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The effects of irradiation upon lipid and carbohydrate metabolism in the rat

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The effects of irradiation upon lipid and carbohydrate metabolism in the rat

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Content T H E EFFECTS O F IRRADIATION U PO N
LIPID A N D C A R B O H Y D R A T E
M ETA B O LISM IN T H E RA T
toy
Ronald L* Searcy
A D isse rta tio n Presented to the
FA CU LTY O F T H E G R A D U A T E SC H O O L
UNIVERSITY O F SO U T H E R N CALIFCENIA
In P a r tia l F u lfillm en t of the
Requirements fo r the Degree
D O C T O R O F PH X LO SO H IY
( Biochemistry)
June 19*>7
UMI Number: DP21576
All rights reserved
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ph. D fi;0 '57 S V39
This dissertation, written by
Ronald L* Searcy
under the direction ofM:?..Guidance Committee,
and approved by all its members, has been pre
sented to and accepted by the Faculty of the
Graduate School, in partial fulfillment of re
quirements for the degree of
D O C T O R O F P H IL O S O P H Y
..JOHN ..............
Acting Dean Dean
u *
(£i f *
Date....
Guidance C om m ittee .
v Chairman
.....|j0rQ4^dL£
S . :
A C K N O W L E D G E M E N T S
I would lik e to express my ap p reciatio n fo r the valuable a id
extended to me lay pr* M * G * Morehouse and the guidance committee*
In ad d itio n I would lik e to thank th e f • S# Atomic Energy Commission
fo r fin a n c ia l support and the A llan Hancock Foundation fo r th e use
of lab o ra to ry f a c ilitie s *
TA B ES O F C O N T E N T S
P A G E
HISTORICAL IN TRO DU CTION . . . . . . . . . . . .......................... 1
Biochemical E ffe cts of Irra d ia tio n • • • • • • • • • • 2
The a c tio n of io n izin g ra d ia tio n upon l ip i d
metabolism 2
The a ctio n of io n izin g ra d ia tio n upon carbohydrate
metabolism • • • ?
S T A T E M E N T O F TH E P R O B L E M A N D PLA N OF ATTACK................ .... 9
M A TERIA LS A N D M E T H O D S • • • • • • • • • • • • • • • • • • • 11
Techniques Employed in Irra d ia tin g R ats ••• • •• • • 11
Methods Used fo r Iso la tin g Tissue L ipids •• ••••• 11
S urgical techniques employed fo r is o la tin g the
organs . . • • • • • • • • • • • • • • • • • • 11
Determ ination of the lip id content of th e liv e r
and in te s tin a l tr a c t • • • • • • • • « . . • • • 12
Methods of A nalysis of Tissue Lipids • • • • • • • • • lit
Techniques employed fo r separating n e u tra l f a t
components * . . . • • . • • • . * lit
Methods of saponifying the n e u tra l f a t and phospho
lip i d samples • • • • • • • • » • • « • • • • • 16
D eterm ination of the ra d io a c tiv ity of th e lip i d
samples 17
Techniques Employed fo r In V itro Studies • •• ••• • 17
Methods Used fo r the D eterm ination of Fecal and In te s -
_______t3.nal_Lxpxd3_»— •__•_• •_* •_* « •___•____18.
iv*
PA G E
Is o la tio n of th e fe c a l n e u tra l f a t . . • • . • • • 13
Is o la tio n of th e in te s tin a l lip i d resid u es • . • • 19
Determ ination of L iver Glycogen;: . . . . . . • . • • • • 20
, Slf’ K R IM E N T A L RESULTS • • .. . . . « . . • • * . • • » « • • 21
The Measurement of Tissue Lipogenesis in Normal and
Irra d ia te d R ats • • • • • • • • • • • . • • • • • • • 21
The incorporation of lab eled molecules in to liv e r
neutral fat •••••••••••••••••« .21
The incorporation of lab eled molecules in to small
in te s tin a l n e u tra l f a t • • ...................................... 35
The incorporation of lab eled molecules in to liv e r
phospholipids • • » » » • • • * • • • . . . • « 1 + 1
The in co rp o ratio n of lab eled molecules in to sm all
in te s tin a l phospholipids . • • • % • • • • • • « . W +
The form ation and Turnover of L iver Glycogen in Normal
and Irradiated Rats ......... 1+6
The incorporation o f a ce ta te -l-C 1^ in to liy e r
glycogen . • • • • • • • • • • • • • • • • • • • I4 . 6
The incorporation of g ly c in e -l-C ^ in to liv e r
glycogen • • • « • • • • • • • • • « • • • • • • 1 + 6
The incorporation of uniform ly lab eled glucose
in to liv e r glycogen . « • • • • • • • • • • • • 1 + 8
DISCUSSION.................................................................................................. 51
S U M M A R Y A N D CONCLUSIONS.................................................... .... . . . . 60
tvtrt.tqgraphy . . . . . . . . . . . . . . . . . o . . ». »_____ 62
LIST O F T A B L E S
TA BLE PA G E
! • The turnover of a ce ta te -l-G -^ in the liv e r n e u tra l
f a t of normal and irra d ia te d r a t s • ••• • •• * • 2$
II* The in v itr o incorporation of a c e ta te -l-C ^ t in to
the liv e r lip id s of normal and irra d ia te d r a ts . • 26
H I* The lip i d fra c tio n a tio n of n e u tra l f a t iso la te d
from the liv e rs of normal and irra d ia te d r a t s * . • 28
IV* The in vivo incorporation of eaproate-l-C 1^ in to
the liv e r n e u tra l f a t of normal and irra d ia te d
r a t S e e * o e * e * « « * • • * * * • * * * . * * • 2^
V * The in v itr o incorporation of e a p ro a te -l-C ^ in to
the liv e r lip id s of normal and irra d ia te d r a ts • • 31
VI* The in vivo and in v itr o incorporation of glucose-
1-G*^ in to liv e r n e u tra l f a t o f normal and irra d ia
ted r a ts • • • « • • * • • • . • • • • • • « * . • 32
VII* The lip i d fra c tio n a tio n of n e u tra l f a t is o la te d from
the in te s tin e s of normal and irra d ia te d r a ts . • • 37
VIII* The is o la tio n of fe c a l f a t from normal and irra d ia
te d r a t s • • • • • • « • • « • • • • * • • • • • • 39
IX* The l ip i d fra c tio n a tio n of n e u tra l f a t is o la te d from
the feces of normal and irra d ia te d r a ts • • • • * • U O
X * The incorporation of a c e ta te - l- C ^ in to the liv e r
glycogen of normal and irra d ia te d r a ts . * • • * • U 7
XI* The in co rp o ratio n of glycine-l-C ^* in to the liv e r
________ glycogen of normal and irra d ia te d r a ts , ** **** h9
v ii*
T A B LE
XII* The incorporation of uniform ly lab e le d glucose in to
the liv e r glycogen of normal and irra d ia te d r a ts • •
PA G E
50
LIST O P F IG U R E S
FIGURE P A G E
1* In vivo incorporation of a c e ta te -l-C -^ and g ly ce ro l-
1 -C ^ in to l iv e r n e u tra l f a t ......................................... 22
2* Glyeine-l-G^* incorporation in to liv e r n e u tra l
f3% o o e e e o w * * • • • • » • • • • • - • • • • • 3 l|»
3* In vivo incorporation of acetate-l-G ^* and g ly eero l-
1-C-^ in to in te s tin a l n e u tra l f a t . « • • • • • • • 36
Incorporation of a c e ta te - l-C ^ and g lycerol-l-G -^
in to in te s tin a l lip i d resid u es i»2
to vivo incorporation of a e e ta te -l-C -^ and g ly ce ro l-
1-G^* in to liv e r phospholipids • • • .................... • i»3
6 . to vivo incorporation of aeetate-l-C ^* and g ly ce ro l-
1-G*^ in to in te s tin a l phospholipids •• • • • • • • 1*5
HISTORICAL IN TRQ D U CTIO N
While cosmic rays have probably accounted fo r irra d ia tio n
e ffe c ts of some 0,3 mr/day and n a tu ra lly occurring ra d ia tio n s have
been in ex istance since creation* only re c e n tly have b io lo g is ts
come to re sp e c t these small* but constant sources of io n izin g ra d i
ations* Today* in a d d itio n to cosmic rad iatio n * we must concern
ourselves w ith even more powerful em ission sources* As yet* no
c le a rly defined concepts have been proposed to adequately explain
th e many responses to irra d ia tio n exposure* Attempts to fin d these
answers have been accelerated w ithin re c e n t years because of the
wide a p p lic a tio n of m aterials and sources of io n izin g ra y s.
Much a tte n tio n of la te has been drawn to the p hysical and
chemical aspects of tis s u e irra d ia tio n * Workers are now more or
le s s agreed th a t the io n iz atio n s produced by x-rays are th e i n i t i
a to rs of the subsequent b io lo g ic a l reactio n s* B utler (1) has
c la s s ifie d th e rea ctio n s occurring a fte r protoplasm ic exposure to
ra d ia tio n in to a ehain of th ree re la te d events* namely* (a*) the
prim ary io n iz a tio n , (b*) subsequent chemical rea ctio n s and, (c*)
the b io lo g ic a l response* The i n i t i a l ionizing substance has been
proposed by Hugh (2) and others (3* it) to be w ater leading to the
form ation of peroxides which are activ e in subsequent b io lo g ic al
oxidations* In ad d itio n to a chain reaction* Hugh has pointed out
th a t ra d ia tio n s may have a d ire c t and instantaneous e ffe c t on genes*
chromosomes and other protoplasm ic constituents* He a lso has postu
la te d an in d ire c t e ffe c t by way of tox in s and hormonal se cre tio n
2
\
which may he somewhat more delayed, As th is worker mentions i t is
o ften d if f ic u lt to separate the two types of e ffe c ts since there
in v ariab ly is a la te n t period before the b io lo g ic a l response mani
f e s ts i t s e l f . For example, a genie m utation may be immediate but
not noticeable fo r sev eral g en eratio n s.
Biochemical E ffects of Irra d ia tio n
The actio n of ionizing ra d ia tio n upon lip id metabolism.
Bacq e t a l . (5) have compared the whole body f a t le v e ls in normal
fa s te d male r a ts and those receiving 1000 r a t periods of seventy
and one hundred f i f t y hours a f te r irra d ia tio n . The f a t contents
of fa s te d x-rayed r a ts remained constant a t about f?0 per cent of
the i n i t i a l value while the f a t le v e ls of the co n tro l groups de
clin ed sharply to about 20 per cent of the i n i t i a l v alu es. These
authors have in te rp re te d th is to in d icate e ith e r an increased syn
th e s is or a decreased u tiliz a tio n of lip id in the irra d ia te d anim als.
A nalysis of to ta l liv e r f a t by these workers showed x-rayed and con
t r o l groups to have sim ila r iodine numbers. However, in g estio n of
_ f a t by the irra d ia te d animals tended to produce hepatic f a t t y in
f i l t r a t i o n a t much lower le v e ls than in c o n tro l groups. From the
gross appearance of the l iv e r , Ord and Stoeken (6) noted a sim ilar
f a t deposition in guinea pigs subjected to 1000 r .
The lip i d metabolism of bone marrow has been in v estig ated
since i t i s p a rtic u la rly se n sitiv e to irr a d ia tio n . Altman e t a l .
(7) by means of acetate-2-C *^ incorporation have measured bone mar
row lip o g en esis in v itr o w ith samples obtained from normal r a ts and
3
those receiv in g 800 r . The incorporation of was observed to
be increased in both sa tu rated and unsaturated f a tty acids immedi
a te ly a f te r irra d ia tio n * 0ne week a fte r roentgen treatm ent labeled
acetate incorporation in to the unsaturated acids remained elevated
while th a t in to the sa tu ra te d acids was reduced in comparison to
controls* Bernheim e t a l . (8) have noted a lte ra tio n s in the per
oxide form ation of bone marrow obtained from x-rayed rab b its*
These workers found irra d ia tio n to lower both the sulfhydryl content
and the an tio x id an t a c tiv ity of bone marrow homogenates leading to
peroxide form ation in the f a t . They have concluded th a t such per
oxides may be responsible fo r marrow changes noted a fte r ir r a d i
a tio n since these compounds have considerable water s o lu b ility and
can re a d ily d iffu se in to the n o n -lip id po rtio n s of the marrow*
Derangements in serum lip id and lip o p ro te in le v e ls have re
ceived considerable a tte n tio n since Rosenthal (9) noted a charac
t e r i s t i c opalescence in the serum of r a ts crying of irra d ia tio n
in ju ry . In th is species the conversion of low d en sity lip o p ro tein s
to high d en sity components appear to be responsible fo r changes in
lip o p ro te in le v e ls a f te r irra d ia tio n (1©). Hewitt e t a l . (11) noted
a re v e rsa l in th is phenomenon when heparin was adm inistered* These
workers have compared lip o p ro te in le v e ls in sev eral species a fte r
irra d ia tio n since considerable v a ria tio n has been found to occur*
The ra b b it showed larg e increases in the concentration of low den
s i t y lip o p ro te in s one day a fte r roentgen treatm ent* An elev atio n
in the highest d ensity c la ss was noted in the dog te n to th irte e n
h
days a fte r exposure while the r a t shewed an increase in these lip o
