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A nutritional evaluation of the essential fatty acids in the rat
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A nutritional evaluation of the essential fatty acids in the rat
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A NUTRITIONAL EV A LU A TIO N 0? THE
ESSENTIAL FATTY ACIDS
IN THE R A T
A D isse rta tio n
Presented to
th e F aculty of th e Department o f B iochem istry and
N u tritio n , the U niv ersity of Southern C a lifo rn ia
In P a r tia l F u lfillm e n t
of the Requirements fo r the Degree
Doctor of Philosophy
*7
Samuel M. Greenberg
June 1951
UMI Number: DP21544
All rights reserved
INFORMATION TO ALL USERS
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a note will indicate the deletion.
Dissertation Publishing
UMI DP21544
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This dissertation, written by
Samuel M. Greenberg
under the guidance of h.%.S.. Faculty Committee
on Studies, and approved by all its members, has
been presented to and accepted by the Council
on Graduate Study and Research, in partial ful
fillment of requirements for the degree of
D O C T O R OF P H IL O S O P H Y
Committee on Studies
Chairdkm
ACKN0WLEDG1 MENTS
I t i s w ith s in c e re g ra titu d e t h a t I acknowledge
th e guidance and in v a lu a b le a s s is ta n c e o f my F a c u lty
Committee. A s p e c ia l d eb t o f g r a titu d e i s owed to
B r. H arry J . D euel, J r . , chairm an of my com m ittee, f o r
h is c o n stan t guidance and in s p ir a tio n .
I w ish to th an k The B est Foods, I n c . f o r i t s
f in a n c ia l support th a t made t h i s work p o s s ib le .
And w ith s in c e re g ra titu d e I w ish to acknowledge
th e a s s is ta n c e g iv en me by B rs. H. W. V a h lte ic h , D aniel
M elniek, and J . B. Beans, who p repared many sam ples of
f a t s and who made physico-chem ical an a ly se s o f th e s e
sam ples f o r th e b io a ssa y pro ced u re.
I am g la d to acknowledge th e h e lp .I have had from
C larence E. C a lb e rt, Tomoko F u k u i, Evelyn E. Savage,
L i l i a A n is fe ld , and H arry McKee ban, who have g iv en gener
ous and v alu ab le te c h n ic a l a s s is ta n c e in c a rry in g o u t
th e b io lo g ic a l ta s k s involved in t h i s stu d y .
I should a ls o l i k e to re c o rd my thanks to M rs.
M arie V isse r, who spared no e f f o r t in ty p in g t h i s
m an u scrip t.
To my w ife , who made th e f ig u r e s , and to a l l th e
members, o f th e D epartm ent of B iochem istry and N u tr itio n ,
who have been b o th p a tie n t and c o n s id e ra te , my a p p re c ia
tio n and s in c e r e s t th an k s.
TA BLE O F C O N T E N T S
!CH A PTER
I
! I . INTRODUCTION . . . . ,
i I I . HISTORICAL REVIEW . ,
| I H . DIET A N D M ATERIALS . ,
i
Casein E x tra c tio n .
!
j R a t s ............................ .
I
Methyl L inoleate • ,
!
M ethyl L inolenate ,
M ethyl Arachidonate
L in o leic Acid . . ,
E th y l Laurate . . ,
Growth Hormone . . ,
Thyroid Powder . . ,
IV. EX PERIM EN TA L RESULTS ,
F at D epletion of R ats hy C la ssic a l
Method ...........................................................
Use of Growth Hormone in D epletion
of R ats ...........................................................
E ffe c t of Thyroid on F at D epletion . . .
U nsaturated F a tty Acid Requirement
of the R at ......................................................
Establishm ent of logarithm ic dose-
weight gain curve with lin o le ic acid .
Dem onstration of sex d iffe re n c e s in
e s s e n tia l f a tt y ac id requirem ents
i of th e r a t
i ..._______________________________
P A G E
1
5
32
32
38
39
39
40
40
40
41
41
42
42
47
55
61
61
71
CH A PTER P A G E :
Growth r a te s w ith methyl lin o le n a te and
in te rre la tio n s h ip o f lin o le a te and
lin o le n a te . . . . . . . . . ................. 74
Growth r a te s w ith methyl araehidonate and
in te rre la tio n s h ip of lin o le a te and
araehidonate . . . . . . . . . . . . . 77
B io lo g ical Assay of E ss e n tia l Acids in
F ats and a Comparison of B iological and
P hysical Methods .......................................... 92
E ffe c t of Methyl L in o leate Supplem entation
on M ineral O il "Fat-D epleted” R ats . . . 97
R elatio n Of E s s e n tia l F a tty Acids to
Gonad Development in th e Rat . . . . . . 100
7 . DISCUSSION O F .RESULTS....................................... 107
7 1 . SU M M A R Y 120
. \
BIBLIOGRAPHY ....................................... . . . . . . 125
LIST O F TABLES
T A B L E P A G E
I . Composition of the D iets Bnployed in the
Study of Casein E x trac tio n Methods and *
in Experiments w ith F at-D epleted Rats . . . S3
I I . Composition of th e D iets Employed in the
Study of M ineral O il and of Chorionic •
Gonadotropic Hormone on F at-D e ficien t R ats 35
I I I . Growth of R ats on F at-F ree D iets w ith
Casein Prepared by Various Means and on
a C ontrol D iet C ontaining 10 P er Cent
Cottonseed O il ..................... 43
IV. Food Consumption of R ats on F at-F ree D iets
w ith Casein Prepared by Various Means and
on a C ontrol D iet C ontaining 10 Per Cent
Cottonseed O il . . . . . . . .......................... 46
V. Average Body Weight Gain and S urvival of
R ats P reviously on a F at-F ree D iet (Diet
69) U n til Depleted and T h ereafter Receiv
ing 1, No Supplement; 2, Growth Hormone
Alone; 3, Methyl L in o leate Alone; 4, Methyl
L in o leate Along w ith Growth Hormone; and
5, Of a C ontrol Group of R ats R aised and
Continuing on a S im ilar D iet Containing
10 Per Cent of Cottonseed O il (Diet 70) • • 49
VI. Average Food Consumption of R ats oh a
F at-F ree D iet U n til Depleted and There
a f te r Supplemented as I n d i c a te d ...................... 54
V II. Mean Weights and Percentage M o rta lity of
Male R ats on Following Groups: Fat-Low
D iet; Fat-Low D iet Containing 0.05 Per
Cent of D esiccated Thyroid Powder; F a t-
Low D iet Containing 0.05 P er Cent of
D esiccated Thyroid Powder and Twice the
W ater-Soluble Vitamin Content; 30 Per Cent
Cottonseed Oil D iet C ontaining 0.05 Per
Cent of D esiccated Thyroid .......................... 57
v i i
TA BLE
T i l l . Mean W eights, Percentage M o rta lity , and
Weight Gain of Female E ats bn Following
Groups: 6, Fat-Low; 7, Fat-Low D iet
Containing 0*05 Per Cent of D esiccated
Thyroid Powder; 8, Fat-Low D iet Containing
0.05 Per Cent of D esiccated Thyroid Powder
and Twice the W ater-Soluble Vitamin Content;
7, 30 Per Cent Cottonseed O il D iet; 10, 30
Per Cent Cottonseed O il D iet Containing
0.05 P er Cent of D esiccated Thyroid . . .
IX. Body Weights of Male Rats a t Weaning,
a f te r a F at-D epletion D iet; and Gain
in Weight Follow ing Supplem entation
w ith L in o leate, w ith L ino len ate, w ith
Equal Amounts o f L in o leate and L in o len ate,
w ithout Supplement, Receiving a. 30 Per
Cent F a t D iet throughout, or R eceiving
a 30 Per Cent F at D iet Following D epletion
X. Body Weights of Female Rats a t Weaning,
a f te r a F at-D epletion D iet; and Gain in
Weight Following Supplem entation w ith
L in o le a te , w ith L in o len ate, w ithout
Supplement, R eceiving a 30 Per Gent F at
D iet throughout, or Receiving a 30 Per
Cent F at D iet Following D epletion . ...
X I. Body Weight Gains fo r Male R ats on Assay
Groups fo r Methyl A raehidonate, M argarine
F a t, and B u tte rfa t • • • * . ......................
X II. Body Weight Gains fo r Female R ats on Assay
Groups fo r Methyl A raehidonate, M argarine
F a t, and B u tte rfa t ...........................................
X III. Potency of Methyl A raehidonate When Fed
Alone to F at-D epleted Male R ats and When
Fed w ith L in o leic Acid as Determined from
th e Logarithm ic Dose-Weight Gain Curves
Obtained from L in o leic Acid Alone .. ..
PA G E
59
63
66
78
80
@ 4
v i i i
TABLE BASE
XIV. Food Consumption Data f o r Male R ats on
Assay Groups fo r Methyl A raehidonate,
M argarine F a t, and B u tte rfa t ................. 86
XV. Food Consumption Data f o r Female R ats on
Assay Croups f o r Methyl A raehidonate,
M argarine F a t, and B u t t e r f a t .................... 88
XVI. T ibia Lengths o f R ats a f te r 8 Weeks on
No F a t, 30 Per Cent Fat,. L in o leic Acid
Alone, Methyl A raehidonate Alone, and
M ixtures of L in o leic Acid and Methyl
A raehidonate ....................................................... 91
XVII. C onstants Obtained in Analyzing Test
G ils Used in B io lo g ical Assay Procedures 93
XVIII. Growth Data fo r Male R ats On F at-F ree
- r and 7 \ Per Gent M ineral O il-F at-F ree
D iets w ith and w ithout Methyl L in o leate 99
XIX. E ffe c t of Chorionic Gonadotropic Hormone
on Male and Female R ats which had been
F at-D epleted and Re-Fed w ith Low or w ith
High Dosages of Methyl L in o leate . .. . 103
LIST O F FIGURES
FIGURE P A G E
1. The average w eights of weanling male r a ts
during 12 weeks on f a t- f r e e d i e t s ................. 4$
2. The average w eights of weanling female
r a ts during 12 weeks on f a t- f r e e d ie ts . . 46
3 . The average gains in weight of male r a ts
re ceiv in g a f a t- f r e e d ie t alone, growth
hormone, methyl lin o le a te , methyl lin o le a te
p lu s growth hormone, or a d ie t containing
10 per cent of cottonseed o i l . . . . . . . 51
4 . The average gains in weight of female r a ts
receiv in g a f a t- f r e e d ie t alo n e, growth
hormone, m ethyl lin o le a te , methyl lin o le a te
plus growth hormone, or a d ie t containing
10 per cent of cottonseed o i l ........................... 52
5. Average gains in w eight of groups o f male
r a ts when supplemented w ith m ethyl lin o le a te ,
w ith methyl lin o le n a te , w ith m ixtures of
lin o le a te and lin o le n a te , w ith eth y l la u ra te ,
o r when fed a d ie t containing 30 per cent of
cottonseed o i l . . . . . .................................. 69
6. Average gains in weight o f groups of female
r a ts when supplemented w ith methyl lin o le a te ,
w ith m ethyl lin o le n a te , w ith m ixtures of
lin o le a te and lin o le n a te , w ith e th y l la u ra te ,
o r when fed a d ie t containing 30 p er cent of
cottonseed o i l ...................................................... 70
7. The gain in weight of p reviously depleted
male r a ts p lo tte d ag a in st th e log dose of
methyl lin o le a te f e d ........................................... 72
8. The gain in weight of previously depleted
female r a ts p lo tte d ag a in st th e log dose
of methyl lin o le a te f e d ............................. 73
9. The gain in weight of previously depleted
male and female r a ts p lo tte d a g a in st the
log dose o f methyl lin o le a te fed fo r 7 or
X
FIGURE PA G E
8 weeks. P ro je c tio n s on tlie curves of
weight gains fo r groups of r a ts © n methyl
araeh id o n ate, m argarine f a t, and b u tte r
f a t a re given ............................. 85
C H A PTER I
INTRODUCTION
The in creasin g use of hydrogenated f a ts in our
d ie ts , and th e g re a te r reco g n itio n of the im portance of
th e e s s e n tia l f a tt y a c id s in n u tr itio n have rendered
necessary a b io lo g ic a l method fo r th e determ ination of the
e s s e n tia l acid content of f a ts . B io lo g ical determ ination
of th e e s s e n tia l f a tt y ac id s has been needed a s a standard
and a check on th e physicochem ical methods. The presence
o f isom ers of th e higher u nsaturated f a tty a c id s in
tre a te d f a ts , which give p o sitiv e speotrophotom etric read
in g s but possess no b io lo g ic a l a c ti v i t i e s (1 ), has p re
sented d if f ic u lty , as y et insurm ountable, in the use of
physicochem ical methods fo r r e lia b le determ inations o f
th ese a c id s.
Burr (3 ), in h is comprehensive review o f the l i t e r
a tu re on th e e s s e n tia l f a tt y a c id s, l i s t s the lin o le ic
acid requirem ents of th e r a t as ranging from 30 mg. per
day to 100 mg. pe'r day; however, he o ffe rs no d ecisiv e
experim ental data to su b sta n tia te any of th e fig u re s
given. Furtherm ore, c o n flic tin g data have been obtained
concerning the r e la tiv e potencies of the most im portant
e s s e n tia l acid s — namely, lin o le ic , lin o le n ic , and
arachidonic a c id s.
Eor th ese reasons t h i s in v e stig a tio n was under
taken to :
1. Study methods o f f a t d ep letio n o f r a ts .
2. Study th e requirem ents fo r p u rifie d lin o le ic ,
lin o le n ic , and arachidonic a c id s, and t h e ir
in te r r e la tio n s h ip s .
3. E sta b lish a method fo r th e b io lo g ic a l assay
o f the e s s e n tia l ac id s in f a t s .
CH A PTER I I
HISTORICAL REVIEW
In 1920, Osborne and Mendel (3) noted th a t r a ts on
d ie ts containing very sm all amounts o f f a t grew about as
w ell as those on h ig h -fa t d ie ts . He concluded th a t " if
tru e f a ts are e s s e n tia l fo r n u tritio n during growth the
minimum neeessary must be exceedingly sm all". About th is
same time Krogh and Lindhard (4) showed th a t a minimum of
f a t and carbohydrate i s required fo r the b e st u tiliz a tio n
of food. In 1926, Evans and Burr (5) rep o rted the e a r l i
e s t work on th e d etrim en tal e ffe c ts o f a continuous fat*?
fre e d ie t on th e h e a lth o f r a t s . However, n e ith e r the
s p e c ific symptoms nor the sp e c ific fa c to rs in f a t which
revealed the e ffe c t on th e h e a lth of th e anim als were
elu c id a te d . S everal y ears l a t e r , McAmis, Anderson, and
Mendel (6) desoribed th e s p e c ific e ffe c ts of a f a t- f r e e
d ie t on th e growth o f r a t s , and the b e n e fic ia l e ffe c ts
of sm all amounts of f a t . However, these authors were
s t i l l in doubt as to whether th e e ffe c ts noted were
brought about by th e in c lu sio n of sm all amounts of v ita
mins (known or unknown) in the f a t , by th e aid which f a t
may have rendered in th e abso rp tio n o f fa t-s o lu b le v ita
mins, o r by th e presence of f a t per s e . C oincidental
4
w ith th e p u b licatio n of McAmis et, a l . , Burr and Burr (7)
described a d eficien cy d isease produced by the r ig id exclu
sion of f a t from the d ie t of r a ts . The d ie t, which i s
i
s t i l l in general use fo r fa t-d e fic ie n c y experim ents, con
s is te d of re p re c ip ita te d and so lv e n t-e x tra c te d casein,
24 to 12 per cent; sucrose, 72.1 to 84.1 per cent; s a l t s ,
3.9 per cen t; e th e r-e x tra c te d y e a st, and vitam ins A and B.
V ariatio n s in casein and sucrose content depended upon the
age o f th e anim als; higher p ro te in percentages were used
fo r young anim als.
Exclusion of f a t from th e d ie t of the growing r a t
lead s to sev e ral ty p ic a l but obvious symptoms and sev eral
more obscure e f f e c ts . The most pronounced symptoms (2)
a re as follow s:
1 . There i s f a ilu r e to m aintain a normal growth
r a te .
2. S calin ess o f th e skin develops, appearing f i r s t
on the pads of th e h in d leg s, then along the t a i l , and on
«
the back in th e form of flak y dandruff. In some cases,
th e t a i l s l a t e r become n e c ro tic , but caudal n ec ro sis i s
v a ria b le and i s u seless fo r assay s, since derm atological
i
symptoms have been found to depend la rg e ly upon the r e la
tiv e hum idities (8 ,9 ,1 0 ,1 1 ). High hum idities a lle v ia te
5
th e dermal symptoms, w hile low hum idities in c re a se th is .
syndrome.
3. Kidney degeneration i s evident in anim als on a
f a t- f r e e d ie t. M aeroseopically n o ticeab le hem aturia
develops l a te and w ithout g re at re g u la rity . The p ercen t
age of anim als showing t h i s sign v a rie s from 16 per cent
to over 90 per cent (2 ). Borland and Jackson (12) con
firm ed e a r lie r re p o rts of re n a l involvement (6,7,13) by
dem onstrating th a t 100 per cent of th e cases of f a t-
depleted anim als had kidney le s io n s a t autopsy. I t i s of
considerable im portance, however, th a t th ese le sio n s could
be prevented or cured by sm all amounts o f f a t o r c o d -liv e r
o il in the d ie t (12). Maeder (14) has rep o rted more or
le s s sp e c ifio h is to lo g ic a l changes in th e o v aries and
u te ru s of the fem ale r a t on a f a t- f r e e d ie t, and d e ta ile d
h is to lo g ic a l change; in th e skin of th e r a t on f a t- f r e e
d ie ts has been recorded by W illiamson (15).
The h is to lo g ic a l fin d in g s previously rep o rted on
th e e ffe c ts o f a f a t- f r e e d ie t on the gonads.of female
r a ts a re borne out by observations of irre g u la r ovulation
(13) w ith prolonged g e s ta tio n , severe hemorrhage during
p a r tu r itio n , and poor la c ta tio n (13,16). The a n a ly sis of
breeding r e s u lts fo r r a ts on a fa t-lo w d ie t in th is
6
lab o rato ry (17) confirms th e previous fin d in g s of severe
hemorrhage during p a r tu r itio n , and poor la e ta tio n r e s u lt
ing in e a rly death of the apparently normal young. In one
l i t t e r th a t liv e d for only 10 days, general peeling of th e
skin of th e young was observed. C ontrary to the re p o rt of
Burr and B urr (13), Quackenbush and co-workers (16)
rep o rted th a t, in 20 weeks on a low -fat d ie t, no f a ilu r e
of e s tru s was noted in th e female r a t . These workers
rep o rted th a t e th y l lin o le a te and e th y l araehidonate p e r
m itted the production of normal young, but th a t eth y l
lin o le n a te did n o t, nor did i t (e th y l lin o le n a te ) cure
dermal symptoms. In a study o f reproduction and la c ta tio n
of r a ts on a f a t- f r e e d ie t, Evans, Lepkovsky, and Murphy
(18,19) rep o rted th a t su ccessfu l g e sta tio n was not p ossible
on th ese d ie ts . I t must be noted th a t our d i e t , which, p er
m itted g e s ta tio n and p a r tu r itio n in most cases, contained
minute amounts of f a t in th e unextracted casein used.
Evans e t a l , (18,19) observed th a t sa tu ra te d f a tty ac id s
in the form of hydrogenated coconut o i l were of no b e n e fit
fo r fem ales bred on f a t- f r e e d ie ts , but th a t su ccessfu l
g e sta tio n was obtained follow ing th e ad d itio n of the essen
t i a l f a tty ac id s (lin o le ic ) to the ra tio n . However, i t
was observed th a t on th is d ie t w ith th e added lin o le ic
a c id , la c ta tio n -ms n o t norm al. O lcott and M a ttill (20)
confirmed th e r e s u lts of Evans and h is oo-w orkers, but
Mackenzie and h is colleagues (21) c r it ic iz e both of the
above groups o f workers fo r considering th e p o s s ib ility
of d e fic ie n c ie s in th e w ater-so lu b le or fa t-s o lu b le v ita
mins in th e i r d ie ts w ithout excluding th e p o s s ib ility of
d e fic ie n c ie s in cy stin e o r m ethionine, b oth of which are
p o ten t lactogogues. These in v e s tig a to rs (21) showed by
interchange of th e young o f f a t- f r e e mothers w ith the
young of stock-fed m others/ th a t th e young th a t survived
p a r tu r itio n were e s s e n tia lly norm al, since those placed
w ith the sto ck -fed mothers liv e d and the young w ith the
f a t- f r e e m others died. The f a u lt was ascrib ed to f a ilu r e
of la c ta tio n in th e lo w -fat r a t s . Decker and Mead (22)
and Decker e t a l . (23) found th a t chronic f a t- d e f ic ie n t
female mice were com pletely incapable of producing young,
even though the e stru s cycle was ap p aren tly normal and
proven males had been used fo r breeding. Their f a ilu r e
to show sig n s of g e s ta tio n in th e mouse i s in c o n tra st to
r e s u lts th a t had been obtained with the r a t (13,16,18,19)
in which g e s ta tio n was a tta in e d on a fa t-lo w d ie t,
although a l l the young died sh o rtly a f te r b ir th .
