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An investigation of carotene metabolism in the alloxan treated rat

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An investigation of carotene metabolism in the alloxan treated rat

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Content A N INVESTIGATION O F C A R O TEN E M ETA BO LISM IN THE
A L L O X A N TREATED RA T
A T hesis
P resented to
the F a c u lty of th e Department of B iochem istry
The U n iv e rsity o f Southern C a lifo rn ia
School of Medicine
In P a r tia l F u lfillm e n t
o f th e Requirements f o r the Degree
M aster o f Science
by
John Rowland Lowry
June 19U8
UMI Number: EP41285
All rights reserved
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This thesis, written by
JO H N H O W L A N D L O W R Y
under the guidance of h.Xs... Faculty Committee,
and a p p ro ved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements for the degree of
1 ^
.m ST £.S .. .OF .. .. aC. XE NCE
IL-W-.-PAT.MOEE.
Secretary
Date 9 M
Faculty Committee
3 ^
/ . Chairman
IX & Z X
..Lgxvx^JIuSL ..
ACKNOWLEDGMENTS
I wish to ex p ress ny thanks to D rs. H. J . D euel, J r . ,
John W. Mehl, and W alter Marx, fo r t h e i r many h e lp fu l suggestions
and invaluable a id during th e course of t h is work.
TABLE OF CO N TEN TS
CHAPTER PA G E
I . INTRODUCTION A N D HISTORICAL REVIEW.......................... 1
I I . STA TEM EN T OF PROBLEM....................................... . ............................. 6
I I I . EXPERIMENTAL..............................................>......................................... 8
M a terial and methods ..................................................................... 8
Vitam in A a n a l y s i s ...................................... 9
Blood carotene method............................................ . . . . . 10
IV. RESULTS A N D DISCUSSION.................................................... 12
Rat experim ents . . . . . . . . . . . . . . . . . . . 12
Experim ents on humans . . . ................................................ 17
V. SU M M A R Y . . ......................................................................................... 29
BIBLIOGRAPHY.......................................................................................................... 30
APPENDIX................................................................................................................... 37
LIST O F TABLES
TABLE PJGE
I . E ffe c t of A lloxan in B ats on the A b ility to Convert
lilO V Carotene in to Vitamin A - 72 Hour
Experiment ...................................... 19
I I . E ffe c t of A dm inistration o f In s u lin on th e S urvival
Time o f Rats In je c te d w ith 350 mg. per Kg.
A lloxan . . . . . . . 20
I I I . E ffe c t o f 200 mg. p e r Kg. Alloxan on the A b ility to
Convert 11*0 V C arotene in to Vitamin A - 1 * 8 Hour » -
Experiment ...................................... 21
IV. E ffe c t of Alloxan on the Storage A fter Feeding in R ats
Fed 300 I.U . Vitamin A - 1 * 8 Hour Experiment . . . . . 22
V. E ffe c t o f Alloxan on R ats on the A b ility to Convert
li*0 ¥ Carotene in to Vitamin A - 72 Hour E xperim ent. • 23
VI. E ffe c t o f A lloxan on Rats Fed 200 I.U . V itam in A
A cetate - 72 Hour Experiment ..................... 2 1 *
V II. E ffe c t of 200 mg. p e r Kg. Alloxan and O.lt U n its
In s u lin on Rats Fed ll*0 1 Carotene o r 200 I.U .
Vitamin A - 72 Hour Experiment • • • • • • . . . . • • Z$
V III. Negative C ontrols - R ats Fed No Carotene o r Vitamin A. . 26
IX. Summary o f Data - 1 *8 Hour E x p e rim e n t................... ....... 27
X. Summary o f Data - 72 Hour E x p e rim e n t................. 28
C H A P T E R I
INTRODUCTION A N D HISTORICAL REV IEW
There has long been thought to be some derangement o f carotene
m etabolism in d ia b e te s m e llitu s . As e a r ly a s 190U von Noorden (1)
noted a yellow pigm entation in p a tie n ts w ith d ia b e te s . Palmer and
E ckles (2) showed th a t th e carotene was p re se n t i n th e blood as a
carotalbum in, and in 1919 Hess and Myers (3 ) recorded in sta n c e s o f
high blood carotene in d ia b e tic s . Rabinow itch (h ,$ ) seemed to fin d
th a t th e more d i f f i c u l t d ia b e te s was to c o n tro l th e more fre q u e n tly
carotenem ia appeared. I n Q $% o f 500 cases o f d ia b e te s , the carotene
v alu es were above norm al. L a te r, t h is same in v e s tig a to r (6) s ta te d
th a t d ia b e tic s f o r some unknown reason r e ta in vegetable pigm ents
(ca ro te n e ) more th an i s normal and th a t t h i s accounts f o r th e caro
tenem ia.
White and Gordon ( 7 ) , employing a dichrem ate standard f o r
carotene d eterm in atio n , observed th a t th e normal range f o r blood
carotene was 2-8 dichrom ate u n its w hile fo r d ia b e tic s th e range was
5-25 u n its , averaging lU . L a te r Stueck e t a l . (8) found in t h e i r
s e r ie s o f d ia b e tic s the average was 26 dichrom ate u n its .
R a ll i , Brandaleone and Mandelbaum (9) gave d ie ts w ith th e
same carotene co n ten t to a group of normal and d ia b e tic p a tie n ts .
W hile th e d ia b e tic s had h ig h er blood carotene values to begin w ith ,
values rose h igher in th ese p a tie n ts than i n th e normals and remained
2
h ig h er lo n g e r. Large amounts o f carotene were found in th e liv e r s
o f d ia b e tic s on au to p sy . . T his le a d to the e x p la n atio n o f high blood
carotene in d ia b e te s as being due to the i n a b i l it y o f th e l i v e r to
convert carotene to vitam in A and th e unused excess th u s being thrown
o ff in to th e blood stream . T his was extended by work o f R a lli,
P a rie n te and Brandaleone (1 0 ).
Haymann (11) a rriv e d a t s im ila r conclu sio n s. He a lso found
th a t d ia b e tic s e x h ib ite d high blood carotene values and when equiva
l e n t amounts o f carotene were fed to normal and d ia b e tic s u b je c ts ,
th e blood carotene in th e d ia b e tic s rose to h ig h er le v e ls and r e
mained high lo n g er th an did th e norm als. I t was f e l t th a t t h is e f f e c t
was n o t d u e t o a hypercholesterem ia o r a hyperglycem ia.
Clausen (12) in 1933 had found th a t th e l iv e r s o f d ia b e tic
in d iv id u a ls , obtained a t autopsy, had a low vitam in A c o n te n t.
However, Moore (13) found the rev erse in a s e r ie s o f an aly ses o f
d ia b e tic l i v e r s . He re p o rte d th a t th e vitam in A content was h ig h er
th an nonnal and th is tended to ru le o u t the e x p lan atio n th a t th e re
was in p a ire d tran sfo rm atio n o f carotene to vitam in A in d ia b e te s .
His ex p lan atio n was th a t carotene tended to accum ulate a s a r e s u lt
o f a g en eral slowing down o f m etabolism .
Bessey and Wolbach (lU) argued that the blood carotene is a
measure of the difference between the rate of intestinal absorption
i
and th e r a te o f conversion o f carotene in to vitam in A. Hence, in
d ia b e te s , th e ra te o f conversion must be low ered.
I f th e id e a o f im paired conversion were sound, th en d ia b e tic
su b je cts should show a t le a s t o ccasio n al evidences of vitam in A
deficiency* Twenty p a tie n ts w ith ,ju v e n ile d ia b e te s m e llitu s were
examined by Braze r and C u rtis (1*>) who employed th e biophotom eter*
A ll were found to have poor l i g h t ad ap tatio n and nine o f th e group
showed cutaneous changes com patible w ith vitam in A d e fic ie n c y . D aily
a d m in istra tio n o f 60,000 U .S.P. u n its o f carotene d id n o t a ff e c t th e
l ig h t a d ap tatio n w h ile d a ily doses o f 60,000 U .S.P. u n its o f vitam in A
caused lig h t a d a p ta tio n to re tu rn to normal in th re e to twenty-one days
These in v e s tig a to rs p laced th e blame on th e i n a b i l i t y of th e d ia b e tic s
to convert carotene in to vitam in A.
M u rrill, H orton, and Lieberman, found in t h e i r s e rie s of p a tie n ts
deranged carotene m etabolism but no im paired form ation of vitam in A
from carotene (1 6 ). In 16 p a tie n ts th e blood caro ten e averaged 291
micrograras p e r cen t w hile th e normal p a tie n ts averaged 213 micrograms
p e r c e n t. "C arotene to le ra n c e eurvesn showed th a t in d iab e te s the
blood carotene value f e l l back to th e i n i t i a l le v e l w ithout delay
a f t e r an i n i t i a l dose o f 130,000 I.U . o f caro ten e .
A s e r ie s o f d ia b e tic and norm al c h ild re n were stu d ie d by
M osenthal and L oughlin (1 7 ). Tw enty-eight p e r cent o f th e d ia b e tic
c h ild re n e x h ib ite d a hypercarotenenda and 63 p e r cent had serum
vitam in A le v e ls below norm al. Nine p e r c en t o f th e c h ild re n had sig h s