p ro tein s in only th ree days subsequent to treatm en t. On the other
hand, a t the l a t t e r in te rv a l the x-rayed mouse exhibited decreases
in the concentration of the high d en sity lip o p ro te in s.
The work of Hewitt e t a l . (11) appears to im plicate the high
lip o p ro te in le v e ls as being diagnostic of irra d ia tio n death in the
rat> ra b b it and dog. S im ilar term inal ris e s in serum lip id le v e ls
have been reported by Goldwater and Entenman (12). These workers
have considered the lik e lih o o d of sp e c ific hormonal d efects in the
adrenal cortex and thyroid glands a ris in g from irra d ia tio n to be
responsible fo r the elev ated serum l ip i d p ic tu re . F u rth er, they
p o stu late a possible lin k between the abnormal lip o p ro te in metabol
ism and the observed irra d ia tio n in ju ry to the liv e r .
Com atzer and co-workers (13) have in v estig ated other lip id
c o n stitu en ts in the blood a fte r subjecting dogs to $00 r whole body
ra d ia tio n . These workers contend th a t reg ard less of re p o rts of ab
normal increases in liv e r phospholipid production a fte r x-ray t r e a t
ment (lit) these changes are* not re fle c te d in elevated plasma phospho
lip id concentrations# However, in the r a t , Neve and Entenman (15)
have shown increases in the le v e ls of phospholipid phosphorous f o l
lowing adm inistration of 7$0 r . P rogressively higher lip i d le v e ls
were noted w ith increasing x -ray dosage up to 500® r . The plasma
volumes and p ro te in le v e ls showed very l i t t l e v a ria tio n during the
time in te rv a ls stu d ied or w ith roentgen dosages employed.
Since the irra d ia tio n response v aries so markedly w ith species,
5
Entenman a t a l . (16) have extended th e ir i n i t i a l stu d ies to include
sev eral other types of anim als. The plasma phospholipids were ob
served to be increased in a l l animals studied although the onset
and degree of ele v atio n d iffe re d w ith the sp e cie s. In ra b b its ,
guinea pigs and mice a r is e in plasma phospholipid was noted w ithin
the f i r s t tw enty-four hours a f te r irra d ia tio n while in the dog the
le v e l dropped a t th is tim e and did not ris e u n til a fte r the nin th
day. In agreement w ith these fin d in g s, Steadman and Thompson (17)
have reported rap id elev atio n s in serum t o ta l lip id and phospholipid
in sev eral species subjected to ionizing ra d ia tio n .
There is evidence, although a t p resen t incom plete, th a t i r
ra d ia tio n may a l t e r liv e r lip id metabolism. Confusion has a rise n
in these stu d ies through the use of a v a rie ty of animal species and
lack of uniform irra d ia tio n dosages. Much controversy thus e x is ts
as to the s e n s itiv ity of the liv e r to ionizing ra d ia tio n . Pohle
and Bunting (18) noted no h epatic tis su e necrosis when 600 to 2500 r
were adm inistered to the upper abdominal area of r a t s . On the other
hand, A riel (19) using sim ila r roentgen doses has observed liv e r
c e lls to be in ju re d or destroyed in proportion to the x -ray in te n
s i t y . E arly work done by E llin g e r (20) using massive roentgen doses
showed increased amounts of sudanophil f a t p resen t in the liv e rs of
m ice, lik ew ise, Entenman and Weinman (21) have rep o rted a lte ra tio n s
in h epatic phospholipid metabolism. An increased incorporation of
p32 in to th is fra c tio n was observed by these workers in liv e r s lic e s
from r a ts given 1000 to 2500 r . However, they could not support
6
these findings by in je c tin g p32 in to the in ta c t rat* Likewise,
Florsheim and Morton (22) have reported no s ig n ific a n t a lte ra tio n
in the incorporation of p e rip h e ra lly adm inistered p32 in to the b rain
and l iv e r lip id s of x-rayed ra ts* The c h o le ste ro l eontent of the
liv e r was observed by Gernatzer e t &U (23) to be elevated in r a ts
i
given 700 r w hile the choline-containing phospholipids were de
creased*
The lip id metabolism of the sm all in te s tin a l t r a c t has not been
in v estig ate d in animals su ffe rin g from irra d ia tio n in ju ry although
i t has been found to be one of the most ra d io se n sitiv e tis s u e s in
the body* Conard (2li) has observed the sm all in te s tin e o f mice to
be damaged by as l i t t l e as $0 r« This worker has suggested th a t
the observed fu n ctio n al changes in to n ic ity and m o tility are prob
ably re la te d to the e a rly ra d ia tio n symptomology of anorexia, weight
lo ss and diarrhea* I n te s tin a l desquamation re s u ltin g from ir r a
d ia tio n has been im plicated by Mead e t al* (2$) in the form ation
of the increased fe c a l f a t noted in mice m aintained upon a f a t fre e
diet* Likewise, Dobbs and Webster (26) in e a rly in v e stig a tio n s,
reported elev atio n s in th e fe c a l f a t of p a tie n ts receiving x -ray
therapy* However, these workers a ttrib u te d th is to an impairment
in in te s tin a l absorption* In c o n tra st to both re p o rts , Coniglio
e t al* (27), employing p a ir-fe d r a t s , have concluded th a t d istu rb
ances in f a t balance do not follow x -irra d ia tio n * Both x-rayed
and p a ir-fe d co n tro ls ex h ib ited decreases in fe c a l f a t excretion*
However, in l a t e r stu d ies by these in v e stig a to rs (28) an increased
7
fe c a l f a t ex cretio n was reported in x-rayed Rhesus monkeys* The
lack of d iffe re n tia tio n between the d ire c t e ffe c ts of irra d ia tio n
and those of the ensuing anorexia appear a t le a s t p a r tia lly re
sponsible fo r these c o n flic tin g r e s u lts .
The actio n of ionizing ra d ia tio n upon carbohydrate metabolism.
There i s no w ell su b sta n tia ted evidence of the in a b ility of the i r
ra d ia te d animal to oxidize glucose by way of pyruvate and the t r i
carboxylic acid cy cle. However, d e fin ite a lte ra tio n s have been
shown to be produced in the carbohydrate metabolism of the x-rayed
anim al. Lourau and L aritique (29), studying the blood sugar p ic tu re s
of irra d ia te d guinea p ig s, have found hyperglycemia and a diminished
glucose tolerance* This phenomenon cannot be explained on the b asis
of a g re a te r absorption of glucose by the in te s tin a l t r a c t since a
60 per cent reduction in glucose absorption was noted in the r a t
sm all in te s tin e by Barron e t a l . (30)* With irra d ia tio n doses
ranging from only 5© to $00 r , these workers noted an abrupt drop
in the in te s tin a l re s p ira tio n ra te to accompany the reduced absorp
tio n capacity* D etriek and co-workers (31) have su b sta n tia ted these
re s u lts by the use of glucose perfusion techniques upon in te s tin a l
segments from normal and irra d ia te d r a t s .
F ish el (32) was f i r s t in 19l*l to rep o rt th a t massive lo ca liz ed
irra d ia tio n doses affe cte d glycogen deposition in the liv e r* P rosser
(33) observed liv e r glycogen le v e ls to be lowered a fte r x -ray ex
posure and a ttrib u te d th is decrease to anorexia* I f t e r tre a tin g
r a ts w ith $00 to 2000 r , Worth and Nims (3U) noted elevations in
8
h ep atic carbohydrate sto re s up to fo rty -e ig h t hours a f te r exposure.
N either shielding of the liv e r or p itu ita r ie s re su lte d in a decrease
in tiie liv e r glycogen le v e ls (3f>) elev ated above c o n tro l le v e ls by
roentgen treatm en t.
Several authors ( 36, 37) have concluded th a t irra d ia tio n must
a ffe c t h ep atic u tiliz a tio n of carbohydrate. During fa s tin g , Denson
e t a l . ( 38) have observed liv e r glycogen le v e ls to be elev ated and
a ttrib u te d such an e ff e c t to an impairment in g ly c o g e n o ly sis. How
e v er, Nims and Sutton (39) have suggested th a t in the x-rayed animal
th ere may be a stim u latio n in gluconeogenesis mediated by th e p itu ita ry
adrenal system . Most re c e n tly , Kay and Entenman (ij.0) have stu d ied
l iv e r glycogen le v e ls in the fa ste d r a t a fte r roentgen treatm en t.
Twenty-four hours a fte r irra d ia tio n the carbohydrate sto re s mere
found to increase in proportion to the x -ray dose. These workers
have suggested th a t the synth esis o f carbohydrate in th e tre a te d r a ts
a ris e s from non-carbohydrate p recu rso rs, e ith e r body p ro tein s o r f a t s .
To t e s t th is p o stu la te , th ey have measured the d a ily ex cretio n of n in -
h y d rin -reactiv e compounds in the urine and plasma of fa s tin g x-rayed
r a t s . I t mas found th a t during the periods of elevated glycogen the
la rg e s t e x cretio n and the hig hest plasma le v e ls of ninhydrin-reactive
\
substances were observed. F urther id e n tific a tio n of these compounds
in the urine of irra d ia te d animals showed th a t valine was the only
glycogenic amino acid excreted in elev ated amounts. Kay and Entenman
have thus concluded th a t glycogenic amino acids rele ased from the i r
radiation -induced p ro te in catabolism are taken out of c irc u la tio n fo r
hepatic glycogenosis and do not appear in the u rin e .___________________
ST A T E M E N T O F T H E P R O B L E M A N D PL A N O F A T T A C K
I t can re a d ily be seen th a t many of the m etabolic aspects of
the irra d ia tio n syndrome are n o t, as y e t, f u lly understood* P a rti
cu lar among, these is the e ffe c t of ionizing ra d ia tio n upon lip id
and carbohydrate metabolism. The purpose of the in v e stig a tio n s pre
sented here was to study irrad iatio n -in d u ced a lte ra tio n s in these
processes by means of the incorporation of lab eled p recu rso rs. The
c a p a b ility of the liv e r and sm all in te s tin e to u tiliz e a c e ta te -C ^
and glycerol-G^* was in v estig ate d over a tw enty-four hour period*
This made possible an accurate assessment of the turnover ra te s of
the tagged molecules in both n e u tra l f a t and phospholipid fractio n s*
In a d d itio n , incorporation stu d ies were c a rrie d out in v itro employ
ing liv e r s lic e s to determine i f the a lte ra tio n s noted in the hepatic
lipogenesis of in ta c t x-rayed r a t were m anifest a t the c e llu la r le v e l.
The form ation and ex cretio n of fe c a l lip id was measured in nor
mal and exposed r a ts to determine whether irra d ia tio n in ju ry to the
in te s tin a l w all might a lso a ffe c t these processes* Fecal lipogenesis
was evaluated in the d iffe re n t animal groups in two ways* F ir s t,
the amounts of lip id excreted in the feces was measured, and second,
the u tiliz a tio n of in tra p e rito n e a lly C ^ -a c e ta te fo r fe c a l lip id
synthesis was compared in normal and x-rayed animals* I t was pos
s ib le to d ire c tly im plicate the in te s tin a l t r a c t in fe c a l lip id
form ation by measuring the a c tiv itie s of the in te s tin a l lumen r e s i
dues a fte r e ith e r lab eled a cetate or g ly cero l in je c tio n .
The glyceride and fre e f a t t y acid composition of th e liv e r and
10
in te s tin a l n e u tra l f a t was stu d ied in normal and tre a te d r a ts to
support in te rp re ta tio n s based upon observed d ifferen ces in G -^-in-
corporation* The ro le played by various lab eled precursors in form
ing f a t t y acid and n o n -fatty acid m oieties of the lip id molecules
was assessed by sa p o n ificatio n analyses of n e u tra l f a t and phospho
lip id s*
Since th ere i s much co n trad icto ry evidence regarding the effe cts;
of ra d ia tio n upon h epatic glyeogenesis, the liv e r glycogen le v e ls of
normal r a ts and those subjected to 1000 and 15>00 r were in v e stig a te d .
Reduction of glycogen le v e ls by fa s tin g p rio r to x -ray exposure was
u tiliz e d to demonstrate whether th e elevated glycogen le v e ls ex
h ib ite d by tre a te d r a ts re s u lte d from synthesis; or a re ta rd a tio n in
glycogenolysis* The u tiliz a tio n of lab eled a c e ta te , glycine and
glucose fo r glyeogenesis was measured to determine the possible gly
cogen precursors in fa s te d normal and x-rayed ra ts*
M /ITERIALS A H D M E T H O D S
Techniques Employed in Irra d ia tin g Rats
Female rats neighing 175 ♦ 15 grains (which had been maintained
on a purina diet) were randomly selected from the albino B*S*S* strain*
S' . . . .
Baring x -ray exposure the animals were placed in a wooden box divided
by means of c e llu lo se a c e ta te s tr ip s in to fo urteen compartments w ith