Quackenbush, Kummerow, and Steenbock (16) found
8
th a t -the te s te s of th e male r a t atrophy on a f a t- f r e e
d ie t; th is observation was confim ed by Evans and h is
co lla b o ra to rs (24), who found th a t th e male r a t on such
d ie ts in v a ria b ly became s t e r i l e . However, th is type of
s t e r i l i t y was rep o rted to be curable by the use of essen
t i a l f a tty a c id s. This r e v e r s ib ility of s t e r i l i t y in the
male i s much lik e th a t observed in vitam in A d eficien cy ,
and u n lik e th a t re s u ltin g from lack o f vitam in E; th e
l a t t e r type i s ir r e v e r s ib le . In c o n tra st to these fin d
in g s, Mackenzie e t a l . (21) have rep o rted fin d in g m otile
sperm in the epididymides of male r a ts a f te r 44 weeks on
a lo w -fat d ie t. In a study of f e r t i l i t y in another
sp ec ie s, R eiser and Gibson (25) have recorded th a t hens on
a f a t- f r e e d ie t show no d iffe re n c e s in egg production from
those on a 4 per cent cottonseed o i l d ie t, b u t th a t the
eggs from th e lo w -fat hens re v eal lower f e r t i l i t y a s meas
ured by lower h a tc h a b ility and a higher m o rta lity of the
embryos•
The observations recorded as to poor reproduction
of lab o ra to ry anim als provide some grounds fo r the s ta te
ment of Maeder (14) th a t f a ilu r e of reproduction in humans
may in some oases be due to a lack of adequate f a t in the
d ie t. A nalysis o f th e lip id s in th e blood is a u sefu l
9
index of u n satu rated f a tty acid d efic ie n cy , since a l l
anim als te s te d have shown a drop in the iodine number of
1 th e plasma lip id s . Such a c r ite r io n of d eficien cy i s one
| of th e few u se fu l t e s t s th a t can be performed on human
su b je c ts (26,27).
An in crease in w ater consumption has been noted fo r
anim als on a fa t-lo w d ie t. Burr and Burr (IS) recorded as
much as 100 per cent in c re a se in w ater in ta k e fo r female
r a ts on a fat-lo w d ie t as compared to those on a d ie t
i
containing f a t . In a l a t e r re p o rt, Burr (2) has s ta te d
th a t th e e ffe c ts of th e ac id s upon w ater consumption p ar
a l l e l th e ir e ffe c ts upon th e skin; j l . e . , i f th e sk in symp
toms a re cured, th e w ater consumption i s reduced to
normal; h is d ata on d a ily w ater consumption a f te r "cu res”
w ith th e follow ing ac id s a re : e s te rs of c o d -liv e r o i l ,
18.6 m l.; e th y l lin o le a te , 11.2 m l.; eth y l lin o le n a te ,
25.5 m l.; and methyl araehidonate, 9.3 ml. Quackenbush
and h is colleagues (16) could fin d no d iffe re n c e s in w ater
consumption f o r female r a ts even a f te r 20 weeks on a f a t -
fre e d ie t; however, some in crease was shown fo r male r a ts .
Excessive w ater consumptions have a ls o been rep o rted fo r
mice on low -fat d ie ts (23).
Among o th er symptoms th a t commonly occur in anim als
10
su ffe rin g from fa t-d e fic ie n c y a re an in ere ase in fo r
r a ts (28) and fo r men (26,27); high, m etabolic ra te s
(26-30); and a new symptom, p o ssib ly aso rib a b le to f a t-
d eficien ey in the chicken, namely, edema and je lly - lik e
i n f i l t r a t i o n of th e peritoneum (31).
The c r it e r ia most commonly considered in q u a n tita
tiv e stu d ie s, in order o f th e ir u se fu ln e ss, a re : e ffe c ts
on body w eight, e ffe c ts on b lo o a -lip id le v e ls , cure or
prevention of d e rm a titis , e stru s cycle d istu rb an ces (32),
and w ater consumption measurements. Great care must be
exercised in th e use of dermal symptoms as a method of
measurement, due to th e s e n s itiv ity o f th is syndrome to
hum idity le v e ls . Extreme v a ria tio n s have also been
recorded fo r changes in the e s tru s cycle and fo r d if f e r
ences in w ater consumption.
The in te rre la tio n s h ip between th e e s s e n tia l f a tty
acid s and the B vitam ins has been a su b ject of extensive
in v e s tig a tio n . Goldberger and L i l l i e (33), in 1926, noted
the im portance of w ater-so lu b le substances in th e preven
tio n and cure o f dermal le s io n s . E arly dem onstrations
th a t w ater-so lu b le and lip id fa c to rs were both involved
in th e eure or prevention of a form o f d e rm a titis now
fre q u e n tly termed " ra t acrodynia” were made by Schneider
XI
and co-workers (34), R ichardson and Hogan (35), B irch
and Gyorgy (36), and Salmon (37). T ests w ith is o la te d
compounds have shown th a t lin o le ic a c id (9 ), pyridoxine
(3 8 ,3 9 ), and pantothenic acid (40) a re in te r r e la te d .
Q,uackenbush e t a l . (41) employed a h ig h ly -p u rifie d
d ie t containing sy n th e tic v itam in s. They showed t h a t, on
a complex d efic ie n cy of lin o le ic a c id , p y rid o x in e, and
pantothenic ac id (which causes aerodynia in th e r a t ) ,
pantothenic acid did not a lle v ia te th e symptoms, pyridox
in e produced some a lle v ia tio n of symptoms h u t no cu re,
w hile th e a d m in istra tio n of eth y l lin o le a te re su lte d in
an apparent cure o f th e d efic ie n cy symptoms. On a sub-
cu ra tiv e le v e l of lin o le a te , pyridoxine was able to com
p le te th e r e l i e f o f th e d e rm a titis . P antothenic acid
and pyridoxine in com bination improved th e dermal condi
tio n , and lin o le a te added l a t e r showed fu rth e r improve
ment. These workers re p o rted th a t su stain ed growth of
young r a ts was accomplished only when a l l th re e substances
were fe d . This o bservation th a t vitam in Bg and the essen
t i a l f a tt y ac id s can each a lle v ia te d efic ie n cy of th e
o th e r, and th a t both a re necessary fo r normal growth, was
confirmed by th e work of Salmon (42), Richardson and
Hogan (43 ), and more re c e n tly by th a t o f V io llie r (44).
12
Emerson (45) found th a t the types of d e rm a titis
produced by th e two d e fic ie n c ie s , pyridoxine and the
e s s e n tia l f a tt y a c id s, were e a s ily d istin g u ish a b le . How
ever, Em erson^ work has been c r itic iz e d because her ob
se rv a tio n s were based on skin symptoms, which are u n re li
able except under th e most r ig id ly c o n tro lle d co n d itio n s.
Medes and her co-workers (46) showed th a t, on an e s s e n tia l
f a tty acid and p y rid o x in e-d e fieien t d ie t, the growth
response to pyridoxine alone was g re a te r than th a t r e s u lt
ing from eth y l lin o le a te alo n e. Sherman (47), in h is
recen t review of th e re la tio n of pyridoxine to f a t metabo
lism , s ta te s th a t each fa c to r (pyridoxine and the essen
t i a l f a tty acid s) must be regarded as "indisp en sab le” ,
since one compound cannot rep lace the o th er but can merely
reduce th e requirem ent fo r th e o th e r, and th a t each has
a s p e c ific fu n ctio n to perform in .th e body. These d if f e r
ences in fu n ctio n a re borne out by a re p o rt of Daniel
e t a l . (48), who observed a convulsive syndrome produced
in young suckling pig s by a BQ ~ d e fie ie n t d ie t. This
syndrome was not a ffe c te d by th e presence or absence of
com o i l in th e d ie t.
A review of th e p o ssib le m etabolic fu n ctio n s of
pyridoxine o ffe rs some b a s is fo r an understanding of the
13
in te rre la tio n s h ip of pyridoxine with the e s s e n tia l
ac id s (49). The f i r s t suggestions of a mechanism of
a c tio n were th a t f a t had a sparing a c tio n on th e pyridox
ine requirem ent (36,50)• B irch (51) concluded th a t
vitam in Bg functioned in th e u tiliz a tio n of th e u n satu r
ated f a tty a c id s, w hile Engel (52) noted th a t pyridoxine
and pantothenic acid in cre ase th e q u an tity of f a t synthe
sized by th e r a t . Engel fu rth e r noted th a t vitam in Bg
and th e e s s e n tia l f a tty ac id s a c t m utually in in flu en cin g
th e lip o tro p ic ac tio n of choline and are complementary in
t h is re sp e c t. Richardson and h is colleagues (53) suggest
th a t th e u nsaturated acid s do not rep lace pyridoxine but
delay th e onset o f th e skin symptoms. B irch (51) had
shown e a r lie r th a t th e a d d itio n of f a t to the d ie t delayed
or com pletely prevented th e onset of th e d e rm a titis ,
caused by pyridoxine d efic ie n cy , even to the time of
d eath , and th e e ffe c tiv e n e ss of the f a t was shown by
Salmon (37) to be d ire c tly p ro p o rtio n al to th e degree of
u n sa tu ra tio n .
McHenry and Gavin (54) have concluded th a t the ro le
of pyridoxine in th e mammalian organism includes th a t of
f a t sy n th esis from p ro te in . Since Umbreit and Gunsalus
(55) have shown th a t pyridoxine i s involved in the
14
enzymatic decarboxylation o f amino a c id s, which i s one of
th e e s s e n tia l ste p s in the production o f f a t from p ro te in ,
th e suggestion of McHenry and Gavin (54) would seem to
gain co nsiderable support.
Medes e t a l . (46) observed th a t th e amount of body
f a t produced in r a ts re lie v e d of a Bg f a t d efic ie n cy by
pyridoxine was approxim ately th e same as in anim als
re lie v e d by e th y l lin o le a te . A ll fra c tio n s o f the f a t
of these anim als were in cre ased , in clu d in g th e highly
u n satu rated f a tty a c id s . On th e pyridoxine supplement
th e in crease was d is trib u te d almost equally among the d i- ,
t r i - , and te tra e n o ic fra c tio n s . When lin o le a te was fe d ,
th e dienoic fra c tio n was more elev ated . These data bear
out th e r e s u lts of sev e ral o th er in v e s tig a to rs (56,57)
which in d ic a te th a t pyridoxine plays a ro le ,b o th in the
sy n th esis of arachidonic a c id and in i t s d e p o sitio n .
; Tulpule and Patwardhan (58) noted th a t the t o t a l
lip o id fra c tio n of th e plasma of th e r a t was not a ffe c te d
by th e presence or absence of e s s e n tia l f a tt y a c id s and/or
pyridoxine in th e d ie t. On the o th er hand, th e average
iodine number of plasma f a tt y ac id s of th e anim als in f a t-
fre e and in f a t - and p y rid o x in e-free groups (96.2 and
77.4 re sp e c tiv e ly ) a re s ig n ific a n tly lower than th e values
15
observed in the p y rid o x in e-free o r in th e p o sitiv e co n tro l
groups. In th is re sp e ct th ese in v e s tig a to rs are in agree
ment with Hansen and Brown (59,60), who noted low iod in e
numbers fo r plasma f a tty ac id s in fa t-d e p le te d r a t s . I t
i s concluded e ith e r th a t th e u tiliz a tio n of f a t in the
absence of pyridoxine i s d efectiv e o r th a t pyridoxine
d efic ie n cy a f f e c ts th e d e sa tu ra tio n process in the liv e r .
Hot only a re the B vitam ins in th e d ie t in te r
re la te d w ith th e requirem ent of th e animal organism fo r
th e e s s e n tia l f a tt y a c id s, b u t th e re a re o th er d ie ta ry
fa c to rs which a ffe c t the requirem ent fo r th ese a c id s.
Hume e t a l . (10) could fin d no evidence th a t the presence
or absence of vitam in E made any s ig n ific a n t d iffe re n c e s
in a growth t e s t w ith r a ts (males and fem ales) i However,
Hove and H a rris (61), in an experiment w ith male r a ts ,
concluded th a t vitam in E did extend the e ffe c tiv e n e ss of
suboptim al q u a n titie s of lin o le ic a c id in preventing or
curing f a t d eficien cy in r a ts . A very extensive study in
th is lab o ra to ry of the p o ssib le in te rre la tio n s h ip between
lin o le a te and vitam in E re su lte d in negative r e s u lts fo r
male and female r a ts over a wide range of dosages of both
fa c to rs (62). H irseh and Jaoquot (63) have reported th a t
very high dosages of the e s s e n tia l f a tty aoids cause a
16
d ise a se , w ith th e o h a ra c te ris tic symptoms of avitam inosis
E, which can be o ffs e t by'adding la rg e amounts of tocoph
e ro l to the d ie t.
MaoKay and Barnes (64) have presented d ata in d ic a
tin g th a t b io tin d eficien cy symptoms caused by a raw egg-
white d ie t can be a lle v ia te d o r delayed e ith e r by pyridox
ine or by corn o i l , which i s an in d ic a tio n th a t some
p o ssib le in te rre la tio n s h ip e x is ts between th e e s s e n tia l
f a tty acid s and b io tin . Cheng, Greenberg, Deuel, and
Melnick have re c e n tly presented data on the r e la tio n of
u n satu rated acid s to b io tin in the n u tr itio n of c e rta in
mi croorganisms (65).
It. has been shown th a t the d e rm a titis produced by r
d eficien cy of th e e s s e n tia l f a tty acid s i s exaggerated
when th e n o n -e sse n tia l f a tty ac id s o r f a ts a re fe d . In
1950, B urr and Burr (13) rep o rted th a t no cure was ob
tain ed by th e in clu sio n of satu ra te d f a tty acid s in the
d ie t of r a t s , and th ese same in v e s tig a to rs w ith M ille r (66)
te s te d th e e ffe c tiv e n e ss of the sa tu ra te d f a tty ac id s in
hydrogenated coconut o i l , as w ell as of o le ic acid and
a lp h a -e le o ste a ric a c id , an isomer of lin o le ic a c id , and
found a l l in e ffe c tiv e fo r curing f a tty acid d efic ie n c y .
More re c e n tly Sarma e t a l . (6?) showed th a t th e a d d itio n
17
of o le ic acid to a f a t- f r e e ra tio n containing suboptimal
amounts o f pyridoxine re su lte d in g re a te r growth in h ib i
tio n . This in h ib itio n o f growth was la rg e ly counteracted
by th e ad m in istratio n o f more vitam in Bg . I t was found
by Evans and Lepkovsky (68) th a t th e g ly cerid e s of s a tu r
ated f a tt y ac id s do not perm it as much growth in the r a t
when they a re the so le source of energy, o ther than the
p ro te in component, as i s th e ease when sucrose i s the sole
source. This same phenomenon has been noted when r a ts
were fed a regimen containing a considerable proportion e£
e la id in (29 ). A nisfeld e t a l . (62) in th is lab o rato ry
have dem onstrated th is depressing e f fe c t on a q u a n tita tiv e
b a s is , using satu ra te d coconut o i l and lin o le ic a c id . I t
has been noted th a t r a ts receiv in g d ie ts high in sa tu ra te d
f a t , but lacking in the e s s e n tia l f a tt y a c id s, a re unable
to e f fe c t an ap p reciab le in crease in the amount of to ta l
body lip id s , but re a d ily exchange d ie ta ry f a tt y ac id s with
those in the tis s u e s (69). More re c e n tly E e ise r (31) has
rep o rted th e negative e ffe c t of sa tu ra te d f a tty a c id s in
chicks on a f a t- f r e e d ie t.
Among th e d i f f i c u l t problems to be solved in a study
of th e e s s e n tia l f a tty a c id s i s one o f d ep letin g anim als
fo r study. The c la s s ic method i s th a t estab lish ed by Burr
18
and Burr (7) except fo r th e s u b s titu tio n of sy n th e tic B
vitam ins fo r ex tracted y e a st in th e d ie t. By adm inistering
a d ie t low in f a t to th e nursing mothers of r a ts which are
to be used fo r d ep letio n s tu d ie s , one i s able to d ep lete
growing r a ts in approxim ately th re e months (9 ,7 0 ,7 1 ). As
an illu s t r a tio n of th e e f fe c t which the mother can have
on th e sto rag e of u n satu rated ac id s in the young, i t has
been shown th a t th e iod in e number of the carcass f a ts of
weanlings could be reduced from 154 to 94 by tra n s fe rrin g
th e mothers during pregnancy from a stock d ie t to a d ie t
c o n sistin g of potato m eal, casein , and s a lts (71).
In view of th e work p reviously c a rrie d out w ith
sa tu ra te d f a tty ac id s and such u n satu rated acid s as o le ic
or e le o s te a rie , i t should be p o ssib le to reduce m a te ria lly
th e time of d ep le tio n o f young r a ts when these in h ib ito ry
lip id s are included in th e f a t- f r e e d ie t. Barki and h is
c o lla b o ra to rs (72) found i t im possible to d ep lete ad u lt
r a ts on a f a t- f r e e d ie t, but they were ab le to develop
symptoms o f d efic ie n cy by starv in g the anim als u n til h a lf
t h e ir normal weight had been l o s t , a f te r which th e anim als
were allowed to e a t the d e fic ie n t d ie t ad lib itu m . I t i s
im portant to note th a t, by t h i s method, approxim ately the
same kind of s tre s s i s placed on the ad u lt anim als as i s
19
placed on th e weanling on a f a t- d e f ic ie n t d ie t, as a r e s u lt
of th e ra p id form ation of new c e lls and tis s u e . The pro
duction of fa t-d e fic ie n c y symptoms by s tre s s has a lso been
recorded by Decker, F ille r u p , and Mead (23) in th e ir work
on th e mouse. Chronic fa t- d e f ic ie n t mice showed none of
th e usu al symptoms of d eficien cy u n til a f t e r blood samples
had been taken by t a i l am putation. These wounds f a ile d to
h eal in the d e fic ie n t mice and concurrently th e ty p ic a l
syndrome of fa t-d e fic ie n c y beeame apparent.
Several methods of hastening th e process of deple
tio n were in v e stig a te d in th e course of th is s e rie s of
experim ents on th e e s s e n tia l f a tt y a c id s . The f i r s t was
by exhaustive e x tra c tio n o f th e casein to be used in the
d ie t. The second method te s te d co n sisted in the use o f
growth hormone (73). This method was suggested by th e
r e s u lts o f previous work with vitam in A (74) in vfcich the
d efic ie n cy was accentuated by growth hormone. N either
th e e x tra c tio n method nor the hormone appeared to a id in
th e fa t-d e p le tio n of r a t s . D esiccated thyroid was added
to th e d ie t as a th ir d method of in creasin g th e r a te of
d ep letio n o f th e r a t (75). A fo u rth method was suggested
by th e work of Bacon (76) in which m ineral o il was in c o r
porated in to th e d ie t o f the d ep letin g r a ts . An apparent
20
a c c e le ra tio n of the development of the d eficien cy m s
noted in th i s case.
Many fa c to rs a f f e c t th e requirem ent of th e r a t fo r
th e e s s e n tia l f a tty a c id s. Some, of these co n d itio n s have
been discussed: fo r example, the previous n u tritio n a l
h is to ry of the mother r a t , th e le v e l of the B vitam ins in
the d i e t , the age o f th e anim als, and s tr e s s fa c to rs added
to the d ie t or imposed upon th e anim als. However, under
f a i r l y standard co n d itio n s one should be able to estim ate
th e d a ily requirem ent o f th e r a t fo r lin o le io a c id . Burr
(2) has reviewed these fin d in g s and has l is te d re q u ire
ments as ranging from 20 mg. per day as a p rophylactic
dose fo r th e growing young r a t (77) to 1©0 mg. per day as
th e optimum fo r th e growth of anim als th a t had reaehed a
p lateau on a f a t- d e f ic ie n t d ie t (32). The comparative
p otencies quoted fo r lin o le n ic and arachidonic acid s vary
w idely, depending upon th e c r ite r io n of measurement.
B urr e t a l . (66) rep o rted obtaining equal growth e ffe c ts
from lin o le n ic and from lin o le io acid s f o r depleted r a t s ,
and fu rth e r reported th a t the e ffe c ts were a d d itiv e when
te s te d by feeding the substances to g e th e r a t a le v e l of
approxim ately 45 mg. each per day. When th ese in v e stig a
to r s replaced 1© per cent of a m ixture of methyl lin o le a te
21
and methyl lin o le n a te w ith methyl araeh id o n ate, i t was
found th a t th e m ixture containing th e araehidonate was
le s s e ffe c tiv e than th e o rig in a l m ixture.
Turpeinen (32) determ ined th a t th e e ffe c tiv e n e ss
of arachidonic acid fo r growth o f d e fic ie n t r a ts was th re e
tim es as g re a t as th a t of lin o le ic a c id . Burr and h is
co-workers (78) rep o rted th a t th ey were unable to confirm
Turpeinen1s work with arachidonic a c id . These in v e s tig a
to r s obtained equal growth e ffe c ts w ith arachidonie and
lin o le ie ac id s a t le v e ls of 20 and 40 mg. per day, and
rep o rted th a t lin o le n ic a c id gave good growth, equal to
th a t o f lin o le ic a c id a t le v e ls of 20, 40, and 60 mg. per
day. However, lin o le n ic acid was found to have l i t t l e
e ffe c t in cu rin g d e rm a titis . Hume e t a l . (79) in v e stig a te d
th e r e la tiv e potencies of th e various e s s e n tia l acid s
about th e same time as Burr (78) and rep o rted th a t methyl
araehidonate was approxim ately tw ice as potent as lin o le a te
in stim u latin g th e growth of depleted r a ts , while lin o le a te
appeared somewhat su p erio r f o r th e r e l i e f of sk in symptoms.
In v e stig a tio n s in th is lab o ra to ry in d ic a te the
in f e r io r c u ra tiv e p ro p e rtie s o f lin o le n a te , w ith somewhat
in f e r io r growth p ro p e rtie s as compared to lin o le a te ;
although, a t a le v e l of 10 mg. each in a m ixture, a d d itiv e
22
growth, e ffe c ts were noted fo r lin o le a te and lin o le n a te
i
combined (80). Furtherm ore, i t was found th a t araehidonate
was approzim ately th re e tim es as a c tiv e fo r growth as
lin o le a te (81).
In a re cen t study of the th ree most common e s s e n tia l
a c id s , Quackenbush and h is co lla b o ra to rs (16) found th a t
e th y l lin o le n a te had n o t cured th e scaly condition of r a ts
a f te r 7 weeks, even a t high dosages, while eth y l lin o le a te
and e th y l araehidonate had re lie v e d th is syndrome in 5
weeks. In a study of the e ffe c t of th ese ac id s on la c ta
tio n , i t was found th a t when lin o le a te and araehidonate
were added to the f a t- f r e e d ie t production of normal young
took place w hile eth y l lin o le n a te did not perm it the main
tenance of normal young by th e r a t. F at analyses of th e
r a ts on the th re e acid supplements revealed a remarkable
constancy both in percentage of to ta l f a t and in the
iodine values of body f a t irre s p e c tiv e o f th e d ie ta ry
supplement.