o f vitam in A d e fic ie n c y . There was a d ir e c t c o rre la tio n o f th e blood
carotene le v e l and th e degree o f cholesterem ia and a p a ra lle lis m
between hepatomegaly and hypercarotenenda — supporting the concept
o f th e dependency o f an in c re a se in carotene and i t s s o lu b ility in f a t .
In a l a t e r re p o rt th e sane w orkers (18) f in d , in an o th er s e rie s
o f d ia b e tic s , high blood carotene in 2k p e r cen t o f th e cases stu d ie d .
But th ey b e lie v e th a t t h i s carotenenda once common i s becoming le s s
and le s s im portant in d ia b e te s . Inproved methods o f treatm en t are
th e reasons f o r t h is b u t th e ex act mechanism i s unknown. Perhaps
carotenenda i s th e r e s u lt o f in p a ire d f a t metabolism which can show
improvement w ith pro p er d i e t .
In a s e rie s o f 116 u n se le c te d d ia b e tic s , Kimble e t a l . (19)
were unable to show high blood carotene in a s ig n if ic a n t number of
t h e i r s u b je c ts . They d id , however, dem onstrate th a t a la rg e number
o f th e d ia b e tic s e x h ib ite d a low vitam in A :carotene r a t i o . Carotenenda
was r a th e r in fre q u e n t. These w orkers a lso a ttr ib u te th e deranged
carotene m etabolism found in d ia b e tic s f o r so many y ears to d ie ta ry
f a c to r s .
\
R e la tiv e ly l i t t l e expe rim en tal work has appeared on t h i s
I
problem . R alH (20) found vitam in A d e fic ie n c y symptoms in depancre-
a tiz e d dogs and th a t th e vitam in A content o f th e l iv e r s was r e la tiv e ly
low. T his was n o t c o rre cte d by feed in g carotene and a deranged f a t
m etabolism was p o stu la te d as th e cause of the observed phenomenon.
T his was follow ed by an endeavor to cure th e synptoms found
in depancreatized dogs by v ario u s d ie ta r y a g e n ts. The observed lo s s
and sc a lin e s s were n o t cured by c y s te in e , y e a s t, l i v e r e x tra c t o r
vitam in A. The l iv e r s o f th e dogs had normal s to re s o f vitam in A.
In no case in any o f the work d id R a lli eaploy p a n c re a tic e x tr a c t as
a cure f o r th e condition observed in th i s : • dogs. T his alone would
c a s t some doubt on the v a lid ity o f t h i s work.
A nother in v e s tig a tio n (21) by H a lli involved th e e f f e c t o f
lig a tin g th e p a n c re a tic duct o r pancreatectom y on the l i v e r sto rag e
of vitam in A a f t e r carotene feeding to dogs* Carotene was fed in
corn o i l over se v e ra l months and the r e s u lts showed th e l iv e r s o f
lig a te d and dep an creatized dogs had le s s vitam in A th an those o f
normal anim als.
C H A P T E R I I
STA TEM EN T OF PRO B LEM
" N
I t i s obvious from tb s preceding c h ap ter t h a t th e r e la tio n
o f pancreas to carotene m etabolism i s n o t w e ll understood. I t was
decided th e re fo re to examine th e conversion o f carotene to vitam in A
in anim als w ith d e f ic ie n t p a n c re a tic fu n c tio n . A unique to o l was
known to be a v a ila b le f o r th e production o f d ia b e te s in t e s t anim als.
T his substance i s a llo x an and ex ten siv e work from many la b o ra to rie s
has shown c o n clu siv ely i t to be able to cause a tru e d ia b e te s alm ost
in d is tin g u ish a b le from th e c lin ic a l type (Appendix).
The r a t was se le c te d a s th e t e s t anim al since i t has been
e x te n siv e ly used in the work leading to th e provitam in concept o f
carotene a c t iv i t y . The r a t can be d ep le te d o f a l l d e te c ta b le l iv e r
vitam in A and upon feeding carotene and subsequent a n a ly s is o f the
l i v e r o f such anim als e x h ib it newly formed vitam in A. T herefore, i f
th e re i s any d e v ia tio n from normal carotene netabolism in d ia b e tic
r a t s p rev io u sly d ep leted o f l i v e r vitam in A, i t should be d e te c ta b le
a s th e abnormal content o f th e l i v e r vitam in A follow ing a t e s t dose
o f carotene adm inistered p e r o s.
Experiments were planned and c a rrie d o u t a t two tim e in te r v a ls .
The f i r s t involved a U8 hour p erio d from th e tim e o f in je c tio n of
allo x an to th e time o f k i ll i n g . Carotene was given 2k hours before
k i l l i n g . The second involved a 72 hour experim ent a s i t was d e sire d
to have a s ex ten siv e a p e rio d o f d ia b e te s as p o s s ib le . I t i s n o t
7
p o ssib le to prolong th e s tu d ie s o f groups o f vitam in A -free a llo x an
in je c te d r a t s lo n g e r th a n 72 hours.
By ex p erim en tatio n , i t was found th a t 200 mg. p e r Kg. o f
a llo x an was an e f fe c tiv e le v e l w hile 3£0 mg. p e r Kg. eaused tis s u e
damage. Data on b o th le v e ls have been o b tain ed .
One study o f t h i s problem has appeared which involved a llo x a n
(2 2 ). B ats, n o t vitam in A -fre e , were given 2000 I.U . o f carotene
d a ily and k ille d by repeated in je c tio n s o f a llo x a n . The tis s u e s o f
th ese r a t s contained le s s vitam in A th an those o f the controls*
A lso, i t was d e sire d to re in v e s tig a te th e o fte n rep o rte d high
blood carotene i n d ia b e te s i n man. A ccordingly, a group of d ia b e tic
p a tie n ts a t th e Rancho Los Amigos o f th e Los Angeles County H o sp ital
were se le c te d along w ith a group of n o n -d iab e tic c o n tro ls . Employing
th e n et hod o f blood carotene determ ination o f Kimble (2 3 ), i t was
found th a t th e d ia b e tic group e x h ib ite d markedly h igher v a lu e s.
Acknowledgement i s made o f the in v alu ab le h e lp in t h is phase o f th e
work to Miss B io le tta Thomas and Mr. Sheldon M. S in c la ir, Chemists a t
th e Los Amigos H o sp ita l.
CHAPTER I I I
EXPERIM ENTAL
I . M ATERIALS A N D M E T H O D S
F or the production of d ia b e te s, allo x an ob tain ed from th e
Edwol L a b o ra to rie s, Chicago, I l l i n o i s was used. I t m elted a t 2$2°-
25ii°C. (L ite ra tu re 256°C.) and assayed 17-18$ n itro g e n (T h e o re tic a l
17.50$). S olutions were fre s h ly prepared in p h y sio lo g ic a l s a lin e ,
*
sin c e , upon s ta n d in g ,,a llo x a n decomposes in to in so lu b le parabanic ,
acid*.
F or the in s u lin experim ents, protam ine zinc in s u lin (Squibb)
was employed a f t e r d ilu tio n to th e d e sire d stre n g th w ith p h y sio lo g i
c a l s a lin e . This was in je c te d subcutaneously.
Carotene (90$ b e ta and 10$ alpha) obtained from the G eneral
I
Biochem icals Company was used. I t was d issolved in a sm all amount
o f d ie th y l e th e r and was added to a known amount o f cottonseed o il*
The e th e r was evaporated and the o i l c en trifu g e d to c a rry down any
undissolved caro ten e. This y ie ld e d an o i l containing lUO of
carotene p e r 0.1 m l.
The vitam in A s o lu tio n used was made by grinding c r y s ta llin e
vitam in A a c e ta te ( D is tilla tio n P roducts) in to cottonseed o i l . The
stre n g th was ad ju sted to 200 I.U . p e r 0*1 ml. The stre n g th o f both
the carotene and vitam in A so lu tio n s were v e rif ie d by a n a ly s is on th e
Beckman spectrophotom eter, and th ey were kept under n itro g e n and in
the cold when n o t in u se .
9
R ats from th e U n iv e rsity of Southern C a lifo rn ia stock colony,
were, in je c te d w ith allo x an in tr a p e r ito n e a lly a f t e r a tw enty-four hour
\
f a s t (2 ii). Urine was c o lle c te d under to lu en e in m etabolism cages.
I t was assumed th a t a high p r o te in , low f a t d ie t would be b e n e fic ia l
t
to th e d ia b e tic r a t s . A ccordingly, th e stan d ard vitam in A -free d ie t
was used w ith th e sta rc h reduced to 8% and th e v itam in .te st c a se in
t
in cre ased to 65%. The f a t percentage was 5%. U nfortunately th e work
o f L az aris e t a l . (25) was n o t known in t in s to be o f u se , f o r th ey
showed th a t a llo x a n -d ia b e tic r a t s e x h ib ite d marked improve merit and
lo n g er l i f e i f p laced on a 70# f a t d i e t . Twenty-four hour samples
o f u rin e were analyzed f o r reducing sugar by th e method o f Hawkins
and Van Slyke (2 6 ). The sugar reagent was stan d ard ized a g a in st known
glucose s o lu tio n s .
The r a t s used f o r th e t e s t s were d ep leted o f vitam in A by
p lacin g them on a vitam in A -free d ie t f o r a t l e a s t te n days b efore
u s e . That t h i s e ff e c tiv e ly dep leted th e vitam in A s to re s in th e l i v e r
can be seen from th e d a ta on l iv e r a n a ly sis o f such r a ts (Table V III).
At th e term in a tio n of an experim ent th e r a t s were k ille d by
chloroform in a clo sed j a r . The l iv e r s were excised and kept fro ze n
u n t il th e analyses were c a rrie d o u t.
I I . VITAMIN A ANALYSIS