a 1/8 inch c ellu lo se a c e ta te top* Each compartment held one r a t com
fo rta b ly and did not perm it any change in position* The top and
bottom of th e box were p e rfo ra te d a t reg u la r in te rv a ls to ensure
adequate v e n tila tio n during x -ray treatm ent* The recep tacle contain
ing the animals was placed on a tu rn ta b le 76 cm* from the tube and
slow ly ro ta te d a t 2 r*p*m* A fan was d ire c te d toward the box to
prevent overheating during irra d ia tio n *
The irra d ia tio n was c a rrie d out by using a G * E* Maximar 250
machine, operated a t 250 k.v* and 15 ma* w ith a 5 mm. copper and a
1 m m * aluminum f i l t e r , plus a copper parabolic f ilte r * The l a t t e r
i s a non-uniform f i l t e r which produces a f l a t isodose surface of
x -ra y in te n sity * The dose r a te was measured w ith a Victoreen ro en t
gen m eter and found to be 17*9 r per minute w ith only n eg lig ib le
d a ily v ariatio n *
Methods Used fo r Is o la tin g Tissue Lipids
S urgical techniques employed fo r is o la tin g the organs*
Both normal and x-rayed animals were in je c te d in fra p e rito n e a lly
w ith one of the various C^k-labeled compounds in the follow ing
amounts; 1*0 jic acetate-l-C ^* (0*01; mg*), 1 .0 pc glyeerol-l-C ^*
12
(3*1 mg»), 1*0 jxc caproate-l-C ^* (1.0 mg.)> 1 .0 ;ac glycine (0.08 m g.),
0.2 )ic glucose-1-0^* (0.2 mg.) o r uniform ly lab eled glucose 1 .0 pc
(0.01 mg*). A fter the p rescrib ed absorption in te r v a ls , the r a ts
uere an esth etized w ith Nembutal. Ihen unconscious, the animals Here
neighed and th e abdomen opened by a m idline incisio n * The v isc era
Here moistened w ith sa lin e and moved out of the abdominal cavity*
The liv e r Has taken out immediately and placed in a m ixture of dry
ice and eth an o l. The in te s tin a l t r a c t Has removed a t th e p y lo ric
and cecal junctions* The lo ss of any of i t s contents was prevented
by the use of hem ostats.
Determ ination o f the l ip i d content o f the liv e r and in te s tin a l
t r a c t . The methods used Here a s lig h t m odification of those pro
posed by Goldman e t al* (i& ). A fter free zin g , the liv e r s and sm all
in te s tin e s were placed in a Waring blender and minced in £0 ml* of
ethanol* S im ilar organs from two animals were pooled and thoroughly
mixed in th is manner*
E ther was then added to a l l samples to approximate a mixture
of aleo h o l-eth er (1 :1 ). The tis s u e minces were tra n s fe rre d q u an tita
tiv e ly from the blender to 2£© ml* Erlenmeyer fla s k s and incubated
fo r two hours in a w ater bath a t J+0° C* The samples were then de
canted through f i l t e r s in to 2£0 m l. c o lle c tio n flask s* With th is
f i r s t decantation as much of the m aterial as p o ssib le was re ta in e d
in the incubation flask s* Approximately £0 ml* of the alco h o l-eth er
m ixture was added and the incubation was repeated* With the next
tra n s fe r to the c o lle c tio n fla s k s a l l of the m ateria l was washed
13
int© the f i l t e r .
The f i l t e r papers containing th e minced samples were placed in
25 cm. lhatm an Soxhlet thim bles and e x tra c te d fo r twelve hours w ith
e th y l ether* The ex tracted lip id m aterial from each sample was then
combined w ith the appropriate alco h o l-eth er f i lt r a t e * The solvent
volumes were evaporated to approxim ately 5 ml* tinder the reduced
pressure obtained w ith a water pump* In atmosphere of carbon dioxide
was used to prevent the oxidation of u n saturated acids* To increase
the speed of the d i s t il l a t i o n the so lu tio n s were warmed in a w ater
bath to 1*0° C.
These concentrates were then e x tra cted w ith sm all p ortions of
petroleum e th e r (Skelly A) using 125 m l. sid e a m f la s k s . Small por
tio n s of petroleum e th e r were sw irled with the concentrate and the
top la y e r was poured o ff in to a weighed 50 m l. centrifuge tube* Ex
tra c tio n s were repeated u n til 50 ml* of petroleum e th e r had been used*
The volume was reduced to 1-2 ml* by allowing the e th e r e x tra c ts to
stand a t room temperature* The tubes were then f i l l e d with anhydrous
acetone to p re c ip ita te the phospholipid. To ensure a complete pre
c ip ita tio n w ithout m oisture absorption, a l l tubes were placed in a.
d e ssic ato r fo r tw enty-four hours.
The acetone soluble m ateria l (n e u tra l f a t ) was separated from
the acetone insoluble fra c tio n (phospholipid) by centrifugation*
The supernatant acetone so lu tio n was decanted in to weighed 125 ml*
Erlenmeyer flask s* The process was repeated a f te r adding another
50 m l. p o rtio n of anhydrous acetone to the centrifuge tu b e. The
lit
p re c ip ita te d phospholipid was placed in the vacuum oven to dry fo r
two to th ree hours* The tubes were then weighed and the amounts of
phospholipid determined* To obtain the n e u tra l f a t , the acetone
washes were placed on hot p la te s a t $0° C and taken alm ost to dryness.
They were then placed in the vacuum oven fo r two to three hours,
weighed and the n e u tra l f a t c alcu late d .
Methods of A nalysis of Tissue Lipids
Techniques employed fo r separating n e u tra l f a t components* An
anionic re s in was used fo r the i n i t i a l separation of free f a t t y acids
from th e acetone-soluble lip i d fra c tio n . P rio r to u se, about 20 grams
of Amberlite IRA-1*O0 was placed in a beaker containing 100) ml* of 12
per cent sodium hydroxide* This m ixture was s tir r e d m echanically fo r
fiv e m inutes. The re s in mixture was then f i lt e r e d through a Buchner
funnel and washed rep eated ly w ith d i s t il l e d w ater u n til a c h lo rid e-
fre e n e u tra l f i l t r a t e was obtained. At th is p o in t, the re s in was
rin se d w ith a sm all amount of wet e th e r and tra n s fe rre d to a beaker
containing a weighed amount of n e u tra l f a t dissolved in wet ether*
.This mixture-was s tir r e d m echanically fo r-fiv e minutes and-again ~
f i lt e r e d through a clean Buchner funnel* Small amounts of wet e th e r
were used to wash any occluding glyceride from the re s in and the
to ta l f i l t r a t e was c o lle c te d , washed w ith w ater and d ried w ith an
hydrous sodium su lfa te * The glyceride fra c tio n was obtained by
evaporating to dryness*
The re s in now containing the f a t t y acids: was tra n s fe rre d quanti
ta tiv e ly to a: beaker which contained 100 ml* of a m ixture of alcohol-
15
concentrated hydrochloric acid (9:1)» The complete mixture was s t i r
red fo r fiv e minutes and th e re s in f ilte r e d o ff as before* The A m
ber l i t e was then washed w ith 100 ml* of alco h o l-eth er (3*1) to re
move any adhering f a t t y acid and the f i l t r a t e s were combined* The
f in a l so lu tio n was made acid by the addition of sm all amounts of con
cen trated hydrochloric acid and was d ilu te d w ith d i s t il l e d water to
approximate a 5© per cent alcohol solution* The f a t t y acids were
then removed from the so lu tio n by eth e r ex tractio n * This e x tra c t
was washed sev eral tim es w ith w ater and d ried over sodium su lfate*
A fter evaporating to dryness, the weight o f the fre e f a t t y acids was
obtained*
The separation of the mono-, di-and triglycerides is based upon
their differential solubility in 80 per cent ethanol and petroleum
ether* This solvent pair for the monoglyceride separation was de
veloped by Bash and Stewart (1*2), while the scheme of separation
termed "completion of squares" was first described by Bush and Densen
(1*3)* Mattson (1*1*) has further modified the procedure to include the
use of four separatory funnels.
Using th is method, equal volumes of the solvents were employed
fo r each e x tra c tio n with a minimum d ilu tio n o f 1 to 1 * 0 of lip id to
solvent* The glyceride sample fre e d from f a t t y acids was dissolved
in 25 ml* of petroleum e th e r and added to the f i r s t funnel containing
25 m l. of 80 per cent ethanol* The two solvents were shaken and
allowed to separate* The lower la y e r of ethanol was added d ire c tly
to the second sep arato ry funnel containing a fre sh amount of p e tro -
16
leum ether* This process was repeated u n til fo u r separations were
obtained* A fter each sep aratio n fre sh 80 per cent ethanol was added
to the f i r s t funnel u n til a l l four funnels contained the solvent
pairs* The ethanol lay e r was drawn o ff the fo u rth funnel and added
to a weighed flask* This process was repeated u n til a l l the alcohol
lay ers had been co llec ted from the l a s t funnel* A t o ta l of six teen
separations were required to obtain th is r e s u l t. In th is procedure,
the monoglyceride s were found mainly in th e alcohol lay ers of the
th ir d and fo u rth funnels* The d i- and trig ly c e rid e s remained p rin
c ip a lly in the petroleum e th e r lay ers of the f i r s t two funnels*
Methods of saponifying the n e u tra l f a t and phospholipid samples*
N eutral f a t samples from the liv e r and in te s tin e were dissolved in
5© ml* ©f I N alcoholic potassium hydroxide and refluxed over b o ilin g
water fo r three to four hours* The m ixture was then a c id ifie d w ith
3 © per cent s u lfu ric acid and d ilu te d w ith 1 0 © ml* of distilled water*
The to ta l f a t t y acids were e x tra cted from the sa p o n ificatio n mixture
by means of th ree 75 ml* p ortions of petroleum e th e r. The combined
petroleum e th e r e x tra c ts were washed w ith w ater, d ried over anhydrous;
sodium s u lf a te , f i lt e r e d and evaporated to dryness to obtain the
t o ta l f a t t y acid weight*
In te s tin a l and liv e r phospholipids were saponified by a. d iff e r
en t method* These samples were i n i t i a l l y hydrolyzed by reflu x in g
with 3© ml* of alcohol containing 1 per eent s u lfu ric acid fo r four
hours to remove the bases* The m ixtures were then made alk a lin e by
the ad d itio n of 3© ml. of 10 per cent alcoholic potassium hydroxide
17
and were refluxed f o r another two hours* The f a t t y acid s were then
iso la te d using methods sim ila r to those described above*
Determ ination of -fee ra d io a c tiv ity of the l i p i d samples* Small
portions of each l ip i d sample were placed on weighed aluminum p lan -
chets to measure the rad io a ctiv ity * In most cases s u ffic ie n t m aterial
was added to o b tain an in f in ite thickness* Ey counting lip i d sanples
weighing from 1 to 100 mg* a c a lib ra tio n curve had been made to de
term ine the amount neeessary to have an in f in ite thickness* From
th is curve, i t was estim ated th a t an in f in ite thickness was obtained
between 50 to 60 mg* of lip id * When in s u ffic ie n t m a te ria l was a v a il-
able to planchet a t in f in ite th ick n ess, the curve was used fo r d e te r
mining the counts present*
Chloroform was found to be the b e st solvent to f a c i l i t a t e tra n s
f e r o f the sem i-solid lip i d to the planchet and fo r obtaining an even
d is trib u tio n on the surface of the planchet* A ll planchets were
d rie d a t le a s t tw enty-four hours under an in fra re d lamp before count
ing* The ra d io a c tiv ity of th e d ried samples was measured by placing
the planchets in a Nuclear gas-flow counter connected to a Berkeley
scaler*
Techniques Employed fo r In V itro Studies
Normal and irra d ia te d animals were s a c rific e d by a sharp blow
on the head* The liv e rs were e x tra cted while the animals were un
conscious and placed in physiological sa lin e kept cold w ith shaved
ice* L iver s lic e s were made using a Stadie-R iggs microtome and were
placed in sm all weighed beakers containing Krebs-Ringer phosphate
18
b u ffe r. These beakers were weighed again to obtain the approximate
wet weight of the tis s u e . In most cases, aliq u o ts of n early 1 gnu
of liv e r s lic e s were used fo r each incubation fla s k . The tis su e was;
tra n s fe rre d to 125 ml. Erlenmeyer fla sk s containing 20 m l. of the
phosphate b u ffer together w ith a measured amount of the rad io activ e
compound being stu d ied . P rio r to th is tra n s fe r, these flasks: had
been brought to 37° C by incubation in a Warburg constant-tem perature
b a th .
Immediately before the incubation of the liv e r s lic e s , a l l flasks;
were f i l l e d with an oxygen-carbon dioxide gas m ixture and tig h tly
corked. They were then immersed in the Warburg tank and shaken con
