significantly different from the
liver lipid of those fed the non-treated used oil diet.
5.7.2) Lipid WeightlUver Weight Ratio
5.7.2.1) First Experiment
Table 9 displays the results obtained for lipid weight/liver weight ratio when rats
are fed 7-DH and 4-:MH diets. In comparison to the control group. the 7-DH diet group of
rats showed a highly significant increase (p< 0.0001) of lipid weight/liver weight ratio.
The 4-MH group did not show any significant difference in liver lipid accumulation. The
increased ratio observed for the 7-DH diet group of rats was significantly different
(p<o.oool) from that of animais fed the 4-MH diet.
5.7.2.2) Second Experiment
In pair-feeding experiment, rats fed 10% or 15% of 7-DH diet' sbowed an
increase in their lipid weight/liver weight ratio in comparison to a control group of rats

---

78
fed NH diet. The increase was highly significant (p< 0.00(1) in the 15% 7-DH diet group
but only at p< 0.01 in the group of animals fcd 10% of7-DH diet. When rats are fcd 5%
of 7-DH diet, no significant difference were noticed. Results are to be seen in Table 10.
5.7.2.3) Third Experiment
AnimaIs fcd 0.15% CFA diet showed in this experiment that cyclic fatty acid
monomers have the potential to induce a highly significant accumulation (p< 0.0001) of
liver lipid in comparison to a control group fcd NH diet. (Table Il).
5.7.2.4) Fourth Experimem
Pair-feeding rats with 15% of T-7DH diet showed a significant increase of lipid
weight/liver weight ratio al p< 0.001 in comparison with a control group of animals fed
NH diet. (Table 12). The decreased level of lipid weight/liver weight ratio observcd for
the group of rats fed the T-7DH diet was significantly different (p< 0.01) from that of
animals fcd the non-treated used oil met.
5.7.3) Liver Fatty Acid Profiles
In all experimental groups of rats. fatty acid profiles obtained from GLe analysis
did not show any significant difference when compared with the fatty acid profùes of the
control group. The trend observed was a non significant increase in palmitic acid (C16:0)
content in livers of rats fed either 4-:tvŒI; 7-DH; CFA, or T-7DH diets in comparison with
their respective control groups. In addition, gamma-linolenic acid (CI8:3n-6) content was
increased but non significantly when animaIs are fed 7-DH, CFA, or T-7DH diets. The
percentages of the other FFAs : stearic (C18:0). oleic (CI8: In_9),linoleic (C18:2n-6), or
linolenic (C18:3n-3) were non significantly decreased when animais are fed the used oils
or 0.15% cyclic fatty acid monomers in the diet. (Sec Appendix for the tables containing
the weight percent of fatty acids of interest).
5.8) LIPID/pROTEIN RATIO
5.S.1) First Experiment

---

79
Resu1ts plotted in Figure 14 show that lipid/protein ratio of rats fed 7-DH diet was
significantly low (P< 0.0(01) when compared ta the control group of rats [ed NH diet.
The increase observed for the 4-MH group of rats was not significantly different from the
control group. However, when the 4-MH diet group of rats was compared to those fed the
7·DH diet, the lowest ratio observed for the latter group was significantly different (p<
0.0001) from that of the fonner group of animaIs.
Figure 14: LipidlProtein Ratio in Liver OfRats Fed NH, 4-MH & 7-DH Diets
(Mean ± SEM)
1.5-
.2
-~
T
" 1 -
1::1
.Q:j
-
rn.. NH
0
"'"
=..
.....
•••
~ 4·MH
"l;l
:ë
--
E2l 7-DH
~
0.5-
... p< 0.0001
0=10
0=10
0=11
Group of Rats
5.8.2) Second Experiment
Lipid/protein ratio when rats are pair-fed graded doses of 7·DH diets showed that
the 15% of7·DH diet induced the lowest ratio with p< 0.01 while groups fcd 5% or 10%

---

80
of 7-DH diets did not show any significant decrease as compared. ta the control animaIs
(Figure 15).There was no apparent dose response.
Fi~re 15: LipidlProtein Ratio in Liver OfRats Fed NH & Graded Doses Of
7-DH Diets
(n=lO & Mean ± SEM)
1.25
.9
1
-CIl01:
•••
l' ,
,
,
••
•••
' ","; , ....
~
&1 NH
•••
............
, , , ,
1::
•••
,;",",,,,
'iij
0.75
0
•••
" '" " .....
, ., , ,
[J'J
-
, , , ,
5% 7-DH
~
•••
" , , "
•••
' .... , "
0..
' , , ,
-..
•••
,",, "
, ,.....
~
, , , "
,
EJ lO%7-DH
Cl.
0.5
•••
" ..... """
•••
.........
' , , ,
::3
•••
.........
, , , ,
, , , ,
pj
•••
.........
15%7-DH
•••
.........
" , , ,
0.25
•••
,' '"
, ,",.....
•••
...
... ... ...
, , , ,
•••
,",",","
•••
...
...
...
, , , ,
•••
,"," "
, ,"
•••
, ,",",
'\\,
"\\
•• p< 0.01
0
" " " ....
Group of Rats
5.83) Third Experiment
When rats are fed 0.15% CFA diet. their lipidlprotein ratio significanûy decreased
(p< 0.0(01) in comparison with a control group of animals fed NB diet. Results are
plotted in Figure 16.

---

81
Fi~re 16: LipidlProtein Ratio in Liver OfRats Fed NH & CFA Diets
(Mean ± SEM)
1.5-
T
1 -
.::
-c-:1:11:
=
•••
'ij:
-
....
0
...
•••••
~
0.5-
-
•••••
"Cl
•••••
'a
•••••
::3
•••••
•••••
•••••
... p< 0.0001
•••••
•••••
0
•••••
D=10
0=11
Group or Rats
5.8.4) Fourth Experiment
Rats pair-fed with T -7DH diet did not show any significant decrease in
lipid/protein ratio when compared to a control group of rats fed 15% of non-heated
partially hydrogenated soybean ail diet. (Figure 17). The increased levei of lipid/protein
ratio observed in the liver of rats fed the T-7DH diet was significantly different (p< 0.05)
from the ratio noticed for those fed the non-treated used oil diet.

---

82
FilUlre 17: LipidlProtein Ratio in Liver Of Rats Fed NH, 7-DH & T-7DH
Diets
(n=lD & Mean ± SEM)
1.25
NS
1
... . .
......"."'....
...............
...".... ....
".
...............
...,/'."'."'....
...............
"'
"' .
0 NH
...............
............ 1' •
...............
"'......."....
...............
..."'
.
...............
ll.lI 15% 7-DH
......J' .......
...............
......J' .......
...............
m T·7DH
"'''''./'0'''''''
...............
... ....
,/'.,/'
"'.
................
".
...............
......"'.......
...............
"."'
.
...............
...............
...............
•• p< 0.01
.........J' ....
...............
""......"'....
...............
......,.."'....
...............
!'Os = Non Significant
"."'
.
Group or Rats
5.9) LIVER GLYCOGEN CONTENT
5.9.1) First Experiment
Totalliver glycogen content expressed in mg/wet liver weight (Table 9) when rats
are fed 7-DH diet showed a highly significant decrease (p< 0.0(01) in comparison with a
control group fed NH diet. Animais fed 4-MH diel showed a slight decrease which was
significant at p< 0.05 when compared ta the control rats. When results are expressed in
mg/g (Table 9), the same trend (p< 0.0001) was observed in the group of rats fed 7-DH

---

83
diet. In this way of expressing the results, no significant decrease was observed when
animaIs were Ced 4-MH diet. Whcn the 4-w-I diet group of rats was compared to those
fed the 7-DH wet, the decreased content of liver glycogen noticed for the latter group was
significantly different (p< 0.05) from that of the fonner group.
5.9.2) Second Experiment
In the pair-feeding experirnent. Table 10 displays the results about liver glycogen
content. Totalliver glycogen in mg/liver wet rissue decreased significantly (p< 0.0001) in
the group of rats fed 15% of 7-DH diet as weIl as in those fed 10% of ?-DH diet in
comparison to the control animals. When feeding 5% of the ?-DH diet to rats, the
decreased liver glycogen content observed was not significantly different from the control
rats. When results are expressed in mglg liver tissue, animais fed 5% of 7-DH diet
showed a slight significant (p< 0.05) decrease of glycogen content in comparison with a
control group fed NH diet. The d.ecreased values observed in groups fed 10% or 15% of
?-DH diets followed the same trends (p< O.OCKH) seen when the results are expressed per
liver content. An apparent dose response was noted.
5.9.3) Third Experiment
Table Il contains the results of animals fed 0.15% CFA diet in comparison with a
control group of rats fed NH diet. The decrease in liver glycogen content observed in
CFA group was significant at p< 0.001 when the results are expressed in mgl1iver wet
weight. However. the decreased glycogen value when results are expressed in mg/g liver
tissue was highly significant (p< 0.0001).
5.9.4) Fourth Experiment
Pair-feeding rats with 15% of the T·7DH diet showed a significant decrease (p<
0.05) when results are expressed in mg/liver weight in cornparison with a control group
of animaIs fed the NH diet (Table 12). The decreased liver glycogen content observed
when results are expressed in mg/g liver tissue was significant at p< 0.01. The lowest

---

84
liver glycogen content observed for rats fed the non-treated. used oil diet was significantly
different (p< 0.05) from that of animais fed the treated used oil diet.
5.10) LIVER MICROSOMAL CYTOCHROMES CONTENT
5.10.1) Cytochrome b5
5.10.1.1) FirstExperiment
The results obtained from the measure of cytochrome bS content of liver
microsomes of rats Ced 4-MH or 7-DH diets are to be seen in Table 13. The 7-DH diet
when fcd to rats induced a highly significant increase (p< 0.0001) of cytochrome bS
content in their liver microsomes in comparison with a control group of rats fed NB diet.
Even though the 4-MH wet fed to rats induced an increased content of cytochrome bS in
microsomal fraction, the difference observed when compared to the control group was
also significant but al p< 0.01. The increased content of Cyt.bS observed for the ?-DH
diet group of rats was significantly different (p< 0.0001) from the level noticed for the
group fed the 4-MH diet. An apparent dose response was observed.
Table 13: Hepatic Microsomal Cytochromes Contents Of Rats Fed NH, 4-MH
& 7-DH Diets
NH
4·MH
7·DH
(n=8)
(n=lO)
(n=l1)
(Mean±SEM)
Cyt.bs Content
0.37±0.01
0.54 ±O.oz'
0.84 ± 0,04···
(nmoVrng micr.protein)
Cyt.P450 Content
1.03±0.03
1.47±0.D6·
1.97 ± 0.08·"
(nmoVmg micr.protein)
·p<0.01
••• p< 0.0001
micr.::: microsomal

---

85
5.10.1.2) Second Experiment
The results of the content of cytochrome bS when rats are pair-fed graded doses of
the 7 days frying used oil are presented in Table 14. The highly significant increase (p<
0.0001) of cytochrome bS is to he seen when animaIs are fed 7-DH diet in comparison
with a control group fed the NH diet. AnimaIs fed 10% of the 7-DH diet aIso showed a
significant increase of the content of cytochrome bS but al p< 0.01. An apparent dose
response was noted.
Table 14: Hepatic Microsomal Cytochromes Contents OfRats Fed NH
& Graded Doses Of7-DH Diets
NH
5% 7-DH
10% 7·DH
15% 7-DH
(Mean + SEM & n=lO)
Cyt.bs
Content
0.64 ± 0.05-
0.75 ± 0.08·"
(nmol/mg micr.protein)
0.46 ± 0.02
0.48 ± 0.03
Cyt.P450 Content
1.04 ± 0.03
1.13 ± 0.06
1.32 ± 0.09'
1.68 ± 0.08'"
(nmol/mg micr.protein)
• p< O.QI
micro = microsomal
••• p< 0.0001
5.10.1.3) Third Experiment
Feedlng 0.15% CFA diello rats 100 to a highly significanl increase (p< 0.00(1) of
the content of cytochrome bS in liver microsomes when compared te the control group
fOO NH diel as evidencOO by the results presented in Table 15.

