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Studies of fatty acids: Effects on nutritional status and tissue cholesterol levels of rats

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Studies of fatty acids: Effects on nutritional status and tissue cholesterol levels of rats

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Content STUDIES OP.PATTY ACIDS:
EFFECTS O N NUTRITIONAL STATUS AND TISSUE
CH O LESTEROL LEVELS O F RATS
by
Esin Ummehan Kent
A T h esis P resen ted to th e
FACULTY O F THE G R A D U A TE SCH O O L
UNIVERSITY O F SO U TH ER N CALIFORNIA
In P a r t ia l F u lfillm e n t o f th e
Requirem ents fo r th e Degree
M ASTER O F SCIENCE
(B ioch em istry and N u tr itio n )
August 1958
UMI Number: EP41338
All rights reserved
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U N IV E R SIT Y O F S O U T H E R N C A L IF O R N IA
GRADUATE SCHOO L
UNIVERSITY PARK
LOS ANG ELES 7
5 1 W 5 1
This thesis, ’ written by
.......................
under the guidance of h&c.....Facuity 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
( f f ' 1
Dean
Date.
Faculty Committee
y I ' .Chairman
' j &f rv' nL& *x-
UT t k Ij B JL
) S t S B 0'
A C K N O W L E D G E M E N T S
I should lik e to ex p ress ray e v e r la s tin g g r a titu d e
and m ost s in c e r e thanks to Dr. R .'B . A lf in - S la te r fo r
her sp o n so rsh ip , encouragem ent, g u id a n ce, and fo r a most
w onderful a s s o c ia t io n throughout the cou rse o f t h is
in v e s t ig a t io n .
I should a ls o l i k e to thank the o th er members o f
ray com m ittee, P ro fe sso r s Mehl and Marx, fo r th e ir
u nd erstan d ing and h e lp fu l c r it ic is m .
TABLE OF CO NTENTS
CHAPTER PAGE
I . INTRODUCTION . . ............................................................... 1
I I . HISTORICAL BACKGROUND................................................. 3
E s s e n tia l F a tty A cids .......................................... 3
Nature and o ccu rren ce ...................................... 3
E s s e n tia l f a t t y a c id d e fic ie n c y . . . . 5
F u n ction s of e s s e n t ia l f a t t y a c id s . . . 6
E ffe c t o f e s s e n t ia l f a t t y a c id on
c h o le s te r o l m etabolism . ............................. 8
R ela tio n o f Fa/fes to C h o lester o l M etabolism 10
F a tty A cid s and A t h e r o s c l e r o s i s ................... 15
I I I . PLAN O F TH E EXPERIM ENT .............................. 16
Feeding Experim ents With E thyl E ste r s o f
F a tty A c i d s ....................... 16
Experiment I ............................................................. 16
Experiment I I ............................ 16
Experiment I I I ......................................................... 16
Experim ents w ith Fat Free and 15$ Hydro
gen ated Coconut O il ............................ 1?
Experiment I V ......................................................... 17
Experiment V . . . . . . . ............................. 18
IV. MATERIALS AND METHODS................................................ 19
Methods o f S a c r i f i c e ................................ 19
D iets . ................................ 20
CHAPTER
P rep aration o f E th y l L in o le a te ........................
E x tra ctio n o f L ip id s ...............................................
Waring Blendor method . . . . . . . . . .
Plasma e x tr a c tio n method .................................
S o x h le t m e t h o d ........................ . ...........................
C h o le ste r o l determ ination' .................................
D eterm ination o f t o t a l l i p i d s . . . . . .
Procedure fo r L ip o x id a se Method fo r th e
D eterm ination o f P o ly u n sa tu ra ted F a tty
A c i d s ...................................................................... . .
Room tem perature s a p o n ific a tio n ...................
Blank . . . ................................. ....
T est sam ple . ..............................................................
In cu b ation . . . . . . . . . . ...................
A bsorption read in g ...............................................
C a lcu la tio n o f P o ly u n sa tu ra ted F a tty Acid
Content .... . ........................ . . . . . .
V. RESULTS.....................................................................................
E ffe c t o f A d m in isterin g F a tty A cid E sters
, to M ale Weanling R ats M aintained on a
Fat Free D iet (Experim ent I) ........................
E ffe c t o f Adding 1$ C h o le ste r o l and 0 .2 5 $
B ile S a lt s to th e Fat Free D ie ts , Supple
mented w ith F a tty A cid E s te r s , of Male
W eanling Rats (Experiment I I ) . . . . . .
iv
PAGE
20
23
I
23 I
2k
2k
2k
26
27
2?
28
28
28
28
29
30
30
35
CHAPTER PACE
E ffe c t o f Supplem entations w ith E th yl
L ln o le a te to W eanling Male R ats Main
ta in e d on a Fat Free D iet Supplem ented
w ith V arious S a tu ra ted or N o n -e s se n tia l
U nsatu rated F a tty Acid E ste rs
(Experim ent I I I ) ................................................... kO
E ffe c t o f Supplem enting C h o le ste r o l and
B ile S a lt s to A dult Male R ats M aintained
on a Fat Free D iet fo r S ix te e n Weeks
(Experiment I V ) .................................................... k6
E ffe c t o f F eeding 15$ H ydrogenated Coconut
Alone or Supplem ented w ith C h o le ste r o l
and/o.r B ile S a lts to A dult Male Rate
P r e v io u sly M aintained on 15$ Hydro
gen ated Coconut O il D iet f o r Four
Months . , . ............................................................. 51
VI. DISCUSSION ................................... . . . . . . . . . 56
V II. SU M M A R Y AN® CONCLUSIONS ...................................... 6k
BIBLIOGRAPHY........................................................ 6?
LIST OF TABLES
TABLE PAG E
I . The Formulae o f the E s s e n tia l F a tty A cids . b
I I . Com position o f D ie ts . . ......................... 21
I I I . C om position o f Vitam in Mix ..................... 22
IV. Growth o f W eanling Male R ats Fed a Fat
Free D iet Supplem ented w ith V arious
F a tty A cid E s t e r s ...................................... 31
V. Plasma and Adrenal C h o le ste r o l L e v els of
W eanling Male R ats Fed a Fat F ree D iet
Supplem ented w ith V arious F a tty A cid
E s t e r s .................... ................................................... 33
V I. C h a r a cter iza tio n o f L iv e r L ip id s o f Wean
lin g Male R ats Fed a Fat Free D iet
^ Supplemented w ith V arious F a tty Acid
E s t e r s ....................... 3 b
V II. Growth o f Weanling Male R ats Fed a Fat
Free D ie t P lu s C h o le ste r o l P lu s B ile
S a lts Supplemented w ith V arious F a tty
A cid E ste r s ............................................... 36
V II I . Plasma and Adrenal C h o le ste r o l L e v els o f
Male W eanling Rats Fed a Fat F ree D iet
P lu s C h o le ste r o l P lu s B ile S a lt s
Supplem ented w ith V arious F a tty Acid
E s t e r s ................................................... 37
v i i
TABLE PAGE
IX. C h a ra cteriza tio n o f L iv e r L ip id s o f Male
W eanling R ats Fed a Fat Free D iet P lu s
C h o le ste r o l P lu s B ile S a lts Supple
mented w ith V arious F a tty A cid E s te r s . . 38
X. Growth o f W eanling M ale Rats Fed a Fat
Free D ie t Supplemented w ith V ariou s
N o n -e s s e n tia l F a tty A cid E ste r s P lu s
E thyl L i n o l e a t e ............................ 41
X I. Plasma and Adrenal C h o le ste r o l L e v e ls o f
Male W eanling Rats Fed a Fat F ree D iet
Supplem ented w ith N o n -e s se n tia l F a tty
A cid E ste r s P lu s E thyl L in o le a te . . . . 44
X II. C h a ra cter iza tio n o f L iv er L ip id s o f Male
W eanling R ats Fed a Fat Free D iet
Supplemented w ith V arious N o n -e s s e n tia l
F atty A cid E ste rs P lu s E th yl L in o le a te . 45
X I I I . Growth o f Adult Male R ats Fed a F at Free
D iet w ith Supplem ents o f B ile S a lt s and
C h o l e s t e r o l ........................................................ 48
XIV. Plasma and Adrenal C h o le ste r o l L ev els o f
A dult Male Rats Fed a Fat Free D iet w ith
Supplem ents o f C h o le ste r o l and B ile
S a lts . . . ..................................... 49
v i i i
TABLE PAG E
XV. C h a r a cter iza tio n o f L iv e r L ip id s o f Male
A dult H ats Fed a Fat Free D ie t w ith
Supplem ents o f C h o le ste r o l and B ile
S a lts . ....................................................................... 50
XVI. Growth o f Adult Male Rats Fed a 15%
H ydrogenated Coconut O il D iet w ith
Supplem ents o f C h o le ste r o l and B ile
S a l t s ................................. 52
XVII. Plasma and Adrenal C h o le ste r o l L e v e ls o f
j
Male A dult Rats Fed a 15% Hydrogenated
Coconut O il D iet w ith Supplem ents o f
C h o le ste r o l and B ile S a lts . . . . . . . 5^
X V III. R e su lts O btained on L iv er L ip id s o f Rats
Fed a 15% Hydrogenated Coconut O il D iet
w ith Supplem ents o f C h o le ste r o l and
B ile S a lts . . ............................................... 55
/
LIST O F FIGURES
FIGURE PAG E
1 . Gain in Weight o f Rats Fed a Fat F ree D iet
Supplemented w ith V arious F a tty A cids
fo r S ix Weeks ......................................................... f y 2
CHAPTER I
INTRODUCTION
i ,
U n til about t h ir t y years ago, most m ed ical s c i e n t i s t s
| b e lie v e d th a t v a r io u s s p e c ie s o f anim als would grow s a t i s - j
f a c t o r i l y w ith o u t e a tin g f a t s . But in 1 9 2 8 ^ th e above
p o s tu la tio n was proved to be f a u lt y s in c e m ice on a
co m p letely l i p i d - f r e e d i e t , adequate in o th e r r e s p e c t s ,
l o s t w eigh t and became in c r e a s in g ly s u s c e p tib le to in f e c
t io n .
i
i The p h y s io lo g ic a l d ata on f a t s and e s s e n t i a l f a t t y
! a c id s in d ic a te th a t fa t i s a req u ired component o f th e d ie t !
i :
■ o f many s p e c ie s o f an im als. In th e ab sen ce o f d ie ta r y f a t , 1
| a d e f ic ie n c y syndrome ap p ears. Burr and Burr^2 '^) f i r s t
d e sc r ib e d th e f a t d e fic ie n c y symptoms in d e t a i l , em phaslz-
I
in g th e poor grow th, th e cau d al n e c r o s is , th e in c r e a se d
1
t
consum ption o f w ater not accom panied by in c r ea se d u rin e
volum e, and th e pregnancy d i f f i c u l t i e s . The p rev en tio n o r
I
a lle v ia t io n o f th e s e d e f ic ie n c y symptoms appeared to be a |
(fu n c tio n o f th e amounts o f l i n o l e l c a c id and, p o s s ib ly ,
o th e r h ig h ly u n sa tu ra ted f a t t y a c id s p resen t in th e d i e t .
A con n ection between th e e s s e n t ia l f a t t y a c id s and th e
r e g u la tio n o f c h o le s te r o l m etabolism has been shown by many
in v e s t ig a t o r s . Current o p in io n on th e e f f e c t o f v a r io u s
f a t t y a c id s on th e c h o le s te r o l c o n c e n tra tio n in serum i s
q u ite c o n tr o v e r s ia l. I t has been su g g ested th a t th e i
2
d ep ressin g e f f e c t on c h o le s te r o l, c o n ce n tra tio n s In serum by-
c e r ta in d ie ta r y f a t s i s r e la t e d to t h e ir p o ly u n sa tu ra ted
f a t t y a c id c o n te n t.
I t i s a ls o b e lie v e d .t h a t th e p resen ce o f n o n -e s s e n tia l
f a t t y a c id s , e it h e r sa tu ra te d or u n sa tu ra ted , p resen t in
th e f a t d ie t prom ote h y p e rch o le ste ro le m ia by in c r e a s in g th e
requirem ent fo r e s s e n t ia l, f a t t y a c id s . However,
S ta r e , H egsted, and G o ts ls ' ' from experim ents in w hich
r a t s were fe d v a r io u s f a t s , b e lie v e th a t sa tu ra ted f a t t y
a c id s are s y n e r g is t ic w ith e s s e n t ia l, f a t t y a c id s in lo w er
in g serum c h o le s t e r o l l e v e l s , and th a t th e u n satu rated
n o n - e s s e n tia l f a t t y a c id s a re r e s p o n s ib le fo r r a is in g serum
c h o le s te r o l, l e v e l s ...
In view o f th e c o n f lic t in g id e a s con cern in g th e
p o s s ib le r o le o f f a t t y a c id s in th e r e g u la tio n o f t i s s u e
c h o le s te r o l l e v e l s , t h i s in v e s t ig a t io n was undertaken to
stu d y th e e f f e c t o f th e e s t e r s o f c e r ta in sa tu ra te d and
u n sa tu ra ted f a t t y a c id s when fe d alo n e a s a pare compound
on th e n u t r it io n a l s t a tu s and on some p h a ses o f c h o le s te r o l
m etabolism in th e r a t .
CHAPTER I I
HISTORICAL B A C K G R O U N D
I . E s s e n tia l F a tty A cids
A. Nature and occurrence
E arly experim ents In d ic a te d th a t th e f a t d e f ic ie n c y
syndrome r e s u lte d from a la c k o f p o ly u n sa tu ra ted f a t t y
a c id s and cou ld he a lle v ia t e d by th e a d m in istra tio n o f
e s t e r s o f l i n o l e l c a c i d . ^7) Further s t u d i e s 8 , 9 » 1 0 , 1 1 )
proved th a t th r e e p o ly u n sa tu ra ted f a t t y a c id s , nam ely,
l i n o l e i c , l i n o l e n i c , and a ra c h ld o n lc co u ld be e f f e c t i v e in
t h i s r e s p e c t. S a tu ra ted , m ono-onsatu rated , and o th e r v ery
h ig h ly u n sa tu ra ted f a t t y a c id s had l i t t l e , i f any,
e f f e c t .
The th r ee e s s e n t i a l f a t t y a c id s are n ot e q u a lly
p o te n t. Deuel and R eiser^ 1^ rep orted th a t i n .s p i t e o f
d iv e r g e n t r e p o r ts o f d if f e r e n t in v e s t ig a t o r s , th e p r e s e n t
con sen su s i s th a t a ra ch id o n ic a c id i s th e most a c t iv e o f
th e e s s e n t i a l f a t t y a c id s fo r man. Quakenbush and co
w orkers (^5) have shown th a t l i n o l e i c a c id i s more e f f e c t i v e
than lin o le n i c a c id as an a n ti- d e r m a t itis fa c to r in th e
r a t .
A lthough l i n o l e n i c a c id p o s s e s s e s a growth a c t i v i t y ,
i t i s unable to cure or p rev en t th e sk in a b n o r m a litie s.
However, Deuel and c o - w o r k e r s i n d i c a t e d th a t alth o u g h
l i n o l e n i c a c id had o n ly a s l i g h t growth prom oting a c tio n
.T A BL E I
-T H E F O R M U L A E O F T H E ESSENTIAL FATTY ACIDS
L in o le ic : CH^(C H 2 ) ^C H =C H C H 2CH*CH(C H 2 ) ?C O O H
(C l7H 3lCOOH)
L in o le n ic : G H 3C H 2C H = C H C H 2C H =C H C H 2CH =CH (C H 2 )?C O O H
( ci 7h29C00H)
Arachldpnlc : C H 3 (G H 2 ) ^CH-CHCH2C H = G H C H 2G H »C H C H 2CH=(C H 2 )3C O O H
(C *^ ^ H 3 j^ C O O H )
; r
when i t was fe d a lo n e to f a t d e p le te d r a t s , when fe d w ith
suboptimum d o ses o f l i n o l e a t e , the r e s u lta n t a c t i v i t y o f
lin o le n a t e e q u a lle d th a t o f l i n o l e a t e . These r e s u lt s su g
g e s t th a t a sp ark in g a c tio n of lin o l e a t e i s req u ired b efore
lin o le n a t e can p la y i t s r o le as an e s s e n t i a l f a t t y a c id .