The v itam in A a n a ly s is enployed was th e method developed a t
th e U n iv e rsity o f Southern C a lifo rn ia ; i t i s d escrib ed in some d e ta il
by Mattson e t a l . (2 7 ).
The samples were p laced In 125 ml. b o ilin g f la s k and 20 ml.
o f 9$% e th a n o l and 1 ml. o f $0% K Q H f o r each gram o f tis s u e was added
and th e m ixture was allow ed to re flu x f o r 30 m inutes. The cooled
sa p o n ifie d s o lu tio n was washed in to a continuous e x tra c to r w ith 20 ml.
o f e th a n o l and 35 ml. o f d i s t i l l e d 1^0 to make th e eth an o l concentrate
50#j care was taken n o t to exceed 80 m l. This was e x tra c te d f o r
th re e hours w ith sk e lly so lv e A. The e x tra c ts were th e n washed w ith
HgO and d rie d w ith sep arate p o rtio n s o f anhydrous sodium s u lf a te . The
samples were made to th e d e sire d volume w ith sk e lly so lv e A. F ive ml.
a liq u o ts were then evaporated to dryness and th e resid u e taken up in
1 m l. o f CHCI3. Vitamin A was determ ined by th e C a rr-P rie e re a c tio n
usin g 5 ml. o f a sa tu ra te d so lu tio n o f antimony tr ic h lo r id e in chloro
form . The rea g en t was added ra p id ly from a p ip e tte care being taken
n o t to cause bubbles and th e maximum fading blue c o lo r was taken as
the vitam in A v alu e. One drop o f a c e tic anhydride was f i r s t added to
preclude th e development o f a cloudy so lu tio n due to w a te r. The con
c e n tra tio n o f vitam in A in th e sample was c a lc u la te d from sta n d a rd i
z a tio n curves prepared w ith known amounts o f vitam in A a c e ta te .
I I I . B LO O D C A R O T E N E M E T H O D
The method o f blood carotene a n a ly sis used was -that o f Kimble
(2 3 ). Three nil. o f blood plasma (lith iu m o x alate was the. a n ti-c o a g u la n t)
were mixed w ith an equal volume o f 9$% e th y l a lc o h o l and 7 m l. of
sk e lly so lv e A. A fte r thorough m ixing, th e m ixture was c e n trifu g e d and
5 m l. o f sk elly so lv e was drawn o f f and read in a Klett-Summe rson
c o lo rim e te r. Blood carotene values were c a lc u la te d by comparison
w ith a c a lib r a tio n curve those a rriv e d a t w ith a stan d ard carotene
s o lu tio n . The f i n a l values are expressed in term s o f whole blood
using th e hem atocrit value o f 0*6.
C H A P T E R IV
RESULTS A N D DISCUSSION
I . R A T EXPERIM ENTS
T his in v e s tig a tio n was undertaken to d isco v er w hether a llo x a n -
d ia b e tic r a t s could e x h ib it a deranged carotene m etabolism . I t th u s
appears from the d ata i n T ables I I I and V th a t r a t s d ia b e tic from a
dose o f 200 mg* p e r Kg* a llo x an can convert carotene in to v itam in A
a s w e ll as norm al r a t s . From th e d a ta obtained i t i s e v id e n t th a t
d ia b e te s does n o t d estro y th e r a t 's a b i l i t y to m etabolize carotene*
The d a ta contained i n Table I I I a re from experim ents on r a ts in je c te d
w ith 200 mg* p e r Kg. a llo x a n and subsequently fe d carotene d isso lv e d
in cottonseed o il* F o rty -e ig h t hours e lap sed from th e tim e o f in
je c tin g a llo x an to th e tim e o f k i ll i n g . A ll th e r a t s e x creted la rg e
amounts o f glucose in a 2k hour p e rio d . R ats $8-62 were weighed a t
th e beginning and end o f th e experim ent and showed an average w eight
lo s s o f 6 grams. This i s due mainly to reduced food in ta k e ; however,
th e l a t t e r was n o t measured q u a n tita tiv e ly * The l i v e r w eights based
on 100 gram body w eight a re normal* The amount o f vitam in A sto re d p e r
gram o f l i v e r a re those o f a normal r a t . These comments can be a p p lied
to Table V w ith the exception th a t th e tim e in te r v a l f o r th e whole
experim ent was 72 h o u rs. The f a c t th a t th e average vitam in A found p e r
gram o f l i v e r i s la rg e r f o r th e d ia b e tic group i s n o t considered sig n i
f i c a n t . The mean value o f the l i v e r w eights o f norm al r a t s was 0*8 grams
h ig h e r th an t h a t o f the w eights of th e d ia b e tic liv e rs * The average
vitam in A v alue in Table I I I i s low er th a n th a t f o r
13
i
Table V b a t th e d iffe re n c e in time in te r v a ls i s most probably the
reason*
I t m ast be emphasized th a t th e lo n g e s t tim e in te r v a l employed
was 72 h o ars. T his can be d escrib ed as an " e a rly d iab etes" as th e re
can be fu rth e r changes in a d ia b e tic anim al i f d ia b e te s i s allow ed
to p e r s i s t fo r weeks o r even months. Then, p o ssib ly deranged caro
tene m etabolism could appear. I t mast be remembered th a t the r a t s
were on a vitam in A fre e d ie t and such a q u estio n could n o t p o ss ib ly
be s e ttle d w ith such anim als.
The im pression was gained from th e p re s e n t work t h a t vitam in
A -d e fic ie n t r a t s are unable to liv e as long as n o re a l r a ts w ith
allo x an d ia b e te s . T his i s in agreement w ith th e o b serv atio n of Wold
(28) who foand th a t J?0# o f h is vitam in A fre e r a t s died w ith in a week
a f t e r receiv in g a llo x a n . Using such a d ie ta r y regime i t i s ap p aren t
th a t th e r a t i s n o t e s p e c ia lly s u ita b le f o r th e study o f long term
a llo x an d ia b e te s . However, i f o th e r anim als are to be used, th e re
rem ains th e problem o f how to ev alu ate carotene metabolism sin ce the
r a t seems to be the only anim al y e t made vitam in A fre e by a standard
la b o ra to ry procedure.
I t i s suggested t h a t the problem m ight be stu d ie d in a llo x an -
tre a te d ra b b its since th ey have been kept a liv e f o r months (2 9 ). I t
i s in te r e s tin g , in t h i s connection, to sp ecu late what e f f e c t th e
th y ro id would have on th e course o f carotene m etabolism in such long
term experim ental d ia b e te s . Koneff (30,31) has shown th a t alloxan
in r a t s causes a ra p id o n se t of hypofunction o f th e th y ro id gland.
A lso, th e re can be no q u estio n t h a t vitam in A and th e th y ro id gland
lU
I
are in some way in te r r e la te d (3 2 ). I t would seem th a t th ese f a c ts
would have to be kept in mind in any work of t h i s n a tu re .
The d iab e te s stu d ied in th e p re se n t work i s an e a r ly one and
would seem to b e ar b u t s lig h t resemblance to the c li n i c a l d ia b e te s
observed in man. I t would be d e sira b le to in v e s tig a te carotene me
tab o lism in long term d ia b e te s.
I t should be p o in ted out th a t th e c r ite r io n f o r th e o n set of
d ia b e te s was a severe g ly c o su ria . In every d ia b e tic r a t th e re appeared
a high glucose output in th e u rin e . T h erefo re, we are d ealin g w ith
a tru e d ia b e te s h e re . I t was n o t considered necessary to determ ine
in a d d itio n , blood su g ar.
When th e d ata from Table V and VI are compared, i t i s apparent
th a t th e re i s no e s s e n tia l d iffe re n c e in th e vitam in A sto re d in th e
l i v e r a f t e r feeding c lo s e ly e q u iv a le n t amounts o f carotene and v ita
min A. Table VI co n tain s d ata a rriv e d a t from 72 hour experim ents
on a llo x a n -tre a te d r a ts fed 200 I.U . vitam in A a c e ta te . The re
s u ltin g vitam in A s to re d in th e l i v e r i s o f the same o rd e r o f magni
tude as th a t o f th e c o n tro l an im als. The a c tu a l mean amount p e r gram
of a llo x an r a t l i v e r i s s lig h tly g re a te r (about 2 I.U .) th an f o r the
norm al, b u t these liv e r s average alm ost a gram h e av ier th an th e norm al
l i v e r s . In some unpublished work on man, McCoord (33) fin d s , th a t
some of h is p a tie n ts su ffe rin g from d ia b e te s responded b e tt e r to th e
fre e v itam in A a lc o h o l th an to th e e s t e r . P o ssib ly th e vitam in A
e s t e r s , i n o rd e r tor be u t il i z e d , must f i r s t be hydrolyzed in v iv o .
■ V.
Vitam in A a c e ta te was used in th e p re se n t work and r a t s , 72 hours
- a f t e r a llo x a n a d m in istra tio n , had no d i f f ic u lt y sto rin g i t in t h e i r
l iv e r s .
Table VII co n tain s d ata ob tain ed from te s tin g tbe p o ssib le
e f f e c t o f in s u lin upon r a t s given a llo x a n , and allow ed to liv e 72
hours. T his experim ent i s analogous to those contained in Tables V
and V I. I t i s shown t h a t th e re i s no change in the vitam in A sto re d
in t h e lliv e r o f th e in s u lin r a ts when compared to those th a t received
no in s u lin (Table V ). Upon considering th e re sp e c tiv e l i v e r w eights
i t can be sa id on th e b a s is o f th e se experim ents th a t in s u lin has no
e f f e c t. A lso, i t i s p la in th a t in th e type of d ia b e te s stu d ie d ,