---

86
Table 15: Hepatic Microsomal Cytochromes Contents Of Rats Fed NH
& CFA Diets
NH
CFA
(n=8)
(n=l1)
(Mean±SEM)
Cyl.bs Content
0.37 ± 0.01
0.73 ± 0.06···
(nmol/mg micr.protein)
Cyt.P450 Conlent
1.03± 0.03
1.86±0.11"·
(nrnoUmg micr.protein)
••• p< 0.0001
micro = microsomal
Table 16: Hepatic Microsomal Cytochromes Contents Of Rats Fed NH, 7-DH
& T-7DH Diets
NH
15%7-DH
T-7DH
(Mean ± SEM & n=lO)
Cyl.bs Content
0.46±0.02
0.75 ±0.08·"
0.49 ± O.03NS
(nmoVmg micr.protein)
Cy/.P450 Conten/
1.04 ± 0.03
1.68 ±O.Og·...
NS
1.15±O.D6
(nmoVrng micr.protein)
••• p<O.OOOl
NS = Non Significanl

---

87
5.10.1.4) Fourth Experiment
Pair-feeding rats with 15% of the T-7DH diet did not show aoy signifieant
increase in the content of microsomal cytochrome bS when compared to the control group
fed the NH diet. The greatest content of Cyl. bS in the microsornal fraction of rats fed the
non-tteated nsed oil diet was significantly different (p< 0.(01) from that noticed for the
group of animaIs fed the treated used oil diet. Table 16 cantains the values.
5.10.2) Cytochrome P450
5.10.2.1) First Experiment
Results of cytochrome P450 content when rats are fcd 4-MH or 7-DH diets in
comparison with a control group fcd NH diet are to he seen in Table 13. The cytoehrome
P450 content in liver microsomes of animaIs fcd the 7-DH diet was increased (p< 0.0001)
as compared to the control group. The group [cd 4-MH diet alsa had increased liver
cytochrome P450 content but at p< 0.01. When the 4-MH diet group of rats was
compared to the group of rats fed the 7-DH diet, the greatest content of cytochrorne P450
observed in the latter group was significantly different (p< 0.(01) from that noticed in
the former group. An apparent dose response was noted.
5.10.2.2) Second Experiment
In the pair~feeding experiment where rats are fed graded doses of the 7 days used
oil diets, the content of liver microsomal cytochrome P450 in the group of rats fed the
15% of 7-DH diet was highly increased (p< 0.0001) when compared to the control
animals fed the non-heated partially hydrogenated soybean oil diet. When animais are fed
10% of the 7-DH diet, the increase observed was also significant at p< 0.01. Feeding rats
with 5% of 7-DH diet did not show any significant increase in the content of liver
microsomal cytochrome P450. Table 14 shows the results. A dose response was also
apparent in this experiment.

---

88
5.10.2.3) Thini Experiment
Table 15 displays the results of liveT microsomal cytochrome P450 content when
rats are fed 0.15% CFA diet. The inerease observed was highly signifieant (p< 0.0001)
when compared to the control rats fed the NH diet.
5.10.2.4) Fourth Experiment
The content of liveT microsomal cytochrome P450 when rats are pair-fed 15% of
the T-7DH diet is te be seen in Table 16. The increase of the cytochrome P450 content
was not significantly diffcrent from the control rats fed the NH diet. The greatest level of
Cyl.P450 ohserved for the group of rats fed the 7-DH diet was signifieantly different (p<
0.0001) from that noticed when animals are fed the treated used oil diet.
5.11) ENZYMES ACTIVITY
5.11.1) Glutamnte Pyruvate Transaminase (Ee 2.6.1.2)
5.11.1.1) First Experiment
L-alanine:a-ketoglutarate aminotransferase, EC 2.6.1.2, activity investigated in
liveTs of rats fed 4-MH or 7-DH diets showed that the increase observed was not
significant when compared to a control group of rats fcd NB diet. There was no
significant difference obsetved between the 4-MH and the ?-DH diet groups of rats. The
results are presented. in Figure 18.

---

89
Figure 18: Glutamate Pyruvate Transaminase Activity in Liver OfRats Fed
NH, 4-MH & 7-DH Diets
(Mean ± SEM)
20-
NS
NS
T
,-,
T
=:.
15 -
--
's:
Ëil NH
;::l
-;
CI
..
4-MH
Cl
10 -
.2
...=
~ 7-DH
Cl
..il.l-Cl 5-
-
NS = Non Significant
0=10
0=10
0=11
Group of Rats
One intemational unit CU) of an enzyme is dermed as !hat amount of enzyme that will
convert IlJlllol of substrate per minute under the spedfied conditions of the procedure.
5.11. 1.2) Second Experiment
The activity of the transferase EC 2.6,1.2 observed in plasma when rats are pair-
fed graded doses of 7-DH diets did not show any statistical signîficance when compared
ta a conrroi group of animals fed NH diet. Figure 19 shows the results.

---

90
Figure 19: Glutamate Pyruvate Transaminase Activity in Plasma Of Rats Fed
NH & Graded Doses Of7-DR Diets
(n=10 & Mean ± SEM)
25
NS
NS
NS
.-..
20
2-
........
'"
\\..
•••
, , ,
•••
... ... " "
[!J NH
-
' , ,
••• ' " '" ,
ï3
' , ,
•••
....... " ....
~
15
' , ,
•••
\\..
\\..
" ....
' , ,
, , ,
0 5%7~DH
l'I;
••• " " " "
.
, , ,
~
••• .... , , ,
, , ,
Cl
•••
" , , ,
l'I;
' , ,
~ 10% 7·DH
-
•••
" " , ....
10
••• .... , , ....
' , ,
Cl
, , ,
"'"
<li
••• .... \\. '\\. "
•••
' '\\, .... ....
-
, , ,
Cl
•••
" , ........
.....
' , ,
' , ,
• 15%7~DH
•••
' " , "
5
•••
....., \\.,", \\.
•••
...,",","
••• "," ,, ,,
, , ,
NS = Non Significant
••• " " , ,
••• ........ , ,
, , ,
" , , ,
0
Group of Rats
5.11.1.3) Thini Experiment
Figure 20 shows an increase in the activity of liver glutamate pyruvate
transaminase when rats are fed 0.15% CFA diet in comparison with a control group of
rats [ed NH diet. However, this increase was not statistically significant.

---

91
Figure 20: Glutamate Pyruvate Transaminase Activity in Liver OfRats
FedNH & CFA Diets
(Mean ± SEM)
20 -
NS
-
T
;:>
--
T
[l[l[l[l[l
[l[l[l[l[l
•• lS
""'O'''';.U;'''
;",N''',..,.''';;
CI:I[:JOI:l
;:>
" ' ; " " " " ; ; ; ;
;u",,,.,,,u,,,
O[][]1:10
N""''''''''''
",,,,,,."U,,,
[][][][][1
";"" .."",,,..
œ
[][][][][]
"";"'1'''''''''';
=
""".N",,,n
n"'...."."n
[l[l[l[l[l
~
0
",,""'''''''',..,
<=
h"''''",,,,,
[l[l[l[l[l
,.. ...."',..;N·".
œ
10
Ol:l[][][]
",......"''',..N''
O N ·.." , . ..
=
.
""",,,,,,,n
[l[l[l[l[l
~
"
.."''''"....",..,..
.!l
OI:lOOO
''''',,'''''''''''''''''
''''",,.'''...,,,.,,
=
,.."",,u."',,.....'"
CODe[]
".." ........"'......
~
,,,,,,,,,',,"',,,
C[]C'CO
""",
"'".", ....
.... "'".."
"'''';,;.,j'
[][][][][]
..
5
,;"" .. "",,..,
",
·Nh'''h'.I'NN
[l[l[l[l[l
0 ;......" ' . 1 0 '
" " ...n ...",..
[l[l[l[l[l
....."u·".",,,
.l'.... ,,.."''''''' ..'
[][][][][l
0/"",.."." ..".
'''.NUnn''·
CO[]CC!
,....,,"''''....,......
"".,,/'N',,....;
O[]I:::I0[]
NS = Non Significant
""N'N'''';'''
o
,"'.."'",....,..,
C[u:n::It:l.
0=10
0=11
Group of Rats
5.11.1.4) Founh Experiment
The slight increased activity of alanine aminotransferase Ee 2.6.1.2, obtained in
plasma, from the group of rats fcd 15% of the T-7DH diet in a pair-feeding experiment
did not have a statistically significant difference in comparison with a control group fed
NH met. There was no significant difference between the non-treated used ail diet group
of rats and those fed the treated used oil diet. Figure 21 displays the histogram of the
report.

---

92
Figure 21: Glutamate Pyruvate Transaminase Activity in Plasma OfRats Fed
NH, 7-DH & T-7DH Diets
(n=10 & Mean ± SEM)
25
NS
NS
20
--..
:J
' - '
•••••
....
•••••
·a
•••••
•••••
0
NH
:J
15
•••••
•••••
-=
•••••
E3 15% 7-DH
1:1
•••••
~
•••••
œ
10
•••••
Il T-7DH
1:1
...Q,>
•••••
....
•••••
1:1
•••••
•••••
- 5 •••••
•••••
•••••
•••••
!"OS = Non Significant
•••••
0
•••••
Group of Rats
5.11.2) Microsomal NADPH-Cytochrome P450 Reductase
(EC 1.6.2.4)
5.11.2.1) First Experiment
The aetivities of the P450 mixed fnoetioo oxidase enzyme EC 1.6.2.4 measured in
1iver microsomes of rats fed 4-MH or 7-DH diets were increased when compared to a
control group of rats fed NH diet. The increase was highly significant (p< 0.00(1) when
animaIs were fed the 7-DH diet. It was also highly significant when rats were fed 4-MH
diet (p< 0.(01). The greatest activity measured for the group of rats fed the 7-DH diet

---

93
w.s signifie.nùy different (p< 0.0001) from th.t of animals fed the 4-MH diet. Figure 22
displays the comparative histogram. An apparent dose response was notOO.
Figure 22; NADPH-CYlOchrome P450 Reductase Activity in Liver Microsomes
OfRats Fed NH, 4-MH & 7-DH Diets
c
.~
(Mean ± SEM)
-c 150
"~
...
•E
c~0".~ 100
a
••
!il NH
..a
1:::1 4·MH
-cËl
111
'"
7·DR
0
a
50
c
.~
•• p< û.ex)!
..-.;:
... p< 0.0001
"'u
."
0
0=8
0=10
D=l1
Group of Rats
5.11.2.2) Second Experiment
Figure 23 displaying the histogram of the enzyme Ee 1.6.2.4 activity when rats
are pair-fed graded doses of 7-DH diets showed an increasing level of the enzyme
response to the diets when compared to a control group fed NH diet. The increase was
highly signifie.nt (p< 0.0001) in 10% and 15% of 7-DH diet groups. 1t was also

---

94
significant at p< 0.001 when animais were fed 5% of the 7-DH d.iet. A dose response was
noted.
Figure 23: NADPH-Cytochrome P450 Reductase Activity in Liver Microsomes
OfRats Fed NH & Graded Doses Of7-DH Diets
(n=lO & Mean ± SEM)
Cl
200
'ij
....o...Co
•••
-;
e
o
li>
o
...
fli NH
y
'ë
0
5%7-DH
CIl
e
---
&'1 10% 7·DH
-=
'ë
'::::l
0
15%7-DH
o
eCl
.S
•• p< 0.01
;0.,
....
.......
....
• •• p< 0.0001
....Col-<
Group of Rats
5.11.2.3) Third Experiment
Feeding 0.15% CFA diet to rats highly induced the activity of NADPH-
cytochrome P450 reductase at p< 0.0001 when compared with a control group of rats fed
NH d.iet. Figure 24 shows the histogram.

---

95
Fimlle 24: NADPH-Cytochrome P450 Reductase Activity in Liver Microsomes
OfRats Fed NH & CFA Diets
(Mean ± SEM)
Cl
150
.~
~
0.
...
•
e~
.J'.J'.J'.",.",.",
..................
100
.......J'."'."'.'"
...................
."'....J'.J'."'.'"
••
..................
...."'."'."'."...
e
..................
."'......."'.,.'"
..................
....".."'.,/'."'.'"
..................
~
~
e
-
',/'0"',,/',"' • .1','"
..................
'J"J"""""""'"
..................
."'."'."
..
.1'.,.
~
••
.
e
·''·.l'·J'·b.!'·'''
..................
:;
50
'''''.1'''''''''''''''''
..................
."'."'...."'."'...
..................
e
...."'."'.,."'.'"
..................
•
'(""""".1""''''
..
'\\0
.
...."'.J'."'."'.'"
..................
• . . p< 0.0001
••
'''''''''1'.",./,.",
..................
.,....."'."'."'.'"
...................
."'."'...."'......
..................
o
......."'.".."'.'"
0=8
0=11
Group of Rats
5.11.2.4) Fourth ExpeIiment
Pair-feeding rats with 15% of the T-7DH diet led 10 a signifieant inerease (p<
0.01) of the liver microsomal NADPH-cytochrome P450 reductase activity when
compared with a control group fed NH diet. The greatest activity of the reductase
observed for the group of rats fed the non-treated used oil diet was significantly different
(p< 0.0(01) from that measured for the group of animais fed the T-7DH diet. Figure 25
shows the histogram.

---

96
Fîgure 25: NADPH-Cytochrome P450 Reductase Activity in Liver Microsomes
OfRats FedNH, 7-DH & T-7DH Diets
1::
(n-10 & Mean ± SEM)
';:;
-0 200
"'"
Q"
0:1
E
•••
0
'"
0
150
""
.=:E
••
~ NH
oc
E
.+••
12 15% 7-DH
- 100
.=
••••
.Ë
••••
'0
CCCCI:l
••••
~
••••
T-7DH
E
tltlCCC
1::
Cl:ltlCC
••••
50
cecce
••••
••••
.=
CCCCC
eCtl8
••••
••••
.. p< 0.01
~
aoe e
-
coee
••••
:~
[loeee
••••
-y
eoecc
••••
0
<
••••
... p< 0.0001
Group 01 Rats
5.11.3) Carnitine Palmitoyltransferase-I (CPT-/)
(Ee 23.1.21)
5.11.3.1) First Experirnent
The activity of CPT-I in liver mitochondrial membrane of rats fed ?-DH diet
significantly decreased with p< 0.01 in comparison with a control group of rats fed NH
diet. There was no data record.ed for the 4-MH diet group of rats. The histogram plotted
in Figure 26 shows the evidence.

