University of Southern California Dissertations and Theses

Studies of the effect of a vegetable fat and an animal fat on cholesterol metabolism

(USC Thesis Other)

Studies of the effect of a vegetable fat and an animal fat on cholesterol metabolism

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content STUDIES OP THE EFFECT OF A VEGETABLE
FAT AND AN ANIMAL FAT ON
CHOLESTEROL METABOLISM
by
Lilia Aftergood
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biochemistry)
June 1956
UMI Number: DP21566
All rights reserved
IN FO R M A TIO N TO ALL U SE R S
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
D issertation Publishing
UMI D P21566
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
Microform Edition © ProQuest LLC.
All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6
UNIVERSITY OF SO U TH ER N C ALIFO R N IA
G R A D U A T E S C H O O L
U N IV E R S IT Y P A R K
LO S A N G E L E S 7
0 6 1 © ^ ^ &
This dissertation, written by
L i l i a Af.terjgopd.................................
under the direction of^L.G uidance Committee,
and approved by all its members, has been pre
sented to and accepted by the Faculty of the
Graduate School, in partial fulfillment of the
requirements for the degree of
D O C T O R O F P H IL O S O P H Y
T y s e / ^iiL
D ean
Guidance Committee
(L. V Chairman
Lhz0ks 7&
ACKNOWLEDGEMENTS
I should like to express my everlasting gratitude
and appreciation to Professor Harry J. Deuel, Jr., for his
sponsorship, encouragement, and enthusiasm throughout the
course of these investigations.
I am indebted to Arthur F. Wells for his sugges
tions and to Dr. Supravat Mukherjee for his cooperation
and help in the chromatographic analysis.
I should also like to thank the other members of my
committee, Professors Mehl, Geiger, and Rittenberg for
their understanding and helpful criticism.
To Dr. R. B. Alfin-Slater— for her guidance and
' generous help and for the wonderful association which made j
: this work a pleasant task— my most sincere thanks. i
TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION AND HISTORICAL REVIEW ..........
Role of Liver in the Maintenance of Choles
terol Levels .............................
Transport of Cholesterol.......... . . . .
Role of Fat ...............................
Human studies........ ..................
Importance of essential fatty acids . . .
Effects of animal versus vegetable fat . .
Effect of Phytosterols and Other Analogs . .
Relation to Phospholipid Metabolism . . . .
Effect of Age and Sex ....................
Fatty Acid Pattern of Cholesterol Esters . .
Statement of the Problem ..................
II. EXPERIMENTAL................. . ..............
Materials and Methods .................. .
Animals . ...............................
Fats .......... .............
Cottonseed oil.................... . .
Lard.................... ..............
Diets . . . ...........................
Extraction of cholesterol and lipids . . .
Waring Blendor method ................
PAGE
1
2
1 +
j
6
ii
13
16
19
2b
25
i
26 I
2 7 ;
30 |
30 ;
3° |
30 |
30 :
3° |
31 !
3^ !
31 * |
CHAPTER
Soxhlet method .......... ..
Plasma extraction ................ . .
Determination of cholesterol ............
Determination of total lipids ..........
Isotopic studies .........................
Incubation procedure ............ ...
Analytical procedure ..................
Study of liver lipids . . . . . . . . . .
Separation of fractions by chromatography
Isolation of free fatty acids .....
Spectrophotometric determination of
unsaturated fatty acids . ..........
Determination of iodine value ........
Thiobarbiturate test for fat oxidation . .
Liver function test .... ............
Plan of Experiment .........................
Plasma and liver cholesterol levels . . .
Cholesterol levels in carcasses . . . . .
The effect of equalizing cottonseed oil
cholesterol level with that normally
occurring in lard ........ ......
The effect of vitamin E on cholesterol
levels in male rats fed a diet containing
PAGE
3^
35
35
37
38
38
38
ko
bo
bl
b2
*+3
Mf |
|
b6 '
b6
b6 |
!
*+6 !
CHAPTER
15 per cent l a r d .......... ..........
Digestibility studies .............. . . .
Cholesterol biosynthesis in the liver . .
Thiobarbiturate test for fat oxidation
Liver function test ....................
Study of fatty acid components of
isolated liver lipid fractions ....
Ill. RESULT'S.....................................
Plasma and Liver Cholesterol Levels ....
Cholesterol Levels in Carcasses ..........
The Effect of Equalizing Cottonseed Oil
Cholesterol Level with That Normally
Occurring in Lard .............. .
The Effect of Vitamin 1 on Cholesterol
Levels in Male Rats Fed a Diet Containing
15 Per Cent Lard .......................
Digestibility Studies ....................
Cholesterol Biosynthesis in the Liver . . .
Thiobarbiturate Test for Fat Oxidation . .
Liver Function Test .......................
Studies of Fatty Acid Components of Isolated
Liver Lipid Fractions ..................
vi
PAGE
b?
k-7
1*8
*f8
b8
^9
k9
56
57
59
62
6b
67
68
69
vii
CHAPTER PAGE
IV. DISCUSSION.................... 77
V. SUMMARY AND CONCLUSIONS.................. 86
BIBLIOGRAPHY ..................................... 91
LIST OF TABLES
TABLE
I.
II.
III.
IV.
V.
VI.
Composition of Diets ....................
The Plasma Cholesterol Levels in Male and
Female Rats Fed a Diet Containing Either
15 Per Cent Lard or 15 Per Cent Cotton
seed Oil for 6, 12, and 2h Weeks ....
The Effect of Age and Cholesterol Feeding on
Male and Female Rats Fed a Diet Containing
Either 15 Per Cent Lard or 15 Per Cent
Cottonseed Oil as Reflected by Plasma
Cholesterol Levels .................. .
The Average Liver Lipid and Cholesterol
Levels in Male and Female Rats Fed a Diet
Containing Either 15 Per Cent Lard or 15
Per Cent Cottonseed Oil Diet for 6, 12,
and 2^ Weeks ...........................
The Effect of Age and Cholesterol Feeding on
Male and Female Rats Fed a Diet Containing
Either 15 Per Cent Lard or 15 Per Cent
Cottonseed Oil as Reflected by Liver Lipid
and Liver Cholesterol Levels .......... ,
The Average Cholesterol Levels in Carcasses
Excluding Liver of Rats Raised on L and
Cs Diets for..?1 * Weeks . . . . . . . . . . ........,
PAGE
31
50
51
53
55
5-&_
TABLE
VII.
VIII.
IX.
(.
X.
XI.
IX
PAGE
The Average Plasma and Liver Cholesterol
Values and Liver Lipid Levels of Male
Rats Kept for 6 Weeks on a 15 Per Cent
Cottonseed Oil Diet Supplemented with
Cholesterol to Equal its Concentration
Normally Occurring in Lard.............. 60
The Average Plasma and Liver Cholesterol
Levels and Liver Lipid Levels of Male
Rats Fed a Diet Containing 15 Per Cent
Lard with and without the Addition of
Vitamin E and with and without the Ad-
!
dition of One Per Cent Cholesterol .... 61 [
|
The Digestibility of Cholesterol in Male j
and Female Rats Fed a Diet Containing :
Either 15 Per Cent Lard or 15 Per Cent
Cottonseed Oil for 2 Weeks 63 :
iL.
The Incorporation of C -Acetate In Vitro
into Liver Cholesterol of Male Rats Prefed
Either a Diet Containing 15 Per Cent Lard
or 15 Per Cent Cottonseed Oil with or j
without the Addition of One Per Cent
Cholesterol 65 .
l U-
The Incorporation of C -Acetate In Vitro
X
TABLE PAGE
into Liver Cholesterol of Female Rats
Prefed Either a Diet Containing 15 Per
Cent Lard or 15 Per Cent Cottonseed Oil
with or without the Addition of One Per
Cent Cholesterol ...... ............ 66
XII. The Percentage Distribution of Cholesterol
Esters, Phospholipids, and Triglycerides
in Pooled Liver Extracts.......... 70
XIII. The Percentage Distribution of Fatty Acids
of Cholesterol Esters in Pooled Liver
Extracts .................... 71
XIV. The Percentage Distribution of Fatty Acids
of Triglycerides in Pooled Liver Extracts. 72
XV. The Percentage Distribution of Fatty Acids
of Phospholipids in Pooled Liver Extracts. 73
! XVI. The Percentage Distribution of Fatty Acids
I of Cholesterol Esters and Phospholipids
i of Pooled Liver Extracts of Animals Fed
i
I
| 15 Per Cent Lard with and without Vitamin
l
| E and with and without One Per Cent
1 Cholesterol................... 75
CHAPTER I
INTRODUCTION AND HISTORICAL REVIEW
As early as 1769 the substance cholesterol was
identified by Poulletier (1) in a pathological tissue,
gallstones. In studies that followed, investigators
continued to consider cholesterol as an abnormal constitu
ent of the animal body, until in 1838 it was found in the
normal brain (2) and, later, in the normal blood (3).
The main interest in cholesterol metabolism, at the
present time, is based on its possible relationship to the
disease, atherosclerosis. This disease, considered to be
a normal result of aging, was first connected with a
derangement in cholesterol metabolism when Anitschkow and
also Aschoff (**•) in 1910-1912 discovered that athero-
sclerotic-like lesions can be produced in rabbits by
feeding cholesterol.
In the years following, numerous investigators in
this field have accumulated considerable knowledge about
cholesterol and its role in animal physiology and bio
chemistry. Cholesterol is a solid alcohol found in every
animal cell. A multitude of compounds, of a similar
molecular structure is found in plant and animal tissues,
among the most important being the plant sterols, the bile
f " ■' ' 2 ;
! acids, hormones of the adrenal cortex and the sex organs,
■ and the provitamins D (5). !
j
i ;
Role of Liver in the Maintenance of Cholesterol Levels. 1
! The liver is the most important organ of the animal '
I
body concerned with cholesterol metabolism. It serves as
i
I the principal, but not the sole, site of cholesterol
t j
I synthesis (6); it is a reservoir where endogenous or !
; I
; exogenous cholesterol may be held for future utilization;
it is the site of oxidation of cholesterol to bile acid and
i also of the disposal by excretion into the bile (7). '
I i
; The amount of cholesterol that is synthesized by the
I i
animal body is inversely related to the amount of exogenous;
cholesterol which is absorbed (8, 9). As early as 1933j >
, Schoenheimer and Breusch (10) observed that when moderate '
: amounts of cholesterol were administered to mice, a smaller
amount of cholesterol was biosynthesized. j
i
Frantz, Schneider, and Hinkelman (11) reported that
in rats fed a diet to which one per cent of cholesterol had
been added there occurred a considerable increase in the
concentration of liver cholesterol, but only a minimal rise
in the concentration of serum cholesterol. The rate of
i
hepatic cholesterol synthesis appeared to depend very
sensitively on the cholesterol content in the liver. The
action of the liver could be viewed as that of a buffering |
3 i
I
mechanism for the serum cholesterol. Tomkins et al. (12), '
in studies of cholesterol synthesis from acetate in rat I
liver slices, found that marked depression in synthesis
occurred when the animals were fed 0.5 per cent cholesterol;
j ■
the synthesis practically ceased when a diet containing 5 I
per cent cholesterol was fed for 8 days. i
It can be assumed, therefore, that the controlling ;
i
factor must be quite sensitive, since an increase of only 1
i
a few milligrams per 100 gm of liver in exogenous free
i
i
cholesterol corresponds to a decrease in synthetic rate to
half of normal (13). But in spite of the excessive amount
of exogenous cholesterol which is absorbed, the animal
j
never ceases to manufacture its own cholesterol (l1 *-).
i
Gould (13) postulated that the response of hepatic choles- j
i
terol synthesis to the increased concentration in the liverj
might be considered a part of the homeostatic mechanism !
which attempts to maintain a constant level of cholesterol J
i
in the liver and plasma despite changes in the dietary
intake. i
l
Another feature of cholesterol metabolism is the i
storage of its excess in esterified form in the liver. 1
These cholesterol esters appear to be metabolically inert !
and in some species, particularly the rat, may increase to
many times its normal concentration without any marked I
i increase reflected in the level of plasma cholesterol or
! • ' |
j in the concentration of free cholesterol in liver. Thus . i
i
^ the liver cholesterol ester concentration appears to be a i
j sensitive index of the presence of excessive amounts of i
j i
i cholesterol in the body. i
I . i
j The liver is the organ which serves as the source j
iof almost all endogenously derived plasma cholesterol (6). ;
1 i
j The rate of hepatic synthesis of cholesterol varies in such'
I
: a manner as to keep the level in liver and in plasma
j I
I constant. !
!
I ■ ■ !
I Transport of Cholesterol.
It has been shown by Biggs et al. (15) and Chaikoff :
et al. (16) that all absorbed cholesterol is transported
i
jvia the intestinal lymphatics. Although almost all dietaryj
; cholesterol is ingested in the free state, most of the i
i I
lymph cholesterol is in the esterified form. Practically J
• I
all of the dietary cholesterol contained in lymph travels ;
in the chylomicron fraction (17). Based on this finding,
/Friedman et al. (18, 19) postulate that dietary-derived
i
cholesterol is not identical with that manufactured by the
i ;
ianimal itself, since the former enters into the blood boundi
1 i 1
to other lipids in an insoluble particle whereas the latter!
I
:is in solution. 1
Heilman et al. (22) observed differences in the rate
of appearance of biosynthetic and dietary cholesterol in
plasma cholesterol, but they concluded that cholesterol
derived from the diet is eventually indistinguishably mixed
with cholesterol synthesized in the body.
Once in blood, the chylomicron-cholesterol is
probably influenced by two different processes; one is the
heparin-, , clearingM factor mechanism (20), and the other is
ingestion by the hepatic reticulo-endothelial cells (21).
Investigations of Friedman and Byers (19) suggest that the
reticulo-endothelial system does not participate in the
egress of endogenously produced cholesterol Treatment of
rats with reticulo-endothelial blocking agents, Thorotrast
(colloidal solution of thorium dioxide) and trypan red,
was found to prevent the characteristic increase in liver
cholesterol normally produced by the administration of high
cholesterol diets, as reported by Rice et al. (23).
Similar treatment had no effect on liver cholesterol in
rats fed normal diets. A decrease of plasma cholesterol
levels occurred in both the Animals fed high cholesterol
and normal diets.
The ultimate fate of the dietary cholesterol is in
the hepatic parenchymal cell (2*+). Here it is stored for
possible subsequent discharge as such into the blood and
6 i
I
into the bile or for conversion to cholic acid. According '
j
to Siperstein and Murray (25), the chief excretory product 1
of cholesterol in human bile is glyeocholic acid, while in
the rat the major bile acid end product of cholesterol j
i
metabolism has been identified as taurocholic acid (26). ;
However, bile cholesterol seems to represent, under i
most circumstances, a portion of that cholesterol newly ;
synthesized and discharged by the liver into the blood,
rather than an excretory product per se of the liver (27, j
28). This seems to account for the finding of Gould and
associates (29) and others (28) who observed that, after i
the administration of radioactive acetate, bile cholesterol
had a higher activity than did the cholesterol from any
other tissue in the body, including the plasma and liver.
The latter tissues perhaps exhibited less activity because
of the diluting effect of the inactive cholesterol present ,
before the administration of the radioactive precursor.
Significant quantities of unchanged cholesterol are ,
lost to the body only via the feces (18).
Role of Fat.
Ever since Anitschkow's pioneer achievement (b )
there have been continuous attempts to influence cholesterol
levels in various organs by dietary means. A well estab
lished effect of dietary cholesterol itself is an increase
of the level of cholesterol in the liver, and thereby a
i decrease of the biosynthesis. In addition, many contro-
, versial findings pertaining to the effect of the dietary
fat have been reported.
It is generally accepted by most investigators that
fat is a required vehicle for the absorption of cholesterol
i in most of the species studied (3. 0, 31).
In 1935j Eckstein (32) studied the effect of high
and low fat diets on the sterol metabolism of the rat. The
amounts of sterol in the livers of rats were greater when
! j
the percentage of corn of soybean oil in their diets was !
increased. Subsequently, the same author observed that the
level of liver cholesterol varied with the nature of
dietary fat (33)• On a 28 per cent fat diet, high liver
, sterol levels were obtained when corn oil was fed than when
i
the coconut oil (a saturated fat) was administered. The
| author concluded that fatty acids of low molecular weight
(other than acetate) are converted to cholesterol with
difficulty.
Kim and Ivy (3*0 have shown that fat in adequate
quantities facilitated the absorption of cholesterol as
determined by the difference between dietary and fecal