L in o le ic a e id e x i s t s in s e v e r a l d if f e r e n t iso m ers,
but i t i s b e lie v e d th a t the n a tu r a lly o ccu rrin g isom er has
th e h ig h e s t p oten cy fo r a l l e v ia t in g the e s s e n t i a l f a t t y
a c id d e f ic ie n c y sy m p to m s.(2 li,l8 ,1 9 )
B. E s s e n tia l F a tty A cid D e fic ie n c y
As was m entioned p r e v io u s ly , Burr and B u r r ^ * ^ were
/
th e f i r s t in v e s t ig a t o r s to observe th e e s s e n t i a l f a t t y a c ic
d e f ic ie n c y in r a t s . They a ls o rep o r ted th a t v e g e ta b le f a t s
had c u r a tiv e and b e n e f ic ia l e f f e c t s on th e r a t s which had
been fe d a f a t f r e e d ie t and which e x h ib ite d th e e s s e n t i a l
f a t t y a cid d e f ic ie n c y symptoms.
E s s e n tia l f a t t y a c id d e f ic ie n c y symptoms have a ls o
been n oted in s p e c ie s o f anim als o th e r than r a ts* B ie r i e t
a i ( 2 0 ) r e port e(i th a t c h ic k s fe d a d ie t c o n ta in in g O.Oij.6 per
c e n t f a t fo r s i x weeks had a reta r d e d growth r a t e , d ep ig
m en tation o f f e a t h e r s , and s c a lin e s s o f th e s k in . When
compared w ith ch ic k s who had a v e g e ta b le f a t in t h e ir
d i e t s , th e f a t d e f ic ie n t m ale ch ic k s had sm a lle r t e s t e s anc
some o f th e f a t d e f ic ie n t fem ale c h ic k s d id n ot show normal
( 2 1 )
developm ent o f th e o v id u c ts., P e ife r and Holman' ' were
6
a b le to a l le v i a t e th e dermal symptoms o f r a b b its fe d a f a t
fr e e d i e t c o n ta in in g c h o le s te r o l by s u b s tit u tin g corn o i l
fo r c h o le s t e r o l.
The dog i s a ls o r e a d ily s u s c e p tib le to th e developm ent
o f th e f a t d e f ic ie n t syndrome when p la c e d on an e s s e n t i a l
f a t t y a c id d e f ic ie n t d ie t . Hansen and W iese^2 2 ^ f i r s t
dem onstrated th a t th e symptoms o f f a t d e f ic ie n c y in . dogs
are s im ila r to th o se produced in o th er s p e c ie s .
There i s a c o r r e la tio n betw een th e e s s e n t i a l f a t t y
a c id con ten t o f th e b lood and th e occu rren ce o f eczem a, not
o n ly in in fa n ts but a ls o in young c h ild r e n and a d u lt s .
G-rupper and Azerad^2^) showed th a t th e r e was a r e la t io n s h ip
betw een th e a d d itio n o f f a t to c h ild r e n 's d ie t and th e
d isap p earan ce o f eczema. Two groups o f w ork ers, Faber and
R oberts^2^ and G onbleet and P ace,^ 2^) have c o r r e la te d th e
appearance o f eczem atous symptoms in a d u lt human s u b je c ts
w ith th e red u ctio n in th e l e v e l o f u n sa tu ra ted f a t t y a c id s ,
e s p e c ia ll y o f l i n o le a t e and a ra c h id o n a te , in th e b lo o d .
F u n ction s o f E s s e n tia l F a tty A cid s
In a d d itio n to a l l e v i a t i n g dermal symptoms, e s s e n t i a l
f a t t y a c id s m ed iate a v a r ie d number o f fu n c tio n s . On a d ie t
adequate excep t f o r e s s e n t i a l f a t t y a c id s th e r a te o f groxcfch
o f r a te can be shown to be a lin e a r fu n c tio n o f th e lo g dose
o f l i n o l e i c a c id .^ k * ^ '^ D euel and co -w o rk ers( 2^) rep o r ted
t h a t w ean lin g m ale r a ts fe d a f a t d e f ic ie n t d ie t d id not
7
respond to growth hormone. A prompt resp on se to growth
hormone was o b ta in e d , however, when the r a t s were su p p le
mented w ith 20 mg o f l i n o l e i c a cid p er day; 60 mg o f
l i n o l e i c a c id produced no fu r th e r a c c e le r a tio n in grow th.
Pat d e f ic ie n t r a t s in je c te d growth hormone and fed m ethyl
li n o l e a t e r e a c te d im m ediately in-, c o n tr a st to th e more tard y
r e a c tio n when m ethyl lin o l e a t e was g iv en a lo n e . Adm inis
t r a t io n o f a 1 0 .0 per cent c o tto n se e d o i l d ie t to r a t s
r e c e iv in g l i n o l e a t e a t an optimum l e v e l r e s u lte d in fu r th e r
a c c e le r a tio n o f grow th.
Deuel e t §1,(28) showed th a t th e r e was a sex d iffe r e n c e
in th e requirem ent fo r l i n o l e i c a c id . The optimum dosage
fo r m ale r a t s ex ceed s 50 mg d a ily , w h ile th a t fo r fem ale
r a t s has been found to be betw een 10 and 20 mg p er r a t p er
day. The growth r a te o f m ale r a ts p lo t t e d a g a in st th e lo g
dose o f lin o le a t e was a lin e a r fu n c tio n when l i n o l e a t e in
th e range o f 5 to 50 mg were a d m in istered .
Kramar and Kovacs^2^ were th e f i r s t I n v e s tig a to r s to
c a l l a tte n tio n to th e fa c t th a t th e c a p illa r y r e s is t a n c e o f
human su b je c ts was h ig h er in p a tie n ts who had r e c e iv e d
v e g e ta b le o i l s than in th o se who had partaken e x c lu s iv e ly
o f a d ie t c o n ta in in g anim al f a t s . Fourteen years l a t e r ,
Kramar and L e v in e(30) were a b le to prove th a t e s s e n t i a l
f a t t y a c id s p r o te c te d r a ts a g a in s t th e c a p illa r y f r a g i l i t y .
The absence o f e s s e n t ia l f a t t y a c id s in th e d ie t o f
r a ts r e s u lt s in a fu r th e r d e f ic ie n c y symptom, c h a r a c te r iz e d
8
by in c r e a se d p e r m e a b ility o f th e sk in to R in g er's s o lu t io n .
I t would seem th a t th e e s s e n t i a l f a t t y a c id s may be
req u ired fo r th e m anufacture o f c e r ta in c e l l la y e r s n e c e s
sary fo r proper sk in fu n c tio n ..(31)
There i s now c o n sid e r a b le evid en ce th a t th e l e v e l o f
d ie ta r y f a t may be o f im portance In p h y s io lo g ic a l fu n c tio n s
such a s pregnancy and la c t a t io n . I t i s c e r ta in th a t a t
l e a s t a p o rtio n o f th e b e n e f ic ia l e f f e c t s o f f a t s on
pregnancy and la c t a t io n can be a sc rib e d to t h e ir e s s e n t i a l
f a t t y a c id c o n te n t. Deuel e t a l/3 2 ) supplem ented fem ale
r a ts w ith s e v e r a l l e v e l s o f c o tto n se e d o i l o r o f m ethyl
l i n o l e a t e during th e p erio d o f b reed in g and la c t a t io n ,
a lth o u g h fa t was not req u ired by th e fem ale r a t fo r concep
t io n . When th e d ie t was o th e rw ise com p lete, In th e absence
o f a l l fa t from th e d i e t , th e young were in v a r ia b ly born
dead o r d ied im m ediately a f t e r b ir t h . Whereas d a ily d o ses
o f c o tto n se ed o i l as low as 10 mg in su red th e s u r v iv a l o f
th e pups fo r th r e e d ays, a s much a s 200 mg o f t h is f a t was
req u ired p er day fo r an optimum e f f e c t on th e rep rod u ction
and la c t a t io n p r o c e s s . S in ce the in v e s t ig a t o r s ob served
com parable r e s u lt s when a t l e a s t 80 mg o f m ethyl l i n o l e a t e
was fe d , they concluded th a t th e e s s e n t ia l f a t t y a c id s
c o n s t it u t e th e p r o t e c t iv e component in th e f a t .
D. E ffe c t o f E s s e n tia l F a tty A cid on C h o le ste r o l
M etabolism .
E s s e n tia l f a t t y a c id s have been shown to be in v o lv e d
j in c e r ta in p h a ses o f c h o le s te r o l m etabolism . As e a r ly as
(•S 'S }
: 1923» B loor in h is s tu d ie s o f u n sa tu ra ted f a t t y a c id s
1
in th e plasm a o f v a rio u s s p e c ie s o f anim als found th a t
j th e s e a c id s e x is t e d m ainly in com bination w ith c h o le s t e r o l.
S u b seq u en tly, B lo o r and h is c o - w o r k e r s e x t e n d e d th e s e
o b se r v a tio n s and found th a t th e u n sa tu ra ted f a t t y a c id
e s t e r , c h o le s te r o l l in o l e a t e , was th e c h ie f e s te r o f
c h o le s te r o l in th e plasm a. K elsey and L on gen ek er^ -^ have
a ls o shown th a t in plasm a o f c a t t l e , 62 p er cent o f
c h o le s te r o l was combined w ith l i n o l e a t e .
1 I t has been rep o rted th a t in th e r a t th e a d m in istra -
I
! tlo n o f a f a t f r e e d ie t r e s u l t s in a r e d u c tio n o f b lo o d
c h o le s t e r o l. Gofman e t a l^ 3 6 ) ob served th a t d ie ta r y
' r e s t r ic t i o n o f f a t and c h o le s te r o l in man s im ila r ly
r e s u lte d in a re d u ctio n o f b lo o d c h o le s t e r o l. However,
J A lfin -S la te r et. a lJ 3 7 ) ob served th a t alth o u g h th e amount o f
c h o le s t e r o l in th e plasm a o f th e r a ts m ain tain ed on a fa t
f r e e d ie t i s lo w er than th a t found in r a t s r e c e iv in g a d ie t
i
c o n ta in in g f a t , a concom itant In crea se in th e l i v e r
c h o le s t e r o l and th e adrenal c h o le s t e r o l o c c u r s.
The r e la t io n s h ip between th e in ta k e o f e s s e n t i a l f a t t y
a c id s and plasm a o r l i v e r c h o le s te r o l c o n c e n tr a tio n s has
been shown by many in v e s t ig a t o r s . The exp erim en tal d a ta
o b ta in ed by Cochrane e t a l , ^ 8 ) by K in s e ll e t a l , ^ 9 )
by K i n s e l l i n d i c a t e th a t e le v a te d l e v e l s o f b lood
10,
I I
I c h o le s te r o l in c o n d itio n s such as d ia b e te s can be g ra d u a lly !
j d ecrea sed to normal when la r g e amounts o f v e g e ta b le f a t s
(c o n ta in in g e s s e n t i a l f a t t y a c id s ) are in clu d ed in th e \
d i e t . M oreover, Mukherjee and A l f i n - S l a t e r f o u n d th a t
th e a d d itio n o f l i n o l e i c a c id to r a ts f e d a f a t f r e e d ie t j
i
m a in ta in s a normal l i v e r c h o le s te r o l l e v e l and normal j
h e p a tic c h o le s te r o l s y n th e s is .
A con n ection between e s s e n t i a l f a t t y a c id s and c h o le s -j
t e r o l m etabolism has a ls o been shown by Bromer and D a y .^ 2 ^
They found th a t fe e d in g c h o le s te r o l to r a t s on an e s s e n t i a l ,
f a t t y a c id d e f ic ie n t d ie t h a sten ed th e appearance o f th e
{d e f ic ie n c y syndrome and in c r e a se d the s e v e r it y o f
!d e f ic ie n c y symptoms. They a ls o observed th a t th e l i v e r ^
i
|t o t a l l i p i d was in c r ea se d to an e x ten t over and above th a t i
i
'a t tr ib u t a b le to th e accum ulation of c h o le s t e r o l.
j - i
j Relation o f F a ts to C h o le ste r o l M etabolism
The n atu re o f th e d ie ta r y f a t has been shown to a f f e c t
c h o le s t e r o l m etabolism . In s t u d ie s on humans, Ahrens e t '
I
\a l}* 3 ) h a v e e h o w n th a t s ix s u b je c ts w ith d ie ta r y o b e s it y ;
showed s ig n if ic a n t red u ctio n in serum f r e e end t o t a l I
I
c h o le s t e r o l c o n c e n tr a tio n s when p la n t f a t s were s u b s titu te d
i s o c a l o r i c a l l y f o r etnimal f a t s . Gordon and co-w ork ers,
who a ls o worked w ith human s u b j e c ts , confirm ed th e f a c t th a t
th e in g e s tio n o f an u n sa tu ra ted f a t , i . e . , su n flow er o i l ,
low ered serum c h o le s te r o l l e v e l . However, hydrogenated
11
coconut o i l fe d under s im ila r c o n d itio n s produced a
su sta in e d r i s e o f serum c h o le s te r o l l e v e l s . When hydro
g en a ted coconut o i l was fe d in la r g e am ounts, the a d d itio n !
o f an equal amount o f su n flow er seed o i l to th e d ie t w ith a
correspon d ing in c r e a s e in c a lo r ic in ta k e le d to a d ecrea se
in c h o le s te r o l serum l e v e l . The in tr o d u c tio n o f su n flow er
seed o i l le d to tr a n s ie n t e x c e s s iv e f e c a l e x c r e tio n o f fat.
Normal f a t a b so rp tio n was m ain tain ed by a s li g h t in c r e a se
o f n e u tr a l s t e r o l s and c o n sid e r a b le In c r e a se o f b i l e a cid s
in th e d ie t . I t was concluded by the in v e s t ig a t o r s th a t
su n flo w er seed o i l prom otes ca ta b o lism and e x c r e tio n o f
c h o le s t e r o l.
I t has been shown^ 5 ) th a t s e l e c t i v e l y hydrogenated
v e g e ta b le o i l s c o n ta in in g some e s s e n t ia l f a t t y a c id s are
f u l l y d ig e s t ib le , h a rm less, and o f n u t r it io n a l v a lu e as
shown by lo n g term s tu d ie s w ith r a t s . However, A lf in -
S la te r e t a l ^ 6 ) rep o rted th a t th e a d d itio n o f hydrogenated
coconu t o i l to th e f a t f r e e , but o th erw ise adequate, d ie t
o f r a t s r e s u lte d in d e p le tio n o f e s s e n t ia l f a t t y a c id s In a
sh o r te r tim e in te r v a l and at a low er anim al w eigh t than when
th e r a t s were fe d a f a t fr e e d ie t a lo n e . In r a ts r e c e iv in g
d ie t s d e f ic ie n t in e s s e n t ia l f a t t y a c id s an in cr ea se d
c h o le s te r o l co n ce n tra tio n in th e l i v e r and a d ecreased
c h o le s t e r o l co n ten t in the. plasm a was found a f t e r one week
o f fe e d in g . However, a lth ou gh th e e s s e n t i a l f a t t y a c id
d e f ic ie n c y symptoms were aggravated in th e. anim als on th e
12
f a t f r e e d i e t , th e plasm a and l i v e r c h o le s te r o l l e v e l s o f
r a ts r e c e iv in g hydrogenated coconut o i l in t h e ir d i e t s
g r a d u a lly retu rn ed to normal ra n g es. The in v e s t ig a t o r s
su g g e st th a t t h i s e f f e c t may be due to th e a v a i l a b i l i t y o f
sh o rt chain f a t t y a c id s co n ta in ed in hydrogenated coconut
o i l f o r e s t e r i f i c a t i o n o f c h o le s t e r o l. S ub seq u en tly,
A lf in - S la t e r e t a l ^ 7 ) in v e s tig a te d th e e f f e c t o f hydro
g en a ted t r io le in : on u t i l i z a t i o n o f e s s e n t i a l f a t t y a c id s in
th e r a t . Male r a t s d e p le te d o f e s s e n t i a l f a t t y a c id s fo r
16 weeks were then given supplem ents o f m ethyl l in o l e a t e
and w ith o u t th e hydrogenated t r i o l e i n p ro d u ct. They found
t h a t , u sin g grow th as a c r it e r i o n , th e r e were no i l l
e f f e c t s due to th e p resen ce o f th e hydrogenated t r i o l e i n
prod uct which co n ta in ed 33 p er cen t o f tra n siso m ers o f
o l e i c a c id and no e s s e n t ia l f a t t y a c id .
