EFFECTS 3? meme FAT .
AND vammze EiEPON me smezm
OF we we FROZEN seeeeee

Thesis fer the Degree of M. S.
MlCR’aGAN STATE UNWERSWY
IGHN DAMIEN {GENE
1976

 

OI“

 

 

 

ABSTRACT

EFFECTS OF DIETARY FAT AND VITAMIN E UPON
THE STABILITY OF MEAT IN FROZEN STORAGE

by

John Oamen Igene

Sixteen 4-day old veal calves were allotted into
four groups and fed solely on filled milk in which half<xf
the calves received a stable saturated fat (coconut oil)
and the other half an unsaturated fat (corn oil). Half of
the claves on each treatment were supplemented with 500 mg
d-a-tocopheryl acetate per calf per day (groups 1 and 3).
whereas. the remainder were unsupplemented. The calves
were slaughtered after 8 weeks and samples of kidney and
omental fat and of meat (longissimug ggggi muscle) were
removed. wrapped in freezer paper. frozen and stored at
~18°C. The initial samples were analyzed for fatty acid
composition. Samples were also analyzed for vitamin E
and TBA values at 0. 1, 3 and 6 months storage.

The average slaughter weight of the calves ranged
from 139 lb for group 3 to 162 lb for group 1. Carcass
yield was about 53% for group 3, and approximately 60%

for groups 1. 2 and h. respectively. Average total lipid

John Oamen Igene

content of the meat was below 2%. while the phospholipid
content ranged from 0.75 to 0.82%. The mean level of fat
in the internal lipid depots varied from 24 to 75% among
groups.

The prOportion of saturated fatty acids ranged from
60 to 70% in the fatty tissues and meat triglycerides from
coconut oil fed calves, while the level varied from 30
to uoz for the corn oil diets. The amount of saturated
fatty acids in the phospholipids (33%) was not influenced
by the different rations. The distribution of monoenoic
acids in the fatty tissues and meat neutral lipids varied
from 23 to 34%. but showed no clear-cut pattern of dis-
tribution. The level of dienoic acid was about 28% in
both the depot fats and meat triglycerides from calves
fed corn 011. whereas. the level was only 5% for the same
tissues from the coconut oil fed calves. The amount of
linoleic acid in the phospholipids was on average about
40 and 27%, in the meat from calves fed corn oil and
coconut oil diets, respectively. The level of polyenoic
acids (9%) in the phospholipids from the meat was not
affected by differences in treatment.

On the average the initial level of vitamin E in the
meat was generally below 6 pg/g of tissue. while the
initial level in the depot fats from calves fed supplemental
vitamin E ranged from about 16 to 42 ug/g of tissue.
Vitamin E declined steadily during storage. but the rate

of decline was not the same in all tissues. In the fatty

John Oamen Igene

tissues, over 60% of the initial toc0pherol was still pre-
sent after 3 months freezer storage. In the meat lipids.
the levels of tocopherol at 3 months of storage had de-
clined to about 40 and 25% in calves fed coconut oil and
corn oil diets.

The stability of the lipid was monitored during frozen
storage by the 2-thiobarbituric acid test. Up to 6 months
of storage. the meat and omental tissues were stable. with
a maximum TBA number of about 0.2 and 0.h, respectively.
However, the kidney fat was less stable. The TBA values
for kidney fat from calves fed corn oil without supplemental
vitamin E had increased to the threshold level for rancidity
at 6 months of freezer storage.

Yesults show that young calves selectively deposited
dietary fats in the tissues without significant altera—
tion. The data also demonstrated that dietary vitamin E
and saturated fatty acids contribute to the stability

of the tissues during frozen storage.

EFFECTS OF DIETARY FAT AND VITAMIN E UPON
THE STABILITY OF MEAT IN FROZEN STORAGE

by

John Oamen Igene

A THESIS

Submitted to

Michigan State University

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1976

ACKNOWLEDGEMENTS

The author is indebted to Dr. A. M. Pearson for
his direction and encouragement throughout his study and
during the preparation of this thesis. The author also
wishes to express his appreciation to Dr. F. B. Shorland.
then visiting professor from Victoria University of
Wellington, New Zealand, for his interests and supervision
of this study. Sincere appreciation is also extended to
the guidance committee, Drs. L. L. Bieber, L. R. Dugan, Jr.,
and J. F. Price for their critical review of this thesis.

Appreciation is expressed to Dr. D. L. McGillard for
his assistance in statistical design and analysis of data;
to Dr. D. R. Romsos for allowing the author use of his
laboratory facilities; to the Distillation Products Indus-
tries (Rochester, New York) for donating the vitamin E
acetate used in this study; and to the Bendel State Gov-
ernment of Nigeria for providing funds to support his
studies.

Finally, the author is particularly grateful to his
family, at large, (especially to his senior brother, Benedy)

for their encouragement, patience. understanding and support.

ii

DEDICATION

To the living memory of my late mother. Oiyimhebedan,

who gave me all my motivation and desire in life and per-

severed through every imaginable problem to educate me.

iii

TABLE OF CONTENTS

INTRODUCTION 0 O O O O O I O O O O O I O O O O O O 0

REVIEW OF IJTERATURE . . . .
Deterioration of Fats. Oils and Fat- Containing
Foods . . . . . . . . . . . . .
Mechanism of Autoxidation . . . . . . . . . .
Hematin Compounds as Pro-Oxidant of Meat
Oxidation . . . . . . .
Role of the Oxidation State of Iron . . .
Mechanism of Hematin Catalysis . . . .
Antioxidant Level of Hematin Compounds
Catalysis by Metal Ions . . . . . .
Action of Antioxidants in Food Lipids . . .
Vitamin E as a Lipid Antioxidant . . . .
Species Differences in the Ability to
Deposit Dietary Toc0pherols in Tissues.
Vitamin E Activity and Product Stability.
The Composition of Animal Fats . . . . . . . .
Influence of Diet . . . . .
Fatty Acid Composition of Animal Fats . .
The Phospholipid Content in Animal
Tissues . . .
Relationship of Fatty Acid Composition to
Autoxidative Stability of Meats . . . .
Changes in Neutral Lipids and Phospho-
lipids During Frozen Storage . . .
The 2- thiobarbituric Acid (TBA) Test as a
Measure of Meat Rancidity . . . . . . .
MA as an Index of Lipid Oxidation in
FOOdS l O O O O O O O O O O O O O 0

EXPERIMENTAL . . . . . . . . . . . .
Materials and Methods . . . . . . . . .
Solvents . . . . . . . . . . . .

Animals and Rations . . . .

Vitamin E (d-a-tocopheryl Acetate) .
Statistical Treatment . . . . . . . .

Extraction of Total Muscle Lipid . . . .

a

Separation of Phospholipid and Neutr l
Lipids . . . . . . . . . . . . .

iv

Rendering of Depot Fats . . . . . . . . .

Preparation of Methyl Esters . .

Analysis of Fatty Acid Composition of the
Triglycerides and Phospholipids . . . .

Determination of Toc0pherol in Meat and
Fatty Tissues . .

TBA Analysis for Lipid Oxidation of Meat
and Depot Fat . . . . . . . . . . . .

ESULTS AND DISCUSSION . . . . . . . . . .
Analysis of Experimental Diets . . . . .
Liveweight, Carcass Weight and Carcass

Yield of Veal Calves . . . .
Fat Content of the Meat and Depot Fat .
Fatty Acid Composition . . . .
Fatty Acid Composition of the Phospho—
lipids. . . . . . .
The Toc0pherol Content in the Meat and Fatty
Tissues. . . . . .

Influence of Storage Time on the Stability

of Vitamin E in Longissimus dorsi . . .
Toc0pherol Content of Kidney Fat . . . .
Toc0pherol Content of Omental Fat . . . .
Prediction of the Stability of Vitamin E.

Lipid Oxidation in Animal Tissues . . . . . .
Lipid Oxidation in Meat . . . . . . . . .
Lipid Oxidation in Depot Fats . . . . . .

SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . .
BIBLIOGRAPHY O O O I I O O O O O O O O O O O I O O O
AI‘PE NDIX O O O I C O O O 0 O O C O O O O C C O O I

Page

Table
l.

10,

ll

12.

13.

LIST OF TABLES

Levels of Coconut Oil and Corn Oil
Filled Milk Fed to the Experimental
Calves . . . . . . . . . . . . . . . . .

Composition of Filled Milk . . . . . . .

The Fatty Acid Composition of the Two
Experimental Diets Determined by Gas
Liquid Chromatography . . . . . . . . . .

Live Weight, Carcass Weight and Carcass
Yield of Veal Calves. . . . . . . . . . .

Fat Content of Longissimus dorsi, Kidney
Fat and Omental Fat . . . . . . . . . . .

Mean Fatty Acid Composition of Kidney Fat

Mean Fatty Acid Composition of Omental

Fat 0 0 O O I O O O O O O O O O O O O O 0

Mean Fatty Acid Composition of Glycerides
in Longissimus Dorsi . . . . . . . . . .

Mean Fatty Acid Composition of Phospho-
lipids of Longissimus Dorsi . . . . . . .

Summary of Average Fatty Acid Composition
of Kidney and Omental Fats . . . . . . .

Summary of Average Fatty Acid Composition
of Meat Lipids . . . . . . . . . . . . .

Mean Tocopherol (Vitamin E) Levels in
Longissimus Dorsi as Influenced by
Storage Time. . . . . . . . . . . . . . .

Mean Toc0pherol (Vitamin E) Levels of
Kidney Fat as Influenced by Storage Time.

vi

Page

34

36

46

#8

50
54

55

56

58

59

6O

63

66

Table Page

in. Mean Toc0pherol (Vitamin E) Levels of
Omental Fat as Influenced by Storage Time 70

15. Mean Thiobarbituric Acid (TBA) Number of
Longissimus Dorsi as Influenced by Storage
Time 0 o o o o o o o o o o o o o o o o o o 75

16. Mean TBA Numbers of Kidney and Omental
Fat 0 O O I O O O O O O O I O O O C O O O 80

vii

LIST OF FIGURES

Figure

1. Changes in Toc0pherol Level of
Longissimus Dorsi During Freezer Storage.

2. Changes in Toc0pherol Level of Kidney Fat

3. Changes in Tocopherol Level of Omental
Fat 0 I O O O O O O I O O O O I O I O O O

A. Changes in TBA Numbers of Longissimus
Dorsi as Influenced by Length of Freezer
Storage 0 o o o o o O I O o O O O I o O O

5. Changes in TBA Numbers of Kidney Fat
During Freezer Storage (-1800) . . . .

6. Changes in TBA Numbers of Omental Fat

During Freezer Storage (-1800) . . . . .

viii

LIST OF APPENDIX TABLES

Page
Calves: Weights at Slaughter (lb) . . . . 99
Veal Lipids: Longissimus dorsi . . ._. . . 100
Veal Lipids: Kidney Fat. . . . . . . . . . 101
Veal Lipids: Omental Fat . . . . . . . . . 102
Veal Vitamin E: ug/g Lipid . . . . . . . . 103
Veal Longissimus dorsi TBA Value: mg/lOOO
g Tissue; and Veal Longissimus dorsi Vitamin

E: ng/g Lipid. o o o o o 0 o o o o o o o 0 101+

TBA Values for Kidney and Omental Fat
Tissues (mg/1000 g tissue) . . . . . . . . 105

Veal Lipids: Fatty Acid Composition of
Kidney Fat (%) o o o o o o o o o o o o o o o 106

Veal Lipids: Fatty Acid Composition of
Omental Fat (%) . . . . . . . . . . . . . . 107

Veal Lipids: Fatty Acid Composition of
Longissimus Dorsi (%) . . . . . . . . . . . 108

Veal Lipids: Fatty Acid Composition of
Phospholipids (Longissimus dorsi) . . . . . 109

ix

Figure
l.
2.

1.3..

LIST OF APPENDIX FIGURES

#350 Kidney fat (group 1) . .
#368 Longissimus dorsi (group 2)
#342 Longissimus dorsi (group 3)
-#364 Kidney fat (group 4) .

Page
112
114
116
118

INTRODUCTION

Increasing awareness that linoleic acid tends to
favor low concentrations of serum cholesterol has prompted
researchers to find ways to increase the levels of PUFAS
(polyunsaturated fatty acids) in the fats of meat animals.
It is well rec0gnized that the composition of the ingested
fat does not appreciably affect the prOperties of the depot
fat of ruminants. primarily due to the hydrogenation of the
unsaturated fatty acids by the rumen microorganisms. To over—
come this effect, ruminants have been fed encapsulated poly-
unsaturated oils. such as safflower oil. coated by a protein,
which is hardened with formaldehyde (Scott gt gl., 1970).
The drOplets, thus protected, bypass the rumen, but are
released on reaching the intestine where they are absorbed
like that of non-ruminants.