in s u lin does n o t p a rtic ip a te in th e metabolism o f carotene and
vitam in A w hile nothing can be s a id as to t h e i r u t i l i z a t i o n .
*
A dm ittedly, the in s u lin dosage was a r b i t r a r i l y s e t a t 0.1 a
u n its p e r r a t b u t t h is amount has been shown by C antor, Tuba and
Capsey (3U) to re s to re blood sugar to norm al w ith in th re e hours in
a llo x a n -tre a te d r a t s .
On the o th e r hand, r a t s were n o t able to m etabolize carotene
when adm inistered 35> 0 mg. p e r Kg. a llo x an (Table I . ) . I t can be
seen th a t in 72 hour experim ents, analogous to those where tr a n s fo r
m ation o f carotene in to vitam in A was obtained no vitam in A was
sto re d in th e liv e r s o f r a t s fed 11*0 caro ten e . This dose o f
a llo x an causes damage to the r a t which i s n o t rev ersed by in s u lin
(Table I I ) . When t h is le v e l of a llo x an i s u sed , some r a t s w i l l die
w ith in 2h h o u rs. Those th a t liv e w ill n o t respond to in s u lin , and
even a f te r se v e ra l in je c tio n s , d ie w ith in 96 h o u rs. Those r a t s given
only one in je c tio n o f O.U u n its died in 72 hours and i t would seem
t h a t , i f t h is were a tru e d ia b e te s w ith o u t o th e r co m p licatio n s, the
16
in s u lin would a t le a s t be expected to prolong t h e i r liv e s .
A lloxan, in la r g e r doses i s known to damage tis s u e s o th e r than
p a n c re a tic i s l e t c e l l s . Kidney damage has been observed in many
a llo x a n -tre a te d anim als (35) (36,37) and t h i s i s a p o ssib le answer.
I t was thought th a t damage to th e gut might e x p la in th e fin d in g s of
Table I . A ccordingly, se c tio n s were nade of th e gut and compared
to th a t o f normal anim als and no pathology could be observed. The
mean l i v e r w eights o f such r a t s are high and p o ssib ly t h is i s patho
lo g ic a l evidence. The g u t washings from r a t s 28, 29, 32, and 3k
(Table I ) were analyzed f o r caro ten e and none could be d e te c te d .
This would appear to e lim in a te f a ilu r e to absorb carotene as an
e x p la n a tio n . The la c k of any evidence f o r h is to lo g ic a l changes in
th e g a s tr o - in te s tin a l t r a c t may only mean th a t a biochem ical d e fe c t
( f a ilu r e to convert carotene to vitam in A) has been produced w ithout
obvious h is to lo g ic a l changes.
B a lli (20, 21) in h is woik w ith pancreatectom ized (togs, seems
to fin d a derranged carotene m etabolism . Some doubt can be thrown on
/
h is r e s u lts since no p a n c re a tic ju ic e was su p p lied in any o f h is
work.
Clausen e t a l . (23) stu d ied a llo x an d ia b e te s and carotene
m etabolism in th e r a t . The co n d itio n s in t h e i r experim ents were more
n e a rly comparable to th e p re se n t experim ents w ith 350 mg. p e r Kg.
a llo x a n , sin ce th ey k ille d t h e i r anim als w ith repeated doses o f a llo x a n .
They found th a t such r a t s w ith a llo x a n d ia b e te s evidenced le s s vitam in
17
A in t h e i r l iv e r s a f t e r being fe d 2000 I.U . of caro ten e th an d id the
c o n tro l anim als.
I I . EXPERIMENTS O N H U M A N S
The r e s u lts o f th e blood carotene d eten n in atio n s upon p a tie n ts
a t th e Rancho Los Amigos H o sp ita l, can be sunm arized:
C arotene ^ p e r c e n t
whole blood
D iab etics 3U 101.9 1 19.2
(36.1 -19it)
Normals 2$ (N on-diabetic) $6 1 27.6
(S .6 -139)
Nothing a c tu a lly new o r unknown i s o ffe re d here b u t i t i s
b e liev ed to be a confirm ation o f an o fte n rep e ate d o b serv atio n (3»
U, 6 , 7 , 8 , 9 , 10, 11, 1 2 ).
I t i s b e lie v e d th a t th e reason the normal blood value i s low
j
i s t h a t the method o f a n a ly sis used (23) allow ed incom plete e x tra c tio n
o f carotene from th e plasm a. I t would th u s throw a l l th e v alu es low
b u t th e s ig n if ic a n t d iffe re n c e o f th e two groups i s marked and must
be ex p lain ed . E v id e n tly , in man, blood carotene i s high in d ia b e te s
m e llitu s . A ll p a tie n ts te s te d were on e s s e n tia lly th e same d i e t .
A ll o r n e a rly a l l the d ia b e tic s were m aintained on in s u lin . I t must
be emphasised th e th e "norm als’ * are only n o n -d ia b e tic s and t h i s does
n o t exclude o th e r p a th o lo g ic a l c o n d itio n s. Some o f th e "normal"
blood carotenes were h ig h (fiv e cases g re a te r th a n 80 ^ p e r cen t) so
th a t p o ssib ly o th e r co n d itio n s e x h ib it high blood caro ten e .
18
I t seems, in view o f so many re p o rts , th a t high blood carotene
i s a fin d in g to be a sso c ia te d w ith d ia b e te s in man, and, the work
o f Kimble e t a l . (19) n o tw ith stan d in g , w i l l s t i l l have to be ex
p la in e d .
19
T A B L E I
EFFECT OF A L L O X A N IN RATS O N TH E ABILITY TO C O N V ER T 11*0 *
C A R O TEN E INTO VITAMIN A - 72 H O U R EXPERIM ENT
Rat
number
W eight Sex L iv er w eight
p e r 100 gm.
body w eight
Brine sugar
gma. p e r 2 1 *
h o u rsl
Vitam in A p e r
gram liv e r
2 1 * hour t e s t
I.U .
In je c te d w ith 350 mg. p e r Kg. a llo x an
17 60 M 10.7
++++
0
20 60 M 7 .0
++++
0
22 61 M 6.1*
++++
0
282
62 F
9.5
++++
0
322
62 F 8.7
++++
0
3 hi 63 F
7.7 1 .5 0
29 62 F 12. ij
2.3 0
Mean
8.91
C ontrols
23 63 M 9.3 0 6.7
2 1 * 63 M
9.05
0 l*.l*
25
6 1 * M 8.1* 0 6.7
26 6 1 * F 6.6$ 0 9 .8
Mean 8.39 6.9±0.98
^ ++++ obtained by Standard B enedict’ s T est
o
Gut washings analyzed f o r carotene*
I
20
TABLE I I
EFFECT O F ADMINISTRATION OF INSULIN O N THE SURVIVAL TIME O F
HATS INJECTED WITH 300 mg. PER Kg. A L L O X A N
Rat Weight Sex In s u lin S u rv iv al
number u n its tim e (h ours)
100 103 F 0 i* & ;
101 91 F 0
i 4
2 1 * 1 102 100 F 0
103 90 F 0.1* 72'
101* 80 F 0.1* 72'
A 78 M 0.1* 72
B 86 M 0.1* 72
C 9 1* M 0.1* 722
90 11*0 M
962
91
11*8 F 96;
92 11*0 M 96?
89 127 F 96
^ Bloody u rin e in 2 1 * hours - d isc a rd .
2
In je c te d January 6* 191*8* 8PM. January 8* a l l r a ts d ia b e tic .
0.1* u n its in s u lin a t 2PM and 8PM. January 9 , 0.1* u n its causing poor
c o n d itio n o f r a t s . January 10, r a t s 90* 91* 92 dead. January 1 1 * *
r a t 89 d ied a f t e r being c o n sta n tly tr e a te d w ith in s u lin ev ery s ix
hours*
21
T A B L E I I I
EFFECT OF 200 mg. PER Kg* A L L O X A N O N THE ABILITY TO C O N V E R T
UlO * C A R O TEN E INTO VITAMIN A - U 8 H O U R EXPERT IE N T
Rat
nunfoer
Weight1 Sex L iv e r w eight
p e r 100 gm.
body w eight
Urine sugar
gins, p e r 2k
hours
Vitamin A
p e r gram
l iv e r - 2h
hour t e s t
I.U .
58 125/119
F i*.30 0.61i U.7
59
120/122 F 3.96 0.96 5.7
60 125/120 F
ii.25 1.51 5.0
6 l 130/122 F lt.51* 1.62 U.3
62
135A25
F li.oU 1.30 5.8
79
118 M 3.91 1.03 6.9
80 101 M li.27 1.86 3.2
81 110 M 2*.10 1.21 9.1
Mean i*.17 5.6+0.569
1 When two w eights a re given they r e f e r to beginning and end
o f h8 hour period*
22
T A B L E IV
EFFECT OF A L L O X A N O N THE STO R A G E AFTER FEEDING IN RATS FED
300 I.U . VITAMIN A - 1 * 8 H O U R EXPERIM ENT
Rat Weight Sex Liver weight Urine sugar Vitamin A per
nunber per 100 gm. gras, per 2 2 * gram liv e r
body weight hours 2k hour t e s t
I.U .
In je c te d w ith 200 mg. p e r Kg*
72 115
F k.36 1.23 27.9
73 12*0 M
1*.93 1.00 11.8
7)4 85
F
3 . n 0.203 2i*.0
75 130 F
U.79 1.33 36.5
77 135
F U.50 0.2*73
18.8
Mean
U.33 23.8+3.77
Controls
93 103
M
2*. 13 0 37.9
9li 93
M 3.86 0 23.7
95 105
M 2**21 0 22*. 2
Mean
28.6+3.78
23
TABLE V
E F F E C T O F A L L O X A N IN R A T S O N T H E ABILITY T O C O N V E R T lUO V
C A R O T E N E IN T O V IT A M IN A - 72 H O U R E X P E R IM E N T
R at
nunber
Weight Sex L iver w eight
p e r 100 gm.
body w eight
Urine sugar
gms. p e r 2 1 *
hours
Vitamin A
p e r gram
l iv e r - 2 1 *
hour t e s t
I.U .
In je c te d w ith 200 mg. p e r Kg.
O U 90 F 5 .8 9 0.26
5.35
029 125 F 3.81* 0.52 6.9
030 125 M lt.10
0.1*7 10.3
OOUi litO M 3.02 0.92 7.8
0016 120 M U.03 0.83 6.2
00U6 110 M 3.90 1.17 7.9
001*7 107 . M It.32 2.16
9.3
Mean
iu l5
7.79±0.556
C ontrols
i
0$ 90 F
5.71 0 5.60
O il 127 F
i*.71 0 7.65
012 151
M It.63 0
7.35
O H * 132 M 5.86 0 3.1t
006 110 M
U.57 . 0 lt.U
007 1U7
•M
5.55
0 3.8
008
119
F lt.27 0
8.5
009 135
M It.66 0 it.0
Mean it.99 5.59l0.61t6
2U
T A B L E VI
E FF E C T O F A L L O X A N O N R A T S FED 200 I.U . V IT A M IN A A C E T A T E
72 H O U R E X P E R IM E N T
Rat Weight Sex L iv er w eight Urine sugar Vitam in A
number p e r 100 gm. gms. p e r 2a p e r gram
body w eight hours l i v e r - 2a
hour t e s t
I.U .
In je c te d w ith 200 mg. p e r Kg.
02 95
F 5.76
0.97 9.80
07 85
F 5.17 1.03 8.7 >
025 133
M
3.79 0.73 11.7
027 115
F U.91 1.92 a.8
001*0 93
F a .la 0.83 a .i
OOUl 111 F 3.71 2.12 10.8
00U2 I3li
F 3.66
1.63 9.2
00U3 12h M 3.6 9