The r e p o r ts o f th e e f f e c t s o f d if f e r e n t f a t s on
c h o le s t e r o l m etabolism end on e s s e n t ia l f a t t y a c id d e f i
c ie n c y symptoms a re very c o n tr o v e r s ia l. As e a r ly as 1932,
Evans and L eprovsky^® ) rep o rted th a t th e f a t d e f ic ie n t
syndrome was aggravated when r a t s on an e s s e n t ia l f a t t y
a c id d e f ic ie n t d i e t were fe d sa tu r a te d f a t s . These
fin d in g s were confirm ed in 1936 by S i n c l a i r ^ 9 ) and by
Deuel in 1 9 5 1 . R ec en tly , se v e r a l I n v e s tig a to r s t e s t e d
a la r g e number o f o i l s fo r t h e ir e f f e c t s on c h o le s te r o l
l e v e l s . H egsted, G o ts is , and S ta r e ^ ) rep o rted th a t non-
e s s e n t i a l u n sa tu ra ted f a t t y a c id s ( o l e i c , o le o s t e a r lc )
13'
: I
prom ote h y p e r ch o le ste r o le m ia in r a t s , w hereas e s s e n t i a l an d 1
I sa tu r a te d f a t t y a c id s reduced h y p e r ch o le ste ro le m ia .
i
j Anderson, K eys, and Grande^ 1 ) observed t h a t , in men, serum'
! c h o le s te r o l resp o n se to v a r io u s f a t s corresponded rou gh ly
to th e p r in c ip le th a t sa tu r a te d f a t s prom ote h ig h e r e h o le s -j
t e r o l l e v e l s than p o ly u n sa tu ra ted fa te ; but n e ith e r th e j
d egree o f u n sa tu ra tio n o f th e f a t nor l i n o l e i c a c id co n ten t
f u l l y ex p la in ed t h e ir r e s u l t s . The in v e s t ig a t o r s a ls o
o b serv ed th a t coconut o i l e le v a te d th e c h o le s te r o l l e v e l s
l e s s than w ould be exp ected from th e t h e o r ie s th a t d eg ree
| o f sa tu r a tio n o r co n ten t o f e s s e n t ia l f a t t y a c id s o f th e
I d ie t i s th e c o n t r o llin g fa c to r o f th e t i s s u e c h o le s te r o l
■ i
l e v e l s . 1
i
; Gerson et. a l f e d a group o f r a t s fo r s e v e r a l weeks
a f a t f r e e d ie t to which th ey added sm all q u a n titie s of
l i n o l e i c a c id , b e e f f a t , o r m ethyl h exad ecan oic a c id and
i
s tu d ie d t h e ir e f f e c t on t o t a l , e s t e r , and fr e e c h o le s te r o l
co n ten t o f c a r c a ss l i p i d s . T otal c h o le s t e r o l co n ten t o f j
i
•the c a r c a ss l i p i d s was in c r e a se d by l i n o l e i c a cid or b e e f |
f a t , but m ethyl h exad ecan oic a c id was w ith o u t e f f e c t . Both
e s t e r and f r e e c h o le s te r o l l e v e l s in th e t i s s u e s o f th e
r a ts were in c r e a se d by l i n o l e i c a c id . With b e e f f a t , th e
e s t e r c h o le s te r o l l e v e l was in c r e a se d ,b u t th e f r e e c h o le s
t e r o l l e v e l was d ecr ea se d . Male r a ts t h a t were fe d
l i n o l e i c a c id o r b e e f f a t showed a much g r e a te r In c r e a se o f
c a r c a ss e s t e r c h o le s te r o l l e v e l s than d id fe m a le s. In !
i»!
: exp erim en ts w ith m ale r a t s , 3hon and K r o b e r '^ ) determ ined I
I I
th e c h o le s te r o l co n ten t o f l i v e r s o f t h e s e anim als fe d
I !
e it h e r a low f a t b a sa l d ie t o r a d ie t co n ta in in g 20 p er |
! I
I cen t by w eight o f th e com m ercial f a t s to be t e s t e d . They
found th a t th e in c r e a s e s in th e c h o le s te r o l co n ten t o f th e j
l i v e r were produced by f a t s w ith a r e l a t i v e l y h igh co n ten t j
o f u n sa tu ra ted f a t t y a c id s such as o l i v e o i l and su n flow er
seed o i l . The in v e s t ig a t o r s ex p la in ed th e e f f e c t o f o liv e
o i l by th e p r esen ce o f sm all amounts o f sq u a len e, a
I
c h o le s t e r o l p r e c u r so r , in th e o i l . j
I t has been su ggested ^ **) th a t th e c h o le s t e r o l d ep r es- j
I i
s in g e f f e c t o f c e r ta in f a t s i s r e la te d to t h e ir p o ly u n sa t
u ra ted f a t t y a c id c o n te n t. Corn o i l has been shown to
I
|r e s u lt in th e lo w erin g o f th e serum c h o le s t e r o l l e v e l s o f ;
I s e v e r a l s p e c ie s o f an im als. B everidge et. al^-55) rep o rted
i th a t corn o i l co n ta in ed a f a c t o r o r f a c t o r s th a t le a d to a
|d e p r e ssio n o f plasm a c h o le s t e r o l in r a t s . They a ls o
!
dem onstrated th a t a l l f a t s o f anim al o r ig in co n ta in v a ry in g :
i
amounts o f a f a c t o r o r f a c t o r s th a t produce and In cr ea se in 1
plasm a c h o le s te r o l l e v e l s . Jon es and h is c o - w o r k e r s ^ 6 ) !
j
have observed th a t in c h o le s t e r o l fed c h ic k s , co m o i l
seemed to produce a low er plasm a c h o le s te r o l l e v e l than d id
c o tto n se e d o i l . I t was p o s tu la te d th a t an u n id e n tifie d
f r a c t io n o f th e co m o i l , n ot e a s ily e x tr a c te d , was
I n h ib it in g th e ex p ected r i s e in c h o le s te r o l co n ce n tra tio n
l
*
o f th e ch ick s fe d th e c h o le s t e r o l c o n ta in in g d i e t . :
i ^
I I I . F a tty A eide and A th e r o s c le r o s is
The Im portance o f e s s e n t i a l f a t t y a c id s in r e la t io n
to a t h e r o s c le r o s is has been dem onstrated by K ritch evsk y and
h is co -w o rk ers. They found th a t th e fe e d in g o f corn
o i l , w hich c o n ta in s a h ig h p ro p o rtio n o f e s s e n t i a l f a t t y
a c id s , was a b le to suppress th e d e p o s itio n of c h o le s te r o l
in th e a o rta o f rs.b b its to marked e x te n t, even when 3 p er
cen t c h o le s te r o l was added to th e d i e t s o f th e se a n im a ls.
On th e o th e r hand, sh o rten in g (a hydrogenated v e g e ta b le
o i l ) d id not appear to a l l e v i a t e th e c o n d itio n . These d ata
lik e w is e r e fu te th e concept th a t f a t p er se has an a th e ro
g e n ic e f f e c t . S u b seq u en tly , the same in v e s t ig a t o r s fe d
r a b b its a d ie t c o n ta in in g 2 p e r cen t o f c h o le s t e r o l
d is s o lv e d in m eth yl s t e a r a t e , m ethyl o l e a t e , o r m eth y l-
9 * £ ll-o c ta d le n a te . They n oted th a t th e s e v e r it y o f a th e ro -
mata d evelop ed by th e above d i e t s v a r ie d in v e r s e ly w ith th e
Io d in e Humber o f th e f a t t y a c id e s t e r . The in v e s t ig a t o r s
su g g e st th a t th e fu n c tio n o f u n satu rated f a t t y a c id s in
t h i s r e sp e c t i s to keep th e c h o le s te r o l a s s o c ia te d w ith th e
f a t t y a c id s In e s t e r form , suspended in th e b lo o d , and thus
a id in i t s rem oval v ia th e b i l e o r o th e r pathw ays.
CHAPTER I I I
PLAN O P THE EXPERIM ENT
F eed in g Experim ents With E th y l E aters Off F a tty A cids
Experiment I . Seven groups o f w ea n lin g male r a te were
m ain tain ed on a f a t f r e e d ie t and supplem ented w ith a f a t t y
a c id e s t e r fo r s i x w eeks, a t th e end o f w hich tim e th e y
were s a c r if ic e d . The d ie t s w ere a d m in istered as f o llo w s :
Supplem ents to f a t f r e e d ie t
\
ate lo o m g /ra t/d a y
e th y l p a lm ita te .
e th y l s te a r a te
e t h y l o le a t e
e th y l e la id a t e
e t h y l l in o le a t e
e th y l b u ty ra te
none
Experiment I I . The same d ie t s were used as in E xp eri
ment I but w ith th e a d d itio n o f 1 p er cen t o f c h o le s t e r o l
and 0 .2 5 p er cent o f b i l e s a l t s to th e f a t f r e e d i e t .
A gain, seven groups o f m ale r a t s , groups 8 to 1^ , w ere
in v e s t ig a t e d .
Experiment I I I . S ix ty w ean lin g m ale r a ts were d iv id e d
in to s i x grou p s. The ex p erim en ta l p e r io d con tin u ed f o r s ix
w eeks, a t th e end o f which tim e th e r a t s were s a c r if ic e d .
Group No. o f
1 10
2
n
3
n
k
i t
5
ii
6
n
7
i i
17
The anim als were fe d and supplem ented a s fo llo w s :
Supplem ents to th e f a t fr e e d i e t
( 1 ) (2 )
' Group 100 m g /d a y /ra t 100 m g/d ay/rat
15 e th y l p a lm ita te e th y l lln o l e a t e
16 e th y l s t e a r a te e th y l l l n o l e a t e
17 e th y l o le a t e e th y l ll n o le a t e
18 e th y l e la id a t e e th y l l l n o l e a t e
t
19 e th y l lln o l e a t e e th y l l l n o l e a t e
20 e th y l b u ty ra te e th y l lln o l e a t e
In exp erim en ts I and I I I th e fo llo w in g were stu d ie d :
1 . Growth .
2 . S e v e r ity o f sk in d e f ic ie n c y symptoms.
3 . F ree, t o t a l , and e s t e r ( t o t a l - f r e e ) c h o le s t e r o l in
b lo o d •
F ree, t o t a l , and e s t e r ( t o t a l - f r e e ) c h o le s t e r o l In
l i v e r .
5 . T otal c h o le s te r o l in adrenal*
i
6 . T otal l i p i d in l i v e r .
7 . L iv er w e ig h t, a s p er cen t o f body w eig h t.
8 . Per cen t o f p o ly u n sa tu ra te d f a t t y a c id s in f a t t y
a c id s o f l i v e r l i p i d s .
9 . The Io d in e Number o f f a t t y a c id s o f l i v e r l i p i d s .
Experim ents w ith Fat Free and 15$ H ydrogenated Coconut O il
Experiment IV . M u lt male r a ts (17 weeks o ld ) were
m ain tain ed on th e fo llo w in g d ie t s f o r s ix w eeks, a t th e end
o f w hich tim e th e y were s a c r i f i c e d . The d ie t s were admin
is t e r e d as fo llo w s :
Group Supplem ents to th e f a t fr e e d ie t
XXIIIa 1% c h o le s t e r o l
X X IIIb 1$ c h o l e s t e r o l p lu s 0 . 2 5% b i l e s a l t s
X X IH e 0 .2 5 ^ b i l e s a l t s
X X IIId none
Experiment V. Adult m ale r a ts (4 months o ld ) w ere fe d
hydrogenated coconut o i l d ie tp a s fo llo w s :
Group Supplem ents to th e 1^% hydrogenated
coconut o i l d ie t
XXIa 1% c h o le s te r o l
XXlb 1 $ c h o l e s t e r o l p lu s 0 . 2 5% b i l e s a l t s
XXIc b i l e s a l t s
XXId none
In e x p e r im e n ts I I , IV , and V t h e f o l lo w in g c r i t e r i a
w ere s tu d ie d :
1 . G row th.
2 . S e v e r i t y o f s k in d e f i c i e n c y sym ptom s.
3 . F ree, t o t a l , and e s t e r c h o le s t e r o l in b lo o d .
I*. F ree, t o t a l and e s t e r c h o le s te r o l in l i v e r .
5 . T o ta l c h o le s t e r o l in a d ren a l.
6 . T o ta l l i p i d in l i v e r .
7* L iver w eig h t as p e r c e n t o f body w e ig h t.
CHAPTER IV
MATERIALS A N D M ETH O D S
Male a lb in o r a t s o f th e U n iv e r s ity o f Southern C a lifo r
n ia s t r a in were u sed .
The w ean lin g r a ts were o b ta in ed from m others w hich
were on a low f a t d ie t from th e tim e o f m ating and during
la c t a t io n . Twenty-one day o ld r a t s w eig h in g 30 to 5© grams
were p la c e d two In each c a g e , w ater and fo o d b ein g su p p lie d
ad lib it u m . The tem perature o f the room in w hich th e r a ts
were k ep t was ap p roxim ately 78° P; how ever, some v a r ia tio n s
o ccu rred .
The anim als w ere supplem ented o r a lly w ith 100 m g /r a t/
day o f a f a t t y a c id e s t e r and th e su pp lem entation p e r io d
co n tin u ed fo r s i x w eeks, a t th e end o f w hich tim e th e y were
s a c r if ic e d .
The a d u lt m ale r a t s used in exp erim en ts IV and V had
been fe d e it h e r f a t fr e e or 15 p er cen t hydrogenated
coconut o i l d ie t f o r fo u r m onths, u n t il th e s t a r t o f th e
p r e s e n t in v e s t ig a t io n . They w ere m ain tain ed on t h e ir d ie t s
fo r s i x w eeks, a t th e end o f w hich tim e th e y were s a c r i
f ic e d .
Methods o f S a c r if ic e
The r a t s w ere a n e s th e tiz e d by in tr a -p e r it o n e a l in j e c
t io n s o f nem butal and b lood was taken by h eart p u n ctu re.
20
The l i v e r and ad ren a l g la n d s were e x c is e d , trimmed,
b lo t t e d , and w eigh ed .
D ie ts
The com p osition o f th e f a t fr e e and 15 p e r cen t hydro
g en a ted coconut o i l d ie t s a re g iv en in T ab les I I and I I I .
The d e sig n a tio n o f th e d ie t s used in T able I I i s as
fo llo w s :
FF m f a t f r e e .
FFG « f a t f r e e p lu s c h o le s t e r o l p lu s b i l e s a l t s .
ICO - 15 p er cen t hydrogenated coconut o i l .
F a ttv a c id s . E thyl b u ty r a te , e th y l o le a t e , e th y l
• \
e la ld a t e , e th y l s t e a r a t e , and e th y l p a lm ita te were k in d ly
p rep ared and su p p lie d by Dr. C. M. Gooding o f th e B est
Foods C orporation.
P rep a ra tio n o f E th yl Lino1 eat e
a . P rep a ra tio n o f e th y l e s t e r s o f m ixed f a t t y a c id s o f
s a fflo w e r o i l .
3 .0 grams o f eth a n o l was added to 100 grams o f s a f -
flo w e r o i l and th e m ixture was h eated to 55°C. I t was then
tr a n s fe r r e d to a sep a ra to ry fu n n e l and a llo w ed to c o o l.
The bottom la y e r (g ly c e r o l) was removed and the e s t e r s were
washed w ith f i r s t c o ld , then h ot w a ter. The s o lu tio n was
d r ie d w ith anhydrous sodium s u lf a t e and f i l t e r e d .
b. S ep aration o f e th y l l l n o l e a t e .