Before entering the stage of rumination, calves do
not hydrogenate unsaturated dietary fat, and consequently.
lay down such fat like non—ruminants. Hence, in the depot
fats of young calves, the dietary fat is laid down without
being subject to significant changes prior to deposition.
The resulting meat and meat products show marked increases

in the linoleic acid content of the depot fats and

phospholipids. Since the unsaturated fats are oxidized
more readily. the stability of the meat products becomes
of primary interest.

Much research on the keeping quality of fats. and
to a lesser extent to that of phospholipids, has been carried
out on rendered or extracted lipids using such criteria as
the peroxide value, the TBA number and the length of the
induction period. Although work has also been done on
cooked meats. less is known about the oxidative changes
in the fats of meat during frozen storage.

The present study was designed to obtain knowledge
about the changes in the lipids during frozen storage of
meat as they occur i3 gigg. Since the content of polyun-
saturated fatty acids markedly influences meat stability,
it was deemed necessary to determine the effect of various
concentrations of linoleic acid and other PUFAS on the
oxidative stability of meat in relation to changes in the
level of tocopherol (vitamin E). The stability of the
lipids during storage at -18°C were evaluated by use of

TBA numbers (Tarladgis gt gl., 1960).

REVIEW OF LITERATURE

Deterioration of FatsL Oils and Fat-Containing_Foods

Although oxidative deterioration of lipids involving

the uptake of atmospheric oxygen is common to most foods
containing plant or animal tissues, the mechanism in-
volved may vary (Tappel, 1953, 1955: Dugan, 1961; Lea,
1962). Invariably. autoxidation (uptake of oxygen) of
the lipids in such foods is promoted by heat, light (es-
pecially of short wave length) and metal catalysts.
especially by copper and iron (Smith and Dunkley. 1962;
Waters. 1971).

There is much evidence in the literature (Dugan.
1961; Lundberg. 1961; Lea, 1961: Ingold, 1967: Waters,
1971) in support of the generally accepted oxidative mech-
anisms for food lipids. These include autoxidation. lipoxi—
dase-catalyzed and hematin-catalyzed oxidation. However,
the mechanisms show differences in the initiation pro-
cess, activation energies and in the rates of oxidation
(Tappel, 1953, 1955: Dugan. 1961; Lea, 1962). Since
lipoxidase does not exist in meats (Banks. 1944; Tappel,

1952, 1953). its role will not be discussed herein.

2+

Mechanism of Autoxidation

The currently accepted mechanism for autoxidation
of unsaturated fatty acids was first elucidated by Farmer
and Sutton (1943) and by Bolland and Koch (1945). They
concluded that fat oxidation is initiated by the abstrac—
tion of hydrogen from a labile methylene group in the
molecule of the unsaturated fatty acid (RH). A free radical
(R') is. thus. formed to which oxygen is attached to form
a peroxy radical (ROO'). The peroxy radical subsequently
abstracts a hydrogen from a nearby site of another unsat—
urated fatty acid molecule to form a hydrOperoxide (ROOH)
and to prOpagate the chain. The hydrOperoxides may then
decompose yielding free radicals. which can initiate new
reaction chains. The steps involved in the mechanism are

schematically shown below.

(I) Initiation:

 

RH i» R' + H'

(II) Propagation:

 

 

 

 

 

 

 

"'""'R' + 02 A, R00'
1200' + RH % ROOH +3;-
- f (P
ROOH energy R0' + 'OH
120' + RH ROH + R'
'OH + RH H20 + R'
t

 

Chain branching occurs in the last three reactions with

a rapid increase in the number of free radicals through

autocalytic interaction.

(III) Termination:

 

 

 

2R“ -=% R-R
2120' 3‘ RO-O-P.
2ROO' 5? various products

According to Dugan (1961). the initiation process
has an activation energy of about 45 KCal/mole, while the
propagation steps have lower activation energies. The
decomposition of the hydrOperoxides to form free radicals.
which further take part in the autocalytic chain reactions is
by either thermal instability or by reaction with other
materials (Farmer and Sutton, 1943).

Mabrouk and Dugan (1960) and Lundberg (1962) have dem-
onstrated that the decomposition of hydroperoxides can take
place by a monomolecular as well as by a bimolecular mech-

anism, as illustrated below:

 

 

Monomolecular:

ROOH } 30° + 'OH
Bimolecular:

2ROOH ‘4, 2RO° + H20

They indicated that monomolecular decomposition takes place
at a lower concentration of hydrOperoxides and at higher
temperatures. Mabrouk and Dugan (1960) have also reported

a lower activation energy for the bimolecular than for the

monomolecular mechanism for hydroperoxide scission.

The unpleasant odors of rancid oils and fats are due
mainly to aliphatic saturated and unsaturated aldehydes
and acids of lower molecular weight produced by the even-
tual oxidative scission of the long hydrocarbon chains of
lipid molecules (Mookherjee and Chang, 1963; Kimoto and

Gaddis. 1970).

Hematin Compounds as Pro-Oxidants

The catalytic effect of the iron porphyrins on the
oxidative deterioration of polyunsaturated fatty acids
(PUFAS) was first described by Robinson (1924), who attri-
buted the catalysis to the iron content of the molecule.
Since then, a number of studies (Kendricks and Watts, 1969;
Fishwick, 1970a, 1970b) on the role of heme-iron in catalytic
oxidation have been reported.

According to Greene (1975), the main forms in which
nvoglobin (Mb) may exist in meats are as Mb. oxymyoglobin
(Mb02) and nitric oxide ferrohemochrome (NOH), respectively,
in whirh the iron porphyrin is in the ferrous (Fe2+) state.
The other forms of Mb include ferrihemochrome and metmyo-
globin (Meth), both of which contain iron in the ferric
(”e3+) state (Kendricks and Watts, 1969: Fishwick, 1970a,
1970b; Greene, 1975).

A number of investigators (Watts and Peng, 1947;
watts, 1954; Lewis and Wills, 1963; Tappel, 1952; Liu,
1970a, b; Liu and Watts, 1970; Greene, 1975) have indicated

that hematin compounds are involved in lipid oxidation

in meat. On the other hand, Sato and Hegarty (1971) indi-
cated that the component of cooked meat responsible for
catalyzing lipid oxidation is a water soluble diffusate,
hence, neither Hb. Mb, MbO2 nor Meth are responsible.

In support of this view, Love and Pearson (1974) reported

2+ to cooked meat

upon addition of purified Meth and Fe
that only the latter compound was effective as a pro-
oxidant. Meth at levels of 1-10 mg/g of meat failed to

catalyze oxidation.

Role of the Oxidation State of Iron

COnsiderable differences in Opinion have been expressed
on the mechanism of hematin-catalysis regarding the impor-
tance of the oxidation state of iron to the catalytic ac—
tivity of the heme pigments. Younathan and Watts (1959).
Kendricks and Watts (1969) and Greene (1975) have prOposed
that the Fe3+ hemes are the active catalysts of lipid
oxidation. Evidence for this was demonstrated by showing
that cooked meat develOped warmed-over-flavor (MOP) during
refrigerator storage more rapidly than raw meat. During
cooking, the Mb of raw meat is converted to Meth.

Smith and Dunkley (1962). Brown gt g1. (1963) and
Hirano and Olcott (1971) found no difference in the rate

2+

of lipid oxidation catalyzed by Fe and Fe3+ hemes.

consequently. Smith and Dunkley (1962) and Safe and Hegarty

e2+

(1971) concluded that :e is the active catalytic agent

rather than the hematin-compounds.

Tarladgis (1961) reported a close relationship be-
tween lipid oxidation, spin state, ligand.field. and pig-
ment changes in animal tissue. He attributed the catalytic
activity of ferric hemOproteins to the paramagnetic char-
acter of the porphyrin bound iron rather than to its
oxidation state. According to Tarladgis (1961) and Fish-
wick (1970 b), the presence of five unpaired electrons
in Meth produces a strong magnetic field. which would
favor the initiation of free radical formation from the
high spin state (Fe3+). Iron porphyrins in the low spin

state (Fe2+) as in fresh and freeze-dried meats. exhibited

no catalytic activity (Fishwick, 1970a, b).

Mechanism of Hematin Catalysis

The oxidation of unsaturated fats in the presence of
hematin compounds is believed to proceed by way of a chain
reaction mechanism (Barrorland Lyman. 1938). Banks (1944)
postulated that preformed linoleate peroxide was necessary
for hematin catalysis. As a consequence, Tappel (1953)
proposed a theory of hematin catalysis aimed at explaining
the data presented by Banks (1944). According to the theory.
the mechanism of linoleate oxidation involves a direct reac—
tion of linoleate peroxide with hematin catalysis. PrOpa-
gation and termination reactions are essentially similar
to the autocalytic mechanism. Hence, the critical reaction

in hematin catalyzed unsaturated lipid oxidation is the

catalytic decomposition of hydrOperoxides to free radicals
(Tappel. 1953).

Tappel (1953) has reported the relatively low activ—
ation energy of 3.3 KCal/g mole for hematin—catalyzed
oxidation. This suggests that rancidity of meat products
would proceed readily at frozen storage. However. Tappel
(1952, 1955) has indicated that frozen fresh meat
is relatively stable despite the presence of hematin
catalysts.

In a later study. Tappel (1955) attempted to broaden
the knowledge of unsaturated lipid oxidation by hematin
compounds and to define more precisely the mechanism of
the reaction. He concluded that Mb has catalytic activ-
ity quantitatively similar to that of hemin, cytochrome
c and hemoglobin. He then suggested that hematin involves
the formation of a lipid peroxide-hematin compound and
its subsequent decomposition into free radicals. which
propagate the chain reactions with the concomittant des—
truction of the catalyst.

The mechanism of hematin-catalyzed unsaturated lipid

peroxidation (Tappel, 1955) is shown on the following

page.

lO

 

 

 

 

LOOH

ID.
LO' + LH tt3'LOH + L.
L. + 02 )LOO'
LOO‘ + LH —) LOOH + L.
Where:

LH = Linoleic acid

IOOH = Hydroperoxide linoleate

Antioxidant Level of_Hemat;ngCompounds
Lewis and Wills (1963) demonstrated that hemoglobin.
cytochrome c. hematin and tissue homogenates at high

concentrations all had inhibitory effects on linoleate

11

oxidation. The concentration of hemoglobin necessary to
show antioxidant activity increased with higher concentra-
tions of fatty acids, which is in agreement with the re-
sults of Kendricks and Watts (1969).

Catalygis by Metal Ions

There is agreement in the literature that metals.
particularly the transition elements (iron and copper).
are powerful oxidative catalysts (Smith and Dunkley, 1962;
Waters, 1971: Sato and Hegarty. 1971: Love and Pearson,
1974: Greene. 1975). Foods usually contain trace elements
of heavy metals. which probably arise. at least in part,
from the presence of metal activated enzymes or their
decomposition products or else through contamination
(Waters. 1971).

According to Wills (1965). inorganic iron is a par-
ticularly notorious oxidative catalyst. Love and Pearson

(1974) have demonstrated the catalytic effects of Fe2+ a

s
a pro-oxidant in meat. Privett and Blanck (1962) and
Sato and Hegarty (1971) reported that at levels below
1 ppm, Fe2+ and other metals are incapable of catalyzing
lipid oxidation.

A number of investigators (Privett and Blanck. 1962:
Ingold, 1967; Waters. 1971) have concluded that the
primary oxidative function of heavy metals. like that

of hematin compounds. is to increase the rate of

12

hydrOperoxide decomposition to free radicals. Wills (1965)
reported that the catalysis of lipid peroxide formation

by inorganic iron is pH-dependent. The Optimum rate

of peroxidation reaches a maximum close to pH 5.5 but
decreases under alkaline conditions. There is strong

evidence that Fe is a more active catalyst than Fe

(Smith and Dunkley, 1962: Sato and Hegarty, 1971: Waters.
1971: Love and Pearson. 1974).
The mechanisms of metal catalysis were reviewed by

Ingold (1967) and Waters (1971). The initial step in the

mechanism is the donation of an electron by the Fe2+ to

the hydroperoxide. The Fe2+ is. thus. oxidized to Fe3+

2+ by the decomposition

state. to be later reduced to Fe
products of the hydrOperoxides (Ingold, 1967: Waters,

1971). This reaction is shown schematically below:

 

 

 

ROOH + 142+ r R0' + ’OH + M3+ (I)
3003 + 113+ ? ROO' M2+ + H+ (II)
M3+ + RH # R' + M2+ + H+ (III)

Metal catalysts also function as oxidation inhibitors.
Smith and Dunkley (1962) and Ingold (1967) have demonstrated
that above a certain critical concentration. ferrous iron

and cobalt may function as inhibitors.