0.95 7.1
00U8 107 M a .12
1.35 10.1
Mean a.8o 8.a7i0.829
C ontrols
001 109 F a .19 0 6.a
002 103 F 3.50 0 11.3
003
80 M a. 26 0 16.9
00U 110 M 3.a6 0 10.3
005 95
M 3.72 0 11.1
01 108 F
a.68 0 6.85
Mean 3.96 10.ii8tL.lOl
2$
T A B L E VII
EFFECT OF 200 mg. PER Kg. A L L O X A N A N D 0.1* UNITS INSULIN O N H A T S
FED 11*0 V CAB3TENE OR 200 I.U . VITAMIN A - 72
H O U R EXPERIM ENT
R at
number
W eight Sex Supplement
fe d
L iv er w eight
p e r 100 gm.
body w eight
Urine
sugar
gm s./
2 1 *
h a irs
Vitamin A
p e r gram
l i v e r - 2 1
hour t e s t
I.U .
09 108 F C arotene 6.11 2.02 U.7
010 107 M
n
iu$2i 0.86 11.7
013 92
F
«
1*.91 1.30 H i.9
01$ 121*
F
it
$.31*
1.78 7 .2
016 130 F
t >
!u31 0.83 7 .0
Mean $.01* 9.0+1.6$
017 10$ F Vitamin A $.6$ 1 .7 0 6.6
021 130 M
n
, 1*.17 2.73 6.2$
023 111*
F
»
$.21 0.86 7.0$
Mean $.00 6 .6 3 i0 .l8
26
T A B U S V III
NEGATIVE CONTROLS - RATS FED NO CAROTENE OR
VITAMIN A
R a t
number
W e ig h t S e x L iv e r w e ig h t
p e r 1 0 0 gm.
b o d y w e ig h t
A llo x a n 1 V ita m in A
p e r gram
l i v e r - 2k
h o u r t e s t
I .U .
T a b le
p e r t a i n
in g
27 70 M 7 .9 0 0 T a b le I
bZ
102 F 3.36 0 0 T a b le I
82
115
F li.30 0 0 T a b le I I I
3 95
F
1 u 87
0 0 T a b le V I
6 - - 95
M li.19 0 0 T a b le V I
96 109 F Ji.37 20 0 T a b le IV
97 90 F 3.76 20 0 T a b le IV
1 R e f e r s t o r a t s w h ic h r e c e i v e d a l lo x a n and f a i l e d t o d e v e lo p
g l y c o s u r i a - i n mg. p e r Kg*
2
No f a d in g b lu e c o lo r w i t h C a r r -P r ic e r e a g e n t , ta k e n a s no
v ita m in A .
2 7
T A B L E IX
S U M M A R Y " O F D A T A - 1 * 8 H O U R EXPERIM ENT
Alloxan
le v e l1
Urine sugar
gms. p e r 2k
hours - Mean
Supplement
fed
Vitam in A per
gram l i v e r -
2h hour t e s t
Mean
200 mg./Kg.
(8)
1.26
(0.61* - 1.86)
lUO y carotene
0.569
(3 .2 - 9 .1 )
200 rag./Kg.
(5)
0.81*
(0 .2 0 3 - 1.33)
300 I.U .
vitam in A
23. 8+ 3.77
(11.8-36.5)
C ontrols
(3)
0 300 I.U .
vitam in A
28.6 i 3.78
(2 3 .7 -3 7 .9 )
Figure in p aren th eses in d ic a te s number of r a ts , in each
group.
28
T A B L E X
S U M M A R Y O F D A T A - 12 H O U R E X P E R IM E N T
A lloxan
le v e l1
Urine sugar
gms. p e r 2k
hours - Mean
Supplement
fed
Vitamin A p e r
gram l i v e r -
2h hour t e s t
Mean
350 mg./Kg.
(7)
i | i .2
1 III lUO 'i carotene 03
C ontrols
(W
0 li*0 'i carotene 6.9 + 0.98
(l*.l* - 9 .8 )
200 mg./Kg.
(7)
0.901*
(0 .2 6 -2 .1 6 )
11*0 carotene
7 .7 9 t 0.556
(5 .3 5 -1 0 .3 )
200 mg./Kg. p lu s
in s u lin
(5)
1.35
(0.8 3 -2 .0 2 )
lUO X carotene 9 .0 + 1.65
(1*.7 -1 2*.9)
C ontrols
(8)
0 ll*0 V carotene 5.59+ 0.61*6
(3.U - 8.$)
200 mg./Kg.
(9)
1.28
(0 .7 3 -2 .1 2 )
200 I.U .
Vitamin A
8.1*7+ 0.829
(l* .l -1 1 .7 )
200 mg./Kg. p lu s
in s u lin
(3)
0 200 I.U .
Vitam in A
6.63+ 0.18
(6 .2 5 - 7.05)
C ontrols
(6)
0 200 I.U .
V itam in A
10.1*8+ 1.1*1
(6.1* -1 6 .9 )
1 F ig u r e i n p a r e n t h e s e s i n d i c a t e s nuntoer o f r a t s i n e a c h g r o u p .
2
O b ta in e d b y S ta n d a r d B e n e d i c t ' s t e s t .
3
No f a d in g b lu e c o l o r d e v e lo p e d up on a d d i t io n o f C a r r - P r ic e
r e a g e n t .
C H A P T E R V
SUM M ARY
V ita m in A - f r e e r a t s w e r e made d i a b e t i c b y i n j e c t i o n w it h
a llo x a n * Upon t h e o n s e t o f d i a b e t e s c a r o te n e w a s g iv e n p e r o s a f t e r
d i f f e r e n t p e r io d s o f tim e (2 ii and U8 h o u r s ) .
I t was found th a t r a t s rec eiv in g 200 mg* p e r Kg. a llo x an can
tran sfo rm carotene in to vitam in A as w e ll as normal vitam in A -free
r a t s . R ats rec eiv in g 350 mg* p e r Kg* a llo x an are unable to m etabolize
caro ten e .
The experim ents show t h a t in s u lin n e ith e r in c re a se d th e amount
o f carotene transform ed to vitam in A n o r keep a liv e th e r a ts receiv in g
350 mg. p e r Kg. alloxan*
In a s e rie s o f blood carotene d eterm in atio n s upon d ia b e tic
p a tie n ts a s ig n if ic a n tly h ig h er average blood carotene value was found
f o r 3h d ia b e tic s when compared to 25 non-diabetics*
B IB L IO G R A P H Y
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l‘
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32
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APPENDIX
A L L O X A N DIABETES
The follow ing se c tio n i s intended a s a reasonably complete review
o f a l l th e work upon allo x an a v a ila b le to the w r ite r . A lloxan has
I
been known f o r more than 100 y e a rs, since W oehler produced i t by
ox i d a tio n o f u r ic a c id . I t i s a c o lo rle s s powder m elting a t 25>6°C.,
which i s e a s ily soluble in w ater and a lc o h o l. I t decomposes on
h y d ro ly sis to u rea and m esoxalic a c id . Alloxan may be reduced to
d ia lu r ic a c id w ith which i t a c ts as an o x id atio n -re d u ctio n system .
The a c tio n o f c e r ta in o x id izin g agents upon u r ic acid g iv e s r i s e to
a llo x an in v i t r o .
I t would be o f g re a t sig n ific a n c e i f allo x an could be demon
s tr a te d as a n o m a l o r abnormal c o n stitu e n t o f anim al tis s u e . L iebig
appears to have d e te c te d i t s presence in th e g e la tin o u s mucus from
an i n te s t i n a l c a ta rrh (37a), w hile Lang (38) seems to have found i t in
th e u rin e o f a h e a rt p a tie n t.
In p la n ts i t has been found in i t s h a lf-red u c e d form ,
a llo x a n tin , in th e h y d ro ly sates of crude b e e t ju ic e (3 9 ), broad (fav a)
beans (1*0) and vetch seeds (i£L). Suben and Tipson (1*2) in working out
a tis s u e t e s t f o r a llo x an b e liev e th ey have dem onstrated i t i n ex
t r a c t s of l iv e r from a v a rie ty of an im als. These authors note th a t
th e y in ten d to in v e s tig a te f u l l y th e V a lid ity o f t h e i r method o f
I
a n a ly s is . T ipson (1*3) s ta te s th a t iro n in tis s u e g iv es r is e to mis
lead in g c o lo r changes which might be m istaken f o r a llo x a n . Br(lckmann
( J U U ) offers a m odified a n a ly tic a l method
38
S tru c tu re and A ction. Alloxan i s not the only compound
p ossessing unique diab eto g en ic p ro p e rtie s . D ia lu ric a c id has been
shown to be a c tiv e (1*5) and L aszt (1*6) showed th a t 20 mg. d ia lu r ic
a c id p e r 100 grams body w eight produced g ly c o su ria , p o ly u ria , and
k e to n u ria in $ < $ > o f th e r a t s in je c te d . A lloxantin and b a r b itu r ic
a c id were both in a c tiv e . The r e la tio n o f s tru c tu re to a c tiv ity was
in v e s tig a te d by Hidy (1*7) who found monomethyl a llo x a n a c tiv e and
dim ethylalloxan, benzoyleneurea, n in h y d rin , i s a t i n , mesoxlamide,
e th y l m esoxalate, and sodium m esoxalate a l l in a c tiv e . H idy.denotes
two n itro g e n atoms of d if f e r e n t b a s ic ity jo in e d by a system of double
bonds a s e s s e n tia l. Koref e t a l . (1*8) confirm th e o b serv atio n th a t
d ia lu r ic a c id i s a c tiv e and p o s tu la te i t s being charged to a llo x an
in vivo b efore producing any a c tio n . Intravenous in je c tio n s of
a llo x a n tin caused d ia b e te s. F erbanic acid and sodium a llo x a n a te were
in a c tiv e . An ex ten siv e in v e s tig a tio n by Brtlckmann and W ertheimer
(1*9) has revealed p ro p y lallo x a n , dim ethyl and d ie th y l a llo x a n tin to
be d iab e to g e n ic. H igher N -su b stitu te d a llo x a n s , or those w ith sub
s t i t u t i o n on th e imino o r C cj p o s itio n , are in a c tiv e . 1 ,2 , Naphtho-
quinone-i*-sulfonic a c id causes deranged carbohydrate m etabolism b u t
no tru e d ia b e te s. D ia lu ric a c id has produced d ia b e te s in ra b b its
in d is tin g u ish a b le from th a t caused by a llo x a n (5 0 ).
39
P ro te c tiv e A c tiv ity , By p ro te c tiv e a c t iv i t y i s meant the
re c e n tly discovered a b i l i t y o f some compounds to p ro te c t the anim al
a g a in st damage by a llo x a n . Lazarow (5>1,£2) has been prom inent in
t h is work. I t was found th a t intravenous in je c tio n s of g lu ta th io n e
o r cy stein e one o r two m inutes b efo re a llo x a n com pletely p ro tec te d
r a t s from d ia b e te s. Amino a cid s and vitam in C had no such power. I t
i s known th a t th e g lu ta th io n e content o f tis s u e ( l i v e r and in te s tin e )
shows a marked f a l l a f t e r in je c tio n s of a llo x a n and Lazarow p o in ts
out th a t p o ssib ly th e i s l e t c e l ls possess le s s of th e p ro te c tin g
substance th an o th e r c e lls and hence are p r e f e r e n tia lly a tta c k e d .
C ysteine p o ssib ly re a c ts w ith a llo x a n , y ie ld in g an in a c tiv e compound.
Banergee (53) in v e s tig a te d n ic o tin ic a c id , p y rid in e d ica rb o x y lic a c id