To each gram o f mixed e s t e r s in ste p a , 125 00 o f 95
TABLE I I
COMPOSITION OF DIETS
Component FF H G O
% %
C asein 20.00 2 4 .0 0
S a lt mix if. 00 if. 00
C e llu flo w e r if. 00 4 .0 0
V itam in s 0.23
0 ,2 3
C holine 0 . 2if 0 .2 4
S ucrose 7 1 .5 3 5 2 .5 3
H ydrogenated
coconut o i l
------
15.00
22
TABLE I I I
COMPOSITION O F VITAMIN M ix i/
Component
G-rams
p-A m lnobenzoic A cid 4 8 4 .0
I n o s it o l 400.0
A sco rb ic Acid 1 6 0 .0
Calcium P an toth en ate 4 8 .0
Thiamine 7 .0
N iacin 4 8 .0
R ib o fla v in ; 22.0
P y rid o x in e H ydrochloride 21.0
F o lic Acid 8.0
Menadione 4.0'
B io tin 2.0
V itam in B-#12, 0 .1 $ T r itu r a tio n 4 8 .0
C r y s ta le ts ^ / 1 0 .4
Alpha Tocopherol A ceta te 2 7 7 .3
V itam in -F ree T est C asein 12270.0
2 grams o f t h is v ita m in mix i s used in 100 grams o f
d ie t ; t h i s In tro d u ces 0.23 grams o f v ita m in s and 1.77 grams
o f e a s e in .
1 gram c o n ta in s 500 U .S .P . u n its o f Vitam in A and
50 U .S .P . u n its o f Vitam in Bg*
23
p er cen t eth a n o l were added. The eth a n o l s o lu tio n was
h ea ted to 50°C and 175 grams o f urea w ere added. The
tem perature was m aintain ed f o r o n e -h a lf hour, a f t e r w hich
th e m ixtu re was a llo w ed to c o o l o v e r n ig h t. The s o lu tio n
was f i l t e r e d and th e p r e c ip it a t e was washed w ith c o ld 95
p er c e n t eth a n o l and then d isca r d e d . To th e f i l t r a t e , in
a la r g e sep a ra to ry fu n n e l, w a ter was added u n t il th e e s t e r
r o se to th e s u r fa c e . The aqueous phase was drawn o f f and
d isc a r d e d . The e s t e r was washed w ith w a ter (c o ld , th en
h o t ) , d ried w ith sodium s u lf a t e and f i l t e r e d .
The e th y l l l n o le a t e was p u r ifie d by vacuum d i s t i l l a
t i o n , th e f i r s t 5 p er cen t o f th e d i s t i l l a t e b ein g
d isc a r d e d .
The s a fflo w e r o i l was k in d ly su p p lie d by th e P a c if ic
V eg eta b le O il Company.
E x tra c tio n o f L ip id s
Waring B lendor m ethod. The e x tr a c tio n method was
b ased on a method o f Thompson e t a l . (58) The l i v e r was
p la c e d in th e Waring B lendor and 30*0 ml o f a 2 :1 eth a n o l
w ater m ixture and 100 ml o f S k e lly so lv e B (b .p . 63»3-69»3°C )
were added and th e m ixture was hom ogenized fo r f i v e
m in u tes. F o llo w in g a s ta tio n a r y p erio d o f f iv e m in u tes,
th e m ixtu re was rehom ogenized fo r an oth er f iv e m in u tes.
Then th e c o n te n ts o f th e Blendor were q u a n tita t iv e ly tr a n s
fe r r e d to a sep a r a to r y fu n n e l, th e la y e r s sep a ra ted , and
2 k
: th e homogenate was reex tra cte& tw ice w ith 100 ml o f
i S k e lly s o lv e B. The e x tr a c ts were p o o led and co n ce n tra ted
by ev a p o ra tio n w ith h eat and f i l t e r e d in to v o lu m etric
1 f l a s k s .
i Plasma e x tr a c tio n m ethod. The b lo o d o b ta in ed from th e
h ea rt was tr e a te d w ith sm all q u a n titie s o f h eparin to
p rev en t c l o t t i n g . The sam ples were c e n tr ifu g e d f o r tw enty
m in u tes a t 2500 rpm. F ourteen ml o f e th a n o l: a ceto n e (1:1
by volum e) were added to each ml o f plasm a by means o f a
s y r in g e , u sin g enough fo r c e to in su re com plete m ix in g .
I
| The plasm a p r o te in s were p r e c ip ita t e d and c h o le s te r o l and
j l i p i d s were e x tr a c te d by th e s o lv e n t . The r e s u lta n t
j
p r e c ip it a t e was c e n tr ifu g e d f o r ten m in u tes a t 3000 rpm,
i
| and th e e x tr a c t o b ta in ed was sto r e d in sto p p ered b o t t l e s
t
■ u n t il th e a n a ly se s were perform ed.
i
S o x h let m ethod. This method was used to e x tr a e t
I l i p i d s from th e a d ren a l g la n d s o f th e r a t s . At s a c r i f i c e ,
th e ad ren al g la n d s were removed, trimmed, p o o led and
j w eigh ed , and th en were p la ce d in an e x tr a c tio n th im b le .
The a d ren a ls were crushed on th e s id e o f th e th im b le by a
s p a tu la . The th im b le and c o n te n ts were p u t in S o x h let
e x tr a c to r s and w ere r e flu x e d fo r e ig h t hours w ith an
e th a n o l:e th e r m ixtu re (3 :2 by volu m e). The e x tr a c t was
then tr a n s fe r r e d to v o lu m e tric f la s k s and made up to th e
a p p ro p ria te volum e.
C h o le ste r o l d e te r m in a tio n . The method i s a
m o d ific a tio n o f th e Sehoenhelm er-Sperry method d evelop ed by
N le ft and D euel.
An a liq u o t o f th e t i s s u e e x tr a c t c o n ta in in g a p p ro x l-
i
I
m ately 0 .1 to 0.5 mg o f c h o le s te r o l was evap orated to
1
d ry n ess in a 60°C co n sta n t tem perature b lo c k under a stream
o f a i r .
One ml o f e th a n o l:a c e to n e s o lu tio n ( l : l by volum e) and
2 drops o f 33 p e r cen t K O H w ere added; th e tu b es were
corked and th e sam ples were k ep t a t 60°0 f o r f i f t y m inu tes
i
w ith freq u en t sh a k in g . The s o lu tio n was then n e u tr a liz e d
I
to th e p h en op h th alein end p o in t w ith 15 p er cen t a c e t ic
a c id . Then 2 ml o f e th a n o l:a c e to n e s o lu t io n ( l : l by
!volum e) were added. The c h o le s t e r o l d ig it o n id e was p r e c ip -
! it a t e d by th e a d d itio n o f 1 ml o f 0 .5 p er cen t d ig ito n in
s o lu tio n (in 50 p e r cent a lc o h o l) , and a llo w ed to in cu b a te
'a t room tem perature o v e r n ig h t. The p r e c ip it a t e was then
I c e n tr ifu g e d a t a speed o f ap p roxim ately 3000 rpm f o r about
tw en ty m in u tes. The su pern atan t liq u id was c a r e f u lly
decanted and th e p r e c ip it a t e washed w ith 3 nil o f anhydrous
e t h e r . This was then c e n tr ifu g e d a t a speed o f a p p ro x i-
(
m a tely 3000 rpm f o r ten m in u tes and th e su pern atan t e th e r
was d isca r d e d . The p r e c ip it a t e was d r ie d in a i r a t 60 °C,
0 .5 ml o f g l a c i a l a c e t ic a c id was added, and th e m ixtu re
was h eated a t 60°C u n t il th e p r e c ip it a t e was co m p letely
d is s o lv e d . Three ml of chloroform were added, and th e
tu b es were brought to ajtem p eratu re o f 35° 0 . _ _ _
j The c o lo r r e a g e n t, p r e v io u s ly p repared by adding 1 ml 1
, o f co n cen tra ted s u lf u r ic a c id to each 9 ml o f c h ill e d
I e-ce tlc anhydride was then added. The tu b e s were Incubated
! i
■ f o r e x a c tly ten m inutes a t 35°C a t which tim e th e tu b es
were p la c e d In an ic e b ath f o r a t l e a s t ten m inutes p r io r j
l
to r e a d in g . The c o lo r i n t e n s it y was th en read in a K le t t -
Summerson p h o t o e le c t r ic c o lo r im e te r a t 620 m illim ic r o n s .
A rea g en t blank c o n s is t in g o f a c e t ic a c id , ch loroform , and
c o lo r reagen t was alw ays used to s e t th e zero p o in t on th e
I
c o lo r im e te r . V alu es were read from a standard curve ;
determ ined sim u lta n e o u sly .
| For th e d eterm in a tio n o f f r e e c h o le s t e r o l, th e same
1 ' I
I p roced u re used in t o t a l c h o le s t e r o l was fo llo w e d w ith th e s e '
|e x c e p tio n s : ( l ) U su a lly a la r g e r a liq u o t was taken fo r
i
a n a ly s is , and (2 ) th e h y d r o ly s is ste p was o m itted .
I :
1 D eterm ination o f t o t a l l i p i d s . An a liq u o t o f 25 ml o f
i
‘l i v e r l i p i d e x tr a c t was evap orated to d ry n ess in an
Erlenm eyer f la s k on a h ot w ater b a th . C harring was ;
i
a v o id ed . Then 30 ml o f S k e lly s o lv e B and ap proxim ately 5 I
!gm o f anhydrous sodium s u lf a t e was added and th e m ixtu re wssj
f i l t e r e d through a Whatman f i l t e r paper in to a ta r e d j
Erlenm eyer f la s k . The o r ig in a l f la s k and co n te n ts on th e I
f i l t e r were r in s e d w ith f i v e s u c c e s s iv e p o r tio n s o f S k e lly
s o lv e B, and were combined w ith th e f i l t r a t e s . The
combined f i l t r a t e s c o n ta in in g t o t a l l i p i d s were then
evap orated to d ryn ess on a ho t w ater b a th , d ried o v ern ig h t J
27
in an oven a t 80°C, a f t e r w hich th e l i p i d s were w eigh ed . !
; D eterm ination o f io d in e number. To an a liq u o t o f a
!
chloroform s o lu tio n o f th e l i p i d , e x tr a c te d from l i v e r , a j
i !
d e f i n i t e amount (10 cc ) o f 0 .1 N o f io d in e m onochloride in
I g l a c i a l a c e t ic a c id (W ijs R eagent) was added. B lanks w hichj
|c o n ta in e d chloroform p lu s W ijs reagen t w ere run sim u lta n - j
e o u s ly . The g la s s stop p ered f la s k s were k ep t in th e dark <
i
i
f o r h a lf an hour. The r e a c tio n was stop p ed by adding 10 ml !
i
o f 10 p e r cen t KI s o lu t io n , and a f t e r a few m inutes th e
jm ixture was t i t r a t e d w ith 0 .0 3 N sodium t h lo s u lf a t e . The
l
,io d in e number was exp ressed a s th e number o f grams o f ;
I io d in e absorbed by 100 gm o f f a t t y a c id s .
I
iProcedure fo r L ip o x id a se Method fo r th e D eterm ination o f
j P o ly u n sa tu ra ted F a tty A cids
^ The lip o x id a s e method was ob ta in ed by p erso n a l commu
n ic a t io n from th e I n v e s t ig a t o r s , Dr. J . MacGee and Dr. F.
i
M attson o f P r o c te r and Gamble. I t i s a sim p le and ra p id
!
method fo r th e d eterm in a tio n o f p o ly u n sa tu r a te d f a t t y
a c id s . The p otassiu m s a l t s o f th e f a t t y a c id s are o x id iz e d :
* I
by atm ospheric oxygen in th e p resen ce o f th e enzyme
lip o x id a s e and th e a b so rp tio n o f th e d le n e h yd rop eroxid es j
a t 23^ m^tUin a Beckmann D U S p ectrop h otom eter. J
Room tem perature s a p o n if ic a t io n . O n e-tenth mg o f
l i v e r l i p i d e x t r a c t , PFA (p o ly u n sa tu ra ted f a t t y a c id s )
co n ten t about 10 p er c e n t, was p la c e d in a 100 ml g la s s
i
t
28
sto p p ered v o lu m e tric f la s k . The s o lv e n t was removed by-
g e n t le heat under a stream o f n itr o g e n . One ml o f 0 .5 per
i
j c e n t a lc o h o lic K O H was added, th e f la s k was flu s h e d w ith
' n itr o g e n , sto p p ered , and th e f la s k was r o ta te d to a llo w
good c o n ta c t betw een th e sam ple and th e r e a g e n t. The
sample was a llo w ed to stand a t room tem perature in th e dark
f o r more than fo u r h ou rs. Then 20 ml o f 1.0M p o ta ssiu m ,
b o r a te b u ffe r , pH 9 . 0 , 1 ml o f 0 .5 N HCL, and w ater to 100
ml a re added and m ixed a f t e r th e s a p o n ific a tio n . The
d il u t e sample co n ta in ed about 5 gammas o f PFA p er ml and
cou ld be a ssayed w ith o u t fu r th e r d ilu t io n . When th e sam ple
!co n ta in ed sa tu r a te d f a t s , g e n t le h e a tin g w ith th e a p p lic a -
1tio n o f n itr o g e n was n e c e ssa r y to a llo w good co n ta ct
Ibetween th e sam ple and th e a l k a l i .
I
j B lank . Three ml o f s o lu t io n to be t e s t e d c o n ta in in g
|5 tf to 25 £ o f PFA in 0 .2 M o f potassium b o ra te b u f f e r , pH
1 9.0 w ere p la c e d in a t e s t tu b e . 0.1 ml o f th e b o ile d
I
id ilu t e lip o x id a s e s o lu tio n was added and th e sample was
I
m ixed.
T est sam ple. Three ml o f PFA as u sed in th e b lan k
sam ple were p la c e d in a t e s t tu b e . 0 .1 ml o f th e d ilu t e
s o lu t io n o f lip o x id a s e was added and th e sample was m ixed.
In c u b a tio n . Both th e b lank and th e t e s t sample were
a llo w ed to stan d a t room tem perature f o r t h ir t y m in u tes.
A bsorption r e a d in g . A fte r t h ir t y m inu tes o f in cu b a
t i o n , th e blank and th e t e s t sample were tr a n s fe r r e d to
29
Beckman quartz c u v e tte s having a 1-em l i g h t p a th , and th e
Beckman D U sp ectrop h otom eter was a d ju sted to a zero reading
a t 23^ m yH w ith th e b lan k . The o p t ic a l d e n s it y o f th e t e s t
sam ple was read a g a in s t th e b lan k zero v a lu e .
C a lc u la t io n o f P o ly u n s a tu r a te d F a t t y A c id C on ten t
% PFA * OP x 3880
j —
Where: 0D = o p t ic a l d e n s ity
^ s t o t a l number of th e sample put in th e
c u v e tte
PFA ss p ercen t p o ly u n sa tu ra ted f a t t y a c id in
th e sample
Source o f f a c t o r (3880):
3880 = U L . x 1 0 0 0 x 100 x 3
3.0 7 9 .9
Where: 3*1 * d ilu tio n f a c t o r , 3 ®1* sample p lu s 0.1
3 .0
m l. enzyme.
79*9 s s p e c if i c e x tin c tio n c o e f f i c i e n t (0D o f
1 gm p er L. PFA in 1-cm . l i g h t p a th a t
23^ mjD .
1000 = c o n v e r s io n from gm/L to $ / m l .
100 = co n v ersio n to p ercen ta g e
3 = volume o f sample
CHAPTER V
RESULTS
E ffe c t o f A d m in isterin g F a tty Aold E ste r s to Male W eanling
Rats M aintained on a Fat Free D iet ( E xperim ent. I )
A ll th e r a t s , w ith th e ex cep tio n o f th o se r e c e iv in g
e th y l l l n o l e a t e , had th e t y p ic a l e s s e n t ia l f a t t y a c id
d e f ic ie n c y symptoms. The syndrome was more sev ere in the
groups r e c e iv in g sa tu r a te d f a t t y a c id e s t e r s than In th o se
groups r e c e iv in g th e u n sa tu ra ted n o h - e s s e n tla l f a t t y a c id
e s t e r s . The e th y l b u ty ra te supplem ented r a t s e x h ib ite d
th e m ost sev ere symptoms, th e e t h y l s t e a r a te fe d group
l e s s sev ere and th e e th y l p a lm lta te group showed th e l e a s t
se v e r e e s s e n t i a l f a t t y a c id d e f ic ie n c y symptoms when
compared w ith th e o th e r two sa tu r a te d f a t t y a c id e s t e r s .