s ta n tly fo r three hours. A fter the incubation, the so lu tio n bathing
t
the tis s u e was; decanted and the s lic e s were washed twice w ith 3© m l.
of so lu tio n containing the non-radioactive compound* Such measures;
ensured removal of a l l rad io activ e compound which might adhere to
the tis s u e s lic e s . A lcohol-ether (1:1) was; then added to h a lt enzy
m atic a c tiv ity and the l iv e r tis s u e was: e x tra cted fo r lip id s; accord
ing to procedures previously o u tlin e d .
Methods; Used fo r the D eterm ination of Fecal and I n te s tin a l Lipids;
Iso la tio n of the fe c a l n e u tra l f a t . Both normal and irra d ia te d •
r a ts were placed se p a ra te ly in cages so designed as not to perm it
aecess to any of the excreted fe c a l m a te ria l. Food was w ithheld from
the animals during the fo rty -e ig h t hour fe c a l c o lle c tio n p e rio d . At
the end of this, tim e, feces from each cage were pooled and d ried in
19
vacuo fox* e ig h t hours* A liquots of from one to two grains were weighed
out and tra n s fe rre d to a m ortar and ground to a fin e powder* th is
m aterial was then plaeed in a 125 ml* Erlenmeyer f la s k and incubated
w ith 75 ml* of aleo h o l-eth er (3:1) a t 1*0° G fo r th ree hours* th is
process was repeated w ith fre s h solvent and the f i l t r a t e s were col
lected* th e ground fe c a l m ateria l was wrapped in f i l t e r paper and
e x tra cted upon a Soxhlet apparatus w ith e th y l e th e r fo r six tee n hours*
the re s u ltin g lip id e x tra c ts were then added to the previously col
le c te d f i l t r a t e s and the t o t a l volume of the so lu tio n s decreased a t
reduced pressure in an atmosphere of carbon dioxide* these concen
tr a te s were e x tra c te d se v era l tin e s w ith petroleum e th e r (S kelly A)*
d ried over anhydrous sodium s u lfa te , f i lt e r e d and evaporated to dry
ness to obtain th e t o ta l n e u tra l f a t weight*
Is o la tio n o f th e in te s tin a l l ip i d re sid u e s* Bats in tra p e rito -
n e a lly in je c te d w ith e ith e r 1*© p.e of a ce ta te -l-C 1^ o r glycerol-l-C ^*
were sa c rific e d a t tim e in te rv a ls ranging from fiv e minutes to twenty*
fo u r hours a f te r isotope adm inistration* Under Nembutal anaesthesia
the in te s tin a l t r a c t was severed a t the p y lo ric and ile o c e c a l Junction
and i t s contents flu sh ed out* This was accomplished by slip p in g a
m etal tube connected to a larg e syringe in to the in te s tin a l lumen*
Warm d is t il l e d w ater was used as the i n i t i a l flu sh in g agent follow ed
by two 2© ml* p o rtio n s of a i r and two 2© ml* portions of aleo h o l-
e th e r (1:1)* The flu sh in g s were combined from two animals sa c rific e d
a t the same time in terv al*
These washings were e x tra c te d se v era l tim es w ith 5© ml* portions
20
of e th y l e th e r, d ried over sodium s u lfa te , f i lt e r e d and the lip id
residues obtained by evaporation of the solvent* A fter weighing,
the samples were tra n sfe rre d to planchets and the ra d io a c tiv itie s
determined*
D eterm ination of L iver Glycogens
Rats were sa c rific e d two hours a fte r an in tra p e rito n e a l in je c tio n
of a lab eled compound, the liv e r s e x tra cted and immediately frozen in
a dry ice-alco h o l mixture* The frozen tis su e was b lo tte d dry and a
wet weight was determined* The liv e rs were quickly tra n sfe rre d to
weighed $0 ml* centrifuge tubes* containing approxim ately 10 ml* of
30 per cent aqueous potassium hydroxide* The tis su e was. so lu b iliz e d
com pletely by incubating in b o ilin g w ater fo r fiv e to s ix hours:*
E thanol was added to the cooled samples to approximate a f in a l a l
cohol concentration of 60 per cent* The glycogen p re c ip ita te was,
allowed to remain a t le a s t twelve hours before centrifugation* The
supernatant so lu tio n was decanted o ff and the p re c ip ita te was resu s
pended in lj? ml* of d i s t il l e d w ater and heated u n til dissolved* A
re p re e ip ita tio n was accomplished by again adding alcohol*. This pro
cess was repeated a t le a s t three times u n til a white p re c ip ita te was
formed* The glycogen was d ried by suspending twice in anhydrous: ace
tone and discarding the supernatant each time* Drying was; completed
in the vacuum oven during a p erio d of two to th ree hours or u n t il a
constant weight was obtained*
EX PERIM EN TA L RESULTS
The Measurement o f Tissue Lipogenesis in Nonaal and Irra d ia te d Rats
The incorporation of lab eled molecules in to liv e r n e u tra l fa t*
Marked incorporation d ifferen ces are observed when the sp e c ific
a c tiv itie s of l iv e r n e u tra l f a t are compared a f te r the adm inistration
of tagged a ce ta te or g ly cero l (Figure 1)* Glycerol-l-G-^* r e s u lts in
higher sp e c ific a c tiv itie s in th is h epatic fra c tio n of normal r a ts
than does lab eled acetate* F u rth e r, the depressant e ffe c t th a t f a s t
ing has upon lab eled a ce ta te incorporation i s not evident w ith tagged
g ly c e ro l. On the co n trary , glycerol-C1^ le v e ls in th is l ip i d fra c
tio n of fa ste d r a ts equals or exceeds th a t observed in fed groups a t
sev eral time in te rv a ls*
In ad d itio n to d ifferen ces observed in normal r a t s , the in
corporation p ie tu re of those subjected to roentgen treatm ent shows
marked a lte ra tio n s in lab eled acetate and g ly cero l u tiliz a tio n *
Dramatic elevations are noted w ith the two carbon precursor a f te r
x -ray exposure which g re a tly exceeds the sp e c ific a c tiv ity le v e ls of
both fa ste d and fed c o n tro l groups* In many in sta n c e s, th e G^*-
a c e ta te incorporation le v e ls in the irra d ia te d groups are more than
s ix tim es g re a te r than those observed in the u n treated animals* H ow
ev er, such i s n o t the ease when h epatic lipogenesis i s measured by
means o f lab eled g ly cero l incorporation* Here the content of the
l iv e r n e u tra l f a t fra c tio n is q u ite sim ilar to o r s lig h tly reduced
from th a t observed in c o n tro ls.
Since these stu d ies stro n g ly suggest a more ra p id isotope tun>-
22
m VIVO INCORPORATION OF ACETATE - I - C 14 AND
GLYCEROL-1-C14 INTO LIVER NEUTRAL FAT
O
2
u
>
>
F -
O
<
o
1 1 1
C L
< 0
30
25
20
1 5
10
U
c=> FASTED RATS
■ ■ FED RATS
CXDFASTED X-RAYED RATS
ACETATE
4
i
GLYCEROL
I
.25 .75 1 .2 5 3.0 6.0 24.0 .25 .75 1 .2 5 3.0 6.0
TIME AFTER INJECTION CHOURS)
24.0
FIG. I NORMAL FED AND FASTED FEMALE RATS ARE
COMPARED WITH FASTED IRRADIATED ANIMALS AFTER
AN IN TR A PER ITO N EA L INJECTION OF I.OJJC OF LABELED
ACETATE OR GLYCEROL. ALL FASTED ANIMALS
RECEIVED THE ISOTOPES FOLLOWING THE STARVATION
PERIOD. IRRADIATION WAS ADMINISTERED PRIOR TO
FASTING IN THE TREATED ANIMALS.
23
over in the case of acetate a f te r x -ray treatm ent, mathem atical com
parisons have been applied to th e incorporation d ata as outlined by-
Tarver (1*5). However, as th is author p o in ts o u t, c alcu latio n s o f th is
type n e c e ssita te c e rta in assumptions* F i r s t , i t must be supposed th a t
th e liv e r n e u tra l f a t is homogeneous in nature and th a t the lo ss of
the la b e l is approxim ately random* The second assumption is th a t a l l
the liv e r n e u tra l f a t i s lab eled p rio r to the time considered fo r
calculation* C ertain ly , n e ith e r of these suppositions are completely
tru e in any b io lo g ic a l system . N evertheless, T arv er's mathematical
treatm ent may be employed to show c e rta in u se fu l iso to p ic re la tio n
sh ip s. A sem ilogarithm ie p lo t of the sp e c ific a c tiv ity versus time
!
was made u tiliz in g the a cetate d ata presented in Figure 1 . This show
ed th a t the major p o rtio n o f the, descending slope occurs between one
and one-quarter and s ix hours a fte r a ce ta te ad m in istratio n . Over th is
time in te rv a l the v e lo c ity constant k can be c alcu lated employing the
re la tio n s h ip ,
k ■ 2^3 log Co
t Ct
Co denotes the sp e c ific a c tiv ity a t th e i n i t i a l time p erio d and Ct
the sp e c ific a c tiv ity a fte r s ix hours* The h a lf - lif e of the isotope
can be c alcu late d from the v e lo c ity constant by the re la tio n sh ip ,
t / x 8 5 I*1 2 ■ Q.69
k k
From e ith e r the v e lo c ity constant o r the h a lf - lif e the turnover time
2k
can be determined by the re la tio n s h ip ,
Tt » 1 * = t 1/ 1
k In 2
R esults obtained from th e above equations are presented in Table I*
A much higher v e lo c ity constant i s ex h ib ited by animals tre a te d with
l£00 r and thus in d ic ate s a more ra p id u tiliz a tio n of the isotope
than i s seen in e ith e r fed or fa s te d groups* Likewise, the h a lf - lif e
of th e lab eled two carbon compound i s shortened a f te r roentgen t r e a t
ment* At the in te rv a ls examined the normal fed animals took n early
nine hours to u tiliz e £© per cent of the la b e l as compared to only
th ree hours in the x-rayed animals*
To support the in vivo e ffe c ts of irra d ia tio n upon C ^ -a c e ta te
incorporation in to liv e r lip id s , a sim ila r stucfcr was performed in
v itr o * R esults obtained from the two methods are in good agreement
as in d icated by Table II* Again, fa s tin g decreases lab eled acetate
incorporation in to the liv e r n e u tra l f a t o f normal r a ts while x -ray
exposure increases the incorporation of the isotope* Nearly a th re e
fo ld ele v atio n in ra d io a c tiv ity i s noted in the n e u tra l f a t from an i
mals tw enty-four hours p o s t-irra d ia tio n w hile a tw o-fold increase i s
noted fo rty -e ig h t hours a fte r exposure*
Since x-rayed r a t s ex h ib ited a stim u latio n in lipogenic a c tiv ity
the glyceride composition of th e hepatic n e u tra l f a t from a ce ta te in
je c te d animals was examined* S eparation of the acetone-soluble fra c
tio n from liv e rs o f normal fed or fa s te d and fa s te d irra d ia te d r a ts
in to fre e f a t t y a c id s, mono-, d i- and trig ly ce rid es: i s shown in
25
TA B LE I
TH E T U R N O V E R O F ACETATE-l-G1* * IN TH E LIVER N E U T R A L
EAT O F N O R M A L A N D IRRADIATED RA TS
Tjpe of
Animal
S p ecific
A c tiv ity
a t l'/f h r .
(C0)
S p ecific
A c tiv ity
a t 6 h r .
(Ct)
k t/&
Tt
Normal
3.1
0.1 8.9 12.8
Normal
Fasted
3-U 1.5 0.2 h»0 5.8
X-rayed
Fasted
29.5 8.9 0.3 2.7 lu©
2 6
T A B L E I I
T H E IN TORO IN CCRPCRA TIO N O F AGETATE-1-C1^
INTO T H E LIVER LIPIDS CF N O RM A L A N D IRRADIATED RA TS
Number F asting Roentgen N eutral F a t Phospholipid
o f Period Bose
Samples (hr*) mg* 1 / c./m« 2 / mg* c./m .
gm. mg. gm. mg*
12 3/ © ©
19*3 2 .9 2 1 .9 1*5
12 2 k © 22.2
1*9
26.8 0.6
6
2 k 1500 21*7 6.1
3 8 .5
1*8
12
m
© 13*2 1 .1 * 2 1 .9 0 . 1 *
6 1500 H i.8 2 .8
28*7 1 .1
1 / The m illigram s of lip i d are given per gram wet weight of liv e r
* “ tis s u e .
2 / These fig u re s denote the sp e c ific a c tiv ity expressed as counts I
~ per minute per m illigram of l ip i d m aterial*
3 / A liquots of approxim ately 1 gm* of tis s u e were incubated w ith
0*03 >ie. of lab eled a ce ta te fo r three hours*
27
Table m. No s ig n ific a n t d ifferen ces between the percentage d is
trib u tio n s of the n e u tra l f a t fractio n s; from normal fa s te d or fed a n i-
*
trials are noted* The reduction in C ^ -a c e ta te incorporation re s u ltin g
from sta rv a tio n is re fle c te d p a rtic u la rly in the fre e f a t t y acids and
monoglyeerides• In addition* s trik in g increases in the re la tiv e
amounts of these two components are observed in the liv e r n e u tra l f a t
iso la te d from the irra d ia te d r a t s . In the case of the free f a t t y
acids th is amounts to an average of 1*>.3 per cent in the x-rayed groups
as co n trasted to an average o f 8.1 per cent in the non-treated r a t s .
The monoglyceride fra c tio n from tre a te d animals i s n early doubled th a t
noted in normal r a t s . This in tu rn re s u lts in a reduction in the per
centages of d i- and trig ly c e rid e iso la te d from the liv e r s of the former
groups. Such a lte ra tio n s in h epatic f a t t y acid and glyceride composi
tio n a f te r roentgen treatm ent may be a re f le c tio n of increased synthe
t i c a c tiv ity as suggested by the a ce ta te incorporation studies;.
The incorporation of caproate-l-C -^ was stu d ied in the in ta c t