and 2 phenylqainoline-l*-carboxylic acid and found th a t th ey afforded
complete p ro te c tio n a g a in st a llo x a n . The substance BAL has been shown
to p ro te c t r a t s from a llo x an when given by in je c tio n and p e r o s .
(5U ,55).
On th e o th e r hand, asco rb ic a c id in c re a se s th e potency o f
a llo x a n when adm inistered one minute b efo re the a llo x a n (5 6 ).
d -Iso a sc o rb ic acid i s in a c tiv e . S u lfan ilam id o -cy clo p ro p y lth iazo le
causes a hypoglycemia in normal ra b b its b u t t h i s a c tio n i s reversed
in allo x an d iab etes a s was shown by Chen (5 7 ). The a n c ie n t remedy
f o r d ia b e te s , syzygium Jambolonum, has been shown to be w o rth less
a g a in st allo x an d ia b e te s (5 8 ).
Species R elatio n s in Alloxan D iab etes. A lloxan has been found
diabetogenic in a v a rie ty of anim als. The d ia b e te s has been stu d ied
in r a b b its (59) which e x h ib ite d a hypercholesterolem ia, hyperlipem ia
and a d re n al c o rtic a l le s io n s . Sheep were stu d ied by J a r r e t t (.60).
The h i s t o r ic a l p ic tu re , blood chem istry and g en eral degenerative
changes in dogs were in v e s tig a te d by Goldner and Gomori ( 6 l ) , Dosne
developed allo x an d ia b e te s in th e toad (6 2 ). Hats were e x te n siv e ly
stu d ie d by Goldner e t a l , (63)* D iabetes was shown in the c a lf by
McCandless (61*), and S c o tt, H arris and Chen wo iked w ith b ird s (6 5 ).
D iabetes was induced by feeding a llo x an to c a ts by Ruben e t a l . (6 6 ),
One p a r t of a llo x an was mixed w ith fiv e p a r ts of d ie t and o ffe re d to
c a ts which had been fa s te d 2 1 * h o u rs. Ten o u t of lit c a ts developed
ty p ic a l d ia b e te s.
The follow ing i s a c o lle c tio n of the diabetogenic dosage of
a llo x an f o r some ty p ic a l anim als:
R at 2 0 0 -3 0 0 m g ./K g . I P
R a b b it 1 0 0 -2 0 0 m g ./K g . IV
Cat 1 5 0 m g ./K g . IV
Monkey 1 0 0 -1 5 0 m g ./K g . IV
Dog 50-100 m g ./K g . IV
P ig e o n 1 2 5 -2 0 0 m g ./K g . IV
T heories of A lloxan A ction. ' S ev eral workers have o ffe re d t h e i r
ex p lan atio n of how a llo x a n e x e rts i t s a c tio n . J . E. Eaulsen (6 7 ), on
h is to lo g ic a l evidence, b e lie v e s a llo x a n produces a spasm o f the p re
c a p illa ry v e sse ls o f th e i s l e t tis s u e and i f t h is i s prolonged c irc u
la tio n to th e i s l e t s i s choked o ff and the i s l e t c e l ls d ie . In o th e r
words, th e mechanism i s t h a t of a v a so c o n stric tiv e e f f e c t upon a
tis s u e th a t i s h e a v ily v a sc u la riz e d . Brtlckmann (h9) d isc a rd s the
com petitive in h ib itio n mechanism now so p o p u lar in ex p lain in g drug
a c tio n . He does t h is by saying th a t no s u b s tra te s im ila r to allo x an
i s known in v iv o . Moreover, the s u b s titu tio n o f a pro p y l group onto
a llo x an must involve considerable s t e i i c s tr e s s y e t t h is compound i s
a c tiv e . A lso, i f 1,2. naphthoquinone-h-sulfonic acid i s f u l ly proved
to be a d ia b e tic a g en t, the com petitive in h ib ito r id ea w ill have to
be abandoned. S e le c tiv e accum ulation o f to x ic amounts of alloxan i n
i s l e t tis s u e i s , of course, p o ssib le b u t hard to prove. In f a c t,
a f t e r in je c tio n , a n a ly sis showed more a llo x an in th e l iv e r s and kidney
than was found in th e whole p an creas. Lazarow (5 l) b e lie v e s th a t
in a c tiv a tio n o f -SH groups o f enzyme p ro te in s concerned in in s u lin
sy n th e sis by a llo x an i s th e mode of a c tio n . Brttckmann r e je c ts t h is
by showing la rg e re se rv e s of g lu ta th io n e in the pancreas a f t e r
a llo x an in je c tio n and th e re i s reason to b e lie v e th a t any oxidized
-SH group would be q uickly reg en erated .
The m ain d i f f i c u l t y i n t h e e l u c i d a t i o n o f t h e mode o f a c t i o n
o f a llo x a n l i e s i n t h e l a c k o f o u r k n ow led ge o f th e s p e c i f i c b i o
c h e m ic a l p r o c e s s e s o f t h e t a r g e t t i s s u e , t h e i s l e t . P r o g r e s s h e r e
s h o u ld th ro w l i g h t on th e m echanism o f i n s u l i n s y h t h e s i s .
Alloxan D iabetes and Enzymes. I t has been known f o r many
years th a t in a d ia b e tic s ta te se v e ra l enzymes e x h ib it changes in
a c tiv ity . Alloxan o f f e r s a new method o f a tta c k on th ese phenomena.
U 2
Seram amylase has been shown to e x h ib it a marked f a l l in r a ts in
je c te d w ith allo x an (6 8 ). Rat l i v e r and muscle c a th e p tic a c t iv i t y
was in v e s tig a te d by M irsky (69) who found th a t muscle a c t iv i t y of
normal and a llo x a n -tre a te d anim als d id n o t d i f f e r b u t th e l i v e r
e x tra c t o f d ia b e tic r a t s showed a marked in c re a s e . In s u lin d id not
rev erse t h i s e it h e r when added in v itr o o r when in je c te d th re e hours
before d e ath .
The v i t a l q uestion o f glycogen phosphorplysis in d iab e te s was
in v e s tig a te d by L aszt and Vogel (7 0 ), They assume t h a t glucose
re s o rp tio n i s dependent upon th e r a te o f phosphorylation and fin d th a t
th e ra te of glycogen phosphorylation in th ei muscle o f a llo x an iz ed r a t s
i s in c re a se d . In s u lin in v itr o dim inished the r a t e . The .v ario u s
phosphorus f ra c tio n s o f th e blood were compared to normals and marked
d iffe re n c e s found. Serum phosphatase in c re a se d .
However, S ta e b e lin (71) was unable to confirm th ese fin d in g s
and he was unable to show any in c re a se d p h o sphorylation. In s u lin d id
have a depressing e f f e c t both i n normal and d ia b e tic an im als,
Gemmill (72) found, in a s e rie s o f Warburg measurements, th a t
fro g muscle g ly c o ly sis i s in h ib ite d by a llo x an and t h i s e f f e c t i s
p ro p o rtio n a l to the a llo x an co n ce n tra tio n and i s reversed by c y ste in e - .
Hexokinase, a t f i r s t g la n c e , could seem to be in h ib ite d o r blocked
in d ia b e te s . T his p o in t was cle are d up by Broh-Kahn and Mirsky who
showed a llo x a n iz e d r a t muscle had f u l l y as a c tiv e hexokinase a c tiv ity
as normal (7 3 ).
1 j3
S t a t i s t i c a l l y s ig n if ic a n t in c re a se s o f 23 and 38 p e r cen t
re s p e c tiv e ly in th e a c t i v i t i e s o f acid and a lk a lin e l iv e r phos
p h atases have been dem onstrated in r a t s w ith a llo x an d ia b e te s (7li).
In s u lin therapy was found to re s to re both a c id and a lk a lin e a c t i v i t i e s
to normal*
A lloxan D iabetes and o th e r Endocrine G lands* As i s to be
expected, much study has been made b o th of the e f f e c t of a llo x a n
d iab e te s upon o th er glands and th e in flu en ce v a rio u s glands e x e rts
upon th e course and s e v e rity o f a d ia b e tic c o n d itio n induced by
a llo x a n . A lloxan has been a p o ten t weapon i n th e hands o f Houssay
in h is stu d ie s on g la n d u la r in te ra c tio n s in d ia b e te s .
T hyroid. Many y ears ago L ic in i observed a f la tte n in g o f the
f o l li c u l a r ep ith eliu m occurred in h is d ia b e tic dogs (7 5 ). The
h is to lo g ic a l p ic tu re of th e r a b b it th y ro id as in flu en ced by la rg e
subcutaneous in je c tio n s of in s u lin was stu d ied in 1933 by Haiha and
U o tila (7.6) and i t was shown in the gland e x h ib ite d evidences o f
h y p erfu n ctio n . In r a b b its , dead from hypoglycemia, hyperemia the
red d ish cytoplasm ic gran u les were very numerous. One month a f t e r
d isco n tin u in g th e in s u lin , th e th y ro id gland f o l l i c l e s appeared f u l l
of rre d -s ta in e d c o llo id ; th e c e l ls and n u c le i were sm a ller and more
deeply sta in e d , w ith numerous u n stain ed cytoplasm ic v acu o les.
An in v e s tig a tio n o f th e h is to lo g ic a l p ic tu re o f th e th y ro id
of r a ts w ith allo x an d ia b e te s has been undertaken by Koneff (30)
(31)* The g en eral conclusions drawn from t h i s work were th a t a llo x an
c a u s e s an a tr o p h y o f t h e t h y r o id and t h i s e f f e c t i s a s e c o n d a r y
r e s u l t o f t h e d i a b e t e s me 1 l i t u s and n o t a d i r e c t a c t i o n o f th e
a l lo x a n g iv e n a t .a d o s e l e v e l o f 200 mg, p e r K g. b o d y w e i g h t . .
The m o st s e v e r e d ia b e t e s p rod u ced t h e m ost e x t e n s i v e c h a n g e s
i n t h e g la n d . A ls o th e G o lg i a p p a r a tu s w a s r e d u c e d i n s i z e an d
c o m p le x it y and th e m ito c h o n d r ia w ere fe w e r i n nunfeer and no lo n g e r
e x h i b i t e d n o rm a l o r i e n t a t i o n . T h e se c h a n g e s w ere i n t e r p r e t e d a s