---

97
Figure 26: Carnitine Palmitoyltransferase-l Activity in Liver Mitochondria Of
Rats Fed NH & 7-DH Diets
(n=8 & Mean ± SEM)
::cs
10 -
0»
e
...
T
.r:
0»
.S
7.5 -
••
.,..
::: 1:
=
s.. .-
0»
[][]t1[][]
~-
(,,>
0
-
r:1I:1I:~[][]
...
~Q,
[][][][][]
[][][][][]
.."::
O.D
5-
[][][][][]
E 8
--
[][][][][1
~ .S
[1[][]C]O
Q,E
[l[][]Gg
"0
[][][]1:1~
[][][]C]C
S
2.5 -
[][][][][]
1:1
[]I:U:][][]
.S
[][][][][]
[I[][][][]
.. p< 0.01
=--.
-
[][][][][]
I:I[][]I:I[]
:E
1:I[]1:I1:10
0
-(,,>
<
Group or Rats
5.11.3.2) Second Experiment
Figure 27 shows the decreased activity of the enzyme CPT-I when rats are pair-
fed graded doses of 7-DH mets in comparison with a control group fed. NH diet. The
decreased value observed in the group fed 15% of the 7-DH diet was significantly
different from the control group at p< 0.01. Even though a decreased activity was
observed. in groups fed. 5% and 10% of7-DH diets, il was not significantly differem from
the control animais. An apparent dose response was noticed.

---

98
Fi~re 27: Carnitine Palmitoyltransferase-I Activity in Liver Mitochondria Of
Rats Fed NH & Graded Doses Of7-DH Diets
(Mean ± SEM)
10
•
o
."';;•·•0-.-.
ONH
~­
0 0
-.-.'"
e ~
~ 5%7-DH
;; e
"'.
E;J 10% 7-DH
'0 Ëà
e
•
!!il 15% 7-DH
.5
,;'
.~
-•..,
•• p< 0.01
n=8
n=7
n=7
D=8
Group or Rats
5.11.3.3) Third Experimen1
When rats are fed 0.15% CFA diet, the decreased activity of CPT-I observed was
significant at p< 0.01 in comparison with a control group of rats fed NH met. Figure 28
displays the histogram.

---

99
Fi~re 28: Carnitine Palmitoyltransferase-I Activity in Liver Mitochondria
OfRats Fed NH & CFA Diets
(Mean ± SEM)
-"l;l 10
~
5
...
oS:
[][][]Ot]
OO[]O[:J
~
.9
ClCOOO
• •
7.5
DO[][]O
.-::
CDCD[]
1::
1::
.. ...
D[][][][1
co ~
[][][][]O
':::0
C[][][][]
>,100
[][][][][]
o
1:1.
-
[]OO[][]
. -
Coll
5
E e
[]O[]OO
='i:l
[JOCCIC
~
~ CFA
1:1. ...
OO[][lC
El
[l[][][][]
0
OOOO[:J
5
OO[][]C
1::
2.5
ClClOOO
OCIODO
.el
or:II::10 CI
•• p< 0.01
[lD[lOO
>.
OCl[][][]
;'::
[l[]Or:lO
.::
O[][][j[]
-....
0
<.
0=8
0=7
Group of Rats
5.11.3.4) Fourth Experiment
The histogram plotted in Figure 29 shows a non significant decreased activity of
the transferase EC 2.3.1.21 in liver llÙtochondrial membrane of rats fed 15% of the T-
7DH diet in comparison with a control group fed NH diet. The lowest activity of CPT-I
measured for the group of rats fed the non-treated used oil diet was not significantly
different from that observed for those fed the treated used ail diet.

---

100
Figure 29: Carnitine Palmitoyltransferase-l Activity in Liver Mitochondria Of
Rats FedNH, 7-DH & T-7DH Diets
(Mean ± SEM)
10 -
...
NS
~
1"
%
'"
,!:
7.5
-13
-
••
~
T
"'"
Ci
, , ,
,
~ ';J
, , , ,",
, , " , ,
, , , , ,
-....o-0
, , , , ,
~NH
...
, , , , ,
~
,
~Q.
" , , ,
, , , , ,
, , , ,
, .... .... .... ....
5
(2]
15%7-DH
.5ë
( l I _
-
"
, , ,
, ,
....
....
....
, ,
" ",
Q"
,
,El
.... .... ,
,
"...."
, ,
.... .... " , .... "
,
, , ",
'0 e
,
m T-7DH
, ,
....
, " ....
, ,
El
....
,
.... "....
....
, , , ",
1:
~
,' , , ,
....
2.5 -
, , , , ,
, , , ,
....
, , , ,
.!:!
, , ,
8:
....
....
, , , ~ "~
•• p< 0.01
~
....,
"
'\\.
..... '\\,
" , , ,
", ,
,
.... "," ",
§
,,,, ",",",
, ,
, ,,,, "
NS
, ,
=Non Siignficant
o
~
n=8
n=8
n=7
Group or Rats
5.11.4) 1socitrate Dehydrogenase (/CDH)
(EC 1.1.1.42)
5.11.4.1) Fust Experiment
The activity of isocitrate:NADP-oxidoreductase, decarboxylating EC 1.1.1.42 is
decreased in liver mitochondria TCA cycle when rats were fed either 4-WI or 7-DH diets
in comparison with a control group fed NH diet. The decreased activity observed in the 7-
DH diet group was significantly different from the control group at p< 0.05. However, the

---

101
decreased activity noticed when animais were fed 4-MH diet was not significant when
compared to the control group. The lowest ICDH activity measured for the 7-DH diet
group of rats still has an apparent dose response although not statistically different from
that of animals fed the 4-MH diet. Figure 30 has the histogram of the report.
Fi&ure 30: [soGitrate Dehydrogenase Activity in Liver OfRats Fed NH,
4-MH & 7-DH Diets
(Mean ± SEM)
0.6
0.5
•
=
.~
-
;..,0
••••
0.4
-"'"
:E CI.,
-
••••
••••
~ NH
~
~
••••
<. El
••••
0.3
••••
~ --=
[] 4-MH
".
ë: .-
••••
... El
••••
~--
110>-
••••
c. 0
ID 7·DH
0.2
••••
V':l
El
••••
::1
••••
.9
••••
0.1
••••
••••
••••
••••
••••
• p< 0.05
0
0=10
0=10
0=11
Group or Rats
5.11.4.2) Second Experiment
ICDH activity is decreased when rats are pair-fed graded doses of 7-DH diets as
evidenced by Figure 31. The decreased activity observed in the group ofrats fed 15% of

---

102
the 7-DH diet was significantly different (p< 0.05) from the control group fed NH diet.
Feeding 5% or 10% of 7~DH diets did not show any significant decreased activity of the
enzyme when compared to the control group.
Fi&1Jre 31: Isocitrate Dehydrogenase Activity in Liver Of Rats Fed NH
& Graded Doses Of7-DH Diets
Group or Rats
5.11.4.3) Third Experiment
Isocitrate dehydrogenase activity is significantly decreased (p< 0.05) when rats
are fed 0.15% CFA diet in comparison with a control group of animals fed non~heated
partially hydrogenated soybean oil. Figure 32 displays the histogram of the results.