13

Action of Antioxidants in Food Lipids

Antioxidants may interfere with or delay the onset
of oxidative breakdown of fats and fatty foods (Blanck,
1955). Primary or phenolic antioxidants (such as toco-
pherols. butylated hydroxyanisole(BHA)or butylated hydroxy-
toluene(BHT))function by breaking the oxidative reaction
chains (Shelton, 1959). In support of this viewpoint,
Cort gt g1. (1974) reported that the phenolic antioxi-
dants act as electron or hydrogen donors to quench elec-
tron mobility with subsequent interruption of free-radical
chain reactions.

According to Uri (1961), the mechanism of antioxi-

dant action is as follows:

 

1200' + AH (antioxidants) ) ROOH + A’

The radical may be stabilized by recombination in either

of two ways:

 

A' + A' s9'A2

 

ROO' + A“ —’ ROOA

This means that during autoxidation. the antioxidants
are converted into dimers and other products (Uri, 1961).
It is also possible that the antioxidant is oxidized dir-
ectly by oxygen. as in the case of toc0pherol. which is
partly oxidized to tocoquinone in fats (Tappel. 1962).

At the end of the induction period, the antioxidants

l4

disappear with little being known as to their exact fate

(Cort gt_g;.. 1974).

Vitamin E ggfia Lipid Antioxidant

The tocOpherols are products of synthesis by plants.
but may occur naturally in animal tissues in the non-
saponifiable portion of the lipid fraction--usually to-
gether with sterols. vitamin A. vitamin K and other naturally
occurring antioxidants (Mervyn andMorton, 1959: Bieri.
1969). Vitamin E is also found in association with phos-
pholipids. occurring largely in subcellular membranes
(Witting, 1975).

There appears to be some limitations in the amount
of tocOpherol that can be accommodated within a normal
membrane (Witting, 1975). Absorption of a-toc0pherol de-
creases inversely with increasing levels in the diet
(Adams gt gt.. 1959: Losowsky gt_g;.. 1971). On the other
hand. the a-tocopherol content of most fats and oils is
a log function of their polyunsaturated fatty acid (PUFA)
contant (Hove and Harris. 1951).

It is believed that the level of a-tocOpherol deposited
in a tissue depends on the particular isomer and homologue.
the dietary level. the duration of the feeding period.
the particular species. the specific tissue and the amount
of destruction in the diet and gut (Adams gt g;.. 1959:
Machlin, 1962: Marusich gt gl.. 1975: Witting. 1975). The

15

storage of the d-form of a-tocopherol is about twice as
great as for the dl-form and the relative efficiency

of absorption of the toc0pherols in decreasing order

is: alpha. beta. gamma and delta. respectively (Machlin.
1962: Parkhurst gt gt.. 1968).

The stability of tocopherol in the diet depends on
the amount of peroxidizable lipid. the temperature. the
length of storage. the concentration of trace metal cata-
lysts. the concentration of hydr0peroxides and the protec-
tive effect of other antioxidants (Adams gt g;.. 1959:
Parkhurst. 1968: Witting. 1975). It is suggested
(Moore and Sharman. 1959: Machlin. 1962) that destruction
of tocopherol in the gut may be considerable when PUFAS

are ingested concurrently with the tocopherols.

Species Differences in the Ability to Depogit Dietary

Toc0pherols in Tissues
All unsaturated lipids require stabilization. hence

the need for vitamin E supplementation in feeds to stabil-
ize the resultant animal products (Burr gt _a_l.. 1946).

The only lipid antioxidant. which is stored in appreciable
amounts by animals. is a-tocopherol (Barnes gt g;.. 1943:
Lundberg gt g;.. 1946). However. marked differences have
been reported in the ability of domesticated species to
deposit dietary tocopherol in their tissues. The ability
of poultry to store dietary a-tocOpherol has been confirmed

(Criddle and Morgan. 1947: Kummerow’gt_gt.. 1948: Mecchi

16

_t _;.. 1953: 1956a: Webb gt g;.. 1973. 1974: Marusich
gt g;.. 1975). Mecchi gt gt. (1956a) and Marusich gt_g;.
(1975) reported a higher level of a—toc0pherol in the
tissues of chickens than in turkeys fed the same diet.
They also showed that turkeys require a higher level of
supplemental a-tocopherol to delay the onset of rancidity
than chickens.

There is little agreement on the ability of the pig to
store dietary vitamin.E. Watts gt gt. (1946) suggested
that the pig could not store vitamin E at a level of prac-
tical value. probably because of inefficient absorption.
However. Astrup (1973) reported absorption and deposition
of vitamin E in the pig. He also indicated that the
dietary vitamin E improved the taste. flavor and the
oxidative stability of the meat.

Deposition of dietary a-tocOpherol is reported to
be very inefficient (Watts. 1946: Caravaggi and Wright,
1969). Caravaggi and Wright (1969) reported that sheep
fed a-tOCOpheryl acetate excreted virtually all of it in
the feces in 4 days. Based on these results. they recom-
mended intramuscular administration. However. Buchanan-
Smith gt gt. (1969) demonstrated positive absorption and
increased tissue levels of a-toc0pherol in sheep upon
oral administration.

The ability of the calf to deposit dietary tocOpherol
has been well documented (Decker and Hill. 1947: Eaton
gt g;.. 1958: Adams gt_gt., 1959: Poukka and Oksanen.

17

1972: Ellis gt gt.. 1974). Similar results have also been
obtained with mature steers (Kimoto gt gt., 1974).
Nevertheless. little has been done in supplementing
ruminants with vitamin E. presumably because of their
ability to alter the lipid fraction of feeds in the rumen
(Shorland gt £11.. 1957). Caravaggi gt _a_;L_. (1968. 1969)
have suggested that vitamin E is more efficiently deposited
in tissues of ruminants by intramuscular injection of
a-tocOpheryl acetate. They concluded that the real differ-
ence between the digestive and injection routes is due
to lack of absorption rather than rumen hydrolysis. How—
ever. intramuscular administration of a-tocopheryl acetate
in calves and mature steers was found to be inefficient

(Blaxter gt gt.. 1953: Adams gt g;.. 1959).

Vitamin E Activity and Product Stability

The principal effect of an antioxidant is the neu-

 

tralization of the free radical in the first stage of the
autoxidative chain reaction (Uri. 1961). Thus. the reac-
tion of the antioxidant and the free radical involves

the transformation of the antioxidant into the unstable
free radical form. which easily undergoes irreversible
changes (Dam. 1953: Cort gt g;.. 1974: Witting. 1975).

Michaelis and Wollman (1949) demonstrated that

semiquinone is the free radical form of vitamin E. They
theorized that a-tocopherol stabilizes lipids through

formation of a semiquinone structure. Although the tp vitro

18

antioxidant effect of the four known tocopherols, increases
in order of alpha. beta. gamma and delta. the activity tp
ytyg increases in the Opposite order (Dam. 1953: Park-
hurst gt_gt.. 1968).

Alpha-tocopherol is regarded as the lipid antioxidant
of nature. and this is believed to be its most important
function (Poukka and Oksanen. 1972: Witting. 1975). The
chemical basis of the action of vitamin E is its combina—
tion with free radical intermediates of lipid oxidation and
lipid peroxides. thus inhibiting further lipid peroxida-
tion (Tappel. 1962).

Tappel (1962) has proposed that a—tocopherol is
oxidized to a number of products in hematin-catalyzed.
peroxidizing linoleic acid. The reactions involved are
shown on the following page.

A number of studies have demonstrated the beneficial
antioxidant activity of a-tocopherol in improving the
stability of carcass lipids of poultry (Criddle gt g;..
1947: Mecchi gt gt.. 1953: Webb gt gt.. 1973. 1974: Marusich
et gt.. 1975). of veal and beef (Lundberg. 1944: Ellis

_t__;.. 1974: Kimoto gt_g;.. 1974). of mutton (Caravaggi

and Wright. 1969) and of pork (Astrup. 1973). Neverthe-
less. some workers regard a-tocopherol as a poor antioxi-
dant. particularly in products containing highly unsaturated
fatty acids (Lips, 1947: Witting gt gt.. 1964: Witting.
1969: Benedict gt_gt.. 1974: Witting. 1975).

The dietary requirement for vitamin E increases with

l9

.sowpmcflxOMOm Owned Scum posponm same one

.mcosH:d_Hmnmzaooopnu u mm was vasomsoo OPMHOOEAOPSH N am “Honmnmooop:u u <

 

 

 

 

mm am a
mzoo mmw
o
\ \x /
I
max as
RSS/ \ e /
> \ a \
mxo mmo
s _ _ _ .

 

20

increasing amounts Of unsaturated fatty acids. which sug-
gests that it plays an important dietary role as an antioxi-
dant (Kimoto gt_g;.. 1974: Witting. 1975: Marusich gt gt..
1975). Alpha-tocopherol has an Optimum concentration for

a minimum rate of oxygen uptake. and increasing the level
beyond this optimum results in an increased rate of oxygen
uptake (Parkhurst gt g;.. 1968: Witting. 1975).

The level of a-tOCOpherOI decreases with aging of
meats (Adams gt g;.. 1959). Hence. fresh meat products
having high levels of linoleic acid require more a-tocOpherol
in the lipid fraction to enhance their stability (Kimoto
gt g;.. 1974: Marusich gt gt.. 1975). Alpha-tocopherol
is destroyed five times as rapidly by the linolenate as by
linoleate (Lips. 1957: Witting. 1969). As the degree of
unsaturation in the fatty acids increases. the induction
period decreases and the fatty tissues eventually become
rancid. despite the presence of a-tOOOpherol (Kimoto gt gt..
1971:. Ellis gt g1” 1974; Witting. 1975).

At low concentrations. a-tocopherol functions as an
antioxidant. but at high concentrations may become a pro-
oxidant (Chipault. 1961). The minimum formation of per-
oxide-free radical initiation occurs at a concentration
of about 1-3 umole of a-tocopherol per g fat (Witting.
1975). Witting (1975) suggested that an increase in
toc0pherol concentration results in increased peroxide
formation through free-radical initiation. an increased

rate of autocatalysis and an increased rate of destruction.

21
The Composition of Animal Fats

Lipids in meat. poultry and fish are often classified
as depot or adipose tissue and as intramuscular or tissue
lipids (Watts, 1962: Love and Pearson. 1971). The depot
fats are largely localized as subcutaneous deposits. al-
though large quantities may be present in the thoracic
and abdominal cavities and between the muscles as inter—
muscular deposits.

The triglycerides are the principal components of
adipose tissue (Watts. 1962) and are deposited largely
as fat globules localized within the individual cells.

On the other hand, tissue lipids are an integral part of
various cellular structures, which include the cell wall
(Kono and Colowick. 1961). the mitochondria (Holman and
Wildmer. 1959) and the sarcoplasmic reticulum (Newbold
gt g;.. 1973). Although adipose tissue is deposited in a
fairly consistent pattern. it is influenced by species.

diet. environment. sex and other factors (Deuel. 1955).

lpttuence of Diet

Although species differences in the composition of
depot fat may be related to the composition of the diet.
dietary influences within a species can be controlled
(Shorland. 1952). The data presented by Ellis and Isbell
(1926 a. b) have clearly demonstrated the influence of diet
upon some measures of carcass firmness and the prOportion

of different fatty acids in the depot fat of the pig. It

22

is well established that soft pork results when a high
level of corn oil is fed to hogs. and that the resulting
fat is more susceptible to autoxidation (Ellis. 1933).
Hence. the composition Of the depot fats of non-ruminants
tends to reflect that Of the dietary fat. On the other
hand. in ruminants the depot fats are not influenced to
any extent by diet (Reiser. 1951: Shorland gt gt.. 1957).
In order for ruminants to be directly reaponsive to dietary
unsaturated fats. it is necessary to by-pass the rumen by
means Of a duodenal fistula (Ogilvie gt gt.. 1961) or
otherwise protect the dietary fat from the action of rumen
microorganisms (Cook gt g;.. 1970: Scott gt g;.. 1971).
However. calves before entering the stage of rumina-
tion do not hydrogenate unsaturated dietary fat. and con-
sequently. lay down such fat like non-ruminants (Holmberg
gt_g;.. 1956). Recent studies (Poukka and Oksanen. 1972:
Wright gt gt.. 1974: Ellis gt gt.. 1974: Kimoto gt g;..
1974) have confirmed the monogastric behavior of the young

calf.