In th e n o n - e s s e n tia l u n sa tu ra ted f a t t y a c id e s te r su p p le
m ented grou p s, o le a t e and e la ld a t e were e q u a lly e f f e c t i v e .
The c o n tr o l group, which r e c e iv e d a f a t f r e e d ie t a lo n e ,
d id n ot show a lo p e c ia as th e r a t s in th e o th e r groups
supplem ented w ith th e f a t t y a c id e s t e r s d id , but d evelop ed
s c a ly t a i l s and paws.
As shown in Table I f , th e anim als fe d th e f a t f r e e
d ie t supplem ented w ith b u ty ra te g ain ed 82 grams, w ith
s t e a r a te 97 gram s, w ith o le a t e 107 gram s, w ith e la ld a t e
128 gram s, w ith l l n o l e a t e 157 gram s, and th o s e on th e f a t
fr e e d ie t a lo n e g a in ed 122 grams during th e supplem entation
I
TABLE IV
G R O W T H O F W E A N L IN G M A L E RATS FED A FAT FREE DIET
SUPPLEM ENTED W IT H VARIOUS FATTY ACID ESTERS
SU M M A RY
Group
M r k
Supplement
to
f a t fr e e
d ie t
I n i t ia l
w eight
Weight in grame at w eekly In te r v a le
during supplem entation
Gain in w eight
during
supplem entation iv O •
1 2 3
4
5
6
v i l i /
(10 )
None
(co n tro l group)
gm
53
78 98
123 135
156
175
gm
122
VI
(10 ) B utyrate 56
79
100 121
135
144 138 82
I I
(9)
S tearate 54
81 101 120 122 145 151 97
(1 0 ) Palm i t a te 53 79 97 109 125
154 160
107
IV
(10 ) E lald ate 58 90 96
135 156 171 186 128
I I I
(10 ) O leate
53
80 100 116 126
152 160 107
V
(10 ) L ln o lea te 53
80
99 145 167 196 210
157
i/Ntamber o f r a ts in th e group. Vji
H
p e r io d .
The exp erim en tal d a ta o b ta in ed on plasm a and ad ren a l
c h o le s t e r o l l e v e l s are l i s t e d in Table V.
In th e t o t a l and e s te r f r a c t io n s o f plasm a c h o le s t e r o l
s ig n if ic a n t d iff e r e n c e s werd o b ta in ed betw een th e g ro u p s.
S te a r a te and b u ty r a te supplem ented groups e x h ib ite d th e
h ig h e s t plasm a c h o le s te r o l c o n c e n tr a tio n s ( t o t a l plasm a
c h o le s t e r o l, 6i . 2 mg % f o r th e s te a r a te g*oup and 7 1 .3 mg %
fo r th e b u ty ra te group) and th e f a t f r e e c o n tr o l group had
th e lo w e st plasm a c h o le s te r o l l e v e l (^3*6 mg % fo r t o t a l
plasm a c h o le s t e r o l) .
The ex p erim en ta l r e s u lt s o b ta in ed on th e l i v e r l i p i d
e x t r a c ts are c it e d in Table V I. In th e l i v e r c h o le s te r o l
c o n c e n tr a tio n s , th e v a r ia tio n s betw een th e v a lu e s o f th e
groups were m ain ly in th e t o t a l and e s t e r c h o le s te r o l
f r a c t io n s ; th e s t e a r a t e , e la ld a t e , and b u ty ra te groups
h a v in g th e h ig h e s t l e v e l s , w hereas p a lr a ita te , o le a t e ,
ll n o l e a t e supplem ented groups and th e f a t fr e e group had
low er l i v e r c h o le s t e r o l c o n c e n tr a tio n s.
As shown in Table V I, th e e la ld a t e supplem ented group
had th e h ig h e s t l i v e r t o t a l l i p i d c o n c e n tr a tio n s (7 1 .8
mg/gm) w ith b u ty r a te , p a lm ita te , s t e a r a t e , f a t f r e e , and
o le a t e supplem ented groups fo llo w in g i t in, d ecr ea sin g
c o n c e n tr a tio n s. The l ln o l e a t e supplem ented groups had the
lo w e s t l i v e r l i p i d co n ce n tra tio n o f a l l th e grou p s, 3^.0
mg/gm.
, : : ---------------------
TABLE V
PLASMA AND ADRENAL CHOLESTEROL LEVELS OF W EA NLING - M ALE RATS FED A
FAT FREE DIET SUPPLEMENTED W ITH VARIOUS FATTY ACID ESTERS
Group
No.
Supplement
to
f a t free
d ie t Free
Plasma c h o le s te r o l
mg % 2/
T otal E ster
Adrenal c h o le s te r o l
mg/gm2/
& t V
VII ,
( i o ) i /
None
(co n tr o l)
5-7
=0.7
|0 .6
± 4.2
37.9 3 .4 2 8 .4
VI
(10)
B utyrate
1 3 .5
±1.3
71.3
*9.3
57.8
4 .9
47.6
II
(9)
S tea ra te 11.0
±1.0
61.2
±4.3
50.2
3 .1
34.2
I
(10)
P alm itate 7.6
± 0 .7
45.3
±1.2
37.7 2 .7 3 4 .8
IV
(10 )
El a i date 10.1
±1.0
4 7 .8
± 0 .7
37 .7 5.3 3 2 .4
III
(10 )
O leate 12.1
± 0.8
57.5
± 1 .9
4 5 .4 3 .0
46.5
V
(10)
L ln o lea te 11.3
± 4.6
59-0
± 3 .4
47.7
4 .9 47.6
l/Number o f r a ts in the group. 2/ f = F ree.
2/ Including standard erro r o f the mean.
y T
= T o t a l.
T A B L E VI
CHARACTERIZATION O F LIVER LIPIDS O F W E A N L IN G M A L E RATS FED A
'FAT FREE DIET SU PPLEM EN TED W IT H VARIOUS FATTY ACID ESTERS
LIVER
Group
No.
Supplement
> to
fa t fr e e
d ie t
Wgt£/ % Body
wgt2/
C holesterol
mg/gm2/
f 5 / ty E1 /
Total l i p i d
mg/gmi/
Iodine
no. of
f a t t y
acid s
% PFA o f
l i p i d s 2/ »W
VII none
(10 )1/ (c o n tro l)
gm
8.2
± 0.8
4.8
+0.6
1.8
+0.1
2.6
±0.2
0.8 47.1
±0.3
66 2.9
VI
(1 0)
Butyrate
+5 ’9
1 0 .3
4.3
±0.4
1.9
±0.1
2.8
±0.1
0.9
6 5*6
+0.2
78
2.7
I I
(9)
S tearate 7.7
t o . 3
4.7
± 0.1
3.2
±0.2
4 .0
±0.3
0.8
.49.5
±0.2
62 3.2
I
(10 )
P alm itate 7.6
t l . l
4.4
±0.2
1 .4
±0.2
, 1.8
±0.1
0.4
57.3
10.2
75 2.3
IV
(10 )
E la ld a te
8.9
±0.6
5.7
±0 .1
2.0
±0 .1
+3.S
±0.3
1.8 71.8
±0.1
64
2.9
I I I
(10 )
O leate
7.7
5.0
±0.2
1.8
- 0.1
+2.5
±0.9
0.7 42.1
±0.1
64 2.0
V
(10 )
Lino1ea te 8.8
+0.7
4 .1
±0.2
+1 -9
±0.1
+2.3
±0.9
0.4 34.0
±1.0
227 8.4
i/Number o f r a ts in th e group.
2/ Including standard error o f the mean.
2/PFA = percen t o f polyunsaturated f a t t y a c id s.
4/t>etennlned by lip o x id a s e method.
5 / f = Free.
§ / t * T otal.
2/ e * E ster. K j3
&
The n atu re o f the f a t t y a c id s in th e l i v e r l i p i d was
ch a r a c te r iz e d by lip o x id a s e and Iod in e Number determ ina
t i o n s . The p ercen ta g e o f p o ly u n sa tu ra ted f a t t y a c id s o f
th e l i v e r l i p i d s o f th e l i n o l e a t e supplem ented group was
8 .4 p e r c e n t, w hereas th e same p ercen ta g e in th e o th er
groups ranged from 2 to 3 p er cen t a s shown in Table V I.
L iv er f a t t y a c id e s t e r s o f l i n o l e a t e supplem ented group
had th e h ig h e st Io d in e Number (2 2 7 ), th e b u ty ra te (78) and
th e p a lm ita te (75) having th e n ex t h ig h e s t , and th e o th e r
f a t t y e s t e r s supplem ented groups had lo w er Iod in e Number
v a lu e s ran gin g from 62 to 66 a s shown in Table VI.
E ffe c t o f Adding 1% C h o le ste r o l and 0 .2 5 # B ile S a lts to th e
Fat F ree D ie t s . Supplem ented w ith F a tty A cid E s te r s , o f
Male W eanling R ats (Experim ent I I )
The e s s e n t i a l f a t t y a c id d e fic ie n c y symptoms d eveloped
by th e v a rio u s gro u p s, w ith th e ex cep tio n o f th e l i n o l e a t e
supplem ented group, were s im ila r (but more se v ere) than
th o se o f th e groups in Experim ent I .
The growth r a te i s shown in T able V II, and th e plasm a
and ad ren al c h o le s t e r o l l e v e l s in Table V III. The r e s u lt s
o b ta in e d on th e l i v e r l i p i d e x tr a c ts are l i s t e d in T able
IX.
Of th e sa tu r a te d f a t t y a c id e s t e r s p a lm ita te , when
supplem ented to a n im a ls, e x h ib ite d a b e t t e r w eight g a in
than th e s te a r a te group and th e b u ty ra te anim als showed th e
. j ; :
TABLE VII
G R O W T H O P W E A N L IN G M A L E RATS FED A FAT FREE DIET PLUS C H O L E ST E R O L
PLUS BILE SALTS SU PPLEM EN TED W IT H VARIOUS FATTI ACID ESTERS
S U M M A R Y
Group
No.
Supplements
to
fa t fr e e
d ie t
In lt i a l
w eight
Weight in grams at w eekly in te r v a ls
during supplem entation
Gain in weight
during
supplem ent at i on
1 2
3
*
5
6
XIV
(1 0)1/
b ile s a l t s
c h o le s te r o l
gm
63
100 13*
155
170 191 202
gm
139
X III
(9)
b ile s a l t s
c h o le s te r o l
b utyrate
61 80 9* 101 138 1*0 161 100
IX
(9)
b ile s a l t s
c h o le s te r o l
ste a r a te
62 86 108 128 1*0 1*8
"163 101
V III
(1 0)
b i l e s a l t s
c h o le s te r o l
p a lm ita te
60 86 101 136
155 171 188 128
XI
(1 0)
b ile s a l t s
c h o le s te r o l
e la ld a te
51
76 101
113 137 15* 167 116
X
(10)
b ile s a l t s
c h o le s te r o l
o le a te
53
78 10* 12* 1*2 162
177 12*
XII
(1 0)
b i l e s a l t 8
c h o le s te r o l
lln o le a t e
60 83
113
1*0 169
18* 209 1*9
1/Number o f rata in th e group
-
s
TABLE V III
PLA SM A A N D A D R EN A L C H O LESTER O L LEVELS O F M A L E W E A N L IN G RATS FED A FA T FREE DIET
PLUS C H O LE ST ER O L PLUS BILE SALTS SU PPLEM EN TED W IT H VARIOUS FATTY ACID ESTERS
Group
No.
Supplements
to
fa t fr e e
d ie t
Free
Plasma c h o le s te r o l
mg % 2/
T otal E ster
Adrenal cho]
mg/gm2/
Free
^ esterol
Total
XIV
( 9 ) i /
b i l e s a l t 8
c h o le s te r o l
10 . 4
* .0 .4
59.8
± 2 .4
49'. 4 3 .7 28.5
X I II
(9)
b i l e s a lt s
c h o le s te r o l
but yrate
20.7
±0.9
68.2
±1.7
47.5 8 .7 35.0
IX
(10 )
b i l e s a lt s
c h o le s te r o l
ste a r a te
14.0
±1.2
51.1
± 3 .4
3 7 .1 4.0 20.0
V III
(10 )
b i l e s a lt s
c h o le s te r o l
p a lm ita te
9.0
*1.2
51.0
±1.6
42.0 2 .4
26 .7
XI
(10 )
b i l e s a lt s
c h o le s te r o l
e la ld a te
11.1
±1.3
4 2 .4
±5.1
31.3
12.0 36.0
X
(10 )
b i l e s a lt s
c h o le s te r o l
o le a te
17.5
±1.0
79.8
±8.6
62.3 2 .9
3 2 .4
X II
(10 )
b i l e s a lt s
c h o le s te r a l
lln o le a t e
12.1
±1.5
43.7
±3.1
31.6 6.8 38.6
1/Number o f r a ts in the group.
2 /In clu d in g standard error o f the mean.
TABLE IX
CH ARACTERIZATIO N O F LIVER LIPIDS O F M A L E W E A N L IN G RATS FED A FAT FREE DIET
PLUS C H O LESTER O L PLUS BILE SALTS SU PPLEM EN TED W IT H VARIOUS FATTY A C H D ESTERS
LIVER
Group
No.
Supplements
to
f a t fr e e
d ie t
W eight!/
% Body
Weight V
C h o lestero l
mg/gm2/
F T E *
T otal lip id
mg/gmW
XIV
( lO ) i/
b ile s a l t s
c h o le s te r o l
±5.1
4 . 6
±0.2
2.0
±0.2
6.2
± 0.9
4 .2 71.2
* 1.1
X III
(9)
b ile s a l t s
c h o le s te r o l
b u tyrate
J a
- ± 0 .4
A. 8
±0.2
2 .7
±0.2
8 .7
±1.1
6.0 66.1
10.6
IX
(9)
b i l e s a l t s
c h o le s te r o l
s te a r a te
. 8.2
to . 5
+ 5.3
t o . 5
.2 .3
10.2
+ 8.9
t o . 3
6.6
3:3
V I I I
(10 )
b i l e s a l t s
c h o le s te r o l
p a lm ita te
8.3
±0.2
t o .3
.3.4
±0.2
a8.6
t o . 5
5.2
S:i
IX
(10 )
b ile s a l t s
c h o le s te r o l
e la ld a te
8 .3
±1.2
4 .8
•to. 2
2.4
± 3 .0
7 .6
±1.6
5.2 66.8
t o . 7
X
(10 )
b ile s a l t s
c h o le s te r o l
o le a te
7 .3
±0.1
.4 .0
±0.2
, 2.6
t o . 2
7.8
±1.1
5.2 69.1
± 0 .4
X II
(10 )
b ile s a lt s
c h o le s te r o l
lln o le a t e
9-3
t o .9
4 .2
±0.2
2.5
± 0.3
4 .8
± 0 .4
2 .3 6 0 .9
±0.2
l/Number o f r a ts in th e group.
^ /in c lu d in g standard error o f the
1
mean.
39
lo w e s t growth r a t e . The two n o n - e s s e n tia l u n sa tu ra ted
f a t t y a c id e s t e r s were not e q u a lly e f f e c t i v e ; o le a t e
r
supplem ented groups e x h ib ite d a b e t te r grow th r a te than d id
th e e la ld a t e supplem ented a n im a ls. The group r e c e iv in g
l l n o l e a t e had th e h ig h e st grow th r a te ,a n d th e f a t f r e e
group had th e n ex t b e s t on e.
The p a lm ita te and s t e a r a te supplem ented groups had th e
h ig h e s t l i v e r w e ig h t, ex p ressed as p erc en t o f body w eig h t
( 5 .4 $ , 5 . 3$» r e s p e c t iv e ly ) , w ith e l a ld a t e , b u ty r a te , fa t
f r e e n ex t (4 .8 $ , 4 .8 $ , 4 .6 $ , r e s p e c t iv e ly ) and o le a t e and
l i n o l e a t e supplem ented groups having th e lo w e st v a lu e s
(4 .0 $ and 4 .2 $ , r e s p e c t i v e ly ) .