---

103
Filrure 32: Isocitrate Dehydrogenase Activity in Liver Of Rats F ed NH
& CFA Diets
Mean ± SEM)
0.6 -
T
0.5 -
............... :;0;;
~,~:.!:..~:.~~rC:.
•
!-:.~(~'-~:.-(:.~~
.!:
rI'·i·-I·~ ..~·~i11
.'\\. ........"'.......
~
-::,.r::..::,'!:,.r::,.r:~
;..,-
0.4 -
~,:,:.~:,.~:.-::.-:~
-
Q
.- ...
.r:~.r::.!:..r::.-::.-:~
.~ c.
!::::''':.-:,~,e:~
-~ !l(I
.r::.-::.!:.:{:..r::..r:~
-<
~
El
0.3-
-::.-:,-::..;,~:.-:(
v--
!:..r::..r::..r::,.r::..r::.
c I:::l
-::::,~:..-:t\\.-: :.'!:..
.-
'v El
-::.'::.-::.!:.-::.'!~
~
~ --
-:,-::.'!:.-::.'!:.-c~
c.'E
0.2-
~,-:;.!~-:,~,-~~
çne
":.-::.!:..~:.~:.~~
:1
-:-..-:~":.-:::':'-c~
re:.-C:.~:.-::,'!,,,~
• p< 0.05
.!3
0.1-
! ;.!:.'!,(:.'!:,.'l~
!:..~:..r::,..r::,.r::..r::
":.:':.tC:,.-::,.-::.-:~
!:.'!:.-::.!::::::~
~~~~~.~.~.~.
0
D=10
D=l1
Group or Rats
5.11.4.4) Fourth Experiment
The decreased activity of the oxido-reduction enzyme EC 1.1.1.42 noticed when
rats are pair-fed 15% of the T-7DH diet was not significantly different from a control
group of rats fed NH diet. The lowest ICDH activity measured in the liver of rats fed the

---

104
non-treated used PHSBO diet was not sîgnificantly different from that observed for the
group of animals fed the treated used oîl diet. Figure 33 has the results of the report.
Fi~ 33: Isocitrate Dehydrogenase Activity in Liver OfRats Fed NH,
7-DH & T-7DH Diets
{n=lQ & Mean ± SEM)
0.6
0.5
NS
•
.:3
•••••
•••••
•••••
.... "'"
•••••
[i]NH
•::
Cl..
•••••
.-
....
•••••
~
< a
•••••
0.3
•••••
~ lS%?-DH
~]
•••••
·0 El
•••••
110'_
•••••
~ T-7DH
Q" ë
0.2
•••••
rn 1:
•••••
::1.
•••••
•••••
.::
0.1
•••••
•••••
•••••
• p< 0.05
•••••
NS = Non Significant
o
•••••
Group or Rats
5.11.5) Glucose 6-Phosphate Dehydrogenase (G 6-PDH)
(Ee 1.1.1.49)
5.11.5.1) First Experiment
D-glucose 6-phosphate:NADP-oxidoreductase, EC 1.1.1.49, measured in the liver
of rats fed either 4- MH or 7-DH diets showed a depressed activity in comparison with a

---

105
control group of rats fed NH diet. When rats were fed the 7-DH diet, the depressed
activity observed for the enzyme was highly significant (p< 0.0001) when compared ta
the control group. Although a depressed activity was observed when animaIs were fed 4-
:MH diet, the difference was not significant when compared to the control group. The
lowest activity of G 6-PDH measured for the group of rats fed the 7~DH diet was
significantly (p< 0.01) different from that observed for the 4-MH dier group of animaIs.
Results are presented in Figure 34.
Figure 34: Glucose 6-Phosphate Dehydrogenase Activity in Liver Of Rats
Fed NH, 4-MH & 7-DH Diets
(Mean ± SEM)
0.02
= 0.015
'Qi
~~
...
.;:
"'"
Q.
D NH
-=t\\l',I
.:;: e
0.01
Il 4-MH
"-
=
Col
t:. .-
....
Col
El
__
~ 7-DH
~o
[J:l
a
::t
O.oos
.:
••• p< 0.0001
0=10
0=10
0=11
Group or Rats

---

106
5.11.5.2) Second Experiment
When rats are pair-fed graded doses of 7~DH diets a depressed activity of the
oxidoreductase EC 1.1.1.49 was noticed when compared with a control group of rats fcd
NB diet. AnimaIs fed 15% of the ?-DH diet displayed the significantly lowesl activity
(p< 0.0001) among groups in comparison to the control group. When rats are fed 10% of
the 7-DH diet, the depressed activity of the enzyme observed was al p< 0.01 when
compared 10 the control group. Feeding of 5% of the ?-DH diet 1ed to a slight depressed
enzyme's activity which was significant al p< 0.05 in comparison to the control group.
The histogram is plotted in Figure 35. An apparent dose response was noted.
Fi~ure 35: Glucose 6-Phosphate Dehydrogenase Activity in Liver OfRats
Fed NH, & Graded Doses Of7-DH Diets
(n=10 & Mean ± SEM)
0.02
•
••••
•
••
.....
0.015
•
-
~o
••••
..
••••
g
~ ~
~ ~
NH
- ~
:E 0.
••••
' "
.........
u
••••
",",","
..........
,. ,
,
~
...........
-
..........
u
...........
0.01
••••
' "
...........
!ill 5% 7·DH
.. a
",. ,
..........
.
U
._ -
•
••••
' "
" "
.......
.".
a
••••
' "
'" ,
...........
..........
~ ....
••••
..
' "
.........
',",',"
...........
El 1O%7-DH
..
~-
••••
..........
u _
, , , ~
",a
••••
,", ,,
..........
.........
~
0. 0
"-
••••
' ", ,,
..........
...........
~
..........
, , ,. ~
~ 15% 7-DH
..........
0.005
••••
' "
...........
..........
.~
••••
' "
...........
"
...........
~ ~ "
••••
' "~ ~
••••
..........
,' , ,
...........
~ "
..........
+ p< 0.05
••••
' "
',',','
'::';'::';':r;.':
•••
, , , ,
...........
...........
0
••••
, , , ,
....
.. ....
".
' "
.........
...........
• p< 0.01
' "
.. p< 0.001
Group of Rats

---

107
5.11.5.3) Third Experiment
The depressed activity of G 6-PDH recorded when rats are fed 0.15% CFA diet
was highly significant (p< 0.0001) when compared with a control group of rats fed NB
diet. Results are to he seen in Figure 36.
Figure 36: Glucose 6-Phosphate Dehydrogenase Activity in Liver Of Rats
Fed NH, & CFA Diets
(Mean ± SEM)
0.02-
or
= 0.015 -
q,l
-
~Q
-
•••
"1'"
...
...
Q"
.::- ~
1.0'
< El
0.01 -
1.0']
S
Col
.El
___
~-
Q,,0
rJ:lS
:::1.
0.005 -
=
...
••• p< 0.0001
0=10
n=11
Group of Rats
5.11.5.4) Fourth Experiment
Pair-feeding rats with 15% of the T-7DH diet led to a significant depressed
activity (p< 0.01) of G 6-PDH when compared with a control group of rats fed NH diet.

---

108
There was no significant difference between the non-treated and the treated used oil diets
in terms of glucose 6-phosphate dehydrogenase activity when these were fed to rats.
Results are displayed in Figure 37.
FijWre 37: Glucose 6-Phosphate Dehydrogenase Activity in Liver Of Rats
Fed NH. 7-DH & T-7DH Diets
(n=lQ & Mean ± SEM)
0.02
=
.;;:
•••••
0.015
>,0
•••••
•
.... -
..
•••••
••
-;: 0.
•••••
•••••
lE NH
~Ol)
•••••
<' e
•••••
-- 0.01 •••••
l§]
15% 7-DH
~_9
•-<J El
__
•••••
4 1 -
•••••
0. 0
•••••
~ T·7DH
cne
•••••
:1.
•••••
0.005
•••••
.9
•••••
•••••
• p< 0.01
•••••
•••••
•••••
•• p< 0.001
0
•••••
Group or Rats

---

109
CHAPTERVI
DISCUSSION OF RESULTS

---

110
The changes which occurred when heated fats were fed to animaIs in this study
confIrm that the nutritional quality of a fat is altered by length of the heating. radine value
of me oils droppe<! from 100 in the non-heated PHSBO (NH) to 94 in oil used for seveD
days frying (7-DH). The decre.,e observed was due 10 the loss of unsaturalion of the
heated ails. This is confmned by the decreased value of oxidative stability index recorded
for 7-DH (Table 3). Also, the results in Table 3 clearly show that the 7-DH was darker
than NH and 4~MH. These two parameters suggest a rise of polymerie products in the
heated. used cils, Indeed, Johnson et a1.(82,83] indicated that thermal oxidation products
from PUFAs are responsible for much of the 1055 of nutritional value of thermally
oxidized ails. The polymerie products forrned upon heating may cause at least part of the
growth depression (Figure 5) observed in animals fcd 7-DH diet. The increased peroxide
value noticed for 7~DH (Table 3) in cornparison to NH, suggests that peroxides are
formed into the used fats upon storage. However, the analysis of the seven days abused
oil showed a decreased peroxide value when compared to the four days used PHSBO.
These results are in accordance with Nawar [84] who stated that the peroxide vaine of
vegetable oils first increases when heated over time and then decreases. In this study,
increased percentages (Table 4) of saturated fatty acids C16:0 and C18:ü in the used oils
as compared to the non-heated PHSBO were also noticed. In addition, a decrease of the
unsaturate<! fatty acids C18:1n-9. CI8:2n-6 and C18:3n-3 was observed. This decrease of
the C18:2n~6 is in accordance with Johnson & Kurnmerow [83] who round that
unsaturated fatty acid content was decreased when corn oil was heated at 200°C. Cyclic
fatty acid monomers were aIso detected in the used ails (0.11 % for the 4 days used ail,
and 0.16% for the 7 days used ail). This finding is in accordance with the literature
[22,85,86] that heating a fat in air causes the fonnation of volatile scission products and
non-volatile derivatives snch as cyc1ie fany acid monomers, dimers, and polymers.
The lower nuUitional quality of thennally oxidized oils as weIl as the compounds
generated in used oils have the potential ta cause harmful effects to rats when inges~

---

111
Even though there were no significant differences in food intake among groups
throughout the experiments, animaIs fed ?-DH or cyc1ic fauy aeid maDamer diets
exhibited haïr 10ss and dermatitis (Figures 6 & 7). This may suggest that the polymerie
compounds generated during frying could be the cause of the ohserved haïr loss and
dermatitis sinee pure cyclic [auy aeid monomers had the same cffeet in rats when they
were ingested. These results are in aecardance with Alexander et a1.[9] who round that
rats [ed diets containing heated corn oil or heated peaout ail developed haïr 10ss and
dermatitis. In this study, rats Ced 7-DH diet grew al a lower rate, as did thase fed pure
cyclic fatty aeid monomers in cornparison to the control group [cd NH met. These
observations may he due to the polyrners generated in the used ails upon heating since the
CFA group of rats grew less than controls but more than the group fod 7-DH diet. (Tables
5 & 7). When rats were pair-fod with 15% of the 7-DH diet, their hody weight gain
deereased at the tennination of the experiment although not in a statistieally signifieant
manner. Rats on the 7 ·DH diet had lower body weight than thase on NH diet whieh are
the control group.
Besides animals' weight gain, those pair-fod with 10% or 15% of the 7-DH diets
appeared ta have significantly less red blood cells per volume of blood. This may suggest
an effeet that might involve vitamin B12 or folic acid metabolism in rats fed the used ail
and lead to a possible anemic s13te for these animals. The red eell decrease also eould be
due ta membrane fragility caused by oxidation or cyclic fatty acid monomers and other
degradation products. When the used fat was treated with magnesium silicate, an
improvement in the red cell count of animals fed the treated ail was observed, however,
the trend was not significant.
When rats were fed 15% of the 7-DH diet, their liver weight/body weight ratio
(Table 9) was a little bit higher than those fed NB suggesting an increase in liver size. As
reported by Nakamura et al.[B7], when they fed autoxidized safflower oi! to rats the liver
weight/body weight ratio was also increased. It can he inferred that the used fats may

---

112
con tain products that lead te an increased liver weight. In addition, the increased level of
protein or lipid content noticed in the liver (Tables 9-12) may contribute to this abnormal
liver weight of animais fed the used ail or cyclic compounds. Liver enlargement was
noticed al the end of lhe 10 weeks experimenlal period. E\\-Shattory et al. [88] also found
enlarged livers in thei! study and attributed il to the increased in the body weight of
animals fed thermally oxidized oils.
The increase of liver protein observed in this stndy is in accordance with that
conducted by Miller & Landes [43] who found an increased content of liver protein when
rats were fed laboratory thennally oxidized fats. This may suggest that less protein i5
being used for somalie growth and more i5 being retained in the liver of animais fed the
used ails when compared to the control rats fed non-heated oil. This increase of protein in
liver of rats fed the used oils may be a way for the body to cope with the adverse
metabolic effects of the heated fats as they reponed in their study. In this present study,
along with the increased level of liver protein observed. the microsomal protein content
was also significanlly increased (p< 0.000\\) when animaIs were fed 10% DH, 15% of the
7·DH or CFA diets. The results are in accordance with Ashwin et al.[89] who reported a
highly increased protein content in the microsomal fraction of rats fed laboratory
thermally oxidized canola oil. The use of a commercial heated PHSBO in this study led
ta the same results suggesting that laboratory or commercial heating have the sarne
effects on liver or microsomal protein. This increased content in the microsomal protein
may suggest a high protein syntbetic rate to come to the aid of the increased participation
of the mixed-function oxidase enzymes involved in xenobiotics detoxification. In
addition. the slight liver tissue damage observed as evidenced by the increased level of
alanine aminotransferase. rnay contribute to the high protein level noticed in liver tissue
as weIl as in microsomes. The signifieant (p< 0.01) increased liver protein content
observed and reponed in Figure 13 when rats are fed 15% of the T-7DH diet may suggest
a recovery process for the damage caused te the liver ceUs by the texie compounds. When_

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113
compared to the control group of rats, a highly significant (p< 0.0001) increase in protein
content was observed for the rats fed 7-DH diet.
The significant (p< OJK>OI) increased level of hepatic lipid noticed when rats are
[ed 15% of the 7-DH diet suggests liver lipid accumulation in these animaIs in
comparison to the control group. This evidence certainly contributed to the increased