A fa c to r which must be considered in a study of
th e e s s e n tia l f a tty ac id requirem ents of the animal
organism i s th e sy n th esis of a compound or compounds
possessing growth or c u ra tiv e a c tiv ity w ithin th e organ
ism. Data have been presented by sev eral in v e s tig a to rs
23
which suggest th a t arachidonic acid can he synthesized by
the growing r a t . E ckstein (82) in 1929, and Spadola and
' E llis (83) in 1956, were th e f i r s t to p resen t such d ata.
i
| S in c la ir (29,84,85) has rep eated ly s ta te d th a t the r a t can
synthesize the e s s e n tia l f a tty a c id s, and in fa c t " a ll the
f a tty ac id s u su ally found in animal tis s u e " .
Evans and Lepkovsky (8), rep o rted th a t, i f r a ts had
access to th e ir fe c e s, sc a lin e ss did not develop although
growth was ju s t as se rio u sly re ta rd e d . Since ex tracted
y east was used, i t i s p o ssib le th a t th i s e ffe c t could have
been due to synthesized B vitam ins, to normal m etabolic
f a t , or to synthesized f a t . In t h i s case, f a ilu r e of
growth appears to elim inate th e p ro b a b ility th a t the e ffe c t
was due to e s s e n tia l a c id s. S in c la ir (86) s ta te s th a t
o liv e o il in the d ie t of experim ental r a ts stim u lates the
production o f arachidonic and lin o le ic a c id , but h is
f a ilu r e to feed a h ig h ly -p u rifie d d ie t, and h is f a ilu re
a t th a t time to recognize th e te n a c ity of the body fo r
u nsaturated acid s (87) in v a lid a te h is in te rp re ta tio n of
th e d a ta .
In 1938, Nunn and Smedley-MacLean (88), in an
extensive study of t h i s problem, concluded th a t th e absence
of any CgQ or Ggg a c id s w ith four o r more un satu rated bonds
24
in r a ts fed f a t- f r e e d ie ts suggests th a t un less lin o le io
or lin o le n ic (or arachidonic) aoid is given, th e r a t i s
unable to sy nthesize th e se a c id s " e s s e n tia l fo r continued
ex isten ce o f th e anim al". I t was fu rth e r p o stu lated th a t,
b esid es any d ir e c t e ffe c t lin o le ic o r lin o le n ic a c id s may
have on the sk in , t h e i r main fu n ctio n i s t o supply m a te ria l
from which h ig h er and more unsaturated acid s may be syn
th e siz e d . An in cre ase in arachidonic acid in r a ts re c e iv
ing methyl lin o le a te in d ic a te s sy n th esis from the lin o le
a te , w hile ra ts ,g iv e n methyl lin o le n a te show In creases in
arachidonic and docosapentaenoic a c id s; trie n o ic acid was
n o t d etected in any case.
A re p o rt from th e M assachusetts I n s titu te o f
Technology (89) s ta te s th a t analyses o f f a tty ac id s from
r a t s fed d e u te rio -is o -o le in suggest th a t arachidonic acid
i s synthesized by th e r a t and th a t t h i s hypothesis i s
supported by the presence of deuterium in th e arachidonic
acid m olecule. R ecently B arki e t a l . (72) found th a t the
skin syndrome o f fa t-d e fic ie n c y sometimes disappeared
spontaneously from r a ts a f te r long periods on a f a t- f r e e
d ie t and concluded th a t th e se r a ts could synthesize some
e s s e n tia l f a tty a c id s. An a n a ly s is of the f a ts of
depleted r a t s , which had recovered spontaneously, in d icated
£5
a lim ite d sy n th esis o f e s s e n tia l ac id s in the mature r a t ,
w hile in another experiment (90) proof was o ffered th a t
arachidonic acid was synthesized from in g ested d i- and
trie n o ic a c id s. These r e s u lts were in complete agreement
w ith those of R ieckehoff, Holman, and B urr (91), who found
th a t th e trie n o ic acid content of th e h ea rt tis s u e o f the
r a t decreased ra p id ly upon supplem entation of the r a t with
corn o i l (lin o le ic acid) or c o d -liv e r o i l (penta- and
hexaenoic a c id s ). I t was found th a t th e d ep o sitio n of the
polyethenoid f a tty a c id s took place p rim arily in the
phospholipid f a tty a c id s , and th a t very l i t t l e change
occurred in the n e u tra l f a t .
C ontrary to the fin d in g s of th e p rev io u sly quoted
in v e s tig a to rs , Bernhard and Sohoenheimer (93), Bernhard
(95), and Bernhard e t a l . (94), using the deuterium tech
nique, showed th a t lin o le ic and lin o le n ic ae id s ©ould
not be synthesized. B ra tz le r and h is co-workers (95)
could fin d no evidence of th e sy n th esis of lin o le io acid
in swine, nor could Hansen and Wiese (96) dem onstrate a
sy n th esis in th e dog. On th e b asis of a study o f the
u n satu rated f a tty acid s of the eggs of hens on f a t- f r e e
d ie ts , R eiser and Gibson (25) concluded th a t hens could
not synthesize polyunsaturated f a tty acid s from n o n -fat
26
precu rso rs but th a t they could synthesize a c id s having
i
3, 4, and 5 (but not 6) double bonds from lin o le ic a c id .
In a la t e r re p o rt (97) th e se in v e s tig a to rs studied carcass
and organ lip id s of chicks on f a t- f r e e d ie ts and found
fu rth e r confirm ation th a t chicks cannot synthesize lin o le ic
o r lin o le n ic a c id s , but th a t they can produce higher poly
u n satu rated acid s from th e se . I t was found th a t in th e
chick on a f a t- f r e e d ie t, as in the r a t, th e organ lip id s
of th e growing chick r e ta in th e higher polyunsaturated
f a tty acid s more ten acio u sly than th e carcass lip id s , and
phospholipids more av id ly than n e u tra l f a t. Dienoie acid
was converted to t e t r a - and pentaenoie a o id s, while
trie n o ic ac id was converted to d i - , t e t r a - , p en ta-, and
hexaenoic a c id s . R eiser e t a l . (98) reported an ad d itio n
a l observation concerning th e in terco n v ersio n of th e poly
u n satu rated a c id s. I t was found th a t, in th e case of the
hen on a f a t- f r e e ra tio n , trie n o ic aeid makes i t s appear
ance in the phospholipids as th e o th er polyunsaturated
acid s d ecrease.
K arrer and Koenig (99) used various acid s as sup
plem ents to a f a t- f r e e d ie t, which might have been ex
pected to provide e s s e n tia l f a tty acid s as in term ed iates
during the process of m etabolism. N either A 1 G , 1 S -
27
nonadeoadienoic a c id , which should y ie ld lin o le ic acid by
th e lo s s of one carbon atom, nor / \ ^ *^ -e ic o s a d ie n o ic
a c id , which should y ie ld lin o le ic acid by oxid atio n ,
showed any a c tiv ity when fed to f a t- d e f ic ie n t r a t s .
The mechanism of a c tio n of th e e s s e n tia l f a tty
a c id s i s an in trig u in g su b ject and i s in essence the
problem f o r which a l l o th er stu d ie s in th is fie ld a re but
stepping sto n es. A number o f th e o rie s are advanced, and
some experim ental evidence has been presented to substan
t i a t e th ese th e o rie s . S in c la ir did an extensive s e rie s
of in v e stig a tio n s of fa t-d e fic ie n c y and i t s in flu en ce on
the phospholipid fra c tio n of the body lip id s of th e r a t .
He showed th a t the com position of tis s u e phospholipids
was markedly influenced by th e d ie ta ry f a t (100) and th a t
the phospholipid f a tty a c id s m aintained a much higher
degree of u n satu ratio n (Iodine number of 100) than did
th e n e u tra l f a t f a tty ac id s (Iodine number of 60) (101).
He noted fu rth e r (84) th a t very sm all amounts of u n satu r
ated f a ts had a marked e ffe c t upon th e le v e l ©f u n satu r
a tio n of the phospholipids but had no dem onstrable e f fe c t
on th a t of th e depot f a t . These tis s u e phospholipids
m aintained th e ir u n satu rated f a tt y acid s d e sp ite prolonged
fa s tin g o r th e feeding of a d ie t rie h in f u lly sa tu ra te d
ze
f a t (hydrogenated coconut o il) (102).
However, S in c la ir (29) l a t e r showed th a t th e re q u ire
m ent of th e e s s e n tia l ac id s i s g re a te r on a f a t- f r e e d ie t.
At th is time he sta te d th a t th e e s s e n tia l f a t t y ac id s are
im portant fo r f a t sto rag e . This view i s in confirm ation
of an e a r lie r statem ent o f Nunn and Smedley-MacLean (88)
th a t th e h ighly un satu rated a c id s may play some ro le in
the sto rag e of f a t, sin ce th e re is evidence th a t f a t may
s t i l l be synthesized from carbohydrates, but th a t on f a t-
fre e d ie ts the anim al i s unable to s to re t h i s f a t . Heves3r
and Smedley-Macl.ean (28), in stu d ie s w ith ra d io a c tiv e
phosphorus, concluded th a t phospholipids were being synthe-
f
sized as a c tiv e ly in r a ts w ith long-standing d e fic ie n c ie s
as in normal r a t s . S tudies of the re s p ira to ry q u o tien ts
(103,104) of r a ts on f a t- f r e e d ie ts have supplied addi
tio n a l evidence th a t f a t i s synthesized on a f a t- f r e e
d ie t w ithout the a d d itio n of d ie ta ry e s s e n tia l f a tty
a c id s. In recen t re p o rts , V io llie r (105) and (Jsehaedler
and V io llie r (106) have concluded th a t the e s s e n tia l
f a tty ac id s promote the absorption as w ell as th e deposi
tio n of f a ts in th e r a t . Barnes e t a l . (107) found th a t
the in co rp o ratio n of lab eled ac id in to liv e r phospholipids
is the same fo r r a ts ra ise d on a f a t- d e f ic ie n t d ie t as had
29
been observed f o r r a ts on a normal d ie t; they th e re fo re
concluded th a t a severe d eficien cy of e s s e n tia l ac id s does
not prevent m o b ilizatio n o f ingested f a tty a c id s.
B esides some d ire c t e ffe c t on the sk in , an im portant
fu n ctio n of the more commonly found lin o le ic and lin o le n ic
ac id s i s to supply m a te ria l from which higher and more
unsaturated a c id s may be syn th esized . Indeed, i t was the
opinion of both Turpeinen (32) and of Smedley-Maclean
and Nunn (108) th a t th e prim ary need of the body i s fo r
arachidonic a c id .
The r e s u lts of Smedley-MacLean and Hume (109,110)
in d ic a te th a t a major fu n ctio n of the acid s th a t r e s u lt
in growth i s f o r the form ation o f new tis s u e . Smedley-
MacLean and Nunn (110) reported th a t th e Walker tumor
tra n sp la n te d to r a ts gave re s u lts th a t in d ic a te d a higher
than normal requirem ent f o r e s s e n tia l f a tty a c id s.
Attem pts by Mead (111) to dem onstrate e ffe c ts of f a t-
d eficien cy on tumor growth were n eg a tiv e.
Wiese and Hansen (112) have reported th a t in dogs
th e c h o le ste ro l e s te r ac id s carry th e h ig h est percentage
of u n satu rated f a tty a c id s in the serum. Hess and
V io llie r (113) found th a t the lip a s e a c tiv ity of th e
plasma of r a t s on a f a t- f r e e d ie t dim inishes to
____
so
approxim ately 50 per cent o f th e normal le v e l. The
ad m in istratio n of sm all amounts o f sunflow er seed o i l
re sto re d th e normal plasma lip a s e a c tiv ity .
A recen t study o f c e rta in liv e r enzyme systems by
Kunkel and W illiams (114) has thrown some new lig h t on
th e p o ssib le biochem ical fu n ctio n of th e e s s e n tia l f a tty
a c id s. Succinic oxidase a c tiv ity did not appear to be
a lte re d by any d ie ta ry p retreatm en ts of the animal (deple
tio n of f a t , a d m in istratio n of methyl lin o le a te , o r of
corn o i l ) . The a c tiv ity of cytochrome oxidase was markedly
increased in fa t-d e fic ie n c y ; methyl lin o le a te , which did
not a ffe c t the o th er enzymes stu d ied , did m aintain the
normal le v e l of a c tiv ity of th is enzyme. The increased
a c tiv ity of liv e r cytochrome oxidase appears to p a r a lle l
th e increased m etabolic r a te (50,104) in f a t d eficien cy ,
and may account in a la rg e p a rt fo r th e increased meta
b o lic ra te of d e fic ie n t anim als. Choline oxidase and
endogenous re s p ira tio n appear to be reg u lated by th e
d ie ta ry in clu sio n of a f a t but n o t by methyl lin o le a te .
The data suggest to th e in v e s tig a to rs (114) th a t c e rta in
of the re sp ira to ry enzymes may be lip o p ro te in s . Of
p a r tic u la r importance in th e study of e s s e n tia l f a t t y acid
a c tiv ity i s the fin d in g th a t c e rta in enzyme systems may be
31
a lte re d by f a ts in th e d ie t but a re not a ffe c te d by
lin o le a te alo n e.
S toerk (115) has desoribed symptoms of acute p y ri-
doxine d eficien cy in the r a t , brought about by th e use of
• *
desoxypyridoxine, th a t a re id e n tic a l w ith those obtained
on a f a t- d e f ic ie n t d ie t. He has found th a t the form ation
of antibody p ro te in i s im paired in t h i s d efic ie n cy . O n
autopsy, B g -d eficien t mice showed f a tt y i n f i l t r a t i o n of
th e liv e r s . Thus, not only does th e vitam in d eficien cy
r e s u lt, as does fa t-d e fic ie n o y , in d e rm a titis , t a i l
n e c ro s is , and weight lo s s , b u t a ls o in distu rb an ces of
p ro te in metabolism and in the appearance o f f a tt y liv e r s .
Some of the experim ental work to be presented can
perhaps b e tte r be in te rp re te d in th e lig h t of data p re
sented in t h i s ra th e r extensive review of the li te r a t u r e
r e la tiv e to th e b io lo g ic a l a c t i v i t i e s of the e s s e n tia l
f a tt y a c id s.
CH A PTER I I I
BUT A N D M ATERIALS
The com positions of th e d ie ts used in th e e s s e n tia l
f a tt y aeid experim ents a re given in Tables I and I I .
These f a t- f r e e d ie ts a re alm ost id e n tic a l w ith th a t used
by B urr and Burr (R) in th e ir o rig in a l experiment on f a t
d efic ie n cy . The o rig in a l Burr d ie t contained ex tracted
y e a st as a source of th e w ater-so lu b le vitam ins; however,
th e use of ex tracted y east as a source of th e B vitam ins
was c r itic iz e d by Quackenbush e t a l . (16), since i t was
p o ssib le in h is lab o ra to ry to dem onstrate the presence of
a considerable amount of u n ex tractab le lip id s in y e a st.
Casein e x tra c tio n . S everal methods fo r th e
e x tra c tio n of f a t from th e casein to be used were tr ie d
in an e f fo rt to hasten th e ra te of f a t d ep le tio n of the
anim als to be te s te d . Five d iffe re n t casein p rep aratio n s
were used in the p relim inary experiment (d ie ts 69-69d)
to determ ine by w eight-gain responses which of th e various
methods employed fo r e x tra c tio n of th e f a t would give the
b est r e s u lts .
D iet 69 contained v ita m in -te st casein as i t was
received from General Biochem icals, Inc.
T A B L E I
Composition of the Diets Employed in the Study
of Casein Extraction Methods and in Experiments with Fat-Depleted Rats
Dietary component
Fat-free dieifcs. Control diets
69 6jte 69f 70 70a 70b
I &
%
t
%
i
Casein, vitamin-test ^ 20.00 19.99 19.99 20.00 27.80 27.7 9
Sucrose 71*67 71.63 71.30 61.67 31.74 31.72
Cottonseed o il ^ — « . —
10.00 30.00 29.98
Cellulose ^ 4.oo 4.00
4.oo 4.00 $.00 $.00
Salt mixture ^ 4.00 4.oo 4.oo 4.oo $.00 $.00
Water-soluble vitamin mixture ^
0.33 0.33
0.66
0.33 0.46 0.46
Thyroid powder, whole, desiccated
— 0.0$ 0.0$ —
—
0.0$
Fat-soluble vitamins
— — -- — — —
34
F O O T N O T E S FOE TA BLE I
General B iochem ieals, In c.
-^Wesson o i l .
5 /c e llu flo u r obtained from th e Chicago D ie te tic Supply
House.
•^/©sbome-Mendel s a lt m ixture (116).
The w ater-so lu b le vitam in m ixture had th e follow ing
percentage com position: choline and in o s ito l, each 36.70;
para-am inobenzoic a c id , 18.35; thiam ine ch lo rid e hydro -
o h lo rid e, 2.20; calcium panto th en ate, 2.06; n ic o tin ic
a c id , 1.83; rib o fla v in and pyridoxins hydrochloride, each
0.85; f o lic a c id , 0.51; 2-m ethylnaphthoquinone, 0.15;
and b io tin , 0 .0 2 .
^A rm our, U .S.P. (C ontrol number F12203)•
7 /
- 'F a t- s o lu b le vitam ins were adm inistered in 95 per cent
e th y l alcohol and were adm inistered o ra lly 5 tim es
weekly. The d a ily dosages on the d ep letio n study with
various e x tra cted casein samples and fo r th e, growth
hormone experim ent were: vitam in A, 21 U.S.P. u n its ;
vitam in Dg, 2 .1 U .S.P. u n its (Nopoo f is h concentrate
containing 800,000 u n its of vitam in A and 80,000 u n its
of vitam in D per gram ), and alpha-tocopherol (Merck),
0.11 mg. ^
The d a ily dosage on a l l o th er experim ents except fo r the
m ineral o i l t e s t were: vitam in A, 50 u n its , vitam in Dg,
5 u n its , and alp h a-to co p h ero l, 0.5 mg. In th ese exper
im ents th e supplements were given o ra lly tw ice weekly,
and th e vitam ins eame from the same sources as fo r
e a r lie r experim ents.
T A B L E IX
Composition of the Diets Employed in the Study
of Mineral Oil and of Chorionic Gonadotropic H orm one
on Fat-Deficient Bats
.Dietary component
Fat-free
diet
Mineral o il
diet
6?g 70c
&
%
Casein, vitamin-test &
20*00 20.00
Sucrose \
71.67 6^.17
Mineral o il ^ -----
7»$0
Cellulose &
U.oo iuO O :
Salt mixture &/ 1 * * 0 0 IuO O
Water-soluble vitamin mixture
0.33 0.33
Fat-soluble vitamins ^ — —
56
F O O T N O T E S F O R TABLE I I
1 /
; General B iochem icals, In c ,
! ^ S q u ib b s m ineral o i l , heavy C a lifo rn ia liq u id petroleum .
^ C e llu f lo u r obtained from the Chicago D ie te tic Supply
House.
^O sborae-M endel s a lt m ixture (116).
^ T h e -w ater-soluble vitam in m ixture had th e follow ing
percentage com position: thiam in ch lo rid e hydrochloride
and rib o fla v in , each 3.34; pyridoxine hydrochloride,
1.25; calcium pan to th en ate, 3.12; n ic o tin ic a c id , 0.46;
in o s ito l, 23.2; para-am inobenzoic a c id , 9.27; fo lic
a c id , 0.46; b io tin , 0.05; and choline ch lo rid e, 55.51.
^ I n the case o f th e experiment w ith m ineral o i l (Croups
37-42), each o f th e follow ing fa t-s o lu b le vitam in sup
plements was adm inistered on th e same day, once weekly
as in d ic a te d : Vitamin K so lu tio n in je c te d in volume o f
0.G5 m l. of propylene g ly co l. The so lu tio n contained
10.5 mg. of 2-m ethyl, 1,4-naphtoquinone p er m l. and was
given subcutaneously. Vitamins A, D, and E were given
in tra p e rito n e a lly to each r a t onee weekly In 0.05 ml.
of e th y l la u ra te . The so lu tio n contained 1929 micrograms
of vitam in A a lc o h o l, 16 micrograms of c a lc if e r o l, and
56 mg. of alpha-tocopherol per m l.
In th e case of th e chorionic gonadotropin experim ent,
the fa t-s o lu b le v itam ins were adm inistered o ra lly tw ice
weekly dissolved in propylene g ly c o l. The d a ily dosage
of vitam in A was 45 U .S.P. u n its , of vitam in Do was
4.5 U .S.P. u n its , and of alp ha-tocopherol was 0 .8 mg.
37
D iet 69a contained tlie v ita m in -te st casein which
Md "been subjected to continuous e x tra c tio n fo r 7 days in
a 1500 ml. Soxhlet e x tra c to r -with a l s l m ixture of 95 per
cent e th y l alcohol and d ie th y l e th e r.
D iet 69b contained v ita m in -te st casein which had
been tre a te d as follow s: 6 batches of 110 grams each of
th e v ita m in -te st casein were washed tw ice with d i s t i l l e d
w ater (24 hours each) and th en were shaken fo r about 8
hours with 1300 ml. of 0.085 N NaOH (4-1 ml. to lu e n e ),
which dissolved th e ca sein . The 6 batches were f i lt e r e d
through g la ss wool in to a 1 2 - lite r b a tte ry j a r . A 1:1
m ixture of 1 N HC1 and 1 N a c e tic acid was added slowly
w ith constant s tir r in g u n til the is o e le c tr ic p o in t (4.7)
was reached. About 1400 ml. o f acid was used and th e tim e
of ad d itio n was about 4 hours. The re p re c ip ita te d casein
was tra n s fe rre d to a 5 -g allo n b o ttle and was washed w ith
w ater u n t i l fre e o f 01 as te s te d w ith AgNGg. The p re c ip i
ta te was then f i lt e r e d through a Bdehner f unnel w ith the
aid of su ctio n . Alcohol (95 per cent) was added to the
casein to aid in th e removal of w ater. A fte r drain in g
o ff the surplus water and a lc o h o l* the casein was d ried
a t room tem perature and was used without fu rth e r ex trac
tio n .