The a d d itio n o f 1 p er c e n t c h o le s te r o l to th e f a t fr e e
but o th e rw ise adequate d ie t y ie ld e d th e ex p ected In crea se
in c h o le s te r o l c o n c e n tr a tio n s o f some o f th e t i s s u e s
s tu d ie d . B u tyrate and o le a t e supplem ented groups had th e
h ig h e s t plasm a c h o le s t e r o l l e v e l s , th e o th e r groups had
low er l e v e l s w ith th e e la ld a t e and l i n o l e a t e having th e
lo w e st v a lu e s .
The in c r e a s e in l i v e r t o t a l and e s t e r c h o le s te r o l
c o n c e n tr a tio n s were two or th r e e tijnes h ig h er than th o s e o f
th e groups n o t r e c e iv in g c h o le s t e r o l in t h e ir d i e t s . In
a d d itio n , in th o s e anim als r e c e iv in g th e th r e e sa tu r a te d
f a t t y a c id e s t e r s , th e l i v e r c h o le s te r o l l e v e l s were
h ig h e s t . The n o n - e s s e n tia l f a t t y a c id e s t e r s supplem ented
groups y ie ld e d in te rm ed ia te l i v e r c h o le s t e r o l con centration s
and th e r a ts on th e f a t f r e e d ie t e x h ib ite d even lo w er
v a lu e s . The l i n o 1 e a te supplem ented group had the lo w e st
c h o le s t e r o l l e v e l s .
The f r e e a d ren a l c h o le s t e r o l l e v e l s w ere more a f f e c t e d
by th e d ie ta r y c h o le s t e r o l than were th e t o t a l ad ren al
c h o le s t e r o l c o n c e n tr a tio n s . R ats supplem ented w ith
e la ld a t e had th e h ig h e st (12.0 mg/@a) a n d 'th o se su p p le
m ented w ith p a lm ita te th e lo w e st (2.k mg/gm) f r e e a d ren a l
c h o le s t e r o l l e v e l s .
The b u ty ra te and s t e a r a te groups had alm ost th e same
l i v e r t o t a l l i p i d c o n c e n tr a tio n s (66.3 and 66.1 mg/gm,
r e s p e c t iv e ly ) , w hereas th e p a lm ita te supplem ented r a t s had
a h ig h e r c o n c e n tr a tio n than th o se fe d th e two o th er
sa tu r a te d f a t t y a c id e s t e r s (7 0 .3 mg/gm). The f a t f r e e
group had th e h ig h e s t t o t a l l i p i d l e v e l s in th e l i v e r , and
th e l i n o l e a t e group had th e lo w e s t l i v e r l i p i d co n cen tra
t io n s o f a l l th e groups s tu d ie d .
E ffe c t o f Supplem entations w ith E thvl L in o le a te to W eanling
Male R ats M aintained on a Fat Free D iet Supplem ented w ith
V ariou s S a tu ra ted o r N o n -e s s e n tia l U nsaturated F a ttv A cid
E ste r s (Experim ent I I I )
j
As was ex p e c te d , none o f th e e s s e n t i a l f a t t y a c id
d e f ic ie n c y symptoms occurred in th e se r a t s . The grow th
r a t e shown in Table X was as fo llo w s : l i n o le a t e supplem ents
a lo n e produced th e h ig h e s t growth r a t e , l i n o le a t e when
T A BLE X
G R O W T H OF W EANLING M ALE RATS FED A FAT FREE DIET SUPPLEMENTED W ITH
* VARIOUS NON-ESSENTIAL FATTY ACID ESTERS PLUS ETHYL LINOLEATE
Group
No.
Supplem ents
to
f a t f r e e
I n i t i a l
w e ig h t
W eight in grams a t w eek ly I n t e r v a ls
d u rin g su p p lem en ta tio n
Gain in w e ig h t
d u rin g
su p p lem en ta tio n
d ie t 1 2
3
4
5
6
XX
{1 0 ) 1/
l i n o l e a t e
gm
56 83
111 12? 179 199
224 168
XIX
(8 )
l i n o l e a t e
b u ty r a te
59
96
119 149
174
193
217 156
XVI
(10 )
l i n o l e a t e
s t e a r a t e 53
81 116 144 172
197 216 163
XV
(10 )
l i n o l e a t e
p a lm ita te 62
99
124
155 175 197 225 163
X V III
(10 )
l i n o l e a t e
e la i d a t e 89
118
143 163 183 206 152
XVII
(10 )
l i n o l e a t e
o l e a t e 55 89 110 138 166 186 201 146
^N um ber o f r a t e in th e grou p .
GRAMS
GAIN IN WEIGHT OF RATS FED A FAT FREE DIET
SUPPLEM ENTED WITH VARIOUS FATTY ACIDS
FO R SIX W EEKS LINOLEATE ADDED
120
100
ui
ui
O Ui
F igu re 1
+STEARATE
! 4 3 !
(su p p le m en ted w ith s t e a r a t e and p a lm it a t e p ro d u ced th e n e x t
i ' i
b e s t g ro w th r a t e , b u t y r a t e and e l a i d a t e when su p p lem en ted
w ith l i n o l e a t e w ere e q u a l l y e f f e c t i v e , and l i n o l e a t e (
(su p p le m en ted w it h o l e a t e c a u s e d th e lo w e s t g row th r a t e .
I
'T h ese d i f f e r e n c e s , h o w e v e r , w ere n o t g r e a t and a r e p r o b a b ly
n o t s i g n i f i c a n t .
R a ts su p p le m e n te d w ith b o th l i n o l e a t e and o l e a t e
e x h i b i t e d h ig h e r p la sm a t o t a l c h o l e s t e r o l l e v e l s th a n d id
th e r a t s su p p le m e n te d w ith l i n o l e a t e a lo n e o r th o s e g iv e n
l i n o l e a t e su p p le m e n te d w ith p a lm it a t e and e l a i d a t e . As
i •
shown i n T a b le XI, a n im a ls g iv e n s t e a r a t e and butyrate
(b o th su p p le m e n te d w ith l i n o l e a t e ) show ed th e lo w e s t p lasm a
c h o l e s t e r o l l e v e l s . j
^n T a b le ,XI a r e r e p o r te d th e v a lu e s o b ta in e d from j
|a n a ly s e s on a d r e n a ls . G roups g iv e n l i n o l e a t e (w ith o u t a n y
i I
su p p le m e n ts) and l i n o l e a t e p lu s o l e a t e had th e lo w e s t
i
(adrenal cholesterol levels. Groups ‘Supplemented with
stearate plus linoleate, palmitate plus linoleate, elaidate
plus linoleate, and butyrate plus linoleate followed in I
decreasing order.
' The v a r i a t i o n s b e tw e e n th e g r o u p s o f th e l i v e r
c h o l e s t e r o l l e v e l s w ere n o t v e r y s i g n i f i c a n t ; th e r e s u l t s
o b ta in e d a r e l i s t e d in T a b le X I I .
As f a r a s l i v e r s i z e i s c o n c e r n e d , r a t s g iv e n l i n o l e
a t e a lo n e show ed th e lo w e s t l i v e r w e ig h t , e x p r e s s e d a s
p e r c e n t o f b od y w e ig h t ( 3 . 1 ^ ) , r a t s su p p le m e n te d w ith
TABLE XI
P L A S M A * A N D A D R E N A L C H O LE ST ER O L LEVELS O F M A L E W E A N L IN G RATS FED A FAT FREE DIET
SU PPLEM EN TED W IT H NON-ESSENTIAL FATTY ACID ESTERS PLUS E TH Y L LINOLEATE
Group
No.
Supplement
to
f a t fr e e
d ie t
Plasma c h o le s te r o l
mg % 2/
Free T otal E ster
Adrenal c h o le s te r o l
mg/gm2 /
f 2/ t y
X X
(10 )1/
lin o le a t e 1 2 .7
t.1.1
59.9
■ fc-3.6
47.2 3 .6
13.3
XIX
(8 )
lin o le a t e
b u tyrate
1 2 .9
■fcl.9
56.9
-L2.3
44.0 4 .8 2 4 .4
XVI
(10 )
lin o le a t e
ste a r a te
8.6
± 0 .7
56.8
±2.8
48.2
2 .5 14.5
X V
(10 )
lin o le a t e
p a lm ita te
14.3
±1.0
58.5
±3.1
44.2
4.3 15 .2
XVIII
(10 )
lin o le a t e
e la id a te
1 2 .4
*1.0
57.2
±1.2
44.8
3..1
15.6
XVII
(10 )
lin o le a t e
o le a te
13.2
± 1 .7
67.7
± 2 .2
54.5
2.6
13.9
^Number o f r a ts in the group.
Including standard error o f the mean.
2/F a Free.
^ / t = T otal.
TABLE XII
CH ARACTERIZATIO N O F LIVER LIPIDS O F M A L E W E A N L IN G RATS FED
A FAT FREE DIET SU PPLEM EN TED W IT H VARIOUS NON-ESSENTIAL
FATTY ACID ESTERS PLUS ETH Y L LINOLEATE
LIVER
Supplements
Group to
No. fa t fr e e
d ie t
Liver
w g ti/
% Body
wgt2/
C h olesterol
mg/gm?/
Free Total E ster
T otal l i p i d
mg/gm?/
Iodine
no. of
f a t t y
a cid s
% PFA o f
l i p i d s !/>^/
XIX lin o le a t e
<10 )1/
gm
.9 .1
- 0.2
+3 *1
±0.2
± 1.9
.2 .5
±2.5
0.6 38.1
±0.2
200
9 .9
X X lin o le a t e
(8 ) b utyrate
,9 .3
±0.6
,3 .9
±0.2
2 .4
±0.1
3 .0
- 0.2
0.6
53-5
±0.3
80 6 .4
XVI lin o le a t e
< 10 ) ste a r a te
+?*1
±1.0
.3 .7
±1.2
+1 *9
±0.1
2 .9
±0.2
1.0 46.0
± 0.9
80 8.1
X V lin o le a t e
< 10 ) p a lm ita te
8 .7
± 0.3
.3 .9
± 1.5
+1 *7
±1.6
+2 .7
±0.6
1.0 48.1
±1.5
100 9 .8
XVIII lin o le a te
(10 ) e la id a te
+8.5
± 0 .4
+3 .7
±0.2
2.2
i o . i
+2 .9
- 0.2
0 .7 42.3
-0 .3
98
11.3
XVII lin o le a t e
(10 ) o le a te
8 .3
±0.2
+3*9
±0.2
2 .4
±0.1
2.8
±0.3
0 .4 52.6
±0.3
100
9 .7
^Number o f r a ts in th e group.
2/ Including standard error o f the mean.
1/PFA = p ercen t o f polyunsaturated f a t t y a c id s.
^ D eterm ined by lip o x ld a se method. ,
2 * 6
e la id a t e p lu s l i n o l e a t e , and s te a r a te p lu s lin o le a t e were
e q u a lly e f f e c t i v e (3*7$) and s l i g h t l y h ig h e r , and r a t s fed
b u ty r a te p lu s l i n o l e a t e , p a lm ita te p lu s l i n o l e a t e , and
o le a t e p lu s l i n o l e a t e , a l l o f which were e q u a lly e f f e c t i v e ,
had th e h ig h er l i v e r w eigh t a s p er cen t o f body w e ig h t.
As shown in Table X II, l i n o l e a t e su p p lem en tation a lo n e
to r a t s caused th e lo w e st t o t a l l i p i d c o n c e n tr a tio n (38.1
mg/gm) whereas b u ty r a te p lu s lin o le a t e g iv e n to r a t s caused
th e h ig h e s t l i v e r t o t a l l i p i d c o n c e n tr a tio n (53*5 mg/gm).
The f a t t y a c id e s t e r s in th e l i v e r s o f a l l th e groups
showed a high d egree o f u n sa tu ra tio n a s determ ined by th e
llp o x id a s e and th e Iod in e Number d e te r m in a tio n s. (T hese
r e s u l t s are l i s t e d in Table X I I ) . The p ercen ta g e o f p o ly
u n sa tu ra ted f a t t y a c id s ranged from 11 . 3# (e la id a te p lu s
l i n o l e a t e group) to 6,k% (b u ty r a te p lu s l i n o l e a t e g ro u p ).
F a tty a c id e s t e r s o f th e l i v e r s o f l i n o l e a t e supplem ented
group had an Io d in e Number o f 200, o f th e p a lm ita te p lu s
l i n o l e a t e and o le a t e p lu s l i n o l e a t e groups a s l i g h t l y low er
Io d in e Number was o b ta in ed , and o f th e b u ty r a te p lu s
l i n o l e a t e group lo w e s t v a lu e (80) was o b serv ed .
E ffe c t o f Supplem enting C h o le ste r o l and B ile S a lts to Adult
Male R ats M aintained on a Fat Free D iet f o r S ix te en Weeks
(Experim ent IV)
A ll th e fo u r groups d ev elo p ed e s s e n t i a l f a t t y a c id
d e f ic ie n c y symptoms. Most o f th e r a t s a ls o had
47
p a th o lo g ic a l lu n g s .
As shown in Table X III, th e groups r e c e iv in g c h o le s
t e r o l , o r c h o le s t e r o l and b i l e s a l t s , o r b i l e s a l t s d id not
gain a s ig n if ic a n t amount o f w e ig h t. The group o f r a t s fed
th e f a t f r e e d ie t w ith no supplem ents g a in ed 26 grams
d u rin g th e ex p erim en ta l p e r io d .
The f a t f r e e d ie t and f a t f r e e d ie t w ith b i l e s a l t s
fe d to r a ts cau sed a low er l i v e r w e ig h t, ex p ressed a s
p e r c e n t o f body w eigh t o f an im als (3 «2$ and 3*4$, r e sp e c
t i v e l y ) when compared w ith th e l i v e r w e ig h ts o f an im als fe d
th e f a t f r e e w ith c h o le s te r o l d i e t s and f a t fr e e w ith
c h o le s t e r o l and b i l e s a l t s d i e t s , w hich cau sed th e h ig h e s t
p e rcen ta g e o f l i v e r w eight (3 *9^)♦
The plasm a and ad ren al c h o le s t e r o l l e v e l s are c it e d in
Table XIV and th e r e s u lt s o b ta in ed w ith th e l i v e r l i p i d s
are l i s t e d in Table XV.
A ll th e c h o le s t e r o l l e v e l s o f th e t i s s u e s examined
were h ig h e r in th e group r e c e iv in g b i l e s a l t s w ith th e fa t
f r e e d ie t than in th o se o f th e f a t f r e e d i e t fe d a lo n e ,
w hereas th e t o t a l l i v e r l i p i d c o n c e n tr a tio n o f r a t s fe d th e
f a t f r e e d ie t was h ig h er than th a t o f th e group su p p le
m ented w ith b i l e s a l t s .
The a d d itio n o f c h o le s t e r o l o r c h o le s t e r o l p lu s b i l e
s a l t s to f a t f r e e d ie t s o f r a t s produced th e ex p ected
in c r e a s e s in th e plasm a and l i v e r c h o le s t e r o l l e v e ls and
t o t a l lip id , c o n c e n tr a tio n s . Both th e plasm a and l i v e r [
T
TABLE XIII
G R O W T H O F A D U LT M A L E RATS FED A FAT FREE DIET W IT H
SUPPLEM ENTS O F BILE SALTS A N D C H O L E ST E R O L
Group
No.
Supplement
to
fa t fr e e
d ie t
I n it ia l
w eight
Weight
1
in grams at w eekly in te r v a ls
daring supplem entation
2 3 4 5 6
Gain in w eight
during
supplem entation
XXIII
a /
(8 )1/
c h o le s te r o l
gm
228 230 217
192 200 213
221 -7
XXIII
b
(8 )
d h o le ste r o l
b ile s a l t s 220 219 219 193
212 211
217 -3
XXIII
c
(8 )
b ile s a l t s 226 223 200 217 223 230 229 3
XXIII
d
<5>
none 205
246 244 229
232
233 231 26
^Number o f r a ts in the group.
i
TABLE XIV
PLA SM A A N D A D R E N A L C H O LESTER O L LEVELS O F " A D U LT M A L E RATS FED A FA T
FREE DIET W ITH SUPPLEM ENTS O F C H O LESTER O L A N D BILE SALTS
Group
No.