liver size ohserved in rats [ed 15% 7-DR or CFA diets. Furthennore, the lipid/protein
ratio (Figures 14-16) of animaIs fed cither 7-DH or CFA diets clearly confirms the
tremendous amount of protein being retained or produced in comparison to that of lipid in
liver tissue of the same group of rats. The ratio was significantly lower in the above cited
group ofrats when compared to the NB or the 4-MH groups of rats showing an increased
amaunt of liver protein when compared to that of lipid. However. rats fed the T-7DH diet
showed a non significant decrease of their lipid/protein ratio suggesting that the treated
oît may have a positive nutritional effeet on improving the accumulation of liver lipid or
protein observed in anirnals fed 7-DH or CFA diets. In ail experiments, diets were
prepared with 15% casein vitarnin free as source of protein. This level was just below the
nonnallevel of casein which is 20% by weight but was above the lower levels (5-12% by
weight) [90].
With regard ta the effeet of the amount of protein on the activity of liver
microsomal rnixed-function oxidase system, numerous studies [91-98] have shown that
diets containing low levels of casein (5-12% by weight) decrease the mixed-function
oxidase enzymes system aetivities such as O-deethylase. cytochrome P450. 0-
demethylase, hydroxylase. and N-demethylase when compared with a diet containing a
normal level of casein (20% by weight). In view of the above infonnation. the highly
significant increase (p< 0.0001) of Cyt.P450 content (fables 13-15) in animais fed 7-DH
or CFA diets clearly demonsrrates the hannful effcets of the cornponents generated in the
used oils as weil as that of cyelie fatly acid monomers. Also. rats fed 4-MH or 10% 7-DH
diets showed increased levels of Cyt.P450 in the microsomal fractions but ta a lesser

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114
1
1
extent (p< 0.01). These resu!ts are in accordance with Siess et a1.[12] who found a
significanûy elevated content in Cyt.P450 with rats fed a diet containing 1000 or 10,000
1
ppm cyclic fauy acid monomers. This explains the increased content of liveT microsomal
1
Cyt.P450 and the reductase activity measured in this study when rats are fecI the used oils
1
that is confinned te con tain substantial arnount of cyc1ic fatty acid monomers (See
1
chromatograrns in Appendix). Therefore, commercial frying operation is sources of CFA
1
generation in the heated fats. This may suggest the incorporation of foreign compounds
1
generated during frying. cooling and reheating cycles into the fried foods ealeo by people.
1
ln addition, Ashwin et al.[89] found in rats fed laboratory thennally oxidized canola oil a
40% enlargement of their pools of hepatic Cyt.P450. When rats are fed 15% of T-7DH
1
diet (Table 16), the increase of Cyt.P450 content was not significant in comparison with a
1
control group of rats fed NH diet. This suggests that sorne of the generated components in
1
the heated used oil have been removed from it by the magnesium silicate treatment of the
1
used oil and thus decreased its toxic effects. Therefore, less Cyt.P450 had been formed in
1
liver microsomes due to less toxic substrates leading 10 a less extensive detoxification
1
process involving the enzyme NADPH-cytochrome P450 reductase whose activity was
1
proponional to the Cyt.P4S0 content of the microsomal fraction.
Besides Cyt.P4S0. liver microsomal cytochrome bS exerts a function in lipid
1
metabolism, desaturatîon and elongation [99] of fatty acids, and cholesterol synthesis. In
1
addition, as described earlier in the background information in chapter III, Cyt.bS acts as
an electron donor to Cyt.P4S0 and is reduced either by NADPH-Cyt.P4Sü reductase or
another microsome-bound flavoprotein NADH-Cyt.b5 reductase (EC 1.6.2.2) which is
specifie for NADH [54]. The highly significant increase (Tables 13-15) observed in rats
fed either the 7-DH or CFA diets suggests that an excess level of components generated
during heating or lhat of pure cyclic fatty acid monomers might be associated with an
enhancement of the functions that Cyt.bS potentially has. This could explain the greater
activity of NADPH-cytochrome P4S0 reductase noticed in this study and thereby that of

---

115
1
1
NADH-eytochrome bS reduetase in the liver microsomal fraction which may participate
1
somewhat indireetly in the detoxifieation already undertaken by Cyt.P450 enzyme.
1
Indeed, the activity of NADPH-cytochrome P450 reductase is concomitantly increased
1
with p< 0.0001 in the group of rats fed the 7-DH or CFA diets in comparison with control
rats fed NB diet (Figures 22-24). Recall that this enzyme receives electrons from NADPH
1
through FAD and FMN as described earlier in the background information in chapter III.
1
When rats are fed 4-MH or 10% 7-DH diets, the significant increase (p< 0.01) observed
1
when compared with the control groups showed that at the recorded leve1s the contents of
1
either Cyt.P450 or Cyt.bS were less than those of animals fed 7-DH or CFA diets. This
1
clearly suggests th.t liver microsomal content of Cyt.b5 and Cyt.P450 is dependent upon
the concentration of the components generated in the used oils during heating. Although
1
the levels of significance of NADPH-Cyt.P4S0 reductase activity in both 4-MH and 10%
1
7-DH diets were different, they were al! highly significant (p< 0.001 & p< 0.0001
1
respectively) from their control groups. The four days used ail may contain about 10% of
1
aIl the components generated during frying that are causes of the deleterious effects
1
noticed when compared with the signific.nt level recorded in the 10% 7-DH diet group of
1
rats. However. when animaIs are fed 15% of me T-7DH diet, neimer the increase of their
CyLP450 nor that of Cyt.bS contents were significant. These results suggest an
1
improvement of nutritional parameters signaling a reduetion of toxie compounds in livers
1
of mose animais in comparison with a control group of rats fed NH diet. In addition. the
signifieant (p< 0.01) inereased activity of NADPH-Cyt.P45ü reductase noticed in me T-
1DH group c1early showed that the liver enzyme was being used less extensively than in
rats fed 7-DH or CFA diets.
Cytochrome bS's implication in the desaturation and elongation of fatty acids may
suggest its involvement in the desaturation of the increased level of palmitic 3cid (CI6:0)
generated in livers of rats fed 7-DH or CFA diets. This may contribute ta the non
significant increased leve1 of gamma-linoIenic acid (CI8:3n-6) observed when rats are

---

116
1
1
fed those diets. Another explanation can be seen by the decrease in carnitine
1
palfiÙtoyltransferase-1 activity in the liver mitochondria of rats fcd 7-DH, graded doses of
the 7-DH, or CFA diels (Figures 26-28). These resulls suggesl that less palmitic acid was
1
being degraded via the mitochondrial ~-oxidation pathway, described earlier in the
1
background information in chapter TIl. Excess palmiric acid were instead conveyed to
1
desaturation with consequent formation of gamma-linolenic acid. Funhennore, gamma-
1
linolenic acid does not appear 10 have any positive nutritional effects on the metabolism
1
of fats fed the used oils or cyc1ic fatry acid monomers. It could be part of the origin of the
poor growth, hair 10ss, dermatitis, or other deleterious effects that OCCUITed in rats fed the
1
thermally oxidized oils or cyclic fany acid monomers. Besides the desaturation
1
mechanism that takes place in the endoplasmic reticulum, another possible explanation
1
for the decreased activity of CPT-I ohtained when rats were fed 7-DH or CFA diets
1
involves the incorporation of fatty acid group into phospholipids and triglycerides thus
1
minimizing the flux of fatty acid oxidation via the mitochondrial pathway. The fauy
1
livers observed in groups fed 7-DH or CFA diets could find here part of its significance.
The non significant decreased activity of CPT-I (Figure 29) obtained for the group of rats
1
fed 15% of the T-7DH diet suggests that the magnesium silicate treatment of the used oil
1
may have positive nutrition al effects on improving the enzyme's activity. Thus, more
1
fattyacids are being oxidized via the rnitochondrial ~-oxidation pathway.
1
Fany acids from the group of rats fed the used oil or those fed cyclic fatty acid
1
rnonomers are Dot weIl degraded via ~-oxidation pathway and so are the one coming from
carbohydrates metabolism. Indeed, the highly significant decreased content of liver
glycogen (Tables 9-11) obtained when rats were Ced the used oil or CFA diets suggests an
increased glycolytic activity leading ta the formation of pyruvate which is subsequently
converted to acetyl-CoA via the pyruvate dehydrogenase complex enzyme in the
mitochondrial matrix. The acetyl-CoA formed could undergo oxidative degradation
through the TCA cycle with production of COZ and HZO with concomitant formation of

---

il7
1
1
reducing equivalent NADH, or it may be utilized for the biosynthesis of long chain fatty
1
acids like palmitic acid via fatty acid synthase.
In regard to the TCA cycle pathway. the significant decreased activity of isocitrate
1
dehydrogenase observed when rats were fed the used oil or the CFA diets (Figures 30-32)
1
suggests an impainnent of TCA cycle enzymes activity. Tbe mechanism of the nonnal
1
reaction of ICDH was described in the background infonnation in chapter III. These
1
results are in accordance with those reported by Yoshioka et al [11] who found a
1
decreased activity of the TCA cycle enzyme succinate dehydrogenase when rats are feci
1
autoxidized safflower oils. They attrihuted this impairment ta the high carbonyl value
detected in the oils. Even though the above cited authors found a decreased activity of
1
succinate dehydrogenase. which was the only one report in the literature. their finding
1
could not lead to better understand the metabolic pathway of fatty liver reported in this
1
study and in the literature because succinate dehydrogenase in the TCA cycle cannot give
1
any information about enzymes al the beginning of the cycle whieh are necessary ta
1
ascertain if acetyl-CoA is fully oxidized in the cycle. Measuring the activity of isocitrate
1
dehydrogenase which catalyzes the Brst dehydrogenase reaction in the TCA cycle is
1
better off to localize where is the key point of fatty and enlarged liver reponed in this
study and in the literature. In this stndy, since the frrst dehydrogenase reaction in the TCA
1
cycle was impaired. it suggested an accumulation of citrate since aIl of the citrate formed.
1
under citrate synthase activity. was not metaholized in the TCA cycle and the excess went
through the mitochondrial membrane under passive diffusion and refluxed or regenerated
the cytosoi with acetyl-CoA under ATP-citrate iyase (EC 4.i.3.6) activity. Then, the
excess of acetyl-CoA in the cytosol could have undergone fatty acid synthesis. The
complementary needed NADPH for fatty acid synthesis was then supplied by L·
mal.te:NADPH oxidoreduct.se (decarboxylating) (EC 1.1.1.40), also c.lled malie
enzyme. The evidence that malie enzyme in addition to the pentose phosphate shunt,
known to be the primary source of NADPH for the conversion of acetyl-CoA ta fatty

---

118
acids, may supply NADPH for lipogenesis was reported by Fitch & Chaikoff [100] and
also by Lowenstein [101]. The matie enzyme reaction itself was fIrst found by Ochoa et
aL [102]. The following Figure 38 schematically describes the reactions related to the
possible pathway described above.
Fiirure 38: Possible Pathway To Cope With An Excess OfAcetyl-CoA
~ Carbohydrates
Glycolysis
It is also known that under high energy conditions (i.e. high ATP/ADP + Pi, and high
NADH/NAD+ ratios), ICDH activity is inhibited in the TCA cycle. On the other hand,
during periods of low energy the activity of this enzyme is stimulated in order to
accelerate energy generation in the TCA cycle. In this study, since a control group of rats
was fed the same level (15%) of the non-heated PHSBO, the energy condition in that

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119
1
1
group remained the same in thase fed the used oils or in those fed CFA diets. Therefore,
1
the deereased aetivity of rCDH obtained in the experimental groups may have other
1
origin that eould be attributed to seeondary degradation products sueh as polymerie
triglycerides, carbon yi, or to the eyelie fatty aeid monomers generated during frying.
1
Even though the impairment of ICDH aetivity was observed when animals were fed the
1
T-7DH d.iet (Figure 33). the effect was lesser than the other experimental groups of rats.
1
This may suggest an irnproved metabolie effeet in those animals when compared te the
1
group of rats fed the 7-DH d.iet.
1
Aeeording to Ashwin et al.[89] who studied the effects of thermally oxidized
1
canola oil, sorne secondary metabolism of thermally oxidized oil decomposition products
1
may raise peroxide levels in the liver. They also reported that sorne of those oxygen-
1