38
D iet 69c contained casein re p re o ip ita te d in ttie
same manner as in 691) t u t with e x tra c tio n in th e Soxhlet
e x tra c to r follow ing th e p re c ip ita tio n . The casein wa.s
ex tra cted f o r 5 days w ith a 1:1 m ixture of eth y l alcohol
and d ie th y l e th e r, a f te r which th e casein was freed of
so lv en ts fcy a i r drying. F in a lly , th e d ried casein was
ground fin e in a coffee g rin d e r before in co rp o ratio n in to
th e d ie t.
D iet 69d contained casein re p re o ip ita te d and
ex tra cted as in 69b and 69c exeept th a t the solvent
e x tra c tio n was continued fo r 7 days.
R a ts. The r a ts used were alb in o w eanlings obtained
from the U n iv ersity of Southern C a lifo rn ia colony. The
m others were kept on a lo w -fat (Sherman) d ie t from the
time o f m ating u n til th e weaning of the young. The wean
lin g s (21 days of age) were assigned to th e various exper
im ental groups. I f th e anim als were to be assigned
d ir e c tly to experim ental groups, the r a ts were assigned
so th a t the groups were balanced as regards litte r m a te s ,
sexes, and w eights. However, when th e anim als were
depleted befo re being assigned to groups, th e balancing
was done a t th e tim e of forming groups from th e depleted
anim als. A ll experim ental anim als were housed in
39
in d iv id u a l, ra is e d , screen-bottom cages, and were supplied
■with, th e b asal d ie t and w ater ad lib itu m .
Methyl lin o le a te . A ll samples of methyl lin o le a te
used were obtained from th e Hormel Foundation, A ustin,
M innesota. The samples were prepared from com or cotto n
seed o ils by sa p o n ific a tio n and brom ination of th e m ixture
of f a tt y a c id s . The tetra b ro m ste a ric a c id obtained was
p u rifie d by repeated c r y s ta lliz a tio n s . The p u rifie d t e t r a
brom stearic acid was then debrominated and e s te r if ie d w ith
m ethanol. The methyl e s te r s , a f te r washing and drying,
were d i s t il l e d a t 1 mm. o f p ressu re. The th e o re tic a l
value is 172.4. The io d in e values reported (Wijs method)
fo r th re e d iffe re n t samples used are lis te d below. The
conjugated polyunsaturated c o n stitu e n ts (from u ltr a v io le t
ab so rp tio n data) expressed as per cent methyl e s te rs of
C 1Q f a tty ac id s are a lso re p o rted .
Lot Iodine M enoio T rienoic T etraenoic
No. value
% $
1 172.4 0.17 0.001 tra c e
2 172.7 0.025 tra c e none
6 172.6 0.04 none none
Methyl lin o le n a te . Obtained from th e Hormel
Foundation, A ustin, M innesota. The hexabrom stearic,acid
obtained by brom ination of th e f a tty acid s of lin seed o i l ,
was tre a te d in th e same manner as was the tetrab ro m stearic
aeid used in preparing methyl lin o le a te . The sample used
had a rep o rted iodine value (Wijs) of 261.4 (th e o re tic a l
value 260.4). The conjugated c o n stitu e n ts (from u ltr a
v io le t absorption data) expressed as p er cent methyl e s te rs
of f a tty acid s were reported a s: d ie n o ic , not more
than 0.05 per cent; trie n o ie , tra c e ; and te tra e n o ic , tra c e .
Methyl arach id o n ate. The methyl arachidonate was
obtained from P ro fesso r J . B. Brown of Ohio S ta te Univer
s ity . The acid was prepared by brom ination of ac id s of
th e suprarenal phosphatides, p u rific a tio n , debrom ination,
and e s te r if ic a tio n . The iodine number was rep o rted to be
301.0 (th e o re tic a l 3 18.8). The m olecular weight was
rep o rted to be 319.0 (th e o re tic a l 318.5).
L in o leic a c id . Obtained from the Hormel foundation.
The a c id , prepared in th e same manner as th e e ste r,h a d an
iodine value (Wijs) of 180.48 (th e o re tic a l 181.03). The
conjugated c o n stitu e n ts (from u ltr a v io le t absorption data)
expressed as percentage of C^g f a tty a c id s were reported
as follow s: d ien o ic, 0.197; trie n o ie , 0.004; and t e t r a
enoic, 0.003 per cen t.
E thyl la u r a te . Obtained from the Eastman Kodak
Company.
41
Growth hormone. Supplied by I . M. Bunding of
Armour and Company. The m aterial was in th e form of
w hite c r y s ta ls . The co n tro l number was 3PKR3 and was
assayed by Armour and Company to have such a potency
th a t §0 m icrogram s/rat/day was s u ffic ie n t fo r a 20-gram
weight in crease over normal in 15 days.
Thyroid powder. Whole, d esiccated , th y ro id powder,
U .S.P. was obtained from Armour and Company.
C H APTER IV
EXPERIMENTAL RESULTS
F a t D ep letio n Of R ats By C la s s ie a l Method
A summary o f grow th r a te s of r a t s , male and
fem ale, on d i e t s p rep ared w ith th e v a rio u s c a se in sam ples
d e sc rib e d in C hapter I I I , i s g iv en in T able I I I . D ata
from Table I I I a r e p re se n te d g ra p h ic a lly in F ig u re s 1 and
2 . R ats fe d a c o n tro l d ie t (D iet 70) were ru n co n c u rren tly
to determ ine th e com pleteness o f th e d ie t a f t e r th e f a t
had been added. A summary o f th e food consum ption d a ta
f o r r a t s on th e d e p le tio n d i e t s and c o n tro l d ie t a re p re
sen ted in T able IV f o r th e f i r s t 10 weeks of th e e x p e ri
m ent.
The symptoms noted were s im ila r to th o se d e sc rib e d
by B urr and B urr (7). The f i r s t and most obvious symptom
noted i s c e s s a tio n of normal grow th; a f t e r a few weeks
th e f e e t begin to c ra c k , e s p e c ia lly th e pads of the hind
f e e t ; s c a lin g of th e t a i l becomes e v id e n t, and in some
c a s e s , th e t a i l ap p ears to become rin g e d and may become
n e c r o tic ; in advanced sta g e s o f d e fic ie n c y , s c a le s
(d a n d ru ff) may be found on th e back; reddening of th e
ey e s, ra is e d inflam ed a re a s covered by c r u s ts around th e
fa c e and head, and some p riap ism may o ccu r; in a few
T A B L E III
Growth of Rats on Fat-Free Diets with Casein Prepared by Various M eans
and on a Control Diet Containing 10 Per Cgnt of Cottonseed O il
Diet
No.
N u m ber
of
rats
Weight
at
21 days
. Body weight at end of following weeks
1 ! 2 3 h 5
6
7
8
9 10 11 12
Male rats
69
69a
69b
69c
69d
70
5
5
5
5
5
39
1 * 0
39
1 * 0
1 * 0
gm.
59
60
63
62
61
70
87
88
89
87
98
gm.
105
112
1 1 1 *
106
111
U9
gm .
122
133
136
130
132
151
gm .
138
15k
155
1 1 * 8
350
187
gm.
1 1 * 7
170
163
163
167
2 3 1 *
181
178
171
179
236
SB-
161
190
181
177
192
250
IS*
167
196
188
1 8 1 *
198
272
m •
171
206
192
192
206
289
is*
166
20 1*
193
192
205
305
IS*
196
185
180
202
3 0 1 *
t
Female rats
69 5 1 * 0
55 80 99 1 1 1 * 128 138 1 1 * 9 356 162 166 170 1 6 1 *
69a
5
ho 59 78 96 115 128
139 1 1 * 1 * 152 158 163 163
162
69b
5 la 59 80 96 113 125
133 1 1 * 0 1 1 * 7 351 3 5 1 *
158 352
69c
5
hO 57 80 103 115 129 iia 1 1 * 3 1 5 1 * 1 6 1 * 166 168
165
69d
5 39 59 8 1 * 103 113 131 138 1 1 * 1 * 153 151 359 362 152
70
5 1 * 0 63 88 i n 132 1 1 * 8 161 172 181 190 195 2 0 1 * 207
■ F X -
U »
T A B I E IV
Food Consumption of Bats on Fat-Free Diets with Casein Prepared by Various M sans
and on a Control Diet Containing 10 Per Cent of Cottonseed O il
Diet
No.
N u m b er
of
rats
Food weight at end of following weeks
Total
1
2
3 u 3 6 7
8 9 10
t
Male rats
69
69a
69b
69c
69d
70
5
5
5
5
$
Wu8
Ii6.lt
1 * 1 .0
51.1*
51.8
1*7.6
gm.
67.0
69.2
70.6
71.1*
70.0
77.0
gm.
7l*.6
87.6
83.1*
89.1*
80.2
76.0
gm.
77.0
81*,6
81.0
85.8
85.1*
81.8
gm .
86.1*
90.1*
85.2
91.0
89.0
9i*.2
ss*
81.2
9l*.0
92.0
89.8
91.0
100.8
gm .
8 1 * * 1 *
96.6
9 1 * . 0
101,0
91.6
96.0
gm .
86.1*
98.6
96.0
87.1*
98.6
9 1 * . 0
g£»
83.8
95.6
92.0
89.2
90.2
100.2,
©a.
79.1*
96.1*
93.2
82.1*
92.1*
99.8
am.
set9
765
859
828
839
8 1 * 0
867
Female rats
69 5
3U.8 '61i.lt 73.2 76.8 85.2 87.2 85.0 83.1*
82.1* 77.0 7U 9
69a 1*1.6 59.2
73.1* 79.1* 81.3 83.5 88.3 85.7
80.0
76.5 7 1 * 9
69b 5 1*9.8 70.8 81.8 76.0 91*.8 81.8 81.8 88.0 81.1* * 88,0
7 9 1 *
69c
5 1*7.0 62.6 7lt.6 76.0 78.2 77.8
79.1*
78.6 76.0. 75.2 725
69d 1 * 1 .1 * 61.1* 78.0 7U.8 86.6 8 1 * . 8 82.1* 83.8 86.8; 88,1* 768
70 5 1 * 7 .1 * 62.0 7l*.6 73.2 70.6 77.0 75.2 79.1* 73.6 72.6 706
4 5
2 6 0 M A L E S
240
10% COTTONSEED OIL
220
200
180
c o
3E
<
c c
©
I
160
140
I-
X
©
1 0 0
• VITAMIN TEST CASEIN
O P P T + E X T 7 DAYS
■ P P T + EXT 3 DAYS
A EXT 7 DAY
▲ P P T ONLY
80
60
40
WEEKS
Figure 1. The average w eights of weanling male r a ts
during 12 weeks on f a t- f r e e d ie ts containing casein
ex tra c te d by v arious procedures, and of a group of r a ts
receiv in g a s im ila r d ie t which contained 10 per cent of
cottonseed o i l .
46
220
F E M A L E S
200
10% COTTONSEED OIL 180
160
V )
2
<
K
O
140
120 t-
I
o
100
w
• VITAMIN TEST CASEIN
O P P T + E X T 7 DAYS
■ P P T + E X T 3 DAYS
A EXT 7 DAYS
A P P T ONLY
80
60
fig u re 2. Tli© average w eights of weanling female
r a ts during 12 weeks on f a t- f r e e d ie ts containing
casein ex tra cted by various procedures, and of a group
of r a ts receiv in g a sim ila r d ie t which contained
10 per cent of cottonseed o i l .
47
advanced cases, hem aturia and death i s observed.
No s ig n ific a n t d iffe re n c e s were noted fo r any of
th e d ep letio n groups w ith in the 12-week period req u ired
to d ep lete th e anim als. The low value observed in the
case of th e male r a ts on D iet 69 i s caused by th e in c lu
sion in th e average of th e r e s u lts one r a t th a t fa ile d to
grow. This r a t weighed only 121 grams by the 12th week.
The depleted anim als were assigned w ithout regard to th e
p a r tic u la r d ep letio n d ie t used to th e d iffe re n t subsequent
te s t s on th e f a t- d e f ic ie n t r a ts , and th ese r a ts were used
on the growth hormone experim ent.
Use Of Growth Hormone In D epletion Of R ats
Since growth hormone had been used by E rsh o ff and
Deuel (74) to accentuate d e fic ie n c ie s in d ie t, i t was our
opinion th a t th is hormone might be used to a c c e le ra te th e
appearance of fa t-d e fic ie n c y symptoms. Furtherm ore, th is
experiment was undertaken to determ ine whether f a t con
trib u te s to n u tr itio n o th er than as a souree of the essen
t i a l u n satu rated ac id s and as a re se rv o ir fo r meeting
c a lo ric needs.
The depleted r a ts from the previous d ep letio n
experim ent were assigned in p a r a lle l groups according to
sexes. Group 1 co n sisted of negative c o n tro ls, which
i 48
i !
* ;
j were continued on tlie f a t- f r e e regimen (D iet 69) and j
i received o ra lly 0.1 ml. of eth y l la u ra te , the d ilu e n t fo r j
; th e methyl lin o le a te adm inistered to Groups 5 and 4. The !
i
I
1 o ra l supplement fo r th is group and o th er groups in th i s \
: experiment were given th ric e weekly. The r a ts assigned to I
I i
I Group 2 received th e same b a sa l d ie t and supplements as |
; did those in Group 1 except th a t they were in je c te d in tr a -
| p e rito n e a lly s ix tim es per week w ith 150 X of growth
- hormone disso lv ed in 0.1 ml. of ph y sio lo g ical s a lin e . J
! Group 5 received th e same treatm en t as Group 1, except
: th a t th e o ra l supplement was made to contain 20 mg. of !
methyl lin o le a te per day, made up in e th y l la u ra te so th a t :
0.1 m l. given th re e tim es weekly supplied th e required ;
i amount of lin o le a te . Group 4 received the same supplements!
I i
i l
! as Group 3, except th a t growth hormone was adm inistered in ,
I 1
' the same manner as to Group 2. Group 5 in th i s experiment J
I |
| i s th e p o s itiv e c o n tro l group used in the e a r lie r depletionj
! i
t e s t and continued w ithout in te rru p tio n on th e 10 per cent J
1 f a t d ie t (D iet 70). !
‘ • - i
j A summary of the weight changes fo r the r a ts , both j
i
, male and fem ale, on the growth hormone experiment i s p re-
I
J sented in Table V and is g ra p h ic a lly presented in F igures
j 3 and 4 fo r th e male and female r a t s , re sp e c tiv e ly . J
T A B L E V
Average Body Weight Gain and Survival of Eats Previously on a Fat-Free Diet (Diet 69)
U ntil Depleted and Thereafter Receiving: 1 , N o Supplement; 2, Growth H orm on e Alone;
3, Methyl Linoleate Alone; kt Methyl Linoleate with Growth Horm one; and $s of a Control
G roup of Rats Raised and Continuing on a Similar Diet Containing 10 Per Cent Cottonseed
O IL (Diet 70).
G roup
No.
3 o • of
rats at
start
Average weight gain at end of following weeks
Period 1 Period 2 Period 3 i / V
Start
1
2 6 10 Hi
18 22-0 125-3 126-1 129-1* 32-7
Male rats
\ %
s
5
5
5
6
5
gm.
186
182
1 7 1 *
173
30U
B S *
-2
3
8
13
5
as*
i
7
7
25
13
££•
-12
17
1 4 6
67
30
-10
21 (1 * )
73-
85
1 * 9
IS*
5(1*)
1*1(3)
87-
1 0 1 *
59
Si*
110
11*0(5)
62
128
156
95
1 1 * 0
169
110
as*
157
183
n 5
g * a «
1 6 1 *
207
130
S i-
192
235
138
Female rats
1
6 158 8 10
5 7 1 1 * (5)
2 | / 6 160 8
9 15(5)
16 18 .
3 K /
6
157 9 13 3 1 * 1 * 9 56 6 1 * 70
73
82
95
108
hS/§/ 6 161
13
26
la 70 82 92 98
97 io5
1 2 1 *
135
. 5 5 207 0
3 1 5
32
3U
U 6 58 68 72 86 96
50
F O O T N O T E S FO R TA BLE V
^ T h e fig u re s in parentheses under w eight gains in d ic a te
the number of r a ts remaining a liv e a t th a t period*
The f i r s t fig u re in each column heading gives th e - to ta l
number of weeks on experim ent, while th e second fig u re
gives th e weeks on period 2 or 3.
^G roups 5 and 4 received 60 mg. of methyl lin o le a te
o ra lly d a ily in s te a d .of 20 rag.
^G roups 3 and 4 received th e d ie t containing 10 p er cent
cottonseed o i l during th is period in ad d itio n to Group
5, which receiv ed th e same d ie t throughout. The growth
hormone was stopped a t th e end of th e 25th week due to
in s u f f ic ie n t supply.
5 /
— '150 Y " Growth hormone in je c te d d a ily (6 tim es weekly)
in tr a p e r it o n e a lly •
g /
- '2 0 mg. Methyl lin o le a te supplemented o ra lly each day.
51
no,
26 0
240
10% COTTONSEED OIL
200
« 180
ME LINOLEATE 4- G.
Z 140
ME LINOLEATE 1 2 0
• 80
10%COTTONSEED OIL 6 0 MG I
LINOLEATE
ME LINOLEATE
6 0
4 0
FAT FR E E +G .H .
20
FAT FREE
-20
1 2 1 3 1 4 IS 1 6 1 7 1 8 1 9 20 2 1 22 23 84 2S 26 27 2B 29 30 3 1 32 33
WEEKS
Figure 3. The average gains in weight of male r a ts
p reviously re ceiv in g a f a t- f r e e d ie t u n til d e p le te d ,
when they were continued on the same d ie t alone (1),
w ith th e growth hormone (2 ), w ith 20 m g* of methyl
lin o le a te and l a t e r 60 mg. d a ily (3 ), w ith 20 mg. of
m ethyl lin o le a te and l a t e r 60 mg. d a ily plu s th e growth
hormone (4 ), o r receiv in g a d ie t containing 10 per cent
cottonseed o i l before and throughout the t e s t (5 ).
Groups 3 and 4 received th e cottonseed o i l d ie t during
the l a s t 7 weeks w ithout a d m in istratio n of growth
hormone.
52
t a o
1 4 0 -
130
120
1 1 0
10% COTTONSEED OIL
100-
80
ME LINOLEATE+ G.H.
60
50
ME LINOLEATE
10% COTTONSEED OIL 60 MG
LINOLEATE
4 0 -
30
T FAT FREE + G. H.
/ J ^ 0 - 0 - < r JO " ° " °
I asx P' ' 0" 0’ . *
W fc-—..* ..# .- * . m- FAT FREE
^ I iTT14 " TTiU 4 k 4 krkbikkL
WEEKS
20
22 23 24 25 28 27 28 29 30 3t 32 33
F igure 4. The average gains in weight of female r a ts
prev io u sly re ceiv in g a f a t- f r e e d ie t u n til d ep leted ,
when they were continued on the same d ie t alone (1 ),
w ith the growth hormone {2), with 20 mg. of methyl
lin o le a te and l a t e r 60 mg. d a ily (5 ), with 20 mg. of
methyl lin o le a te and l a t e r 60 mg. d a ily plu s the growth
hormone (4 ), or re ceiv in g a d ie t containing 10 per cen t
cottonseed o i l before and throughout the t e s t (5).
Groups 3 and 4 received the cottonseed o i l d ie t during
the l a s t 7 weeks w ithout adm inistered growth hormone.
53
Food consumption data were c o lle c te d fo r th e 13th
week o f th e experim ent, and the re s u lts a re presented in
Table ¥1.
The negative co n tro l groups (Groups 1 and 2) were
discontinued a f te r 14 weeks. A fte r 22 weeks, when the
e ffe c t of the lin o le a te had la rg e ly worn o ff, th e lin o le a te
supplement was increased from 20 to @ 0 mg. d a ily in
Groups 3 and 4. When th is in crease in lin o le a te in tak e
(23rd-25th week) was shown to r e s u lt in no augm entation
of growth, the anim als were tra n s fe rre d to co n tro l d ie t
70, on which they were continued fo r 7 weeks (26th-32nd
week), a t which tim e th e experiment was term inated.
Because of the exhaustion of th e supply of growth hormone,
in je c tio n s of th e hormone were stopped a t th e end of th e
lin o le a te a d m in istratio n (end of 25th week).
In both, th e negative co n tro l group (Group 1) and
the linoleate-supplem ented group receiving th e growth
hormone (Group 2 ), th e weight gain was g re a te r, and the
appearance of th e r a ts in th e negative group (Group 2)
was b e tte r than fo r the p a r a lle l groups not receiv in g th e
hormone. The male r a ts appeared to be unaffected by the
in cre ase in th e dosage o f methyl lin o le a te a t the end of
th e 22nd week; however, th e fem ale r a ts appeared to have
5 h
T A B L E VI
Average Food Consultation of Rats on a Fat-Free Diet
u n til Depleted and Thereafter Supplemented as Indicated
(The Data Presented are for the 13th W eek o f the Experiment)
Group
No.
Dietary
supplement
.Food eaten daring 13th week ^
or
treatment M ales Females
fig.
• ££•
1 N one 91.5(2) - 7.8 81.8(5) ± 1.9
2
2/
Growth hormone -/ 92.7(3) t 6.7 79.0(U) t 2.6
3
Ifethyl linoleate ^
86*5(1*) t $.h 79.3(6) t 2.9
h
Methyl linoleate ^ .
and growth hormone
n.5(k) ± 5 .1 * lk.5(5) t k.l
5
Control diet ^
86.5(10 1 iuS 72.0(U) t 1.U
Figures in parentheses give number of rats averaged. Includes
standard error of the m ean calculated by the formula
j/£ d^/n-l / l/"n where w dM is the deviation from the m ean
and M nn is the number of observations.
2/.
l£0 Y daily administered intraperitoneally.
I/,
y
20 mg. daily administered orally.
Diet No. 70 containing 10 per cent of cottonseed o il.