i n d i c a t i n g a h y p o fu n c tio n in g g la n d .
Thyroid w eights decrease in p a r t i a l l y depancreatized r a ts
(77) and t h is was a lso a fin d in g o f Koneff in h is a llo x a n -tre a te d
anim als (3 0 ).
Houssay and coworkers have d ire c te d t h e i r a tte n tio n on t h is
problem (77 >78). They showed th a t thyroidectom y prevented d iab e te s
in r a t s w ith 95 p e r cen t o f the pancreas removed and t h is e f f e c t
was rev ersed by th y ro x in . I f th e thyroidectom y was perform ed th re e
months a f t e r th e pancreas was removed (9 5 ^ ),th e re was no e f f e c t . In
o th e r w ords, thyroidectom y a f t e r d ia b e te s has s e t in has no e f f e c t .
Houssay a ls o dem onstrates th a t hyperthyroidism in cre ases and th y ro i
dectomy decreased th e s e n s itiv ity to to x ic and diabetogenic dosage
of a llo x a n (7 9 ). T his wasoonfirmed by M artinez (8 0 ). Dosne (62) on
the o th e r hand found th a t thyroidectom y in no way in flu en ced the
d iab e te s caused by allo x an in the to ad .
The A d r e n a ls . I t h a s b e e n lo n g r e c o g n iz e d t h a t t h e a d r e n a ls
h S
e x e rt t h e i r in flu en ce in d ia b e te s and t h i s has been stu d ie d w ith
a llo x a n . AUcxxan causes a hypertrophy o f th e a d re n al in th e r a t
(3 0 ). R abbits receiv in g 125 mg. p e r Kg. o f a llo x an e x h ib ite d le s io n s
of th e adrenal c o rtic e s in tw elve out o f 25 cases (8 1 ). A drenalec
tomy g re a tly in cre ased th e s e n s itiv ity o f r a t s to a llo x a n ( 8 l ) .
Normal and hypophysectondzed r a t s d id not respond to a s im ila r dose.
Iv ersen (82) seems to prove th a t th e a d re n al c o rte x i s resp o n sib le
f o r th e i n i t i a l hyperglycem ia in r a b b its . T his was a lso stu d ie d in
th e c a t by P e rae ta R (83) sho found th e i n i t i a l hyperglycem ia d is
appeared when th e ad ren als were removed o r in a c tiv a te d b u t not when
th e h e p atic nerves were c u t, and th e ad ren als l e f t i n t a c t . A drenalec
tomy follow ed by co rtin d eso x y -co rtic o ste ro n e a c e ta te has been found
to reduce uzine sugar 50-80 p e r cen t i n r a t s (1|6). Janes (81*) c a rrie d
o ut p a ire d -fe e d in g experim ents w ith "normal* d ia b e tic and a d re n a le c -
tom ized r a t s and th ey a sc rib e any a m elio ratio n o f d ia b e te s which occurs
follow ing adrenalectom y to be a sso c ia te d w ith reduced food in ta k e . A
tw en ty -fo u r hour f a s t la r g e ly d e p le te s th e carbohydrate s to re s o f an
a d re n a le c t onrl ze d anim al and th e glycogen le v e l averaged only 15$ o f
th a t found in th e c o n tro l d ia b e tic s .
M is c e lla n e o u s G la n d s . T h ym ectom ized r a t s sh ow ed g r e a t l y
in c r e a s e d s e n s i t i v i t y t o a l lo x a n g iv e n i n t r a v e n o u s l y . S p le n e c to m y and
c a s t r a t i o n h a d no com parable, e f f e c t (8 5 ). I n r a b b i t s , i n j e c t e d w it h
a l lo x a n in t r a v e n o u s l y , h y p o p h y s e c to c y p r o lo n g e d th e i n i t i a l h y p o -
i
g ly c e m ia and i n som e c a s e s r e s u l t e d i n d e a th ( 8 6 ) . I t i s w e l l known
t h a t t h e p i t u i t a r y p l a y s a d e c i s i v e r o l e i n b lo o d s u g a r r e g u l a t i o n .
1 * 6
Pancreatectoiqy coupled w ith a llo x an in dogs has been stu d ie d
by Thorogood and Zimmerman (87) • Such treatm en t r e s u lts in decreased
in s u lin requirem ents b u t any r e s u ltin g com i s not c o n tro lla b le w ith
in s u lin . A second p a n c re a tic s e c re tio n i s p o stu la te d which fu n ctio n s
to in cre ase blood sugar and a c ts in preventing k e to s is in in s u lin -
d e fic ie n t dogs.
M iscellaneous Work on Alloxan D ia b etes. Janes and Dawson
(138), u sing p a ire d feed in g , in v e s tig a te d th e e f f e c t of feed in g d i
e th y ls t i l b e s t r o l to allo x an d ia b e tic r a t s . The data in d ic a te d th a t
th e substance was n o t a diabetogenic agent n o r th a t i t am elio rates
d ia b e tic symptoms.
That th e kidney i s somehow involved i s in d ic a te d by th e work
o f Jimenez and Grande-Covian (8 9 ). They found th a t when the kidney
blood supply was cu t o ff ten m inutes b efo re allo x an i s in je c te d , no
hyperglycem ia r e s u lts . Dogs were used and the work arose from th e
o b serv atio n t h a t death in a llo x a n d ia b e te s in dogs was c h a ra c te riz e d
by a re n a l d istu rb a n c e .
A llo x a n p r o d u c e s c y s t o x i c damage n o t o n l y t o t h e b e t a c e l l s
b u t a l s o th e a d r e n a l c o r t e x , l i v e r and k id n e y when a d m in is te r e d p e r
r ectu m ( 1 8 0 - 2 5 0 mg. p e r Kg* t o r a b b i t s (3 1 * )). T h ese i n v e s t i g a t o r s
o f f e r t h e s u g g e s t io n t h a t p o s s i b l y a llo x a n i s a p r o d u c t o f n u c l e o -
p r o t e i n breakdow n and i s in v o lv e d i n c l a s s i c d i a b e t e s .
B e n n e tt e t a l . (35) fo u n d no d i f f e r e n c e i n th e d ia b e t o g e n ic
and nephrotoxic dosage o f a llo x a n in r a t s a s judged by hyperglycem ia
and azotem ia. As d ia b e te s p e r s i s t s , th e re i s alm ost co u p lets func
tio n a l recovery of the kidneys. Kidney damage has a ls o been noted
in b ird s (6 $ ). Dunn e t a l . (3?) noted th a t r a ts in some cases,
though not in v a ria b ly , showed n e c ro sis in th e re n a l tu b u le .
The rep roductive behavior of r a t s su ffe rin g from a llo x an
d ia b e te s has been re p o rte d by Davis e t a l . (9 0 ). I t was found th a t
only fiv e out o f th ir ty - th r e e r a t s survived th e whole g e s ta tio n period
and th ese c a s t only m ascerated p la c e n ta e .
The blood co ag u latio n time in a llo x an d ia b e tic anim als has
been rep o rted (91) to e x h ib it a marked in c re a s e .
N ic o tin ic a cid fed to d ia b e tic r a ts (1 gram p e r Kg. d ie t)
gave r is e to a marked in c re a se in k e to s is (9 2 ). A high choline
in tak e d id not a l t e r th e p ic tu r e .
S te tte n has employed deuterium in obtain in g some inform ation
as to the source o f u rin e sugar in a llo x an d ia b e te s (9 3 )r He fin d s
r „ . ’
t h a t a b o u t o n e - f o u r t h t h e u r in e s u g a r i s s y n t h e s iz e d i n v iv o and t h e
r e m a in d er i s d i e t a r y . The l i v e r g ly c o g e n d e r iv e d from fr a g m e n ts
s m a lle r th a n C£ w as more i n t h e d i a b e t i c r a t s and th e s y n t h e s i s o f
f a t t y a c i d d e c r e a s e d a b o u t 5% .
B rain s lic e s of a llo x a n d ia b e tic r a t s o x id ize g lu co se, pyruvate,
la c ta te and su ccin ate as w e ll as normals (91;) • ' E ighteen amino acid s
a ls o showed no a b n o rm a litie s. The l i v e r o f the a llo x a n r a t s could
n o t oxidize glutam ate w hile th e kidney could, and n e ith e r tis s u e
could handle g lu co se. C a n za n elli, G uild and Rapport (9k) made th ese
d eterm inations employing stan d ard Warburg tec h n iq u e s.
U 8
K ap lan e t a l , (95) have s t u d ie d t h e m e ta b o lism o f r a t s i n
a l lo x a n d i a b e t i c com a. The a n im a ls show ed a r i s e o f p la sm a i n
o r g a n ic p h o s p h a te and a d e c r e a s e i n th e t o t a l a c i d - s o l u b l e p h o sp h a te
o f l i v e r . An i n c r e a s e i n l i v e r in o r g a n ic P and a f a l l i n a d e n o s in e
pyrophosphate w ere a l s o show n . I n s u l i n , when g iv e n i n e x c e p t i o n a l l y
la r g e d o s e s , te n d e d t o r e v e r s e t h e s e t r e n d s .
B lo o d and b lo o d g l u t a t h io n e o f d i a b e t i c r a b b i t s h a s b e e n
s t u d ie d ( 9 6 ) ,
Two i n v e s t i g a t i o n s o f t h e r e l a t i o n o f t h e d i e t t o t h e s u s
c e p t i b i l i t y t o a llo x a n (9 7 ) and t h e c o u r s e o f a l lo x a n d i a b e t e s (25>)
have a p p e a r e d . I n th e fo r m e r , H o u ssa y e t a l . h a v e shown t h a t r a t s
on a h ig h l a r d , h ig h l a r d and h ig h p r o t e i n , an d h ig h l a r d p l u s
c h o l in e d i e t a l l d i e fro m d o s e s o f a l lo x a n t h a t n o r m a lly p r o d u c e
s t a b l e d i a b e t e s . I n th e l a t t e r i n v e s t i g a t i o n i t w as shown t h a t
a l l o x a n - t r e a t e d r a t s p la c e d on a s e v e n t y p e r c e n t hog t a l l o w d i e t
show ed m arked im p rovem en t. , D i u r e s i s d e c r e a s e d an d g lu c o s e d is a p p e a r e d
fro m t h e u r i n e .
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A study of the mechanism of the protective action of cholesterol in hyperthyroidism