containing constituents whieh reach the liver (including fatty alkyl alcohols, aldehydes.
1
ketones. monamerie. and dimeric acids) were patential sources of reactive oxygen species
1
through their metabalism by cellular oxidases. In addition. the authors reported that a
1
diversion of protein to accommodate the increased liver size, possible synthesis of
1
peroxisomes. and endoplasmic reticulum and their associated enzymes. may dioûnish the
1
eapacities of other enzymes. This statement cou Id he seen as another explanation to the
1
decreased activity of ICDH measured in this study.
1
Irnpairments of the activities of the enzymes CPT-I and ICDH in animals fcd the
used oil or cycUe compounds led to an excess of acetyl-CoA in eytosol and triggered fatty
acid synthesis in liver tissue. In order for fatty acid synthesis to oceur. the availability of
the reducing equivalent NADPH must be effective. NADPH supply for fatty acid
biosynthesis is made possible through me pentose phosphate shunt where glucose 6-
phosphate dehydrogenase and 6-phosphogluconate dehydrogenase produce the needed
reducing equivalent for the synthesis. The first NADPH generating reaction of the
pentose phosphate pathway is described in the background information in chapter ID. The
significantly decreased activity of glucose 6-phosphate dehydrogenase (G 6-PDH)

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observed in this sludy (Figures 34-37) for all of the experimental groups of rats in
comparison with the control group, may suggesl a high demand for the reducing
equivalent NADPH 50 that the excess NADPH produced in tum depressed the enzyme's
activity. Indeed, according to Rawn [103], NADPH is a potent inhibitor of G 6-PDH so
that a sUght increase in the cellular concentration of NADPH severely depresses the rate
of the pentose phosphate pathway. Also. the enzyme is depressed by the intennediates of
fauy aeid biosynthesis, soch as fauy acyl-CoA molecules, which by binding to the
enzyme dîmer dissociate it iota inactive monorners. It is notewonhy to recaI! that eartîer
in this stndy it was reported that NADPH-cytochrome P450 reductase was fully active
when rats were fcd the used oil or the cyclic fauy aeid monorners. This rnixed function
oxidase enzyme, as described earlier in the background infonnation in chapter III, must
receive the two electrons that it needs for its activity from NADPH. Therefore, NADPH
produced in the pentose phosphate shunt is not only used for fatty aeid biasynthesis but
alsa far the mixed function oxidase system involved with detoxification processes in liver
microsomes. The highly signifieant inereased activity of the enzyme NADPH~
cytaehrome P450 reductase noticed in this study confmns the inereased utilization of
NADPH ta cape with the high level of toxic compounds ingested by rats when they are
fcd the used oil or the cyeUe fany aeid monomers. It is aIso possible ta assume that, the
liver G 6-PDH of those animaIs was in high demand 50 that its production of NADPH
became less and less effective when faced with the high level of taxie compounds that
made NADPH-cytochrome P450 reductase utilize more NADPH than fatty acid synthase.
Moreover, the toxie cornpounds generated during frying or the eyclic fatty aeid
rnonomers when ingested by animaIs did not help their whole metabolism and thus might
have reduced the function of G 6-PDH. Furthermore, it is possible to assume that the
enzyme G 6-PDH might become defieient in a such used oil or cyclic fatty aeid
monomers feeding for a prolonged study that will go over 10 weeks of experiment. As
reported earlier in tbis study. rats fed the used oil exhibited a significant low number of

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red blood cells when compared with thase fed the non-heated PHSBO. The low red cell
couot could lead to an anemic state in these animals. When rats are fed 15% of T-7DH
diet. the significant depressed activity of G 6-PDH obtained suggests that the treatment of
the used oil with magnesium silicate had a certain effeet on recovering the hepatic
activity of the enzyme but not completely.
From this study, il can he inferred that irnpairment of liveT enzymes ICDH, G 6-
PDR, CPT-l, were observed in rats fed heated PHSBO or cyelie fatty aeid monomers
diets when compared with a control group of animals fcd a non-heated PHSBO diet.
However, when the used oil of partially hydrogenated soybean oil was treated with 10%
magnesium silicate, an improvement on the activities of the studied liver enzymes was
noticed.
In view of the results obtained from this study. an investigation of A1P-citrate
lyase and malie enzyme activities should he performed. In addition, acetyl-CoA
carboxylase activity should also be measured along with the expression of the gene
coding for fatty acid synthase (Ee 2.3.1.85). Fatty acid synthase is a multifunctional
enzyme that catalyzes the conversion of acetyl-CoA and malonyl-CoA to long-chain fatty
acids, in the presence of NADPH. Indeed, acetyl-CoA carboxylase (BC 6.4.1.2) is the
enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Moreover. 514
rnRNA content of livers and its gene expression should also be investigated in order to
give a complete enzyme study in research on heated fats. According to Clarke et a1.(104],
numerous studies [105-109] found that the rnRNA coding for the liver S14 protein
(mRNA~S 14) exists in high abundance in lipogenic tissues and its regulation of
ex.pression shares many characteristics with adaptive proteins involved in lipid
metabolism. Therefore. any changes in the S14 rnRNA levels, should be similar to thase
in f.tly .cid synthase rnRNA.
_

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122
SUMMARY

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123
Four sets of experiments were conducted in this study to examine the effeets of a
commercial dietary heated partially hydrogenated soybean oil (PHSBO) on rat liver
enzyme activity. The frrst experiment was based on the effects of PHSBO used 4 days (4-
WI) and that used 7 days (7-DH) for frying fish, French fries and chicken. The second
experiment focused on pair-feeding rats diets containing graded dose of the same batch of
7-DH oil. The third experiment was based on feeding rats diet containing pure cyclic fany
acid monomers (CFA) sinee these compounds have been isolated from the commercial
heated PHSBO used in this research. The last experiment was based on treating the
commercial heated PHSBO with magnesium silicate which removes from the used ail
sorne of the polyrners and sorne of the free fany acids generated during deep-fat frying.
AH the four sets of experiments described above were conducted in each case in
conjunction with a control group of rats fed non~heated PHSBO (NH) diet. AU diets were
isocaioric with 15% of the fat leve!. In the case of CFA diet, 0.15% of CFA (>98% pure)
was mixed into 14.85% of the same batch of the nonvheated PHSBO. In each of the four
experiments, the different diets were fed to animaIs randomly distributed in cages for 10
weeks and free access 10 tap water was provided. At the end of the experimental period, a
group of 6 rats per day was randomly chosen and killed by Guillotine after brief C02
inhalation. Blood was collected and liver was quickly excised, weighed and about 2 gram
portions of the liver were introduced ioto the appropriate buffer solution for the purpose
of enzyme analysis. The rest of the liver was frozen in liquid nitrogen for future analyses.
Data were analyzed by analysis of variance (ANOVA) for a completely randomized
design using StatView statîstical software package (1988. Abacus Inc. CA). When
significant (p< 0.05) F test were detected, pairwise comparisons of mean among groups
were perfonned by Fisher's protected least significant difference (PLSD).
In the first study in comparison to the control group of rats, significant increased
contents of cytochrome P450 (p< 0.(001) and cytochrome b5 (p< 0.00(1) as weil as a
significant (p< 0.(001) increased activity of NADPH-cytochrome P450 reduclase were

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124
noticed in the group of rats fed 7~DH diet. Wheu the 4-MH diet was fed to rats significant
increasedcytochromes P450 and bS contents were recorded but al p< 0.01 while p< 0.001
was measured for the reductase activity. An apparent dose response was noted for
cytochromes contents of liver ITÛcrosomes as weil as the activity of the reductase. It was
also seen with the other enzymes investigated in this research. In addition, carnitine
palmitoyltransferase-I (CPT-I), isocitrate dehydrogenase (lCDH) activities were
significanùy decreased (p< 0.01 & p< 0.05 respectively) in liver of animaIs fcd cither 7-
DH diets suggesting an impairment of metabolic pathways. Therefore, citrate
accumulates and more fauy acids are available leading to accumulation of lipid
synthesized in liver (fauy liver). Moreover, decreased liver glycogen in those animals
were observed suggesting a release of greater amount of glucose in the blood.
Furthermore, glucose 6-phosphate dehydrogenase (G 6-PDR) activity was significantly
depressed (p< 0.0001) in experimental group of animals fed 7-DH diet. This may suggest
an inhibition of the enzyme by the high level of NADPH being produced and the level of
fatty acid present since the enzyme is mediated by NADP+/NADPH ratio and by fatty
acyl-CoA. The decreased activity observed when rats were fed the 4-MH diet was not
significanùy different from the control group of animals. The 4-MH diet was less hannful
when fed to rats than the 7-DR diet.
The analysis of the 4-MH and 7-DH oils showed that these commercial heated
PRSBO contain 0.11 % and 0.16% CFA respectively.
In the second study, pair-feed.ing rats with graded doses of the 7-DH diet gave
similar results to those obtained in the frrst study when the 7-DH diet was fed to rats.
In the third study, feeding rats with pure cyclic fatty acid monomers in the met
(0.15% diet) led to sunHar resulls when compared to animais fed the 7-DR diet. This may
suggest an involvement of CFA in the impainnent of rat liver metabolism when such
used ails are fed to animals even though other generated components in the heated oils
(carbonyl, polymen;, elc.) are also implicaled.

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125
In the fourth study, rats were fcd diet containing 15% of the 7-DH ail treated with
10% magnesium silicate. Although the same trend ofresults seen in the previous studies
were noliced, higher enzyme activity was recorded. The decreased activities observed for
CP'f-I and ICDH were not significantly different from the control group of rats fed the
NB diet. However, the decreased activity noticed for G 6-PDH was significant al p< O.Ol.
This may suggest that the treatmcnt of the 7-DH ail with magnesium silicate had a
positive effeet on recovering enzyme activity of severalliver metabolic pathways such as
those ofePT-I, ICDH, and G 6-PDH in rats fed a ,uch treated oil.
Key words: rat; heated fats; cyelle fauy acid monomers; liver enzymes.

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126
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127
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73- A.D.C.S. official method Tg 1a-64 (1973)
74- A.D.C.S. official method Cd 8-53 (1973)
ModifiOO by Oil-Dri Corporation, Chicago.
PresentOO at A.D.C.S. Convention in Baltimore (1990)
75- A.D.e.S. official method Ca 5a-40 (1977)
76- A.D.C.S. official method Cc 13b-45 (1982)
77- Perkins, E.G. (1978)
Analysîs and standards of fatty acids.
in "Encyclopedia of Chemical Technology", 3rd ed.
(Kirk-Dthmer, 00.), volA, pp. 845 - 853
John Wiley & Sons, Inc., New York, U.S.A.
78- A.D.e.S. recommendOO practice Cc 17-79
79- A.D.C.S. official method Ce 2-66 (1973)

---

134
80- StatView SE + Graphies"" (1988)
Abaeus Concepts, Ine., P.O. Box 3086
Berkeley, CA 94703, U.S.A.
81- Steel, R.GD. & Torrie, J.R. (1980)
Principles and Procedures of Statisties. A Biometrical Approach 20d ed.
(Napier, C. & Maisel, J.W. eds.), pp. 172 - 194
MeGraw-Hill Publishing Company, New York, U.S.A.
82- Johnson, O.C.; Perkins, E.; Sugai, M. & Kummerow, F.A. (1957)
Studies on the nutritional and physiologie al effeets of thennally oxidized ails.
J.A.O.C.S., 34: 594 - 597
83-Johnson, O.C & Kummerow, FA (1957)
Chemical changes which take place in an eddible oil during thermal oxidation.
J.A.O.C.S., 34: 407 - 409
84- Nawar, W.W. (1984)
Chemical changes in lipids produced during thermal processing.
J. Chem. Edueat., 61: 299 - 302
85- Paulose, M.M. & Chang, S.S. (1973)
Chemical reactions involved in deep fat frying of foods:
VI. Characterization of nonvolatile decomposition prooucts of trilinolein.
J.A.O.C.S., 50: 147 - 154
86- Waltking, A.E.; Seery, W.E. & Bleffert, J. (1975)
Chemical analysis of polyrnerization products in abused fats and oils.
J.A.O.C.S., 52: 96 - 100
87- Nakamura, M.; Tanake, H.; Hattori, Y. & Watanabe, M. (1973)
Biological effecls of autoxidized safflower ails.
Lipids, 8: 566 - 572
88- El-Shanory, Y.; Hegazy, S.; Soliman, M.M. & Aly, S.M. (1991)
Heated fats.