55
su ffe red a growth in h ib itio n , during th e same p erio d . The
in c lu sio n of cottonseed o i l in to th e d ie t did not change
th e slope of th e male growth curve ap p reciab ly , but i t
did in cre ase the slope of the growth curve of the female
r a ts to a considerable e x te n t, l a t e r experim ents show
these phenomena to be rep ro d u cib le.
Itffe c t Of Thyroid On F a t D epletion
The f a ilu r e of the growth hormone to accentuate
f a t d eficien cy in th e r a t led to the use of another method
of producing s tr e s s upon th e m etabolic system o f the r a t .
The use of th y ro id to a c c e le ra te the appearance of d e fi
ciency symptoms in experim ental anim als has been reviewed
in a re cen t p u b lic a tio n from th is lab o ra to ry (75).
Six weanling male and 6 weanling female r a ts were
assigned to each of 5 groups. The com position of th e
d ie ts used in t h i s experiment a re recorded in Table I .
Group 6 received the f a t- f r e e b a sa l D iet 69, w hile
Group 7 received th e same d ie t to which was added 0.05
per cent of d esiccated th y ro id powder (D iet 6 9 e). Group
8 received a f a t- f r e e d ie t (Diet 69f) w ith the w ater-
so lu b le vitam in content double th a t received by th e r a ts
on Groups 6 o r 7. A h ig h -fa t b asal d ie t (D iet 70a) was
fed to Group 9, and t h i s same d ie t w ith 0.05 p er cent of
§6
th y ro id powder added (D iet 70b) was given to Group 10.
The recorded w eights of the anim als and th e p er
centage m o rta lity up to 11 weeks a re given in TablesVII
and V III. The standard e rro r of th e mean weight was
determined a t th e end o f the 6th week in stead of a t a
l a t e r p erio d , since a la rg e number of th e anim als in each
group were s t i l l a liv e a t th a t stage of th e experim ent.
Even as e a rly as 6 weeks, a s ig n ific a n t d iffe ren ce ean be
observed between th e male anim als on th e f a t- f r e e d ie t
(Group 6) and th a t containing an added 0.05 per cent
th y ro id powder (Group 7) or th a t containing th e same amount
of th y ro id w ith double the B vitam ins (Group 8 ). There is
t
lik ew ise a s ig n ific a n t d iffe re n c e between Group 7, which
had th y ro id added to the f a t- f r e e d ie t and Group 8, which
had th e same d ie t w ith double the amount of the w ater-
soluble vitam ins.
The m o rta lity record re v e a ls th e same re la tio n s h ip
between th e groups as i s shown by the weight record a t the
6th week. The p o s itiv e c o n tro ls (Groups 9 and 10) show
th a t th e th y ro id a t th e le v e l fed i s n o t to x ic in a normal,
fa t-c o n ta in in g d ie t.
T A B L E V II
M ea n Weights, Percentage M ortality, and Weight Gain of Male Eats on Following Groups:
6, Fat-Lowj 7 , Fat-Low Diet Containing 0,0? Per Cent of Desiccated Thyroid Powder;
8, Fat-Low Diet Containing 0.05 Per Cent of Desiccated Thyroid Powder and
Twice the Water-Soluble Vitamin Content; 7, 30 Per Cent Cottonseed O il Diet;
10, 30 Per Cent Cottonseed (HI Diet Containing 0.05 Per Cent of Desiccated Thyroid.
(6 Animals in Each Group)
Category
Group 6
Diet 69
G roup. 7
Diet 69e
G roup 8
Diet 6?f
Group 9
Diet 70a
G roup 10
Diet 70b
Body weight, gm .
Start
. i
1*1.1 1*3.7 1 * 1 * .0 1*1.8 1*3.8
Weeks on d iet
1 6l*.0 61.8 62.8 70.2
73.7
2 92.7 85.7 85.8 102.8 106.8
3
112.8 10l*.8 102.2
13l*.5 138.0
1 * 132.7 120.5 105.0 159.2
167.7
\ y
133.7 127.5 106.5(1*) 187.9 198.2
173.3 138.7 lllt.O 207.0 216.3
+ it.o
± 2.1* ± 3.1 t il* .9 ±7.5
7 181.8 139.7 116.5 223.2 231*.2
8
187.3 138.0(5) 117.7 21*0.7 251.8
9 190.3 135.2 130.0(3) 251.5
260.8
10
200.5 137.0(1*) 139.5(2) 26i*.2 271.2
11 203.3 11*1.7(3) 33l*.0(l)
271.7 280.2
Total gain in weight, gm. 162.2 98.0 90.0
229.9 236.1*
M ortality, %
0 5° 83 0 0
vn
5©
F O O T N O T E S F O R T A B L E VII
1 /
F igures in parentheses rep resen t number of anim als in
group surviving a t end of week in d ic a te d .
Zj
“ 'In c lu d e s th e s t andard erro r of th e mean calcu lated by
th e form ula l/4d2/n -1 / ! n , where '*dn is the devia
tio n from th e mean and ”n" is the number of observations*
T A B IE VIII
lea n W eights, Percentage M ortality, and Weight Gain o f Female Rats on Following Groups:
6 , Fat-Low j 7 , Pat-Low D iet Containing 0.0$ Per Gent, o f D esiccated Thyroid Powder;
8 , Fat-Low D iet Containing 0.0$ Per Cent o f D esiccated Thyroid Powder and
Twice the W ater-Soluble Vitamin Content; 7 , 30 Per Cent Cottonseed O il D iet;
10, 30 Per Gent Cottonseed O il D iet Containing 0.0$ Per Cent o f D esiccated Thyroid.
(6 Animals in Each Group)
Category
Group 6
Diet 69
G roup .7
Diet 69e
G roup 8
Diet 6?f
Group 9
Diet 70a
G roup 10
Diet 7© b
Body weight, gm .
Start 1*6.3 1*$.3
1*3.8
1 * 1 * .7 1*3.8
Weeks on d iet i /
1
6$.0 61*.0 60.8
69.3
70.2
2
8$.8 81*.$ 7$ .7 9it.8 96.0
3
106.7 103.2 89.$ 122.0 12l*.7
1 * 111*.3
11$. 2 102.$ 137.0
11*0.7
$ 0/ 12U.3 122.1*($)
Hi* .0(1*) 1$1**2 l6 l.$
6 l ! 13U.7
13$.2. . 117.$
166 ,$ 17$. 8
1 7.1t ± k .l tlO .$ ± 3.9 1 7.1*
7 11*2.$ 139*0 110.$ 173.3 1 8 1 * .7
8 11*8.7 137.2 128.$(2) 181.3 190.3
9 1$3.7
126.8 127.$
188.8 191*.8
10 1$7.3 117.1* 119 .$ 19$ .0 201.0
11 161.0 121.0(3) 13$.0(1) 200.7 208.3
Total gain in weight, gm . lli*.7 7$.7 91.2 1$6.0 161*.$
M ortality, % 0 $0 83 0 0
vn.
v o
60
FOOTNOTES FOR TABLE VITI
^ F ig u re s in parentheses rep resen t number of anim als in
group surviving a t end of week in d ic a te d .
^ I n c lu d e s the standard e rro r of the mean ca lc u la te d by
the formula l/£d2/ n - l / J~n , where w dw is th e devia
tio n from th e mean and "n" i s th e number of observations.
61
U nsaturated F a tty Acid Requirement Of th e R at
E stablishm ent o f logarithm ic dose-w eight gain curve
•with lin o le ic a c id . The procedures follow ed in th is
s e rie s of t e s t s i s sim ila r to those follow ed in th e t e s t s
w ith growth hormone. The anim als were continued during
th e experim ental perio d on the same b a sa l d ie t (D iet 69)
as was used in th e d ep le tio n period except fo r the p o sitiv e
c o n tro l groups, one of which was continued on D iet 70a
from weaning.
The dosages of vitam ins A and D were doubled over
th a t used w ith the growth hormone t e s t s , w hile th e amount
of alp h a-to co p h ero l fed was quadrupled. A ll supplements
of substances to be te s te d were fed biw eekly. The t o t a l
volume of each dose was ad ju sted to 0 .1 ml. w ith e th y l
la u ra te w ith th e exception of Group 15, where the t o t a l
dose fed amounted to 0 .4 m l. A ll supplements were admin
is te r e d o ra lly w ith a 0.25 m l. tu b e rc u lin syringe c a l i
b rated to 0.01 m l. having a blunted number 18 needle.
The groups used were as fo llo w s: 11, neg ativ e con
t r o l s (e th y l la u ra te on ly ); 12, 13, 14, and 15, methyl
lin o le a te in d a ily doses of 5, 10, 20, and 50 mg. respec
tiv e ly ; and a p o s itiv e c o n tro l group (30 per cent f a t d ie t)
supplemented w ith 0 .1 m l. of e th y l la u ra te biw eekly. A
62
second co n tro l group (Group 20) received th e h ig h -fa t d ie t
from weaning (minus e th y l la u ra te ) w ithout any d ep letio n
1 period having interv en ed .
In order to show whether th e v a ria tio n s in body
j
weight observed represented tru e growth or weight g ain , a
determ ination of ti b ia len g th was made from x -ray photo
graphs by a m odification of th e procedure described by
Deuel e t a l . (117). This involved measurement of the bone
photograph on a screen with a 20-fold enlargement as
determ ined from the a c tu a l length of a sim ultaneously pro
jected centim eter sc a le . These tib ia e measurements as
w ell as the weight gains of the male and female r a ts w ith
values fo r the body w eights a t th e s t a r t , a t th e end of
d e p le tio n , and a t the conclusion of the te s t s a re given in
Tables IX and X re sp e c tiv e ly . The gains-in-w eight of the
male r a ts receiv in g th e sev eral supplements are shown in
F igure 5, while th e corresponding data on th e female r a t s
a re presented g ra p h ic a lly in F igure 6.
In order to determine whether the re la tio n s h ip
between growth and lin o le a te in tak e is a q u a n tita tiv e
one, th e weight gains have been p lo tte d ag ain st th e
logarithm ic dose of th e lin o le a te fed . F igures 7 and 8
give th e r e s u lts when th e d ata fo r male and female r a ts
T A B IE IX
Body Weights o f Male Eats a t Weaning, A fter a F at-D epletion D iet and Gain in Weight
Following Supplementation w ith L in o lea te, w ith L inoien ate, w ith Equal Amounts of
lin o le a te and L inoienate, w ithout Supplement, R eceiving a 30 Per Gent D iet Throughout
(D iet 7 0 a ), or R eceiving a 30 Per Gent Fat D iet Following D ep letion . (5 Rats Per Group)
D aily supplements Body w eight a t
Gain in body w eight a t end o f
follow in g weeks of supplem entation
{ r T Y Y t l ’ n
Jfethyl
lin o le a te
Methyl
lin o ien a te
End o f
depletion No. Weaning
3
6
9 y
11
2£*
0
mg.
0
m •
1*3.6
m*
181.8
as«
- 0 .8
fig*
1. 1 *
gm.
- 6 .2 1 6.8
12
5 0 1 * 3 .1 *
181*.0 1 2 .0 22.8
1 * 1 .1 * i 6.7
10 0 h k.6 186.6 20.8 37.6
52.0tU *.3
ll*
20 0 1*0.6 183.6 26.8 1*7.6 61*.8 ± 2.5.
15
50 0
1 * 5 .1 *
19l*.6 32.6 6 0 .0 80.1* t 1*.6
16 0 10 1*1.2 181*.8 0.1* 6.2 . 9.6 1 2.7
17 5 5
39.2 192.2 12.6 22.6 3l*.8 ± 3.0
18 10 10 1*2.0 I8i*.0 22.2 50.2 69.0+10.6
19
30$ f a t < 3
a fte r dep
lie t
le t io n
ia .8 183.0 36.0 72.1* 98.1*+ 13.1*
20
30$ f a t d ie t
from weaning
itl.8
271.7 288.1* 3H *.0^ 328.3 ^
T A B IE IX (contd)
G roup
No.
Gain in body weight at end of
following weeks of supplementation
Body weight
at 15 weeks
Tibia * .
length MW
12
15
- g£.
ffl* m *
m m .
11 -10.8 -26.6 355.2 37.07 ± 0.80
12 52.1* 58.0 21*2.0 38.80 i 0.62
13 6 1 * .l* 7 U .1 * 261,0 38.67 t o .5 l
U *
78.1* 85.6 269.2 38.61 i 0.37
15 99.1* 117.8 312.1* 39.82 i 0.71*
16 2.2 7.2 192.0 37. 8 1 * ± o.51t
17
1*0.2 1 * 1 * . 2 231*.0 38.95 t o.86
18 77.0 93.1* 277.1* 38.91* t 0.25
19 U 3.2 127.6 310.6 39.08 t 0.38
20 31* 0 .7 y 3 1 * 2 .8 y 31*2.8
—
Sr
65
FO O TN OTES FOR TA B LE IX
1 /
S tandard erro r of the mean ca lc u la te d by the formula
V t& 2/i*-l / i/n" , where "d" i s the d ev iatio n from the
mean and "n” is th e number of o b serv atio n s.
^ S i x r a ts .
^B ody w eight.
^ D eterm in atio n s made a t end of 18 weeks.
T A B U S X
Body W eights o f Female Rats a t Weaning, A fter a F at-D epletion D iet and Gain in Weight
Following Supplementation w ith lin o le a te , w ith L inolen ate, w ith Equal Amounts o f
L inoleate and L inolen ate, w ithout Supplement, R eceiving a 30 Per Cent D iet Throughout
(D iet 70a), or R eceiving a 30 Per Cent Fat D iet Following D epletion , (5 Rats Per Group)
D aily supplements Body w eight a t
Gain in body w eight a t end of
Group
No.
Methyl
lin o le a te
Methyl
lin o len a te
End o f
dep letion
A V I J L A U n J H i f e Vfeo^ovx 9 Uj p j j e iU S U W O V J k U A A
Weaning 3
6
O s
U
2g*
0
H L -
0
gm.
1*5.2
m *
167.6
as*
1.2
g£.
-1 .0
gm.
-l*.t* ± 5 .2
12
5
0 1*3.6 161.8 5 .2
10.1* 17.1* ± u.o
13
10 0
l£.l*
167.6 11.1* 21*.8 33.6 i 1 *.7
Ik
20 0 1*2.2 162.2 22.5 27.0 36.8 ± 3.2
1$
50 0 1*5.0 161*.6 20.3
2 1 * .2 29.8 ±2.6
16 0 10 1*0.0 173*0 7.1* 8.0 8 .0 ± 5 .0
17 5 5 i*l*.2 162.1* 8.0 17.0 20.0 ±2.1*
18 10 10
1 * 5 .1 *
161*.2 21.8 31.8 38,8 ±5.7
19
30$ f a t d ie t
a fte r d ep letion
1*0.2
169.9
33.6 1*0.6 51.2 ±7.5
20 &
3 0 5 6 f a t d ie t
from weaning
1 * 1 * .7 200.7 215.7 & 223.1* 231.2
O
O s
T A B L E X (contd)
Group
No.
Gain in body w eight a t end o f
follow in g weeks o f supplementation
Body w eight
a t 1$ weeks
T ibia t/ j /
len gth 12 35
ffl*
m m .
11 -7.2 -8.1* 159.2
3 1 * * 7 7 i 0.51*
12 21.6 23 .U
185.2
35.32 t 0.55
13
1*3.0 1 * 9 .1 *
217.0 35.78 t o.l*i
1 1 *
Ii5.8 53.8 216.0 36.00 t o.35
35
38.0 1*6.8 231,1* 35.82 t Q.23
16 6.2 17.2 190.2
35.27 t o.53
17
26.2 30.2 192.6
3l*.9l* t 0.29
18 1*3.6
1 * 5 .5 209.7 35.86 1 0.1*8
19 53.1* 62.1* 232.3 35.96 i 0.33
20 233.0 2 / 231*.2 y 2 3 1 * * 2
68
FO O T N O T E S FO R TA BLE X
^ S t a ndard erro r of th e mean ca lc u la te d by th e formula
l/ld 2/ n - l / ] / ~ ~ n , where "d” i s th e d e v iatio n from the
mean and R n" i s th e number of o b serv atio n s.
^ / Six r a ts .
^B ody w e i^ it.
^D eterm in atio n s made a t end of 18 weeks.
69
1 2 0
100
90
V) 70
60
50
H7
5 4 0
1 K 30
I ca
;
- 1 0
-2 0
WEEKS
F igure 5. Average gains in weight of groups of male
r a ts , p reviously depleted of f a t fo r 11 weeks, when
supplemented over a 15-week period w ith methyl lin o le a te
(12, 5 m g.; 13, 10 m g.; 14, 20 mg.; 15, 50 mg. d a ily ) ,
w ith methyl lin o le n a te (16, 10 mg. d a ily ) , or w ith equal
doses of methyl lin o le a te and methyl lin o le n a te (17,
5 mg. each; 18, 10 mg. e a ch ). The neg ativ e co n tro l group
(11) received no f a t except e th y l la u ra te , w hile the
p o s itiv e co n tro l group (19) received the 30 per cent
f a t d i e t .
70
6 0
50
40
I - 20
- 1 0
WEEKS
fig u re 6. Average gains in weight of groups of female
r a t s , p rev io u sly depleted of f a t f o r 11 weeks, when
supplemented over a 15-week period w ith methyl lin o le a te
(12, 5 mg.; 13, 10 mg.; 14, 20 m g.; 15, 50 mg. d a ily ) ,
w ith methyl lin o le n a te (16, 10 mg; d a ily ) , o r w ith equal
doses of methyl lin o le a te and methyl lin o le n a te (17,
5 mg. eaeh; 18, 10 mg. each ). The negative co n tro l group
(11) received no f a t except e th y l la u r a te , w hile the
p o s itiv e co n tro l group (19) received th e 30 p er cent
f a t d ie t.
71
a re expressed in t h i s manner. For th e sake of sim p lic ity
only the fig u re s fo r re g u la rly spaced periods a re included
in F ig u res 7 and 8. The d ata are eq u ally s a tis fa c to ry §1.
th e case of th e male r a ts fo r a l l th e in terv en in g weeks
4
follow ing the 4th week. The fa c t th a t a l l th e p o in ts f a l l
on a s tr a ig h t lin e in d ic a te th a t th e growth response to
lin o le a te i s a q u a n tita tiv e one fo r d a ily doses in the
range of 5 to 50 mg.
Inasmuch as th e re la tio n s h ip "between gain in "body
weight and lo g . dose of lin o le a te fed i s c le a re s t a t 9
weeks, the standard e rro r of th e mean i s calciliated fo r
th a t p erio d .
Dem onstration o f sex d iffe re n c e s in e s s e n tia l
f a tt y a c id requirem ents of th e r a t . The r e s u lts obtained
w ith lin o le a te d if f e r considerably fo r the male and the
•female r a t . These d iffe re n c e s a re most evident from an
in sp e c tio n of F igures 7 and 8. In F igure 7, the male
r a ts p resen t s tra ig h t lin e logarithm ic dose-w eight gain
curves fo r lin o le a te dosages ranging from 5 to 50 mg. p er
day. However, in F igure 8, th e lo g . dose a p p ra isa l fo r
fem ale r a ts , a s tra ig h t lin e fu n ctio n i s obtained f o r the
th ird week fo r doses of 5, 10, and 20 mg. but not fo r
50 mg. o f lin o le a te per day. This re la tio n s h ip i s main
tain ed through th e 5th week but i s no longer evident a f te r
72
1 2 0
15 WEEKS
1 1 0
100
12 WEEKS
90
80
9 WEEKS
6 WEEKS
40
& 3C
3 WEEKS
20
-20
LOG DOSE - MILLIGRAMS
i.e
F i g u r e 7. T h e gain in weight of p r e v i o u s l y depleted
male r a ts p lo tte d a g a in st th e log dose of methyl
lin o le a te fed f o r 3, 6, 9, 12, and 15 weeks. P o in ts A,
B, C, D, and E re p re se n t th e weight gains of r a ts on a
30 p e r cent f a t d i e t f o r c o r r e s p o n d i n g p e r i o d s , w h i c h
c a n be u s e d f o r d e t e r m i n i n g t h e o p t im u m lin o le a te
r e q u i r e m e n t by e x tra p o la tio n .
7 3
so
50
15 WEEKS
40
12 WEEKS
9 WEEKS
6 WEEKS
2 0 3 WEEKS
LOG D O S E - MILLIGRAMS
i
1
i 0.4 to 0. 2 0.6 0. 8 ( .0
:
fig u re 8. The gain in weight of p reviously depleted
female r a ts p lo tte d a g a in st th e log dose of methyl
lin o le a te fed fo r 3, 6, 9, 12, and 15 weeks. P o in ts A,
B, C, D, and 1 re p re se n t th e weight gains o f r a ts on a
30 per cent f a t d ie t fo r corresponding p erio d s.
74 i
th is p erio d , sin ce the growth e x h ib ited by th e r a ts on ;
! th e 20 mg. dose no longer f a l l s on th e " s tra ig h t lin e "
curve and approxim ates th a t of th e 10 mg. dosage r a t s .
There seems to be an a c tu a l suppression in th e growth of
th e female r a t s w ith th e 50 mg. le v e l of lin o le a te under
th e experim ental cond itio n s employed.
Growth r a te s w ith methyl lin o le n a te and in te r -
1 re la tio n s h ip of lin o le a te and lin o le n a te . When methyl
! j
' lin o le n a te was fed a t a le v e l o f 10 mg. p er day in the
same manner as was m ethyl lin o le a te , p r a c tic a lly no growth
occurred in male o r fem ale r a ts {Group 16 on Tables IX
I
and X re s p e c tiv e ly ). An average t i b i a len g th f o r th e |
J
male r a t s on th e lin o le n a te equal to no more than th a t
; obtained fo r th e n eg ativ e co n tro l group, lends support
to th e n egative weight gain fo r th is group. When 5 mg.
each of m ethyl lin o le a te and methyl lin o le n a te were fed
sim ultaneously d a ily , th e re s u ltin g growth was le s s in
the case of the male r a t s than was th e growth fo r 5 mg.
of lin o le a te alone but was higher than was the growth fo r j
5 mg. of lin o le a te alone fo r th e female r a ts (Group 17, ,
i Table X ). However, when 10 mg. each of m ethyl lin o le a te
1 and methyl lin o le n a te were fed sim ultaneously, th e
I
I re s u ltin g growth was id e n tic a l f o r both male and female j
r a ts w ith th a t noted fo r the groups receiving 20 mg. of
| m ethyl lin o le a te d a ily (Group 18, Tables IX and X).