r a t as another source of tw© carbon fragm ents. R esults obtained from
the l iv e r lip id s; a f t e r a two or four hour absorption period are pre
sented in Table IV. F asting i s seen to depress G-^-caproate incorp-
»
o ratio n in to liv e r n e u tra l f a t of normal r a ts to a sim ila r e x ten t a s
th a t observed w ith C ^ -a c e ta te . Although a t the two hour absorption
in te rv a l the isotope le v e l noted in x-rayed animals i s increased* the
extreme heights of a c tiv ity observed w ith tagged acetate are never
rea d ied . However* a f te r fo u r hours an even more pronounced stim ula
tio n in caproate incorporatio n i s noted in x-rayed groups. Here
T A B LE I I I
T H E LIPID FRACTIONATION O F N E U T R A L FAT ISOLATED
F R O M T H E LIVERS O F N O R M A L A N D IRRADIATED BA TS
/ •
F asting Roentgen F a tty Acids Monoglyeerides D i- and Triglycerides.
Period Dose
% of T otal V % of T otal % of T otal
(h r.) T otal Count
(X 1©2)
T otal Count
(x 1©2)
T otal Count
(x 102)
O £ /
© 9.2 0.8
25.9 1.1* 6l*.9 3.6
1 * 8
0 7.1 ©.1
30.3 0.5 62.9 3.2
1 * 8 . 1500 16.3 1.1* 56.3
17.2
27.1*
7.8
’ 1 * 8 1500 1U.3 0.6 1*6.1 12.8
39.6 li*.3
1 / The t o ta l count i s expressed p er $0 mg. of lip id m a te ria l.
2 / Each fig u re rep resen ts th e average of fo u r determ inations•
ro
09
25
T A B L E I?
T H E IN VLW INCCEPCRATION O F CAPRQATE-1-G1^
INT© T H E LIVER N E U T R A L FAT G F N O R M A L A N D IRRADIATED RA TS
Number
of
Animal a
Absorption
Period
(hr*)
F asting
Period
(h r.)
Roentgen
Dose
N eutral F at
T otal 2 / e./m . ' y
Count m ig *
(x 103)
k 3 /
6
2
2
0
% B
0
0
3 .6
1 .7
15.1*
1 0 .6
5
2 kB 150© 2 .1 1 7 .8
k k
0 0 2 .0 1 6 .2
k kB 0 0 .5 3 .3
k k kB 1500 1 .5 lU.U
1 / These fig u re s denote the t o t a l count contained in the complete
— l ip i d sample*
2 / These fig u res denote th e sp e c ific a c tiv ity expressed as counts
~ ~ per minute per m illigram of lip id m aterial*
3 / Each animal was in je c te d in tra p e rito n e a lly w ith 1*0 p.e+ of
lab eled caproate*
30
n e a rly fo u r tim es as much la b e l i s found in the n e u tra l f a t of the
irra d ia te d animals as in the fa s te d c o n tro ls. N evertheless, a t no
instance does the t o ta l a c tiv ity in th is fra c tio n from the tre a te d
group ever exceed th a t observed in normal fed animals* Table V shows
th a t these r e s u lts obtained w ith C ^-cap ro ate in v itro when incorpora
tio n is measured in liv e r slic e s*
S aponification analyses were u tiliz e d to determine the r e la tiv e
amounts of a c tiv ity lo c a liz e d in the f a t t y acid p o rtio n s of liv e r
glyceride molecules* Approximately 90 per cent of the G ^ - a c tiv ity
was found to resid e in the f a t l y acids derived from liv e r n e u tra l f a t
of animals in je c te d w ith lab eled acetate* However, th is was found
not to be th e case a fte r caproate adm inistration* C alculations show
th a t in these r a t s from 30 to 50 per cent of to ta l a c tiv ity must be
presen t in the glycerol m oieties of the n e u tra l f a t molecules* Such
re s u lts suggest th a t caproate can be u tiliz e d fo r n o n -fatty acid syn
th e s is to a la rg e r ex ten t than acetate* However, these a c tiv ity
values fo r the g ly cero l p o rtio n of the molecule are not as high as
those obtained a f te r g ly c e ro l-l-C ^ adm inistration* In th is instance
\
approxim ately 90 p er cent of th e la b e l was confined to the n o n -fatty
acid moiety*
The p o s s ib ility arose -that the irrad iatio n -in d u c ed stim u latio n
in lipogenesis might be re fle c te d in an a lte re d carbohydrate and amino
a cid u tiliz a tio n fo r f a tty acid formation* To in v estig ate the ro le
of g lycolysis in supplying precursors fo r lip id synthesis glucose-1-
incorporation was measured in vivo and in v itro * Table VI shows
31
T A B L E V
T H E IN "V IT R O IN CO RPO RA TIO N O F CAFRQATE-1-O^*
INTO TH E LIVER LIPIDS O F N O R M A L A N D IRRADIATED RA TS
Number F asting Roentgen N eutral F a t Phospholipid
of
inrples
Period
(hr*)
Dose
mg * j /
gm *
c*/m*
mg.
mg*
gm *
• e./m«
3 2 / 0 0 10.9 125.5 27,9 I5.it
3
0 0
8.5 135.5 31.1 3lt.9
3 hB
0 10.lt it.? 26.7 3.1
3 m
0 10.3 It.?
21.1 5.8
3 kB 1500 11.3 168.5 3 lt.l 2it.9
3 . kB 1500 12.5 125.7
36.2
ltl.3
1 / The m illigram s of lip id are given per gram tret weight of liv e r
~ tissu e*
2 / These fig u res denote the sp e c ific a c tiv ity expressed as counts
“ per minute per m illigram of lip id m aterial*
3 / A liquots of approxim ately 1 gm. of tis s u e mere incubated w ith
~ 1 ,0 ^ic* of lab eled caproate fo r three hours*
32
T A B L E 71
TH E IN V IVO A N D IN VITRO IN CO RPO RA TIO N O F
GUJGOSE-l-C1^ INTO LIVER N E U T R A L FIT O F N O R M A L A N D IRRADIATED
R A TS
Number F asting Roentgen L iver N eutral F a t V
of
Animals
Period
(h r.)
Dose
In Vivo
c./m . £ /
mg.
In V itro
e./m .
mg.
k
1 * 8
©
2.1* 2.0
k 1 * 8 0 2.7
1*5
k
1 * 8 1#30
2.3 1.9
k
1 * 8 1$00 2.1
1.7
1 / Each in vivo and in v itro determ ination was made with. 0*2 /no*
“ of lab eled glucose *
2 / These fig u re s denote th e sp e c ific a c tiv ity expressed as counts
"" per minute per m illigram of lip id m aterial*
3 3 ?
th a t liv e r n e u tra l f a t e x h ib its no s ig n ific a n t irra d ia tio n d if f e r
ences in C ^ - a c tiv ity when studied e ith e r in the whole animal o r with
liv e r slic e s* A nalysis of the f a t t y acids derived a f te r saponifica
tio n demonstrated th a t approxim ately 10 per cent o f the to ta l a c tiv ity
was p resen t in th e f a t t y acid m oieties from normal animals while a l
most 30 per cent of the a c tiv ity was lo cated in th is fra c tio n from
exposed animals* Such re s u lts suggest the p o s s ib ility of a somewhat
g re a te r u tiliz a tio n of the rad io activ e carbon fo r f a t t y a cid forma
tio n a f te r x -ray treatm ent*
U tiliz in g g ly c in e -l-C ^ as a lip id precursor presented what ap
peared to be a depression in C ^-im corporation follow ing irra d ia tio n
when stu d ied a t a sin g le two hour absorption period* This process
was then stu d ied over a s e rie s of time in te rv a ls as demonstrated in
Figure 2 . These r e s u lts suggest a depression in C ^ -g ly cin e in
corporation immediately follow ing irra d ia tio n * In normal r a ts the
peak of incorporation in to liv e r n e u tra l f a t is; reached approxim ately
s ix hours a f te r isotope adm inistration and i s follow ed by & ra p id
turnover in the next th ree hours* In c o n tra s t, the incorporation
maximum a fte r irr a d ia tio n does not occur u n til nine hours; a fte r in
jectio n * Here the sp e c ific a c tiv ity averages only 1*1} as compared
to the maximum value of h*l in normal ra ts * However, i t can be seen
th a t a fte r twelve hours the sp e c ific a c tiv ity of the n e u tra l f a t from
normal animals is decreased while th a t from the x-rayed groups is in
creased* In the l a t t e r case the C ^ -co n ten t r is e s in tw enty-four
hours to a value almost equal to the maximum a tta in e d nine hours a f te r
in je c tio n .
SPECIFIC ACTIVITY C C /M /M G )
M
GLYCIN EH-C14 INCORPORATION INTO LIVER
NEUTRAL FAT
• NORMAL RATS
— o IRRADIATED RATS
o-o
20 8 24 4 12 1 6 0
TIME AFTER INJECTION C HOURS)
F I G .2 THE ABILITY OF NORMAL RATS AND
THOSE S U B JE C T E D TO 1 5 0 0 r TO INCORPOR
ATE I.OJULC OF LABELED GLYCINE J S COM
PARED OYER A TW EN TY FOUR HOUR PERIOD.
The Incorporation of lab eled m olecules in to sm all in te s tin a l
n e u tra l fa t* Lipogenesis ■ w as stu d ied in the in te s tin a l -wall by means
of lab eled a ce ta te and g ly cero l since th is organ is extrem ely activ e
in th is process* The a b il i ty of the in te s tin e to u t il i s e lab eled
acetate fo r n e u tra l f a t synthesis d iffe rs markedly from th a t of la b e l
ed glycerol (Figure 3)* G lycerol-l-C^* a c tiv ity is higher in the gly
cerides of normal r a t s th a t is a c e ta te , w hile th is s itu a tio n i s re
versed a f te r irr a d ia tio n . £ f te r lab eled a c e ta te adm inistration the
sp e c ific a c tiv ity i s found to be higher in th e n e u tra l f a t of the
irra d ia te d animal than th a t noted w ith C ^ -g ly cero l* Likewise, these
animals m aintain higher le v e ls of a c tiv ity in th is lip id fra c tio n a t
the longer absorption in te rv a ls than do th e controls* In normal r a ts
the c ^ - a c t i v i t y of in te s tin a l f a t begins to decline about th ree hours
a f te r a ce ta te adm inistration* I t f a l l s from an average sp e c ific
a c tiv ity of h*l a t the former p eriod to 1*7 a t the end of tw enty-four
hours* However, a f te r roentgen treatm ent the sp e c ific a c tiv ity aver
ages £ * 5 > a t th e th ree hour in te rv a l and i s increased to even higher
le v e ls a fte r tw enty-four hours* This apparent build-up of rad io
a c tiv ity i s not evident when lip o g en esis i s measured w ith C ^ -g ly -
cerol* The f a c t th a t isotope le v e ls observed in th e l a t t e r case are
much reduced in the x-rayed groups suggests th a t g ly cero l u tiliz a tio n
fo r th is process may be rad io sen sitiv e*
Samples of the in te s tin a l n e u tra l f a t fra c tio n from anim als i n
je c te d with acetate-l-C ^k were separated in to fre e f a t t y acids and
glyceride components (Table V fl). Ho d is tin c t a lte ra tio n s in the
36
r\
o
2
u
k J
>-
>
o
<
u
U J
CL
< /)
m VIVO INCORPORATION OF ACETATE-1-C*^ AND
GLYCEROL-I-C14 INTO INTESTINAL NEUTRAL FAT
c=3 FASTED RATS
■ ■ F E D RATS
C O FASTED X-RAYED RATS
10
ACETATE
j
J
GLYCEROL
I
.25 .75 1 .2 5 3.0 6.0 24.0 .25 .75 1 .2 5 60
TIME AFTER INJECTION CHOURS)
24.0
F I G . 3 NORMAL FED AND FASTED FEMALE RATS ARE
COMPARED WITH FA STED IRRADIATED ANIMALS AFTER
AN IN TR A PER ITO N EA L INJECTION OF 1.0 UC OF LABELED
ACETATE OR GLYCEROL. ALL FASTED ANIMALS
RECEIVED THE ISOTOPES FOLLOWING THE STARVATION
PERIOD. IRRADIATION WAS ADMINISTERED PRIOR TO
FASTING IN THE TREATED ANIMALS.
TA B LE VII
T H E LIPID FRACTIONATION W N E U T R A L FAT ISOLATED
F R O M T H E INTESTINES GF N O R M A L A N D IRRADIATED RA TS
T asting Roentgen F a tty Acids Monoglyeerides D i- and T riglycerides
Period
(hr*)
Dose -
% of T otal V
T otal Count
(x l©2)
% of
T otal
T otal
Count
(x 102)
% of
T otal
T otal
Count
(x 102)
0 y
0 5*1 0*9 21.7 U.7
72.8 12.7
1 * 8 0 7.6 0.9
10.7 2.3 81.7 5.1*
1 * 8 15©0 8.7 0.8 15.5
2.6 75.8 6 .0
1 * 8 1500 9.8 1*2 ll* .l 2.1 76.1 6.6
1 / The to ta l count i s expressed p er 50 mg* of lip id m aterial*
2 / Each fig u re rep resen ts the average of four determ inations*
38
composition of the in te s tin a l n e u tra l f a t are evident in the ir r a d i
ated animals ’ when compared to controls* Such re s u lts might be ex
pected i f ir r a d ia tio n m erely le d to an impairment in glyceride u t i l i
z atio n ra th e r than an a lte ra tio n in lip id synthesis*
A Study 'Has made of fe c a l f a t ex cretio n in fa s te d normal and
x-rayed r a ts to determine i f irra d ia tio n damage to the in te s tin e might
be re fle c te d in th is process* AH animal groups were fa s te d since
x-ray s have been shown (1*6) to produce a severe anorexia la s tin g fo r
se v era l days* Thus, any v a ria tio n in fe c a l lip id content due to
d ifferen ces in food consumption was elim inated* R esults obtained
from the eth e r-so lu b le fra c tio n of the feces are shown in Table ¥111*
normal animals averaged 56*1* mg* of f a t excreted per gram of feces;
while those subjected to 1000 and 1500 r excreted la rg e r amounts of
lip id , namely values of 60*2 and 82*7 respectively* A cetate-l-G -^
was in je c te d in tra p e rito n e a lly in to se v era l r a ts p rio r to fe c a l col
le c tio n to determine i f irra d ia tio n would stim ulate i t s use fo r fe c a l
l ip i d formation* In th is manner i t was demonstrated th a t x-rayed ani
mals u tiliz e d more lab eled acetate fo r fe c a l lip o g en esis as w ell as
ex cretin g more fe c a l fa t* This i s re fle c te d in both the t o ta l count
and sp e c ific a c tiv ity observed in th is fractio n * Separation pro
cedures were employed to analyze the fre e f a t t y acid and glyceride
content o f the fe c a l f a t so as to compare i t s composition in both
groups (Table IX)* O verall, the f a tty acids in the normal animals
average some 39*2 per c e n t, the monoglyeerides 20*2 per cent and d i-