Part 3. Biological effeet and effect of heating and tempering cils on fatty acid
composition of liver, heart and serum lipids of rats.
Die Nahrung, 35: (lO) 1007 - lO12
89- Ashwin, J.L.; Harris, P.G. & Alexander, J.C. (1991)
Effects of thennally oxidized canola ail and chronic low ethanol consumption on
aspects of hepatic oxidative stress in rats.
Nutr. Res., Il: 79 - 90
90- Nutrient Requirements of Laboratory Animais 3rd 00. #lO (1978)
National Academy of Sciences, Washington, D.C., U.S.A.
91- Campbell, T.C. & Hayes, J.R. (1976)
The effects of quantity and quality of dietary protein on dmg metabolism.
Fed. Pme., 35: 2470 - 2477

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135
92- Hayes, J.R.; Mgbodile, M.U.K. & Campbell, T.C. (1973)
Effects of protein deficiency on the inducibility of hepatic microsomal drug-
metabolizing enzyme system.
I. Effeet of substrate interaction with cytochrome P4SQ.
Biocbem. Pharrnacol., 22: 1005 - 1014
93- Bidlack, W.R.; Brown, R.C. & Moban, C. (1986)
Nuttitional parameters that alter hepatic drug metabolisrn, conjugation, and
toxicity.
Fed. Proc., 45: 142 - 148
94- Mgbodile, M.U.K.; Hayes, J.R. & Campbell, T.C. (1973)
Effects of protein deficiency on the inducibility of the hepatic microsomai drug-
metabolizing enzyme system.
II. Effeet on enzyme kinetics and electron transport system.
Biochem. Pbarrnacol., 22: 1125 - 1132
95- Kato, N.; Tani, T. & Yoshida, A. (1980)
Effeet of dietary level of protein on liveT microsomaI drug-rnetabolizing enzymes,
urinary ascorbic acid and lipid rnetabolism in rats fed PCB-containing diets.
J. NUIT., 110: 1688 - 1694
96- Clinton, S.K.; Truex, C.R. & Visek, W.J. (1977)
Effects of protein deficiency and excess on hepatic mixed function oxidase
activity in growing and adult [ernaIe rats.
Nutr. Rep. lnt., 16: 463 - 470
97- Antal, M.; Nagy, K. & Bedo, M.B. (1982)
Effect of dietary protein and lipid on the activity of hepatic mixed function
oxidase system in young and adult rats.
Ann. NUIT. Metab., 26: 393 - 399
98- Butler, L.E. & Dauterman, W.c. (1988)
The effect of dietary protein levels on xenobiotic biotransfonnation in F344 male
rats.
Toxicol. Appl. Pbarrnacol., 95: 301 - 310
99- Jeffcoat, R. & James, A.T. (1984)
The regulation of de saturation and elongation of fatty acids in mammals.
in "Fatty Acid Metabolism and its Regulation"
(Numa, S. 00.), vol.7, pp. 85 - 112
Elsevier Science Publishers B.V., New York, U.S.A.
100- Fitcb, W.M. & Chaikoff, LL. (1960)
Extent and patterns of adaptation of enzyme activities in livers of nonnal rats fed
diets high in glucose and fructose.
J. Biol. Chem., 235: 554 - 557
101- Lowenstein, J.M. (1961)
The pathway of hydrogen in biosynthesis.
J. Biol. Chem., 236: 1213 - 1216

---

136
102- Ochoa, S.; Mehler, A.H. & Kornberg, A. (1948)
Biosynthesis of dicarboxylie acids by carbon dioxide fixation.
1. Isolation and properties of an enzyme from pigeon liver catalyzing the
reversible oxidative decarboxylation of L-malic acid.
J. Biol. Chem., 174: 979 - 1000
103- Rawn, J.D. (1989)
Glycogen metabolism, giuconeogenesis, and the pentose phosphate pathway.
in "Proteins, Energy, and Metabolism"
(Carolina Biological Supply Comp. 00.), pp.385 - 420
Neil Patterson Publishers, Burlington, Ne, V.S.A.
104- Clarke, S.o.; Armstrong, M.K. & Jump, D.B. (1990)
Nutritional control ofral liveT fatty acid synthase and S 14 rnRNA abundance.
J. Nutr., 120: 218 - 224
105- Perez-Castillo, A.; Schwartz, H.L. & Oppenheimer, J.H. (1987)
Rat hepatic rnRNA-S 14 and lipogenic enzymes during weaning: role of 514 in
lipogenesis.
Am. 1. Physiol., 253: E536 - 542
106- Liaw, C.; Seelig, S.; Marash, C.; Oppenheimer, J. & Towle, H. (1983)
Interactions of thyroid honnone, growth hormone, and high carbohydrate fat-free
diet in regulating severa! rat messenger ribonucleicacid species.
Biochemistry, 22: 213 - 221
107- Jump, D.B.; Narayan, P.; Towle, H. & Oppenheimer, J.H. (1984)
Rapid effects of triiodothyronine on hepatic gene expression.
J. Biol. Chem., 259: 2789 - 2797
108- Jump, D.B.; Veit, A.; Santiago, V.; Lepar, G. & Herberholz, L. (1988)
Transcriptional activation of the rat liver S 14 gene during post-natal development.
J. Biol. Chem., 263: 7254 - 7260
109- Jump, D.B.; Tao, T.Y.; Towle, H.C. & Oppenheimer, J.H. (1987)
Dissociation of hepatic messenger ribonucleic acid S 14 levels and nuclear
transcriptional rates in suckling rats.
Endocrinology, 118: 1892 - 1895

---

137
APPENDIX

---

138
Preparation of Whole Homogenate, Post-mitochondrial Supernatant
and Microsomal Fractions
Lake, B.G.
Through Ihis procedure, keep tissue and tissue fractions cold (0-4"C) hy using
precooled homogen;z;ng medium and centrifuge rotors. Also keep tissue
homOf!enizers measurinf!cvlindi!N and centrlrUf!i! tubes in ice.
1-) Remove excess blood from the liver sample by washing il in the
homogenizing medium.
Theo, dissect away any pieces of connective tissue etc.
Gently blct the liver sarnple dry with tissue paper.
11-) Weigh the liver sample (record the weight).
Quickly transfer the liver to a homogenizing vessel which is kept in ice.
For a 25% homogenate, add approximately 3ml/g tissue.
(O.05M Tris-HCI buffer, pH 7.4 containing 1.15% KCI)
Theo, homogenize the liver sample with five relurn strokes of the motor-
driven pestle.
Homogenates which contain large amounts of connective tissue may
require to be filtered through nylon mesh or surgical gauze before
centrifugation.
III·) Careful1y decant the homogenate whilst examining il for pieces or
undisrupted tissue, into a rneasuring cylinder.
Make up the desired volume with washings of homogenizing medium
from the homogenizer tube.
In the case of rat liver, adjust the final homogenate to approximately
O.25g fresh tissue/ml (Le. a 25% homogenate).
Record the final volume of the homogenate.
Cover the top of the measuring cylinder with parafilm.

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139
Then, transfer a desired amount immediately to pre-cooled centrifuge
tubes.
IV-) Cap and balance the tubes according to the centrifuge manufacturers
instructions.
Preparation ofPost~MitochondrialSupernatant
Capped and Balanced
Homogenate
Centrifugation 10,000 g; 20 min. at2 - 4 degree C.
Then, carefully remove tubes from centrifuge and
decanl
_---..1....--_. (Tbis is Post-mitocbondrial Supernatant)
Residue
Must he taken in a pre-cooled cylinder or other
Supernatant
suitablc reccptacle. REMOVE TIŒ
Known Volume
FLOATING LIPID LAYER on the top
with the aid of a Pasteur pipette and discard il
Centrifugation 105,000 g; 60 min.
Supernatant
Microsomal Pellet
Cytosolic fraction is carefully removed
with a Pasteur pîpette whiIst discarding
any floating lipid layer.
Can be used at tbis step.

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140
Preparation of Washed Microsornes
( Microsomal pellet)
Resuspend lhe Pellet in a known volume of
1.15% KC\\ solution containing in addition
10 mM EDTA with the motof-driven homogenizer.
Theo, Centrifugation 105,000 g ; 60 min.
Washed Microsomal
Resuspended in
Microsornal
~
Suspension
)
Pellet.
0.25 M Sucrose.
Rapidily Frazen in a dry
ice/acetone bath or liquid
Nitrogen and stored at
-7rY'C.
( Frozen Microsomes )
Freshly thawed microsomes will be used. for the experirnents.
Washed microsomal pellet
must be suspended by homogenization In fresh
homogenizing medium (O.05M Tris-HCl buffer, pH 7.4 containing 1.15% Kel) to an
appropriate tissue concentration and record the volume of the suspension.

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141
Calculation Of the Tissue Concentration (i.e. g fresb tissue/mI) of Whole Homogenate,
Post-mitochondrial Supernatant and Microsomal Fractions
Cane. (g/ml) of whole
Weill'h! of Iiver (Ir)
homogenate & Post-
mitoch. supemat.
Volume ofPost-mitoch.
supemaL spun (ml)
Cane. (Yml) of
Cane. ofPost-milOch. X
microsomal fraction
supcmal. (g/ml)
Volume of microsomal fraction (ml)
The microsomal fraction is DOW ready for use.
*** First,the proteio content must he determined and the microsomal fraction
diluted to a known protein concentration before use.
*** ff the preparation cannat be used immediate/y, it shouid be stored as the
following:
Storage of microsomai suspensions in:
• 0.154M KC/ containing 10 mM HEPES-HC/
bzifjer,pH 7_6 containing 1 mM EDTA and 20%
g/ycero/ at -20"C to -70"C or preferab/y in liquid
nitrogen.

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142
Determination Of Cytochrome P450 Content
(Lake, B.a.)
(Keep Microsomal Suspensions in jee)
PRINCIPLE
The principle of the spectral detennination of total cytochrome P450 content (i.e.
the SUffi of aIl the varions isoenzymes in the sample) is that the reduced haemoprotein
combines with carbon monoxide (CO) to give a characteristic absorption spectrurn with a
maximum at 450 nm.
EQUIPMENT
.. Dual beam recording spectrophotometer.
.. Matched glass or quartz 1 Cm pathlength cuvettes .
.. Stop-dock or Timer.
* Varions pipettes. parafllm & tissues.
.. Glass or plastic disposable test tubes.
MEDIA
• Homogenizing medium (0.05 M Tris-HCl buffer, pH 7.4)
e
0.2 M Phosphate buffer, pH 7.4. This buffer is stable for severa! weeks at room
temperature.
_ Carbon monoxide (house cylinder in an efficient fume cupboard).
_ Sodium hydrosulfite (i.e. Sodium dithionite).
PROCEDURE
~ Prepare a suitable dilution of the microsomal fraction in 0.2 M phosphate
buffer, pH 7.4. (For example, WÎth rat liver microsomes: use 0.25 g or rresh tissue/ml to obtain the
microsomal suspension).

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143
Add small quanlity of solid
Sodium Hydrosulfite
•
Add 2 ml of
4 ml of 0.2 M Phosphate
........t--'-----microsomal suspension
buffer. pH 7.4
By using parafl1m, mix genlly
Ihe lest tube by inversion
Test Tube
Divide lhe sample
between 2 malched
ICm Spectrophoto~
meter cuvettes.
Wilh a Dual Beam
Spectrophotometer,
Scan the cuvettes
between 400-500 nm.
Clean Test
Clean Reference
Cuvette
Cuvette
Ideally this should result in a
perfect flat baseline or at least
in a baseline with no appreciable
absorbance difference between
400 & 500 nm.
Should a poor baseline be obtoined check thtJI the cuvettes are a
malched pair and are clean.
Then, in an efficient fume
cupboard, gently bubble the contents of the
Test Cuvetle
wilh CO for about 1 min.
Carbon Monoxidc
bubbled Test Cuvette
Record the difference spectrurn between 400 & 500 nm.
The spectrum should comain a prominent absorption maximum at 450
nm.

---

144
llï? CALCULATION OF RESULTS
From the spectrophotometer chart detennine "'A(450-490) nm, that is the
difference between the absorption maximum at 450 nm and the baseline at 490 nm,
correcting if necessary for any absorption difference between these wavelengths priar to
bubbling the test cuvette with CO.
The extinction coefficient of Cyt.P450 has been detennined to he 91 Cm2{mmol.
Hence the Cyt.P450 concentration (nmoVml) of the sample is calculated by the fannula:
Vol. of micro. sample (ml) + Vol. of Phosphate buffer (ml).
t,M450-490)nm X
lOOQX
1
91
Vol. of microsomal sample (ml)
The Cyt.P450 concentration of the sample may he expressed as either nmol/mg
microsomal protein or nmol/g liver by simply dividing by either the sarnple protein
(mg/ml) or tissue (g liver/ml) concentration, respectively.
Determination Of Cytochrome bS Content
(Lake, B.O.)
(Keep Microsomal Suspendons in ice)
PR/NC/PLE
Cytochrome bS is the other haemoprotein present in microsomal fractions from
mammalian tissues and is readily estimated by the difference spectrum of NADH-reduced
versus oxidized cytochrome. The reduction of cytochrome bS is catalyzed by the presence
of a rnicrosomal flavoprotein enzyme namely NADH-cytochrome bs reductase (EC
1.6.2.2.)
EQUlPMENT
* Dual beam recording spectrophotometer.
* Matched glass or quartz lcm patWength cuvettes.

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145
* Stop-dock or Timer.
* Various pipettes. parafùm & tissues.
* Glass or plastic disposable test tubes.
MEDIA
• Homogenizing medinm (0.05 M Tris-HC1 buffer, pH 7.4)
- 0.2 M Phosphate buffer, pH 7.4. This buffer is stable for several weeks al room
temperature.
Sodium hydrosulfite (i.e. Sodium dithionite). This is used if the same
sample is to be held for the determination of cytochrome P450 content. After cytochrome
bS has been detennined. simply reduce both cuvettes with sodium hydrosulfite and then
proceed as described in Cyt.P450 content procedure.
PROCEDURE
··Prepare a suitable dilution of the microsomal fraction in 0.2 M phosphate
buffer, pH 7.4. (For example, with rat Iiver microsomes: use 0.25 g of rresh tissue/ml to obtain the
microsomal suspension. TheD, add 2 ml of suspension to 4 ml of buffer. Mix & keep in ice until
analysis.)
**Then, divide the sample between two matehed lem spectrophotometer cuvettes
and with a dual beam spectrophotometer scan lhe cuvelles belween 400 & 500 nm.
••Add a small quantity of solid NADH [or if preferred 25 III of a freshly prepared
2% (w{V) solution in buffer] ta the lesl cuvelle and using parafilm mix gently by
inversion.
**After approximately 2 min record the spectrum between 400 & 500 nm.

---

146
Ir§>CALCULATION OF RESULTS