sterol levels. The fatty acid portion of the neutral fat
! molecule was found to be the active factor. Serum
8 :
cholesterol levels were significantly higher in the groups
of rats receiving cholesterol and oleic acid, than in
those receiving cholesterol and corn oil, in which there
is little free fatty acid.
Swell and Flick (35) fed to rats diets containing
25 per cent lard* oleic acid, or stearic acid with or with
out added cholesterol. The blood cholesterol rose sharply ;
in both groups on the lard diets. When oleic acid was
added to the diets, the total and ester cholesterol of
plasma of both groups declined slightly. On the stearic
!
acid diet the total and ester cholesterol in plasma I
declined more sharply in the cholesterol group. The ■
authors suggested that incomplete absorption of cholesterol
took place when a high per cent of saturated fatty acid was
present in the diet.
More recently, Swell et al. (36) reported that when
cholesterol-free diets, containing soybean oil hydrogenated,
to varying degrees, were fed to rats, the highest degree of
unsaturation of oil in the diet produced the highest blood
cholesterol levels. It was also observed that fat need not
be present in the diet for the absorption of dietary
cholesterol, provided a sufficient amount of bile salts
were present. The fatty acid necessary for cholesterol
esterification could arise from endogenous sources. It was
suggested that dietary fat serves a dual purpose: a) it
|stimulates the flow of bile necessary for cholesterol
iesterase activity, and b) it provides the fatty acid
'necessary for cholesterol esterification.
j Kritchevsky et al. (37), working with rabbits, have
•shown that the presence of a fat in diet significantly
i
influenced cholesterol levels in serum and liver. Highest
'serum levels were obtained when partially hydrogenated
}
ivegetable shortening was used, while the corn oil, which
1
contains approximately 85 per cent of unsaturated fatty
acids, caused highest cholesterol levels in the liver.(
i
Alfin-Slater et al. (38) studied the effect of low
fat and high fat diets on the synthesis of cholesterol in
■rats. The uptake of deuterium in cholesterol was measured
iat the end of definite time intervals. It was found that
!
ithe amount of newly formed cholesterol present in the liver
»
jand plasma of rats prefed a low fat diet was unchanged when
t
■the animal was placed on a high fat diet. A high fat diet
seemed to be more efficient in removing the cholesterol
which had accumulated in the liver during previous
cholesterol feeding.
! The effect of mineral oil as a vehicle for choles
terol transport has also been investigated (39) • The
I
‘ addition of light mineral oil at a level of 6 per cent in
10 r
j the diet fed to rats increased the fecal excretion of
t
; endogenous cholesterol by 39 per cent when there was no j
! cholesterol in the diet, and increased the total excretion !
i
! of cholesterol by 30 per cent when cholesterol was added
! i
! to the diet. This might be due to the solubility of cho- |
|
! lesterol in mineral oil and also.to the laxative properties'
1
» ;
Jof mineral oil itself. !
i j
I Dubach and Hill (kO) have found that cholesterol was!
J !
; absorbed by the rabbit whether or not neutral fat was fed i
i i
with the cholesterol. \
In the experiments of Bollman and Flock (*+1) , the I
i
i absorption of cholesterol, as measured by the concentration:
I • t
in the lymph, appeared to be as good in the rat when free
cholesterol was administered with the fat-free diet as when
; it was mixed with oleic acid or a diet containing neutral
i
fat. However, the volume of lymph on a fat-free diet was
!definitely decreased. i
! 1
i The effects of cholesterol feeding in the rat seem :
to be dependent on the nature of fat in the diet. Raulin
and his group (4-2, ^3) reported that an addition of 3 per
cent cholesterol to the diet containing 15 per cent total
fatty acids of sunflower seed oil caused high mortality,
eye trouble, and sterility. The cholesterol or the fatty
acids fed individually did not cause such disturbances.
Human studies. There have been a great many in
vestigations of the effect of dietary fat on blood
; cholesterol levels in humans.
I Throughout the 19^2-19^7 period in Germany a general
i
j decrease in serum cholesterol levels was observed in men
| (M+). The cholesterol level did not, however, change in
| the erythrocytes. The reason for this decrease was
I
! believed to be an effect of the fat-deficient diet which
I
j was prevalent at this time. The authors agreed with the
j theory of Schramm and Wolff (**5) according to which,
| cholesterol plays an important part in the fat absorption.
i
I When the diet supplies only minimal quantities of fat, the
total fat exchange is greatly reduced and less cholesterol
is necessary. Thus its low level is not to be regarded as
| a pathological condition by itself, but is to be related
l !
: to a diminished fat turnover.
i
i
Mellinkoff et al. (**6) studied the effect of a fat--
i
free diet on patients with gastric ulcers. A fall in serum
cholesterol occurred in thirteen out of fourteen cases
tested. The decrease was greater when the initial value
was relatively high.
i On the other hand, Wilkinson et al. (*+7) reported
that in essential familial hypercholesterolemia the
'absolute amount of carbohydrates, fat, protein, and
............................ .......... — . . . . . ...- - ---■ ~2I
cholesterol in the diet had no relation to the level of :
i
total serum cholesterol. '
Keys and collaborators (*f8, ^9, 50, 51, 52) have
performed studies on the serum cholesterol level of healthy;
men in different geographical locations. They concluded
that dietary cholesterol per se had no important influence ;
on the serum cholesterol concentration but that the latter ■
i
was markedly affected by the total fat content of the diet.)
The effect of the diet tended to be more pronounced in ,
middle-aged men than in younger men. The average concen-
!
tration of cholesterol in the serum of men in areas where !
I
the diets were very high in fats was 25 to 50 per cent
greater than the average in areas where the diets were low
in fat. A significant effect of dietary fat level on the
serum cholesterol concentration was evident in man in a few;
!
weeks on a changed diet. •
i
Gofman et al. (53) also reported that partial dietaryj
i
restriction of fat and cholesterol in man resulted in a
)
gradual decrease in serum level of cholesterol-bearing
lipid and lipoprotein molecules. j
Hatch et al. (5^) performed controlled observations !
of serum lipid levels in a series of hospitalized patients ;
with severe hypertension, undergoing both partial and
practically complete restriction of dietary fat and
cholesterol. The results of drastic dietary restriction of
fat and cholesterol showed great variability among patients
as far as the effects on the serum lipid patterns were
concerned. Serum cholesterol ester concentration usually
declined with restriction of dietary fat intake.
Wollaeger et al. (55) in a study of two normal
individuals found that the total plasma fatty acids and
cholesterol decreased somewhat during a period on a lipid-
free diet. However, Brown et al. (56) found no change in
the serum cholesterol or total fatty acids in one subject
subsisting for 6 months on a diet extremely low in fat.
Importance of essential fatty acids. A new light
has been thrown on this question through studies of a fat-
deficient diet on the liver cholesterol levels. Alfin-
Slater, Aftergood, Wells, and Deuel, Jr. (57) reported that
weanling rats placed on a fat-free diet developed an
increased level of cholesterol in the liver as early as one
week, when compared with rats placed on Purina Chow or a
15 per cent cottonseed oil diet. The presence of a
saturated fat in the diet yielded results similar to the
fat-free diet after the first week but after b weeks the
amount of cholesterol in the livers of these animals was
considerably less than those on a fat-free diet, although
still increased over normal. These authors postulated that
1 essential fatty acids are necessary for the proper ester-
! j
; ification and transport of cholesterol from the liver. I
i |
; In a subsequent paper (58), the same authors con-
i
I
i firmed the influence of the essential fatty acids on the !
I 1 :
! course of cholesterol metabolism. The increased choles- j
| !
!terol concentration in the liver of rats on essential fatty!
i 1
: acid-low diets was found to be confined almost exclusively
j to the ester fraction. j
i i
I Shapiro and Freedman (59) reported recently that the!
i I
, hypercholesterolemic response to cholesterol feeding in j
| i
i rats on a sulfur-deficient protein diet was largely pre- j
! I
vented by the use of dietary fat containing high levels of 1
essential unsaturated fatty acid and supplementation with :
methionine. Substitution of a hydrogenated fat under the
same experimental conditions did not effectively prevent
jhypercholesterolemia. I
1 - !
Peifer and Holman (60) studied the effect of cho- j
t i
ilesterol feeding on diabetic rats on essential fatty acid- i
deficient diet. Cholesterol did not significantly
'influence the development of the deficiency in diabetic
i
‘animals. In the nondiabetic, the deficiency was produced
within 2 weeks by the addition of one per cent of choles-
•terol to the fat-deficient diet. Both syndromes might be
: i
j the reflection of the same phenomenon. Accelerated
15
,transport of essential fatty aeids due either to endogenous
hypercholesterolemia in the diabetic animal or to exogenous
hypercholesterolemia in the nondiabetic animal leads to the
more rapid depletion of body stores of essential fatty
i
acids.
The importance of the degree of unsaturation of the
jdietary fat in influencing blood cholesterol levels in
i * '
(chickens was indicated by Chahine and Kummerow (61).
,Chickens fed 0.5 per cent cholesterol and 10 per cent of
'hydrogenated cottonseed oil (I.V. 93) in addition to the
i
(standard poultry ration had a higher blood cholesterol
level than those fed highly Unsaturated cottonseed oil.
Furthermore, chickens which had been fed hydrogenated
vegetable fat had as high a cholesterol level as those fed
(animal fats such as lard or butter fat.
Bromer and Day (62) observed that the hypercholes
terolemia and the increase in liver cholesterol were much
greater in essential fatty acid deficient rats given
cholesterol than in controls. Small supplements of methyl
linoleate caused a markedly lower level of cholesterol
esters in the blood and liver.
Experiments in which the type of dietary fat and not
only its quantity were considered, indicate that the
; important factor might well be the fatty acid composition
■'............... ' 16:
of the fat.
Effects of animal versus vegetable fat. Although
many workers 0+7, H-8, 53, 63) consider that serum choles
terol levels are controlled by fat intake, regardless of
j
source, evidence to the contrary has been reported by •
Kinsell and co-workers (61 *, 65, 66, 67). These investi- j
gators substituted vegetable fat for animal fat in the dietj
l
of diabetic and nondiabetic patients with vascular diseases
and this alone resulted in rapid and maintained fall of j
serum cholesterol and phospholipids. A high vegetable fat
diet was also effective in presence of animal fats. When
the fats were alternated (66), a decrease in phospholipids
and cholesterol resulted on the vegetable fat diet and a
i
rise to average levels when animal fat was fed. In
general, Kinsell and Michaels (68) observed that "factors
which cause elevation or depression in plasma cholesterol
produce the same type of effect upon plasma phospholipids."
A statistical study by Hardinge and Stare (69)
demonstrated that strict vegetarians have lower cholesterol
levels than those vegetarians who eat dairy products, and
that both groups have lower cholesterol levels than the
general population.
It has also been reported by Tsai et al. (70) that
dogs have lower serum cholesterol levels on a vegetable fat
j"‘: " ..... - T?1
: diet than when fed a commercial diet containing variable ;
; amounts of animal fat and cholesterol. j
i
! Ahrens et al. (71) reported that six subjects with j
' obesity of exogenous origin showed significant reductions J
i !
j in serum concentrations of free and esterified cholesterol j
i
- and of phospholipids when plant fats were substituted iso- 1
i
j calorically for animal fats. The approximate magnitude }
i !
I of the change was a 20 per cent decrease. Neutral fat ;
i
] levels showed no significant change.
I
! Another group of workers (72) studied the effects of!
i i
; varying the level of dietary cholesterol and the level and ,
; i
| type of fat. In agreement with Hildreth et al. (63), Mayer!
et al. (72) found that an increase in the level of dietary
fat as vegetable fat led to an increase in plasma choles- i
i
, terol levels. Subsequently, to obtain better controls, thej
' same workers (73) incorporated cholesterol and vegetable !
' and animal fats into homogeneous formula diets. Under the !
' conditions of this study, there was a highly significant
i
I
statistical difference between the effects of vegetable fat1
!and animal fat on the blood lipids. Diets containing 1
vegetable fat, comprising either 28.b or 58.5 per cent of 1
total calories, with'or without supplementary cholesterol, '
led to decreases in plasma lipid levels. A hypothesis has
'been presented that although vegetable fat per se has no
: tendency to increase the concentration of plasma lipids, inj
, conjunction with some unknown substance of animal origin ;
; usually present in a non-vegetarian diet, it does raise ;
the levels of these components.
i
Very few investigations on comparisons of the
1 effects of animal and vegetable fat on cholesterol metabo-
' lism have been performed with animals. However, as early I
I |
| as 19^8, a paper by Schettler appeared (7*+) in which mice i
I
were fed sesame oil with the addition of phytosterol,
jLiver and plasma cholesterol levels at 1, 3, and 6 weeks |
1 were compared with those of a group of mice fed depot fat !
! with phytosterol. Significant increases were found in
total plasma cholesterol and in total and free liver cho
lesterol in the mice on the animal fat diet. An additional
!observation was that, even though the diets were iso-
:caloric, the first group of animals lost weight, while the :
second group gained. The author concluded that the
:phytosterol content of plant oils is not the cause of lower
cholesterol levels.
A year later, the same author published the results
of the further studies in this field (75). Sesame, olive,
and linseed oil were compared with depot Tat, lard, and
I tallow. Definite differences in liver cholesterol levels
obtained in the mice fed these experimental diets. An
initial increase occurred on the plant oil diets with '
subsequent leveling off at 21 days. On the animal fat i
diets, the liver levels continued to increase. Most
changes occurred in the esterified portion of cholesterol. 1
When dietary cholesterol was added to the various experi- !
mental fat diets (76), an increase in cholesterol levels I
was observed in blood as well as in other organs with j
higher levels obtaining in the animals fed animal fat, i
supplemented with cholesterol.
i
Effect of Phvtosterols and Other Analogs. 1
Almost concurrently with Schettler, studies were j
reported which differentiated between the origin of fat. >
Investigations were directed toward elucidation of a factor
responsible for the differences obtained on vegetable and ;
animal fat diets. >
i
One of the differences in the chemical composition
1
between the two types of fats is their sterol content.
I
Vegetable fats contain phytosterols, as opposed to cho- :
lesterol present in animal fats. Thus the effect of dietaiy
phytosterols as such on plasma and liver cholesterol levels
was investigated.
In 1951 Peterson (77) reported that the addition of
mixed soybean sterols to a cholesterol-enriched diet fed
to chicks prevented the hypercholesterolemia which
otherwise occurs on a high cholesterol diet. That sito
sterol might interfere with reabsorption of cholesterol
secreted into the intestinal tract in mice had previously
been suggested by Sperry and Bergmann (78). Subsequently, '
\
)
; Peterson et al. (79) showed that mixed soy sterols, mixed i
i
sitosterols, beta-sitosterol. stigmasterol, and ergosterol j
were effective inhibitors of the progressive rise of plasma
cholesterol and of deposition of cholesterol in the liver j
I of Chicks, which normally occurs when cholesterol is fed. i
i Esterification of phytosterols with capric acid destroyed
1 ,
I their ability to prevent an increase in plasma and liver |
, cholesterol levels.
1
The prevention of hypercholesterolemia in
cholesterol-fed rabbits also has been successfully ae-
1 complished by the concomitant feeding of soybean sterols
( 80) .
Using cholesterol-^f-C-*-1 * in rats, Hernandez et al.
"rmBI j
i (81, 82) demonstrated that either mixed soybean sterols or '
i
beta-sitosterol resulted in a marked decrease in the amount