and trig ly c e rid e s 1*0*9 per cent while those in irra d ia te d r a ts aver-
39
T A B L E VIII
TIE ISOLATION m FECAL FAT F R O M N O R M A L A N D
IRRADIATED RA TS
dumber
o f
Animals
Number of
Analyses
Roentgen
Dose
Fecal
.Fat
mg. 2 /
gm.
T otal
Counts £ /
c./m . 3 /
mg.
S pecific
A ctiv ity
C * / t O l*
mg.
6k
31
0
56.k 75 1.3
3k
1 7
1000 60.2
— —
39 21 150© 82.7 3k8 3.5
1 / F eeal f a t weights are expressed as m illigram s of l ip i d m aterial
* " per gram dry weight of fe c e s.
2 / The ra d io a c tiv ity fig u re s r e f e r only to six tee n normal and
— seven tre a te d r a t s which were in je c te d in tra p e rito n e a lly with
0.1 /lc . of lab eled ace ta te p rio r to the fe e a l c o lle c tio n .
3 / The to ta l counts are those contained in the l ip i d m aterial is o r
~ la te d from 1 gm. of fe c e s.
TA ELS IX
TH E LIPID FRACTIONATION O F N E U T R A L FAT ISOLATED
F R O M TH E FECES OF N C R H A L A N D IRRADIATED R A TS
Roentgen . F a tty Acids Monoglyeerides 'D i- and T riglycerides
Bose
% of
T otal
T otal i /
Count
(x 1© 2)
% of T otal
T otal Count
(x 102)
% of
T otal
T otal
Count
(x 102)
0 £ /
U2.9 0.6 16.8 0 .5 ¥>.3 0.7
0
0
3U.9
39.9
0.5
0.6
23.9 0.U
19.9 0.5
Ul.2
1*0.2
0.8
0.6
1500
\
hZ.9 1.1 l6 .ii 0.9
Uo.7 1*7
1500 U3.8 1.2 22.6 0.8 33.6 1.8
l / The t o ta l count i s expressed p e r 50 mg* of lip id m aterial*
2 / Each fig u re denotes one determ ination upon fe e a l lip id is o la te d from animals In je c te d w ith
* ” lab eled acetate*
£
la
age h3*3$ 19*5 and 37*1 per cent re sp e c tiv e ly . The composition of
the fe c a l f a t from the ttro groups thus appears q u ite sim ila r and
suggests a common o rig in .
In order to e s ta b lis h a more d ire c t re la tio n sh ip between the
✓
in te s tin a l t r a c t and fe c a l f a t form ation, the lip id residues contain
ed in the sm all in te s tin a l lumens were examined. This was done in
normal and irra d ia te d r a ts a fte r the adm inistration of lab eled ace
ta te o r g ly cero l (Figure U). No s ig n ific a n t a lte ra tio n s were noted
in the amounts of the l ip i d residues iso la te d from e ith e r normal or
x-rayed groups. In a d d itio n , fa s tin g does not appear to be an im
p o rtan t fa c to r in reducing the isotope incorporation in normal r a t s .
The fa s te d n o n -treated animals incorporate e ith e r C ^ -a c e ta te or C ^ -
glycerol as w ell or b e tte r than the fed groups. However, th e in
creases in sp e c ific a c tiv ity are most s trik in g ly e x h ib ited in the
lip id resid u es is o la te d from animals subjected to 1500 r . I r r a d i
a tio n , th e re fo re , appears to stim u late the u tiliz a tio n of labeled
precursors fo r the synthesis of lip id contained in the in te s tin a l
lumen as w ell as th a t iso la te d from th e fe c e s .
The incorporation of lab eled molecules in to liv e r phospholipids♦
Figure 5 shows th a t in normal animals a fte r g ly c e ro l-l-C ^ adminis
tra tio n g re a te r amounts of a c tiv ity are found in hepatic phospho
lip id s than are noted w ith lab eled a c e ta te . In a d d itio n , w ith la b e l
ed g ly ce ro l, the sp e c ific a c tiv ity observed in the fa s te d animal ex
ceeds in most eases th a t of the fe d , while fa s tin g reduces acetate
incorporation in to th is h epatic fra c tio n . X-rayed r a t s , on th e other
INCORPORATION OF A C E T A T E -I-C 14 AND
g l y c e r o l - i- c I 4 in t o INTESTINAL LIPID RESIDUES
O
2
2
N
o
> ~
>
H
O
<
O
u.
u
UJ
C L
in
FIG. 4 NORMAL FED AND FASTED FEMALE RATS ARE
COMPARED WITH FASTED IRRADIATED ANIMALS AFTER
AN INTRAPERITONEAL INJECTION OF 1.0JiC OF LABELED
ACETATE OR GLYCEROL. ALL FASTED ANIMALS
RECEIVED THE ISOTOPES FOLLOWING THE STARVATION
PERIOD. IRRADIATION WAS ADMINISTERED PRIOR TO
FASTING IN THE TR£ATED ANIMALS.
15
10
iFASTED RATS
■ FED RATS
CX3FASTED X-RAYED RATS
ACETATE
1
I
GLYCEROL
.2 5 .7 5 L25 3X)6X> 2 4 .0 .2 5 .7 5 3 .0 6.0
TIME AFTER INJECTION CHOURS)
24.0
IN VIVO INCORPORATION OF ACETATE-1-C14 AND
GLYCEROL-I-C1 * INTO LIVER PHOSPHOLIPIDS
15
O
2
2
U 10
> -
>
h *
o
<
o
t 5
o
U J
0.
w
0
.25 .7 5 125 6.0 240 .25 .7 5 1.2 5 3.0 6j 0 24.0
TIME A FT E R INJECTION CHOURS)
FIG 5 NORMAL FED AND FASTED FEMALE RA TS A RE
COMPARED W IT H F A S T E D IRRADIATED ANIM ALS AFTER
AN IN T R A P E R IT O N E A L IN JE C T IO N OF 1 . 0 JUC OF LABELED
ACETATE OR GLYCEROL. ALL FASTED ANIMALS
RECEIVED THE IS O T O P E S FOLLOWING THE STARVATION
P E R IO D . IR R A D IA T IO N WAS ADM INISTERED PRIOR TO
FA ST IN G IN THE T R E A T E D ANIMALS.
I FASTED R A TS
M U FED RATS
C D FA STED X-RAYED RATS
' V r -
ACETATE GLYCEROL
b h
hand* do not show th e impairment in tagged a ce ta te u tiliz a tio n fo r
phospholipogenesis o rd in a rily caused by starv atio n * This e f f e c t,
though not as pronounced, i s q u ite sim ila r to th a t noted in the liv e r
n e u tra l f a t of the roentgen tre a te d animal (Figure 1 ). In v itr o r e
s u lts (Table I I ) have served to su b sta n tia te these fin d in g s.
Irra d ia tio n anomalies re la te d to tagged g ly cero l p a rtic ip a tio n
in phospholipid form ation are not as c le a rly defined. In most in stan
c es, i t appears th a t the C ^ - le v e ls a tta in e d in x-rayed groups a f te r
g ly cero l ad m in istratio n are reduced as compared to fa s te d c o n tro ls.
T herefore, irra d ia tio n e ffe c ts noted w ith lab eled g ly cero l are quite
d iffe re n t from those obtained with lab eled acetate* I t would thus
appear th a t the e ff e c t of x-rays upon the u tiliz a tio n of liv e r phos
p h olipid precursors i s dependent upon which moiety of the molecule
i s concerned. The incorporation of G ^ -a e e ta te which is p rim arily
concerned w ith f a t t y acid synthesis is found to be increased while
th a t of g ly cero l which i s concerned w ith the form ation of the non
f a t t y acid p ortions i s reduced.
The inco rp o ratio n of lab eled molecules in to sm all in te s tin a l
phospholipids. The t o ta l incorporation of both lab e le d a ce ta te or
g ly cero l (Figure 6) is dram atically reduced in to the in te s tin a l phos
pholipids of roentgen tre a te d r a t s . These r e s u lts are expressed in
terms of to ta l a c tiv ity ra th e r than sp e c ific a c tiv ity since the
amounts of in te s tin a l phospholipid were markedly decreased by x -ray
exposure. In absolute amounts the normal animals averaged 121 mg.
of t o ta l in te s tin a l phospholipids w hile th is amount was lowered to an
j J N VIVO INCORPORATION OF ACETATE-I-C
.14
14
GLYCEROL -l-C
AND
NTO INTESTINAL PHOSPHOLIPIDS
CZD FASTED RATS
FED RATS
O H FASTED X-RAYED RATS
GLYCEROL
.25 .75 .25 .75 1.25
25 3.0 6.0 24.0
TIME AFTER INJECTION CHOURS)
FIG. 6 NORMAL FED AND FASTED FEMALE RATS ARE
COMPARED W ITH F A S T E D IRRADIATED ANIMALS AFTER
AN IN T R A P E R IT O N E A L IN JEC TIO N OF 1 .0 JJC OF LABELED
ACETATE OR GLYCEROL. ALL FASTED ANIMALS
RECEIVED THE ISO T O PE S FOLLOWING THE STARVATION
PE R IO D . IRR A D IA TIO N WAS ADM INISTERED PRIOR TO
FA STIN G IN THE T R E A T E D ANIMALS.
U6
average o f ill mg. a fte r x -ray exposure* Since th e incorporation
le v e ls are reduced to about the same ex ten t as the t o ta l amounts the
irra d ia tio n e ffe c t is not evident when the sp e c ific a c tiv itie s of
th is fra c tio n are considered*
The Formation and Turnover of Liver Glycogen in Normal and Irra d ia te d
Rats
The incorporation of aeetate-l-G *^ in to liv e r glycogen* Normal
r a ts a fte r fa s tin g fo r fo rty -e ig h t hours, e x h ib it a marked reduction
in liv e r glycogen le v e ls (Table X), while animals given 1500 r before
fa s tin g show liv e r glycogen contents sim ila r to th a t of fed ra ts*
In a d d itio n , the x-rayed animals incorporated n e arly th ree tim es more
lab eled ace ta te in to liv e r glycogen than did controls* F asting p rio r
to roentgen treatm ent was employed to determine whether or not the
elev ated glycogen sto re s o f x-rayed r a ts re s u lte d from a n et synthe
s is or a re ta rd a tio n in glycogenolysis* The l a s t animal group shown
in the ta b le were fa s te d fo rty -e ig h t hours before exposure to 1500 r
and an a d d itio n a l tw enty-four hours a fte r* The glycogen le v e ls of
these animals are noted to be in creased from the p re -irra d ia tio n
value of 0*5 p er cent noted in fa s te d animals to a p o s t-irra d ia tio n
value of 3»7 per cent* T herefore, th is increased glycogen le v e l sub
sequent to the irra d ia tio n of fa s te d r a ts could only have re s u lte d
from a n e t synthesis*
The incorporation of gly cine-l-C^* in to liv e r glycogen* Labeled
glycine was employed to evaluate the ro le played by an amino acid in
the form ation of glycogen by normal and x-rayed ra ts* F astin g is
T A B L E X
TH E IN CO RPO RA TIO N W ACETATE-l-C1* 1 IHTG
T H E LIVER G L T G O G E N W N G RU A LA N D IRRADIATED R A T S;
Number of
Animals
Roentgen
Dose
F asting
Period
(h r .)
Liver Glycogen
lifeight V
T otal
Count £ /
(x 1 © 2 )
8 0
A
©
lift
3 . 4
A £
0 . 7
A O
o
8
u
i 5 o o
4 0
4 8
U o
3 . 4
WeO
2 . 0
8 1 5 0 0 4 8 / 2 4 I t 3 . 7
1 . 8
1 / The amounts of liv e r glycogen are expressed as the p e r cent
by wet Height of tissu e *
2 / These fig u re s denote ■tee t o ta l a c tiv ity observed in the complete
~ glycogen samples two hours a f te r the ad m inistration of 0*2 /ac.
of lab eled acetate*
3 / This animal, group was fa s te d 48 hours p rio r to irra d ia tio n and
“* zk hours a fte r*
1*8
noted to again lower carbohydrate sto re s in normal r a ts (Table XI)
although i t does not im pair the capacity of liv e r to u tiliz e lab eled
glycine* Both normal fa s te d groups incorporate more than th ree to
fo u r fo ld the amounts of glyeine-G -^ than do fed animals* The i r
ra d ia te d animals have an even g re a te r capacity fo r the u tiliz a tio n
of th e lab eled amino acids f o r glycogen* This e ffe c t appears im
m ediately since animals examined rig h t a f te r exposure show an average
of 12*5 x 102 counts in the glycogen as compared to the c o n tro l value
of 3*9 x l©2 counts* In a d d itio n , the stim u latio n in lab e le d glycine
inco rp o ratio n i s of long duration since elev ated le v e ls are evi
dent tw enty-four and fo rty -e ig h t hours p o st-irra d ia tio n *
The incorporation of uniform ly labeled glucose in to liv e r gly
cogen* A more immediate precursor of liv e r glycogen was stu d ied to
determine i f i t s u tiliz a tio n was a ffe c te d by irra d ia tio n * The sm all
amounts of p resen t a t th e time of s a c rific e (Table X II) in d icate
th a t the conversion of lab eled glucose to glycogen i s n o t ra p id in
the fed ra ts* However, i t s u tiliz a tio n i s increased in normal fa s te d
animals where th e t o ta l a c tiv ity average 6*6 x I©2 counts in compari
son to the fed le v e l of 0*3 x 102 counts* Although not of the same
magnitude as noted w ith glycine-l-G 1^ , the irra d ia te d animals again
show an increase in ra d io a c tiv ity immediately a f te r treatm ent* H ow
e v er, a f te r a period of fo rty -e ig h t hours w ithout food, th e x-rayed
groups e x h ib it an isotope content in the liv e r glycogen quite sim ila r
to th a t observed w ith the lab eled amino acid*
49
T A B L E XI
T H E IN CO RPO RA TIO N G F GLTCIUE-1-C1^ INTO
T H E LIVER G LICO G EN 0F N O R M A L A N D IRRADIATED RATS
Number of
Animals
Roentgen
Dose
p astin g
Period
(hr*)
L iver Glycogen T otal
Count 2 /
(x 102)
6 0 ©
4.1 3.9
4 1500
0
3.7 12.5
4
©
24 0.2: 15.8
4 1500
24
4.0 30.5
ll
©
48 0.1
13.7
4 1500 48 2*7 28.8
1 / The amounts of liv e r glycogen are expressed as the per cent
“ by wet weight of tissu e*
£ / These fig u re s denote th e t o t a l a c tiv ity observed in the complete
~ glycogen samples two hours a fte r the adm inistration of 1*0 /ae.
of lab eled glycine*
T A B L E XII
TH E IN CO RPO RA TIO N O F U N IFO R M LY LA B ELED G L U C O SE
INTO TH E LIVER G L Y C O G E N O F N O R M A L A N D IRRADIATED R A TS
Number of
Animals
Roentgen
Dose
Fasting
Period
(h r.)
L iver Glycogen
Weight 2 /
T otal
Count £ /
(x 102)
3
0 0 1 .2 0.3
k
1500 0
1*S 1.9
k
0
m ©.5
6rn 6
k
1500
k* i.U 29.3
1 / The amounts of liv e r glycogen are expressed as the per eent
* “ by wet weight of tissu e*
2 / These fig u re s denote the to ta l a c tiv ity observed in the complete
glycogen samples two hours a fte r the ad m in istratio n of 1*0 juc#