The growth of th e r a ts adm inistered lin o le n a te
alone (Group 16), lin o le a te and lin o le n a te to g eth er a t
!
5 mg. le v e ls each (Group 17 ), and lin o le a te and lin o le n a te
to g e th e r a t 10 mg. le v e ls each (Group 18) i s presented
i g ra p h ic a lly in F igures 5 and 6. The potency of th e l i n -
1 |
j o le a te a t le v e ls of 5 mg. each per day and of 10 mg. each
' p er day i s obtained from F igure 7 in which male r a ts alone
are used. The potency v alu es a re obtained by p ro je c tin g
th e gain in weight of th e t e s t group (the lin o le n a te
group) on th e lo g . dose-w eight gain curve fo r lin o le a te
alone and o b tain in g th e eq u iv alen t lo g . dose from th e
i ab sc issa and from t h i s value the equ iv alen t dosage in
lin o le a te i s obtained. The potency of the lin o le n a te
alone, f o r growth, was p r a c tic a lly n i l , while a t a 5 mg.
lin o le a te -5 mg. lin o le n a te m ixture dosage per day, the
| average potency value was 5 .3 mg. of lin o le a te and ranged
1 from 2.7 mg. to 3.6 mg. per day fo r 5 determ inations based:
i on values a t 3, 6 , 9, 12, and 15 weeks. The potency of
; the lin o le a te -lin o le n a te m ixture a t a le v e l of 10 mg. of
| each per day was 20.8 mg. of lin o le a te equivalence p er
day based on 5 determ inations made a t 3, 6, 9, 12, and
76
15 weeks, w ith values ranging from 15 to £3 mg. per day.
The r e s u lts a re somewhat le s s d ec isiv e on th e ac tio n
of lin o le n a te as a c u ra tiv e agent fo r th e skin symptoms.
Although th e hum idity of the r a t rooms could not he con
tr o l le d , c e rta in in te re s tin g observations could he gleaned
on th e dermal symptoms. I t i s of in te r e s t th a t the nega
tiv e c o n tro l r a ts (Group 11) m aintained th e d eficien cy
skin symptoms throughout the 15 weeks of th e experim ental
p erio d , w hile as low a dosage a s 5 m g* of lin o le a te per
m itted improvement of t h i s syndrome in th e males and
re su lte d in a complete cure of th e symptoms in the fem ales.
Higher dosages of 10 to 50 mg. per day gave cures in both
sexes in sh o rte r len g th s of tim e. The low est cu ra tiv e
tim es fo r a l l anim als on a group were a t 9 weeks f o r
males on 50 mg. of lin o le a te per day and a t 9 weeks fo r
fem ales on both th e 20 and 50 mg. dosages of methyl l i n
o le a te per day. Ten mg. of methyl lin o le n a te d a ily
caused no improvement in th e skin condition of the r a ts ;
th e sim ultaneous a d m in istra tio n d a ily of 5 mg. each of
r
lin o le a te and lin o le n a te perm itted s lig h t improvement in
15 weeks; w hile 10 mg. per day of each of these two acid s
cured a l l fiv e males by 15 weeks and a l l fiv e fem ales by .
15 weeks.
77
Growfeh r a te s with methyl araohidonate and in t e r
re la tio n s h ip of lin o le a te and arao h id o n ate. The ex p eri
ment w ith methyl araohidonate was conducted in the same
manner as was th e determ ination of th e potency of methyl
lin o le n a te . Male and female r a ts were depleted on P ie t 69
and then were assigned to t e s t groups c o n sistin g of a neg
a tiv e co n tro l (Group 21) receiv in g 0 .1 ml. of eth y l la u ra te
biweekly and Groups 22 through 25, which received 10, 20,
50, and 100 mg. of m ethyl lin o le a te d a ily re sp e c tiv e ly .
A ll groups were adm inistered th e ir supplements biweekly;
a l l groups received th e ir supplements made up to a volume
of 0.1 m l. w ith eth y l la u ra te except Groups 24 and 25,
which received 0.25 and 0.50 ml. re sp e c tiv e ly . Groups 26
and 27 received 10 and 20 mg. re sp e c tiv e ly of methyl
araohidonate d a ily , and Group 28 received d a ily a m ixture
of 5 mg. eaeh of methyl araohidonate and methyl lin o le a te ,
w hile Group 29 received 10 mg. of each a c id . Group 30
served as a p o sitiv e co n tro l in K&ieh th e depleted anim als
were placed on a 30 per cent cottonseed o i l ^ d ie t (P ie t
70a) concurrently w ith the assay groups. Summaries of th e
weight gains of the male and female r a ts on th e several
groups a re given in Tables XE. and XEI re sp e c tiv e ly .
^ B la c k and White w interized cottonseed o i l . Opalousas,
L ouisiana B efinery.
T A B L E X X
Body W eight G ains f o r Male R ats on A ssay Groups fo r . ,
ife th y l A raoh id on ate, M argarine F a t, and B u tte r fa t =/
DailF dosage
Body
weight
at
start
end of following wee
Group
No.
Linoleic
acid
Ifethyl
araohidonate
Body gain in weight at cs
1 2
3 It 5
6
7
8
mg. mg.
S2*
gm.
gm « gm. g£.
ss* a *
gm.
SS-
21 0 0 198.8 -6.8 -10.0 -18.8 -21.8 -23.0
(1)
-30.0
(1)
-38.0
(2)
-3U.0
(3)
22 10 0
207.5
It.2 18.8
21.5 29.5 32.5 37.5 1*3.5 1*8.I t
23 20 0 208.2 -2.0 2.6 11.6 26.2 33.8 39.0 50.lt 55.8
2 1 * 50 0 208.2 10.lt l8.lt 3lt.8 lt2.8 1*7*0 1*7.6 6l.it
25 100 0 19it.8 11.8 26.0 35.2 ii3.2 51.6
63.3 69.3 7it.5
26 0 10 203.2 13.0 12.2 25.6 1*3.2 1*7.2 1*9.7 58.0 61*.0
27 0 20
197.3 10.0 llt.7 22.3 36.7 lilt.O 5it.3 62.0 67.0
28
5 5 198.0 11.5 2lt.8 28.5 36.3 37.0
51.5 56.3 61.0
2 9 10 10 222.6
13 J i
28.2 39.8
it7.lt 52.6
59.it 65.7 71.0
30 20 with
hydros.
117.5 mg.
coconut o il
217.2
-1*.3 12.2 21.0 27.3 1*0.7 1*9.0
56.3 60.0
31 125 mg. margi A | / 2Q5.8 -0.8 9.8 11.6 19.6 26.0 23.2 31.8 3it.2
32 250 mg. marg. A
125 nig. marg. B * *
2 $ Q mg. marg. B 2 /
213.2 -2.8
19.2 22.lt 30.6 33.2 31.2 36.it ltl.8
33 200.6 -8.lt 15.0
19.it 25.0 30.it 31.1* 30.6 36.2
3 1 * 198.0 9.5
20.8 2lt.0 27.6 3lt.O 33.2 35.2 1*2.2
36 30$ cottonseed o il
(Diet 70a)
203.lt lt0.2 67.8 86.8
105.lt 115.5
(1)
13U.2
(1)
UtO.5
(l)
11*7.7
(1)
co
FO O T N O T E S FO R TA BLE X T
- / a11 groups s ta rte d with 5 r a ts except fo r Groups 22,
27, and 30 which s ta r te d w ith 4, 3, and 6 r a ts
re sp e c tiv e ly . F igures in parentheses give number
of anim als th a t had died up to the time of weighing.
y
M argarine A i s unsupplemented m argarine o i l . A nalysis
presented in Table XVII.
3 /
-'M arg arin e B i s supplemented with 1.25 p er cent non
hydrogenated cottonseed o i l . A nalysis presented in
Table XVII.
T A B L E X II
Body W eight G ains fo r Female R ats on A ssay Groups fo r .
Ife th y l A raoh id on ate, M argarine F a t, and B u tte r fa t =f
Group
No.
Dailjf dosage
Body
weight
at
start
Body gain in weight at end of following weeks
Linoleic
acid
Ifethyl
araohidonate 1 2 3 it 5
6
7
8
2S*
mg.
ss* s s .
gm. 12*
gm.
J a 2 *
g£. g£.
go.
21 0 0 17U.0 -1.6 1.6 -2.8 -5.2 -7.lt
-9.6 -8.lt -13.lt
(1)
22 10 0 161.0 6.8 15.8 20.8
2it.5
27.2 29.0 33.8 3U.0
23 20 0 163.6 7.8 12.2 I8.it 21.6 26.2 30.6 33.8 36.8
2 1 * 50 0 161.1* lit.2 26.2 3li.2 ii0.2 itit.it 1*8.2 1 * 9 .1 * 50.8
25 100 0
167.7 1.5 16.7 21.7 25.3
26.2
30.7 3U.5 36.0
26 0 10 161*. 2 6.8 ll.lt 16.8 20.2 19.2 2lt.lt 30.6 29.2
27 0 20 159.8 ll.lt 15.0 19.6 25.0 26.0 29.6 30.8 31.6
28
5 5
159.6 12.8 18.8
2it.5
30.8 33.8
ill. 3 it3.3 50 ;5
29 10 10 162.0 10.0 17.lt 26.0 33.lt 35.2 38.6 it0.6
itit.it
30 20 with 117.5 mg.
hydrog. coconut o il
153.0 — 5.0 l8.lt 22.lt 26.0 28.8 32.8 3U. 8
31 125 mg. marg. A
250 mg. marg. A &
125 mg. marg. B &
250 mg. marg. B 2 /
168.6 it.8 7.8 15.6
19.lt 23.6 2U.6 26.2 27.2
32 160.0 6.0 13.8 19.8 26.lt 30.0 31.2 35.6 37.0
33 155.8 2.6 2.2 5.2 8.8 13.0 lit.2
l5.lt 21.0
3k 170.8 It.2 7.2 10.3
(1)
16.5
(1)
17.3
(1)
17.2
(1)
20.2
(1)
25.2
(1)
35 125 mg. butterfat I63.it 2.1t 5.2 10.2 13.0 io.5 15.2
15.2 19.5
36 30$ cottonseed o il
(Diet 70a)
170.2 2l*,0 38.2 ltO .it 51.8 55.8 62.8 61t.2 66.2
F O O T N O T E S FOE TA BLE X II
A ll groups s ta rte d w ith 5 r a ts except fo r Groups 25
and 54, which s ta rte d w ith 6 and 4 r a ts re sp e c tiv e ly .
F igures in parentheses give number of anim als th a t
had died up to the tim e of weighing.
2 /
“^Margarine A i s unsupplemented m argarine o i l . A nalysis
presented in Table XVII.
5 /
M argarine B i s supplemented with 1.25 per cent non
hydrogenated cottonseed o i l . A nalysis presented in
Table XVII.
j 82 j
| Logarithm ic curves drawn fo r tlie 7th and 8 th weeks j
| of th e feeding experim ent a re given in F igure 9. Both
male and female growth r e s u lts a re p lo tte d in th is fig u re .
P ro je c tio n of th e growth obtained w ith th e methyl arach id - :
I
| onate perm its the c a lc u la tio n of i t s potency in term s of
| lin o le a te equivalence. T abulations of th e p otencies e a l-
| culated a t the 7-week and a t th e 8-week period fo r the
! i
araohidonate alone and when fed sim ultaneously with l i n -
i :
o le a te are recorded in Table XEII. On th e 10 mg. le v e l j
I of araohidonate alo n e, the potency as ca lc u la te d fo r the j
! j
j 7th and 8th weeks i s 4 .0 tim es as g re a t as th a t obtained I
' w ith lin o le ic acid alone; on th e 20 mg. le v e l, the
I
araohidonate had a potency 2.7 tim es th a t obtained w ith
1 lin o le ic a c id . The average potency based on th ese two
I values i s 5 .4 tim es th a t obtained w ith lin o le ic a c id . I
I
j This potency value agrees very c lo se ly w ith th a t found by
| I
! Turpeinen (52), who found th a t arachidonic ac id was 5
i
t ;
tim es as e ffe c tiv e fo r growth of depleted r a ts as was
I i
lin o le ic ac id . ;
The growth obtained with sim ultaneous d a ily dosages
!
of 5 mg. each of lin o le ic acid and methyl arachidonate,
> when p ro jected on th e 7th and 8th week re fere n ce curves
in F igure 9, re v eals an a c tiv ity 5 .2 tim es th a t fo r
83
2
o
z
<
o
X
o
UJ
80
7 0
6 0
5 0
4 0
3 0
20
10
o-
8 WEEKS
MALES
7 WEEKS
CP, 2 9
CP. 2 7
GP. 2 6
GP. 2 8
B-i 8 WEEKS
7 WEEKS
FEMALES
A-l
C
0 . 4 0 .8 1.2
LOG DOSE, MG.
1.6 2.0
F igure 9. The gain in weight of prev io u sly depleted
male and fem ale r a ts p lo tte d a g a in st the log dose of methyl
lin o le a te fed fo r 7 and 8 weeks. P ro jectio n s of weight
gains of Groups 26, 27, 28, and 29 re p re se n t d a ily dosages
of 10 mg. of m ethyl araohidonate, 20 mg. of m ethyl arach-
id o n ate, 5 mg. each of lin o le a te and araohidonate fed
sim ultaneously, and 10 mg. each o f lin o le a te and arach-
idonate fed sim ultaneously re sp e c tiv e ly . P o in ts A -l, B -l,
and C re p re se n t 125 mg. d a ily dosages of M argarine A,
M argarine B, and B u tte rfa t re sp e c tiv e ly , w hile P oin ts A-2
and B-2 re p resen t M argarines A and B re sp e c tiv e ly a t
250 mg. dosages d a ily .
T A B L E m i
Potency o f Ifethyl Araohidonate W hen Fed Alone to
Fat-D epleted Male Bats and W hen Fed w ith L in o leic Acid
a s Determined from the Logarithmic Dose-Weight Gain Carves
. Daily dosage
Equivalent potency in ...
terms of lin oleic acid
Methyl
araohidonate
Linoleic
acid 7th week 6th week
SS*
S£.
mg.
10 0 38 U
20 0
$h 53
5 5
32 31
10 10 75 76
’lin o le ic a c id alo n e. ffhen 10 mg. of each acid was fed as
i i
| a m ix tu re, th e potency of the m ixture was ca lc u la te d to be
3.8 tim es th a t of lin o le ic ac id alo n e. I f the methyl
’ arachidonate m oiety i s considered as possessing the a c tiv - '
i t y remaining a f te r the su b tra c tio n of the mg. of lin o le ic
acid a c tu a lly fed from the lin o le ic ac id potency ca lc u la te d
: fo r th e m ixture, one o b tain s a potency much g re a te r than ;
; l
j th a t obtained fo r th e arachidonate alone. In th e case of 1
i
j the 5 mg. dosage of araohidonate fed w ith 5 mg. o f l i n - j
i o le ic a c id , th e potency ca lc u la te d fo r th e arachidonate
I
| m oiety i s 5 .4 tim es th e a c tiv ity of lin o le ic a c id , w hile
the arachidonate, fed a t a 10 mg. dosage with an equal !
' amount o f lin o le ic a c id , has a ca lc u la te d m oiety potency
th a t i s 6.6 tim es the a c tiv ity of lin o le ic ac id alone. ,
The r e s u lts obtained with th e sim ultaneous ad m in istratio n i
of both a c id s a re evidence of a sy n e rg istic a c tiv ity fo r
I
th e two a c id s.
I
i Food consumption d ata fo r male and female r a ts on
th e referen ce lin o le ic ao id groups, th e various methyl j
I arachidonate groups, and the negative and p o sitiv e co n tro ls
! are given in Tables 2XV and X V re sp e c tiv e ly . The d if f e r
ences between any of th e groups of female r a ts on th e f a t -
fre e d ie t, irre s p e c tiv e of th e type or amount of
T A B L E XIV
Food Consumption D ata fo r Male R ats on A ssay Groups
fo r M ethyl A rach id on ate, M argarine F a t, and B u tte r fa t — '
Group .
No. ±/
N um ber
of
rats
Food consumed in grains at end of following weeks
Total 1 2
3 1 * 5
6
7
8
21
5
81 J r 7i*.0 73.6 72*.0 72.5 66.3 66.3 77.0
585
22 1 * 83.0 97.3 93.5
99.8
96.3 91*. 3
99.8
99.5
7 6 2 *
23 5 83.2*
81.6 91.6 96.0
97.2* 95.2*
98.6 96.6 7 1 * 0
2i* 5
95.6 90.6 100.0 93.8 91.1*
82.8 79.8 8 9.0
723
25 5 97.1*
9 1 * . 2 9l*.l* 83.6. 98.0 92.0 93.2 91.2* 7 2 * 1 *
26
5 Jh.h
88.8
102.5 101.3 100,0 98.3 9l*.0 96.7 756
27 3 80.3 96.0
97.3 100.7 96.3 89.0 97.3 98.3 755
28
5
7lu8 99.6 96.6
97.7 83.7 9l*.i* 92*. 2 96*2 737
2? 5 95.2* 105.8 110.2* 108,2 102,6 102.6
101.5 98.8
825
30 6 80.8
101.3 95.2 90.7 93.3 95.3
100.8
91.7 7 1 * 9
31 5
98.8 91.2
77.2* 83.2 88.2 85.2 90.2 87.8 702
32
5
102.2* 100.8
93.1* 95.2
91.0 88.0 91*.0 86.0
751
33 5
10i*.2 91.0 92.8 92.8
91.5
90.6
90.5 89.8
7 2 * 3
3k 5 88.0 101.6 100.2 91.8 100.2 92.2* 96.0 96.2*
767
35 5 77.0' 99.0
96.3 80.7 83.0 96.0
91.3
89.0 712
(1) (1) (1) (1)
36 5 96.2* 81.5 78.5 77.5 86.0 80.2 76.2 73.0 6 2 * 9
CO
O N
87
F O O T N O T E S F O R T A B L E XIV
t
F igures in parentheses give number of anim als th a t had
died up to the time of weighing.
f
Supplements adm inistered to groups l is te d a re described
in Table XX.
TABIE X V
Food Consumption Data f o r Fem ale B ats on A ssay Groups _ ,
f o r M ethyl A raeh id on ate, M argarine F a t, and B u tte r fa t
Group .
no. y
N um ber
of
rats
Food consumed in grams at end of f ollcwing "w eeks
Total 1 2
3 U 5 6 7
8
21 5
8^.6 78.7
76.2 8It.lt 79.2 73.8 77.0 82.6 638
(1)
22
5
80.2 83.0 79.2 78.8
77.3 78.3 8U.3V 77.5 639
23 5 77.h
77.2 ?5.2 76.8 77.8 76.8 72.0 7U.8 6o8
2U 5
85.2 88.6 89.8 88.it
80.8 77.6 77.0 80.3
668
25 6
7U.3
89.2 88.2
75.5
83.8 78.5 79.0 79.2 6U8
26
5
75.0 77.8 88.2 71.6 68.it 75.6 8U.8 69.8 611 '
27 5
83.8 86.2 79.8 77.6 7U.lt 76.6 71.2 72.6 622
28
85.3
82.8 87.0 72.0 77.0 78.0 72.8 80.0
635
29 5 . 93.ii
82.)* 87.8
83.lt
81.2
7 7.U 71.8
76.U 65U
30 5 101.5 90.0 86.0 81.2 78.2 75 .U
70.6
75.U 658
31
83.8 83.2 82.8 78.8 80.8 8U.6 80.8 81.0 656
32 5
90.2 93.8 82.6 81.8 8U.6 81.8 79.2 78.6 672
33 5
78.0 82.0 82.2 72.8 78.2 77.6 7U.6 82.2 628
3U h 86.5 87.0 89.0 86.2 83.2 75.8 82.5 80.0 670
(1) (1) (1) (1) (1) (1)
35 5 72.8 85.0
83.3
8it.O 81.0 83.2
93.7 93.0 676
36 5
77.2
70.h 66.0 55.lt 5U.0 53.8 U9.2 55.6 U82
F O O T N O T E S FO R TA BLE XV
F igures in parentheses give number of anim als th a t
had died up to th e tim e o f weighing.
Supplements adm inistered to groups lis te d are described
in Table XI.
! supplem entation used, a re sm all, The male r a ts on the
| neg ativ e eo n tro l group (Group 21) a te le s s of the b asal
d ie t than did any of th e groups supplemented w ith essen
t i a l a c id . Except fo r the p o sitiv e co n tro l group (Group
, 36), which received a high c a lo rie d ie t, th e re a re no
t
»
s ig n ific a n t d iffe re n c e s in the food consumptions except
i
fo r th e group th a t was supplemented w ith a m ixture of
; 10 mg. eaeh of methyl arachidonate and lin o le ic a c id d a ily
1 (Group 29). Although th e food consumption of Group 29
1 (males) i s g re a te s t, th e t o t a l gain in weight over a
period of 8 weeks was g re a te s t in Group 25 w ith an average
gain of 74.5 gm. versus 71.0 grams f o r Group 29.
T ibia len g th s of th e r a ts on the negative co n tro l
d ie t (Group 21), on th e p o sitiv e co n tro l d ie t (Group 30),
| and on the lin o le ic a c id and arachidonate supplement
i
| d ie ts (Groups 22-29) were determined as p rev io u sly de-
i
! scrib ed (117) a t the end of the assay period. The re s u lts
> of th e measurements of the tib ia e a re ta b u la te d in
Table XVI. No d iffe re n c e s in tib ia len g th s were observed
fo r male or female r a ts whether the anim als were on 0 f a t ,
3© per cent f a t , or on any o f th e le v e ls of lin o le ic acid
o r methyl arachidonate fe d .