.of cholesterol recovered from thoracic duct lymph. This
finding has been challenged by Rosenman et al. (83). How
ever, studying the effect of the soy sterols on liver
storage of the marked sterol as well as a fecal excretion
of labeled material, Burke and his co-workers (8*f)
; substantiated the findings of Hernandez et al. (81).
Ilfin-Slater et al. (85) reported that simultaneous i
i
feeding of plant sterols and cholesterol evoked no change
j in plasma levels of cholesterol or in the carcass. How- j
j ;
I ever, total cholesterol and lipid contents of the liver |
i t
1 were both markedly less than when cholesterol was fed j
j ' l
} alone. The addition of one per cent plant sterols at the !
! j
! k- per cent fat level reduced the cholesterol content of thej
t
i liver from 16.5 to 2.9 mg per gm of liver. Raising the ;
! I
j level of sterols to 3 per cent brought the cholesterol j
i !
\ f
i values to within the normal range. The same effect was j
|
observed at a higher intake level of fat.
More recently, the same workers (86) reported
i further studies on the mechanism of action of soy sterols
I
i
i in cholesterol absorption. Plant sterols were fed alter- ;
! [
' nately with a high cholesterol diet to rats. It was found «
t
that the cholesterol content of the liver was much ;
’ i
| decreased over a control group, which was given a high
cholesterol diet alternately with a cholesterol-free diet, j
<
but containing no plant sterols. In an experiment in which
: various cholesterol esters— such as cholesterol linoleate,
oleate, and acetate— were fed to rats with and without
plant sterols, it was found that 1) cholesterol linoleate
I and cholesterol acetate were better absorbed than
cholesterol oleate, and 2) the absorption of the esters in
I
jail cases was inhibited by the presence of plant sterols in
the diet.
A number of investigators have been exploring the
effects in man of the administration of soy sterols, with
I conflicting results. Wilkinson (87) reported that gamma-
|
!sitosterol has not been effective in reducing the concen-
I
jtration of blood cholesterol. Kinsell et al. (88) have
!
jreported variable results, while Poliak (89) and Best
!
et al. (90) have shown a lowering of serum cholesterol by
the prolonged administration of sitosterol. As early as
, 19l f1 +, Msoya lecithin’ 1 which is composed of lecithin,
cephalin, oil, phytosterol, inositol, and carbohydrates
was reported to lower serum cholesterol level (91).
; Generally it has been suggested (79, 90, 92) that
jsitosterol interferes with cholesterol absorption by
^competing for esterification, a step in the transport
mechanism by which cholesterol is absorbed (79, 93).
Schoenheimer (9^) failed to demonstrate by chemical methods
j
:any absorption of sitosterol, and it was long considered to
!be essentially non-absorbable. Recently, Gould (95),
employing tritium-labeled sterols, has restudied the
absorption of cholesterol and sitosterol. Sitosterol was
absorbed though much less readily than cholesterol. It
; also disappeared from the blood much more rapidly and did
not accumulate in the body. According to Gould et al.
; (96), this might account for its harmlessness, although
: actually this finding could lend support to the concept
' that it acts by competing with cholesterol for absorption
(90). Swell et fO.. (92) have shown that plant sterols and
' cholesterol are esterified under the same conditions by
I pancreatic cholesterol esterase in vitro. It is probable
that the soybean sterols compete with the cholesterol for
. the enzyme, bile salts, and fatty acids. i
\
■ In line with the studies of the action of phy
tosterols on cholesterol accumulation in the body, other
analogs have been investigated. Thus, Beher and Anthony
(97) reported that the addition of dihydrocholesterol to
' diets containing cholic acid and cholesterol significantly ;
i
decreased mouse liver cholesterol levels. However, feeding
. a diet containing dihydrocholesterol to mice with elevated
; liver cholesterol, caused by prefeeding a cholesterol diet,
did not result in an increased cholesterol mobilization
(98). Siperstein et al. (99) prevented plasma cholesterol
elevation in the cholesterol-fed bird by the simultaneous
administration of dihydrosterol.
Other methods for interfering with cholesterol
! absorption, such as adding iron salts to the diet for the
purpose of precipitating bile salts, have been foimd to be ,
more or less effective in experimental animals (100).
Relation to Phospholipid Metabolism. '
| Many investigators believe that the metabolism of j
: phospholipids is connected, at least in part, with that of ;
! ■ i
j cholesterol. There is a tendency for a reduced phospho- j
i I
j lipid phosphorus to total cholesterol ratio in athero- I
! I
j sclerosis (101). The phospholipids of the atheromatous ;
i '
! aorta of a cholesterol-fed rabbit exhibit a five- to j
. I
! sixfold increase in turnover rate (102). The phospholipid
1
. content of the liver of cholesterol-fed rabbits was nearly j
!
* twice as that of a control (103, 10*+). The authors
speculated that a relation exists between the accumulation
of lipids in a tissue and stimulation of phospholipid
1 turnover. Tissue may respond to excess lipid by increasing'
i its rate of lipid oxidation, a process in which phospho-
j lipids participate. Alternatively, one might think of j
• i
phospholipids as emulsifying agents and colloid stabilizers;
Ridout et al. (105) observed that in rats increasing
;
amounts of dietary cholesterol caused a progressive
• increase in the accumulation of both glycerides and cho
lesterol esters in the livers. However, when increased
J
i
■ amounts of choline were administered, liver glycerides were
! maintained only slightly above the normal range.
:Cholesterol esters in the liver were slightly elevated.
' Pilgeram and Greenherg (101), employing an in vivo
I
system derived from the livers of rats which had been fed
j
, a diet containing cholesterol and cholic acid, demonstrated
!that these dietary components stimulated the formation of
t
:phosphatidyl choline. The authors suggested that
|phosphatidyl choline was necessary for normal cholesterol
!metabolism and that the difference in species suscepti-
jbility to cholesterol-induced atherosclerosis might be
icorrelated with differences in their capacity for phos-
i
!phatidyl choline formation.
i
In contradiction to the above mentioned reports were
the results of Clement et al. (106), who found that feeding
of cholesterol to rats depressed the synthesis of liver
phospholipids.
I
i
»
:Effect of Age and Sex.
j Some studies of blood cholesterol levels in humans
as related to age and sex have been undertaken. Thus,
Garcia et al. (10?) observed that in women, serum choles-
1terol levels are not necessarily related to the pattern of
jfat absorption, and that concentrations of serum choles-
I
terol increase with age up to the seventh decade, when a
I decrease occurs. Other investigators (108) have reported
i
jthat between the ages of 60 to 80 years women had
26
i
1
j significantly higher levels than the men. In both sexes a ;
■ sharp drop occurred at 75 or 80 years.
t
In the rat, the hepatic synthesis of cholesterol has
j
| been studied at different ages (109). It appears that the !
!synthesis depends upon the age of the animal, with a con- ,
| •:
lsiderably greater synthesis occurring in the younger rat.
Fatty Acid Pattern of Cholesterol Esters.
i ■
I There are comparatively few reports in the litera- j
! ■
jture concerning the fatty acid make up of cholesterol
jesters present in plasma or liver. Bloor (110) noted that !
; I
!the fatty acids in combination with cholesterol have the j
!highest iodine number of those in any fraction of blood of ,
various species.
In the case of human blood, Bloor and co-workers
(111) reported that the unsaturation of the fatty acids was
i
jlowest in neutral fat, Intermediate in phospholipids, and '
i 1
!highest in the case of those combined with cholesterol.
; Channon and Collison (112) found that arachidonic j
i
and linoleic acids occur in the acetone-soluble fraction
t
of beef blood, along with palmitic and stearic acids. In
observations on cow plasma, Kelsey and Longenecker (113)
noted that 62 per cent of the fatty acids combined with
jcholesterol in plasma were dienoic acids as contrasted with
only 18 per cent of this type in triglyceride fat.
• However, Keegan and Gould (llH-) have reported the isola- j
r
tion of cholesterol oleate from the plasma of man and dog i
i with a yield corresponding to more than 50 per cent of the ■
i i
j total cholesterol esters present. More recently, Achaya
I
I et al. (115) determined the component fatty acids in plasma
i
! lipid fractions of the rat. Normal rat plasma contained j
j
! j
i about equal concentrations of cholesterol esters and I
' I
i
1 phospholipids. The cholesterol ester fraction had the
! i
' highest iodine value. In rats on a fat-deficient diet,
j the iodine value of the cholesterol ester fraction of
i
> whole blood was decreased, due to a decrease in tetraenoic !
; ' I
; acid and a concomitant increase in dienoic acid. The
, addition of 200 mg linoleate per rat per day to the fat- i
deficient diet restored the blood lipid picture to within
i normal ranges. '
I j
Hammond and Lundberg (116) observed that blood
i
; samples from normal humans on a low fat diet had a greater ;
* ,
j j
; amount of trienoate and hexaenoate than normal blood
1
! donors. Atherosclerotic subjects had greater amounts of i
!
tri, penta, and hexaenoate.
Statement of the Problem.
The foregoing reports all seem to point in one '
I
{ direction— dietary fat is of primary importance in choles- :
i
terol metabolism. At the same time the majority of !
28
investigators agree that the effect of fat might be not
only quantitative but also qualitative. However, in many
; cases the results are inconclusive if not controversial.
j
!
< At the same time, even if the difference between the
| action of fats from various sources is established, there
I is no acceptable explanation for the obtained findings.
This investigation was undertaken in an attempt to
i
j clarify the role of dietary fat in cholesterol metabolism
, and to offer a possible explanation for this action.
i
The species of animal, albino rats of the U. S. G.
! strain, was chosen which, although resistant to the
disease atherosclerosis, presents certain advantages in
this study, namely: They are easily available in our
/ ^
laboratory; they are resistant to most infectious diseases;
their nutritional requirements are known and easily
i 1
controlled; and deficiency syndromes are apparent within
, short periods of time.
Two fats, one of animal, the other of vegetable
' origin, were used and their effect on cholesterol metabo-
, lism was investigated. Once differences were established,
certain problems could be stated more clearly.
a) Are the differences which occur due to changes in
the distribution of cholesterol in the animal body?
b) Is there an effect on cholesterol biosynthesis
29
after feeding the different diets?
c) Is the effect due to differences in cholesterol
absorption on the two dietary regimes?
d) Does the chemical composition of the fat exert an
influence on cholesterol metabolism as a whole,
i.e.,
1) Cholesterol concentration of lard versus
plant sterol concentration of cottonseed
oil.
2) Infinitesimal quantities of vitamin E in
lard versus rather large concentration in
cottonseed oil.
3) Saturated fatty acids present in lard
versus polyunsaturated fatty acid concen
tration in cottonseed oil.
A hypothesis involving the observations obtained is
proposed.
CHAPTER II
EXPERIMENTAL
Materials and Methods.
In order to investigate the effect of the type of
fat on some aspects of cholesterol metabolism, the follow
ing materials were used.
Animals. Male and female albino rats of the
/
University of Southern California strain were used in all
experiments.
throughout the experiment was the refined, winterized
Salad Oil, kindly supplied by Best Foods, Inc. The
analysis of fatty acids present in this cottonseed oil
gave the following results:
Fats
1. Cottonseed oil. Cottonseed oil used
saturated
monoenoie
dienoic
trienoic
31. 2$
19.6
*f9.2
I.V. (iodine value) 106.6
2* Lard. Lard was obtained from Swift Co. It
was an uniform sample with no more than 0.01 per cent
propyl gallate, 0.02 per cent butylated hydroxyanisole,
31
and 0.005 per cent citric acid in propylene glycol added
as preservative. The analysis of fatty acids present in
this lard gave the following results:
saturated 38.9$
monoenoic *+7.8
dienoic 12.1
trienoic 1.2
I.V. 68.1
Cholesterol content in lard 0.08$
Diets. The composition of the diets used is given
in Table I.
The fats were kept in the cold room (3-5°c) prior
to mixing of diets; diets themselves were stored similarly
when not in use.
The designation of diets used throughout the thesis
will be as follows:
Cs - 15$ cottonseed oil
L - 15$ lard
CsC - 15$ cottonseed oil + 1% cholesterol
LC - 15$ lard + 1$ cholesterol
TABLE I
COMPOSITION OF DIETS
Component LC L CsC CS
% %
% %
Lard 15.00 15.00
- -
Cottonseed oil
- - 15.00 15.00
Cholesterol-^ 1.00 - 1.00
Bile salts-/ 0.25 0.25 0.25 0.2 5
Celluflour*^/ ^.00 ^.00 k.00 *+.00
k /
Salt mixture- k.qo 00 ^.00 k. 00
Casein, commercial-^/ 2k. 00 2^.00 2^.00 2M-.00
Sucrose 51.30 52.30 51.30 52.30
Choline-/ 0.2^- 0.2*+ 0.2*1- 0.2*f
Vitamin mixture-/
0.19 0.19 0.19 0.19
Nopsol^/ 0.012 0.012 0.012 0.012
Alpha-T'o copherol-/ 0.012 0.012 0.012 0.012
Footnotes to Table I
-^U. S. P.; Merck Co.
-^Difco Laboratories, Inc., Detroit, Mich.
2/solka-Floc, Brown Co., San Francisco, Cal.
-^Osborne and Mendel (Science 75*339 (1932)) Wesson
Modification; Nutritional Biochemicals Corp.,
Cleveland, Ohio.
■^Lactic casein, Challenge Dairy Co., Los Angeles, Cal.
£/The vitamin mixture consisted of 38.57$ p-amino-benzoic
acid, 31*88$ inositol, 12.75$ ascorbic acid, **.59$
thiamine hydrochloride, 3*82$ niacin, 3*82$ Ca-
pantothenate, 1.72$ riboflavin, 1.72$ pyridoxine,
6^6^$ folic acid, 0.32$ menadione, 0.16$ biotin, and
p. 000,0^$ vitamin Bi2» Merck Co. and Nutritional
Biochemicals Corp.
Z/The Nopsol solution contains 100,000 I. U. of vitamin A
per gm and 20,000 I. U. of vitamin D per gm. Nopco
Chemical Co., Harrison, N. J.
/
-^Nutritional Biochemicals Corp., Cleveland, Ohio.
J ■ ' ".........“ $ * ’ ■
I
j Extraction of cholesterol and lipids. |
j 1. Waring Blendor Method. Animals were j
| anesthetized by Nembutal injection. The liver was quickly i
! !
extirpated, trimmed, and blotted. The following procedure j
based on a method of Thompson et al. (117) was then \
i
! employed. The tissue together with 10 ml of distilled ;
{ water and 20 ml of 95 per cent ethyl alcohol was placed in j
j
I
j the Waring Blendor, then 100 ml of Skellysolve B (b.p. j
I o 1
; 63.3-69.3 C) were added and the mixture was homogenized for
1 .
5 minutes. Following a 5 minute stationary period, the
mixture was rehomogenized for 5 minutes. Then the contentsj
i '
were transferred quantitatively to a separatory funnel, the
layers separated, and the homogenate layer was re-extracted
twice with 100 ml of Skellysolve B. The extracts were
' pooled, concentrated by evaporation with heat in vacuo,
! filtered into volumetric flasks, and adjusted to a definite!
i volume. Subsequently aliquots were taken for lipid and
1 *
cholesterol determination.
2. Soxhlet Method. This method was used for :
! extraction of lipids and cholesterol from carcasses and j
, also from feces. Total carcasses which were frozen follow-j
ing liver extirpation, were ground finely and thoroughly ;
i
homogenized in an electric meat grinder. Duplicate ;
I ' 4
: aliquots of 9 gm each were placed in a Soxhlet thimble and 1
35
refluxed for 8 hours with 95 per cent ethyl alcohol, then
for 8 hours with anhydrous ether. Most of the alcohol
extract was evaporated and the rest was combined with the
ether extract and finally transferred into a volumetric
flask and made up to a definite volume.
In the case of feces, the material was dried at
60°C, separated from adhering particles, weighed, and
thoroughly ground in a mortar. Duplicate aliquots of one
gm were extracted in Soxhlet thimbles as above.
3» Plasma extraction. The blood obtained from
the heart was treated with small quantities of heparin to
prevent clotting, and the plasma was obtained by centrifu
gation of the treated blood. Fourteen volumes of ethyl
aleohol-acetone (1:1 by volume) were added to each volume
of plasma by means of a syringe under sufficient pressure
to facilitate mixing, to precipitate the plasma proteins,
and to extract the cholesterol. The resulting precipitate
was centrifuged for 10 minutes at 3000 RPM and the extract
obtained was stored in a stoppered bottle.
Determination of cholesterol. The method used is a
modification of the Schoenheimer-Sperry method developed by
Nieft and Deuel (118).
An aliquot of the tissue extract containing approxi
mately 0.1 to 0.5 mg of cholesterol was evaporated to
36:
I
!dryness in a 60°C constant temperature apparatus by means !