of uniform ly lab eled glucose*
DISCUSSION
R ats su ffe rin g from acute irra d ia tio n in ju ry have demonstrated
a lte re d lip id m etabolic p a tte rn s in the liv e r and in te s tin e when com
pared w ith normal animals* In addition* th e lipogenic response of
these two organs to io n izin g ra d ia tio n i s quite d ifferen t* The liv e rs*
a f te r x -ray exposure, e x h ib it increased lip id synthesis while such
activ ity - appears reduced in the in te stin e * The d iffe re n t organ re
sponses may r e f le c t d ir e c t and in d ire c t e ffe c ts of ionizing ra d ia tio n
upon th e body* since liv e r lesio n s re s u ltin g from massive roentgen
doses (1*7 ) are u su a lly la te in appearing* the e a rly abnorm alities in
lip i d and glycogen synthesis may b e st be re la te d to an in d ire c t i r
ra d ia tio n e ffect* This could p o ssib ly r e s u lt from the re le a se of
hormones or to x ic substances from rad io se n sitiv e s ite s elsewhere in
the body* C m the o th er hand* the severe a ctio n of ionizing rays
d ir e c tly upon the In te s tin a l t r a c t has been w ell established* Conard
(2U) and o thers (1*8* 1*9) have rep eated ly demonstrated the in te s tin e
to be p h y sio lo g ically and biochem ically se n sitiv e to even minute doses
of irra d ia tio n *
L iver l ip i d metabolism as judged by the incorporation of various
lab eled precursors has been found to be se n sitiv e to both fa s tin g and
x -ray exposure* Several in v e stig a to rs (50* 51) have demonstrated
th a t s ta rv a tio n per se depressed C-^- a ce ta te incorporation in to to ta l
h ep atic f a t t y a d d s* The d ata presented here not only demonstrate
such a depression* but also in d ic ate the p a rtic u la r l ip i d fra c tio n s
concerned* Among normal animals lab eled a ce ta te incorporation in to
52
liv e r n e u tra l f a t was n o ticeab ly decreased by fa s tin g fo r fo rty -
e ig h t hours. I t would appear th a t scone mechanism i s av ailab le in the
fed animal to f a c i l i t a t e ra p id incorporation and u tiliz a tio n of la b e l
ed a ce ta te which i s e ith e r absent or in h ib ite d by f a s tin g . However,
lip o sy n th e tic co n tro llin g fa c to rs which are se n sitiv e to sta rv a tio n
in the normal r a t are not observable a f te r irra d ia tio n . On th e con
tr a r y , the incorporation of tagged ace ta te in to the n e u tra l f a t fra c
tio n of fa s te d tre a te d groups g re a tly exceeds th a t o f normal fed
anim als. Both sa p o n ific atio n analyses and g ly eero l-i-G -^ incorpora
tio n stu d ies in d ic a te th a t the irrad iatio n -in d u c ed lipogenesis in the
n e u tra l f a t may be confined to the synthesis of the f a t t y acids of
these m olecules. Is o la tio n o f'th e f a tty acids has demonstrated th a t
the increases in a c e ta te incorporation are confined alm ost exclusively
to th is f ra c tio n . F u rth e r, no pronounced in creases in n e u tra l f a t
a c tiv ity were noted in the l iv e r n e u tra l f a t fra c tio n when synthesis
was judged by the incorporation of lab eled g ly c e ro l. This lab eled
compound has been shown to be p rim a rily concerned w ith the turnover
of n o n -fatty acid p o rtio n s of l ip i d m olecules.
Although an irra d ia tio n linked stim u latio n in h ep atic n e u tra l
f a t fra c tio n i s indicated by the incorporation of lab eled a ce ta te no
in creases in the weights of th is liv e r l ip i d have been observed.
However, much higher percentages of f a t t y acids and monoglyeerides
have been found to be p resen t in liv e r n e u tra l f a t samples from x-ray
ed anim als. This may r e f l e c t g re a te r glyceride a c tiv ity since
lorehous e t a l . (52) have shown monoglyeerides to p lay an im portant
53
ro le in in te s tin a l lip id synthesis* An acc elera te d u tiliz a tio n of
n e u tra l f a t fo r re p a ir in tis s u e s other than th e h ep atic tissu e
might also explain the lack of build-up of th is fra c tio n in the liv er*
In support of th is p o stu la te , Bacq e t al* (5) have noted increased
le v e ls of t o t a l carcass f a t of irra d ia te d r a ts when compared to con
t r o l s . Such data may be in te rp re te d as an expression of g rea ter ,
liv e r lipogenesis*
A fter roentgen treatm ent h epatic phospholipid form ation i s stimu
la te d to a somewhat le s s e r e x te n t than th a t of the n e u tra l f a t as
judged by lab eled a ce ta te incorporation* This i s the f i r s t d ire c t
evidence e stab lish ed both in vivo and in v itro which can be used to
lin k the irrad iatio n -in d u c ed liv e r phospholipogenesis to the increase
serum phospholipid le v e ls noted in se v era l irra d ia tio n stu d ies (15)*
In normal animals: th is plasma lip id fra c tio n has been shown to be
derived mainly from the liv e r in varying amounts according to species
(53) * Entenman and Weinman (21) using were able to demonstrate
an increased liv e r phospholipogenesis in v itr o but n o t in vivo a fte r
subjecting r a ts to doses of 1000 or 2500 r* The workers have con
cluded th a t c e rta in reg u lato ry mechanisms may e x is t in vivo which
m aintain phospholipid synthesis a t a constant rate* A prime objec
tio n to these in vivo experiments is th a t P^2 incorporation levels;
were examined a t only one in te rv a l a fte r isotope adm inistration*
L ittle inform ation concerning synthesis and turnover can be drawn
from a sin g le time period study*
In c o n tra st to re s u lts obtained w ith increased h epatic phos-
&
pholipogenesis i s m anifest in vivo as w ell as in v itro when judged
by C ^ -a c e ta te incorporation* This suggests; th a t u tiliz a tio n o f lab
e le d a ce ta te as a measure of f a t t y a cid turnover in liv e r phospho
lip id s may be a b e tte r in d ic a to r of phospholipogenesis than the in
corporation o f inorganic phosphate* The l a t t e r i s so w idely d is trib u
te d among c e llu la r co n stitu en ts th a t numerous exchange meehanisms not
d ire c tly concerned w ith phospholipid synthesis are more lia b le to
occur*
In v itr o techniques have shown "the increased capacity of the
irra d ia te d animal to synthesize hepatic f a t t y acids from lab eled ace
ta te to be a c e llu la r phenomenon* However, the nature of the c e llu
la r changes re s u ltin g in such a lipogenic response are highly specu
la tiv e * A hormonal stim u latio n of lip o g en esis may p lay a s ig n ific a n t
ro le in th is resp ec t although l i t t l e i s known concerning th e ir secre
tio n in the x-rayed animal* Adrenal c o rtic a l hormones known to be
elab o rated by a s tre s s such as irra d ia tio n are tempting to assig n to
th is mechanism, although th ey have been shown to in h ib it the in
corporation of c ^ - a e e ta te in to liv e r lip id s (5U)* On the o th er hand,
though in s u lin has been demonstrated to have a d e fin ite lipogenic
e ffe c t ($$) i t s ro le in irra d ia tio n syndrome has not been e sta b lish e d .
In ad d itio n to possible hormonal a lte ra tio n s the e ffe c ts of irra d ia
tio n upon c e llu la r enzyme le v e ls has been in v estig ate d both in vivo
($6) and in v itro (57)* Such stu d ie s , however, have not presented
conclusive evidence th a t any enzymes d ire c tly concerned w ith the bio
synthesis o f lip id s are a lte re d in animals su ffe rin g from irra d ia tio n
in ju ry * ______________________________________________________________
55
A lteratio n s in su b tra te concentrations may be im portant in the
irra d ia te d animal in providing carbon sources fo r increased lip o -
g en esis, The ro le of glycolysis to provide lip i d precursors was in
v e stig a te d by the in je c tio n of glucose-l-C-^* in to normal and ir ra d ia
te d r a t s . However, a fte r roentgen treatm en t, glycolysis apparently
provided no increased amounts of carbon as judged by h ep atic incor
p o ratio n measured both in vivo and in v itr o . On th e other hand, a
g re a te r re le a se of p ro te in c a ta b o lite s may provide lipogenic sources
subsequent to ir r a d ia tio n . T herefore, i f lipogenesis was measured in
the x-rayed r a t by means of a lab eled amino acid an isotope d ilu tio n
e ffe e t might be pronounced* This appears to be th e ease, since a
marked reduction in g ly c in e -l-C ^ incorporation was noted in the
liv e r n e u tra l f a t of r a ts tre a te d w ith l£O0 r as compared to controls*
Supporting t h i s , S eh reier e t al* (58) have demonstrated w ith G ^ -
glycine an enlargement in the siz e of the pool of th is amino acid in
x-rayed anim als.
The d ire c t in ju rio u s e ff e c t of x -ray s upon the in te s tin a l tr a c t
i s markedly re fle c te d in i t s lip id metabolism. These e ffe c ts however,
d if f e r ra d ic a lly from those observed in h epatic tissu e* Unlike the
l iv e r , fa s tin g does not im pair the in te s tin a l a b ility to incorporate
lab eled ace ta te in to lip id s . F u rth e r, a more ra p id turnover of C ^ -
a ce ta te i s not evident in th is l ip i d fra c tio n from irra d ia te d rats*
On the co n trary , the build-up of ra d io a c tiv ity in th e irra d ia te d
groups a t the l a t e r periods suggests a possible impairment in the
f a t t y acid u tiliz a tio n of in te s tin a l g ly cerid es, A decreased energy
56
requirem ent in irra d ia te d in te s tin a l c e lls may reduce u tiliz a tio n of
glyceride sto re s fo r th is process* This i s in agreement with workers
(59) who have observed m ito tic a rre s t and the lack of c e llu la r matura
tio n in tis s u e exposed to x-rays*
A ltera tio n s in in te s tin a l glyceride metabolism subsequent to i r
ra d ia tio n may be re la te d to the form ation and ex cretio n of fe c a l f a t
in th is syndrome* P a rt of the in te s tin a l n e u tra l l ip i d le v e ls b u ilt
up in the x-rayed animal could be u tiliz e d fo r th is process* Animals
subjected to 1000 and 1500 r have demonstrated s ig n ific a n t elevations
in the lip id content of the feces over th a t found normally* In addi
tio n , tre a te d animals are capable of u tiliz in g la rg e r amounts of in -
tra p e rito n e a lly in je c te d labeled acetate fo r fe c a l lipogenesis than
normal r a t s . Mead (25) has a ttrib u te d the increased fe c a l f a t le v e ls
as being derived from in te s tin a l desquamation, desp ite the f a c t th is
has been shown by other in v e stig a to rs (59) n o t to provide f e c a l lip id
in the normal animal* Assuming fe c a l lip id to a ris e from d iffe re n t
sources in the normal and x-rayed animal i t might be expected to have
a somewhat d iffe re n t glyceride composition in each case. However,
fra c tio n a tio n of fe c a l n e u tra l f a t samples from normal and x-rayed
animals have shown both fe c a l lip id s to contain approxim ately the same
amounts of fre e f a t t y a cid s, mono-, di-and trig ly c e rid e s* Therefore,
fe c a l lip id in both normal and irra d ia te d animals may be considered
to have a sim ila r origin*
Measurements of sm all in te s tin a l lip id residues in normal and
tre a te d animals a f te r C -^-acetate and C ^ -g ly c e ro l in je c tio n were
57
Bade to d ire c tly t e s t th is postulate* Both lip id precursors were
incorporated to maeh la rg e r ex ten ts in to the in te s tin a l l ip i d r e s i
dues is o la te d from x-rayed animals* The sm all in te s tin e , th e re fo re ,
appears to he d ire c tly involved in the form ation of excreted lip id s
as suggested by Deuel (6 0 ), F u rth e r, the form ation o f l ip i d a t th is
s i t e i s accelerated a f te r roentgen treatm ent when judged by the in
corporation of the two tra c e r compounds*
X-ray treatm ent has been demonstrated in these and previous
stu d ies (61) to s trik in g ly a lt e r in te s tin a l phospholipid metabolism*
The amounts of phospholipid p resen t in th is organ are ra d ic a lly re
duced by irra d ia tio n together w ith th e ir c a p a b ility to incorporate
lab e le d precursors* Both these conditions obtained in x-rayed r a ts
when lip o g en esis was assessed a f te r the adm inistration o f lab eled
a c e ta te , g ly ce ro l, caproate, glucose or glycine* Synthesis of these
lip id s may be c u rta ile d because of widespread c e llu la r breakdown re
su ltin g from x -ra y exposure as rep o rted by sev eral in v e stig a to rs (15)*