T A B L E XVI
Tibia Lengths of Bats after 8 Weeks on N o Fat,
30 Per Cent Fat, Linoleic Acid Alone, Methyl Arachidonate Alone,
and Mixtures of Linoleic Acid and Methyl Arachidonate
.Daily dosage
Tibia length
Group
No,
Linoleic
acid
Methyl
arachidonate M ale Female
26*
ra m . m m .
21 0 0 38,14(2) 35.8(5)
22 10 0 39.2(U) 36.3(14)
23
20 0 38.2(5) 35.5(5)
2h 50 0 39.5(5) 35.5(5)
25
100 0 38.7(14) 36.0(5)
26 0 10 39.3(U) 35.3(5)
27 0 20 38.6(3) 35.3(5)
26
5 5 38.5(5) 35.3(5)
29 10 10 39.1(14) 35.U(3)
36 30$ cottonseed o il
(Diet 70a)
39.7(14) 36.0(5)
Figures in parentheses represent number of rats measured
92
: B io lo g ical Assay Of E s s e n tia l Acids In E ats and a Compar-
! iso n Of B io lo g ical and P hysical Methods j
Using th e logarith m ic curves obtained with lin o le ic '
acid fed to male r a ts (Figure 9 ), a b io lo g ic a l assay was
run on 3 samples of f a ts furnished by The Best Foods, In c .
C onstants obtained by an a ly sis of th e te s t o i ls are summa
riz e d in Table XVII. These values were determ ined by
i !
. P ro fesso r J . B. Beans (The B est Foods', I n c .) , who used a
i
I m odificatio n of the spectrophptom etric method of B rice i
; c t a l . (118) in determ ining t h e ir f a tty acid com positions.
f
I t may be noted th a t th e io d in e values r e f le c t th e
d ilu tio n of th e m argarine o il (A) w ith 1.25 per cent of
I
, cottonseed o i l (non-hydrogenated) to make m argarine o i l
: sample B. The cottonseed o i l has an io d in e value of about
108 and according to th e li te r a t u r e contains 45 per cent
lin o le ic ac id and 2 p er cent lin o le n ic a c id . T h e o re tic a lly
th e re should have been a d iffe re n c e o f approxim ately 0 .5 j
i
per cent in th e lin o le ic acid value rep o rted fo r the *
I
' m argarine o i l before and a f te r supplem entation w ith th e j
1 lim pid cottonseed o il ; however, th is d iffe re n c e was appar-
i
' e n tly too sm all to be d e te c ta b le by p h y sical means.
The f a ts were fed to groups of r a ts co ncurrently
with the lin o le ic acid supplemented groups and c o n tro ls !
T A B L E XVII
Constants Obtained in Analyzing Test Oils Used in
Biological Assay Procedure =/
Composition of to ta l ^ .
. mixed, fatty acids . &
Sample
Melting
point .
%*. I I
Setting
^ 2 /
Free
f a t t y .
acidss'
Peroxide .
value W
Iodine.
No. y
Lino
le ic
acids
Lino-
lenic
acids
Arachi-
donic
acid
Oleic
acids
Satur
ated
acids
%
m.eq./kg.
* •
% % % % %
Margarine
o il (A)
9 1 * .1 25.7 0.01 0.0 71.8 6.6
(6.3)
6 6 70.1
(67.0)
23.3
(22.3)
Margarine
o il + 1.25$
cottonseed
o il (b)
9l*.3 25.7 0.01 0.0 72.9 6.6
(6.3)
0 0
71.1*
(68.3)
22.0
(21.0)
Butter
o il (C)
93.9 23.1* 0.25 0.0 3U.6 2.1
(2.0)
1.1*
(1.3)
trace
31.7
(30.2*)
61*.8
(62.0)
1/ -
-'Sandies analyzed by Professor J. B. Beans.
^ O fficia l and Tentative Methods of the American Oil Chemists’ Society, 191$.
^Method of H. W . Vahlteich et a l. (119).
^Ifethod of B. H . Wheeler (120).
^Figures in parentheses are for values calculated on a triglyceride basis (Fatty acid percentage
6 / -x 0.951*) (121).
-'Determined spectrophotometrically (118). 'O
94
used in Tables XI and X II, in which weight gains fo r
males and fem ales, re sp e c tiv e ly , a re recorded. The food
consumption data fo r th e fa t-fe d r a ts a re presented on
Tables XIV and XV. In a d d itio n to the neg ativ e co n tro l j
group (Group 21) and th e p o sitiv e co n tro l group (Group 36),
a c o n tro l group (Group 30) was used in which the supple
ment was 20 mg. of lin o le ic acid combined w ith 117.5 mg.
of hydrogenated coconut o i l p er day. This control was !
included to a id in evaluating th e e ffe c t of "n o n -e sse n tia l”
f a t in th e supplem entation of th e o i l s . U nfortunately, i t
was im possible to include more groups of c o n tro ls due to
th e shortage o f depleted r a ts . The potencies of th e th re e !
o i l samples in term s o f lin o le ic acid equivalence a re c a l
culated from p ro je c tio n s of th e weight gains of th e o ils
on the lin o le ic ac id weight g ain -lo g arith m ic dose curves
(Figure 9 ). The assay of the two samples of m argarine
f a t a t 125 and 250 mg. le v e ls per day gave comparable ,
I
r e s u lts of 2.3 and 2.3 per cent of lin o le ic acid respec
tiv e ly fo r sample "A” , and gave 2.7 and 2.3 per cent
i
re sp e c tiv e ly f o r sample "B". These m argarines gave values !
of 6.3 per cent of lin o le ic acid fo r each when assayed
spectrophot© m etrically (Table XVII). The b u tte r f a t
assayed b io lo g ic a lly a t 1.6 per cent e s s e n tia l f a tty acid ;
95
as co n trasted w ith a spectrophotom etric value of 3.3 per
ce n t. The values obtained fo r th e m argarines and b u tte r -
f a t may be in v a lid sin ce considerable ex tra p o la tio n of the
logarithm ic curve had to be made. Experience (see F igure
7) has shown th a t th e lin e a r r e la tio n continues through
th e 5 mg. dosage of lin o le a te fo r male r a ts , and these
f a ts were fed a t dosages in th a t range. The e x tra p o la tio n
of th e logarithm ic curve f o r the lin o le ic acid data (F ig- 1
ure 9) during th e 8 th week to 0 mg. dosage of lin o le ic
ac id in tak e gives a value of 22 grams growth increm ent,
whereas a c tu a lly th e anim als (males) lo s t about 54 grams
in w eight. I f one were to draw a s tr a ig h t lin e from th e
lo g . dose value o f 1*0 to 0, th e l a t t e r p lo tte d ag a in st
a weight lo s s of 34 grams and read o ff the values on t h i s !
p o rtio n of the curve, the responses of the anim als on the
m argarines and th e butter-supplem ented ra tio n s , one would
o b tain average fig u re s fo r the m argarine samples o f about •
i
4.5 per cent and a fig u re fo r th e b u tte rfa t of about 4 .3
per cent b io lo g ic a lly a c tiv e f a tty a c id s. I t may be j
concluded from th ese c a lc u la tio n s th a t the b io lo g ic a l j
potencies of the m argarines l i e somewhere between 2,7 and '
4.5 p er cent (most probably close to 2.7 per cent) and
th a t of th e b u tte r f a t sample between 1.6 and 4.3 per cent
! 96
j
i
(most probably clo se to 1,6 p er c e n t).
The r e s u lts of th e bioassay on female r a ts a re not
s a tis fa c to ry sin ce th e lin o le ic acid lo g . dose-w eight gain ;
curves a re n o t s tra ig h t lin e fu n c tio n s.
Group 50, Tables XE and X II, a re male and female
: r a ts re sp e c tiv e ly on dosages of a m ixture of 20 mg. of
i
lin o le ic acid and 117.5 mg. o f hydrogenated coconut o i l '
p er day. Somewhat in creased growth (60.0 gm. in 8 weeks) '
I
was obtained fo r the male r a ts on th is supplement over
th a t found when 20 mg. of lin o le ic acid alone was given
(55.8 gm. o f growth in 8 weeks). The growth of th e m ixture
of acid and hydrogenated coconut o i l p ro jec te d on the I
lin o le ic acid curve (Figure 9) i s equ iv alen t to the growth
obtained w ith 29 mg. o f lin o le ic a c id . Reeent experim ents
i
by o th er workers in t h i s lab o ra to ry (62) have shown th a t
low dosages of hydrogenated f a t fed w ith lin o le a te do not
reduce the potency of th e e s s e n tia l f a t t y a c id , but th a t
as th e ,r e la tiv e q u an tity of th e sa tu ra te d f a t i s increased '
to 250 to 500 mg. d a ily , th e potency of th e lin o le a te is !
decreased. In the ease of the female r a t s , th e same
growth was obtained w ith both th e 20 mg. of lin o le ic acid
alone and th e m ixture of 20 mg. o f lin o le ic ac id w ith
i
117.5 mg. of hydrogenated coconut o i l d a ily . I
97
No n o tic e a b le d iffe re n c e s were observed fo r the
food consumption of th e b asa l d ie t irre s p e c tiv e of the
| le v e l o f th e m argarine, b u tte r f a t, o r coconut o i l supple
ments fo r e ith e r sex (Tables 2U and XV).
E ffe c t Of Methyl L inoleate Supplem entalon On M ineral O il
"gat-D epleted" R ats
I t has been shorn by Bacon (76} th a t m ineral o il
»
causes fa t-d e fic ie n c y symptoms to appear ra p id ly in r a ts
placed on a m ineral o i l - f a t fre e d ie t a t weaning. I t was
a lso shown th a t th ese symptoms could be reversed to some
degree. In o rd er to determ ine th e e ffe c t of th e ro u te of
a d m in istratio n of e ss e n tia l f a tty acid s in re lie v in g the
e ffe c ts of th e m ineral o i l , and to determ ine th e e ffe c t of
v a ria tio n of th e dosage on the r a ts , an experiment was
planned in which weanling r a ts were placed on d ie ts (Table
I I ) which were sim ila r to those o f Bacon. The d ie ts
employed in t h i s experiment d iffe re d from those o f Bacon
and from those in a l l previous experim ents reported in
t h i s paper by changes in the w ater-soluble vitam in complex
added to th e d ie t. The anim als (male) were divided in to
six groups (Groups 57-42). Group 57 was placed on the
f a t- f r e e d ie t (D iet 69g) while Groups 58-42 received the
7§ per cent m ineral o il d ie t (D iet 73). Group 38 served
98
as th e negative co n tro l w ith th e m ineral o il d ie t, while
Groups 59-42 served as t e s t groups fo r the e f fe c t of the
lin o le a te . Groups 39 and 40 received re sp e c tiv e ly 20 mg.
of methyl lin o le a te d a ily (6 days a week) in tra p e rito n e a lly
and o ra lly , w hile Groups 41 and 42 were re sp e c tiv e ly given
50 mg. of lin o le a te d a ily in tra p e rito n e a lly and o ra lly .
The weight gains of the r a ts on the various groups
a re presented in Table XVIII. The presence of the m ineral
o i l in th e d ie t r e s u lts in an ea rly p lateau in the weights
of th e r a ts , w hile lin o le a te , irre s p e c tiv e of the mode of
a d m in istra tio n , prevents o r delays the harmful e ffe c ts
of th e m ineral o i l . Somewhat g re a te r growth i s observed
w ith th e 50 mg. le v e l of lin o le a te than w ith the 20 mg.
le v e l. I t is p o ssib le th a t one o r more w ater-soluble
vitam ins i s a lim itin g fa c to r,, since th e d iffe re n c e in
growth between the 20 and 50 mg. dosages i s n o t very
g re a t. The p o s s ib ility th a t a B vitam in is im plicated in
th e syndrome caused by m ineral o i l l i e s in the fa c t th a t
i
t h i s syndrom p a r a lle ls c lo se ly th a t seen in th e l a s t
stag es o f b io tin d eficien cy in th e r a t . These symptoms
include generalized brown, greasy scaling and a lo p e c ia ,
and ty p ic a l sp ectacle eyes. The animals a lso assume th e
abnormal humped posture and sp a stic g a it noted in r a ts
T A B L E XVIII
Growth Data for M ale Hats on Fat-Free and 7*5 Per Gent Mineral Qil-Fat-Free Diets
With and Without Methyl linoleate (9 Eats per Group)
Group
No.
Diet
fed
Daily
dose
methyl
linoleate
Route
of
admin.
Starting
weight
. Weight gain at end of following weeks
1 2
3 1 * 5 6 , 7
37 69s
3£.
0
__
J 2 2 »
31*.1* 13.1*
IS*
1*0.1* 61*. 1 *
j£5«
81*.6 101.2
s *
103.7
n .
115.1*
38 70c 0
36.7
21.1 1*0.2
5 l . l 57.1 55.5 56.9 56.1*
39
70c 20 i.p . 39*0 13.1 37.8 53.6 61.3 76.8
79.7 87.1
1 * 0 70c 20 oral
1*0.U 18.9 1*2.5
50.6 66.7 73.0
79;5
86.6
la
70c 50 i.p . 36.6 16.5 1*3.8 51**8 70.8 82.2
89.2 93.0
1 * 2 70c 5o oral 31**8
19.3
1*1*.6
57.3 73.2 86.7 89.8 9l*.6
V O
V O
su ffe rin g from b io tin d eficien cy .
In order to t e s t the p o s s ib ility th a t th e vitam ins
are d e fic ie n t in th e m ineral o il d ie t, th e amounts used
by Bacon (76) and by us in previous experiments reported
h ere, have been doublted in te s t s under -way. I t may be
noted th a t sev e ral of th e B vitam ins have been changed in
the m ineral o i l experim ent from those used in a l l preced-
i
ing experim ents; namely, th e le v e ls per kg. of d ie t fo r
th e follow ing vitam ins were changed: rib o fla v in increased
from 27 to 72 mg., n ic o tin ic acid reduced from 60 to 10
mg., in o s ito l reduced from 1200 to 500 mg., para-am ino-
benzoic ac id reduced from 600 to 200 mg., and b io tin re - j
duoed from 2 to 1 mg. These changed le v e ls of the B
vitam ins were used in a l l experim ental f a t- f r e e d ie ts used
by A nisfeld e t a l . (62) w ithout any n o ticeab le e ffe c t
upon th e expected r e s u lts w ith various supplem ents.
d e la tio n Of E sse n tia l F a tty Acids To Gonadal Development
In th e Rat
The e ffe c t of f a t d eficien cy upon the gonads of
r a ts as w ell as th e p o ssib le lo c a tio n of any disturbance
of the development of the gonads was studied w ith a
group of depleted as w ell as re -fe d fa t-d e p le te d r a ts .
R ats, which had been depleted by A nisfeld e t a l , (62)
1 0 1
were used in th is experim ent. These anim als were depleted
in previous experim ents except th a t the B vitam ins of
D iet 69e were changed as in d icated in th e previous ex p e ri
m ent w ith m ineral o i l . A fte r 12 weeks of d e p le tio n , male
and female r a ts were continued by A nisfeld on experim ents
in which one group of each sex was m aintained on th e deple
tio n d ie t fo r 8 a d d itio n a l weeks ( to ta l of 20 weeks) and
another group of each sex was given 100 mg. of m ethyl l i n
o le a te d a ily . Included w ith th e depleted anim als were
anim als th a t received only 5 mg. of lin o le a te d a ily . No
n o ticeab le d iffe re n c e s could be observed between com pletely
d e fic ie n t r a ts and those receiv in g a very low dosage (5 mg.
of lin o le a te . These r a ts were given subeutaneously e ith e r
s a lin e (co n tro ls) or 20 u n its d a ily of chorionic gonado
tro p in made up to 0.1 m l. in s a lin e . Male r a ts were
adm inistered th e hormone o r sa lin e w ithout hormone fo r a
t o t a l of 7 days, while th e female r a ts were given in je c
tio n s fo r 5 days. The r a ts were continued on f a t- f r e e
d ie ts alone or on f a t- f r e e d ie ts supplemented with methyl
lin o le a te fo r the d u ratio n of the experim ent. I t i s
p o stu lated th a t th e hormonal treatm ent would rev eal whether
th e gonads th sn selv es were a ffe c te d by th e d eficien cy of
f a t in the d ie t, o r whether the p itu ita r y fu n ctio n had been
1 0 2
in te rfe rre d w ith by th is d efic ie n cy . I f th e sexual d is
turbances a ttrib u te d to f a t d eficien cy are due to a d ire c t
e ffe c t o f th e d eficien cy upon the gonads them selves, no
stim u lato ry e ffe c t should be noted upon th e ad m in istratio n
of th e chorionic gonadotropin.
A fte r the ad m in istratio n o f th e t e s t substances, the
anim als were s a c rific e d and the t e s te s , sem inal v e sic le s,
and p ro sta te (a n te rio r lobes) of th e male r a ts were excised
and weighed. The o varies and u te ru s of the fem ale r a ts
were removed and weighed. Since no data were found in the
l i te r a tu r e concerning th e e f fe c t of f a t d efic ie n cy on the
thymus w eight, those o f th e female r a t s were weighed and
included in th e weight exam ination. Bata were a lso r e
corded of food consumption during th e hormonal t e s t period.
The weights of th e organs te s te d and th e food consumptions
a re presented in Table X U .
The ad m in istratio n of th e hormone appears to have
had no e ffe c t oh the weight gain of the anim als during the
experim ental period nor does i t appear to have a ffe c te d
the food consumption, i t i s o f in te r e s t to note th a t th e
food consumption of th e r a t i s not a ffe c te d by the le v e l
of lin o le a te given th e depleted r a t . This equal food con
sumption by both th e low o r negative lin o le a te r a ts and by
103
T A B L E XIX
E ffe c t o f Chorionic Gonadotropic Hormone on Male and Female Rats
" W h ich Had Been Fat-D epleted and Re-Fed -
With L ow or With High Dosages o f Jfethyl lin o le a te
Methyl lin o
le a te supple
mented d a ily
0 - 5
»
100
m g*
0 -
. m
5
«
100
mg.
Sex m£L e male female female
Chorionic
gonadotropin
(u n its) M
0 20 0 20 0 20 0 20
Humber o f
ra ts
7
6 6 7 8 7 8
Weight, gm.
. S ta rt „ /
End &
197
199
166
165
259
268
261
273
158
158
1 5 1 *
155
202
202
202
200
Food consumption,
gm.
— ■ —
. — —
6 1 * 57 5 1 * 55
T estes, p a ir , gm. 2.22
1.71
2.68 2.57
Seminal v e s ic le s ,
p a ir , gm.
0.51
0.88 0.86 1.26
P ro sta te, an terior
lo b e s, gm.
0 .1 6 0.29
0.22 0.31*
O varies, p a ir , gm. .Oljl .068 .050 .077
U terus, gm. .260 .285 .391 .1*36
Thymus, gm. ■ *
.126
.113 .193 .200
1
F O O T N O T E S F O R T A B L E TJX
1 /
Parke, Davis and Co. d esiccated chorionic gonadotropin
"A ntuitrinU ^ obtained from pregnancy u rin e . D issolved
in p h y sio lo g ical sa lin e so th a t each 0 .1 m l. contained
20 In te rn a tio n a l U n its. The animals were in je c te d
subcutaneously with 0 .1 ml. of the so lu tio n d a ily . The
negative co n tro ls received equal subcutaneous in je c tio n s
of s a lin e .
2 /
Male r a ts received th e hormone in je c tio n s fo r 7 days;
female r a ts received th e in je c tio n s fo r 5 days.
th e high lin o le a te -fe d r a ts i s evidence th a t th e growth
f a ilu r e of th e f a t- d e f ic ie n t r a ts is not th e r e s u lt of
in a n itio n hut ra th e r a dim inished u tiliz a tio n of th e food
eaten . The w eights of th e te s te s o f the male r a ts are
d ir e c tly re la te d to th e weights of th e animals and are
not a ffe c te d in re sp e ct to weight by the ad m in istratio n
of th e gonadotropic hormone. I t would th ere fo re appear
th a t th e fo llic le -s tim u la tin g hormone se c re tio n of the
p itu ita r y appears to have been unaffected by the f a t d e f i
ciency. However, a considerable in crease in weight of
th e secondary sex organs of th e male r a t was noted in both
th e d e fic ie n t and linoleate-supplem ented groups upon
ad m in istratio n o f the hormone. B est and Taylor (1B2)
s ta te th a t dosages of the chorionic gonadotropin cause
enlargement of th e p ro sta te and seminal v e sic le s and to
a le s s extent th e te s te s o f both normal immature r a ts and
of a d u lt r a ts ; th e percentage in c re a se in the siz e of the
seminal v e s ic le s and p ro s ta te of the f a t- d e f ic ie n t males
i s 73 per cent and 77 p er cent re sp e c tiv e ly , a f te r admin
i s tr a tio n o f the hormone. In th e case o f the lin o le a te -
fed r a ts , th e sem inal v e s ic le s and p ro s ta te s were enlarged
47 and 56 per cent re sp e c tiv e ly .
B est and Taylor s ta te th a t in the fem ale, th e
106
in je c tio n o f gonadotropic hormone causes enlargement of
th e © varies. The percentage in crease in siz e of th e
o varies of the female r a ts on d e fic ie n t and on lin o le a te -
supplemented d ie ts i s almost id e n tic a l, while th e re i s no
e ffe c t of the hormone on the size of the u te ru s. I t i s
d i f f i c u l t to a s c e rta in whether th e g re a tly increased
weight o f th e u teru s of linoleate-supplem ented female
r a ts over th e weights of the u te r i of the d e fic ie n t r a ts
i s due to weight d iffe re n c e s or to distu rb an ces in th e
estrogen se c re tio n of th e r a t . I t would appear from the
data th a t the d iffe ren ce s were due to weight d iffe re n c e s,
since in cre ases in siz e and presumably in a c tiv ity of the
o varies did not a f fe c t th e weights of the u te r i w ithin
e ith e r group.
The weights o f the thymus glands seem to be a
fu n ctio n of body w eight under the conditions of t h i s
experiment and a re unaffected by the use of the chorionie
gonadotropic hormone.