i I
I of a stream of air. ' ■
I :
! l
One ml of ethyl alcohol-acetone solution (1:1 by (
i i
I volume) and 2 drops of 33 per cent potassium hydroxide were
added; the tubes were corked and the samples were kept at
'60 G for *+5 minutes with frequent shaking. The solution J
j !
jwas then neutralized to the phenolphthalein end point with I
I 1? per cent acetic acid. Then 2 ml of ethyl alcohol- !
i i
1 i
|acetone mixture (1:1 by volume) were added. ;
j The cholesterol digitonide was precipitated by the
I !
addition of one ml of 0.5 per cent digitonin solution (in i
\
50 per cent ethyl alcohol), and allowed to incubate at room !
temperature overnight. The precipitate was then centrifuged,
at a speed of approximately 3000 RPM for about 20 minutes. <
j
■The supernatant liquid was then carefully decanted and the
i
Iprecipitate was washed with 3 nil of anhydrous ether. This ;
i :
was then centrifuged at approximately 3000 RPM for 10
I !
minutes and the supernatant ether was discarded. The j
1 o !
Iprecipitate was dried with air at 60 G, 0.5 ml of glacial i
s
i o
acetic acid was added, and the mixture was heated at 60 C
j
juntil the precipitate was completely dissolved. Three ml of
{chloroform were added, and the tubes were brought to a
i
'temperature of 35°C.
j The color reagent, previously prepared by adding one ;
37
ml of concentrated sulfuric acid to each 9 ini of chilled
acetic anhydride, was then added. The tuhes were incubated
with occasional shaking for 10 minutes at 35°C at which
time the tubes were placed in an ice bath for at least 10
minutes prior to reading. The color intensity was then
read in a Klett-Summerson photoelectric colorimeter at 620
millimicrons. A reagent blank consisting of acetic acid,
chloroform, and color reagent was always used to set the
zero point on the colorimeter. Values were read from a
standard curve determined simultaneously.
For the determination of free cholesterol, the same
procedure used in total cholesterol was followed with
these exceptions: 1) Usually a larger aliquot was taken
for analysis, and 2) the hydrolysis step was omitted.
Determination of total lipids. An aliquot (25 or 50
ml) of tissue extract was evaporated almost to dryness in'
an Irlenmeyer flask on a hot plate. Charring was avoided.
Then 30 ml of Skellysolve B and approximately 5 gm of
anhydrous sodium sulfate were added and the contents well
shaken. The solution was filtered through a Whatman #+2
filter paper into a tared Erlenmeyer flask and rinsed with
five successive portions of Skellysolve B, passing rinsings
through filter paper and combining filtrates. The filtrate
was evaporated to dryness on a hot plate, held overnight in
38i
| an air oven at 88°C, and the lipids weighed. |
Isotopic studies. The method of Hotta et al. (119) j
j was adapted in the following way. j
1. Incubation procedure. The rats were j
I sacrificed by skull fracture. Their livers were quickly
I ■ 1
I excised, trimmed, blotted, and weighed. A portion of the j
liver was frozen and stored for further cholesterol
analysis. From the remainder, slices were prepared using
i
, a Stadie-Riggs microtome. Meanwhile, duplicate portion of \
I
liver awaited slicing in a Krebs-Ringer phosphate buffer
I
‘on an ice bath. The buffer solution was prepared according
i
: to Umbreit et al. (120). The slices were placed in the
I
beakers tared with a small amount of ice-cold buffer. The i
I
i beakers were reweighed with the slices and then the slices,1
i 1
j usually weighing between 300 and 600 mg, were transferred J
| into Erlenmeyer flasks containing 5 ml of buffer and one ml!
’ ill.
of sodium acetate-C which were equilibrating in a water
, bath at 37.5°C. The flasks containing the liver slices
were flushed with 95 per cent 0£ - 5 per cent C02 mixture,
I I
! stoppered, and placed back in the water bath where they
! J
;were shaken for 3 hours. f
2« Analytical procedure. The medium was
1
j decanted and the liver slices were rinsed with distilled I
water and hydrolyzed with 2 ml of a freshly prepared
solution of sodium ethylate (0.25 gm of Na dissolved in
10 ml of 95 per cent alcohol) for 30 minutes on the steam
bath. After the addition of a little water,, the flask was j
placed on a steam bath to evaporate the alcohol.. The ;
■ ’ i
contents were then acidified to the brom cresol green end i
point with concentrated HC1. Exactly 10 ml of chloroform j 1
i
t
were added, and the contents were vigorously shaken and j
transferred into a centrifuge tube. The chloroform phase |
j
was clarified by centrifugation. An aliquot of this j
chloroform solution was evaporated to dryness at 60°C in a
stream of air, and the residue redissolved in 5 ml of
ethyl alcohol-acetone mixture (1:1 by volume). About 5 ml !
of an alcohol-acetone solution of unlabeled cholesterol <
(10 mg per ml) were added and total cholesterol digitonide
was precipitated with a one per cent digitonin solution in |
50 per cent ethanol and allowed to digest overnight. The
t
following day the precipitate was centrifuged down at 3000 J
\ |
RPM for 30 minutes and washed three times with anhydrous
ether. The washings were repeatedly checked for radio
activity and only negligible counts were detected. Finally,
a definite volume of an ethanol suspension of the digitonid^
was prepared and duplicate planchets were prepared. They ;
were dried overnight in a desiccator and the radioactivity j
i
measured using an automatic Geiger counter with a mica
ko :
! window (Nuclear, Chicago). j
! ' • i
' A self-absorption curve was obtained by plancheting ,
! j
I increasing weights of radioactive cholesterol digitonide j
! from to 30 mg. The results on the biosynthesized cho- !
• I
i lesterol were corrected for background, self-absorption, <
I and expressed as counts per total liver.
!
I
|
! Study of liver lipids.
‘ i
J 1. Separation of fractions by chromatography. |
j The method was that of Fillerup and Mead (121), as adapted j
| by Achaya (122). :
i ;
! A silica gel column was prepared and washed ;
respectively with 3 column volumes each of methanol,
acetone, ether, and pentane in that order. Liver lipid
extracts were pooled, evaporated in vacuo. dried with
; anhydrous sodium sulfate, and redissolved in a small, j
! l
: definite amount of pentane. Duplicate aliquots were placed,
. on two prewashed columns which were run simultaneously. i
The first fraction— cholesterol esters— was collected by
ipassing through the column 10 volumes of pentane containing
one per cent ether.
Triglycerides and free cholesterol were eluted
together by washing the column with 10 volumes of pentane ;
plus 25 per cent ether, while the third fraction, phospho
lipids, was obtained by eluting with methanol-ether
(1:3 by volume).
I
All the three fractions were evaporated, dried with
anhydrous sodium sulfate, and weighed.
Isolation of free fatty acids. The choles
terol esters were refluxed with 10 ml of 0.25 N alcoholic
i
| sodium ethylate for k hours. Then the saponified mixture
I
}
| was diluted with *f0 per cent ethyl alcohol and left in the
»
; refrigerator overnight. Cholesterol crystals were filtered
; off. The filtrate was extracted with chloroform-ether-
! Skellysolve A (1:3:6 by volume) three times, extracts
i
| discarded and the soap acidified with 10 per cent sulfuric
‘ acid, and swirled gently. The acids were extracted three
, times with ether; ether extracts were washed with distilled
water, dried over anhydrous sodium sulfate, and filtered
! into a weighed flask. After the removal of solvent in
1 vacuo. fatty acids were weighed.
The triglycerides and free cholesterol fraction were
refluxed with 5 ml of 0.25 N sodium ethylate for 2 hours,
, then with 10 ml of 10 per cent alcoholic KOH for 3 hours.
, They were diluted with *+0 per cent ethanol and treated as
1 the first fraction.
The phospholipids were refluxed with 15 ml of one
'per cent alcoholic acid for one hour, then with 15 ml 10
per cent alcoholic KOH for 3 hours. After acidification
with 10 ml of 10 per cent sulfuric acid, the acids were
extracted three times with Skellysolve A, washed with
distilled water, dried over anhydrous sodium sulfate, and J
i
weighed following the removal of the solvent. '
3* Soectrophotometric determination of un
saturated fatty acids. Fatty acids originating from all '
three fractions were dissolved in chloroform, and a
definite aliquot was taken for further analysis. The pro- 1
cedure used is that of Herb and Riemenschneider (123). The
principle of it is that on treatment at high temperatures
with strong alkali, the double bonds rearrange themselves
alternately to give a conjugated system. These compounds
absorb light quantitatively in the ultraviolet region at
definite wavelengths and hence can be estimated. The
reagent was prepared by weighing out 24-. 7 gm of KOH adding .
t
to it 101 gm of ethylene glycol, heating over a burner to
190°C and cooling, following 15 minutes. Eleven gm of the ,
reagent was placed in long pyrex test tubes and kept in a
thermostatic bath at 180°C for at least one hour.
An aliquot of the chloroform solution of fatty acids;
was put in a small glass vial and evaporated on a water
bath. Caution was observed to assure the presence of the
residue in the lower part of the vial exclusively by
repeated rinsing of the sides with chloroform by the aid of
" ' ^3'
1 i
a dropper. Finally, the top of the glass vial was cut off
using oxygen flame and the resulting capsule containing th
| residue was placed in the tube with the reagent. An empty .
capsule was added to the blank tube. The contents were !
{
, agitated frequently with the glass rods. After exactly 15 '
I minutes the tubes were inserted into a beaker of overflow- ,
* ‘ l
ing cold water. Subsequently, the contents of the tubes j
: l
were carefully transferred to volumetric flasks and made up.
>to volume with absolute methanol. Usually a tenfold re-
dilution was required.
The solutions were read at specified wave lengths on
a Beckman DU spectrophotometer. The following specific
extinction coefficients were used in calculations:
Acid I.V.
233
268
315
3*f6 375
dienoic 181.0 91.6 - - - -
trienoic
273.5 ^7.5 90.5
- - -
tetraenoic
333.5 39.7
if 8.2 60.6 - -
pentaenoic 38*f .6
**3.5
if 6.0
56.9 50.if
-
hexaenoic ^ . 3 ifO.O *f9.if
27.8 26.2 28.1
if. Determination of iodine value. To an
aliquot of chloroform solution of fatty acids, a definite
amount of 0.1N iodine monochloride in glacial acetic acid
(Wijs reagent) was added. Blanks were run simultaneously.
; " " . . . . . . . . . ~ M T i
I . i
: The glass-stoppered flasks were kept in the dark for one
I
; hour. The reaction was stopped hy adding 15 ml of 10 per !
I i
cent KI solution, and after a few minutes the mixture was |
i
titrated with 0.1 N sodium thiosulfate. The iodine value j
; was expressed as the number of grams of iodine absorbed by
!
j 100 gm of fatty acids.
| Iodine value contributions of polyunsaturated fatty
i
I acids were calculated from percentages of these acids as j
; i
obtained from the spectrophotometric data and known iodine j
i values for each acid. This allowed the calculation of j
! iodine value contribution of oleic acid by difference and I
l
in turn the percentage of oleic acid in the mixture. i
Eventually the content of saturated fatty acids was
obtained by difference between total weight of fatty acids
: and the weight of unsaturated fatty acids.
This method was used for the analysis of the two !
f
1
| dietary fats, lard and cottonseed oil, used in the follow- i
; I
! ing investigation.
Thiobarbiturate test for fat oxidation. The method
of Patton and Kurtz (12^) was used. Melted fat (3.0 gm)
was weighed into a 50 ml Florence flask and combined with
7-5 ml of distilled water, 6.0 ml of reagent, and 3»0 ml
of 20 per cent trichloracetic acid solution. The flask was,
i
fitted to a condenser and gently refluxed for 10 minutes. ;
:
An aliquot of the aqueous layer was read in the Klett-
Summerson colorimeter with a #52 filter. Distilled water
was used as the blank solution.
The reagent consisted of thiobarbituric acid
solution combined with citrate buffer in proportion 2:1 by I
volume and pH adjusted to 2 ,b , Thiobarbituric acid i
solution was prepared by dissolving 2 gm of acid in 193 ml
of water with 6.6 ml of 2 N sodium hydroxide; after heatingj
0.7 ml of b N HC1 was added and the mixture decolorized by
shaking with charcoal.
Liver function test. The procedure of Frommer (125)'
was adapted as follows. Bromsulphalein solution was
injected intraperitoneally into rats to supply approx
imately 0.125 mg bromsulphalein per gm of body weight.
After exactly 20 minutes, a few drops of blood were
collected from the tail vein of the lightly ether-
anesthetized rat. To 0.30 ml of whole blood, one ml of 2
per cent saline solution of potassium oxalate was added and
the mixture was centrifuged at 3000 RPM for 10 minutes.
Diluted plasma (0.5 ml) was removed and placed in a" tube to
which subsequently one ml saline, 0.5 ml 10 per cent NaOH,
and 5 ml distilled water were added. The color was read
after 10 minutes in a Coleman Junior spectrophotometer at
565 millimicrons. The readings were referred to a standard
curve previously prepared with known amounts of brom
sulphalein and expressed as mg per 100 ml of plasma.
Plan of Experiment.
Plasma and liver cholesterol levels. Male and
female rats were kept after weaning on 15 per cent lard
and 15 per cent cottonseed oil diets. Some of them were
fed LC and CsC diets for 6 weeks prior to sacrificing. At
6, 12, 18, and 2k weeks certain groups (6 to 10 rats per
group) were sacrificed and cholesterol levels in plasma
and liver, and also total liver lipids were determined.
Liver extracts of each group were pooled for further study
of fatty acid pattern (see below).
Cholesterol levels in carcasses. Male and female
rats, which had been fed L and Cs diets for 2*+ weeks,
following sacrificing and removal of liver were analyzed
for the total amount of cholesterol and lipids present in
the carcasses.
The effect of equalizing cottonseed oil cholesterol
level with that normally occurring in lard. Lard was
analyzed for its cholesterol content. Cholesterol was
added to cottonseed oil in an amount to equal the choles
terol content of the lard, and the diets were mixed using
i the regular cottonseed oil (Cs) and the cholesterol- 1
> I
i
'enriched oil. Following 6 weeks on these diets, male rats J
1 i
!(ten animals per group) were sacrificed and analyses
I
!performed as above. i
j
1 The effect of vitamin E on cholesterol levels in male
'rats fed a diet containing 15 per cent lard. Male weanling j
i ” ' !
I rats, ten per group, were fed for 6 weeks 15 per cent lard j
'diet containing no vitamin E with and without one per cent j
cholesterol, with a fivefold amount of alpha tocopherol and j
» j
without any tocopherol added. After sacrificing, plasma ;
i
and liver cholesterol levels and liver lipids were
I
determined.
Digestibility studies. Male and female rats which
were fed L and Cs diets for about 10 weeks were given diets ,
* ' i
;LC and CsC. After a 2 weeks' period of orientation, food !
consumption was measured and feces collected individually i
t
‘ for a period of 2 weeks. Finally, cholesterol and lipids !
were determined in the diets and in the feces, and the per j
cent absorbed was calculated. j
Cholesterol biosynthesis in the liver. Male and
female rats fed L and Cs diets for approximately 2b weeks ,
and LC and CsC diets for approximately 8 weeks were used for.
these studies. Cholesterol biosynthesis was studied in :
| liver slices by incubating the slices with radioactive :
i
I t
j acetate and checking the total counts incorporated. The '
I i
| results were expressed as total counts per liver. Total
| cholesterol was also determined in these livers. j
! ■ 1
Thiobarbiturate test for fat oxidation. The thio- j
I
barbiturate test was performed on lard which had been j
stored in the cold room for 2 months and on cottonseed oil
!
j which had been stored in the same conditions for the same
1 length of time. 1
! Liver function test. A bromsulphalein retention j
i
' test was performed on male rats (five per group) which had !
■ j
been fed L and Cs diets for about 2b weeks and LC and CsC '
. diets for 6 weeks.
- Study of fatty acid components of isolated liver :
• fractions. Pooled liver extracts from rats kept on 15 per !
i
■ cent lard and 15 per cent cottonseed oil diets and from
rats kept on 15 per cent lard with and without the addition:
I
of vitamin E were evaporated and after removal of the
solvent, they were fractionated into cholesterol esters,
triglycerides plus free cholesterol, and phospholipid
fractions. These, in turn, were analyzed for their fatty
; acid composition.
CHAPTER III :
* *
i
j RESULTS l
I t
i
I I
I Plasma and Liver Cholesterol Levels.
The results of the plasma cholesterol determination
i
jin male and female rats fed L, Cs, LC, and CsC diets and
I sacrificed at various intervals are presented in Tables II
I . |
;and III. In all cases where the animals were fed the lard
: j
diets, there is a noticeable effect of dietary cholesterol;