Supplement
to
f a t free
d ie t
Plasma c h o le s te r o l
mg % 2/
Free Total E ster
Adrenal cho]
mg/gm 2/
Free
s te r o l
Total
XXIII
a
(8 )1/
c h o le s te r o l 17.0
± 2 .7
101.2
±6.1
81*. 2
1*.7 5 0 .7
XXIII
b
(8 )
c h o le s te r o l
b i l e s a lt s
18.2
-3*7
101.9
47.2
83.7
!*.0
51*3
XXIII b i l e s a lt s 8 . 1 *
+ A ft
1-7*0 1 *8.6
1*.3
52.1*
c
(8 )
-0 *9
; 33.5
XXIII
d
(5)
none
+ 5 *1
± 1.2 '
J
5 0 .3
± 7 .7
1*5.1* 2 .1
1*8.9
^Number o f r a ts In the group.
Including standard error of th e mean.
r
i
I
TA BLE X V
CHARACTERIZATION OP LIVER LIPIDS OF M ALE ADULT RATS FED A FAT
FREE DIET WITH SUPPLEMENTS OF CHOLESTEROL AND BILE SALTS
/
Group
No.
Supplements
to
f a t fr e e
d ie t
L iver
w eight
% Body
w eight
Free
C h olesterol
mg/gm2/
Total E ster
Total llp l d
mg/gmi/
XX III
a
(8)1/
c h o le s te r o l
gm
. .8.6
- 0 .3
+3.8
10.6
+3 *5
t o .2
1^.9
±1.4
11. k 9 5 .2
±1.0
X X III
b
(8)
c h o le s te r o l
b ile s a l t s
+9 *5
to . 3
3.7
±0.9
3.9
±0.i*
15-3
± 2 .3
11.4
109.5
to . 5
X X III
c
(8)
b i l e s a l t s t o . 2
J2.7
to . 2
+ ^ 1
±1.0
^8.2
±1.5
4.1 61.
±0.5
XX III
d
(5)
none
+ 7 .3
t o . 6
+ 3 .3
t o . 5
+3-3
±1.0
+6.2
±1.0
2 .9 78.6
-0.2
^Number o f ra te In the group.
^ In c lu d in g standard error of the mean.
51
c h o le s t e r o l l e v e l s were a t l e a s t tw ice a s h ig h a s th o s e o f
th e groups n ot r e c e iv in g d ie t a r y c h o le s t e r o l. However,
a d ren a l c h o le s te r o l l e v e l s w ere n ot a f f e c t e d s i g n i f i c a n t l y
by th e d ie ta r y c h o le s t e r o l.
T o ta l l i p i d c o n c e n tr a tio n s were h ig h e r in groups th a t
r e c e iv e d c h o le s te r o l in t h e ir d ie t ( f a t f r e e p lu s c h o le s
t e r o l : 95.2 mg/gm t o t a l l i p i d ) and th e f a t f r e e group
w ith o u t any supplem ents (6l mg/gm) had lo w er co n ce n tra tio n
o f l i v e r t o t a l l i p i d s .
E ffe c t o f F eedin g 15$ Hydrogenated Coconut O il Alone or
Supplem ented w ith C h o le ste r o l an d /or B ile S a lts to Adult
Male R ats P r e v io u s ly M aintained on 15$ Hydrogenated Coconut
Q ll D ie t fo r Four Months
The r a ts in a l l th e fo u r groups showed sev ere e s s e n
t i a l f a t t y a c id sk in d e f ic ie n c y symptoms. Most o f th e r a ts
in th e s e grou p s, to o , had p a th o lo g ic a l lu n g s .
The growth r a te as shown in Table XVI was as fo llo w s :
th e groups r e c e iv in g su p p lem en tation s o f c h o le s te r o l and
b i l e s a l t s and b i l e s a l t s a lo n e gain ed more w eigh t than
groups not fe d b i l e s a l t s .
The c h o le s te r o l supplem ented groups had alm ost th e
same l i v e r w e ig h t, ex p ressed a s p ercen t o f body w eig h t
(3 . 8$ ) , th e group having no supplem ents in th e d ie t was
n e x t ; (3 *3$ ) , and b i l e s a l t s supplem ented group had th e
lo w e s t v a lu e o f l i v e r w eigh t ( 2 . 7$ ) .
T
TABLE XVI
G R O W T H O F A D U LT M A L E RATS FED A 15% H Y D R O G E N A T E D C O C O N U T OIL
DIET W IT H SUPPLEM ENTS O F C H O LE ST ER O L A N D BILE SALTS
S U M M A R Y
Group
Supplements
to 15% I n i t i a l
Weight in grams a t weekly in te r v a ls
during supplem entation
Gain in w eight
during
No. hydrogenat ed
coconut o i l
d ie t
w eight
1 2
3
4
5
6
s uppl em en t at ion
XXI
a ,
< 5)1/
c h o le s te r o l
gm
242 248 260 256 261 258
271 29
XXI
b
<5)
c h o le s te r o l
b ile s a l t s 236
253 263 257 275
286 281
45
XXI
(5)
b ile s a l t s 238 233 260
249 273
278 271 33
XXI
d
(2 )
253 243 215
248
267 280 274 21
^Number o f r a ts in the group.
53
In group XXIa one r a t had lip e m la and had v ery h ig h
c h o le s t e r o l l e v e l s . The r e s u lt s o b ta in ed on t h is anim al
were not In clu d ed In the average o f th e group.
The e x p e r im e n ta l r e s u l t s o b ta in e d a r e sum m arized in
T a b le s XVII and X V III.
The a d d itio n o f c h o le s t e r o l r e s u lte d in s t r ik in g
in c r e a s e s o f a l l th e c h o le s t e r o l l e v e l s o f th e t i s s u e s
exam ined and th e t o t a l l i p i d c o n c e n tr a tio n s . For exam ple,
when c h o le s te r o l was supplem ented to th e d ie t th e fo llo w in g
r e s u l t s were o b ta in ed : plasm a t o t a l c h o le s t e r o l 1 0 7 .5 mg % >
ad ren a l t o t a l c h o le s t e r o l 87.6 mg/gm, t o t a l l i v e r l i p i d
co n c e n tra tio n was 8 2 .1 m g/go, and l i v e r t o t a l c h o le s t e r o l
co n c e n tra tio n was 17.& mg/gm. Whereas when th e d ie t d id
n ot co n ta in any supplem ents th e r e s u lt s w ere as fo llo w s :
t o t a l plasm a c h o le s t e r o l 4 0 .5 mg ad ren al t o t a l c h o le s
t e r o l 23.7 mg/gm, l i v e r t o t a l l i p i d co n ce n tra tio n was 4- 1 .9
mg/gm. Supplem entation o f b i l e s a l t s to anim als fe d th e
hydrogenated coconut o i l d i e t s d id n ot ca u se an in c r e a s e in
t i s s u e c h o le s te r o l o r t o t a l l i p i d c o n c e n tr a tio n s.
TABLE XVII
PLA SM A A N D A D R E N A L C H O LE ST ER O L LEVELS O F M A L E A D U LT MTS
FED A 15% H Y D R O G E N A T E D C O C O N U T OIL DIET W IT H SUPPLEM ENTS
O F C H O LESTER O L A N D BILE SALTS
Group
No.
Supplements
to 15%
hydrogenated
coconut o i l
d ie t
Free
Plasma c h o le ste r o l
mg/gmZ/
T otal E ster
Adrenal eho]
mg/gm 2/
Free
? e s te r o l
T otal
XXI
a
(5 )1 /
c h o le s te r o l 3A.1
±6.2
1*9.5
±5.1
115.*- 8.3 87 .6
XXI
b
(5)
c h o le s te r o l
b ile s a l t s
iB .k
-2 .7
.9 8 .7
±10.5
80.3
6.0 8*.2
XXI
c
(5)
b i l e s a l t s
+ 6 .5
* 1 .*
36.0
*6.2
3 0 .5 * .7 63-1
XXI
d
( 2 )
none
+5' 5
±1.0
*0.5
± 1 .5
35.0
6.1
23.7
i/Number o f r a ts In the group.
2 / Including standard error o f the mean.
•r
TABLE XVIII
RESULTS OBTAINED O N LIVER LIPIDS O F RA TS FED A 1 H Y D R O G E N A T E D C O C O N U T
' . OIL DIET W ITH SUPPLEM ENTS O F C H O LESTER O L A N D BILE SALTS
LIVER
Group
No.
Supplements
to 15$
hydrogenated
coconut o i l
d ie t
L iver
- w eig h t? /
% Body
w eig h t? /
Free
C h o lestero l
mg/gm?/
Total E ster
T otal lip id
mg/gm?/
XXI
a ,
(5)1/
c h o le s te r o l
.9.7
t o . 5
.3.9
±0.5
3.9
±0.4
12.4
±3.1
8.5
82.2
*0.2
XXI
b
(5)
c h o le s te r o l
b ile s a l t s
1 0 .1
t o . 4
,3.9
± 0 .2
3.2
±0.3
17.4
42.2
14.2
75.5
±0.1
XXI
(!)
b ile s a l t s
7.3
±0,9 ±0.3
,2.6
±0.2
+3.1
±0.2
0.5 JO .7
±0.1
XXI
d
(2)
none .7.7
±1.0
+3.4
±0.2
.2.3
±0.2
+3-2
±0.2
0.9 42.0
40.1
^Number o f r a ts In th e group,
^ /in c lu d in g standard er ro r o f th e mean.
CHAPTER VI
DISCUSSION
L iv er p la y s an Im portant r o le in th e in term ed iary
m etabolism of th e l i p i d s . I t I s a tem porary sto r a g e organ
fo r f a t s and I t s l i p i d com p osition may f lu c t u a t e r a p id ly
depending upon th e com p osition o f d ie t and subsequent
in term ed ia ry m etabolism o f th e c o n s titu e n ts con tain ed
th e r e in . I t has a ls o been shown th a t f a t t y a c id s o f l i v e r
l i p i d s in terch a n g e r a p id ly w ith th o se In th e b lo o d .(63)
The requirem ent o f r a ts fo r e s s e n t i a l f a t t y a c id s has
been dem onstrated by t h i s la b o r a to r y and by o th er i n v e s t i
g a to r s as m entioned p r e v io u s ly . I t has a ls o been shown
th a t f a t s c o n ta in in g n o n -e s s e n tia l f a t t y a c id s are unable
to s u b s t it u t e f o r e s s e n t ia l f a t t y a c id s in a l l e v i a t i n g th e
d e f ic ie n c y symptoms produced by an e s s e n t i a l f a t t y a c id
d e f ic ie n t d i e t . (6^*65)
The p resen ce o f th e tr a n s form o f c e r ta in f a t t y a c id s
p r e s e n t in m argarin e(66 ) ^<1 in a hydrogenated t r i o l e i n
product has a ls o been shown n o t to e x h ib it any d e le te r io u s
e f f e c t on n u t r it io n a l s t a tu s o f th e r a t s . (**6 ) However, in-
an exam ination o f l i v e r l i p i d s o f r a ts p la c e d on d if f e r e n t
f a t d i e t s , i t has been shown th a t an in c r e a s e in sa tu r a te d
f a t t y a c id s in a fa.t y ie ld s an in c r e a se d l i v e r c h o le s te r o l
c o n c e n tra tio n and changes th e com p osition o f c h o l e s t e r o l ^ 2 )
e s t e r s so th a t th e f a t t y a c id s a s s o c ia te d w ith th e l i v e r
57
c h o l e s t e r o l w ere m ore s a t u r a t e d even when s u f f i c i e n t
e s s e n t i a l f a t t y a c i d s w ere a l s o p r e s e n t . The i n d i c a t i o n
t h a t c e r t a i n s a t u r a t e d f a t t y a c id e s t e r s e le v a t e d th e
p la sm a and l i v e r c h o l e s t e r o l c o n c e n t r a t io n s and a c c e n t u a te d
j t h e e s s e n t i a l f a t t y a c id d e f i c i e n c y sym ptom s h a s b e e n
c o n fir m e d in t h i s s tu d y w h ere p u re f a t t y a c id e s t e r s h ave
b een f e d to r a t s on an o t h e r w is e a d e q u a te b u t a f a t f r e e
d i e t .
O f th e s a t u r a t e d f a t t y a c id e s t e r s s t u d i e d , n a m e ly ,
b u t y r a t e , s t e a r a t e , and p a l m i t a t e , i t w as o b s e r v e d t h a t
! b u t y r a t e and s t e a r a t e c a u se d a m ore m arked d e f i c i e n c y
i
! syndrom e th a n d i d p a l m i t a t e . The f a t f r e e d i e t c a u se d l e s s
m arked d e r m a t it is th an d id t h e f a t d e f i c i e n t d i e t s s u p p le
m en ted w it h t h e e x p e r im e n ta l s a t u r a t e d f a t t y a c id e s t e r s .
E s s e n t i a l f a t t y a c id d e f i c i e n c y sym ptom s in th e a n im a ls f e d
th e n o n - e s s e n t i a l u n s a tu r a te d f a t t y a c id s w ere l e s s s e v e r e
th a n t h o s e f e d t h e s a t u r a t e d f a t t y a c id s a lth o u g h t h e
'syndrom e w as s t i l l m ore m arked th a n in t h e a n im a ls r e c e i v -
I
|in g no f a t a t a l l in th e d i e t .
I
I t h a s p r e v io u s l y b een shown t h a t e s s e n t i a l f a t t y
a c id s w ere n e c e s s a r y f o r norm al g row th o f t h e r a t s . In
t h i s i n v e s t i g a t i o n i t w as o n c e m ore o b s e r v e d t h a t , t h e
e s s e n t i a l f a t t y a c i d , l i n o l e a t e , p r o d u c e d t h e b e s t g ro w th
r a t e . B u ty r a te and s t e a r a t e su p p lem en ted a n im a ls g a in e d
th e l e a s t w e ig h t . The g ro w th r a t e in t h e p a lm it a t e s u p p le
m en ted grou p w as s l i g h t l y b e t t e r th an t h e g r o u p s f e d t h e
o th e r sa tu r a te d f a t t y a c id s . The growth o f r a t s fe d th e
f a t f r e e d i e t , alth o u g h b e t t e r than th a t o f th e an im als
^ supplem ented w ith sa tu r a te d f a t t y a c id e s t e r s and o le a t e ,
was s t i l l not a s good as e la id a t e w hich gave th e h ig h e s t
igrow th r a te n ex t to l i n o l e a t e .
i
I The d iff e r e n t f a t t y a c id e s t e r s a ls o in flu e n c e d
I 1
I c e r ta in p hases o f c h o le s te r o l m etabolism . B u tyrate and
I
s t e a r a te e le v a te d plasm a and l i v e r c h o le s t e r o l l e v e l s
1
iab ove th o se o f u n sa tu ra ted f a t t y a c id s and f a t f r e e d i e t s .
f
'A lthou gh p a lm itic a c id a cc en tu a ted th e e s s e n t i a l f a t t y a c id
d e f ic ie n c y symptoms, in s te a d o f e le v a tin g th e c h o le s t e r o l !
' l e v e l s o f th e t i s s u e s exam ined, a s was ob served w ith th e
o th e r sa tu r a te d f a t t y a c id s , th e r e was no in c r e a se in
I
plasm a c h o le s t e r o l, and th e l i v e r c h o le s t e r o l l e v e l s
o b ta in ed were s im ila r to th a t ob served in anim als supplemen
ted w ith th e e s s e n t i a l f a t t y a c id , l i n o l e a t e . The r e s u l t s
!o b ta in ed by p a lm itic a c id approxim ate th o s e o b ta in ed w ith
o l e i c a c id . A p o s s ib le ex p la n a tio n may b e th a t s in c e th e
ibody i s capable o f d e s a tu r a tln g p a lm itic a c id in to a non- I
!
1 e s s e n t i a l u n sa tu ra ted f a t t y a e id ( 6 7 ) , a la r g e p o r tio n of
p a lm itic a c id may be co n v erted to p a lm ito le ic a c id and j
{
th e r e fo r e produce e f f e c t s s im ila r to some o f th e non- |
e s s e n t i a l u n sa tu ra ted f a t t y a c id s . The l i v e r l i p i d s o f th e
p a lm ita te supplem ented group had an io d in e number o f 7 5 ,
w hich would a ls o in d ic a te th e p resen ce o f c o n sid e r a b le
amount o f m ono-ethanold f a t t y a c id s .