However, another p o ssib le explanation of th is phenomenon a ris e s from
some re c e n t work of Z ilversm it e t al* (62)* These workers have demon
s tr a te d in hepatectom ized r a ts in je c te d w ith p32 th a t the in te s tin e
may provide appreciable amounts of plasma phospholipids* I t is tem pt-
i
ing to p o stu late th a t the in te s tin e of the x-rayed r a t may supply a
p a rt o f the elev ated plasma phospholipid le v e ls noted by sev eral work
e rs (15) to occur in th e irra d ia tio n syndrome*
In a d d itio n to the irra d ia tio n anomalies noted, comparisons
demonstrate th a t the liv e r and in te s tin a l t r a c t d if f e r in th e ir a b il-
$9
Amino acids are f e l t to be the most av ailab le glycogen pre
cursors since x-rays have been shown to e x e rt a pronounced proteoly
t i c e ffe ct* A study of the a b ility to u tiliz e C ^ -g ly c in e fo r g ly -
cogenesis has demonstrated x-rayed animals b e tte r able to incorporate
th is la b e l in to th is liv e r fra c tio n than controls* This is evident
immediately a f te r exposure to l£OQ r as w ell as tw enty-four and f o r ty -
eig h t hours la te r* In support of th is finding* Say and Entenman (6?)
have shown the blood and u rin e amino acid le v e ls to be elev ated in
x-rayed ra ts*
The a v a ila b ility of glycogenic precursors may not be th e only
fa c to r reg u latin g the liv e r glycogen le v e ls a f te r irra d ia tio n * Kay
and Entenman (1*0) have demonstrated liv e r glycogen d eposition to in
crease w ith the in te n s ity of the applied irra d ia tio n w hile urea ex
c re tio n reaches a maximum le v e l and remains constant* Thus* a ra d ia
tion-induced hormonal stim u latio n may be responsible fo r glycogen
deposition in ad d itio n to the increased a v a ila b ility of precursors*
Several in v e stig a to rs (68 , 69) have shown irra d ia tio n to re s u lt
in a marked hyperglycemia* Kay and Entenman (1*0) have concluded th a t
the elevated blood sugar le v e ls are not re la te d to th e accumulation
of liv e r glycogen a f te r roentgen treatm ent* However, in our labora
to rie s a f te r the ad m in istratio n of uniform ly lab eled glucose the i r
ra d ia te d r a ts incorporated much la rg e r amounts of ra d io a c tiv ity in to
liv e r carbohydrate sto re s than d id controls* These r e s u lts th erefo re
suggest th a t follow ing x -ray exposure a t le a s t a p a rt of the liv e r
glycogen i s formed from blood glucose*
SUMMARY M m C O N C L U S I O N S .
The incorporation of lab eled ace ta te and g ly cero l in to tissu e
lip id s of the r a t demonstrates th a t the f a t t y acid and n o n -fatty
acid m oieties of l ip i d molecules turnover a t ra te s independent
of eaeh other* Thus, measurements o f the sy n th esis of the com r*
p le te lip id molecule may not n e ce ssa rily be re la te d to the tu rn
over of a p a rtic u la r moiety*
Exposure to io n izin g ra d ia tio n has been shown to stim ulate the
incorporation of lab eled a ce ta te in to the f a t t y acids of both
liv e r n e u tra l f a t and phospholipids* F asted x-rayed r a ts have
ex h ib ited an increased capacity fo r acetate-C -^ incorporation
as compared to fed r a t s , desp ite th e f a c t th a t fa s tin g per se
depresses the inco rp o ratio n of th is lab eled compound*
The irrad iatio n -in d u c ed stim u latio n in hepatic lipogenesis
demonstrated in the in ta c t r a t was evident when G -^-acetate
incorporation was measured in v itro w ith liv e r s lic e s . This
c le a rly shows th a t the irra d ia tio n a ffe c t upon lipogenesis is
operative a t the c e llu la r level*
Incorporation stu d ie s w ith o th er th an d ire c t lipogenic pre
cursors im plicate amino acids as p o ssib le carbon sources fo r
increased lip id synthesis observed in th e liv e r s of x-rayed r a t s .
The re la tio n sh ip of p ro te in catabolism to the increased lip id
form ation in the irra d ia te d r a t i s discussed*
The marked e le v a tio n in the f a t t y acid and monoglyeeride com
p o sitio n of hepatic n e u tra l f a t from x-rayed r a ts as compared
to co n tro ls p resen ts fu rth e r evidence of an irrad iatio n -in d u c ed
lipogenesis*
6* A general impairment in the f a t t y acid u tiliz a tio n of the in
te s tin a l glycerides of tre a te d r a ts i s in d icated from the de
creased turnover and build-up o f lab eled a ce ta te in th is lip id
fractio n * This was accompanied by pronounced reductions in the
phospholipid contents of the in te s tin a l w alls o f x-rayed r a ts
which was re fle c te d in a decreased incorporation of a l l la b e ls
stu d ied .
7* Roentgen treatm ent has been shown to r e s u lt in elev ated fe c a l
f a t levels* That these le v e ls r e s u lt from a stim u latio n in
sy n th e tic mechanisms i s evidenced by the increased u tiliz a tio n
of in tra p e rito n e a lly adm inistered a ce ta te fo r th is process a fte r
irra d ia tio n * The s i t e of fe c a l l ip i d form ation i s discussed in
normal and irra d ia te d r a ts in lig h t of d ire c t measurements made
upon th e u tiliz a tio n of lab eled precursors fo r the synthesis of
in te s tin a l lumen lip id *
8* Irra d ia tio n has been shown to induce elev ated liv e r glycogen
le v e ls which could only r e s u lt from synthesis ra th e r than a re
ta rd a tio n in glyeogemesis* The increased capacity of irra d ia te d
animals to incorporate lab eled a c e ta te , glycine and glucose
serves to fu rth e r demonstrate the x -ra y stim u latio n in glyco-
genic a c tiv ity *
BIBLIOimAPHY
1 . B u tle r, J . A. V ., Radiation. Research, U, 20 (1956)*
2* Rugh, R ., The M ilita ry Surgeon, 112:, 395 (1953).
3* Limperos, G ., and Mosher, A* A*, Science, 112, 86 (1950)*
I*. Smith, B« £ • , P a tt, R. M.,' and Tyree, E. B ., Argonne N ational
L aboratories Report - 1&01, 72 (191*9).
5 . Bacq, Z. M., Burg, 6 ., C heV allier, A*, and Heusghem, G.,
£ . P hysiol*, P a ris , 1*3, 61*0 (1951)*
6* (3rd, 1* S ., and Stocken, L. A ., P hysiol. R ev., 33, 356 (1953)*
7 . Altman, K ., Richmond, J . , and Salomon, K«, Biochim. e t biophys.
a c ta , 7, ¥ © (1951).
8 . Bernheim, P ., Athos, 0 ., and W ilbur, K. A ., R adiation Research,
ky 132 (1956).
9 . R osenthal, R. L«, Science, 110, U3 (191*9).
L0. Steadman, L. T«, and Grim aldi, H ., Univ. of Rochester Atomic
Energy Report - 227 (1952).
11. H e e itt, J . E ., and Hayes, T. L ., Am. J . P hysiol. , 185, 257 (1956).
12. Goldwater, W. H ., and Entenman, G ., A m . Chem. Soc. Meeting,
Los Angeles, March, 1953*
13. G ornatser, W * E*, Engels ta d , 0 ., and Davison, J . P ., Am. J#
P h y sio l., 175, 153 (1953). “ “*
II*. Buchanon, D. J . , Darby, W. & . , B ridgforth, C. B ., Hudson, S . ¥ .,
and E fn er, J . A*, Am. J . P hysiol. , 17l*, 336 (1953).*
15. Neve, R. A ., and Entenman, C«, U. S. Naval R adiological Defense
Laboratory R eport, A D 307 (B ), I95I«
16. Entenman, C ., Neve, R. A*, Supplee, H ., and Olmstead, G. A .,
Aroh. Biochem. and B iophysics. , 59, 97 (1955) •
17. Steadman, „L. T«, and Thompson, H. E ., Quart. Tech. Rept. Univ.
Rochester, W R ; 103, 195©.
18. Pohle, E ., and Bunting, C. H«, Acta R adiol. , 13, 117 (1932).
|L9. A rie l, S . M., Radiology, 57 , 561 (1951).
63
20. E llin g e r, F ., ib id . , 2 * 1 # , 2l#l (19i#5).
21. Entenman, C ., Weinman, E . 0 ., and L erner, R. L ., Arch. Biochem.
and Biophysics, 61#, 161# (1956).
22. Florsheim , ¥ • H ., and Morton, M. E ., Am. J . P h y sio l., 176, 15
(1951#). ------ -----
23. C om ataer, ¥ • E ., Davison, J . P ., and Simonson, e . , Federation
Proc. , 13, 656 (195U). “ *
2 1 # .. Gonard, R«, A m * 2* Physiol. , 165, 375 (1951)•
25# le a d , J . F ., Becker, A ., and B ennett, L* R ., J . N u tritio n 1#3,
m ( i9 5 i) .---------------------------------------------------- -----------------------
26. Dobbs, E. C ., and W ebster, J . H ., B r it. R a d io l., 29, 3lj0 (1922#)*
2?. C oniglio, J* G«, Darby, ¥ • J . , W llkerson, M. C ., S te s a rt, R .,
S tockm ill, A*, and Hudson, G. W., Am. J . P hysiol. , 1?2, 86 (1953).
28. C oniglio, 2* G ./D a rb y , W. J* , E fner, J . A ., Fleming, J . , and
Hudson, G. ,¥ ., ib id . 181#, 313 (1956).
29. Lourau, M., and L aritiq u e , 0 ., Arch, s c i. p h y sio l. , 5 , §3 (1951).
30. Barron, E . S . G«, Wolkcwitz, ¥ ., and Muntz, J . A*, Atomic Energy
Commission Document, M D D C - 12l#l, 19k7*
31. D etrick, L. E ., Dpham, H. G ., Higfeby, D ., Debley, V ., and
Haley, T ., R adiation Research, 2 , 1#83 (1955).
32. F is h e l, E ., Sehneiz. med. Wochsohr., 71, 761# (191*1).
33. Warren, S ., Arch. P a th ., 3U, 27© (191*2).
3l*. North, N ., and N ias, L . F ., F ederation Proc. , 8 , U 9 (19l#9).
35. McKee, R. ¥ . , F ederation Proc. , 11, 256 (1952).
36. P ro sser, G. L ., quoted by Lourau e t a l . , J . p h y s io l., 1#3, 593
(1951). ~ :
37. Ross, M. H ., and E ly, J . 0«, 2* C e llu la r Comp. P h y s io l., 1*3,
593 (1951). ~ *”*
38. Denson, J . R ., Gray, E . J . , Gray, J . L ., H erbert, F . G ., Tew, J . T.
and Jensen, H«, Proc. Soc. Exp. B iol, and Med. , 82, 707 (1953).
39. Nims, L . F ., and Sutton, E ., Am. J . Physiol. , 177, 51 (1951#).
t
6k
b©» Supplee, H*, Uteinman, E . 6*, and Entenman, C*, U*S* Naval
R adiological Defense Laboratory R eport, TR-18, 3555*
bl* Goldman, B* S ., Chaikoff, I* L*, R einhardt, W * O*, Entenman, C*,
and D ailben, W * C ., J . Biol* Chem*, 18b, 727 (1 9 0 )*
k2* Bosh, M * T .j and Stew art, R ., Anal* Ghem*, 19, lb3 (19b7)*
b3* Bush, M * T*, and Densen, P* M*, Anal* Ghem*, 20, 121 (19b8).
bb* M attson, F* H*, P ersonal Communication*
b5* T arver, H*, The P ro te in s, Vol H , p a rt B, Academic P ress, 195b*
k6* Smith, D* E*, and T yrse, E* B*, R adiation Research, b , b35 (1956).
1 * 7 .* Low-Beer, A*, S trah len th erap ie, b6, k69 (1933)*
b8. Bond, 7* P*, S w ift, M * N*, A lien, A* C*, and F is h ie r, M * C*,
A m * J . Physiol* 21, 321 (195©).
b9* Q uastler, H* E«, Laugl, R*, K e lle r, M * E*, and Osborn, J* ¥ .,
ib id , , 161, 323 (1951).
50. F e lts , J • M*, Chaikoff, X * L*, and Osborn, M * J * , J* Biol* Ghem*
191, 683 (1951).
51* Chernick, S* S*, Masoro, E« J* , and C haikoff, I* L*, Proc* Soc*
Exp. Biol* and Med., 73* 3b8 (1950)*
52* Morehouse, M. G ., S k ip sk i, W * P ., Searcy, R*, Federation Proc*,
13, 586 (195b). ~~
53* Steadman, L* T*, Quart* Tech* R ept. Univ. R ochester, DR-70, lb
, (19U9). ; “ ‘ *
5b* Gurin, S ., in C lin ic a l Symposium Upon L ipid Metabolism,
Johns Hopkins, 1953'*
55* Brady, R* 0*, and Gurin, S ., J* B iol* Chem*, 187, 589 (195©)•
56* Pow ell, ¥* F*, and P o lla rd , Phys* Rev*, 91, b9b (1953)*
57* Pow ell, W * F*, and P o lla rd , R adiation Research, 2, 109, (1955).
58* S ch reier, K ., D iFerrante, N ., Gaffney, G * ¥ * , Hempelmann, L* H*,
and Altman, K * I* , Arch* Biochem* and Biophysics, 5© » b !7 , 195b*
59* Sperry, W. 1 * , J* B io l* Chem*, l i b , 125 (1936)*
65
60. Deuel, H. J . , The L ip id s, Vol. I I , In te rsc ie n c e , 1951#
61. Morehouse, M. G ., and Searcy, R. L ., R adiation Research, k ,
175 ( i 956). r .
62* Z ilv ersm it, D. B ., Arch* Biochem* and Biophysics, 63, 6U (1956)*
63* Artom, C ., B oll* Soc. I t a l . B io l, sp e r. , 13, 580 (1938).
6k* Chaikoff, I . L ., Physiol. Rev. , 22, 291 (19lt2).
65. T o lb ert, 1 . E ., and ©key, R. J . , J . B io l* Chem. , 19U, 755 (1552).
66. Morehouse, M « G ., and Searcy, R* L ., Science, 122, 158 (1955)*
67. Kay, R. E ., and Entenman, 0 ., Proc. Soc* Exp. B iol# and Med. ,
91, 1 1 * 3 (1956). ■ “ ‘ —
68. Lourau, M. and L aritiq u e , Arch, s c i . p h y sio l. I 4 ,, 197 (1950).
69. Buchwald, K. ¥ . , J . E a p tl. le d . 53, 827 (1931).
Gniverslty of Southern California

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Asset Metadata

Creator Searcy, Ronald L. (author)

Core Title The effects of irradiation upon lipid and carbohydrate metabolism in the rat

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1957-06

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-604009

Unique identifier UC11354559

Identifier DP21576.pdf (filename),usctheses-c17-604009 (legacy record id)

Legacy Identifier DP21576.pdf

Dmrecord 604009

Document Type Dissertation

Rights Searcy, Ronald L.

Type texts

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

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

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