C H A P T E R V
DISCUSSION O F RESULTS
The problem of th e in v e s tig a tio n of methods of
reducing th e tim e of d ep letio n of r a ts to be used in
e s s e n tia l f a tty ac id assay work has been a pressing one.
A lite r a tu r e survey of fa c to rs a ffe c tin g the d ep letio n
time in d ic a te s th a t th e use o f sa tu ra te d f a t or of such
n o n -e sse n tia l a c id s as o le ic or e le o s te a ric may m a te ria lly
hasten th e r a te of d ep letio n of r a ts . Work done re c e n tly
in th is la b o ra to ry (62) w ith sa tu ra te d coconut o il as a
supplement in d ic a te s th a t such a method o f d ep letio n may
be of p ra c tic a l im portance. Methods of exhaustive e x tra c
tio n of th e easein used in the d ep letio n d ie ts had no
apparent e ffe c t on the growth r a te s of th e d ep letin g r a ts
(Tables I I I and IV ). Although growth hormone was not used
in th e d ep letio n period w ith th e r a ts te s te d (Table Y), i t
i s p o ssib le to determ ine the e ffe c t th a t th e hormone might
have had on d ep letin g r a ts by observing the e ffe c t i t had
on alread y depleted r a t s . The growth hormone sample used
in th is experiment caused an increased growth of r a ts on
a f a t- f r e e d ie t supplemented w ith lin o le a te in the same
manner as had been observed by Deuel, Hendrick, and
C rockett (123) w ith f a t d ie ts and growth hormone. However,
108
contrary to ex p ectatio n s, the growth hormone did not cause
th e negative co n tro ls to lo se w eight more ra p id ly than
f
did those not in je c te d . B ather, th e hormone appears to
have sustained th e r a ts fo r 12 to 14 weeks a t a somewhat
elevated maintenance le v e l over th e negative co n tro l
groups. Since Pearson and Panzer (124) re p o rt th a t the
e s s e n tia l f a tt y ac id s have a sparing ac tio n on th e essen
t i a l amino ac id s in r a ts as measured by follow ing u rin ary
and fe c a l ex c retio n , i t may be p o ssib le th a t th e growth
hormone, which has a sto rin g a c tiv ity or sparing a c tio n
on p ro te in (122), i s supplementary in th is re sp e ct to the
a c tio n reported by Pearson and Panzer fo r the e ss e n tia l
f a tty a c id s.
I t may be observed from fig u re 3 th a t no change
occurs in th e slope of th e growth curve fo r male r a ts
when th e dosage i s increased la te in th e experiment (22nd
week) from 20 mg. to 60 mg. per day, although data
gathered l a t e r on th e optimum le v e l of lin o le a te f o r
growth in d ic a te th a t th e le v e l of 20 mg. per day, used
as th e supplement in th e growth hormone experiment i s too
low. In th e case .of th e growth curves of th e female r a ts ,
presented in fig u re 4 , “a n o ticeab le re ta rd a tio n of growth
occurs from the, 22nd week, which i s a phenomenon th a t
109
occurs in fem ales on higher dosages o f lin o le a te in l a t e r
experiments* When th e d ie t was ehanged on the 25th week
! from a f a t- f r e e d ie t supplemented w ith lin o le a te to a
10 per cent cottonseed o i l d ie t , the ra te of growth of
th e males was l i t t l e a ffe c te d as determ ined by the slope
of th e growth curve; however, the growth r a te of the
female r a t was g re a tly in creased on th is diet* These d a ta ,
to o , are su b sta n tia te d by data obtained in l a t e r ex p eri
ments; th a t i s , th a t th e female r a t i s able to grow b e tte r
on a d ie t containing f a t (cottonseed o il) than on a sim i
l a r , f a t- f r e e d ie t supplemented with lin o le a te . liven
though th e le v e l of alpha-tooopherol in the growth hormone
experiment was low (0.11 mg. per day), lik e r e s u lts were
obtained when th is le v e l was increased approxim ately fo u r
fo ld . An extensive study of the r e la tio n of varying le v e ls
of tocopherol and of lin o le a te -with th e growth of male and
female r a ts re v e a ls no in te rre la tio n s h ip between th e two
fa c to rs and the growth of th e anim als (62).
The use of very sm all amounts o f d esiccated thyroid
in a f a t- f r e e d ie t caused rap id growth c e ssa tio n and e a rly
death of male and female r a ts (Tables VII and V III respec
tiv e ly ) . These e ffe c ts were not caused by increased
requirem ents fo r the B vitam ins, since no a lle v ia tio n of
th ese symptoms was a ffe c te d by doubling th e w ater-so lu b le
vitam ins in -th e d ie t. In f a c t, th e d e le te rio u s e f fe c ts of
th e th y ro id were accentuated by th e increased dosage of B
vitam ins (Group 7, Table V II). When generous amounts of
cottonseed o i l were added to the d i e t , no d e le te rio u s
e ffe c ts of d esiccated th y ro id on growth or su rv iv al time
were no ted . The amount of f a t needed to overcome the
adverse e f fe c ts of the th y ro id p re p aratio n must be very
low, since E rshoff (125) did not observe th ese e ffe c ts on
a f a t- f r e e d ie t containing a thyroid p rep aratio n which was
supplemented with 215 mg. o f cottonseed o il d a ily . I t i s
p o ssib le th a t fu rth e r work w ith th y ro id hormone w ill prove
t h i s method to be p ra c tic a l fo r the reduction of th e f a t -
d ep letio n time of r a ts .
The ad m in istratio n of m ineral o i l to r a ts on a
f a t- f r e e d ie t causes e a rly ce ssa tio n of growth (Table
X V III). Bacon (76) showed th a t th e tim e of p lateau of
weight was in d ir e c t re la tio n to th e le v e l o f th e m ineral
o i l in the d ie t. However, i t i s doubtful th a t t h i s method
could be of p ra c tic a l use, since th e syndrome of the d e fi
ciency i s q u ite unlike th a t obtained fo r r a ts on a f a t -
fre e d ie t but i s exactly lik e th a t observed fo r r a ts on a
v b io tin - fre e (raw egg-white) d ie t. However, th e re i s some
I l l
re la tio n between th e m ineral o i l induced syndrome and th e
e s s e n tia l f a tt y a c id s, sin ce th e e ffe c ts of the m ineral
o il can be la rg e ly reversed by the use of methyl lin o le a te .
An exam ination of the d ata obtained on the growth
of r a ts supplemented with dosages of m ethyl lin o le a te ,
a f te r th e d ep letio n period, ranging from 5 mg. to 50 mg.
per day re v eals th a t a logarithm ic dose-w eight gain curve
can be p lo tte d fo r th e male r a ts (Table V III, F igure 7 ).
This re la tio n s h ip e x iste d throughout the experim ental
period from 3 to 15 weeks. The augmented growth of male
r a ts on a 50 per cent fa t. d ie t over th a t obtained on th e
50 mg. of linoleate-supplem ented d ie t in d ic a te s th a t the
d a ily requirem ent of the male r a t fo r lin o le a te is higher
than 50 mg. In a l a t e r experiment (Table ZE, F igure 9)
a logarithm ic curve was obtained fo r male r a ts through
d a ily dosages of 100 mg. of lin o le ic a c id . S t i l l l a t e r
work by A nisfeld e t al* (62) in d ic a te s th a t dosages of
200 mg. per day may n o t be optimum under th e conditions of
th is type of experim ent.
As had been observed in a prelim inary way in th e
growth hormone experim ent, female r a ts reached a p lateau
in weight a t r e la tiv e ly low dosages of lin o le a te (Table
X, F igure 8 ). I t would appear from th e curves th a t th e
maximum growth was obtained with d a ily dosages of 10 to
20 mg. of lin o le a te . This observation was confirmed by
1 data obtained fo r female r a ts on lin o le ic acid in a l a t e r
I
I
experim ent (Table 211, F igure 9 ), in which maximum growth
was obtained on 50 mg. per day, but as e a r lie r shown,
higher dosages fa ile d to in crease the growth and h in ts of
an a c tu a l depression o f growth as compared w ith lower
dosages. Mead (111) has obtained id e n tic a l re s u lts with
female mice, in which optimum growth w ith fa t could not
be d u p licated w ith a lik e f a t- f r e e d ie t supplemented w ith
lin o le a te .
There i s no ready explanation fo r th e sex d if f e r
ences in e s s e n tia l f a tty acid requirem ent. However, i f
th e lin o le a te requirem ent can be re la te d to th e in te r
mediary metabolism of f a t , one might expect such d if f e r
ences to occur. I t has been found by Deuel and Gulick
(126) th a t th e fa s tin g ketonuria in th e human subject and
in r a ts w ith f a tt y liv e r s (127), as w ell as th e exogenous
ketonuria in r a ts and guinea pigs (128) is a fu n ctio n of
sex. The le v e ls of ex c retio n of th e ketone bodies are.
much higher in a l l cases in th e fem ale than in the male.
Deuel e t a l . (129) s ta te th a t th is v a ria tio n i s apparently
re la te d to th e more ra p id disappearance of glycogen and
113
tlie g re a te r proportion of f a t in th e tis s u e s of the female
r a t .
I t i s p o ssib le th a t th e apparen tly normal growth of
female r a ts on a f a t d ie t, which cannot be d u p licated on
a sim ila r d ie t supplemented with lin o le a te may be due to
th e need of the female fo r d ie ta ry f a t o th er than l i n
o le a te , fo r lin o le n a te in ad d itio n to lin o le a te , or fo r
some fa c to r in f a t n o t a t present id e n tifie d . Refined
cottonseed o i l , such as used in th e p o sitiv e co n tro l d ie ts
contains approxim ately 45 p er cen t of lin o le ic and about
2 per cent of lin o le n ic a c id s . Mead (111), in r e la tio n to
th e p o ssib le e ffe c t of lin o le n ic ac id , has observed th a t
th e presence of a trie n o ic (conjugated) aoid w ith dienoic
or w ith n e u tra l f a t in c re a se s g re a tly the ra te of absorp
tio n of th e dienoic or n e u tra l f a t from th e sm all in te s
tin e o f the mouse. F uture work on th is problem of sex
d iffe re n c e s in th e requirem ents o f the r a t fo r th e essen
t i a l f a tty ac id s i s w arranted by th e re s u lts obtained.
L inolenic ac id i s markedly in f e r io r to lin o le ic
a c id , both in growth-promoting a c tiv ity and in i t s cura
tiv e e ffe c t on the dermal symptoms of fa t-d e fio ie n c y
d ise a se . These r e s u lts a re a t variance w ith those of
B urr and co-workers (78), who rep o rted th a t lin o le n ic acid
114
was a c tu a lly su p erio r to lin o le ic acid in i t s a n tid erm ati-
t i s a c tio n and equal to i t in i t s a b i l i t y to produce growth,
i B urr afe a l . (6$) had p rev iously found th a t th e growth-
promoting e ffe c ts of lin o le ic and lin o le n ic ac id s are ad d i
tiv e ; however, M artin (77) has disagreed w ith th is view.
In the present t e s t s , methyl lin o le n a te was found,
when fed as th e only un satu rated f a tt y acid a t a le v e l of
10 mg. d a ily (Tables IX and X and F igures 5 and 6 ), to
have l i t t l e growth-promoting or cu ra tiv e a c tiv ity . When
a m ixture of lin o le a te and lin o le n a te was fed a t a le v e l
of 5 mg. each per day, growth of both male and female
r a ts was approxim ately th e same as fo r 5 mg. of th e l i n
o le a te ; however, a t a le v e l o f 10 mg. of each per day,
growth equal to th a t observed fo r' 20 mg. o f lin o le a te (
alone was obtained in d ic a tin g a d d itiv e growth e ffe c ts , a t
l e a s t , a t th e le v e l fed . This e f fe c t i s in agreement w ith
th a t observed by Burr e t a l . (66). The fa c t th a t no
growth was obtained on the 10 mg. le v e l of lin o le n a te ,
although th is acid may fu n ctio n in growth when lin o le a te
i s concom itantly given, in d ic a te s th a t each a c id plays a
sp e c ific ro le in th e animal organism. I t would thus
appear th a t lin o le ic acid i s req u ired in order to obtain
growth-promoting a c tiv ity from lin o le n ic a c id ; This
115
fa c to r may be of considerable im portance in fu rth e r in v es
tig a tio n o f the problem o f obtaining optimum growth of
female r a ts on a f a t- f r e e d ie t supplemented w ith e s s e n tia l
f a tt y a c id s.
The su p erio r growth e ffe c ts of methyl arachidonate
a re c le a rly shown by c a lc u la tin g the lin o le a te equivalence
of th e arachidonate by use of the logarithm ic dose-w eight
gain curve shown in F igure 9. The equivalent potencies
found a re tab u lated in Table X III. When methyl a ra c h i
donate was used as the so le u n satu rated f a tty acid supple
ment, growth was equal to 2.7 to 4 tim es th a t obtainable
w ith lin o le a te alone. These r e s u lts a re in clo se agree
ment w ith those of Turpeinen (32) who obtained growth
a c tiv ity w ith arachidonate equal to 3 tim es th a t of lin o
l e a te . The growth obtained w ith m ixtures of lin o le a te
and arachidonate are equal in magnitude to th e growth ex
pected from an equal amount o f arachidonate; however, i f
one su b tra c ts th e lin o le a te from th e lin o le a te equivalence
ca lc u la te d fo r th e m ixture, the a c tiv ity of the a ra c h i
donate moiety i s g re a tly enhanced (5.4 to,,,6.6 tim es the
''■ 4
a c tiv ity o f lin o le a te a lo n e ).
These r e s u lts , both w ith lin o le h a te and w ith arach
id o n ate, a re in agreement w ith m etabolic changes found in
th e anim al organism a f te r supplem entation w ith th e various
e s s e n tia l f a tty a c id s. V arious in v e s tig a to rs (88-91,97,98)
have found th a t lin o le ic and lin o le n ic ac id s a re converted
to arachidonic acid in the animal tis s u e s . Both Turpeinen
(32) and Smedley-MaoLean and Nunn (108) have s ta te d th e
©pinion th a t th e prim ary need o f the body is fo r a ra c h i
donic a c id . I t i s lo g ic a l th a t th e supplem entation of a
fa c to r req u ired per se by th e organism would produce
g re a te r a c tiv ity than would a precursor o f th a t fa c to r.
Sinoe evidence is. accum ulating (98) th a t th e trie n o ic acid
probably i s d esatu rated to th e dienoic before being con
v erted to th e te tra e n o ic a c id , the in f e r io r growth obtained
w ith th e lin o le n ic ac id , which must go through a t le a s t
one more step than lin o le ic before being converted to
arach id o n ic, i s understandable.
I t would seem, to o , since the animal organism does
not appear to be ab le to d e sa tu ra te more than one p a ir of
carbon atoms p er m olecule, th a t a t le a s t two m olecules of
lin o le ic (dienoic) a c id must be u tiliz e d to sy nthesize a
m olecule of the arachidonic a c id , a te tra e n o ic a c id . I f
th is i s so, th e higher a c tiv ity of the arachidonate and
th e lower a c tiv ity of lin o le n a te , when compared w ith th a t
o f lin o le a te , would be understandable from th e present
1 1 7
knowledge of th e in te rn e d ia ry metabolism of th e e s s e n tia l
f a tt y a c id s.
I t i s almost axiom atic th a t m etabolic systems in
th e body a re re v e rsib le under conditions which upset the
equilibrium . Thus, i t i s highly im probable th a t lin o le ic
acid would add a 2-carbon fragment to give a 20-earbon
u n it, which i s then fu rth e r d esatu rated to arachidonic
a c id , since Earner and Koenig (99) were unable to obtain
th e rev erse re a c tio n . These workers were unable to demon
s tr a te th a t / \ ^ *~ * ~ 4-eico sad ien o ic aeid ean lo se 2 carbon
atoms to give r is e to lin o le ic a c id .
P o sitiv e evidence th a t th e logarithm ic growth curve
obtained w ith lin o le ic ac id supplem entation using f a t -
depleted male r a ts can be used fo r th e b io lo g ic a l assay
of the e s s e n tia l f a tty ac id s p resen t in n a tu ra l and
tre a te d f a ts and o ils i s presented in Table X III, and in
F igure 9. The physico-chem ical methods have fa ile d to
perm it sep aratio n o f the n a tu ra l, b io lo g ic a lly a c tiv e ,
isom ers from the b io lo g ic a lly in a c tiv e isom ers of the
e s s e n tia l f a tt y a c id s. This shortcoming makes th e in tr o
duction of a b io lo g ic a l assay method fo r measurement of
th e b io lo g ic a lly a c tiv e isom ers very im portant, e sp e c ia lly
under present co n d itio n s, in which much of th e f a ts to be
118
used fo r d ie ta ry purposes undergo treatm en ts which cause
iso m erizatio n of u nsaturated f a tt y ac id s (1 ). One p a r ti
c u la r v a ria b le th a t must be co n tro lle d i s th e e ffe c t of
n e u tra l f a ts or of o th er f a tty acid s on the potenoy o f the
e s s e n tia l f a tty acid co n ten t. This problem has been solved
to a la rg e ex ten t in re cen t work in th is lab o ra to ry (62).
Assays of two hydrogenated vegetable f a t samples give
r e s u lts th a t in d ic a te th a t about h a lf of th e dienoic con
te n t of th e f a t , as determ ined spectro p h o to m etrio ally , is
b io lo g ic a lly a c tiv e .
The re s u lts obtained w ith chorionic gonadotropin
(Table XIX) in d ic a te th a t fa t-d e fic ie n c y does not a f fe c t
to any ap p reciab le ex ten t th e a b i l i ty of the gonads to be
stim ulated by a gonadotropic hormone. I t would th e re fo re
appear th a t any reduction in s iz e o r a c tiv ity of th e sex
organs are a r e s u l t 'o f u n d erstim ulation by th e p itu ita r y
gonadotropic hormones under, a condition of fa t-d e fic ie n c y .
A review of a l l th e accumulated data on food con
sumption under conditions of fa t-d e fic ie n c y and of varying
le v e ls of e s s e n tia l f a tt y acid supplem entation in d ic a te
th a t th e weight a tta in e d by th e anim als i s not a fu n ctio n
of th e le v e l of food eaten but i s more probably a fun ctio n
of th e degree of u tiliz a tio n re a liz e d from th e food
consumed. The tib ia e data accumulated fo r the various
experim ents in d ic a te th a t th e la rg e increm ents of weight
gain of r a ts on various f a tty acid supplements are hot
functions o f growth, as measured by t i b ia le n g th s, but
are probably re la te d to d ep o sitio n of body tis s u e in ste a d .
C H A P T E R VI
S U M M A R Y
! The e ffe c ts of exhaustive e x tra c tio n of foods, and
!
of such " s tre s s " fa c to rs as growth hormone, d esiccated
powdered th y ro id , and o f m ineral o i l on th e ra te o r degree
of fa t-d e p le tio n of the r a t have been stu d ied and evalu
a te d . E x trac tio n of th e food, and th e use of growth
hormone had no e ffe c t in reducing th e tim e of fa t-d e p le tio h
of r a ts . Thyroid and m ineral o il caused e a rly growth
i
re ta rd a tio n and e a rly death of fa t-d e p le tin g r a t s . How
ev er, fa c to rs o ther than fa t-d e p le tio n alone seem to be
involved and both methods would re q u ire much more in v e s ti
g atio n before e ith e r could be used to reduce the d ep letio n
tim e.
The in te rre la tio n s h ip o f lin o le ic , lin o le n ie , and
arachidonic acid s have been in v e stig a te d in re la tio n to
t h e i r e ffe c t on th e growth of fa t-d e p le te d r a ts . L inolenie
aeid was found to have th e poorest growth and cu rativ e
p o te n tia l except in the case where i t was fed sim ultan
eously a t a 10 mg. le v e l per day w ith an equal amount of
lin o le ic a c id . A rachidonic acid was found to possess 2.7
to 4 tim es th e growth a c tiv ity of lin o le ic ac id , when fed
alo n e, and may have as much as 6 tim es th e a c tiv ity of
1 2 1
lin o le ic acid when the two a re fed to g e th e r. An examina
tio n of the p o ssib le m etabolic re la tio n s h ip s of the th re e
most im portant e s s e n tia l f a tty ac id s has been d iscu ssed .
Sex d iffe re n c e s in r a ts a re rep o rted in th e re q u ire
ment fo r and in th e u t iliz a t io n of lin o le ic a c id . L inear
re la tio n s h ip i s obtained between weight gain and the lo g
arithm!/. of the dose of lin o le ic acid , fo r male r a ts . The
accumulated data show th e requirem ent fo r lin o le ic acid fo r
th e fa t-d e p le te d male r a t to be in excess o f 100 mg. per
day. The female r a t experiences growth re ta rd a tio n a t the
hig h er dosages of lin o le ic a c id , but th e optimum dosage
fo r th e female r a t appears to be under 50 mg. o f lin o le ic
ac id per day.
A p relim inary bioassay o f th e e s s e n tia l f a tty acid
content of f a ts i s presented w ith physico-chem ical analyses
of th ese f a ts fo r comparison of the two methods.
A study of th e e ffe c ts of chorionic gonadotropin on
th e sex organs of male and fem ale, fa t-d e p le te d , and
e s s e n tia l f a tty a c id -re fe d r a ts , re v e a ls no apparent
damage to th ese organs by fa t-d e fic ie n c y . I t i s probable
th a t th e re is some depression o f ex cretio n of p itu ita r y
gonadotropic hormone caused by fa t-d e fic ie n c y in th e r a t .
The gain in weight experienced by dep leted r a ts ,
1 2 2
when fed supplements of th e e s s e n tia l f a tt y a c id s, does
not appear to be re la te d to th e amount of food consumed,
nor does th e ■weight-gain seen to he re la te d to growbh as
measured by tib ia len g th . R ather, th e w eight-gain would
appear to r e s u lt from tis s u e d ep o sitio n .
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JUffliversfcy o f Southern CaSfofnf®
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Greenberg, Samuel M
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Core Title
A nutritional evaluation of the essential fatty acids in the rat
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Doctor of Philosophy
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Biochemistry and Nutrition
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health sciences, nutrition,OAI-PMH Harvest
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