5 9 1
i
| The l i v e r t o t a l l i p i d co n ce n tr a tio n o f the groups ;
i
I m ain tain ed on e s s e n t i a l f a t t y a c id d e f ic ie n t d ie t were a t '
j l e a s t 13 mg h ig h e r than th e group supplem ented w ith e th y l j
' l i n o l e a t e , and t h i s group a ls o had th e lo w e s t p ercen t o f
l i v e r w eight when compared w ith o th e r g ro u p s. T h erefore,
i t can be assumed th a t th e l i p i d s accum ulate in th e l i v e r
in th e absence o f th e e s s e n t i a l f a t t y a c id s .
The a d d itio n o f 1 p er c e n t c h o le s t e r o l to an e s s e n t i a l
f a t t y a c id d e f i c i e n t , but o th e rw ise ad eq uate d ie t fe d to
r a t s , h asten ed th e appearance and in c r e a se d th e s e v e r it y o f
th e e s s e n t i a l f a t t y a c id d e f ic ie n c y dermal symptoms,
co n firm in g th e r e s u l t s o b ta in ed by Bromer and Day(**3) and j
j
Holman and P e i f e r . (68) T his e f f e c t was a ls o n oted in th e
i
I
|groups o f anim als supplem ented w ith th e n o n - e s s e n tia l f a t t y !
a c id e s t e r s . However, th e growth r a t e s in th e groups fe d
I I
t h e f a t fr e e d ie t e it h e r a lo n e o r supplem ented w ith p alm i
t a t e , o le a te and b u ty r a te w ere s l i g h t l y enhanced by th e |
p resen ce o f d ie t a r y c h o le s te r o l; th e re was a s li g h t j
d e c r e a se in grow th in th e l i n o l e a t e group and no e s s e n t i a l |
i
j e f f e c t in th e an im als supplem ented w ith e la id a t e and
s t e a r a t e .
»
In g e n e r a l, o f the organs examined fo r c h o le s te r o l
c o n te n t, th e l i v e r was found to show th e g r e a te s t in c r e a se
a s a r e s u lt o f th e in c lu s io n o f c h o le s te r o l in th e d i e t .
Plasm a c h o le s te r o l l e v e l s w ere e le v a te d in th e r a t s fe d th e
60 i
j I
• f a t f r e e d ie t and th e d ie t supplem ented w ith o le a t e o n ly . !
I
The lo w est l i v e r c h o le s t e r o l c o n c e n tr a tio n s were
I
. o b ta in ed in th e l i n o l e a t e supplem ented group. T his i s in !
I
| agreem ent w ith th e work o f A lf ln - S ia t e r e t a l ^ 9 ) W ho
i
o b served th a t when lin o l e a t e was added to f a t f r e e , but j
o th e r w ise ad eq u ate, d ie t c o n ta in in g c h o le s t e r o l th e con cen - j
tr& tio n o f c h o le s t e r o l in th e l i v e r was l e s s than i f th e |
li n o l e a t e was o m itted from th e d i e t . In th e p resen t
in v e s t ig a t io n , i t was found th a t the th r e e sa tu r a te d f a t t y
i
a c id s produced much h ig h er t o t a l l i v e r c h o le s t e r o l con cen - i
t r a t io n s than d id th e u n sa tu ra ted f a t t y a c id s and th e f a t j
i
f r e e d i e t . This i s in agreem ent w ith th e r e s u lt s o f
!A fterg o o d e t a l( 7 0 ) who found th a t when o h o le s t e r o l was fe d !
to r a t s on a c o tto n se e d o i l d i e t (u n sa tu ra ted f a t ) th e
c h o le s t e r o l l e v e l s were low er than th o se o b ta in ed from
» i
an im als fed a la r d (a more sa tu r a te d f a t ) d ie t c o n ta in in g
I c h o le s t e r o l.
I
I t was o f in t e r e s t to fin d out w hether th e In cr e a se d i
I
!
c h o le s t e r o l c o n c e n tr a tio n ob served in th e l i v e r o f an im als
i
fe d th e e s s e n t ia l f a t t y a c id d ie t supplem ented w ith non- J
i
e s s e n t i a l f a t t y a c id s cou ld be d ecrea sed by fe e d in g
l i n o l e a t e s in c e th e l i n o l e a t e had been shown to b e e f f e c t i v e
in a ll e v ia t in g th e symptoms o b ta in ed in anim als fe d a f a t
f r e e d i e t . L in o le a te n ot o n ly d ecrea sed th e l i v e r c h o le s
t e r o l co n ten t but a ls o th e a d ren a l c h o le s t e r o l co n cen tra -
i
t io n s o f a l l th e groups under stu d y was s t r i k i n g ly _______ }
61,
d ecrea se d . The t o t a l l i p i d co n ce n tr a tio n in a l l groups
ex cep t th e one in which o le a te was th e supplem ent was l i k e -
; w ise d ecrea se d . !
The e f f e c t o f l in o le a t e on th e plasm a c h o le s te r o l
i
i l e v e l s when compared w ith the s im ila r groups n ot r e c e iv in g |
l i n o l e a t e was n o t s t r ik in g , ex cep t in th e b u ty ra te group j
where a marked d ecr e a se was o b served . A lf in - S la t e r e t
a i(6 9 ) rep orted th a t th e plasm a c h o le s te r o l l e v e l s o f r a t s j
were n o t d ecrea sed when l i n o l e a t e was added to t h e ir f a t
i
I d e f ic ie n t d i e t , but were a c t u a lly in c r e a se d .
J The f a t t y a c id s o f th e l i v e r l i p i d s o f a l l th e groups j
stu d ie d showed a h ig h d egree o f u n sa tu ra tio n when compared
w ith th e low er u n sa tu ra tio n o f th e corresp on d in g groups not 1
r e c e iv in g l i n o l e a t e supplem ents. T h erefo re, th e l i v e r
c h o le s t e r o l was combined w ith e s t e r s o f th e more u n satu
ra ted f a t t y a c id s . S in c e th e l i v e r l i p i d c o n c e n tr a tio n s o f
th e se groups w ere low , i t was th e o r iz e d th a t l in o l e a t e had
i
I
;an e f f e c t on th e tr a n sp o r t and m o b iliz a tio n o f c h o le s t e r o l
!from th e l i v e r .
i
i
The l a s t p a r t o f th e in v e s t ig a t io n was concerned w ith |
comparing th e e f f e c t s o f f a t f r e e and hydrogenated coconut \
o i l d i e t s , supplem ented w ith b i l e s a l t s and c h o le s t e r o l, on
th e e s s e n t ia l f a t t y a c id d e f ic ie n c y symptoms and c h o le s te r o l
l e v e l s of th e t i s s u e s examined in th e ex p erim en ts.
Hydrogenated coconut o i l i s d evoid o f e s s e n t ia l f a t t y
a c id s , but i t c o n ta in s r e l a t i v e l y sh o rt chain sa tu r a te d
62
! f a t t y a c id s . The e s s e n t ia l f a t t y a c id d e f ic ie n c y dermal
| symptoms occurred more s e v e r e ly in th e group o f r a ts
r e c e iv in g th e hydrogenated coconut o i l d ie t s than in th o s e j
i
r e c e iv in g f a t f r e e d i e t s . However, th e hydrogenated
i :
coconut o i l d ie t caused a b e t t e r growth r a te than d id th e
f a t f r e e d ie t ; t h i s e f f e c t was s im ila r to th a t o b ta in ed on
c h o le s t e r o l su p p lem en tation . Both groups e x h ib ite d low
plasm a l e v e l s on th e c h o le s t e r o l f r e e d i e t . However, th e J
l i v e r and t o t a l ad ren al c h o le s t e r o l l e v e l s o f th e f a t f r e e ^
I I
fe d group were alm ost tw ice a s g r e a t a s th a t o f th e hydro- j
I
g en a ted coconut o i l fed group. The r a t s used in th e s e
J exp erim en ts had been on a f a t fr e e or hydrogenated coconut
] I
:o i l d ie t fo r fo u r months p r io r to th e p r e s e n t exp erim en tal 1
{p er io d . A lf in - S la t e r et. a l ^ 7 ) have shown th a t plasm a and
!l i v e r c h o le s t e r o l l e v e l s o f r a t s r e c e iv in g hydrogenated
I coconut o i l d ie t were a lte r e d during th e f i r s t week o f d ie t
but a f t e r s ix te e n weeks g r a d u a lly retu rn ed to norm al. I t
has been su g g ested th a t perhaps th e a n im a ls adapt and j
p o s s ib ly use th e sh o rt ch ain f a t t y a c id s in th e absence o f !
i
th e e s s e n t i a l f a t t y a c id s . T h erefo re, in a hydrogenated
coconu t o i l fe d d ie t th e same mechanisms were n ot r e s p o n s l- j
b le f o r b oth c h o le s te r o l accum ulation and d e fic ie n c y sk in
symptoms.
The r e s u lt s o b ta in ed w ith th e f a t f r e e group a ls o
confirm th o se o f A lf in - S la t e r e t a l (38) W ho found th a t th e
a d m in istra tio n o f a f a t f r e e , but o th e rw ise ade q u a te, d ie t
r e s u lte d in th e lo w erin g o f plasm a c h o le s t e r o l l e v e l s , w ith
a concom itant in c r e a s e in th e l i v e r and ad ren al c h o le s t e r o l
c o n c e n tr a tio n s.
CHAPTER V II
SUM M ARY AND CONCLUSIONS
1 . Plasm a, l i v e r , and adrenal c h o le s t e r o l l e v e l s ,
l i v e r t o t a l l l p l d c o n c e n tr a tio n , d egree o f a n sa tu ra tio n o f
the l i v e r l i p i d s and growth r a te o f w ea n lin g male r a t s
m ain tain ed on a f a t fr e e d ie t w ith or w ith o u t th e a d d itio n
o f f a t t y a c id e s t e r s o r c h o le s te r o l and b i l e s a l t s xfere
determ ined a f t e r a s i x week fe e d in g p e r io d . The s e v e r it y o f
e s s e n t i a l f a t t y a c id symptoms o f r a ts on th e above d i e t s
was o b serv ed .
T yp ical e s s e n t i a l f a t t y a c id d e f ic ie n c y symptoms were
ob served in; r a ts fe d a f a t f r e e d ie t and a fa t-sfr e e d ie t
supplem ented w ith a n o n - e s s e n tia l f a t t y a c id e s t e r . The
d e f ic ie n c y syndrome appeared much more r a p id ly and s e v e r e ly
when sa tu r a te d f a t t y a c id s w ere g iv en a lo n g w ith th e f a t
f r e e d i e t than when r a ts fe d a s im ila r d i e t were su p p le
m ented w ith a n o n -e s s e n tia l u n sa tu ra ted f a t t y a c id . The
f a t f r e e d ie t supplem ented w ith b u ty r ic a c id caused th e
most se v er e e s s e n t i a l f a t t y a c id d e f ic ie n c y syndrome.
Of th e sa tu r a te d a c id e s t e r , b u ty r a te and s te a r a te
when supplem ented w ith a f a t f r e e d ie t cau sed m arkedly
h ig h e r c h o le s t e r o l l e v e l s in th e t i s s u e s stu d ie d than d id
th e p a lm ita te and th e o th er exp erim en tal f a t t y a c id e s t e r s
and th e f a t f r e e d i e t .
2 . A dd ition o f c h o le s te r o l and b i l e s a l t s to th e
65
I
! above d ie t s cau sed e le v a t io n s in plasm a t o t a l c h o le s t e r o l
i i
and in th e l i v e r t o t a l and e s t e r c h o le s t e r o l f r a c t io n s .
I i
j The o n se t o f th e d e fic ie n c y syndrome was h asten ed and th e
j dermal symptoms w ere more se v e r e than in th e groups fe d th e
junsupplem ented f a t f r e e d i e t .
R ats supplem ented w ith e th y l li n o l e a t e and fe d a f a t
f r e e d ie t o r a f a t f r e e d ie t to which n o n -e s s e n tia l f a t t y
a c id s were added d id not e x h ib it e s s e n t ia l f a t t y a c id
d e f ic ie n c y symptoms and had low er l i v e r and ad ren al c h o le s - [
|t e r o l l e v e l s , and t o t a l l i v e r l i p i d c o n c e n tr a tio n s than
!r a t s on an e s s e n t i a l f a t t y a c id d e f ic ie n t d i e t ,
j L in o le a te m o b iliz e d and tra n sp o rted th e c h o le s te r o l
j e s t e r s from th e l i v e r and th u s p o s s ib ly a f fe c t e d th e in t e r - !
i
m ediary m etabolism o f l i v e r l i p i d s .
3* Adult m ale r a ts m ain tain ed on 15 p er cen t hydro-
i
I
Igenated coconut o i l d ie t e x h ib ite d more se v e r e d e r m a titis
I syndrome but had a b e t te r grow th r a te than d id a d u lt m ale
r a ts r e c e iv in g a f a t f r e e d i e t . j
R ats on b oth th e f a t f r e e d ie t and th e hydrogenated j
coconut o i l d ie t e x h ib ite d r a th e r low plasm a c h o le s te r o l j
i
l e v e l s . However, th e l i v e r c h o le s t e r o l c o n c e n tr a tio n s of
th e r a t s on f a t f r e e d ie t w ere ap p roxim ately tw ice as high
as th e l i v e r c h o le s te r o l co n ce n tra tio n o f th e r a ts
r e c e iv in g th e hydrogenated coconut o i l d i e t .
The a d d itio n o f c h o le s te r o l g r e a tly in cr e a se d th e
t i s s u e c h o le s te r o l c o n c e n tr a tio n s o f r a t s fe d a f a t f r e e
o r a hydrogenated coconut o i l d i e t .
The t i s s u e c h o le s t e r o l l e v e l s o f r a t s fe d th e hydro
g en a ted coconut o i l d ie t were low er than would be exp ected
from r a t s fed a d ie t c o n ta in in g a sa tu r a te d f a t . Two
f a c t o r s may be r e sp o n sib le fo r t h i s e f f e c t , f i r s t l y , th e
a n im a ls may be a b le to adapt to th e ty p e o f sa tu r a te d fa t
p r e s e n t in th e hydrogenated coconut o i l and, se c o n d ly , th e
p r esen ce o f f a t t y a c id s o f medium chain le n g th (C1 2 , C-^)
may d i f f e r in t h e ir a c t i v i t y from f a t t y a c id s o f lo n g e r
ch ain le n g th (C1 6 , O^g).
I t i s con clu d ed th a t a r e la t io n s h ip e x i s t s betw een th e
t i s s u e c h o le s te r o l l e v e l s and both th e n a tu re and th e
q u a n tity o f d ie ta r y f a t s .
BIBLIOGRAPHY
68
BIBLIOGRAPHY
1 . J a f f e . R ., Arch. E x p tl. P ath . Parm akol. 1 3 2 . 8k
(1 9 2 8 ).
2 . Burr, G. 0 . , and Burr, M. M ., J . B i o l * Ohem. . 8 2 . 3k$
(1929)*
3* Burr, G. 0 . , and Burr, M. M ., J . B i o l . Cham., 8 6 , 587
(1 9 3 0 ).
A. A ftergood , L ., B eu el, H. J . , J r . , and A lf in - S la t e r ,
R. B ., J . N utr. . 6 2 , 129 (1 9 5 7 ).
5 . A lf ln - S la t e r , R. B ., J . Am. O il Ohem. Soo. , 2 k , S7k
(1957)•
6 . H egsted, D. M ., G o tls , A ., and S ta r e , F. J . , J . N u tr. ,
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Creator Kent, Esin Ummehan (author)

Core Title Studies of fatty acids: Effects on nutritional status and tissue cholesterol levels of rats

Degree Master of Science

Degree Program Biochemistry and Nutrition

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-778465

Unique identifier UC11347706

Identifier EP41338.pdf (filename),usctheses-c17-778465 (legacy record id)

Legacy Identifier EP41338.pdf

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Rights Kent, Esin Ummehan

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