however, in the animals on the cottonseed oil diet, the J
i
effect of dietary cholesterol is evident only in the
females after 18 weeks. A sex difference is obvious,
therefore, in the rats fed the supplemented cottonseed oil
I
.diet. The effect of the dietary cholesterol seems to be
reflected more in the esterified cholesterol content of the
i l
plasma than in the unesterified portion. There is an ;
increase in esterified cholesterol in the plasma of animals :
on the lard diet as early as 6 weeks after animals are
placed on the experimental diet. In three cases there is
a significant increase in free cholesterol— males and
females fed lard and cholesterol for 18 weeks and females
fed cottonseed oil and cholesterol for 18 weeks. The ef-
i
feet of cholesterol supplementation on the free cholesterol :
content of the plasma is less marked and does not occur
TABLE II
THE PLASMA CHOLESTEROL LEVELS IN MALE AND FEMALE RATS FED
A DIET CONTAINING EITHER 15 PER CENT LARD OR 15 PER CENT
COTTONSEED OIL FOR 6, 12, AND 2k WEEKS
Lard Cottonseed oil
Weeks Sex
Plasma cholesterol Plasma cholesterol
Totali/ Freei^ % Free Totali^ Freei/ % Free
6 M
mg%
80.5*3.2
mg %
23.8±1.8 29 . k 8M-.1+M-.2
mg%
20.9tl.5 2M-.6
12 M 75.k±k.? 23.6*1.2 31.6
79.6*5.5
22.9*1.2
28.9
F 88.M-*M-.8 28.3tlA 32.0 8l.M-±2.8 22.1*1.8 26.9
2k M
77.9*3.5
2M-.0tl.M- 30.8
61.9+3.9 22.M-+1.7 36.1
78.0*M-.8 21.3*1.M-
27.3 69.M*M-.5
22.9*1.M
32.9
-^Including Standard Error of Mean.
TABLE III
THE EFFECT OF AGE AND CHOLESTEROL FEEDING ON MALE AND FEMALE
RATS FED A DIET CONTAINING EITHER 15 PER CENT LARD OR 15 PER
CENT COTTONSEED OIL AS REFLECTED BY PLASMA CHOLESTEROL LEVELS
Plasma
Cholesterol
Sex
Lard Cottonseed oil
LC 6 wks. L 12 wks.
LC 6 wks.
CsC 6 wks. Cs 12 wks.
CsC 6 wks.
Total, mg$-^ M
118.1±9.1 10^.7±11.7
85.6tl f.l f 89.^16.8
F
165. *+±19.3 1^1.6116.7
Free, mg^/ M 26.612.0 37.116.2 23. Oil. 26.511.8
57.11*+. 7
3l +.Vl5.0
%
M 23.3
35.**
26.7 29.6
F
3
2^.2
-^Including Standard Error of Mean.
52 ;
until much later.
I
The average lipid and cholesterol levels in the j
I
liver of male and female rats kept on L and Cs diets for
6, 12, and 2*f weeks are presented in Table IV. j
Cholesterol levels in rats of both sexes fed Cs !
diet are lower than the levels found in L fed animals. In !
t
<
contrast to the results obtained in the plasma, the per- ;
i
centage of free cholesterol is fairly high on both diets, |
reaching over 90 per cent on Cs diet, which is normal for
this species. The sex difference is evident, with females
having lower cholesterol levels, contrary to the situation
in plasma (see Tables II and III).
As the animal matures the effect of age shows a
slight influence on the cholesterol values. There is a ;
slight increase in the total cholesterol of the liver which
j
finally levels off. The liver weights on both diets are ;
quite similar, thus the differences obtained as a result of;
diet are not a function of the size or weight of the
livers.
The total liver lipids seem to be higher on the
lard diet; however, the differences are not statistically
significant. It is of interest that the weights of male
rats at 12 weeks on Cs diet were slightly above those on L
diet (the average values being 329 gm versus 303 gm),
TABLE IV
THE AVERAGE LIVER LIPID AND CHOLESTEROL LEVELS IN MALE AND FEMALE
RATS FED A DIET CONTAINING EITHER 15 PER CENT LARD OR
15 PER CENT COTTONSEED OIL FOR 6, 12, AND 2*- WEEKS
Category Sex
Lard Cottonseed oil
6 wks 12 wks 24- wks 6 wks 12 wks 2*- wks
No. rats M 10 6 ‘ 10 10 6 10
F
9 9
-
9
10
Liver wt, gm
M 9.8 10.*- r 12.5 9.8
10.7
12.2
F ■ -
6.9
8.8
-
7.1 7.5
Lipids, .
mg/gm liver*'
M 4*9.1*2.0 58.*+3.6 58.b±3.6 *■8.6*2.8 53.2*1*. 2 38.9*2.0
F - 52.7*2.0 50.1*2.0 ■-
*-9.6±1.7
b 6 .0*2.0
Cholesterol,
mg/gm liveri/
Total M
2.77±0.39
3.25*0. lb
3.18*0.17
2.76*0.26 2.66*0. 1*- 2. *-8+0.12
F -
2.58*0.03 2.65+0.05
-
2.1 +0+0.06
2.37*0.09
Free M 2.22+0.28
2.*+2*0.09 2. *-3*0.10 2.15*0.20 2.36*0.12 2.15*0.0*-
F - 2.27*0. 0*-
2.13*0.05
- 2.22*0.06
2.07*0.07
! *
M
80.5
75.0 7 8 .b 78.0
88.7
86.6
i
i
F — 88.0
80.3
— 92.8
87.3
-^Including Standard Error of Mean.
■ while at 2b weeks the respective values were 392 gm versus
'353 gm despite the fact that the diets were isocaloric and
'the amount of food intake was approximately the same. This
i
would indicate that the male rats were able to utilize the
!
i Cs diet more effectively than the L diet. Female rats did
i
'not show this difference.
!
The average liver lipid and cholesterol levels of
male and female rats kept on L and Cs diets for 6 and 18
weeks with cholesterol supplemented to the diet for the
last 6 weeks only are presented in Table V.
Here, again, dietary differences are reflected in
both cases and in both sexes. However, the differences are
more striking in the case of females. In the animals on
the lard diets, it can be seen that the female rats show
lower levels in all categories: total liver lipids, where
the value for the males is 131.5, for the females 119.7;
total liver cholesterol, where the values for males are
39.8, and females 31*6; and in the free cholesterol, where
the values are for males 20.1, and females 7*30. The same
sex difference obtains in the animals fed the cottonseed
oil diets supplemented with cholesterol and is, in fact,
more striking except in the case of the free cholesterol
content of the liver. The other values, however, are liver
lipids, males 127.6, females 97*1> and total cholesterol,
TABLE V
THE EFFECT OF AGE AND CHOLESTEROL FEEDING ON MALE AND FEMALE RATS
FED A DIET CONTAINING EITHER 15 PER CENT LARD OR 15 PER CENT COTTON
SEED OIL AS REFLECTED BY LIVER LIPID AND LIVER CHOLESTEROL LEVELS
Category Sex
Lard Cottonseed oil
LC 6 wks. L 12 wks.
LC 6 wks.
, CsC 6 wks. C 12 wks.
CsC 6 wks.
No. rats M
F
10
9
9
9
10
10
Liver wt, gm M
F
11.7
11.6
7.9
12.2 12 A
7.8
Lipids,
mg/gm liverA'
M
F
117. **8.8
131.5*8.1
119.718.1
100.717.^ 127.6*9.^
97.1+6.1
Cholesterol .
mg/gm liver-'
Total M
F
26.912.9
39.813.0
31.6+3.7
22.011.9 32.113.5
16.^±1.7
Free M 7.^10.8 20.111.
7.311.1
^.J+to.1 * 5.710.6
5.6+0.8
%
M 26.2 51.8
25.^
21.U-
I8.7
3^.6
. . i/'lncluding-Standard Error of Mean.
'males 32.1, females 16. if.
' A comparison of liver cholesterol values of animals ;
i fed the different diets shows that in each case the values j
jobtained when the animals are fed the lard diet supplemented
|with cholesterol are higher than the values obtained when !
ithe fat is cottonseed oil, supplemented with the same ;
,amount of cholesterol. In male rats on a lard diet, total '
, / i
! J
liver cholesterol at 6 weeks is 26.9, and at 18 weeks 39.8; :
while on cottonseed oil diets these values are 22.0 and
32.1, respectively. In the case of female rats the values
are, on LC 31*6 and on CsC 16.if.
It is noticeable that in both cases most of the
cholesterol is stored in the form of ester, contrary to the
normal picture when no exogenous cholesterol is present,
i There is an obvious difference in the way that
animals of different ages handle exogenous cholesterol |
since there is an increase in accumulation of liver choles
terol and total liver lipids with age of the animal,
although, actually cholesterol was fed for identical
periods.
Liver weights are similar on both diets.
Cholesterol Levels in Carcasses.
When the carcasses of male and female rats kept for
2*f weeks on L and Cs diets were analyzed for cholesterol,
51
the results presented in Table VI were obtained.
Each value represents an average of five determina
tions done in duplicate on aliquots of the hydrolyzed
carcass. It can be seen that the total cholesterol con
tent of the carcass is lower in the female rat than in the
male; however, when the weights of the rats are considered
and cholesterol values reported per weight of animal, no
differences are observed.
The effect of diet is significantly exhibited here
in cholesterol levels, where higher values obtain in the
animals fed the lard diet as compared with the animals fed
the cottonseed oil diet for the same length of time.
Thus, total cholesterol in male rats on a lard diet is
2.06 per cent of carcass, while on a cottonseed oil diet
it is I.69 per cent. For the females these values are
2.02 and 1.88, respectively.
When the total lipids were determined, the values
for males and females on a lard diet were 12. k and 19.8
and on a cottonseed oil diet 16.9 and 23.3 per cent,
respectively.
The Effect of Equalizing Cottonseed Oil Cholesterol Level
with That normally Occurring in Lard.
The cholesterol level in the lard used for feeding
was determined by the methods previously described and was
TABLE VI
AVERAGE CHOLESTEROL LEVELS IN CARCASSES EXCLUDING LIVER
OF RATS RAISED ON L AND Cs DIETS FOR 2b ¥EEKS-/
Group Sex
Average
body wt
Cltiolesterol
Total
per carcass
Free
% of total
Total ?/
% of carcass-'
gm mg
L M 358 67*+l 81+. 1
2.0610.09
L F 2¥+
*+571 82.3 2.02±0.03
Cs M
375 5969
87.0 1.6910.06
Cs F 230 • ' 398*+
87.^ 1.88+0.09
i^Five rats per group.
-^Including Standard Error of Mean.
59
found to be 0.08 per cent. This value closely agrees with
that of 0.09 per cent reported in the literature (75).
When male rats were fed cottonseed oil to which 0.08
per cent cholesterol had been added, for 6 weeks and the
cholesterol values of plasma, liver, and liver lipids were
compared with those obtained when unsupplemented cottonseed
oil was fed, no differences in the levels were observed.
The results are presented in Table VII.
It can also be noticed that these values compare
quite well with results previously obtained for diet Cs as
shown in Tables II and IV.
The Effect of Vitamin E on Cholesterol Levels in Male Rats
Fed a Diet Containing 15 Per Cent Lard.
Since the lard used in this investigation was
vitamin E-deficient, whereas vitamin E normally occurs in
cottonseed oil, an experiment was undertaken to determine
the effect of vitamin E on the cholesterol levels of
plasma and liver on male rats fed a diet containing 15 per
cent lard with and without the addition of one per cent
cholesterol. The results of this experiment are presented
in Table VIII.
It can be observed that the effect of vitamin E on
endogenous cholesterol is scarcely evident in the liver.
Thus, the values for total cholesterol without vitamin E
TABLE VII
THE AVERAGE PLASMA AND LIVER CHOLESTEROL VALUES AND LIVER
LIPID LEVELS OF MALE RATS KEPT FOR 6 WEEKS ON A 15 PER
CENT COTTONSEED OIL DIET SUPPLEMENTED WITH CHOLESTEROL
TO EQUAL ITS CONCENTRATION NORMALLY OCCURRING IN LARD
Category Cottonseed oil Cholesterol supplemented
cottonseed oil
No. rats 10 10
Liver lipids
mg/gmi'
52.013.5
*+9.512.if
Plasma cholesterol
mg 0/
Total 83.716.0 87.*+±1.5
Free 28.212.2 29.611.^
%
33.6 33.8
Liver cholesterol
mg/gmi/
Total 2.^-710.11 2.ifl!0.13
Free
1.9710.13
1.8910.08
%
79.7
78 A
'^/'including Standard Error Mean.
TABLE VIII
THE AVERAGE PLASMA AND LIVER CHOLESTEROL LEVELS AND LIVER LIPID
LEVELS OF MALE RATS FED A DIET CONTAINING 15 PER CENT LARD WITH
AND WITHOUT THE ADDITION OF VITAMIN E AND WITH AND WITHOUT THE
ADDITION OF ONE PER CENT CHOLESTEROL
Category
- Cholesterol + Cholesterol
- Vit. E + Vit. E - Vit. E + Vit. E
No. rats 10 10 10
9
Liver lipids
mg/gmi'
if8.2+2.0
^•3.8*2.0 118.^*7.^ 100.6±6.7
Liver cholesterol
mg/gmi/
Total 2.76+0.2^ 2.50*0.02
37.3*^.9 23.3*3.3
Free
2.30+0.19
2.22*0.02 if. 00*0.02 3-50*0.0
%
83.3
88.8
10.9
15.0
Plasma cholesterol
mg$i/
Total 69.5*1.0
62.8±3 •*+ 93.9*7.9
92.if±2.1
Free
18.9*1.5
17.if*1.8 19.3*1.8 22A+3.2
*
27.2 26.1
20.5
2*f.2
Including Standard Error of Mean,
is 2.76 whereas with an amount of alpha-tocopherol equal
to five times that normally occurring in cottonseed oil
added to the lard diet the total liver cholesterol is 2.50. ,
This rather insignificant effect is also reflected in total
plasma cholesterol levels (69.5 and 62.8) and total liver i
lipid (W.2 and ^3.8)'.
However, a significant reduction of cholesterol
level in the liver obtains when cholesterol is added to the
diet with vitamin E as compared with the cholesterol in
the liver when cholesterol is fed without the addition of
vitamin E (37*3 versus 23.3 for vitamin E-supplemented
diet). In addition, there is a similar reduction in liver
lipids (118.^ versus 100.6). However, there is no effect
on plasma cholesterol levels, the values being much the
same in both cases (93-9 and 92. b ) .
It should be noted here that the total tocopherol
content of lard is 0.0005-0.0029 per cent, while that in
cottonseed oil is 0.087-0.095 per cent (126).
Digestibility Studies.
The digestibility of cholesterol in the presence of
the two fats was determined to discover whether an
increased cholesterol digestion on the lard diet could be
responsible for the higher cholesterol values obtained.
The results are shown in Table IX. It can be seen that in
TABLE IX
T H E D I G E S T I B I L I T Y O F C H O L E S T E R O L I N M A L E A N D F E M A L E R A T S F E D A D I E T C O N T A I N I N G j
E I T H E R 1 5 P E R C E N T L A R D O R 1 5 P E R C E N T C O T T O N S E E D O I L F O R 2 WEEKS*'
Group Sex
Lipids Cholesterol i
Ingested '
gm
Excreted
gm
% Absorbed^/ Ingested
gm
Excreted
gm
% Absorbed^/;
LC M
2 8 . 5 2 . 7 9 0 . * + + 0 . 7 1 . 9
1 . 0
* + 8 . 2 + 1 . 5 j
F 2 * + . 9 1 . 7
92. 8 + 0.8
1 . 5 0 . 7
5 5 - 5 + 2 . 6 j
CsC M. 3 0 . 2 2 . 2 9 2 . 3 + 0 . 1
1 . 9
1 . 0
* + 7 . 0 + 2 . 5 i
1
F 2 1 . 2
1 . 3
9 3 . 7 + 0 . 2
1 . 3
0 . 6 5 * + . 2 + 2 . 2
-^Five animals per group.
2/
- Including Standard Error of Mean.
male rats the amount of lipid absorbed was approximately
the same on both diets (90.*+ per cent and 92.3 per cent)
and the cholesterol absorbed was also unaffected (*+8.2 per
cent and *f7»0 per cent). In the case of female rats, the
amount of total lipid absorbed and cholesterol absorbed
was also unaffected by the type of fat in the diet (92.8
per cent and 93*7 per cent for total lipid and 55*5 per
cent and 5^*2 per cent for cholesterol). However, it is
interesting to note that the female rats digested higher
quantities of cholesterol on both diets, i l lthough the
amount of lipid absorbed by female rats is only slightly
higher than the per cent absorbed by male rats (127), the
per cent digestibility of cholesterol is considerably
higher in the female than in the male. Here, again, a
sex difference is observed.
Cholesterol Biosynthesis in the Liver.
Tables X and XI show the results of an experiment
to determine biosynthesis of cholesterol in the livers of
male and female rats fed a diet containing 15 per cent lard
of 15 per cent cottonseed oil with and without one per cent
cholesterol. This experiment was performed using liver
slices incubated with CH^C^OONa (about 13,000 counts per
minute per ml). The values presented in Tables X and XI
represent an average of very closely matching duplicate
TABLE X
THE INCORPORATION OF C^-ACETATE IN VITRO INTO LIVER
CHOLESTEROL OF MALE RATS PREFED EITHER A DIET
CONTAINING 15 PER CENT LARD OR 15 PER CENT
COTTONSEED OIL WITH OR WITHOUT THE ADDITION
OF ONE PER CENT CHOLESTEROL
Group Liver wt.
Total
liver cholesterol
cpm/Liver
x 103
Average
cpm/liver
gm
mg/gm
mg/liver
x 103
L 10.0 . 2.3
23.0 56.8 28. * 4 -
l*4-.*4- 2.*4-
3*+. 6 6.9
lb.2 2.2
31.3
16.2
11.9 2.3
27.b *4-0.0
l*f.9 2.5
37.2 22.0
Cs 12.1
2.3 27.9 7.5
*4-1.0
13.0 1.9
2 b.7 1*4-. 5
12.0 1.9
22.8 19.0
12.H- 1.8
22.3
80.0
8.0 2.7
21.6 8*4-.0
LC 1*4-. 8 37.0 5b7.6 8.5
* 4- . 1
12.2
£8.9
596.6 2.*4-
10.8 55.9 603.7
l.*f
CsC 10.6 k&.5 516.5 3.5 2.3
10.1 bo.5
*4-09.0 1.8
12.2
70.3
858.0
1.6
66
TABLE XI
THE INCORPORATION OF C^-ACETATE IN VITRO INTO LIVER
CHOLESTEROL OF FEMALE RATS PREFED EITHER A DIET
CONTAINING 1 5 PER CENT LARD OR 1 5 PER CENT
COTTONSEED OIL WITH OR WITHOUT THE ADDITION
OF ONE PER CENT CHOLESTEROL
Group Liver wt.
gm
Tc
liver c
mg/gm
>tal
iholesterol
mg/liver
cpm/Liver
x 1 0 3
Average
cpm/liver
x 1 0 3
L 9 . 2 2.k 2 2 . 1 5 5 . 0 6 2 . 0
7 . 9
2.k 1 9 . 0 5 6 . 0
9 . 5 2 . 5 2 3 . 9
7 5 . 0
Cs 8 . 8 2 . 2 15.0 17.0
2 5 . 7
7 . 0 2.b 1 6 . 8 3 5 . 0
8 . 3 2.b
1 9 . 9
2 1 . 0
7 . 2 2.b 17.2 3 0 . 0
LC 8 . 3 5 3 . * +
¥ * 3 . 2
2 . 7
2 . 6
9 . 1
¥).*+ 3 6 7 . 6 1 . 8
1 1 . 5
78.6 9 0 7 . 8
2 . 7
1 1 . 1 8 2 . 3 9 1 3 . 5 3 . 3
CsC
7 . 7
3 * + . 2
2 6 3 . 3
not 0 . 8
significant
1 0 . H - 6 1 . 2
6 3 6 . 5 1 . 3
9 . 9
8 2 . 0 8 1 2 . 0 1 . 2
planchets of fairly similar duplicate portions of the liveri
It appears that the presence of large amounts of choles- ;
i
terol in the liver, for example, in the livers of rats ,
prefed a one per cent cholesterol diet, inhibits the in- i
corporation of acetate into cholesterol. There is no !
significant difference in the biosynthesis of cholesterol ,
i
as related to the dietary fat in male rats. In the case :
of female rats, however, the animals fed lard seem to have 1
an accelerated biosynthesis over that of female rats on
the cottonseed oil diet.
In general, there is a great variability in values
among the animals tested, which not always can be related
to the cholesterol level in the liver.
Thiobarbiturate Test for Fat Oxidation.
In order to determine the degree of oxidation in
the two fats used, both fats were tested for oxidation
using the thiobarbiturate test. The Klett readings for the