Fatty Acid Composition of Animal Fats

Natural fats are composed mainly of the straight chain
even numbered carbon fatty acids. typically containing 16
and 18 carbon atoms (Dugan. 1971). Animals tend to be
more uniform in their fatty acid composition than those
of plants. though the range of fatty acids encountered

is still very wide (Hansen gt gt.. 1958).

23

The most abundant and widespread fatty acid in animal
fat is oleic (octadec—ci -9-enoic) acid. Other unsaturated
fatty acids. which are prominently distributed (though not
so uniformly) include linoleic (Octadec-gtge9-gtg-12—cuenoun
and palmitoleic (hexadec-gtg-9-enoic) acid (Hilditch and
Williams. 1964). Of the saturated fatty acids. palmitic
(hexadecanoic) acid is the most prominent. and like oleic
acid. it is seldom absent in any Of the natural animal fats
(Hilditch and Williams. 1964).

In animals. the endogenous fat contains about 25%
palmitic acid, the remaining fatty acids being mainly
oleic with minor amounts of stearic. myristic and palmitoleic
(Shorland. 1952). When animals have access to dietary fat.
the fatty acids may be reflected in the composition of the
depot fat. Animal fats frequently contain linoleic acid and
are often accompanied by linolenic (octadec-gtg-9-gtg-12-
gtg715-trienoic) acid (Gunstone. 1967). By elongation and
desaturation. these acids provide the 020 and 022 polyunsatura-
ted fatty acids Of animal phospholipids (Gunstone. 1967:
Poukka and Oksanen. 1972).

It has been established that minor amounts of odd
numbered fatty acids. especially of saturated C15 and C17
as well as Pentadec-gtgr9-enoic and heptadec-gtg-9-enoic
acids. and branched chain fatty acids occur in animal fats.
including those from ruminants (Shorland. 1962). Shorland
gt gt. (1957) concluded that hydrogenation Of fatty acids

by rumen microorganisms results in diversification Of the

24

fatty acid composition of the dietary unsaturated fatty
acids. They also demonstrated that linolenic acid may be
saturated to give high levels of stearic acid and of tpgpg
and positional isomers of oleic and linoleic acid not found

elsewhere in natural fats.

The Phospholipid Content in Animal Tissues

Many studies (Hornstein gt g;.. 1961: Watts. 1962:
Kinsella. 1972) have shown that the phospholipids are
integral parts of the cellular membranes and may be pre-
sent in tissues as phospholiproteins. The phospholipids
comprise a relatively constant proportion (< 1%) in most
animal fats and contain a high content of polyenes (Watts.
1962: El—Gharbawi and Dugan. 1965: Hornstein gt g;.. 1967:
Turkki. 1967: O—Keefe gt g;.. 1968: Keller and Kinsella.
1973: Body and Shorland, 1974).

There is broad similarity in the composition of phos-
pholipids in the tissues of a variety of mammals and birds
(Ansell and Hawthorn. 1964: Body gt g1.. 1966). More is
known about the pattern of phospholipid distribution in
the tissues of sheep than in other species. Body gt_gt.
(1966) reported the following pattern of phospholipid dis-
tribution in the total tissues of maternal and fetal sheep:
phosphatidyl choline (PC) - 45%: phosphatidyl ethanolamine
(PE) - 25%: sphingomyelin - 11%: phosphatidyl serine (PS)
- 7%; phosphatidyl inositol (PI) - 4% and all others - 8%.

According to Gunstone (1967). each type of phospholipid

25

tends to have its own characteristic fatty acid composition.
He reported that in animal tissues. PE is notably rich in
polyunsaturated C20 and C22 fatty acids. which are derived
from dietary linoleic and linolenic acids. Body and Shor-
land (1974) reported that the amounts and kinds of PUFAS
recorded in the PE fractions may vary with the level and
ratios of linoleic and linolenic acid in the diet. as well
as by the conditions of the analysis.

Hornstein gt gt. (1961) first reported that 20:4. w6
was the only polyunsaturated components besides 18:2. w6
in the phospholipids from pork and beef muscle. Later.
Hornstein gt gt. (1967) reported that beef muscle phospho-
lipids included 22:6. w3: 22:3. O6 and 22:4. w6 in addi-
tion to 20:4, N6. This discrepancy appears to be the result
of using poorer analytical procedures in the earlier study.
In recent studies. Body and Shorland (1974) have shown that
the level of PUFAS from the PE fraction of the rumen and
abomasum of fetal and maternal sheep ranged from 17-43%
of the total fatty acids. In comparison. the levels Of
PUFAS in PC were 7-25%. and in spingomyelin. 1-4% (Body
and Shorland. 1974). The main PUFA components (Body and
Shorland. 1974) in PE were 20:4. w6 and 22:5. O3. with lesser
amounts of 20:5. w3. 22:6. w3. 18:2. N6 and 18:3, w3. res-

pectively.

26

3gtationship of Fatty Acid Qgppgsition tquutogtggtive
Stability Of Meats

The autoxidative stability of meats depends on the
degree of unsaturation (Ellis gt gt.. 1974: Kimoto gt gt..
1974). Consequently. meats. such as pork and poultry.
which have high levels of PUFAS. are very susceptible to
oxidation in frozen storage (Ellis. 1933: Watts. 1962).
Intracellular lipids contain a higher percentage of phos-
pholipids. have higher PUFA levels than the depot fats
and are believed to be more susceptible to autoxidation

(Govendarajan gt_g;.. 1973).

Changes in Neutral Lipids ang_Phospholtpids Durtpg Frozen
Storage

It is commonly assumed that tissue lipids are quite
stable during freezer storage (Cadwell gt_gt.. 1960:
Keskinel gt gt.. 1964: Witte gt g;.. 1970: Kimoto gt gt..
1974). Several workers (Lea. 1957: Younathan and Watts.
1959. 1960) have suggested that negligible changes occur
in total lipids during frozen storage of raw beef. be-
cause the predominant neutral lipids oxidize slowly com-
pared to the phospholipids. However. recent studies
(Keller and Kinsella. 1973: Kimoto gt_gt.. 1974) indicate
that the oxidation of neutral lipids may be a factor in
the deterioration of beef carcasses during freezer storage.

According to Kimoto gt gt. (1974) microbial growth

does not occur in meats stored below -9°C. They further

27

stated that polar lipids are more stable than the neutral
lipids in frozen storage. In contrast. other investigators
(Sulzbacher and Gaddis. 1968: Keller and Kinsella. 1973)
have suggested that the triglycerides from adipose tissue
are the primary cause Of meat deterioration during freezer
storage.

Muscle phospholipids are believed to be the major
contributors to oxidative deterioration of cooked meats
(Younathan and Watts. 1960: Love and Pearson. 1974) and
of freeze-dried meats (El-Gharbawi and Dugan. 1965: Chipault
and Hawkins. 1971). Lea (1957). Caldwell gt gt. (1960). and
Greene (1971) have reported that breakdown of phospholipids
during frozen storage of raw meats results in rancidity
and browning. However. other researchers (Keskinel gt gt..
1964: Evans gt gl.. 1967: Terrel gt g;.. 1968) have reported
negligible changes in the fatty acids of the phospholipids
in beef during freezer storage. On the other hand. Keller
and Kinsella (1973) have reported major changes in the fatty
acids of phosphatidyl choline (lecithin). more especially
in arachidonic acid.

Lipolysis of phospholipids during freezer storage has
been implicated in oxidative degradation of bovine. fish
and chicken muscle (Awad gt g;.. 1968: Bosund and Ganrot.
1969). The recent findings of McMurry and Magee (1972)
that phospholipases occur in mammalian tissues and may
release fatty acids from phosphoglycerides could lend support

to this view.

28

The 2—thiobarbitpric Agtd_jTBA)_Test as a Measgpe of Meat
Rancidity

Malonaldehyde (MA) and similar substances occur in
foods as decomposition products of oxidizing unsaturated
fatty acids. and in the presence of water. exist mainly
as the non-volatile. bound enolate anion (Kwon and Watts.
1964: Kwon gt gt.. 1965). The reaction of MA with TPA,
which has been a useful index for measuring rancidity in
foods was first reported by Kohn and Liversedge (1944).

According to Sinnhuber gt gt. (1958). the principal
reactant is MA. a water soluble substance formed or re-
leased upon heating the sample in an acid medium. However.
Tarladgis gt gt. (1964) have demonstrated that MA can be
measured without the acid treatment. The red pigment ob-
tained in the reaction occurs as a consequence Of the con—
densation of two moles of TBA with one mole of MA (Sinn-
huber gt gt.. 1958). The intensity Of color is a measure
of MA concentration. which has been organoleptically cor-
related with rancidity (Zipser gt gt.. 1964: Kwon and Watts.
1964: KWon gt gt.. 1965).

According to Sinnhuber gt gt. (1958). the proposed TBA
reaction is as shown on the following page.

Kwon gt gt. (1965) have claimed that the reactions
of MA and 2-thiobarbituric-acid-reactive-substances (TBRS)
in moist foods are similar to the reaction of the pure
compounds. However. Slaslaw and Waravdeckar (1965). from

TLC studies of extracts of irradiated fatty acids claimed

29

sommsonno sme pmmm s2 dma

mo

 

 

 

30

that none of the TBRs was MA. Apparently. both MA and

other aldehydes (especially 2.4-a1kadienals and to some
degree 2-a1kenals) are capable of producing the red pigments
with maximum absorbance at about 530 nM (Marcuse and

Johanssen. 1972).

MA as an Index of Lipid Oxidation ip_FOOg§

The formation of MA as a product of lipid oxidation
is generally accepted as the basis of the TBA test (Mar-
cuse and Johanssen. 1972). Numerous techniques have been
used in applying the TBA test to assay for MA in foods.
A number of investigators (Turner gt gt.. 1954: Yu and
Sinnhuber. 1957) have heated the macerated food directly
with an immiscible solvent. Others have applied the test
to a metaphosphoric or trichloroacetic extract of the food
(Tappel and Zalkin. 1959) or to a distillate from the acidi-
fied food (Sidwell gt gt.. 1955: Tarladgis gt gt.. 1960).

All modifications of the method employ acid-heat treat-
ment of the food (Kwon and Watts. 1964). Where distillation
is employed to separate the MA from other food constituents.
maximum volatilization (even of free preformed MA) would not
be expected at pH values above 3.0 (Kwon and Watts. 1964).
This is because the volatile. hydrogen-bonded ring compound
formed undergoes progressive ionization as the pH increases
from 3.0 to 6.5 (Tarladgis gt gt.. 1960: Kwon and Watts.
1964).

A major feature of the TBA test is the fact that the

31

acid reagent can be applied directly to food lipids without
prior extraction of the fat (Lea. 1962). In order to ob-
tain high correlations between TBA values and rancidity
(Lea. 1962: Kwon and Watts. 1964: Zipser gt gt.. 1964:
Kwon gt gt.. 1965: Pearson. 1968) it is necessary to use
moist foods. especially animal tissues and dairy products
(Patton and Kurtz. 1951).

According to Kwon and Watts (1964). in some cases
lipid oxidation in dehydrated foods may be far advanced with
little or no accumulation of MA. since the MA would be in
the volatile. metal-chelated form. Pearson (1968) pointed
out that the TBA test apparently measures the deterioration
in both the extractable and non-extractable lipids. However.
he further reported that relatively high TBA values may
be found in some fresh samples. and yet in advanced stages
of rancidity. the TBA values may actually fall to zero or
remain constant after reaching a maximum value.
Wills (1964: 1966) Observed that the presence of Fe2+
and Fe3+ in small concentrations markedly increased TBA

3+ readily forms a colored com-

values. Presumably the Fe
plex with an organic compound on heating the oxidized lipid
with TBA (Wills. 1964). She has. therefore. recommended
the addition of EDTA to complex the Fe3+ during the blending
process.

The presence of ascorbic acid in the distillate can

also result in high TBA values (Wills. 1966). It has also

been reported that little or no color is produced

32

by oxidized linoleic or oleic acids. but oxidized linolenic
and arachidonic acids give an intense color reaction
(Wills. 1964: 1966): hence. the TBA values for rancidity

in foods may be somewhat empirical (Lea. 1962).