Cytochrome bS is determined by the absorption difference between the peak at
424 nm & the trough at 409 nm in the spectrurn.
The extinction coefficient of Cyt.bS has been detennined to he 185 Cm2/mmoI.
U,lng the t.A(424-409)nm value Cyt.bS concentration (nmoVml) of the ,ample i,
calculated by the fannula:
Vol. of micro. sample (ml) + Vol. of Phosphate buffet (ml).
tl,A(424-409)nm X lQQQ
X
1
185
Vol. of microsomal sample (ml)
The cytochrome bS content may be expressed as either nmoU mg microsomal
protein or nmoVg liver by simply dividing by cither the sample protein (mg/ml) or tissue
(g liver/ml) concentration, respectively.

---

147
SU1lllllary OfTlte Determination OfCytochro11les bS & P4S0 Contents
Scan again
Addition of NADH to-tlTest"
between 500-400 om.
Geutly mix the solution by
inversion. Wail for 2 min.
CylP450
at450nm.
CO bubb!ed of
Reducing of beth
Prior to NADH
the "Test" onIy
cuvettes w\\Ùl
ad ding to the
for 1 rnill.
Sodiwn hydrosulfitc.
lest cuvelle.
400 Dm.
SOOnm.
4OOnm.
SOOnm.

---

148
Determination OfNADPH-Cytochrome C (P4S0) Reductase Activity
(Be 1.6.2.4).
(Lake, B.G.)
"MEDIA
* Homogenizing medium
* 0.1 M Phosphate buffeT, pH 7.6 { N~HP04 anhydr. and NaHl P04. H20}.
, 15 mM KCN (97.7 mg of KCN/lOO ml of ultra-pure H20)
• 0.125 mM Cytochrome C
(Freshly prepared by dissa/ving 1.55 mg/ml Cy/o.C in buffer).
'lOmMNADPH
( Freshly prepared by dissoMng 8.3 mg/ml ofNADPH in buffeT).
"EQUIPMENT
* Dual beam recording spectrophotometer complete with a thennostatted cell
companment, heater and pump to maintain cuvette contents al 37 Oc.
* Matched glass or Quartz 1 Cm pathlength cuvettes.
"PROCEDURE
Into each of 2 matched 1 Cm spectrophotometer cuvettes:
* Pipette 1 ml of Cytochrome C solution
0.2 ml of 15 mM KCN (DO NOT PIPETTE TIDS REAGENT BY
MOurH).
(KeN is added to preven/ erTors due to possible mi.tochondrial
contamination of the microsomal suspension).
* Pipette (for rat liveT microsomes use):
0.1 ml of 50 mg fresh tissue/ml suspension into each cuvette.
* Add 0.1 M Phosphate buffer. pH 7.650 that the total volumes of the test and
reference cuvette contents are 2.4 and 2.5 ml, respectively.

---

149
* Gently, mix the cuvette contents by inversion (using parafilm).
* Transfer the cuvettes te the 37°C thennostatted cell compartrnent of the
spectrophotometer.
* Wait for 3 min.
* Initiale the reaction by adding:
0.1 nù of 10 mM NADPH to the test cuvette ooly.
* Mix the cuvette contents.
* Record the increase in absorbance with time (S min.) al 550om.
WCALCULATION OF RESULTS
* From the spectrophotometer chart. determine the initial rate of cytochrome C
reduction in units of M 550 nrn/min.
* The extinction coefficient of reduced Cyto.C al 550 nro has becn detennined to
he 21 Cm2/mmol.
Hence NADPH-Cyto.P450 reductase activity (nmol/min/ml rnicrosomal suspension) is
calculated by the formula:
A SSO nmfmin. X lWl.Q.. X =-:-_-'2".5'-_-.,._ _--:-'
1
21
Volume ofmicrosomal suspension
(ml) added to cuvette.
*Enzyme aCtlvlty is expressed as either nmol/min./mg microsomal protein
(mg/ml) or tissue (g liver/ml) concentration respectively.

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150
Determination Of Carnitine Palmitoyltransferase 1 Activity
CEe 2.3.1.21).
Mc Garry et al.
[Biochem. J. 214.21-28 (1983)].
I-)Preparation Of LiveT Mitochondria.
(Extraction is accomplished by using: Method "C".)
According to the au thor, the following rnethod Il C" which will be described
yields minimal contamination from other subcellular fractions.
PROCEDURE
(isamPle )
1 Homogenized in a glass Dounce
+homogenizerin9vol.orcold0.25M Sucrase.
Homogenate
Centrifugation (1,000 g for 15 min.)
in a refrigerated centrifuge.
Discarded Supematant
(notice the volume)
(
Nudear pellet)
~ Addanequivalentvolume
of me discarded supematant
by 0.25M Sucrose.
Rehomogenized
Pellet
Rehomogenized pellets are then centrifuged as follows:

---

151
Rehomogenized
Pellet
Centrifugation 600 g; 10 min
(p
Residue
)
Centrifugation: 15,000 g; 15 min.
Supematant
Successive washes and recentrifugation
at 15,000 g for 15 min. in 0.25M
Sucrose and then in 0.15M KCI.
Supematant
( i pellet)
Resuspended in 1
O.I5I'tt KCI.
,.
Mitoc hondria/KCl
Suspension
* II-) Assay of CPT...[ Activity.
Assay II is the one which will he descrihed for this purpose.
Reactions are carried out at 30 oC in 100 mm x 10 mm Pyrex screw cap
glass tubes.
Standard incubation mixture:
0.9 ml ofthe foUo wing:
*105 mmol Tris/HCl pH 7.4
*0.25 mmol Reduced Glutathione

---

152
* 4 mmoi A TP (ATP is used to allow the re-fonnation of Palmitoyl-CoA
from the free Palmitic acid and CoASH generated in the deacylase reaction).
* 4 mmol MgCI2
*15 mmo1 KCl
* 2mmolKCN
* 40 mg Rotenone
(KCN & Ratenone black the oxidation of [l.14C] Palmitoylcamitine to acid soluble products.)
* 10 mg Defatted Albumin (Bovine Serum Albumin Fatty Acid Frcc).
* 50 nmol Palmitoyl-CoA
* 200 nmol L-Camitine
* 1 mCi DL-[Methyl-14C] Carnitine.
Initiation of the Reaction:
It consists of adding 0.1 ml of the mitochondria/KCl suspension (0.2 - 0.5
mg of protein) to the incubation mixture.
Then, incubate for a short time but no more than a maximum of 8
minutes.
Finally, tenninate the reaction by adding: 1 ml of 1.2 M-HCI.
(i.e. 1.2 N-HCl).
lIn Extraction and Counting ofthe Labeled Palmitoylcarnitine.
Mc Garry el al. [1978]
J. Biol. Chern. 253. 4128 - 4136
And
McGarry et aI.[19831
Bioch. J.• 214:, 21 - 28
PROCEDURE
(See next page)

---

153
Terminate Reaction
Sample
tAM1mlufbutaoui.
Suspension in
Butanol
~ Blendedon a Voncx mixer.
Blended Suspension
Centrifuge 600 g; 10 min.
,Ir
,
Transfer of O.5ml of the
Residue
butanollayer ioto a lest
tube.
(Con tains radioactive
Palmitoylcarnitine)
Add O.lml of water
and 0.5 ml of watcr-saturated butanol.
Mixing and Centrifugation
(600 g ; 10 min)
Residue
Supernatant
Butane! Phase
Takc 0.2 ml of
butanol phase and
add 10 ml of
Aquasol.
ASSAY FOR RADIOACTIVITY
in a liquid scintillation counter
(Results are expressed in temIS 01nanomoles ofPalmitoylcarnitine lormed 1min Img protein)

---

154
First Experiment: Fatty Acid Composition Of Liver Tissue (Weight %)
(5730 A OLC Hewlett Packard)
FFA Of Interest
NH (n-l0) *
4·MH (n-9) *
1
7·DH (n=l1) *
C14:0
0.24±0.04
0.26±0.05
0.26 ± 0.11
C16:0
15.34 ±0.69
15.87 ± 0.72
15.91 ± 0.85
C16:1n-7 c
1.27 ± 0.22
1.30 ± 0.19
1.23 ± 0.49
C18:0
10.99 ± 1.17
11.74 ± 0.75
9.86± 0.64
CI8:1n-9 c
12.04 ± 1.32
10.81 ± 2.20
1O.80± 3.10
CI8:1n-9 t
4.47 ± 0.33
3.35 ±0.85
2.73 ± 0.77
C18:1t2 (N.I) +
3.05 ± 0.15
2.49 ± 0.33
2.35 ±0.32
C18:1t3 (N.I) +
1.14 ± 0.05
0.92 ±0.17
0.68±0.13
CI8:2n-6 c
18.29 ± 1.08
17.84 ± 1.34
17.06 ± 1.28
CI8:2n-6 t
0.46± 0.08
0.43 ± 0.06
0.40± 0.09
CI8:3n-3 c
0.36 ± 0.03
0.44 ± 0.12
0.35 ±O.IO
CI8:3n-6 c
0.41± 0.06
0.43 ±0.06
0.46± 0.08
C20:3 c
0.85 ± 0.10
0.95 ±0.10
0.86 ± 0.09
C20:4n-6c
16.24± 1.81
17.98 ± 1.68
15.44 ± 1.01
C20:4n-3 c
0.14±0.02
0.15 ±0.02
0.10 ± 0.05
C22:4n-6 c
0.39 ± 0.04
0.41 ± 0.05
0.30±0.03
C22:4n-3 c
0.28 ±O.IO
0.29±0.07
0.28 ± 0.11
C22:5n·3 c
0.92 ± 0.11
0.89 ±0.16
0.61 ± 0.19
C22:6n-3 c
3.41 ± 0.42
3.39 + 0.40
2.76+0.22
SumPeak, Of
90.30
89.95
82.44
Interest
Other Peak,
5.77
4.08
3.73
% Total Peak,
96.07
94.03
86.17
Eluted
% Total Peak,
3.93
5.97
13.83
Non-Eluted**
• Mean ± STDEV
(c: cis & t=trans)
+ N.I =Non-Identified
•• A measure of non volatile melhyl ester such as hydroacid ester, dimeric & polymerie
compounds & ather like malerial.

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155
Second Experiment: Fatty Acid Composition Of Liver Tissue (Weight %)
(5890 Series n GLC Hewlett Packard)
FFA Of Interest
NH 'D"6\\"
10% 7-DH (0=6)·
15% 7-DH iD"6\\"
C14:0
0.13 ± 0.02
0.18±0.01
0.06 ± 0.00
C16:0
15.75 ± 0.47
16.48 ± 0.60
15.99 ± 0.33
CI6:1n-7 c
1.31 ± 0.30
1.36 ± 0.06
1.61 ±0.54
C18:0
11.56 ± 0.95
11.46 ± 0.87
10.49 ± 0.58
CI8:1n-9 c
10.50 ± 2.00
11.55 ± 1.56
15.27 ± 1.34
CI8:1n-9 ,
4.69 ± 0.80
4.09±0.27
3.55 ±0.11
C18:1t2 (N.I) +
3.36 ±0.35
2.90 ± 0.03
2.55 ±0.12
C18: 1'3 (N.I) +
1.47 ± 0.15
0.68 ±0.07
0.60±0.05
CI8:2n-6 c
18.10± 1.14
17.77 ± 1.33
16.77 ±0.39
CI8:2n-6 ,
0.89 ± 0.11
0.55 ±0.07
0.58 ±0.01
CI8:3n-3 c
0.34± 0.02
0.54 ± 0.03
0.31 ±0.08
CI8:3n-6 c
0.43 ±0.06
0.45 ± 0.03
0.48 ±0.06
C20:3 c
0.72±0.06
0.75 ± 0.04
0.43±0.05
C20:4n-6c
16.28 ± 1.18
15.03 ± 0.85
14.92 ± 0.55
C20:4n-3 c
0.5 ± 0.05
0.10 ± 0.00
0.42 ±0.01
C22:4n-6 c
0.37 ±0.04
0.38 ± 0.11
0.46 ± 0.28
C22:4n-3 c
1.74 ±0.02
1.59 ± 0.44
0.31 ±0.14
C22:5n-3 c
1.63 ± 0.11
1.40± 0.10
0.61 ±0.06
C22:6n-3 c
3.43 + 0.05
2.84+0.17
2.84 +0.03
SumPeaksor
93.20
90.11
88.25
Interest
OtherPeaks
4.18
3.66
2.17
% Total Peaks
97.38
93.77
90.42
Eluted
% Total Peaks
2.62
6.23
9.58
Non-Eluted**
• Mean ±STDEV
(c: cis & t=trans)
+ N.I = Non·ldentified
.* A measure of non volatile methyl ester such as hydroacid ester, dimeric & polymerie
compounds & other like material.

---

156
Third Experiment: Fatty Acid Composition Of Liver Tissue (Weight %)
(5730 A OLC Hewlett Packard)
FFA Of Interest
NH (n-lO) -
CFA (n=l1)-
C14:0
0.24 ± 0.04
0.32 ±0.04
C16:0
15.34 ± 0.69
15.62± 0.70
CI6:1n-7 c
1.27 ±0.22
1.59 ± 0.17
C18:0
1O.99± 1.17
7.69 ±0.91
CI8:1n-9 c
12.04 ± 1.32
13.90 ±0.97
CI8:1n-9 t
4.47 ±0.33
4.\\3 ±0.24
CI8:1l2(N.I)+
3.05 ±0.15
3.23 ±0.34
CI8:1t) (N.I)+
1.14 ± 0.05
0.51 ±0.44
CI8:2n-6 c
18.29 ± 1.08
18.21 ± 1.42
CI8:2n-6 t
0.46 ± 0.08
0.72±0.09
CI8:3n-3 c
0.36±0.03
0.35±0.14
CI8:3n-6 c
0.41±0.06
0.48 ±0.08
C20:3 c
0.85±0.10
0.59 ±0.11
C20:4n-6c
16.24 ± 1.81
16.39 ± 1.51
C20:4n-3 c
0.14±0.02
0.25±0.03
C22:4n-6 c
0.39 ± 0.04
0.31 ±0.04
C22:4n-3 c
0.28 ±0.10
0.19±0.04
C22:5n-3 c
0.92±0.11
0.87 ±0.1O
C22:6n-3 c
3.41 + 0.42
2.88 +0.34
SumPeak, Of
90.30
88.23
Interest
Other Peak,
5.77
5.23
% Total Peak,
96.07
93.46
E1uted
% Total Peak,
3.93
6.54
Non-Eluted**
... Mean ± STDEV
(e= cis & t=trans)
+ N.I = Non·IdcntifJed.
•• A measure of non volatile methyl ester such as hydroacid ester, dimeric & p:Jlymeric
compounds & other likc matcrial.

---

157
Fourth Experiment: Fany Acid Composition Of Liver Tissue (Weight %)
(5890 Series Il GLC Hewlett Packard)
FFA Of Interest
NU (n-6) *
15% 7-DH (06). 15% T-7DH (0=6)'
C14:0
0.13 ±0.02
0.06±0.00
0.06 ± 0.00
C16:0
15.75 ± 1.96
15.99 ± 0.33
15.83 ± 1.58
CI6:10-7 c
1.31 ± 0.30
1.61 ±0.54
1.43 ± 0.54
C18:0
11.56 ± 0.95
10.49 ± 0.58
11.81 ± 1.14
CI8:ln-9 c
1O.50±2.00
15.27 ± 1.34
13.75 ± 2.91
CI8:ln-9 t
4.69 ±0.80
3.55 ± 0.11
3.90±0.15
CI8:l t2(N.t)+
3.36 ± 0.35
2.55 ± 0.12
2.85 ± 0.13
C18:1 t3 (N.t) +
1.47 ± 0.15
0.60±0.05
0.62 ± 0.77
CI8:2n-6 c
18.1O± 1.14
16.77 ± 0.39
17.04± 0.51
CI8:2n-6t
0.89 ±O.II
0.58 ± 0.01
0.35±0.03
CI8:3n-3 c
0.34±0.02
0.31 ±0.08
0.42 ± 0.18
CI8:3n·6 c
0.43±0.06
0.48±0.06
0.52 ±.0.33
C20:3 c
0.72 ± 0.06
0.43 ± 0.05
0.59 ± 0.06
C20:4n-6c
16.28 ± 1.18
14.92 ± 0.55
15.56 ± 0.21
C20:4n-3 c
0.5 ±0.05
0.42± 0.01
0.39±0.01
C22:4n-6 c
0.37 ±0.04
0.46 ± 0.28
0.54 ± 0.21
C22:4n-3 c
1.74 ±0.02
0.31 ±0.14
0.66 ± 0.04
C22:5n-3 c
1.63±0.11
0.61 ±0.06
1.60±0.14
C22:6n-3 c
3.43 + 0.05
2.84 + 0.03
2.71 +0.87
SumPeaks Of
93.20
88.25
90.64
Interest
Other Peaks
4.18
2.17
2.74
% Total Peaks
97.38
90.42
93.38
Eluted
% Total Peaks
2.62
9.58
6.62
Non-Eluted**
• Mean ± SillEY
(e= cis & t=trans)
+ N.I = Non·Identified
.... A measure of non volatile melhyl ester such as hydroacid ester, dimeric & polymerie
compounds & oilier like material.

---

158
Gel Permeatian Chramatagraphy Analysis OfOils Fed Ta AnimaIs
TdCI)'ccddcs
NHSample
...~.
'"
4-MH Sample
Polymers \\ .
7-DH Sample
DiglyccridcJ:
~~
T-7DH Sample
Frcc FaitY AciJs
~/
;..(

---

159
Detemtination OfCyc!ic F atty Acid M onomers in P H8BO Samples
(Mter Methylation & Urea Fractionation Of Non-Polar Fraction)
Cyclic Fatty Acids (CFA): *
Pure CFA Profiles
(> 98% pure)
/
GLC: 5890 Hewlett Packard
(24 psi-H2; Column: CPSIL88
60 m x 0.25 mm x 0.20 pm; Cond:
16Q°C (0), 2°C/min, 220°C)
4-MHSample
CFA Profiles in
1
I.S :::: Phenanthrene
4·MH SampI,
7·DH Sample
CFA Profiles in
1 7·DHSampI<
*J-L Sébédio, LN.R.A., 17 rue Sully, 21034, Dijon, France.

---

160
Vita
Courdjo Lamboni was born on Dctober 5. 1949 in Pono-Novo, republic of Bénin
(fonner Dahomey). He graduated from his country university (Université du Bénin,
Lomé-Togo) and was awarded his "Licence ès Sciences Naturelles" (LICEN.) degree in
1978. In Fall1978, he entered the Graduate College of the University of Dijon, France
where he was awarded his "Maîoise de Biologie Animale" (MAITR.) degree in 1980. He
University in Athens, Ohio, for one year. After that, he entered by FaU 1990 the Graduate
College of the University of Illinois at Urbana-Champaign, in the division of Nutritional
Sciences of the College of Agriculture to work towards his Ph.D. degree under the
direction of Dr. Edward G. Perkins in Bumsides Research Laboratory. During that rime,
he was the recipient of Graduate Fellows Recognition of the Illinois Chapter Gamma
Sigma Delta in 1991 and 1992. He was then initiated and recognized as membership of
the Honor Society of Agriculture Gamma Sigma Delta on April 8, 1993. Courdjo
gmduated from the University of Illinois at Urbana-Champaign with the degree of Doctor
ofPhilosophy in Nutritional Sciences irr.l993.
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