lard sample were much higher than those for cottonseed oil,
namely **35 versus b j. Both fats had been stored in the
cold room for about 2 months. There was a negligible
difference between the lard from the surface and lard from
the inside of the container, namely ^35 versus b-25. This
high value results despite the fact that an antioxidant had
been added to the lard samples.
68 I
I
Liver Function Test.
In order to determine whether the two fats had
i
caused any liver damage in the animal, the Bromsulphalein
!
Retention Test was performed on five male rats picked at j
!
random from each of the L, Cs, LC, and CsC diets. The rats;
in groups L and Cs had been on their respective diets for
2b weeks; whereas in the case of LC and CsC groups,
cholesterol was included in the diets during the last 6
weeks.
The average results obtained are as follows:
Group Bromsulphalein
mg per 100 ml plasma
L
7.7
+
1.9
Cs 6.3 + 2.1
LC 9.6
2.7
CsC
8.7
+ b.O
It can be stated that if any differences in liver
damage are present, these are barely significant, although !
all values are higher than those which obtain in normal
Purina-fed rats, namely zero. However, a true damage to
the liver which occurs in rats given chloroform or carbon ,
tetrachloride yielded values of 26-28. Therefore, it may
be concluded that a change in liver function in all cases
is slight.
: Studies of Fatty Acid Components of Isolated Liver Lipid '
! Fractions. I
i ’ 1
j Pooled liver extracts of male and female rats on L ;
i
and Cs diets at 2k weeks and LC and CsC diets at 18 weeks j
I
were analyzed for their fatty acid content. The results
i are presented in Tables XII, XIII, XIV, and XV. !
! j
In Table XII is presented the percentage distribu- i
tion of the three lipid fractions: cholesterol esters, [
t •
■ triglycerides, and phospholipids in pooled liver extracts
of rats on four different diets.
i
| A sex difference is apparent on the diets without
1 cholesterol, where the cholesterol esters are less abundant
‘ at the expense of triglycerides and phospholipids which j
i
, show increases in the case of females. In addition, males '
< \
| and females on the lard diet have considerably more choles-1
‘ terol esters than males and females on cottonseed oil l
• i
i • \
diets. Also, it can be seen that the concentration of j
phospholipids is significantly greater in the liver of *
animals fed the cottonseed oil diet. ;
The distribution of fatty acids of cholesterol
esters, triglycerides, and phospholipids in pooled liver
extracts is shown in Tables XIII, XIV, and XV.
3 7 i
It can be seen that whereas the unsaturation level ■
(particularly the dienoic acid) of cholesterol esters and ,
70
TABLE XII
THE PERCENTAGE DISTRIBUTION OF CHOLESTEROL ESTERS,
PHOSPHOLIPIDS AND TRIGLYCERIDES IN POOLED LIVER EXTRACTS
L LC Cs CsC
Category
M
F M F M F M F
Cholesterol
esters, %
63 32 1*0+-^ 5k
3° 19
61 kk
Triglycerides,
% 31 **3 36 37
k6
55 25 36
Phospholipids, % 5
2k 12 9 21
25 13 19
Some of the cholesterol esters were lost in
transfer.
TABLE XIII
THE PERCENTAGE DISTRIBUTION OF FATTY ACIDS OF
CHOLESTEROL ESTERS IN POOLED LIVER EXTRACTS
Category
. L LC Cs CsC
M F M F M F M F
Saturated
53 *+9 15
21 60
53 23
22
Monoenoic b5 b? 82 76 22 b?
b9 b?
Dienoic 2 2 2
3
11 - - 26 26
Trienoic - 1 1 2 - - 2
Tetraenoic 1 - -
5
- 1
3
Pentaenoic
- - - - - - - -
Hexaenoic
- - - - -
1 -
I.y. 50 5b 80 72 59 ^3
9b 103
72
TABLE XIV
THE PERCENTAGE DISTRIBUTION OF FATTY ACIDS
OF TRIGLYCERIDES IN POOLED LIVER EXTRACTS
Category-
L LC Cs CsC
M F M F M F M F
Saturated k6 32 27 **3 55
¥f 28 2*f
Monoenoic ho hQ
67 *+9
-
23 37
32
Dienoic 8 8 if 6
27
26
31 37
Trienoic 2 1 1
-
1 if 1 2
Tetraenoie 2
7
1 2 12
3 3 5
Pentaenoic 1 2
- -
3
- - -
Hexaenoic 1 2 - - 2 - - -
I.V. 70 101
67
60 108
103 103
/
117
73}
TABLE XV
THE PERCENTAGE DISTRIBUTION OF FATTY ACIDS
OF PHOSPHOLIPIDS IN POOLED LIVER EXTRACTS
Category
L LC Cs cse
M' F M F M F M F' 1
t
i
Saturated
79 63
38 ,70
73 69
70
i
29,
Monoenoic - 16 b$ b - 18 - 21 ’
\
Dienoie
7 7
8 11
9
8 l*f 21
Trienoic
3 3 3 3 5
-
5 3
Tetraenoic
3
if 3 5 b
-
b 23 ;
Pentaenoie
3
2 2 2
3
2 2 1
Hexaenoic
5 5
1
5
6
3 5
2
I.V. 60
77 91
78 88 50 7b
3-53 ;
i
i
triglycerides is significantly increased on Cs and CsC
diets over that of fractions isolated from L and LC
groups, that of phospholipids is much less influenced by
dietary fat. The penta- and hexaenoic acids though are
present almost exclusively in the phospholipid fraction in •
all groups.
When cholesterol is added to the diet, a consider
able quantity of it becomes associated with dienoic acid
if available. This occurs in the case of rats fed the diet
containing cottonseed oil (26 per cent)} whereas, since
lard contains much less dienoic acid than cottonseed oil,
the dienoic ester of cholesterol is reduced to 2 and 3 per
cent, and the monoenoic acid increases from by and b7 per
cent on cottonseed oil to 82 and 76 per cent on the lard
diet. A similar although not as striking effect is
observed in the triglyceride fraction.
Sex differences in the fatty acid pattern are not
evident.
Pooled extracts of livers of rats fed the lard diet
to which vitamin E and/or cholesterol had been added were
also subjected to analysis for fatty acid distribution,
and separation of the three lipid components. The values
presented in Table XVI were observed.
The presence of exogenous cholesterol caused the
TABLE XVI
THE PERCENTAGE DISTRIBUTION OF FATTY ACIDS OF CHOLESTEROL ESTERS AND
PHOSPHOLIPIDS OF POOLED LIVER EXTRACTS OF ANIMALS FED 15 PER CENT LARD
WITH AND WITHOUT VITAMIN E AND WITH AND WITHOUT ONE PER CENT CHOLESTEROL
Category
■SBSSM. .^.======a=--: .J U S J S S S S S B S S S S . -----
Cholesterol esters Phospholipids
L-E L+E LC-E LC+E L-E L+E LC-E LC+E
Saturated 30 27
26
13
68 b9
53
bQ
Monoenoic
59
61 *
67
82
-
11 16 12
Dienoic
9 5 5
b 12
7
12 11
Trienoic - 1 1 1 b
5
6
7
Tetraenoic 2
3
1 ■ - 10 18 . 8 16
Pentaenoic - - - -
_ _
1 2 1 1
Hexaenoic
- - - -
5
8 b
5
I.V. 78 79 75
. 8**- 96 lb? 107
136
increase in the cholesterol ester fraction which has been j
observed before (Table XII) with a concomitant decrease in -
the phospholipid fraction.
The presence of large amounts of tocopherol induced j
I
higher unsaturation in the fatty acids, particularly in
phospholipid fraction (I.V. of l*+7 as compared with 79
without tocopherol). At the same time, although the con
centration of dienoic acid did not change in the choles- •
terol esters fraction, the amount of monoenoic fatty acid
increased at the expense of the saturated fatty acids.
CHAPTER IV
; DISCUSSION
The major observation which has resulted from this
I investigation is that the type of dietary fat is an
important factor in influencing cholesterol metabolism in
, the rat. As a result of experiments comparing the effects
1 of a vegetable fat, such as cottonseed oil, with an animal
fat, such as lard, on cholesterol levels in plasma and
liver, with and without the addition of cholesterol to the
diet, on cholesterol distribution in the animal body, on
cholesterol digestibility, on cholesterol biosynthesis in
the liver, on cholesterol concentration in total liver
t
lipids, and on the chemical composition of cholesterol
esters in the liver, certain definite differences were
found to obtain.
The rats fed cottonseed oil showed lower plasma
»
cholesterol and lower liver cholesterol levels than the
animals fed lard. In each case, males had lower plasma
cholesterol and higher liver cholesterol as compared with
the females on the same diet. This effect was noticeable
as early as 6 weeks after the onset of the feeding period
and became even more pronounced with time.
The addition of cholesterol to the diet had a
marked effect on the cholesterol levels on both diets
which was particularly pronounced in the liver but also
observable in the plasma cholesterol levels. In the liver,;
!
cholesterol level was greatly increased in the animals fed I
the diet containing lard; the increase was not as great in ■
»
the animals fed the diet containing cottonseed oil. Here,
again, sex difference was noticed on both diets, males had ;
lower plasma and higher liver cholesterol levels than the {
females.
These differences were not confined to the liver
and plasma. In the carcasses of the animals (with the
liver removed) the cholesterol values also reflected the
effect of the type of the dietary fat. Analyses of
carcasses of animals fed the diet containing cottonseed oil
yielded lower cholesterol values than carcasses of animals
on the lard diet. Therefore, this influence of dietary fat
is more widespread than the production of change in the
cholesterol content of the two tissues examined in detail.
At the same time these differences cannot be explained by
a redistribution of cholesterol in the animal body.
Schettler (75* 76) made some similar observations in
his studies on mice. This author found that the intro
duction of animal or vegetable fat into the diet, with and
without the simultaneous addition of cholesterol,
i influenced the plasma and liver cholesterol levels with
I 1
| the lower values found in the animals on the vegetable oil |
j ' • i
! diet. In other investigations of similar nature he reached
| the conclusion that the phytosterol content of vegetable
j oil is not the cause of lower cholesterol levels.
| An obvious conclusion to be drawn from these dif- !
• ferences in, cholesterol content of the tissue of animals
: fed vegetable or animal fats, would be that the digesti-
: bility of cholesterol may be dependent on the type of fat '
: . i
; fed. Therefore, an experiment was performed in which the j
i
possible influence of the dietary fat on cholesterol j
, digestibility was tested. Equal amounts of cholesterol
were absorbed on both diets. However, a sex difference
was clearly shown, with females absorbing significantly
; higher amounts of cholesterol than males. This finding
i
: does not necessarily contradict previous observations :
about the role of plant sterols in cholesterol absorption
1 since the amount of phytosterols in cottonseed oil is
actually quite small when compared with the rather high
percentage of these plant sterols which has been found to
interfere with cholesterol absorption (85). The capacity
of the intestinal mechanism for the absorption of choles
terol in a 250 gm rat is 90 mg per day, as was recently
i
reported by Lin et al. (128); thus, the level fed in the
experiments reported here (one per cent) was well within a
range which could be handled by the intestine.
The possibility would also seem to exist that the
cholesterol content of the animal fat itself might be
responsible for the changes reported. In experiments
designed to test this hypothesis, it was found here that
cholesterol content of the lard did not affect cholesterol
levels in the rat, since the same amount of cholesterol,
when included in the cottonseed oil diet was without effect
in increasing the cholesterol level of plasma and liver.
It is still somewhat in doubt as to whether the rate
of cholesterol biosynthesis in the liver is the primary
site to be affected by the different fats in the diet,
which, in turn, would be reflected in differences in the
levels of accumulated cholesterol. In studies with liver
slices, a significant increase in the incorporation of
labeled acetate into liver cholesterol was noticed in the
female rats fed the diet containing lard over that occur
ring in females fed the cottonseed oil diet. The
individual variability of the results in male rats did not
permit any valid and final conclusions, although it might
be inferred from the results on the female rats that the
male rats would behave similarly, although to a lesser
degree.
The inhibition of biosynthesis of cholesterol in the
. liver by the presence of excess dietary cholesterol was
; confirmed in both male and female rats on both diets.
i ,
The vitamin E content of the fat could be a factor
I
' responsible for at least part of the effect on cholesterol
levels, as shown by the experiment in which the action of
jlard highly enriched with vitamin E was compared with that
iof lard unsupplemented. The addition of large amounts of
alpha-tocopherol to the lard had a significant effect de
creasing the liver cholesterol levels, especially in the
1
case when exogenous cholesterol was administered. The
accumulation of cholesterol esters in the liver of these
cholesterol-fed rats was markedly lowered. The resulting
level was comparable to and perhaps even slightly lower
than that obtained when cholesterol was fed to rats in the
presence of unsupplemented cottonseed oil.
The relationship of vitamin E to lipid metabolism is
not a new concept. Fundamental to the theory that vitamin
E may act in the body as an antioxidant was the demonstra
tion by Dam (129) that lipid peroxides occurred in
measurable amounts in body lipids of vitamin E-deficient
animals. Recently, Hove and Seibold (130) reported that
muscle of vitamin E-deficient hogs had lower concentrations
of dienoic, tetraenoic, and pentaenoic acid than did that
!of vitamin E-supplemented controls; liver fat showed
i
|similar changes.
|
Cholesterol metabolism appears to be closely related
jto that of unsaturated fatty acids. Essential fatty acid
I deficiency causes a significant increase in liver choles-
i
|terol levels in more than one species (58, 59» 61). Lard
|does contain a low but potentially effective concentration
'of unsaturated fatty acids, but without the presence of a
;powerful antioxidant these unsaturated fatty acids may be
:easily oxidized either while in the diet or in the animal
body. Cottonseed oil normally contains relatively large
I
quantities of vitamin E in addition to a much higher con-
,centration of unsaturated fatty acids than is present in
lard.
Lack of natural antioxidants in lard could be
responsible per se for the much higher level of fat oxida
tion here than that which occurs in cottonseed oil as
evidenced by the thiobarbiturate test.
The greater oxidation of the lard, however, did not
seem to have a direct damaging effect on the liver since in
the test for liver damage by the bromsulphalein retention
tests, the results were essentially negative in all cases.
The presence or absence of large amounts of un
saturated fatty acids in the diet was reflected in the
1 composition of liver lipids as studied in pooled extracts.
I It was observed and established that the fatty acids in
! combination with cholesterol differ on the two diets. The
; fatty acids were more saturated in the cholesterol esters
i
! obtained from the animals fed the lard diet than in the
i cholesterol ester fraction obtained from the animals fed
| cottonseed oil. The more saturated fatty acid esters of
;cholesterol have a higher melting point than the un-
i
saturated fatty acid esters and presumably, therefore are
;metabolized with more difficulty and are also less labile,
i Widmer and Holman (131) studying the deposition of
polyunsaturated fatty acids in fat-deficient rats after a
single fatty acid supplementation, reported an increase in
highly unsaturated (tetraenoic) fatty acid in the liver
,when linoleic acid was fed.
j In the present study, the effect of dietary fat is
quite striking. When cholesterol was fed, the amount of
i
'dienoic acid combined with it in the liver was much greater
on the cottonseed oil diet than on the lard diet. Similarity
dienoic acid was also significantly increased in the phos
pholipid fraction on the same diet, while otherwise the
composition of phospholipids was the least affected by
dietary factors. In the triglyceride fraction, dienoic
acid was markedly higher on the cottonseed oil diet.
The protective effect of tocopherol was evident
i particularly when the unsaturation of the fatty acids in
■ the phospholipid fraction was considered (Table XVI). The
synthesis of phospholipids requires large amounts of un-
: saturated fatty acids. These fatty acids are tenaciously
‘ associated with this fraction. If reduced amounts of
i
i essential fatty acids are available, it will be the
phospholipid fraction in which they will be found at the
expense of the other fractions present. This fact can be
correlated with the finding that a larger phospholipid
; fraction occurred on cottonseed oil diets since there were
more unsaturated fatty acids available in cottonseed oil
than in lard to permit synthesis of phospholipids.
Slightly higher fractions of phospholipids in the liver
were also obtained when lard enriched with large amounts
1 of vitamin E was fed, since more of the unsaturated fatty
acids became available due to lack of oxidation.
It has been suggested by several investigators (101,
103) that phospholipids are necessary for normal choles
terol metabolism. It was observed that phospholipids
increase when there is a stimulation of cholesterol
metabolism. This increase may serve as an attempt by the
tissue to rid itself of excess of cholesterol by keeping
the latter in solution. Phospholipids are known to be
85
emulsifiers and carriers of cholesterol.
In this investigation it was observed that the