EXPERIMENTAL

Materials and Methods

Solvents

All solvents. chemicals and reagents were of analytical

grade.

Animals and Rationg

Four groups of 4-day Old Holstein bull calves with four
in each group were used in this experiment. They were
supplied and reared to the completion of the experiment
by the Department of Dairy Science at Michigan State Univ-
ersity.

The calves were fed with colostrum and whole milk during
the first week of life. After 1-week of age. the calves
were fed with milk replacer (filled milk) for 57 days.
at the end of which time they were slaughtered. In groups
1 and 2. the fat was supplied by 15% coconut oil. In
groups 3 and 4. the fat was supplied by 15% corn oil. In
addition. groups 1 and 3 received 500 mg d-d-tocOpheryl
acetate/calf/day. whereas. groups 2 and 4 were unsupple-
mented. The rate of feeding was varied with age as shown

in Table l.

33

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Am
o.m o.m o.o mo:am
m.m m.~ m.m om:om
H.N H.m m.: manna
a.a a.H o.s «anon
m.a m.H m.m mmumm
om.o om.o m.~ wN:N~
mw.o no.0 m.N Hm:ma
m.o m.o o.~ dalm
nVA: cam n masonwv OVAN can H masonwv
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pH a : Add: macs: 5::
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hwn\wamn\com assess ems no mama

 

 

.moeamo Adedosanonxm one on com as“: ooaaam ado choc one ago esnoooo no maoeoa .H edema

35

The filled milks were prepared by Milk Specialties
Company. Dundee. Illinois. Except for the source of fat
(coconut or corn oil). the rations were identical and con-

tained the ingredients shown in Table 2.

Vitamin E (d-a—tocopheryl Acetate
The vitamin E acetate was donated by Eastman Kodak

 

Company of Rochester. New York. The vitamin was dissolved
in hydrogenated vegetable oil containing minimal quantities
of linoleic acid to give 250 mg d-a-tOCOpheryl acetate (w/v)
per m1 of Oil. The calves in groups 1 and 3 were given a
dosage of 2 ml/calf/day of vitamin E-oil. which was thor-
oughly mixed with the milk ration before feeding. The
vitamin E concentrate was made up weekly and stored at

room temperature.

Statistical Treatment

Statistical analysis was calculated using STAT
SERIES developed by the Michigan State University Agri-
cultural Experiment Station and run on Control Data
Corporation (CDC) 6500 computer. Analysis of variance was
carried out for fatty acids. TBA numbers and vitamin
E levels. Standard deviation. correlation and regres-
sion coefficients were calculated using a Cognito 1016
PR programmable electronic printing calculator (Cognito

1016 PR. Smith Corona Merchant. 299 Park Avenue. New
York).

36

Table 2. Composition Of Filled Milk.

 

 

 

Ingredients Percent Composition

Whey - dried 49.2

Non-fat dry milk 25.0

Lactalbuminw-dried (55% protein) 10.0

Oil (coconut or corn oil) 15.0

Calcium carbonate 0.625

MSU vitamin-mineral premix 0.175
Total 100.00

The premix provided the following nutrients per lb of

product:

Vitamin A
Vitamin D3
Riboflavin
Panthotenic acid
Niacin
Vitamin 312
Thiamine
Folic acid

Choline

Magnesium

16.250 I.U.
5.000 I.U.
4 mg
5 mg

18 mg
7 HS
2 mg
1.25 mg

85 mg

141 mg

37
Extraction Of Total Muscle Lipid

The procedure for extraction of total lipid from
muscle tissue was a modification of the technique des-
cribed by Folch gt gt. (1957). After removing all visible
fat and connective tissue. the weighed samples were homo-
genized in a Waring blender and extracted three times with
2:1 (v/t) chloroform-methanol mixture. The weight of the
extracted tissue varied from 60 to 90 g. The extract
and tissue residue were then transferred to a medium
grade sintered glass funnel and filtered under vacuum.

The filtrate was collected in a 500 ml graduated Erlenmeyer
flask. The homogenizer and the residue in the funnel were
washed with an additional volume of chloroform-methanol
and filtered. The final extract was quantitatively trans-
ferred into a 1000 ml separatory funnel and 1Q% by volume
of distilled water was added and thoroughly mixed. The
mixture was allowed to separate into two phases until

the interface was clear. The lower phase was transferred
to a 500 ml volumetric flask and evaporated in a vacuum
Rotavapor-R (Buchi. Switzerland) at 20-30°C. The upper
layer was similarly evaporated. but usually contained a
negligible level of fat.

When the volume of the total lipid extract was re-
duced to 10 to 20 ml. the extract was quantitatively
transferred to a previously tared 100 m1 volumetric flask

by washing witheuiadditional quantity of chloroform-methanol.

38

The final extract was further evaporated until it reached
a constant weight. The weight of the residue (lipid) was

then Obtained by difference.

§gpgpation of Phospholipid gpd_Neutra1 Lipidg

The phospholipid was separated from the total lipid
using the method of Choudhury gt gt. (1960). This method
involves separation on activated silicic acid.in which
neutral lipids are preferentially removed by washing with
chloroform. The phospholipid combines with the activated
silicic acid and is solubilized and extracted with methanol.

A weighed amount Of silicic acid (20-25 g) was activated
for at least 12 hours by drying in a 100°C oven. The lipid
sample was then quantitively transferred to a 125 ml Erlen-
meyer flask containing the activated silicic acid. The
contents were shaken for at least 10 minutes and allowed
to settle. The mixture was then thoroughly stirred and
filtered through a sintered glass funnel under vacuum. The
silicic acid was washed six times with 50 ml portions of
chloroform. The filtrate and washings were combined and
evaporated using the Rotavapor-R as described previously.

The phospholipid fraction was determined by washing the
silicic acid residue with six 50 ml portions of methanol.
The filtrate and washings were combined and evaporated to
a constant weight using the Rotavapor-R. The combined
weight of the phospholipid and neutral lipid was closely
equivalent to the initial level of total lipid. The fat

39

samples were kept in teflon stOppered test tubes and stored
at -l8°C until removed for fatty acid analysis within a

one week period.

Rendertggtof_pepot Fat

Fatty tissues (1-10 g) were transferred to a Thomas
Teflon Pestle Tissue grinder (Thomas Company. Philadelphia.
Pennsylvania) in a steam bath. The fat was ground while
being rendered and extracted with a 2:1 (v/v) chloroform-
methanol mixture. The extract was dried over anhydrous
sodium sulfate. filtered and washed with an additional
volume of chloroform through glass wool into a previously
tared 100 m1 beaker. The rendering and extraction proce-
dure was continued until the residue was completely free
of any lipid material. The solvent was partially evaporated
on a steam bath and the extract was evaporated to a constant

weight in a vacuum oven.

Preparation of Methyl Esters

The technique for preparation of methyl esters
was a modification Of the method described by Morrison
and Smith (1964). A total of 2.5 ml of 14% Boron trifluor-
ide-methanol (Bf3-MEOH) was added to 100-200 mg of lipid
material in a 20 x 150 mm test tube containing 1 ml Of
benzene. The tube was sealed with teflon-lined screw caps
and heated in a steam bath for 40 minutes. After cooling
the sample to room temperature. the esters were extracted

by adding 2 volumes of hexane and then 1 volume Of water.

40

The mixture was shaken vigorously in a Vortex-Genie mixer
until both layers were clear. An aliquot of the upper
layer was transferred to a 5 m1 graduated volumetric flask

and dried with about 0.3 g of anhydrous sodium sulfate.

Analysis of Fatty Acid Compogition of the Triglycgrides
and Phospholipids

ChromatOgraphic analysis of methyl esters was per-

 

formed using a Beckman-GC-4-Gas Chromatograph equipped with
a hydrogen flame detector. The glass column. 6 ft x 2 mm:
(i.d.) was packed with 10% (w/w) diethylene glycol succinate
(DECS) on 100/120 mesh supelcoport (Supelco. Inc.). The
column had been previously cleaned. silanized and packed
under suction. The column oven temperature was 100°C.

the injection port was maintained at 210°C and the detector
at 185°C. The helium carrier gas flow rate was adjusted

to 40 ml/minute or 60 ml/minute. depending on the separa-
tion achieved. The flow rates of hydrogen and oxygen

were 30 ml/minute and 300 ml/minute. respectively. Vary-
ing quantities of sample (0.5-5 n1) were injected.

The emerging peaks were identified by comparing re-
tention times to those of standard mixtures of known fatty
acid methyl esters. Peak areas were calculated by multi-
plying peak height times peak width at half-height and
the percentages of the total fatty acids were determined.

41

Determination of Toc0pherol in Meat anngatty Tissues

The tocOpherol content of the meat and fatty tissues
was determined by the spectroPhotometric method of Erickson
and Dunkley (1964). The procedure involves the extraction
of tocopherol from the tissues using ethanol and hexane.
followed by a separation of the extract from interfering
compounds by silicic acid chromatography. The tOOOpherol
level in the purified extract was determined spectrophotometri-
cally after reacting with 4.7 diphenyl-l-10-phenanthroline.

Duplicate meat samples (3-5.0 g) and fat samples (1-3.0 g)
were homogenized in a Virtis homogenizer (Virtis Research
Equipment. Gardiner. New York) and transferred to a 50 ml
centrifuge tube. Distilled deionized water was added to
bring the volume to 10 ml. A total of 15 ml of absolute
ethanol and 1 m1 of 1 N HCl was added and mixed thoroughly.
The tubes were heated in a water bath for 5 minutes at 60°C
with intermittent mixing. Then 10 m1 of hexane were added
while still warm and the mixtures were shaken for 20 min-
utes in a Vortex-Genie manual shaker (Fisher Scientific
Industries. Massachusetts). The samples were centrifuged
for 5 minutes at 2.000 x G. The upper hexane layer was
removed by pipet and dried over 0.5 g of anhydrous sodium
sulfate.

Removal of interfering substances from the toc0pherol
was accomplished by column chromatography. Glass tubes
(9-10 mm. i.d.) were cut to size and melted to the

apprOpriate dimension with a Bunsen burner. Each glass

42

column was fitted with glass wool and packed with 2 g of
activated silicic acid which had been previously heated

for 12-16 hours at 100°C. The columns were washed with

5 ml of hexane and 5 ml of the extracted tocOpherol ex-
tract were added immediately. The extract was allowed to
drain into the column. after which it was rinsed into the
silic acid by adding 4 m1 of benzene. The eluent was col-
lected in a 10 ml volumetric flask and was made up to volume
by adding an additional 1 ml of benzene.

The eluent was thoroughly mixed and pipetted in 3 ml
volumes into three separate test tubes. In a stepwise
manner. 0.5 ml of 6 x 10'3 M banthOphenanthroline (4.7-
Diphenyl-l-lO-phenanthroline) solution was added and mixed.
This was followed immediately by addition of 0.5 m1 of
1.0 x 10"3 M ferric chloride solution. After allowing 2
minutes for color develOpment. 0.5 m1 of 0.1 M orthophos-
phoric acid was added and mixed thoroughly. The addition
of reagents was carried out in a darkened environment to
prevent photo reduction of ferric ions. Thereafter. ex-
clusion of light was no longer necessary. These reagents
were prepared in absolute ethanol and were stored at 5°C in
an amber bottle. Only freshly prepared ferric chloride
solution was used for the determinations.

The absorbance was determined at 534 nM using a Gil-
ford spectrOphotometer. against a 3 m1 blank of benzene
treated in the same manner as the sample. Tocopherol

content was calculated using the equation:

43

. . _ Absorbance at 534 nM_.
g tocopherol/g llpld - 0.032 (g fatiml in original extract)

TBA Analysis for Lipid Oxigation of Meat and pgpottgat
The steam distillation method of Tarladgis gt gt. (1960)

 

was used to analyze for thiobarbituric reactive material.
The distillation apparatus consisted of a 250 m1 round bottom
flask. which was attached to a Friedrick condensor with a
three-way connecting tube. Electric heating mantles were
used as the source of heat.

A duplicate 10 g sample of meat or fat was homogenized
with 50 ml of distilled. deionized water for 2 minutes in
a Virtis homogenizer at low speed. The homogenate was
transferred quantitatively into a 250 m1 round bottom flask
by washing with 47.5 ml of distilled. deionized water.
The pH of the meat or fat slurry was lowered to 1.5 by
the addition of 2.5 ml of 4 N HCl. Boiling chips were added
and a small amount of Dow antifoam was sprayed into the
flask to prevent foaming. The slurry was steam distilled
using the highest setting on a Powerstat (The Superior
Electric Company. Bristol. Connecticut). until 50 ml of dis-
tillate was collected.

The distillate was mixed and 5 ml were transferred to
a 50 ml test tube. Then 5 m1 of TBA reagent (0.02 M 2-
thiobarbituric acid in 90% glacial acetic acid) were added.
The tubes were stOppered and the contents mixed. The tubes
were heated in a boiling water bath for 35 minutes. After

cooling to room temperature for 10 minutes. absorbance was

44

read at 538 nM using a Gilford spectrophotometer against a
blank containing only distilled. deionized water and TBA
reagent. Absorbance readings were multiplied by a factor
of 7.8 (Tarladgis gt gt.. 1960). TBA values are expressed
as mg malonaldehyde per 1000 g sample.

RESULTS AND DISCUSSION

Analygtg of Egpeptmeptat nigtg

The filled milks were stored at room temperature and
analyzed at the termination of the feeding experiment. Analy-
sis was carried out for vitamin E level. fat and dry matter
content. The ration containing coconut oil contained 26 ug/g
of vitamin E. 80% dry matter and Z5 fat. while the corn Oil
ration had a vitamin E level of 322.6 ng/g. a dry matter con-
tent of 70% and 9% fat.

The diets were also analyzed for fatty acid composition
as shown in Table 3. The data show that the diet with coconut
oil contained appreciable quantities of saturated C8:0 to
014:0 fatty acids. whereas. the corn oil ration contained low
levels of the saturated fatty acids but high levels of 018

unsaturated fatty acids.

‘ .‘ ‘111

 

Liveweights were taken shortly before slaughter. Car-
cass weight was determined after envisceration. both before
and after removal of the hide. The carcass was chilled for
a period of 24 hours at about 33°F. Carcass yield was
determined by expressing the carcass weight (cold) as a

prOportion of the liveweight. Table 4 shows the mean values

45

46

 

 

.psoouom Pawns; mm so>wm mum mosam>Am

 

mm.mm

0.00H

o~.o no.0n oe.mm mn.n oe.o ma.¢a

o sm.~ em.s Ho.n o am.~a

$0.0 mm.o o o sowpmm
HMO

snoo

ma.sa os.oo ea.m o~.o coaemm
Hao

essoooo

 

HMPOB

llllll‘ll J1

médo «.mHo .136 0.36 .136 oéao

0.36 0.20 cacao 0.00 mag—Mom

 

m .hnnmawopmaougo Owsdwn

use an oosflanopoa meowa Hmvsosfluonxm osa one no Cow Hmogaoo Odo< hppmm one .m «Home

a?

for these parameters.

Average liveweight ranged from 139 1b. for.group 3 to
162 lb. for group 1. There was great variability in live-
weight. and hence. in carcass weight and yield. The varia-
tion in liveweight was probably due to the fact that some
of the animals were very unthrifty. especially on the corn
oil diet. Seven of the original calves died during the
early phases of the experiment and had to be replaced. Thus.
some of the animals were fed at different times. The resulting
differences in environmental factors may have affected growth
rate. Unsatisfactory weight gains. poor physical condition
and deaths occurring in calves fed rations containing highly
unsaturated vegetable fat have been reported by Adams gt gt.
(1959)-

The lowest carcass yield amounted to an average of 53.30%
for group 3. which was significantly less than any of the
other group. Groups 1.32 and 4 had carcass yields of 63.03.
58.60 and 61.13%. respectively. with none of these groups
varying significantly from each other.

th Contgnt of thg Meat gpd Qgpgt Fat
The total fat content of the Lopgtagimug dorgi was.

on average. below 2% and is shown in Table 5. There was a
tremendous variation in group 3. probably as a result of varia-
bility in growth rate. In contrast to mature beef. the flesh
of veal is low in fat (Moulton and Lewis. 1940). The data

of Post £3.31- (1972) as quoted by Wrenn gt gt. (1973) indicate

l 1] Ill 1‘

 

 

48

 

. ensues noegwsdam :
ooa x P: we: mmmohmo goo: caofin mmmOnmvo
.nmo ooamfin
.mhzos ma 90% some “moan
H.m H nH.Ho m.am H om.mm 0.5m H m.mea s m caempa> -
Hao anon .e
m.o_H om.mm o.mm H mo.ea H.m¢_u m.mna a m casdpa> +
see cnoo .m
m.n H oo.mm o.eH H me.so ~.oH H m.ooa e m daamea> -
Mao Pssoooo .N
m.m H mo.~o ~.H~ H m.mm a.o~ H. mos e m enamea> +
Hwo pssoooo .H
Aav Andy Anav mamaac< endogenous
owoaoa» ov eased: d noeewsmam em no nooasz
m momma mmmOHMU Ngmfioz o>wq
sues coo: sums

.mo>amo Hmo> mo pawn» mmmonmo Osm .Pzwfimz mmwonmc .Psmfloz o>dq .3 manna

49

that the intramuscular fat content ranges from 0.67% for
6-8 week old calves to 4.77% in mature cattle. Thus. the
data from the present study are consistent with the values
in the literature.

It is known that intramuscular lipid increases with
age and weight. but the phospholipid fraction per gram of
muscle appears to be relatively constant (Link gt gt.. 1967).
Table 5 shows the level of phospholipids. which was relatively
constant and generally below 1%. This is consistent with
the observations of other workers (Watts. 1962: Hornstein
gt gt.. 1967: Turkki. 1967: Body and Shorland. 1974). who
found but little variation in the composition of the phos-
pholipids in muscle.

The levels of depot fat (kidney and omental) are also
shown in Table 5. There was considerable variation in the
level of fat deposited (24-75%). with the minimum level for
the calves on the corn Oil diet. It is well known that the
level of adipose tissue increases with age and weight. and
that it is influenced by species. diet. environment. sex

and other factors (Deuel. 1955).

Fatt id O o it on

The average fatty acid composition and the standard
deviations for kidney fat. omental fat and meat lipids from
calves on the various diets are shown in Tables 6. 7. 8 and 9.
respectively.

The prOportion Of linoleic acid (018:2) was essentially”Uuasame

 

.psooson Panama mm so>Hw Ohm mosam>nm

 

50

 

m.a~ H mm.:: m.oa H ma.ma no.0 H no.0 o~.o H ma.a m neeoea> -
HHO CHOU 3:
n.mm_H m~.a~ m.mm H e.m~ ao.o_H ma.o 0H.H.H on.a m caemea> +
HHO choc .m
a.n H mm.mo n.sH H mo.oo mo.o H ca.o om.o H ::.H .m :Hsmpa> :
HHO vssoooo .m
~.~ H nn.oo H.ma H 53.:a oo.o H em.o mn.o H oo.a a deadea> +
HHO Pssoooo .H
van emu hosOHM Hmnoo Hmaoo mesoapmone
ampsoso 9mm msaHmmesoq mo msaHmmesoA
emu Hmeoa ecoezoo eanaaoenmonm no nee pen
Hmeoa Hmpoa zoo: Hmpoa duos

mv.emm Hmecoso odd pom segues .Hmnoo ousammamcoq no edoezoo was .m manna

51

in the kidney, omental and meat triglycerides from the calves
in groups 3 and 4. and ranged from approximately 26 to 30%
as shown in Tables 6. 7 and 8. respectively. Ellis gt gt.
(1974) have reported similar observations. although the level
of 018:2 was only 12 to 15% in their study of calves fed diets
high in linoleic acid. The values obtained in the present
investigation are also higher than those reported by Wrenn
gt gt. (1973) for calves fed milk fat high in linoleic acid.

The levels of 018:2 in the kidney. omental and meat tri-
glycerides from calves on treatments 1 and 2 ranged from 4 to
8% as shown in Tables 6. 7 and 8. respectively. Ellis gt gt..
(1974) reported a level of about 5% linoleic acid in a commer-
cial sample of veal. I

Vitamin.E supplementation appeared to have a slight
influence on the amount of 018:2 in the various tissues. In-
creased amounts of linoleic acid were found in nearly all the
lipid fractions of all tissues from vitamin E supplemented
animals. However. the differences in the level of 018:2
between vitamin E enriched and nonsupplemented diets were not
statistically significant. Poukka and Oksanen (1972) have
reported decreased levels of 018:2 in some tissues of vitamin
E deficient calves. In the present study. the rations not
supplemented with additional vitamin E were naturally rich
in vitamin E. This is borne out by the fact that the coconut
Oil and corn Oil rations contained 26.0 and 322.6 ug/g of
vitamin E. respectively.

Appendices l. 2. 3 and 4 show representative chromatograms

52

from different animals in groups 1. 2. 3 and 4. respectively.
There was no significant difference in the fatty acid composi-
tion of animal tissues from treatments 1 and 2 and of treatments
3 and 4. However. there were some significant differences
between the coconut Oil (groups 1 and 2) and corn Oil diets
(groups 3 and 4). The level of oleic acid (018:1) was about

30% in the tissues Of calves on corn oil diets. while the
average value of the coconut oil treatments was 23% (Tables

6. 7 and 8).

The amount of palmitic acid (016:0) in the depot fat
and in the neutral lipids of meat from calves on the coconut
oil diet was about 30% (Tables 6. 7 and 8). However. the
amount of 016:0 in the phospholipids from meat of calves on
the coconut oil ration amounted to 17% (Table 9). In treat-
ments 3 and 4 (corn Oil rations). the level of palmitic acid
in the depot fats was 20% (Tables 6 and 7). whereas. it was
17% in the meat neutral lipids and 13% in the meat phospho-
lipids (Tables 8 and 9).

The depot fats and meat neutral lipids contained on the
average 10% stearic acid (018:0) for both the coconut and
corn oil diets (Tables 6. 7 and 8). Likewise. the phospho-
lipid from the meat contained a mean value of 13% stearic
acid on both rations. The kind of oil did not seem to affect
the level of stearic acid in the tissues. However. vitamin
E supplementation slightly increased the level of stearic
acid in the tissues of animals fed both diets.

The high level of lauric acid (012:0) in the coconut

53

oil ration (Table 3) was not reflected by its level in the
omental. kidney and meat triglycerides from calves fed this
ration (groups 1 and 2). The level of lauric acid in these
tissues ranged from 5 to 8% (Tables 6. 7 and 8). On the

other hand. the level of myristic acid (014:0) in the depot
fats and meat triglycerides from calves in groups 1 and 2
greatly increased. ranging from approximately 18 to 23% (Tables
6. 7 andi3). However. Vitamin.E supplementation of the coco-
nut oil ration decreased the amounts of lauric and myristic
acids in the depot fats and meat triglycerides from calves

on this ration (group 1).

Fatty Acid Composition of the Phospholipids
The fatty acid composition of the phospholipids from

 

the longissimus ggpgt muscle are shown in Table 9. The level
of 018:2 comprised slightly over 40% of the fatty acids in the
phospholipids from calves on the corn oil diets. Kimoto gt gt.
(1974) reported a slightly lower level of 018:2 in the meat
phospholipids from calves fed safflower oil. The level of
018:2 in the phospholipids of the meat from groups 1 and 2
(coconut oil diets) was approximately 27%. This is in good
agreement with the data for commercial veal samples reported
by Kimoto 2£.§l- (1974).

The levels of 016:1. 018:1 and 020:3 in the phospholipids
were significantly higher for meat from calves on coconut oil
diets than for those on corn Oil rations (Table 9). How-

ever. the level of 020:4 was not affected by the differences

54

 

.Psoonoa Hanos mm so>Hw mam mosam>

fl

 

 

 

 

Am
am.~.H om.o H om.o H. Hm.o.H o.m H. mm.o H. oH.H H m~.o_H m enamea>
no.om mm.om we.HH mm.H mo.mH na.o ~a.m mH.H - Hao cnoo .:
om.n H. HH.¢ H HH.:_H mw.o_H oo.~_H os.H H Hm.o H. a :Hempa>
an.m~ em.~n mm.ma mA.H mo.mH o.o Ho.m mH.H + HHo anon .m
~m.n_H oo.~ H HA.H_H mm.o H. oH.H H. oH.o H om.H.H a.« H. m :Hampa> -
oo.: no.m~ mo.m oa.H ms.o~ om.o wn.sm He.n Hao escoooo .N
oa.n_H mm.n H mm.o H mm.o H o.m_H Ho.o H ~.m_H o.H_H m :Hsmea> +
oo.a Ha.m~ m:.HH NA.H sm.on mo.H H~.mH o~.m Hao eseoooo .H
«.mHo H.mHo o.mHo H.eao o.oao H.3Ho o.:Ho o.~Ho ecoaemona
noHeHoonsoo eaoe seems ado: .o manna

mv.emm Aeneas no

55

 

.Psoonom inHos mm so>Hw mum mosHm>Am

 

 

 

my

H0.¢.H :m.H H on. «H mm.0 H. oa.m H as. 0_H a~.m_H mm.0 H. m :HemeH>
m0.0m 0:.0m 0. 0 :0.H m0.m~ :0. 0 0H.a 0~.H : HHo dnoo .0

am.m H 0:.3 H 00. H H mm.0 H ~0.~_H NH. 0.H nn.0_H em.0 H. m eHsmpH>
am.0~ 00.0m on. «H 0~.H He.0~ AH. 0 m~.m 00.0 + HH0 0000 .m

0m.~_H 0H.s H n~.~ H em.0.H 0m.: H no. 0_H so.m H 03.0 H. m :HameH> -
0:.0 eH.0~ a:.0 0~.~ 00.Hm 0:. H 0m.m~ 0m.0 HHo encoooo .N

no.H H ne.~ H m0.0_H 00.0.H ~0.m H. on. 0.H 00.~ H 0m.H.H m :HampH> +
00.0 -.H~ no.0 mm.H 00.Hn 0n. H 0H.m~ No.0 HHo escoooo .H

~.0H0 H.0H0 0.0H0 HioHo 0.0H0 H.3H0 0.:H0 01~H0 edosemona
.pmm Hdecoso Ho :oHeHmonsoo 0Ho¢ spews duos .A oHome

56

 

.psoonom panos mm so>Hw one mosH0>

 

 

 

Am
00 H 00.0 0 0 0 0.0H0
00.0 H 00.00 00.0 H 00.00 00.0 H 00.0 00.0 H 0.0 0003
0H.0 H 00.00 00.0 H 00.00 00.0 H H000 00.0 H 00.00 H00H0
00.0 H 00.0H 00.0 H 00.0H H0.H H 00.0 00.H H 0.0 008
00.0 H 00.0 00.0 H 00.H 00.0 H 00.0 00.0 H 00.0 H.0H0
00.0 H 00.2 00.0 H 00.0H 00.0 H 00.00 00.H H 00.00 0.03
00.0 H 00.H 00.0 H 0H0 0 0 . 0003
H0.0 H 00.0 00.0 H 0H.0 0H.0 H 00.H 00.0 H 00.H H.0H0
00.0 H 00.0 00.H H 00.0 00.0 H 00.00 00.0 H 00.0H 0.03
00.0 H 00.0 3.0 H 00.0 00.H H 00.0 00.H H 00.0 0.03
00.H H H00 0.00 H 00.H 00.0 H 00.0 0 0008
0 game; m flame; 0 £500; on 0 find»: + 0080 030.0
on .HHO £000 + HHO :noo HHO Pssoooo HHO pssoooo
0.. n .0. .H.
psOstOue

 

 

.Hmnon msSHmmesoq :H moowsoohHu mo soHPHmomsoo OHo< hppmm sum: .0 OHpme

a

57

in the rations. Kimoto gt gt. (1974) have also presented
data showing that differences in dietary fat did not affect
the content of 020:4 in the phospholipids.

Tables 10 and 11 summarize the fatty acid composition of
kidney. omental and meat triglycerides including meat phos-
pholipids. The data show that there was a significantly
higher level of saturated fatty acids (60 to 70%) in the
fatty tissues and meat triglycerides from calves fed coconut
oil rations than those fed corn oil rations (30 to 40%).

The level of saturated fatty acids in the phospholipids was
about 33% and was not influenced by the different rations.

The amount of monoenoic fatty acids ranged from about
23 to 34% in the kidney. omental and meat neutral lipids
from calves in groups 1. 2. 3 and 4. The distribution of
monoenoic acids in the fatty tissues and meat triglycerides
from calves in the various treatments seemed random. In
the phospholipids. however. meat from the calves in groups
1 and 2 (coconut oil) contained a higher level of monoenoic
acid than meat from calves on the corn oil rations (groups
3 and 4).

There was a significantly higher level of dienoic
acid in the fatty tissues and meat neutral lipids from calves
on treatments 3 and 4 (about 28%) than for those on treat-
ments 1 and 2 (about 5%). In the phospholipids. the amount
of dienoic acid greatly increased to about 40% in the meat
from calves fed corn oil rations. while the level was about

27% in the meat tissues of calves fed the coconut Oil diet.

:11

Treatment

NI

HI

 

 

 

Table 9. Mean Fatty Acid Composition of Phospholipids of Longissimus Dorsi.(a

Corn oil. no
Vitamin.E

Corn oil +
Vitamin E

Coconut oil
+ Vitamin E no Vitamin E

Coconut oil

Fatty Acids

 

58

30““
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0.36 g; 0.27
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C1631

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a)Values are given as weight percent.

 

 

59

 

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60

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61

The level of polyenoic acid was about 9% in the phospho-
lipids of the meat and was the same for all treatments.
Representative meat samples from groups 3 and 4 were
analyzed for possible changes in the fatty acid composition
at 6 months of storage. No changes in the fatty acid com-
position of neutral and meat phospholipids were found to

occur as a result of freezer storage.

The TonQherol Content in the Meat and Fagty Tiseues

Influence of Storage Time en the Stability of Vitamin E in
Longiseimue gorsi.

The levels of tocOpherol in the meat were determined
for fresh samples and again after 1 and 3 months of storage
as shown in Table 12. In the coconut oil fed animals.
Vitamin.E supplementation did not greatly improve the amount
in the meat tissues. At the end of 1 month. the levels of
Vitamin.E in both groups 1 and 2 had declined to about 78%,
but at the end of 3 months. the levels in group 2 (coconut
oil - vitamin.E) was only Shfl of the initial value. whereas.
there was little change in group 1 (coconut oil + vitamin
E).

The initial levels of vitamin E in treatments 3 and 4
(corn oil diets) were 5.19 and 3.44 ug/g of tissue (Table
12), respectively. The level of vitamin E at the end of
1 month represented about 70% of the initial level for each
treatment, but at the end of 3 months the levels had declined

62

to about #2 and 25%. respectively. Thus. supplementation
of corn oil with Vitamin.E influenced the level and stability
of vitamin E in the meat.

Figure 1 shows the effect of storage time on the stability
of vitamin E in the meat. The rate of decline in the meat
from calves fed coconut oil diets + vitamin E (group 1) was
very gradual (b =-O.27). whereas. the decline was rather
sharp (b =-O.97) in the meat from calves fed corn oil diets
+ vitamin E (group 3). Thus. both storage and the type of
oil in the diet significantly (P < 0.01) influenced vitamin
E stability in the tissues.

Adams et el. (1959) have reported that aging of meat
results in the loss of tocOpherol. The greater loss of
vitamin E in meat from the corn oil fed calves supports
the contention that tocopherols are very unstable in storage.
especially in products containing highly unsaturated fatty
acids. which is in agreement with reports by.1ips (19u7.
Keating e3 e1. (1965). and Witting (1975). A significant
interaction (P < 0.05) between diets and length of storage
indicates that the stability of vitamin E in the meat behaved
differently according to the kind of oil used. Thus. vitamin
E was more stable in meat from calves fed coconut oil.

The low level of tocopherol obtained in longiesimus
gege; muscle is in agreement with the values reported by
Kimoto et‘el. (197a). They found u.u and 6.6 ug/g of
tissue of vitamin E in meat from calves fed safflower oil

supplemented with #86 mg of a-tocopheryl acetate for 10

63

 

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35 H 36 3.0 + Sta. EH + Sam Tm H mm.~ mficos m
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3.0 H .36 3.m H 31m To H min Em H $6 3::
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an cmoCozHMsH mm Hmnoa mzawmmflmsoa CH mao>oq Am GHEmHH>V Houonnoooe smmz .NH magma

Figure 1.

64

 

 

Mean vitamin.E (ug tocopherol/g lipid)

 

 

o 1 2 3

Storage Time in Months

Changes in tocOpherol level of longissimus dorsi
during freezer storage. group 1 = coconut oil +
vitamin B; group 2 = coconut oil - vitamin.E;
group 3 = corn oil + vitamin E; group 4 = corn oil
" Vitamin E.

65

weeks and then fed unprotected and protected oil for another

8 weeks. respectively.

Toc0pherol Content of Kigney Fat

The tocOpherol level in the kidney fat was monitored
over a period of 6 months freezer storage as shown in Table
13. The initial levels were 43.68. 19.09. 16.43 and 18.30
u g/lipid for groups 1. 2. 3 and 4. respectively. Supple—
mental vitamin E feeding markedly increased its level in
the fatty tissues of the coconut oil fed group. At any
.given storage time. there was a significant (P < .05)
difference in the tocopherol content between groups 1 and 2;
however. groups 2. 3 and 4 did not differ significantly.

Toc0pherol supplementation of the corn oil diet (group
3) did not improve retention (Table 13). Three of the four
calves in this treatment were unthrifty. and had unsatis-
factory growth rates (Appendix Table l) . The fat content of
perinephric fatty tissues for this group was only about 24%
(Table 5) compared to 75 and 68% for groups 1 and 2 (coconut
oil). respectively. The low level of vitamin E retained by
treatment 3 may be due to the low level of fat in the
tissues since tocOpherol is a fat soluble vitamin. Moreover.
some of these calves had diarrhea which may have caused a
marked decrease of vitamin E in the blood plasma. Thomas
and Okamoto (1955) have reported that diarrhea causes the
loss of vitamin.E from the plasma. The variability in this

group is illustrated by calf No. 371. which had a slaughter

66

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l

 

 

 

 

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6?

weight of 210 lb. a high fat content and a high initial
tocopherol level of 34 ug/g of tissue.

The rate of vitamin.E loss in kidney fat with storage was
generally similar for all treatments as shown in Figure 2.

At 1 month of storage. the toc0pherol retained was over 75%
of the initial value for all treatments. At 3 months. the
level retained was about 60% for all treatments. but at

6 months. the level varied between 26 and 43% for all treat-
ments. A faster and significantly greater rate of decline
(b =-4.9) was observed for treatment 1 than for treatment

3 (b =-l.88). Analysis of variance of the factors that in-
fluence vitamin.E stability in kidney fat indicated a highly
significant storage effect (P‘< 0.01). The differences

in vitamin E retention due to the type of oil in the diet
were also statistically significant (P <0.05). There were
also significant (P < 0.05) interactions between vitamin x
oil. and of oil x vitamin x storage. which indicated the
effects were not always in the same direction.

The levels of toc0pherol found in kidney fat from calves
on treatment 1 are similar to values reported by Ellis e3,el.
(1974). who fed calves safflower oil supplemented with
vitamin.E for a period of 10 weeks followed by feeding a

protected safflower oil diet for another 8 weeks.

Toc0phere;;Content of Omente; Fat
In Table 14. the stability of tocOpherol in omental fat

during 6 months freezer storage is shown. The initial levels

68

 

 

 

 

50
:8
or!
.3! 40 ..
._.
no
>
o
u
2 30
8. P
O
O
.p
3? group 1
an 20
a
"s3
is group 2
§ 10 group 4
g group 3

O W

0 l 3 6

Storage Time in Months (-18°C)

Figure 2. Changes in tocOpherol level of kidney fat.
group 1 = coconut oil + vitamin.E; group 2 =
coconut oil - vitamin.E; group 3 = corn oil +
vitamin.E; group 4 = corn oil - vitamin.E.

69

were 41.09. 14.95. 22.96 and 23.46 ug/g of tissue for treat-
ments 1. 2. 3 and 4. respectively. As in the case of kidney
fat. vitamin E supplementation of the coconut oil diet
significantly (P < 0.01) increased its level in omental
fat. There was a significant (P < 0.05) interaction between
vitamin.E supplementation and the type of oil in the diet.
Supplemental vitamin E in the corn oil diets did not sig-
nificantly influence the content of tissue vitamin E.
probably due to the environmental factors that have been
previously discussed.
Figure 3 shows the decline in vitamin.E levels in
omental fat during storage. As with kidney fat. there was
a significant decline during storage (P < 0.01). The rate
of vitamin E disappearance in the omental fat of group 1
was much slower (b =-2.7) than that of kidney fat (b =-4.9).
The regression coefficient for the disappearance of vitamin
E in group 3 (corn oil diet) was b =-l.95. 'At the end of
1 month's storage. the levels of vitamin E were from 72 to 87%
of the initial value. At 3 months. the levels were from 62
to 74% and at 6 months. residual levels of vitamin.E were
58. 51. 46 and 47% for treatments 1. 2. 3 and 4. respectively.
The data obtained in this study indicate that the
initial levels of tocopherol in kidney and omental fat
were essentially similar on the same treatment. Ellis et,el.
(1974) did not report any significant differences in the
tocOpherol level in the kidney and omental fats of calves.

In the present study. however. there was a significantly

7o

 

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71

 

 

 

 

50.-
E
‘m 40
.H
r4
3’
'3
u
'2 30 _ group 1
p.
o
o
o
.p
to
3
m 2° group 3
.fi \“.-" ‘ group 4
5 ' -
25 group 2
:> 10 . .
a O
o
S

O l —L ’#

o 1 3 6

Storage Time in Months (-18°C)

Figure 3. Changes in tocopherol level of omental fat.
group 1 = coconut oil + vitamin E; group 2 =
coconut oil - vitamin.E; group 3 = corn oil
+ vitamin E; group 4 = corn oil - vitamin E.

73

stability. it is important to be able to predict the
stability of toc0pherol in frozen animal tissues. This is
more important as a result of the ever-growing intercontin-
ental trade in frozen meat and dairy products.

The relationship between length of storage and the
level of vitamin E follows a simple linear regression equa-

tion:
y = a + bx

where y = dependent variable. a = intercept. b = slope.
and x = independent variable.

This regression equation can be used to predict storage
stability of vitamin E. Thus. it is possible to predict
with some degree of confidence the corresponding mean of
y. for a given value of x. We can also predict what a
single observed value of y would be for a given x. but with
less confidence. Hence. the point estimate for either the

mean or a single value is:

>

y = a + bxo

A
where y is the predicted value corresponding to a given

value xo. and xo = storage time.

 

A A ‘2
The standard error of y (Sey) = : Mse (1/n + 159—3531—)
x

where: Mse = standard error of mean. n = number of treat-
ments. x0 = storage time. and i'- mean of treatment.

The standard error for a given value depends upon xo.

7n

Lipid Oxidation in Animal Tissues

Lipid ggigation in Meat

The data for TBA numbers of longissimus gege; muscle
are shown in Table 15. The relationships among the treat-
ment groups are illustrated in Figure 4. Up to 6 months
of freezer storage. all treatments were stable since the
maximum TBA number was below 0.3. According to Watts (1962).
the threshold for rancidity detection in meats occurs at a
TBA value between 1.0 and 2.0.

There were.however. differences in the relative rates
of fat oxidation among the treatments (Figure 4). Analysis
of variance indicated a highly significant (P < 0.01)
effect of vitamin E on the rate of oxidation in meat. Storage
time also had a highly significant (P < 0.01) effect on
the rate of lipid oxidation. However. differences in the
type of oil did not seem to have any significant effect
on the stability of the meat as determined by TBA values.

One major point of interest is that after 6 months of
freezer storage. oxidation was still below the induction
stage (Figure 4) as indicated by the low regression coeffi-

cient.

Lipid Oxidation in Depot Fats

TBA values for lipid oxidation in kidney and omental
fats are shown in Table 16. The relative rates of lipid
oxidation among the treatment groups are illustrated in

Figuresfiand 6. respectively. Analysis of variance

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76

 

 

 

 

0'3 -

I}
m
0)
8
ho
Io
c:
o»
04
73 group 4
t: 0.2 l.
>.
.0
8
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C - M
o
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m
8
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" group 1
u
d)
g 0.1
2:
'd
a:
6+
5
.2

0 W

0 l 3 6

Storage Time in Months (~18°C)

Figure 4. Changes in TBA numbers of longissimus dorsi
as influenced by length of freezer storage.
group 1 = coconut oil + vitamin E; group 2 =
coconut oil - vitamin E; group 3 = corn oil +
vitamin.E; group 4 = corn oil - vitamin E.

77

indicated a higher rate of lipid oxidation in kidney fat
than in omental fat. In addition. the type of oil used in
the ration also significantly influenced (P < 0.01) the
rate of lipid peroxidation in kidney fat (Table 16). The
rate of oxidation of omental fat was influenced less by
the type of oil in the diet than was kidney fat.

The rapid disappearance of vitamin E from the kidney
fat was apparent and is related to increased oxidation. On
the other hand. vitamin E was more stable in omental fat during
storage. and greatly improved its stability. There was also
a strong interaction (P < 0.05) between the type of oil in
the diet and the level of vitamin E on the rate of oxida-
tion in kidney fat. with the corn oil without added vitamin
E being oxidized most rapidly.

There were no significant differences in the levels of
PUFAS between the kidney and omental fat (Tables 6 and 7).
Thus. the overriding factor influencing the rate of oxida-
tion seems to be the level of available vitamin E. This
correlates with a similar report by Lea (1953) showing
that chicken fat was more stable than turkey fat. although
both fats have similar fatty acids. The difference has
been attributed to variations in tocOpherol content. Ellis
et,el. (1974) have reported that the oxidative stability
of rendered depot fats was inversely related to the 018:2
content if the tocopherol levels were similar. The data
from the present study essentially confirm the results of

Ellis e1 e1. (1974). Hence. adipose tissue may not be

78

 

 

 

 

I}

w

‘H

h” 2.0 ..

O

O

O

H

\

0

3.

:33 1.5

H

«I

S:

O

H

3 group 4

ho 1.0 .

5

54

m

,0

S

:3

z 0.5 group 3

.¢

m

9' group 2 4-9

0

S '-——--———r ' group 1
0 '~’ 1 L
o 1 3 6

Storage Time in Months

Figure 5. Changes in TBA numbess of kidney fat during
freezer storage (-18 C). group 1 = coconut oil
+ vitamin E; group 2 = coconut oil - vitamin E;
group 3 = corn oil + vitamin E; group 4 = corn
oil - vitamin e.

79

0.5,

00“ p

0.31.

group (4' /

group 3
group 2

 

group 1

 

Mean TBA Number (mg malonaldehyde/1000 g fat)

0 l ' 3 6

Storage Time in Months

Figure 6. Changes in TBA numbers of omental fat during
freezer storage (~1800). group 1 = coconut oil
+ vitamin E; group 2 = coconut oil - vitamin.E;
group 3 = corn oil + vitamin E; group 4 = corn
oil - vitamin E.

80

 

 

 

 

 

 

 

 

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81

homogeneous in bovine animals as shown by the differing
rates of lipid oxidation in kidney and omental tissues.
Ingle e3 e1. (1972 a. b) have reported that the internal
adipose tissues (kidney and omental) were most active in
younger animals (lambs and calves) and possess considerably
greater activity than that of the subcutaneous depots.
However. they also reported that lipogenesis and lipolysis
are more active in the perinephric than in omental fat.
which is in agreement with the results from the present

study.

SUMMARY AND CONCLUSIONS

It was found that young calves selectively deposit dietary
fats in the tissues without significant changes in the fatty
acid profile. Coconut oil markedly increased the levels of
myristic and palmitic acids as well as the level of saturated
fatty acids in the depot and tissue lipids. Corn oil increased
the level of polyunsaturated fatty acids (PUFAS). especially
the level of linoleic acid. in the depot fats. meat trigly-
cerides and in the meat phospholipids. The level of arachi-
donic acid in the meat phospholipids was fairly constant on
both diets.

Supplementation of coconut oil diets with vitamin.E
significantly elevated its level in the depot fats. but had
less effect upon the meat lipids. Vitamin E supplementation
of the corn oil ration did not significantly improve the
level retained in the depot fats or meat lipids. Vitamin E
declined steadily during frozen storage. The rate of vitamin
E loss in storage was most rapid in kidney fat followed by
the meat lipids. and omental fat. in that order. Losses of
about one-third of the tocopherols occurred in the depot
fats within 3 months of storage at -18°C. Thereafter. a

rapid rate of decline was observed. especially in the kidney

82

83

fat.

By 6 months storage. the TBA values had increased to
the threshold level for rancidity in the kidney fat from
calves fed corn oil without supplemental vitamin E. However.
TBA values were below threshold levels in the omental fat
and meat tissues. regardless of whether they were derived from
calves fed vitamin E or not. The poor stability of kidney
fat is theorized to be related to its faster lipid turnover
rate and a higher rate of metabolic activity.‘

Results indicate that the fatty acid composition of
the ration can alter the fatty acid profile in veal tissues.
The data also show that dietary vitamin.E and saturated fatty

acids contribute to meat stability during frozen storage.

B IBLIOGRAPHY

BIBLIOGRAPHY

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86

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APPENDIX

 

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100

 

 

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0000.0 00 0.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 000.0 0000.0 0.00 0.00 000
000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
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0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
0000.0 0000.0 0000.0 0000.0 0000.0 0.00 0.00 000
00000000230050 0 Amy Amv 3033 000 0.0.0 Amy 03 magnum 0080000

0000000000000 0000000000000 000 0 00 000000 000000 00 000000 0000
00 000003 00 000000 00000 000

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101

 

 

 

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102

 

 

 

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103

 

 

 

 

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105

TBA Values for Kidney and Omental Fat

Appendix Table 7.
Tissues (mg/1000 g tissue).

 

 

TBA Values at
6—Months of Storage

TBA Values at
O—Month of Storage

 

Calf Kidney Omental Kidney Omental
Number Fat Fat Fat Fat
Coconut oil +
Vitamin E
350 0.00 0.00 0.23 -—--
367 0.00 0.10 0.18 0.27
373 0.00 0.05 0.20 0.13
050 0.00 0.00 ---- ---—
Coconut oil, no
Vitamin E
355 0.06 0.06 0.20 0.19
368 0.16 0.16 0.02 0.37
369 0.08 0.13 0.27 0.37
Corn oil +
Vitamin E
302 0.05 0.00 ---- ----
309 0.00 0.00 ---- 0-39
351 0.00 0.00 0.30 0.32
371 0.00 0.09 1.16 0.28
Corn oil. no
Vitamin E
3&8 0004 0000 -""""‘ ""'-
360 0.13 0.12 1.17 0.30
365 0.00 0.09 1.83 0.39
372 0.00 0.09 1.77 0.02

 

106

 

 

 

 

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107

 

 

 

 

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109

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111

Appendix Figure l. #350 Kidney fat (group 1)

Peak No.
l = 012:0
2 = 014:0
3 = 014:1
4 = C1630
5 = C16:1
6 = 018:0
7 = 018:1
8 r 018:2

Instrument: Beckman GC-u chromatograph
Detector: Hydro en flame

Column: Glass ( ft x 2 mm i.d.)
Carrier gas flow rate: 40 ml/minute
Column temperature: 100°C

Injection temperature: 210°C

Oven temperature: 185°C 3
Sensitivity (attenuation) of 5 x 10

 

 

 

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Minutes

113

Appendix Figure 2. #368 Longissimus dorsi (group 2)

Peak No.
l = ClO:0
2 = C12:0
3 = 010:0
4 = 010:1
5 = C16:O
6 = 016:1
7 = C18:O
8 = 018:1
9 = 018:2

Instrument: Beckman GC-h chromatograph
Detector: Hydro en flame

Column: Glass ( ft x 2 mm i.d.)
Carrier gas flow rate: 40 ml/minute
Column temperature: 100°C

Injection temperature: 210°C

Oven temperature: 185°C

Sensitivity (attenuation) of 5 x 103

 

114

 

 

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. g m:

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115

Appendix Figure 3. #3fl2 Longissimus dorsi (group 3)

Peak No.

\OmVOKn-P'WNH
II II II II II II I! H ll
0
|...u
O\

O

Instrument: Beckman GC-4 chromatograph
Detector: Hydro en flame

Column: Glass ( ft x 2 mm i.d.)
Carrier gas flow rate: 40 ml/minute
Column temperature: 100°C

Injection temperature: 210°C

Oven temperature: 185°C 3
Sensitivity (attenuation) o‘ 5 x 10

 

116

 

 

 

 

 

 

 

 

 

 

 

 

 

oncommom nopomvma

Minutes

117

Appendix Figure 4. #364 Kidney fat (group 4)

Peak Q9.
1 012:0
2 014:0
3 t 014:1
4 = 016:0
5 = 016:1
6 z 018:0
7 = 018:1
n = 018:2

Instrument: Beckman GC-4 chromatograph
Detector: Hydro en flame

Column: Glass ( ft x 2 mm i.d.)
Carrier gas flow rate: 40 ml/minute
Column temperature: 100°C

Injection temperature: 210°C

Oven temperature: 185°C

Sensitivity (attenuation) of 5 x 103

 

118

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