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C14 stearic acid metabolism during fat absorption in the rat.

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A study of the ketone body metabolism of excised rat tissues with respect to labile phosphate content

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A comparison of glucose tolerance in normal rats and those with fatty livers

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A comparison of the turnover of cholesterol in rabbits on a normal and high cholesterol diet

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Cholinesterase levels in the sera of rabbits with livers experimentally poisoned by carbon tetrachloride

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Comparison of results of phasic and chromatographic methods for carotene analysis.

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Chemical, physicochemical, and biological studies on the mucoproteins of plasma and serum

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Competitive antagonists of thyroxine and structurally related compounds

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An experimental study of certain factors affecting calcification of bone

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Carbohydrate metabolism in thiamine deficiency.

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A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

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A method for the analysis of deuterium-labeled tissue cholesterol: Attempts to study the relation of the thyroid to the turnover of cholesterol in rats

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Adrenalectomy and fat absorption

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A study on the comparison of the cholesterol content of rat liver homogenates before and after incubation

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A study of the alterations in the amount of cholesterol present in rat liver homogenates after incubation

Asset Metadata

Creator Lowry, John Rowland (author)

Core Title An investigation of carotene metabolism in the alloxan treated rat

Degree Master of Science

Degree Program Biochemistry

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Deuel, H.J. (committee chair), Mark, Walter (committee member), Wehl, John W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-775789

Unique identifier UC11348091

Identifier EP41285.pdf (filename),usctheses-c17-775789 (legacy record id)

Legacy Identifier EP41285.pdf

Dmrecord 775789

Document Type Thesis

Rights Lowry, John Rowland

Type texts

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

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

Repository Name University of Southern California Digital Library

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