decrease in phospholipid fraction (on the lard diet) was
accompanied by higher liver cholesterol levels. One of the
causes of higher liver cholesterol levels in rats fed the
lard diet could be an inadequate supply of phospholipids.
The supply of phospholipids is, in turn, dependent on the
availability of unsaturated fatty acids. Thus, it can be
seen that the unsaturated fatty acids play an extremely
important role in cholesterol metabolism, both by uniting
with cholesterol to form cholesterol esters which are
easily metabolized and which are more labile than the
saturated fatty acid esters of cholesterol, and, secondly
by supplying a pool of fatty acids available for the
synthesis of phospholipids which are necessary for proper
metabolism and mobilization of the cholesterol esters ■
present in the animal body.
CHAPTER V
: i
i
! SUMMARY AND CONCLUSIONS i
: i
t
Plasma and liver cholesterol levels and liver lipids.
I ^ !
: of male and female rats maintained on 15 per cent lard or '
I 15 per cent cottonseed oil diet with and without eholes-
| terol were determined at.6, 12, 18, and 2b weeks. Higher j
i liver cholesterol levels were found in all animals on the I
1 I
, lard diet. Differences in plasma cholesterol levels and 1
1 ' i
total liver lipids were not apparent. A sex difference in
cholesterol metabolism was established; female rats j
! exhibited higher plasma and lower liver cholesterol levels
than male rats on both diets.
i
The possibility that the small quantities of cho
lesterol present in lard might account for the increased
1
: liver cholesterol in the animals on this diet was
eliminated since when the cholesterol content of cottonseed!
; oil was equalized with that normally present in lard, no
effect on liver and plasma cholesterol levels as compared
with regular cottonseed oil was observed. ,
Increased cholesterol concentrations were also noted
in the carcasses (excluding liver) of animals of both sexes
which had been fed the diet containing lard as compared
1
with the cholesterol content of carcasses of animals on the
87 :
i
cottonseed oil diet.
i
The effect of vitamin E in which lard is practically:
i
deficient was studied by adding a fivefold amount of |
!
vitamin E to the lard diets with and without cholesterol, j
i
The increased quantities of vitamin E definitely decreased !
I 1
the liver cholesterol concentration of cholesterol fed rats
to a point where the differences in liver cholesterol con- .
I
tent of animals on both the lard diet and the cottonseed
oil diet were non-existent. Vitamin E, however, was with
out effect in the rats on the lard diet from which
exogenous cholesterol was omitted.
The digestibility of cholesterol when accompanied
by the lard or the cottonseed oil diets in male and female
rats was studied and found to be the same regardless of the
vehicle. However, a sex difference in lipid metabolism was
again exhibited since on both lipid diets, females absorbed
more cholesterol than did the male rats. j
A study of cholesterol biosynthesis in liver slices
1U-
from CA -labeled acetate yielded the observation that
although the results on the male rats were inconclusive
due to a large variability in individual determinations,
by far the greatest synthesis was found in the females on
the lard diet. The fact that the biosynthesis of choles
terol in liver slices is greatly inhibited in the presence
88
t
of large amounts of exogenous cholesterol has been
confirmed.
It has been found that fat oxidation level as
indicated by thiobarbiturate test was much higher in lard
sample than in cottonseed oil sample when both fats were
stored in the cold for the same length of time. This is
probably due to the fact that the lard is not protected by
antioxidants normally present in cottonseed oil.
Rats fed the lard diet showed the same bromsul
phalein retention as did rats fed the cottonseed oil diet,
indicating no difference or impairment in liver function.
When the liver lipid fractions of various groups
were studied, the effect of dietary fat was noted. The
presence of cottonseed oil increased unsaturation of the
fatty acids associated with the triglyceride and choles
terol ester fractions. The concentration of the phospho
lipid fraction was higher in cottonseed oil fed rats. The
addition of large amounts of vitamin E yielded slightly
higher unsaturation of fatty acids even in lard fed rats.
The effect of cholesterol feeding was to increase the
dienoic acid associated with cholesterol esters in livers
of animals on the cottonseed oil diet and to increase the
monoenoic acid associated with cholesterol esters in liver
of animals on the lard diet.
The important factor in the difference in choles- j
terol metabolism on the two fat diets appears not to be the,
difference in the type of sterols usually present in the
particular fat, but rather the degree of unsaturation of I
the fatty acids present in the fat. Both the effect of j
vitamin E and composition of the liver lipid fractions j
substantiate this theory.
I
i
It is assumed that the unsaturated fatty acids which!
are present in abundance in cottonseed oil cause a higher |
lability of cholesterol esters, and, possibly, at the same ;
time allow a more abundant quantity of phospholipids to be
synthesized. Phospholipids are necessary for the proper
metabolism of cholesterol. Vitamin E, which is present in
cottonseed oil and virtually absent from lard, is also a
factor in maintaining the unsaturation level of the fatty
acids through its antioxidant properties.
It is concluded that the biological origin of
dietary fat has a decisive effect on cholesterol metabolism
in the rat.
BIBLIOGRAPHY
r
?
BIBLIOGRAPHY
! 1. Poulletier, de la Salle, circa 1769* Cited by Bills,
| C. E., Phvsiol. Revs., l£, 21 (1935)
1 2. Couerbe, J. P., Ann, chim. et phvs.. 2, 56 (1838)
J 3* Lecanu, L. R., Ann, chim. et phys.. 2, 67 (I838)
»
*+. Anitschkow, N., Experimental Arteriosclerosis in
Animals, in Arteriosclerosis: A Survey of the
I Problem, ed. by E. V. Cowdry, New York, The
MacMillan Co., 1933, p. 271.
j 5* Okey, R., J. Am. Dietet. Assoc.. 30. 231 (195*+)
: 6. Friedman, M., Byers, S. 0., and Michaelis, F.,
M- I - Phvsiol.. 16|+, 789 (1951)
1
; 7• Siperstein, M. D., Jayko, M. E., Chaikoff, I. L., and
Dauben, W. G., Proc. Soc. Exp. Biol. Med.. 78.
I 61*1 (1952)
8. Gould, R. G., Am. J. Med., 11, 209 (1951)
9. Gould, R. G., Taylor, C. B., Hagerman, J. S., Warner,
I., and Campbell, D. J., J. Biol. Chem.. 201. 519
(1953)
10. Schoenheimer, R., and Breusch, F., J. Biol. Chem.,
103. **39 (1933)
11. Frantz, I. D., Jr., Schneider, H. S., and Hinkelman,
B. T., J. Biol. Chem.. 206. 1*65 (195*0
12. Tomkins, G. M., Sheppard, H., and Chaikoff, I. L.,
J. Biol. Chem.. 201. 137 (1953)
13. Gould, R. G., J. Am. Geriatrics Soc.. 2, 61*0 (195*+)
ll*. Taylor, C. B., and Gould, R. G., Circulation. 2, 1*67
(1950)
15. Biggs, M. W., Friedman, M., and Byers, S. 0., Proc.
1 Soc. Exp. Biol. Med.. Z8, 6**1 (195D
16.
17.
18.
19.
20.
21.
22.
23.
2V.
25.
26.
27.
28.
29.
30.
92
Chaikoff, I. L., Bloom, B., Siperstein, M. D.,
Kiyasu, D. Y., Reinhardt, ¥. 0., Daiiben, W. G.,
and lastham, J. F., J. Biol. Chem.. 19*+. V07
(1952)
Byers, S. 0., and Friedman, M., Am. J. Physiol.. 179.
79 (195V)
Friedman, M., Rosenman, R. H., and Byers, S. 0.,
J. Gerontol.. 10. 60 (1955)
Friedman, M., and Byers, S. 0., Circulation. 10. *+91
(195V)
Anderson, N. G., and Fawcett, B., Proc. Soc. Exp.
Biol. Med.. Zit, 768 (1950)
Friedman, M., Byers, S. 0., and Rosenman, R. H.,
M - J- Phvsiol.. 177. 77 (195V)
Heilman, L., Rosenfeld, R. S., lidinoff, M. L.,
Fukushima, D. K., and Gallagher, T. F., J. Clin.
Invest.. 3V, V8 (1955)
Rice, L. L., Schotz, M. C., Powell, J. R., and Alfin-
Slater, R. B., Am. J. Phvsiol.. 178. V83 (195V)
Friedman, M., Byers, S. 0., and Shibota, E., J. Exp.
Med.. 98. 107 (1953)
Siperstein, M. D., and Murray. A. W.. J. Clin. Invest.
av, 1W 9 ( 1955) ----------------------------
Siperstein, M. D., and Chaikoff, I. L., Federation
Proc.. IV, 767 (1955)
Byers, S. 0., and Friedman, M., Am. J. Phvsiol.. 168.
297 (1952)
Landon, E. J., and Greenberg, D. M., J. Biol. Chem..
209. V93 (195V)
Gould, R. G., Campbell, D. J., Taylor, C. B., Kelly,
F. B., Jr., Warner, I., and Davis, C. B., Jr.,
Federation Proc.. 10. 191 (1950)
Cook, R. P., Biochem. J., 30, I630 (1936)
93
31. Sano, M., Tohoku J. Ex^. Med., b , b l7 (192*0
32; Eckstein, H. C., and Treadwell, C. R., J. Biol. Chem..
112. 373 ( 1935)
33. Eckstein, H. C., J. Biol. Chem.. 12£, 99 (1938)
3**-. Kim, K. S., and Ivy, A. C., Am. J. Physiol.. 171. 302
(1952)
35* Swell, L., and Flick, D. F., M. J. Physiol.. 17*f. 51
d953)
36. Swell, L., Flick, D. F., Field, H., Jr., and Tread
well, C. R., Am. J. Phvsiol.. 180. 12b (1955)
37. Kritchevsky, D., Mayer, A. ¥., Tesar, ¥. C., Logan,
J. B., Brown, R. A., Davies, M. C., and Cox, H. R.
Am. J. Phvsiol.. 178. 30 (195*0
38. Alfin-Slater, R. B., Schotz, M. C., Shimoda, F., and
Deuel, H. J., Jr., J. Biol. Chem.. 195. 311 (1952)
39. Karvinen, E., Lin, T. M., and Ivy, A. C., Am. J.
Phvsiol.. 181. **39 (1955)
*+0. Dubach, R., and Hill, R. M., J. Biol. Chem.. 165. 521
(19* + 6)
*fl. Bollman, J. L., and Flock. E. V.* Am. J. Phvsiol..
16W W o (1951) — ” ----
b2. Raulin, J., Compt. rend.. 233. 1318 (1951)
*+3. Mawas, J.. Jacquot, R., and Raulin, J., Compt. rend.,
23*f. 665 (1952)
*+**. Schmidt-Thome, J., Schettler, G., and Goebel, H.,
2. Phvsiol. Chem.. 283. 63 (19*+8)
**5- Schramm, G., and Wolff, A., Z. phvsiol. Chem.. 263,
61 (19*f0) “
*+6. Mellinkoff, S. M., Machella, T. E., and Reinhold,
J. G., Am. J. Med. Sci., 220, 203 (1950)
*+7. Wilkinson, F. C., Jr., Blecha, E., and Reimer, A.,
Arch. Intern. Med.. 6, 389 (1950)
. . ^
1+8. Keys, A., Anderson, J. T., Fidanza, F., Keys, M. H.,
and Swahn, B., Clinical Chem., 1, 3*+ (1955) j
1+9. Keys, A., and Keys, M. H., Brit. J. Nutrition. 8, 138
(195^)
50. Keys, A., Vivanco, F., Rodriguez Minon, J. L., Keys,
M. H.. and Castro Mendoza H., Metabolism. 3L, 195
(195^)
51. Keys, A., Circulation. 115 (1952)
52. Keys, A., Science. 112. 79 (1950)
53* Gofman, J. W., Jones, H. B., Lindgren, F. T., Lyon,
T. P., Elliott, H. A., and Strisower, B.,
Circulation. 2, 161 (1950)
5k, Hatch, F. T., Abell, L. L., Kendall, F. E., Am. J.
Med., 12, 1+8 (1955)
55. Wollaeger, E. I., Lundberg, ¥. 0., Chipault, J. R.,
and Mason, H. L., Gastroenterol.. 2 k , *+22 (1953)
56. Brown, W. R., Hansen, A. E., McQuarrie, I., and
Burr, G. 0., Proc. Soc. Exp. Biol. Med.. 36. 281 1
(1937) !
|57» Alfin-Slater, R. B., Aftergood, L., Wells, A. F., and i
Deuel, H. J., Jr., Federation Proc., 13, 17*+
j \ (195*0 " |
58. Alfin-Slater, R. B., Aftergood, L., Wells, A. F., and
Deuel, H. J., Jr., Arch. Biochem. Biophvs.. 52.
180 ( 195*0
159• Shapiro, S. L., and Freedman, L., Am. J. Physiol.. !
181, 1 + 1 +I (1955) * — --------
60. Peifer, J. J., and Holman, R. T., Arch. Biochem.
| Biophys., 2Z, 520 (1955) |
;6l. Chahine, M. H., and Kummerow, F. A., Abstracts, Am.
Oil Chem. Soc.. October, 1955*
62. Bromer, ¥. ¥., and Day, H. G., Abstracts, Am. Chem.
I Soc.. September, 1953*
95
63. Hildreth, E. A., Mellinkoff, S. M., Blair, G. ¥., and
Hildreth, D. M., Circulation, 1, 6*fl (1951)
6*4-. Kinsell, L. W., Partridge, J., Boling, L., Margen, S.,
and Michaels, G., J. Clin. Endocrine Metabolism.
12, 909 (1952)
65. Kinsell, L. W., Michaels, G., DeWind, L., Partridge,
J., and Boling, L., Calif. Med.. Z8, 5 (1953)
66. Kinsell, L. W., Michaels, G. D., Partridge, J.,
Boling, L., Balch, H. E., and Cochrane, G. C.,
J. Clin. Nutrition. 1, 22b (1953)
67. Cochrane, G. C., Michaels, G. D., and Kinsell, L. ¥.,
J* Clin. Nutrition. 1, 295 (1953)
68. Kinsell, L. ¥., and Michaels, G. D., Federation
Proc.. lb . 661 (1955)
69. Hardinge, M. G., and Stare, F. J., J. Clin.
Nutrition. 2, 83 (195*4-)
70. Tsai, S. Y., Futch, E. D., Kobayashi, M., May, L. G.,
and Gregory, R. L., Texas Reports Biol. Med.. 12,
*4-23 (195*4-)
71. Ahrens, E. H., Jr., Blankenhorn, D. H., and Tsaltas,
T. T., Proc. Soc. Ex£. Biol. Med., 86, 872 (195* 4 -)
72. Mayer, G. A., Connell, W. F., DeWolfe, M. S., and
Beveridge, J. M. R., J. Clin. Nutrition. 2, 316
(195*4-)
t
^73. Beveridge, J. M. R., Connell, ¥. F., Mayer, G. A.,
Firstbrook, J. B., and DeWolfe, M. S., J. Nutri
tion, i6, 311 (1955) ~
7b. Schettler, G., Klin. ¥0chenschr.. 26. 566 (19*4-8)
75. Schettler, G., Biochem. Z., 3bk2» 3*4-9 (19^9)
76. Schettler, G., Bio chem. Z^., 31£, W + (19*+9)
77. Peterson, D. ¥., Proc. Soc. Exp. Biol. Med.. 78. 1*4-3
(1951)
96
78. Sperry, W. M., and Bergmann, ■ W., J. Biol. Chem..
112, 171 (1937)
79- Peterson, D. W., Shneour, E. A., Peek, N. F., and
Gaffey, H. W., J. Nutrition. £0, 191 (1953)
80. Poliak, 0. J., Circulation. Z, 696 (1953)
81. Hernandez, H. H., Peterson, D. ¥., Chaikoff, I. F.,
and Dauben, W. G., Proc. Soc. Ex p. Biol. Med..
8^, >*98 (1953)
82. Hernandez, H. H., and Chaikoff, I. L*, Proc. Soc.
Exp. Biol. Med.. 82, 5>*1 (195*0
83. Rosenman, R. H., Byers, S. 0., and Friedman, M.,
Circulation Res., 2, 160 (195*0
8>+. Burke, K. A., McCandless, R. F. J., and Kritchevsky,
D., Proc. Soc. Exp. Biol. Med.. 8Z, 87 (195*0
85. Alfin-Slater, R. B., Wells, A. F., Aftergood, L.,
Melnick, D.and Deuel, H. J., Jr., Circulation
Res.. 2, >*71 (195*0
86. Alfin-Slater, R. B., Wells, A. F., Aftergood, L., and
Deuel, H. J., Jr., Abstracts, Am. Oil Chem. Soc..
• Philadelphia, 1955 " I
I 1
87. Wilkinson, C. F., Jr., J. Am. Geriatrics Soc.. 3, 381 !
0.95*0 I
1
88. Kinsell, L. W., Cochrane, G. C., Smyrl, S., Fukayama,
G., and Coelho, M., Circulation. 10, 593 (1953)
89. Poliak, 0. J., Circulation. Z, 702 (1953)
i ' ■ i
!90. Best, M. M., Duncan, C. H., VanLoon, E. J., and
Wathen, J. D., Am^ J. Med., 12, 61 (1955) ;
i91. Steiner, A., and Domanski, B., Proc. Soc. Ex p. Biol. I
j Med-, 15, 236 (191 * 1 *) ■ ” !
'92. Swell, L., Boiter, T. A., Field, H., Jr., and
Treadwell, C. R., Am. J. Phvsiol.. 180. 129 (1955),
: 1
93- Swell, L., Boiter, T. A., Field, H., Jr., and Tread- j
well, C. R., Proc. Soc. Exp. Biol. Med.. 86, 295
----------m f r )............ J
9 * k
9 5 .
9 6 .
9 7 .
9 8 .
9 9 .
100.
101.
102.
1 0 3 .
10* * .
1 0 5 .
106.
i
107.
108.
1 0 9 .
110.
“ 97
Sehoenheimer, R., Science. 7b , 579 (1931)
Gould, R. G., Circulation. 10. 589 (195*0
Gould, R. G., Lotz, L. V., and Lilly, E. M.,
Federation Proc., 1^, **87 (1955)
Beher, ¥. T., and Anthony, ¥. L., J. Nutrition. 52,
519 (195*0
Beher, ¥. T., and Anthony, W. L., Proc. Soc. Exp.
Biol. Med.. 86, 589 (195*0
Siperstein, M. D., Nichols, C. W., Jr., and Chaikoff,
I. L., Circulation. Z, 37 (1953)
Siperstein, M. D., Nichols, C. ¥., Jr., and Chaikoff,
I. L., Science. 117. 386 (1953)
Pilgeram, L. 0., and Greenberg, D. M., Circulation
Res., a, **7 (1955)
Zilversmit, D. B., Shore, M. L., and Ackerman, R. F.,
Circulation. % 581 (195*0
Zilversmit, D. B., Shore, M. L., and McCandless,
E. L., Federation Proc.. 1|±, I67 (1955)
Zilversmit, D. B., McCandless, E. L., and Shore,
M. L., Proc. Soc. E x p . Biol. Med.. 89, *f8 (1955) i
Ridout, J. H., Lucas, P., Patterson, J. M., and Best, :
C. H., Biochem. J., ^2, 79 (1952)
Clement, G., LeBreton, E., Pascaud, M., and Tubiana,
> Compt. rend.. 236. ^12 (1953)
Garcia, P., Roderick, C., and Swanson, P., J. i
Nutrition. 55, 601 (1955)
Gillum, H. L., Morgan, A. F., and Jerome, D. ¥., i
J. Nutrition. 55, *0-9 (1955) |
Rosenman, R.H., and Shibota, E., Proc. Soc. Exp. Biol.
Med.. 81, 296 (1952)
Bloor, ¥. R., J. Biol. Chem.. 56. 711 (1923)
98
111. Bloor, W. R., Blake, A. G., and Bullen, S. S.,
J. Allergy, % 227 (1938)
112. Channon, H. J., and Collison, G. A., Biochem. J.,
21, 1212 (1929)
113. Kelsey, F. E., and Longenecker, H. E., J. Biol.
Chem.. 139, 727 (19^1)
11**. Keegan, P., and Gould, R. G., Federation Proc.. 12.
228 (1953)
115. Achaya, K. T., Mukherjee, S., Alfin-Slater, R. B.,
and Deuel, H. J., Jr., To be published.
116. Hammond, E. G., and Lundberg, ¥. 0., Arch. Biochem.
Biophys., 5Z, 517 (1955)
117. Thompson, S. Y., Ganguly, J., and Kon, S. K., 1
Brit. J. Nutrition. 1* 50 (19^9) i
j
118. Nieft, M. L., and Deuel, H. J., Jr., J. Biol. Chem.. I
1ZZ, 1^3 (19^9)
119. Hotta, S., Hill. R., and Chaikoff, I. L., J. Biol. |
Chem.. 206. 835 (195^) ~ I
; 120. Uinbreit, ¥. ¥., Burris, R. H., and Stauffer, J. E., j
! Manometric Techniques and Tissue Metabolism.
Minneapolis, 19^9, p. 119*
121. Fillerup, D. L., and Mead, J. F., Proc. Soc. Ex p.
Biol. Med.. 8a, 57*+ (1953)
122. Achaya, K. T*, Personal communication.
! 123. Herb, S. F., and Riemenschneider, R. ¥., J. Am. Oil
Chem. Soc.. 22, **56 (1952)
; 12**. Patton, S., and Kurtz, G, ¥., J. Dairy Sci.. 3*fr.
i 669 (1951) T j
! 125. Frommer, J., Proc. Soc. Exp. Biol. Med.. 86, %7
(195*0
; 126. Lange, ¥., J. Am. Oil Chem. Soc.. 27. *fl** (1950)
! ■ i
99
127. Severance, E. J., The Digestibility of Some Peanut
Gils in the Bat, Thesis, University of Southern
California, 1952.
128. Lin, T. M., Karvinen, E., and Ivy, A. C., Proc. Soc.
Exp. Biol. Med.. 8£, *+22 (1955)
129- Dam. H., Ann. New York Acad. Sci.. 52. 195 (19^9)
130. Hove, E. L., and Seibold, H. R., J. Nutrition. 56.
173 (1955)
131. Widmer, C., Jr., and Holman, R. T., Arch. Biochem..
1 (1950)
1
Ofll ver slt_yL_or_s-G y-ts e-rn--ear iro rsiiir

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Some studies on the effects of dietary choline on the plasma cholesterol concentration in the rat

PDF

The effect of neoplastic tissue on the metabolism of formic acid by the host

PDF

Studies on fat digestibility: The effect of melting point and dietary calcium and magnesium level

PDF

Intestinal bacteria in the metabolism of dietary cholesterol of animals

PDF

Some aspects of the carbohydrate metabolism of Endamoeba histolytica

PDF

Studies on the metabolism of carotene

PDF

The influence of K3O4 on the regulation of cholesterol side chain cleavage by ACTH and cycloheximide

PDF

Studies on hypervitaminosis A in the rat

PDF

An investigation of the distribution and mechanisms of deposition of exogenous cholesterol in the rat

PDF

Studies on cholesterol metabolism in the rat

PDF

Studies on the in vivo metabolism of thyroxine

PDF

Some aspects of the metabolism of Endamoeba histolytica

PDF

Studies on the separation and turnover of ribonucleotides

PDF

A nutritional evaluation of the essential fatty acids in the rat

PDF

Studies on the mechanism of fat absorption using isotope labeled compounds.

PDF

The half-life of plasma albumin and the possibility of its partial conversion into globulin as studied in the normal adult dog

PDF

A comparative study of the chemistry and biology of Heparin

PDF

Metabolic studies of active rat myocardium

PDF

The influence of the thyroid hormone on cholesterol metabolism in rats and mice.

PDF

Studies of carotenoid and vitamin A complexes with protein in plasma and tissues

Asset Metadata

Creator Aftergood, Lilla (author)

Core Title Studies of the effect of a vegetable fat and an animal fat on cholesterol metabolism

Degree Doctor of Philosophy

Degree Program Biochemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-603030

Unique identifier UC11354755

Identifier DP21566.pdf (filename),usctheses-c17-603030 (legacy record id)

Legacy Identifier DP21566.pdf

Dmrecord 603030

Document Type Dissertation

Rights Aftergood, Lilla

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA