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

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Content STUDIES OP CAROTENOID AND VITAMIN A COMPLEXES
WITH PROTEIN IN PLASMA AND TISSUES
A Dissertation
Presented to
the Faculty of the Graduate School
University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Norman Irving Krinsky
August 1953
UMI Number: DP21553
All rights reserved
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This dissertation, written by
.............. N. ORMN. . . IR.V. I M. . ERI KSKZ..... ..........
under the direction oflY&S.. Faculty Committee, fyvf) I
on Studies, and approved by all its members, has j
been presented to and accepted by the Council
on Graduate Study and Research, in partial ful-
fillm ent of requirements for the degree of
DOCTOR OF P H ILO S O P H Y
D ate.
Committee on Studies
TABLE OP CONTENTS
PAGE
INTRODUCTION ....... *.......♦................ 1
Absorption ................................ 1
Transportation ............................. 7
Storage ......... l l j .
Statement of the Problem 20
MATERIALS AND METHODS ........ 22
Animals 22
Chickens *«...* 22
Rats 22
Beef ........ 22
Hogs 23
Supplements •••••••••••••••••••••••.•*•••«•• 23
Lutein • 23
Vitamin A • • ..... 25
Carrier 25
Route of administration 25
Solvents 26
Plasma Fractionation 26
Ammonium sulphate fractionation •••••••••• 26
Dialysis 27
Solvent extraction 27
Rat Liver Fractionation 28
Ill
FAGE
Schneider-Hogeboom procedure 28
Modified procedure 33
Beef Liver Fractionation ...... 36
Treatment of Plasma ••«•••••••••••••••••••*••• 2^2
Treatment of Liver «•••••••••••••••••••••••• 1^2
Ether extraction ••«*••••••• «• •«••••••••••*• ? j J | .
Alkaline digestion •••••••••••• •• •••«••••• • • l j . 5
Salt extraction .....•»••..*••••••• I 4 . 5
Butanol extraction •*•••••••••••••••••••••#• 2 f . 6
Waring blendor extraction i f . 7
Hypotonic, extraction •••••*•••••••••»« •••••« l j . 7
Ghromatographic Analysis ..................... J j . 8
Chemical Analysis *«»•••••••• •*«•••«•••••••••• l j . 9
Vitamin A •*••••••••••••••••••••••••••••••»•
p-Carotene «• • 5C
Lutein 5l
i
Nitrogen 51
EXPERIMENTAL RESULTS 53
Plasma Fractionation ••••••••••••••••••••»•••• 53
Solvent extraction •«•••••••••••••••*••••••• 33
Ammonium sulphate fractionation ....... 53
Dialysis •••«*•«*•«•••••*••••••••••••••••••• 66
Rat Liver Fractionation »•••»••••••••«••••*•♦ . 70
iv
PAGE
Schneider-Hogeboom procedure ••.••••.•.•«•«. 70
Partial modified procedure •••••••.......♦ 73
Complete modified fractionation ..... 76
Beef Liver Fractionation 80
Distribution ••••••••••..................... @0
Ether extraction 88
Effect of salts on ether extraction 92
Butanol extraction ,•••••••••••••••••«•••.*•• 94
Alkaline digestion 96
Waring blendor homogenization 98
Hypertonic and hypotonic extractions ••«•••• 100
Nitrogen distribution 100
DISCUSSION ................. 102
SUMMARY AND CONCLUSIONS ........................ 11$
BIBLIOGRAPHY ............ 119
LIST OF TABLES
TABLE PAGE
I.
Ether Extraction of ^-Carotene, Lutein,
> 4
Vitamin A Alcohol and Vitamin A Ester
from Chicken Plasma ....................
Sb
II. Distribution of Vitamin A Alcohol and
Vitamin A Ester in Plasma Fractions
Obtained by Ammonium Sulphate Fraction
ation from Carotenoid-depleted Chickens
Supplemented with Vitamin A Ester ••••. 56
III. Distribution of /*-Carotene, Vitamin A
Alcohol and Vitamin A Ester in Plasma:;
Fractions Obtained by Ammonium Sulphate
<
Fractionation from Chickens Raised on a
Carotenoid- and Vitamin A-low Diet, and
Supplemented with ^-Carotene •••*«•«.•• 58
IV. Distribution of Lutein, Vitamin A Alcohol
and Vitamin A Ester in Plasma Fractions
Obtained by Ammonium Sulphate Fractions
from Chickens Raised on a Carotenoid-
and Vitamin A-low Diet and Supplemented
with VItamin A and/or LuteIn ••••••..•*
1
6o
TABLE
V.
VI.
VII.
VIII.
IX.
vl
PAGE
Summary of the Distribution of Lutein,
Vitamin A Alcohol and Vitamin Ester in
Fractions of Chicken Plasma Obtained
by Ammonium Sulphate Fractionation ....
Distribution of p -Carotene* Lutein, and
Vitamin A Alcohol in Fractions of Beef
and Hog Plasma Obtained by Ammonium
Sulphate Fractionation
Distribution of Lutein, Vitamin A Alcohol
and Vitamin A Ester in Fractions
Obtained by Dialysis Followed by
Ammonium Sulphate Fractionation of
✓ - .
Normal Chicken Plasma
Distribution of Lutein, Vitamin A Alcohol
and Vitamin A Ester in Chicken Plasma
as Determined by Dialysis 67
Distribution of Vitamin A Alcohol and
Vitamin A Ester in Rat Plasma Fractions
Obtained by Dialysis ..... ••••••••• 69
Distribution of Vitamin A Alcohol and
Vitamin A Ester in Fractions of Rat
Liver Homogenates Prepared in Hypertonic
Sucrose Solutions and Obtained by
Differential Centrifugation 71
TABLE
XI*
XII.
•XIII*
XIV.
I XV.
i
XVI.
!
|
i
!
i
Partial Fractionation of Rat Liver
Homogenates Prepared in Isotonic
Sucrose Solution .....
Intracellular Distribution of Vitamin A
Ester and Vitamin A Alcohol in Rat
Liver Homogenates
Intracellular Distribution of yB-carotene,
Vitamin A Ester and Vitamin A Alcohol
in Beef Liver Homogenates from Stored
Livers ...
Intracellular Distribution of p-Carotene,
Vitamin A Ester and Vitamin A Alcohol
in Beef Liver Homogenates from Fresh
Livers
Intracellular Distribution of ^-Carotene,
Vitamin A Ester and Vitamin A Alcohol
in Beef Liver Homogenates before and
after Storage for i j .8 Hours •.«••.•••••
Summary of the Intracellular Distribution
of yP-Carotene, Vitamin A Ester and
Vitamin A Alcohol in Beef Liver Homo
genates • «*••«•••
viii
TABLE PAGE
XVII. Ether Extraction ofp-Carotene and
Vitamin A Ester from Beef Liver Homo*
genates of Fresh (F) and Stored (S)
Liver 91
XVIII. Ether Extraction of ^-Carotene and
Vitamin A Ester from Beef Liver Homo
genates of Fresh (F) and Stored (S)
Liver before and after Treatment with
M Potassium Chloride •••••••••..«••«••• 93
XIX. Butanol Extraction of p -Carotene and
Vitamin A Ester from Beef Liver
Homogenates .... 95
XX. Effect of Alkaline Digestion on the
Distribution of ^g-Carotene and Vitamin
A Ester in Isolated Beef Liver
Microsomes •. .. ,...... 97
XXI. Degree of Solubilization of -Carotene and
Vitamin A Ester from Isolated Beef Liver
Mitochondria ••.•••••«•••••••••••••«..• 99
XXII. Distribution of Hitrogen in Beef Liver
Homogenates 101
LIST OF FIGURES
FIGURE PAGE
1. The Fractionation Scheme for Rat Liver
Homogenates 32
2, Modified Schneider-Hogehoom Procedure for the
i Isolation of Various Cell Fractions of Rat
Liver Homogenates Prepared in Isotonic
Sucrose Solution • • • • ••••• • • • ••••• • ••••••••• 3^
3* Harris-Mehl Continuous Homogenizer •••«••*••*. 38
! { . • Fractionation Scheme for Beef Liver Homogenates
Prepared in Isotonic Sucrose Solutions ...... i j . 1
5* A Schematic Representation of the Light-
; Scattering andiFluorescent Regions Resulting
- from the Centrifugation of a Rat Liver
Homogenate Prepared in 0*88 M Sucrose
Solution and Layered under Various Lower
Density Solutions; *•••••••«••••••••••«««•••• 77
I i .*•.• l
: * ■ : T , 1
4
ACKNOWLEDGEMENT
I would like to take this opportunity of expressing
my appreciation to my Faculty Guidance Committee for their
encouragement and advice during the period in which this
work was done. I am particularly indebted to Dean Harry J.
Deuel Jr. for giving me the opportunity to undertake my
graduate studies, and to both him and Professor John W.
Mehl for the many suggestions that have helped mold this
work.
In addition, I am indebted to my fellow graduate
students who have contributed to much of my understanding
of biochemistry through various informal discussions and
seminars.
I am also pleased to acknowledge the financial
assistance of the Proctor and Gamble Company, and the
laboratory space made available by the Allan Hancock
Foundation.
Finally, I take great pleasure in thanking
Dr. Jagannath Ganguly for all that he has done for me
during our association together. I have found it very
rewarding and stimulating, and shall always be grateful
for his guidance and cooperation.
INTRODUCTION
The identification of some of the pigments found in
blood with the earotenoids of plants by Thudiehum in 1869
(1) stimulated considerable Interest in the factors
controlling the absorption, transport and storage of these
pigments. In due time, these Investigations were expanded
to include similar studies on the best known metabolite of
the earotenoids, namely, vitamin A. Various aspects of the
work on these three processes have been reviewed by
Zechmeister (2), With (3)» Clausen (i f . ) and Sobel (5); it is
felt that the following brief review will suffice to point
out the trends in this field.
Absorption
The expression ”species selectivity” has been
overworked In the past in trying to explain the wide
variation observed between different animals in their
ability to absorb the carotenold pigments. With respect to
vitamin A however, a species selectivity does not seem to
exist, for vitamin A appears to be absorbed by almost all
animals- that find .it in their diet. The ability to select
which earotenoids are absorbed or rejected ean be used as
the basis for classifying animals into four groups. (6, 7). ,
2
Group I. Absorb carotenes and carotenols equally
well.
Example: Men, frogs.
Group II, Absorb carotenes preferentially.
Example: Cattle, horses.
Group III. ' Absorb carotenols preferentially.
Example: Fowls•
Group IV. Negligible absorption of all earotenoids.
Example: Rodents, sheep, pigs,
goats, etc.
Once this classification has been accepted, It becomes
necessary to seek another explanation for the absorption
of the earotenoids than the obvious one based on their fat
solubility. Neither the lipolytic (8) or particulate (9)
theories of fat absorption can serve as a rational basis
for explaining this species selectivity. Indeed, these
theories would predict little or no difference In the
degree of absorption between carotenes and earotenols.
As early as 19ll^, Palmer and Eckles (1©) proposed
that in cows, carotene could be absorbed chiefly by virtue
of its ability to form a compound with one of the blood
proteins. The earotenols, on the other hand, being of a
different nature, could not form this compound and there
fore could not be absorbed.
By emulsifying carotene with mineral oil and human
\ serum* Molander (11) was able to induce the absorption of j
tremendous quantities of carotene into rats* which normally
reject the majority of earotenoids in their diet. He
concluded that in the presence of fat, carotene is absorbed
through the lymphatics into the systemic circulation,
whereas when fatty acids were used instead of the fat, the
J
(majority of the carotene was found in the liver, indicating
I
the possibility that absorption had taken place through the
portal circulation. These results are difficult to recon
cile with the work reported by Drummond, Bell and Palmer
(12), who administered carotene and vitamin A dissolved in i
I
I fat to a patient with ehylothorax. When the chylous fluid
was collected, they found that it contained 80% of the
administered vitamin A, but only 2% of the administered !
I
carotene. Unfortunately, there was no mention of whether j
the remaining carotene was absorbed as vitamin A, whether
it was excreted or whether it was possibly absorbed through j
the portal system, a view that has received some support |
recently from the works of Kowalewskl et al. (13) in dogs, j
!
Woytkiw and Esselbaugh ( li j. ) in guinea pigs, and Radice and !
jHerraiz (15>) in rats. In addition, Gribatz and Kanof (l6) 1
have demonstrated the absorption of an aqueous preparation
of vitamin A in chylous ascites, in which there is a j
I lymphatic block, with no absorption of an oily preparation . j
I of vitamin A. Marder et al. (17) have reported the
presence of a yellow unsaturated hydrocarbon in fat obtained
i i
i from human cbylomicra.
The above evidence suggests the possibility that the
mechanisms of absorption for the earotenoids and vitamin A
may not necessarily be identical. The many papers that !
have appeared dealing with the correlation between fat
|
| absorption and vitamin A absorption in premature infants
| (18), new born infants (19)> patients with steatorrhea
1 (20, 21), cystic fibrosis (22, 23, 2l j . ) , chylous ascites
(l6) and other conditions seem to indicate however, that
! a very close relationship exists between these two pro- 1
i |
cesses. This relationship between fat and vitamin A has j
i
been extended by Josephs (25) who proposed that the rate
i
j of absorption of vitamin A was related to the blood lipid
| level. This same author later proposed (26) that the
blood and tissue lipid levels were directly dependent on
i
the vitamin A level in the body, a view which received ,
support from Kagen et al. (27). I
The absorption of vitamin A has been shown to be \
I I
greatly enhanced in most of the conditions listed above j
Jby using a finely-emulsified or water-soluble form of the
j vitamin, rather than the usual oily preparation. This 1
i effect though is not seen at very low levels of vitamin A
!_(0.3-3.0 jig./day) for then the utilization, as measured by |
the gain in weight and disappearance of xerophthalmia, is
identical for aqueous or oily preparations (28), That this
merely facilitates the absorption, and does not alter its
basic mechanism, has been given support by the observations
of Popper and Volk (29) that both the oily and aqueous
preparations of vitamin A were absorbed through lacteals,
as shown by fluorescence microscopy. In fact, only the dog
seems to be able to absorb vitamin A through the capillaries
of the small intestine (if), all other species using the
lacteals almost exclusively, (Rat (30, 31, 32), guinea
pig (llf), pig (30, 32), man (12) and sheep (31)),
In the lumen of the Intestine, hydrolysis of vita
min A esters occurs prior to absorption of the vitamin.
This was first observed by Gray, Morgareidge and Gawley
(33), aud the complete pattern was worked out by Clausen
(if) who showed that after hydrolysis and dispersion with
bile acids, reesterification took place in the wall of the
Intestine. The esters then reach the blood stream, via
the thoracic duct, by way of the lymphatics. This work
has since been confirmed by Eden and Sellers (3if) and by
Thompson et al, (32), although Popper and his associates
(35) did suggest that vitamin A ester could be absorbed
without lipolysis, based on the observations of Increased
plasma levels of vitamin A following the administration
6
of vitamin A as the ester, compared to the alcohol*
Very little is known of the remaining portion of
the absorption process, viz., from the intestinal wall to
the site of deposition. Prom the studies of Drummond
et al. (12), it may be concluded that both carotene and
vitamin A were in some manner, bound in the chylous fluid
of man. Neither component was ultrafilterable, and only
traces could be extracted with diethyl ether, although the
addition of ethyl alcohol rendered them readily extractable.
(
It is difficult to assume that these components are associ
ated with chylomicron-like particles, for then one would
expect to find them distributed throughout the systemic
circulation and the fat depots, as was demonstrated for
various fat-soluble dyes by Mendel and Daniels (36) and
by Gage and Pish (37)* This does not occur with either
vitamin A or carotene, although whether their pathway
differs from the fat-soluble dyes during the actual
absorptive process or later on during distribution is still
not known. It may be that the form In which they are
distributed from the intestine is somewhat analogous to
the colloidal suspension of. carotene used by Drummond
et al. (38), which was rapidly taken up by the liver,
apparently’ by the Kupffer oelis.
7
Transportation
Following Thudichum*s observations (1) on the
similarity of the blood pigments with the plant earotenoids,
Krukenberg, in 1886 (39) showed that the plasma earotenoids
could not be present in a simple fat solution, for direct
extraction of the plasma with organic solvents yielded no
llpochromes. This has been repeatedly confirmed since
that time, the only exception being a report of Palmer (ij.0)
that he was able to extract xanthophylls from chicken
plasma merely by shaking with diethyl ether.
Several reports have mentioned the association of
yellow pigments with various serum protein fractions, such
as that of Hardy and Gardner (i|_X), who crystallized a
yellow albumin from horse sera, and Has lam (i^2) who ob
tained a yellow-fatty extract after treating ox-serum
eugiobin with alcohol-ether. However, it was Palmer and
his associates ( i j . 3) who first proposed that the carotenoid
pigments were present in plasma as protein complexes. They
isolated, from cow plasma, a carotalbumin, which contained,
in addition, cholesterol and phosphorous, presumably as
phospholipids. Palmer could not isolate a similar complex
from chicken plasma ( i j . 0), although he did show that the
xanthophyll was not present in the globulin fractions.
Since Palmer*s proposal, there have been many
8
scattered reports relating carotenoid pigments and the
plasma proteins* Bennhold, in 1935 (Ml*) while investi
gating the combination of plasma proteins with various
♦
dyes and small molecules, observed that the earotenoids
in human plasma migrated in a catophoretic apparatus with
the globulins. In'conjunction with this work, he presented
a theory that one of the major functions of the blood
proteins was to serve as a vehicle for the transport of
smaller molecules and ions. Pett.and*LePage (10) came to
the same conclusion for vitamin A from work done on the
partial alcoholic fractionation of plasma (apparently
human), during a study of the relationship between blood
vitamin A levels and dark adaptation.
In 1942, Mehl (I4 . 6) observed that the plasma carote-
noids were largely concentrated in a fraction rich in
^-globulin, isolated by alcohol fractionation of human
plasma. Using a more refined fractionation procedure,
One ley, Surd and Melin (i j. 7) observed the presence of
earotenoids in the /3-lipoprotein of human plasma. More
recently, One ley (l\B) has reported the unpublished work
of Ames, who found significant amounts of vitamin A
associated with this same protein. Troitskil ( i | . 9 ) also
reported the association of earotenoids withp -globulin,
both in electrophoretic and salting out experiments.
i 9 j
Dzialosynski et al« (50) presented results which
| . i
■ would indicate that both the vitamin A and carotene present1
in human plasma are associated with the albumin, and not j
j the globulin fractions, for no vitamin A or carotene were j
f
precipitated with proteins precipitating at half saturation.
|
with ammonium sulphate, and only when the concentration of j
ammonium sulphate was raised to 75-100$ saturation did they |
; |
| precipitate, j
1 i
Other proposals that the earotenoids and vitamin A j
; are protein bound in plasma have been made by Chevallier j
! and Choron (5l)* who postulated a hemovitamin A present in !
I blood, which was the active form of the vitamin, and a
| 1
J hepatovitamin A in the liver, which was the reserve
material. Glover and Morton (52) made an interesting
j proposal that during absorption, serum lipase or esterase '
i I
! hydrolyzed some of the incoming vitamin A ester to the j
1 alcohol, which could then form a complex with protein
capable of diffusing into the liver storage cells. The i
! ' '
Jremaining ester would enter the liver through the phago- 1
I
t <
J cytie action of the Kupffer cells. Retinene, the aldehyde
of vitamin A, has also been shown (53) to form a complex
i
I with bovine plasma albumin, as indicated by a shift in its
i
absorption maxima after combining it with the protein.
J Chalmers, Goodwin and Morton (54) reported on the !
,effect of ionizing radiation in destroying vitamin A and
10
carotene in non-aqueous media such as butter, but in human
sera the pigments were protected in some way, possibly
through association with proteins*
As can be expected, several groups have, presented
evidence which tends to disprove the existence of protein-
eomplexes in plasma. The first evidence of this nature was
presented by van den Bergh and his associates (55* 5&) who
confirmed Palmer’s studies on the specificity of carotene
in cows and of carotenols in fowls, but ascribed their
behavior in plasma as being due to their presence in a
colloidal solution* This was based on the similarity in
behavior between plasma earotenoids and colloidal solutions
which they prepared.
Emulsification of earotenoids was shown by Danielli
and Pox (57) to shift the absorption maxima of the carote-
noid solutions to longer wavelengths, as had been shown
for colloidal solutions of earotenoids and serum albumin
by Karrer and Straus (58). Sexton (59) has presented
evidence however, that suggests that there is a difference
in the spectral behavior of carotene present normally in
human serum, and a carotene-sol stabilized by human serum*
The strongest evidence against the presence of
protein complex formation of the pigments in plasma is
based primarily on several reports of the almost identical
behavior of plasma lipids and vitamin A and the earotenoids*
Cayer et al* (60) observed that during pregnancy, j
:the presence of hyperlipemic plasma was invariably associ- J
I I
j ated with the highest vitamin A levels, from which they j
i
I concluded that the plasma lipids serve as the vehicle for
vitamin A and allow its accumulation in the blood. As J
mentioned earlier, Kagen et al* (27) found a concomitant j
i
j rise in plasma lipids as the plasma vitamin A increased in
I children with the nephrotic syndrome. Kimble and her
j
I associates (6l) also reported that high vitamin A levels
I I
I were found associated with nephrotic hyperlipemia, as did j
! Gottfried jet al. (62) with respect to carotene levels in
the nephrotic syndrome. All of the above are in line with j
I i
Josephs (25) proposal that there is a direct relationship
i
between plasma lipids and vitamin A and the earotenoids. |
I 1
Another side of this issue is presented by those J
who reject the concept of a direct relationship between the j
plasma lipids and the levels of vitamin A or carotene,
although not claiming that these pigments are protein ;
'bound. Steigmann and Popper (63) hold this view, and also |
! ' '
| support the hypothesis that the liver regulates the plasma
vitamin A level, as normal plasma levels are maintained
!even when vitamin A is removed from the diet in individuals !
1 i
|with healthy livers, whereas in conditions where there is
any liver malfunction, there is a rapid fall in plasma
vitamin A* This view that the plasma vitamin A level
remains fairly constant at the liver's expense is shared
by many other investigators, including Lewis et al. (6i j . ) ,
Josephs (65), Horton and associates (66), Brenner at al.
(67), Lindquist (68) and Ralli et al. (69).
This point of view has been well stated recently
by Urbach, Hickman and Harris (70)• They administered
over 6,000,000 units of vitamin A during a 21 week period
and observed a rise of only 16$ in the plasma vitamin A
level. They concluded that:
MThere seems little doubt that each individual
possessed a plasma level of the fat-soluble
vitamins characteristic of himself, and that
these levels tended, in general, to be main
tained constant, and after supplementation to
return to the characteristic norm.”
In contradistinction to the above, Glover, Goodwin
and Morton (71) proposed that there is a rough linear
relationship between the concentrations of vitamin A
alcohol in the liver and in the .blood. Some support is
given this by Almquist (72) who found a logarithmic
relationship between plasma vitamin A and liver vitamin A
Another view is presented by Krause (73), who found an
13
inverse relationship between plasma and liver vitamin A,
when the liver stores were above 600 units/gram*
Ganguly and Krinsky (71}-) proposed that, while the
liver served as the source of plasma vitamin A aleohol,
even to the extent of completely depleting its own stores
j
to maintain a normal plasma level, the actual concentration
in plasma was dependent on and would be limited by the
degree of association with a plasma protein carrier. There
are exceptions to the usual picture of a fairly constant
plasma vitamin A alcohol level. A recent report (75) of
an individual with hypervitaminosls A disclosed a fasting
vitamin A level of 835Jag. %» with two-thirds of the
vitamin present in the free form. Also, In certain
pathological conditions, normal liver stores may be found
associated with extremely low plasma levels (7&).
The view that the plasma vitamin A level is a more
sensitive index of vitamin A status than either the dark
adaptation test or liver stores has been supported by
Popper and his associates (77)* Using fluorescence
microscopy, they showed that during liver damage, the
vitamin A fluorescence shifts from its normal sites In
the Kupffer cells and in the fine droplets at the edge of
the epithelial cells, to pathologic sites, represented by
large fat droplets. These shifts were indicative of
2k
severe disturbances in vitamin A metabolism within the
liver, for patients with normal liver stores and low plasma
levels of vitamin A usually showed some hepatic damage on
histologic examination.
Storage ;
Palmer, in 1922 (78), reviewed the wide distribution
of carotenoids in such tissues as corpus luteum, plasma,
milk fat, adipose tissue and various internal organs*
Van den Bergh et al. (56) however, were the first to
remark on the fact that certain of the internal organs of
mammals appear to have a selective affinity for carote
noids which is greater than can be explained by their fat
content. In the following years, and particularly after
the reports by Moore in 1931 (79) aud by Baumann, Riisimg
and Steenbock in 193l|- (80) that liver served as the main
storage depot for vitamin A, the question has been debated
as to the degree of or lack of association between the
carotenoids and vitamin A and the tissue lipids. Both
points of view have been amply represented.
A technique for the localization of vitamin A in
tissues was developed by von Querner (81) and utilized
extensively by Popper and his associates (82). This
procedure, fluorescence microscopy, makes use of the
fading green fluorescence that vitamin A exhibits under
15
ultraviolet radiation, in microscopic tissue sections. In
normal liver tissue, this fluorescence is usually concen
trated in the fine fat droplets which fill the cytoplasm
of the Kupffer cells, and in the small peripheral fat
droplets in the epithelial cells. These latter cells also
contain larger centrally loeated fat droplets which show a
variable fluorescence, although always less than the
smaller droplets. In addition, a rapidly fading fluo
rescence, can be observed In the small rod-shaped particles,
believed to be the mitochondria, and a diffuse green fluo
rescence is present throughout the cytoplasm. Hyper-
vitaminosis A shows a general increase in fluorescence,
particularly in the Kupffer cells. In vitamin A depletion,
the first loss of fluorescence is observed in the cytoplasm
,and next in the fine fat droplets; finally, the fluorescence
In the Kupffer cells disappears. These findings correlate
remarkably well with the proposals of Glover and Morton
(52), Glover, Goodwin and Morton (71)> and Krause (73),
that the early and great loss of vitamin A on a vitamin A-
free diet may involve a specific labile form of the vitamin,
stored in the liver as a protein complex, and only later is
the vitamin depleted from the less active or storage form
in the Kupffer cells. In starvation, Popper (82) observed
little change in the vitamin A concentration in the liver,
16
although there was a marked change In the distribution of
the fluorescence. As starvation progressed, all the fluo
rescence became associated with the large, centrally
located fat droplets. Eventually, when fat was no longer
>
morphologically demonstrable, the cytoplasm was found to
t
impart a strong green fluorescence. In all of these
studies, the fluorescence that they observed was associated
with lipids demonstrable by sensitive stains. However,
since only certain lipids revealed this vitamin A fluo
rescence, Popper (82) concluded that the presence of the
fluorescence was due not t© the non-specific solubility
of the vitamin in the available fat, but to a specific
affinity of certain lipids for vitamin A.
That vitamin A and carotenoids found in tissues are
not deposited there as a result of a non-specific solubility
in fat has received support from a large number of investi
gators. Palmer and Kempster (83) presented histological
evidence that the xanthophyll in the shank skin of laying
hens was not dissolved in the fat. Scheer (8i j . ) reported
that thei concentration of carotenoids in California sea
mussels was independent of the lipid concentration.
Lovern, Edisbury and Morton (85) showed that the viscera
of fish frequently contain oils with a higher, vitamin A
concentration than the liver oils, and that denaturation
1?
of the liver protein was necessary for complete extraction
of the vitamin. A* Lease and Steenbock (86) studied the
influence of high- and low-fat diets and the effect of
rapid;fat depletion of the liver, on the hepatic vitamin A
levels. No distinctions were observed in the liver vitamin
A levels in either of these conditions. Horton et al. (66)
were also unable to find' any correlation between the liver
fat and vitamin A concentrations, and similar results were
reported by Barnes (87)* Glayton and Baumann (88) by alter
nating high- and low-fat diets, with and without lipo
tropic supplements, were able to change the fat content
of mice livers from 5$ to 2%% within one week, without
observing any alteration in the liver vitamin A level.
However, a relationship between vitamin A and fat
storage has been noted in rat liver, for Thornbjarnason
and Drummond (89) found greater liver stores of vitamin A
in animals receiving high-fat diets, and decreased storage
of both fat and vitamin A following choline administration.
Depletion of vitamin A from the liver also occurred more
rapidly on a low-fat diet than on a high-fat diet.
Williamson (90) proposed that the decreased plasma vitamin
A level in thyroidectomized rabbits treated with estradiol
was due to the increased deposition of body fat, carrying
with it some vitamin A. Dann (91) suggested that the
transfer of vitamin A from the doe to the foetal rat was___
18
also dependent on the fat content of the diet.
On the other hand, there are several investigators
who do not believe that an affinity for fat is of any
importance in binding vitamin A in the liver. Bukin and
Areshkina (92), using fish livers from which the fat was
removed, fractionated, and the vitamin A determined in the
various fractions, concluded that the vitamin was associated
or adsorbed to proteins. The singular view that vitamin A
is associated with a non-protein-like hydrophillic compound
present in mitochondria was proposed by Ernster,
Zetterstrom and Lindberg (93)» who observed the vitamin in
a trichloraeetie acid extract of rat kidney mitochondria.
In an attempt to explain the decrease in vitamin A
concentration found in livers which have been subjected
to the action of eertain carcinogens, both Abels et al.
(94) and Baumann and his associates (95) postulated a
competition between the carcinogen and vitamin A for a
particular liver protein.
In addition to investigations concerning the storage
of vitamin A and the carotenoids, several workers have made
attempts at localizing these components within various
tissues, besides the earlier mentioned fluorescence studies.
The first attempt was made by Joyet-Lavergne (96, 97), who
observed vitamin A in particles corresponding to mito-
chondria in the liver cell, and this was soon confirmed____
! 19
i
by Bourne (98). Both of these Investigators used the ;
:Carr-Price (99) reagent on histological sections to demon-
| strate indirectly the association of vitamin A with the ,
‘mitochondria. More recently, using the same technique,
Jones (100) has also demonstrated that the mitochondria of j
embryonic erythroblasts obtained from rat yolk sacs also j
i contained vitamin A. However, this procedure has been
j claimed to be of only questionable specificity when used
| histochemically (82), j
1 I
j Shortly after these histological demonstrations of 1
! vitamin A in the mitochondria, and in one of the earliest j
| applications of the technique of differential centrifuga- 1
tion, Goerner and Goerner (101, 102, 103) Isolated the !
I
mitochondria from normal rabbit and rat livers and from
J their corresponding hepatoma tissue. After a thorough
| extraction with fat solvents, the vitamin A was found in ;
1 >
the lipids of the mitochondria isolated from the normal j
livers, but not in the mitochondria isolated from the J
hepatoma tissue. They also observed the marked decrease j
j in the total vitamin A stored in the liver after the
j administration of certain carcinogens (lOij.), !
i
I The most recent study on the distribution of vitamin
•A in liver was made by Collins (105), who used the technique
t ,
j of differential centrifugation, and found 20# of the total |
20
liver vitamin A in the mitochondria, and the remainder
with the exception of the whole cell contamination of the
nuclei, in the supernatant fraction*
Statement of the Problem
With the exception of their role in the visual
processes, the functions ascribed to the carotenoids and
vitamin A have been merely speculative* One reason may
be that too much emphasis has been placed on the fact that
these compounds are fat soluble* While this is certainly .
true for the Isolated pigments, their behavior in vivo
may be entirely independent of this property, provided
that they can enter into the aqueous milieu where our
metabolic reactions occur by complexing with proteins*
That is why the emphasis in this review has been placed
on the reports dealing with the protein complexes of these
pigments. On analysis of these reports however, it is
apparent that the actual mechanisms of absorption,
transportation and storage of these essential dietary
components are still poorly defined* Although I do not
believe that an investigation of these mechanisms consti
tutes the primary problem of vitamin A biochemistry, namely,
the elucidation of the remaining function or functions of
vitamin A aside from its role in the visual system, still
a more precise pattern of these three processes might_______
21
eventually prove of some assistance in the solution of the
more basic problem.
This study was therefore undertaken as an attempt
at extending the present knowledge of the relationships
between the carotenoid pigments, vitamin A and the tissue
proteins, with respect to the absorption, transportation
and storage of these components. It has involved an
investigation of the association of both the free and
esterified forms of vitamin A and the carotenoids, in
different fractions of the plasma proteins and of the
liver cell.
MATERIALS AND METHODS
Animals
Chickens. Both normal and carotenoid-depleted
ehiokens were used. In these experiments. The normal
chickens were either white Leghorns or RhoderIsland reds,
©f unknown history, which were purchased at several local
poultry houses. These chickens were used without reference
to sex. In addition, chicken plasma was also obtained
directly during slaughter at these poultry houses.
The carotenoid-depleted chickens consisted of male
white Leghorns that had been placed on a restricted diet
(106) at 6 weeks of age. As judged by the loss in color
of their shanks and their low plasma carotenoid values,
1 j . to 6 weeks on this regimen were sufficient to markedly
deplete them of their carotenoid stores.
Rats. Normal albino rats of the University of
Southern California strain were used in these studies.
These animals were obtained from the stock colony and had
been maintained on Purina chow or other suitable mixed
diets. Both sexes were used.
Beef. Fresh beef blood ^ was obtained directly
1/ The beef blood was obtained through the
______ courtesy of Swift and Company.___________________
23
from a local packing house, and oxalated immediately to
prevent clotting. It was transported in dark-brown bottles
to decrease any destruction of the carotenoids and vitamin
A by light.
Beef liver was obtained directly from a packing
house as soon as possible after slaughtering, and the
various fractionation procedures were started within 1$
minutes of obtaining these samples. In addition, beef
liver was also purchased from retail markets. In the case
of the latter samples, an unknown amount of time had
elapsed between the time of killing and fractionation.
Hogs♦ Fresh hog blood 2/ was oxalated as soon as
it was obtained and transported in dark-brown bottles* ,
Supplements
Lutein. The lutein was prepared from a crude
concentrate of leaf xanthophylls obtained from the Valley
Vitamin Corporation, McAllen, Texas. The crude material
was saponified with 20% alcoholie potassium hydroxide (w/v)
for 2 days at room temperature, and was then extracted
with diethyl ether. After washing the ether solution with
water and drying over anhydrous sodium sulphate, the
2J The hog blood was obtained through the
courtesy of Swift and Company.
2k
solution was evaporated to dryness In vaeuo, and taken up
in Skellysolve B. This was then chromatographed on an
alumina column (2.3 x 23 cm.) and all the carotenes, as
well as the diesters of lutein were eluted with 20$ acetone
in Skellysolve B. The monoesters of lutein, plus any
monohydroxy-carotenoids, were eluted with 35$ acetone in
Skellysolve B. The main pigment zone remaining on the
column was cut out and eluted with 8$ ethanol in Skellysolve
B. This solution was refrigerated overnight, which caused
a colorless material, presumably sterols, to precipitate.
The precipitate was removed by filtration, and the solution
after transferring to Skellysolve B, was chromatographed on
a Ca(0H)2:celite (3il) column (2.3 x 23 cm.) and developed
with 2$ acetone in Skellysolve B. The main pigment zone
was eut out and used without further purification, as it
would be expected to contain only dihydroxy-carotenoids,
and showed the typical spectrum of lutein, with an
absorption maxima of -Ij 46 mp in hexane.
Known quantities of the stock lutein solution were
transferred to diethyl ether, and peanut oil containing
other supplements, was also dissolved in the ether. The
ether was then removed in vacuo, and additional peanut oil
was added to give the concentration of lutein desired,
usually 1 mg./ml.
25
Vitamin A. The vitamin A used for supplementation
in these studies was a commerical mixture of natural
vitamin A esters obtainable from the Hope© Chemical
Company* Harrison* New Jersey* Weighed amounts of this
concentrate were dissolved directly in peanut oil, and used
after thorough mixing.
Carrier. In all experiments involving the admini
stration of supplements of earotenoids or vitamin A, the
supplements were administered orally dissolved in a
refined peanut oil, which had previously been shown to be
adequately absorbed (107)• In addition to the supplement,
each ml* of oil contained 5 mg. mixed tocopherols and l6 mg.
lecithin*
Route of administration. For chickens* the
supplements were administered by means of a rubber tube,
especially designed for stomach tubing (A. S. Aloe, Sands,
10 Fr.)* This tubing was passed down the chickens throats
for approximately 25 cm. Although this procedure is
t
referred to as ”stomach tubing”, there is little doubt
that the tube only proceeds as far as the crop* The
animals were fasted for 12 to 18 hr. before dosing, in an
attempt to empty the crop, and facilitate absorption.
Supplements were fed t© rats with the aid of a
syringe and a specially constructed #15 hypodermic needle.
26
This needle, 8 cm. long, had a copper jacket attached at
the tip, which facilitated its passage down the rat*s
throat. A maximum of 1 ml. of supplement was given to
a rat with this method.
Solvents
A wide variety of organic solvents have heen used
In these experiments, and unless otherwise indicated, were
of the C. P. grade.
Ethanol refers to 95% ethyl alcohol.
Diethyl ether was maintained peroxide-free by
storage over ferrous wire.
Two types of light petroleum ether have been used.
One, Skellysolve A, has a boiling range from 30° to 1^.0°.
The other, Skellysolve B, boils over the range 6©° to 70°.
Both are obtainable from the Skelly Oil Company, Lyman,
Oklahoma.
Plasma Fractionation
Beef and hog blood, obtained from the packing house,
were eentrifuged and the plasma used directly.- Freshly
withdrawn chicken and rat blood were similarly treated to
obtain the plasma.
Ammonium sulphate fractionation. For this purpose,
27
a solution of ammonium sulphate saturated at room tempera
ture, was added in the ratio of 1 volume to 3 volumes of
plasma to obtain 2$% saturation, or 1 volume to an equal
volume of plasma to obtain 5®% saturation* The ammonium
sulphate solution was adjusted to pH 6*3 with sodium
hydroxide before being used. In order to avoid excessive
destruction of vitamin A, the chilled ammonium sulphate
solutions were added slowly, with constant stirring, to
the plasma, the entire procedure taking place in an
ice-bath. The ammonium sulphate-treated plasma wa3 then
immediately centrifuged for 30 minutes at f>°. Although
this did not allow equilibrium to be reached in the
precipitation, it seemed an unavoidable choice, due to the
rapid.destruction of vitamin A under these conditions*
Dialysis* Fractionation by means of dialysis was
carried out by dialyzing the plasma samples in Visking
sausage casing against cold (2j.°) distilled water. Usually
10 to 20 ml. plasma were dialyzed against 1$ to 18 liters
of water, for 12 to l6 hr. in the dark. The dialysis
tubing was carefully rinsed out to remove adhering material,
and the precipitate was then obtained by centrifugation.
Solvent extraction. A conventional ether extraction
procedure (108) which consisted of shaking plasma with
28
2 to 3 volumes of diethyl ether in the cold ( I | _ ° to 7°)
for five minutes was used. This extraction was repeated
twice*
An additional solvent extraction procedure involved
using fat in an attempt to fractionate the carotenoids
and vitamin A of plasma. A colloidal fat suspension,
prepared by dissolving palmitic acid in peanut oil with
the aid of diethyl ether, removing the ether in vacuo,
and adding water and sodium hydroxide till just alkaline,
was thoroughly mixed with plasma, and then centrifuged,
which resulted in the accumulation of fat at the top of
the centrifuge tube. This was removed and analyzed
independently of the remaining plasma*
Rat Liver Fractionation
Both male and female stock colony rats were used
in these experiments. In the earlier fractionations, the
animals were fasted for 18 to 2i j . hr. before being
sacrificed, but this procedure was discontinued when the
importance of the fat fraction was realized.
Schneider-Hogeboom procedure. Initially, the
procedure of Hogeboom, Schneider and Palade (109),
utilizing 0.88 M sucrose solution as the homogenizing
medium, was used. However, this procedure did not permit
29
the complete fractionation of the liver cells, so an
alternative method described by Schneider and Ho'geboom
(110), in which isotonic 0.25 M sucrose solution is used
as the homogenizing medium* was followed*
The animals were placed under light sodium pento
barbital (Nembutal) anesthesia, and a small flap of skin
was removed over the sternum. An incision was made through
the skin and diaphragm, and as much blood as possible was
withdrawn directly from the heart with an oxalated syringe.
The liver was then immediately excised, blotted to remove
excess blood, and placed in a beaker over chopped ice.
All visible connective tissues were cut away, the livei*
cut into small pieces, and 1 to 2 gm. aliquots were
homogenized with 9 volumes of ice-cold sucrose solution
in a modified Potter-Elvehjem (111) homogenizer, while
Immersed in an ice-bath. The homogenizer consisted of a
thick-walled glass tube, 2© x 1.8 cm. and a lucite plunger
(112) having a 7 cm. grinding surface, which was ground
into the tube with a glycer©1-carborundum mixture. As
the 1 1 beads” which are ordinarily found on glass pestles
could not be constructed using the lucite pestle, the
tip of the plunger was scored to produce better homogen
ization. In some of the earlier experiments, a glass
pestle had been used with similar results.
Histological analysis %/ indicated that i j . 0 t© £0
s
strokes of the pestle were necessary to obtain the minimum
of 80$ cell disruption which many workers consider complete
homogenization (113)* In the experiments in which the
lucite plunger was used, it was obvious that more than 80$
of cells had been disrupted, for the nuclear fractions,
which would include whole cells, never contained more than
20$ of the total vitamin A recovered from the various
fractions.
In some of the later experiments, especially those
using isotonic sucrose solution as the homogenizing
medium, the tissue was homogenized with only % volumes of
this solution in order to decrease the time neeessary for
complete homogenization of larger quantities of tissue.
In all cases, homogenization was carried out In the
cold, with the homogenizing tube immersed in an ice-bath,
and after every ten strokes, the homogenization was stopped
for a few minutes, to permit the contents of the tube to
attain the temperature of the ice-bath.
An International Refrigerated Centrifuge, Model
PR-1, equipped with a high-speed head, was used for these
y The author acknowledges the cooperation of
Dr. M.C. Schotz and Dr. J. L. Mohr In these
analyses.
experiments* This is an angle centrifuge, and all
centrifugal forces have been expressed as the average
centrifugal force, i,e., calculated from the center of
the centrifuge tube. The following formula was used to
calculate this relative centrifugal force:
O * (r)(1118)(rpm)^ g
where is the relative centrifugal force, ”r, f is the
distance from the axis of rotation to the center of the
tube, and Mg”is the force due to gravity.
As it was not possible to attain centrifugal forces
greater than 22,000 x g with this centrifuge, longer
periods than originally suggested were used to isolate
the mitochondria. Figure 1 illustrates the method used
for these fractionations.
HOMOGENATE
10* 10*
Residue
10* 10*
Supernatants
22,000 x g
30* T
Residue
22,000 x g
30*
MITOCHONDRIA MICR0S0HES NUCLEI
+
SUPERNATANT
FIGURE 1# The fractionation scheme, for rat liver
horaogenates. The figures to the left and right of the
vertical lines indicate the centrifugal forces and centrifu-
f
gal times, using hypertonic or isotonic sucrose solutions,
respectively.
33
Modified procedure. As the results from the above
experiments were obtained, it became obvious that the
centripetally-migrating material, hereafter referred to as
the cream fraction, was important insofar as vitamin A
distribution was concerned, Therefore, the procedure
described above was modified to permit the quantitative
isolation of this centripetally-migrating fraction. This
material is quite viscuous at the low temperatures used
for the fractionation, and it became obvious that during
decantation, contamination from this material was
practically unavoidable. It was felt advisable, for these
reasons, to effect the separation at the earliest stage
of the fractionation. As there was no provision for the
segregation of this material in the original procedure,
the following modification was adopted.
The liver homogenate prepared in either hypertonic
(0.88 M) or isotonic (0.25 M) sucrose solution was
carefully layered under a more dilute sucrose solution
(O.ijij. to 0.15 M, respectively) and centrifuged for 1 hr.
at 105,000 x in a pre-eooled rotor (5°). A Spinco
Preparative Fltracentrifuge, Model L, was used for this
and all subsequent centrifugations. After this centrifu
gation, three zones were visible. The turbid creamy layer
at the top was removed with a syringe and needle, as was
3 1 * .
the clear solution lying immediately below it containing
the cytoplasmic non-sedimenting material. This will be
referred to as the supernatant fraction. The residue was
resuspended In sucrose. This contained supposedly all
the nuclei, mitochondria and microsomes.
Figure 2 represents the procedure followed for the
complete fractionation of rat liver homogenates. It Is
similar to the procedure described In the previous para
graph, except that only homogenates prepared in an isotonic
sucrose solution were used. In addition, the tightly-
packed residue was rehomogenized with Isotonic sucrose
solution (5 to 10 strokes in the homogenizer) and
differential centrifugation of this resuspended residue
according to Schneider and Hogeboom’s (110) procedure,
with minor variations, yielded the various particulate»
fractions of the liver cell. The nuclear fraction contained
some cell debris, connective tissue, whole cells and red
blood cells, in addition to the nuclei. The mitochondrial
fraction was essentially free of nuclei when stained with
Wright*s stain. The final residue, which contained little
material staining specifically with Janus green B, was
ealled the microsomal fraction.
HOMOGENATE
x g
Residue
10*
Residue
10*
^Supernatant
10*
Residue
10*
^Supernatant
Residue
CREAM NUCLEI MITOCHONDRIA . . . MICROSOMES SUPERNATANT
FIGURE 2. Modified Schneider-Hogeboom procedure
for the isolation of various cell fractions of rat liver
homogenates prepared in isotonic suerose solutions
36
Beef Liver Prac11onation
Using, the modification described for rat liver, the
following procedure was utilized for the fractionation of
beef liver.
Both fresh and aged beef liver samples were
fractionated. Portions of liver used were obtained from
the center of the liver, the tissue near the surface being
trimmed away. This tissue was cut into small cubes,
approximately 2 x 2 x 2 cm., and as much connective tissue
and blood vessels as possible were trimmed off. These
cubes were then pulped with the aid of a stainless steel
tissue press, which forces the tissue out of a disc per
forated with 1 mm. holes, while under pressure. This was
collected directly into a tared beaker containing ice-cold
isotonic sucrose solution. When a sufficient quantity of
material had been so pulped, additional sucrose solution
was added to give a 1:1 ) . suspension of tissue in sucrose
solution. In several experiments, this ratio was changed
to 1:3 ©r 1:2, due to the large quantities of tissue
handled, and the inability to centrifuge this large volume
at one time.
The suspension obtained was then passed through a
modified Potter-Elvehjem homogenizer, developed by Harris
and Mehl (111).). Homogenization is effected by the
37
continual flow of the suspension between the rotating body
©f the plunger and the ground-glass wall of a stationary
tube* Preliminary investigations indicated that 3 passes
of the suspension through this homogenizer resulted in
almost complete homogenisation. Any residual whole eells
and connective tissue was removed by filtering this
suspension through flannelette, as suggested by Schneider
and Petermann (115). This homogenizer is illustrated in
Figure 3.
STAINLESS
— STEEL
LUCITE ROD
150 ML
RESERVOIR
GROUND INNER
SURFACE---
THICK-WALLED
GLASS TUBING
!!< L
'it t t
PIGURE 3. Harrisr-Mahl-Goatlnuom„.Hotnogenl zer
39
The actual centrifugation scheme was as follows:
26 ml# of the homogenate was carefully layered under 9
of 0.125 M sucrose solution, and centrifuged for 60 min.
at 78*i}-00 x g in a pre-cooled rotor (5°) • Three zones
were visible after this centrifugation. The upper turbid
zone, containing the cream fraction, was removed with the
aid of a syringe and a blunt needle, and any material
adhering to the sides of the centrifuge tube was removed
with a cotton swab. A similar syringe and needle were
used to remove the clear-red supernatant fraction. The
tightly-packed residue was then resuspended in approximately
10 ml. of isotonic sucrose solution, and rehomogenized in
the Potter-Elvehjem homogemizer. The resulting suspension
was centrifuged twice for 10 min. at 600 x g, and the
residues obtained, after rehomogenization, were also
centrifuged for 10 min. at 600 x g» The resulting
residue was designated as the nuclear fraction. It was
light pink when suspended in either isotonic sucrose
solution or distilled water, presumably due to the presence
of red blood cells remaining in the original liver tissue.
The supernatant fractions from the above centrifugations
were then pooled, and centrifuged twice for 10 min. at
5»©0O x g• The residues obtained, after rehomogenization,
were centrifuged for 1© min. at 5,000 x g. The tannish-
colored residue from this centrifugation was called the
mitochondrial fraction. The pooled supernatant fractions
from the above centrifugations were centrifuged for 60
min. at 78,i|.00 x g, and the opaque, reddish residue was
called the microsomal fraction. Usually, a small amount
of eentripetally-migrating material appeared during this
centrifugation, and it was added to the cream fraction.
The supernatant obtained from this last centrifugation was
combined with the supernatant of the Initial centrifugation*
the resulting mixture comprising the entire supernatant
fraction. The entire scheme Is illustrated in Figure If.
HOMOGENATE
Residue
10»
Supernatant
10*
Residues
10*
Supernatant
x g 10*
10*
SUPERNATANT*-
NUCLEI MITOCHONDRIA MICROSOMES CREAM
FIGURE i f . . Fractionation scheme for beef liver
homogenates prepared in isotonic sucrose solutions.
kz
Treatment of Plasma j
Plasma was obtained from the various oxalated blood
samples by centrifugation of the blood for 15 min* at
700 x jg. The clear plasma could then be readily removed
from the tightly packed cells*
The procedure described by Kimble (ll6), with
slight modifications, was used for the extraction of whole
plasma and the various fractions obtained from it* To
10 ml. of plasma (when necessary, water was added to bring
it to this volume), an equal volume of ethanol was added
slowly, with constant swirling, while in a separatory funnel.
Two to three volumes of Skellysolve A was then added, and
the material shaken for 5 min. After settling, the aqueous
phase was drawn off, and reextracted with the same volume
of Skellysolve A* These two extracts were pooled, and
after evaporation to dryness in vacuo, while in a warm
water bath, were redissolved in approximately 5 ml. of
Skellysolve B for further analysis.
Treatment of Liver
Portions of whole liver, as well as the fractions f
obtained by centrifugation, were extracted according to
the procedure of Thompson, Ganguly and Kon (117). The
liver tissue was extracted in a Waring blendor with
^3
Skellysolve A, ethanol and water, In a ratio of 12:2:1.
When the liver fractions, suspended in either suerose
solution or water, were extracted, the volume of their
solutions were treated as an equivalent volume of water,
and the above ratio was maintained. Before each homogeni
zation in the blender, the blender cups were flushed with
nitrogen gas, to prevent undue oxidation.
After $ min. of homogenization in the Waring blendor
the solution was transferred to a separatory funnel, and
the lower alcohol-water phase was removed and rehomogenized
for another 5 > min. period, after adding the same volume
of Skellysolve A used originally. This mixture was poured
into a separatory funnel, following which the alcohol-water
phase was discarded. This method of extraction was found
to give 90-95^ of the total vitamin A present, which
compares well with the alkaline digestion procedure (118)
and has the added advantage of extracting the free and
esterified forms of vitamin A, without any hydrolysis of
the ester.
It has been found advisable to allow the mixture to
remain in the separatory funnel for a few minutes, with
occasional gentle swirling, to effect a better separation
of the two phases. On those occasions where emulsions
formed, a few ml. of ethanol added to the funnel, followed
kk
by swirling the contents, usually resulted in breaking the
emulsion. When this failed, more petroleum ether was added
and the solution was rehomogenized for 1 to 2 rain, in the
blendor, a procedure which invariably gave rise to two
distinct phases.
These extracts were pooled, evaporated to dryness
in vacuo while immersed in a warm water bath, and
redissolved in approximately 5 ml* of Skellysolve B for
chromatography.
Ether extraction. In addition to the above procedure
which was used routinely for liver extractions, several
other procedures were utilized. One of these was the
following eold diethyl ether extraction. Aliquots of the
homogenates, or fractions obtained therefrom, were
suspended in sucrose solution or water, and shaken with
three volumes of diethyl ether for 2 to 3 rain, in the cold
(0° t© 5°). The aqueous phase was reextracted in a similar
manner, and the two extracts pooled.
The relative instability of the isolated particulate
fractions, with respect to their binding affinities for
the carotenoids and vitamin A, was demonstrated by the
bther extractability of these pigments from these fractions
before and after storing them at 0G for 12 hr.
After drying these extracts over anhydrous sodium
sulphate, they were evaporated to dryness in vaeuo and
redissolved in approximately £ ml* Skellysolve B for
further analysis*
Alkaline digestion* In an attempt to bring the
carotene found in various particulate fractions of beef
liver into solution, a procedure described by Claude
(119# 120) was used. This involved alkaline digestion of
the fractions, followed by partial precipitation of the
proteins by acidification, and separation of the insoluble
and soluble proteins.
An aliquot of the microsomal or mitochondrial
fraction, resuspended in Isotonic sucrose, was brought to
approximately pH 12 by the dropwise addition of N sodium
hydroxide. The clear solutions resulting from this
procedure were digested for I 4 . 8 hr. at J j . ° , and the pH was
then readjusted to 4*75 with JJ acetic acid. The precipi
tate obtained was separated by centrifugation, and the
soluble and insoluble proteins were analyzed independently
for vitamin A and caroteneIds.
Salt extraction. Concentrated salt solutions were
also used to disrupt, and possibly solubilize the proteins
of the isolated beef liver fractions. For this purpose,
an equal volume of M potassium chloride was added to the
l j . 6
isolated cell fractions suspended in an isotonic sucrose
solution, giving a final concentration of 0*5 J§ potassium
chloride and 0*125 M sucrose. These were then used
immediately for ether extraction, or stored for 12 hr. at
0° .
t
One portion of these salt-treated fractions was
centrifuged twice for 10 min. at the same centrifugal
force used to sediment the fractions initially. The residue
and supernatant fractions were then analyzed individually.
Butanol extraction. Recently, organic solvents have
been used quite extensively as a means of solubilizing
proteins from various tissues or tissue fractions (121).
The following procedure, adopted from Harris, Bavetta,
Bergren and Mehl (122) has been used with beef liver
fractions.
The cell fractions, suspended in water, were
immersed in a constant temperature bath (38°) for 5 min.
to attain temperature equilibrium. An equal volume of
water-saturated butanol was then added dropwise, with
constant stirring, and the mixture was then stirred for an
additional 30 min. It was then centrifuged, resulting in
the formation of two distinct zones. The upper clear
butanol solution contained the tissue lipids; the bottom
clear solution contained the water-soluble proteins. At
Vr
the Interphas© between these two zones, the precipitated
protein accumulated* The butanol extraet was drawn off*
and the pigments transferred to Skellysolve B by the
addition of water and ethanol* The insoluble and soluble
proteins were pooled and treated as the butanol residue
fraction.
Waring blendor extraction. Various workers (123)
have utilized partial homogenization of tissue fractions
in a *Waring blendor to effect solubilization of proteins*
Aliquots of the particulate fractions in isotonic sucrose
solution were homogenized for 2 min* in a Waring blendor,
at 5°* The resultant solution was centrifuged at the
same centrifugal force and the same centrifugal time as
was used to obtain the fraction originally* The supernatant
and residue fractions of this centrifugation were then
analyzed individually*
Hypotonic extraction, de Duve and coworkers (12ij.)
have proposed that semi-permeable membranes exist around
the particulate material found in tissue homogenates* If
this is true, placing the partieulate material into a
hypotonic solution should result in the liberation of the
contents into solution* This procedure was followed for
the particulate fractions of beef liver homogenates. The
I k 8 1
, i
j fractions, after their last centrifugation, were suspended j
i ^
I in distilled water, and stirred for 10 min. They were
then centrifuged for 10 min. at the same centrifugal field
originally used to sediment these fractions. The super-
natant and residue were then extracted separately. \
i
Chromatographic Analysis
All of the tissue and plasma fractions, obtained
from the various fractionation procedures, after trans
ferring to Skellysolve B, were chromatographed on ad
sorption alumina, 80-200 mesh, obtainable from the Pisher
Scientific Company, as described by Ganguly, Krinsky, Mehl i
and Deuel (12f>) and by Thompson, Ganguly and Kon (117)*
As each new lot of alumina may show different adsorptive
| characteristics, every new lot was tested with a standard
solution of vitamin A ester and alcohol to determine the
strength of eluents necessary for complete elution of the
two fractions. I
The alumina, suspended in Skellysolve B, was poured \
into a 1.5 x lif cm. chromatographic tube, which contained
a cotton plug in the neck of the shaft. Sufficient j
1
alumina was added to give a column height of approximately i
If to 5 cm., and the adsorption affinity was then weakened j
by the addition of a few ml. of 8% ethanol in Skellysolve
1
B. The tissue extraet in approximately 5 ml* of Skellysolve
k-9
B was then added to the column and the vitamin A ester
fraction was eluted using \$ acetone in Skellysolve B as
the eluent* This fraction also contains the carotene, and
any completely esterified carotenoids, if present. When
the tissue fractions contained little or no free carotenols,
such as those from rats and beef, 8% alcohol in Skellysolve
B was routinely used to elute the vitamin A alcohol
fraction. However, in the extracts from chicken plasma,
which contained considerable amounts of free lutein, the
vitamin A alcohol fraction was eluted out first with 35%
acetone in Skellysolve B, after which the lutein could be
eluted with 8% alcohol in Skellysolve B. In all cases,
about 30 to 35 ml. of eluent was used to remove each
fraction.
Chemical Analysis
Vitamin A. Both the free and esterified vitamin
were determined in a similar manner, following their
chromatographic separation. The eluted fractions were
evaporated to dryness _in vacuo (water aspirator) while
immersed in a warm water bath. They were then taken up
in 1 ml. of chloroform and if necessary diluted further
with chloroform. For the determination of vitamin A,
0.5 ml. of this chloroform solution, together with 1 drop
of acetic anhydride were transferred to a 10 x 75 ran* test
tube, and 2 ml. of the Carr-Price (99) reagent were added
by syringe.t© effect rapid mixing. The reagent consisted
of a 20% solution of antimony trichloride in chloroform
(by weight). The color intensity was determined in the
Goleman Junior Spectrophotometer, at 620 mp,. The nature
of the transient blue color formed with vitamin A under
these conditions is such that a definite time interval
cannot be used for determining the maximal optical density
of the solution, so that it was necessary to use the
stable reading which occurs between $ and 10 sec. after
adding the reagent. The actual concentration was calcu
lated with the aid of a calibration curve prepared with
the aid of pure vitamin A aleohol.
When carotenes were present in the vitamin A ester
fractions, a correction factor was applied to the optical
density readings.obtained. This factor was obtained by
treating known concentrations of p-carotene dissolved in
chloroform with the Carr-Price reagent, and measuring the
optical density at 620 mji 8 sec. after mixing*
p -Carotene. This was determined in the \ \ f o acetone
in Skellysolve B fractions, using the Coleman set at
mji. A calibration curve, using pure p-carotene whose
concentration had been determined in the Beckman speetro-
51
photometer, was used to determine the concentration in the
various tissue fractions.
Lutein, Lutein was determined in the Q% alcohol in
Skellysolve B fractions, using the Coleman set at mu,
1%
The concentration was determined using the E„ value of
lenu
Nitrogen, The nitrogen content of the fractions of
beef liver homogenates were determined following a combina
tion of the procedures of Ma and Zuazaga (126) and Ghibnall
et al. (127), Portions of the whole homogenate ©r its
fractions were suitably diluted to contain approximately
1 to 10 mg, protein/ml, 0,1 Ml, aliquots of these solutions
were then charred with 1 ml, of concentrated sulphuric acid
on an electric heating unit, and after cooling, 100 mg, of
a catalyst, containing potassium sulphate:copper sulphate
(penta-hydrate): selenium dioxide in the ratio of ij.00:100:1
was added. The mixture was then digested for 1 } . to 6 hr.
after clearing, which usually occurred within a few minutes
of adding the catalyst. The entire digestion was run in
KJeldahl flasks, with a bulb capacity of 15 ml.
The digested samples were then distilled through a
Kirk still into a 2% boric acid solution. Using a mixed
indicator, bromcresol greentmethyl red (5:1), the solutions
were titrated with 0,0100 N hydrochloric acid.
EXPERIMENTAL RESULTS
The results obtained have fallen into two main
categories, those from plasma and those from liver* In
addition, the liver results have been further subdivided
according to the species investigated, viz., rat or beef
liver.
Plasma Fractionation
Solvent extraction. The first attempt made t©
observe any difference in behavior of the carotenoid pig
ments was a study of the ability of these pigments to be
extracted from chicken plasma with diethyl ether. The
results are presented in Table I. Although these results
were variable from experiment to experiment, no definite
difference could be observed in the relative extractability
of the pigments under these conditions.
TABU I
HBBt UTRACTIO* OP p-CAROTMB, LOTBH, VITAJCI A ALCOHOL AID VTTAIOI A E8TBR ROM CBICXS PLA8MA
Expt. Regimen of oarotenoid- u d Fraotlon
no» Tit— In Ai-dopletod ohlolcsns analysed
Carotene "Tut e in' Vitamin A alcohol
m % ----- --------------------- ? ---------------1 -------
T/100 ml. total r/100 ml. total tAQO ml
Vitamin A ester
% ~ T ”
total T/100 ml. total
Vitamin A ester (66 mg.)
given 4 a hours boforo
Ho supplement
High p-oarotene for
throo days
Lutein (276 r ) and
4 vitamin A ostor (66 mg.)
given 6.8 hours before
Vitamin A ester (66 mg.)
6a given 4.6 hours before.
Ammonium oltrate added
to plasma.
Vitamin A ester (66 mg.)
6b given 4.6 hours before.
Ammonium ehlorlde added
to plasma.
Vitamin A ester (66 mg.)
6o given 4.5 hours before.
Ammonium carbonate added
to plasma.
Plasma
Ether extract
Ether residue
Plasma
Ether extract
Ether residue
Plasma
Ether extreot
Ether residue
Plasma
Ether extraet
Ether residue
Plasma
Sther extreot
Ether residue
Plasma
Ether extreot
Ether residue
Plasma
Ether extreot
Ether residue
66.8
12.8
16.1
66.8
42 J8
46.6
7.6
88.0
16.3
70.8
8.2
3.7
8.0
1.8
1.8
O J O
2 6 J !
14 J t
9JB
31 a
8.3
19.7
90.7
0.0
7 0 JO
90.7
OJO
64 a
90.7
Oa
79a
46.1
60.9
100 J O
OJO
6 4 J
56 Jt
0.0
77 J t
OJO
60*4
OJO
87 J6
82 J t
34 SO
68 a
7 J 0
8 J6
OJO
17 A
6 JO
8*9
166
26.7 23 JO
63.8 119
418
24.7
84 2
416
16.7
171
6*6
18.3
860
41 a
67a
97a
oa
84a
6i a
ua
76.7
ea
82 a
4a
4 i a
sa
sea
55
Another method of extraction, which involved the
addition of a fat emulsion to plasma, followed by the
centrifugal removal of the added fat, was also utilized.
This method did not prove successful, as no means were
available at the time for a quantitative removal of the
added fat.
Ammonium sulphate fractionation. As solvent
extraction of plasma did not lead to any definite con
clusions, the classical salt fractionation procedure, which
involves the precipitation of various plasma protein
fractions by increasing the concentration of ammonium
sulphate, was used. In Table II, the results of three
different salt concentrations on the distribution of free
and esterified vitamin A in chicken plasma are presented.
Marked destruction of vitamin A ester occurred when the
salt concentration was above half saturation and as this
concentration gave some indication of a difference in the
distribution of the ester and alcohol, it was used as the
maximum salt concentration for the remaining experiments.
TABLE II
DISTRIBUTION OP VITAMIN A ALCOHOL AND VITAMIN A ESTER IN PLASMA FRACTIONS OBTAINED BY AMMONIUM
SULPHATE FRACTIONATION FROM CAROTENOID-DEPLETED CHICKENS SUPPLEMENTED WITH VITAMIN A ESTER
Experiment
number
0®4)2S04
eonc.
...*:
Frnotion
analysed
Vitamin A alcohol Vitamin A ester
r/lOO' ml. % total
0 Whole plasma 80*0 196
.6
50 Supernatant 51*7 39.6 41.8 21.3
Precipitate 38*0 47.5 100 61.0
0 Whole plasma 84*6 127
7 60 . Supernatant 23*4 27.6 36.5 28.7
\
Precipitate 74.1 BIS 29.8 23.4
0 Whole plasma 54*5 207
8 75 Supernatant 9.7 17 S
24 £ 11.7
Precipitate 42.8 78.4 66.9 52.3
vn
O'
Table III lists the distribution of |3-carotene,
vitamin A alcohol and vitamin A ester in chicken plasma
fractions obtained by precipitating the plasma proteins
with quarter and half saturated ammonium sulphate* These
results demonstrated that different amounts of the carote
ne id pigments could be found with the proteins precipi
tating under these conditions whieh was indicative of the
fact that the pigments were closely associated with the
plasma proteins*
58
TABLE III
DISTRIBUTION OF ^-CAROTENE, VITAMIN A ALCOHOL AND VITAMIN A ESTER
. IN PLASMA FRACTIONS OBTAINED BY AMMONIUM SULPHATE FRACTIONATION
FROM CHICKENS RAISED. ON A CAROTENOID- AND VITAMIN A-LOff DIET,
i AND SUPPIEMENTED WITH 0-CAROTENE>
Experiment number 3
(NH4)gS04
oono.
%
Fraction
Carotene
r/ioo ml •
Vitamin A
aloohol
r/ioo ml.
Vitamin A
eater
t/100 ml•
0 Whole plasma 35.3 26.2 17.4
25 Supernatant 18 .9 14.2 7.3
Precipitate 7.6 0.0 4.6
50 . Supernatant 18.4 12.7 10.8
Preoipitate 17.6 11.6 8 .6
59
Several carotenoid- and vitamin A-depleted ehlckens
were then orally supplemented with either vitamin A ester,
lutein, or a combination of both, and sacrificed five to
seven hours later. The plasma samples obtained were
fractionated with quarter and half saturated ammonium
sulphate. In these experiments, an attempt was made to
minimize the destruction of the pigments by carrying out
the precipitation in the cold, and then immediately centri
fuging the samples in the cold, and analyzing the super
natant and precipitate. This procedure diminished the
losses in the majority of cases, with the notable exception
of the vitamin A ester remaining after treatment with half
saturated ammonium sulphate. An Important observation
that could be drawn from these results, was that the per
centage distribution of the individual pigments in the
various fractions was approximately the same, regardless
of whether the animals had been supplemented with these
pigments dr not. These results are tabulated in Table IV
and summarized in Table V.
6o
TABLE 17
DISTRIBUTION OP LUTEIN, VITAMIN A ALCOHOL AND VITAMIN A ESTER IN PLASMA FRACTIONS OBTAINED BY AMMONIUM
3UIPEATE FRACTIONATION FROM CHICKENS RAISED ON A CAROTENOID- AND VITAMIN A-LOW DIET AND SUPPLEMENTED
WITH VITAMIN A AND/OR LUTEIN
(Valves expressed es r/lOO ml. plasma)
Lutein Vitamin A aloohol Vitamin A ester
Rxpt. Supplement and £ ( nh4)2S04 riNH4)VsO, ^ W 4 TTS0l-
no* fraotion anelyeed *
26 60 O 26 60 0 26 60
9 Lutein
Whole plasma 98*2 24 *2 6*0
Supernatant 96*2 55*8 22*0 10*4 6*7 8*1
Precipitate 6.2 20*4 1.7 9.6 2.1 0.8
10 Vitamin A
Whole plasma 29.8 117 459
Supernatant 27.0 19*0 96*0 41.8 576 194
Precipitate 8.7 12*7 17*1 68*4 68*4 209
11 Vitamin A
Whole plasma 19*6 68*1 128
Supernatant 19.4 18*2 62.7 31*5 107 67*0
Preoipltate 1.4 1.6 4.1 36.6 9.2 31.4
12 Vitamin A
Whole plasma • 42 *4 816
Supernatant • • 69.2 53*4 671 106
Precipitate - - 6.6 42.0 69.6 290
4 Lutein and Vitamin A
Whole plasma 46.6 24.2 166
Supernatant 53*0 16*0 13.3 11.6 91*4 25.8
Preoipltate 0.0 16.3 7.6 17.6 71.2 66.8
IS Lutein and Vitamin A
Whole plasma 260 68.9 160
Supernatant 280 168 41.9 17,9 75.7 29.0
Preoipltate 0.0 102 7.0 55.3 12.8 90.8
14 Lutein and Vitamin A
Whole plasma 127 63.8 25*0
Supernatant 153 93*6 45.7 19,4 16.9 7*2
Preoipltate 4.4 60.6 0.4 29*6 6.1 14*7
6i
TABLE V
SUMMARY OP THE DISTRIBUTION OP LUTEIN, VITAMIN A ALCOHOL AND VITAMIN A
ESTER IN FRACTIONS OP CHICKEN PLASMA OBTAINED BY AMMONIUM
- - . ...
SULPHATE FRACTIONATION
Constituent
(NH4)2S04
Fraction Conoentration Whole plasma
oono. r/100 ml•
recovery
% . %
0 Whole plasma 100.2
25 Supernatant 97.9 97 .6
Precipitate 3.4 3.4
Lutein
Total 101.3 101.0
5Q Supernatant 61.6 61.4
Precipitate 33.8 33.6
Total 95.4 95.0
0 Whole plasma 56.5
25 Supernatant 50.0 90.1
Precipitate 6.4 11.5
Vitamin A Total 56.4 101.9
alcohol
50 Supernatant 23.7 42.7
Precipitate 34.2 61.6
Total 67*9 104.3
0 Whole plasma 248
25 Supernatant 178 71.7
Preoipltate 34.2 13.8
Vitamin A Total 212.2 85 .6
ester
50 ' Supernatant 59 .9 24.1
Precipitate 98.9 ‘ 39.8
Total 158.8 63.9
- ✓
62
Prom this summary, it can be seen that almost two-
thirds of the vitamin A alcohol is associated with the
proteins precipitating at half saturation with ammonium
sulphate, whereas only a third of the lutein is associated
with the same protein precipitate. The results with the
vitamin A ester are difficult to interpret, due to the
large losses, but they suggest that the distribution of
the- ester is not identical to that of the free vitamin.
In addition to the experiments on chicken plasma,
Samples of beef and hog plasma were similarly fractionated*
Although no difference obtains at quarter saturation, a
variation In association was observed between the p-caro
tene and lutein on the one hand, and vitamin A alcohol on
the other hand when the proteins were precipitated at
half saturation. In this latter ease, the vitamin A
alcohol appeared to be associated with proteins whieh
precipitate to a greater extent at half saturation than
did either p-carotene or lutein. These results appear in
Table VI.
TABLE VI
DISTRIBUTION OP p-CAROTENE, LUTEIN, AND VITAMIN A ALCOHOL IN FRACTIONS
OF BEEF AND HOG PLASMA OBTAINED BT AMMONIUM SULPHATE FRACTIONATION
(nh4)2so4
oone.
%
Fraction
Beef
Experiment number 15
t/100 ml. % recovered
Hog
Experiment number 16
y/100 ml. f o recovered
{3-Carotene
© Whole plasma 689
_
25 Supernatant 646 93,6
m
Precipitate 5.7 0.5
m
50 Supernatant 499 72.4
-
Precipitate 177 25.7 -
.
Lutein
0 Whole plasma 12.0
25 Supernatant 14.1 117.0 -
Precipitate 0.0 0.0
«•
50 Supernatant 10.9 90*8 -
Precipitate 3.5 29.1
«»
Vitamin A aloohol
'
0 Whole plasma 28.4 22.0
25 Supernatant - 27,5 96.9 21.3 96.7
Precipitate 0 .0 0 .0 0.0 0.0
50 Supernatant 17.9 63.0 12.8 58,2
Preoipltate 11.4 40.1 9.0 40*9
O
U>
6V
The plasma samples of several normal chickens were
subjected to a modified fractionation procedure. The
plasma was first dialyzed against distilled water in the
cold, and after centrifuging off any precipitate, the
supernatant was fractionated by means of quarter and half
saturation with ammonium sulphate. These results appear
in Table VII.
TABLE VII
DISTRIBUTION OP LUTEIN, VITAMIN A ALCOHOL AND VITAMIN A ESTER IN FRACTIONS OBTAINED BY DIALYSIS
FOLLOWED BY AMMONIUM SULPHATE FRACTIONATION OF NORMAL CHICKEN PLASMA
(NH4)2S04
cono.
—
Lutein Vitamin A alcohol Vitamin A ester
Fraction
Experiment number
%
17 18 19 17 18 19 17 18 19 i/
r/i©o ml.
0 Whole plasma 386 451 84.9 42.7 50.8 46.1
m-
-
185
Dialysis
preoipitate 5.7 34.4 8.4 6 .9 3.3 6.1 - 110
Dialysis
supernatant 372 - 80.1 - «e
25.1
«» m e
68,5
26 Supernatant
-
AAA
T Z 7 72.0 - 45.6 23.3
m m
41.7
Precipitate - 17.3 5.2 - -
m
-
- -
50 Supernatant' 252 308 51.0 24.2 29.4 11.3 -
«■»
14.0
Preoipitate 6.8
i H
I H
I ^
23.6 16.5 17.3 13.8
«* «D
33.7
Received an oral supplement of 66 mg* vitamin A ester 6 hours before analysis .
vn
Whereas no more than a fifth of either lutein or
vitamin A alcohol was found in the precipitate brought down
by dialysis, approximately three-fifths of the vitamin A
ester was found associated with this precipitate. It was
therefore felt advisable to turn to dialysis as a means of
studying the distribution of these components in plasma.
Dialysis. Table VIII gives the results of several
experiments on dialyzed chicken plasma.
TABUB VIII
DISTRIBUTION OP LUTEIN, VITAMIN A ALCCHOL AND VITAMIN A ESTER IN CHICKEN PLASMA AS DETERMINED BT DIALYSIS
Experiment
number
Lutein Vitamin A alcohol Vitamin A ester
Whole Dialyied plasma
_W'°^e Dialysed plasma
Vihole
plasma
Dlalyied plasma
piasma Supernatant Precipitate p eama Supernatant Preoipitate
Supernatant Frecipitate
r/loo ml.
Previously on a oarotenold-Ie* diet and given luteln-vltamin A solution 6-8 hours before sacrificing
20
21
22
168
120
84.0
154 17.8
97.2 6.4
69.7 2.7
41.1 56.8 7.5
46 A 10.6 0.0
60.8 26.1 4.6
217
126
106
22.2
7.4
10.5
84.1
50.6
25.8
Per oent recovery 81.0 7 .0 62.8 8.6 8.9 51.4
Previously on a earotenold-loar diet and given vitamin A solution 6-8 hours before sacrificing
25 0.0 0.0 0.0 45,1 26.4 0 JO 101 49.0 18.0
Per cent reoevery 68.9 0.0 48.5 17 .8
Previoualy on a nomal diet and given lutein-vitamin A solution 6-8 hours before saorlflolng
24 84.9 80.1 8.4 46.1 26.1 6.1 186 68.6 110
26 540 296 21.0 17 .6 19.4 0,0 66.6 52.4 19.4
26 500 248 59.8 41.0 24.6 4.8 77 A 6.8 31.9
Per cent recovery 86.0
9*6
66.0 10.4 29.8 49.2
Previously on a normal diet and given no supplement
27 572 252 5.7 42.7 40.7 6.9
_
28 461 444 34.4 60.8 46.6 3.5 - - -
Per cent reoovery 84.6 4.9 92.4 10.9
Average per oent reoovery 84.5 7.0 67.0 8.6 21.4 56.6
ON
“> J
Although interpretation of these data is hampered by losses
in.both vitamin A alcohol and vitamin A ester, they are
strongly suggestive that vitamin A ester is associated with
a fraction which precipitates more readily on dialysis than
does the fraction carrying vitamin A alcohol* This is
based on the fact that in those experiments where the
recovery of vitamin A alcohol was essentially complete, no
more than of the total vitamin A alcohol was associated
with the precipitate, whereas the opposite effect was seen
with vitamin A ester, in that no less than 18$ of the total
ester was associated with this precipitate, regardless of
the recovery. The averages of all of these experiments
(bottom row, Table VIII) shows the marked difference in
the percentile distribution*
Lutein, even more than vitamin A alcohol, appears
to be associated with a more soluble plasma protein frac
tion* In addition, no earotenolds were present in the i j .
or 3$% acetone fractions, indicating that esters of lutein
were not present six to eight hours after the administration
of lutein.
Similar studies on the distribution of both forms
of vitamin A in rat plasma following dialysis were carried
out. These are presented in Table IX.
69
TABLE IX
DISTRIBUTION OP VITAMIN A ALCOHOL AND VITAMIN A ESTER IN RAT
PLASMA FRACTIONS OBTAINED BY DIALYSIS
Expt,
no •
Fraction
analyzed
Vitamin A alcohol Vitamin A ester
ml • i * whole r/lOO ml • % whole
plasma plasma
No supplement
29 Whole plasma 21 .5
Dialyeed plasma
Supernatant 13 *6
Preoipitate 0*0
63 *3
0.0
0.0
0.0
0.0
Supplemented with vitamin A ester
30 Whole plasma 85.0
Dialyzed plasma
Supernatant 24.5
Precipitate 33.3
31 Whole plasma 72.4
Dialyzed plasma
Supernatant 46.1
Precipitate 14.2
Average recovery of
experiments number
30 and 31.
Dialyzed plasma
Supernatant
Precipitate
28.8
39 .2
63 .6
19.6
44.9
30 .3
571
6.9
256
31©
22 .2
98.4
1.2
44.8
7.2
31.7
3.3
40.2
70
These male rats were given 16,000 jig, of vitamin A
eater three to four hours before dialysis of the plasma.
Although the recoveries were quite poor, variations in
the distribution of the two forms of the vitamin were
observed. Whereas of the vitamin A aleohol was present
in the plasma supernatant after dialysis, only 3*3% of the
ester could be found in this fraction.
Rat Liver Fractionation
Schnelder-Hogeboom procedure, The initial pro
cedure used for the fractionation of rat liver homogenates
was that described by Hogeboom, Schneider and Palade (109),
which utilizes a 0*88 M sucrose solution as the homogen
izing medium. The results of these experiments are pre
sented in Table X,
TAB 12 X
DISTRIBUTION OF VITAMIN A ALCOHOL AND VITAMIN A ESTER IN FRACTIONS OF RAT LIVER HOMOQSNATES
PREPARED IN HYPERTONIC SDCROSE SOLOTIONS AND OBTAINED BY DIFFERENTIAL CENTRIFUGATION
Experiment
number
Vitamin A eater
Total
raoOT.
Vitamin A aloohol
Total
rseov.
l/gm. Whole Hear % Dlatrlbutlon 1/
r/s». Whole liver % Dlatrlbutlon 1/
A B C D E B C D E A B C D B B C D B
52 2060 1091 821 349 484 36.3 15.4 2261 184 176 99.1 564 53.1 29,9 164 331.3
SS 16.9 7.7 1.7 6.3 49.0 10.8 40.1 15.7
- - - -
54 38 4 23.6 6.4 9 JO 60.5 16 A 23.1 39.0 - ' - -
56 244 223 69.5 214 44.0 13.7 42 4 5064 13,2 2.7 0 ft 3/) 47 4 0.0 52.6 6.7
S6 788 215 56.1 192 464 12.1 41.4 463,1 8.6 4.4 1.1 5.3 40.7 104 48.9 104
ST - 29.3 6.3 4.8 744 13.5 4.6 394 - - - -
S8 171 185 132 28 4 118 394 28 4 6.1 25 .6 4634 6.6 2.4 3.1 0.4 1.8 314 40 4 34 234 7.7
39 229 32.7 23.3 44.2 60 4 20.3 144 27.5 37 .8 181.0 5.2 0.9 0/> 0.6 ' 1.7 28.1 0.0 18.7 53.1 34
40 164 17.3 5.4 29.4 35 A 194 64 33.6 40.4 87 4 2.9 0.0 1/1 0.8 0/) 0/) 55.6 444 0.0 14
A. Whole henogenate D. Ultoohondrla
B. Cream E. Nuolal
C. Supernatant and mtoroaomes i/ Expreaaed ag total reoovery * 100 %
-0
H
I 72 i
i
These fractions were obtained with the aid of an
; International centrifuge, and due to the limitations of j
I 1
i this instrument with respect to the gravitational fields j
I I
| produced by it, the supernatant and microsomal fractions
i
1 i
could not be separated, and were therefore analyzed to- j
gather as shown in Figure I. The rather high values found j
i
in the nuclear fractions can be attributed to the poor !
j disruptive powers of the glass pestle, used for these
i
homogenizations. These experiments indicated the presence
of an additional fraction in liver homogenates, namely,
i
| the material which migrated centripetally during the j
! various centrifugations. For simplicity, this material j
will hereafter be referred to as the ”cream” fraction,
although its exact composition is still unknown. A great j
j deal of difficulty was encountered initially, in trying
to relate this cream fraction to one of the conventional
fractions found in liver homogenates. This material was
quite viscous at the low temperatures used during centri- j
[
fugation, and it became apparent that, during decantation, !
j
contamination by this fraction was practically unavoidable.
i ' I
j However, the importance of this contamination, with respect j
j r
j to vitamin A distribution, was not appreciated until
■ attempts were made to determine the vitamin A content of
|the cream fraction. In experiments 38, 39, and IfO (Table X)
73
the combined supernatant and microsomal fractions were
centrifuged for a considerable time, following which the
centrifuge tubes were immediately frozen in a dry iee-
bath, and then cut into an upper and lower fraction.
Analysis of these fractions indicated that the upper
section, which presumably included the eream fraction,
contained considerable quantities of vitamin A,
Partial modified fractionation. Before the eream
fraction could be quantitatively separated from the
remaining fractions of the liver homogenates a centrifuge
giving rise to considerably higher gravitational forces
during centrifugation was required. With the availability
of the Spinco ultracentrifuge, several partial fractiona
tions were carried out using the modified procedure. The
modification involved layering of the homogenates, prepared
in isotonic sucrose solutions, under a 0.125 M sucrose
solution. These were then centrifuged at maximal centrifu
gal forces (105»000 x _g) for one to two hours, and the
cream layer was then carefully removed with a syringe. The
supernatant fraction was decanted from the particulate
residue, yielding three distinct fractions which were
analyzed separately. Prom the results of these experiments,
which appear in Table XI, it can be readily seen that the
cream fraction contained most of the vitamin A ester stored
in the liver.
TABLE XI
PARTIAL FRACTIONATION OF RAT LIVER HOMOGBNATES PREPARED IN ISOTONIC SUCROSB SOLOTICH
Fraction
analysed
Vitamin A ester Vitamin A alcohol
* * *
Experiment number
41 42 45 44a 44b 44c 44d 41 42 45 44a 44b 44o 44d
Amount present. r/gn.
Whole homogenate 106 221 147 268 268 258 268 2.7 1.7 1.6 5.8 5.8 S.8 5.8
Cream 74.6 176 106 148 146 166 166 0.7 0.7 0.7 1.7 0.9 1.5 1.6
Supernatant 9.6 S.0 9.5 25.0 24.2 51.6 29.1 2.6 0.0 0.6 1.0 1.5 1.6 0.9
Residue 17.6 7.6 18.0 60.1 71.0 49.4 85 A 4.6 0.7 0.9 1.6 1.7 1.4 1.6
Total recovered 101.6 166.6 152.5 251.1 240.2 247.0 268.5 7.9 1.4 2.1 4.5 5.9 4.5 4.0
% reoovery 95.8 84.4 90.0 89.6 95.1 95.6 104.0 295.0 82 A 151.0 115.0 102.7 115.0 106.5
Percentage distribution 1 /
Cream 75.4 95 A 79.6 64.0 60.4 67.1 68.2 8.9 50.0 55.4 59.6 25.1 50.2 57 Jb
Supernatant 9.4 1.6 7.0 9.9 10.1 12.8 10.8 52.9 0.0 25.8 25.5 55.4 57 A 22.6
Residue 17 £ 4.0 15.6 26.0 29.6 20.0 51.0 58.2 60.0 42.9 57.2 45.6 52.5 40 J O
l/ Expressed as total recovery ■ 100??.
vn
One© it was apparent that the cream fraction
contained the majority of the vitamin A ester, it was
thought advisable to determine whether or not the ester
was associated with material which floated only in solutions
with a density equal to or greater than that of a 0.12f> M
sucrose solution. Experiment I | i | . was undertaken, in which
the liver homogenate, prepared in a 0.88 M sucrose so
lution, was centrifuged for one hour at 105,000 x j a j , after
being layered under various solutions of decreasing
density* The solutions used were Q.^, 0*22 and 0*11 M
sucrose, and distilled water* These tubes were then
inspected for light scattering regions and for material
fluorescing under ultraviolet light. As the density of
the upper phase decreased, a more pronounced layer of
light scattering material was observed at the boundary
between the eream and supernatant fractions, but there
was no appearance of fluorescent material at this boundary*
When analyzed, the results were consistent enough to
indicate that the vitamin A ester was associated with a
fraction that migrated centripetally, not only in a 0.88 M
sucrose solution, but also in distilled water. These
results are schematically represented in Figure *>•
77
FLUORESCENT REGION
LIGHT-SCATTERING' AREA
W S / / / / / A
CREAM
SUPERNATANT-
RESIDUE
LAYERED UNDER: 0-44M 0 22M O HM DISTILLED
SUCROSE SUCROSE SUCROSE WATER
FIGURE 5. A Schematic Representation of the Light
Scattering and' Fluorescent Regions Resulting from the
Centrifugation of a Rat Liver Homogenate Prepared in 0*
Sucrose Solution and Layered Under Various Lower U&ftsity
Solutions,
78
gomplete modified fract1onatlon. Once a method for
removing the cream fraction quantitatively had been de
veloped, i.e., by layering the homogenate under a lower
density solution, a complete fractionation of rat liver
homogenates was undertaken, as described in Figure 2.
This fractionation gave, in addition to the cell fractions
previously described in the literature (128), the centripe-
tally migrating, or cream fraction. The results of these
experiments are presented in Table XII. On the average,
over 86# of the recovered vitamin A ester was present in
the cream fraction. While half of the vitamin A alcohol
was also present in this fraction, a considerable per
centage was found in the supernatant and microsomal
fractions•
TABLE XII
INTRACELLULAR DISTRIBUTION OP VITAMIN A ESTER AND VITAMIN A ALCOHOL IN RAT LIVER HOMOGENATES
Fraotion
Vitamin A ester Vitamin A aloohol
Average
e/a I?
Experiment number
45 46 47 48 49 Average 46 46 47 48 49 Average
Amount present, Y/ga.
Whole homogenate 606 416 460 300 98.0 376 7.7 6.5 2.8 6.7 3.8 5.3 71.0
Cream 357 261 358 248 88.3 262 2.3 1.0 1.8 4.4 1.9 2.3 114
Supernatant 63.1 5.7 2.8 5.8 1.6 13.8 1.6 1.4 0,4 0.9 0.6 0.9 15 .5
Mlorosomes 24.0 31.1 3.2 4.5 1.9 12.9 0.9 1.2 0.5 0.7 0.4 0.7 18.4
Mitochondria 7.2 13.2 0.7 1.1 0.7 4.6 0.5 0.5 0.0 0.6 0.0 0.3 16.3
Nuolel 25.1 23.1 2.0 1.2 0.8 10.4 0.5 0.3 0.0 0.2 0.0 0.2 52.0
Total reoovered 466.4 334.1 366.7 260.6 93.3 303.7 6.7 4.4 2.7 6.8 2.8 4.4
< recovered 77.0 80.4 79.6 86.7 95.2 81.0 74.0 80.0 104.0 101.7 73.6 83.1
Percentage distributionH
Cream 76.0 78.0 97.6 95.2 94.6 66.4 40.3 22.7 66.7 64.6 67 & 62.2
Supernatant 11.4 1.7 0.8 2.2 1.7 4.5 26.3 31.8 14.8 13.2 17,8 20.6
Microsomes 5.2 9.3 0.9 1.7 2.0 4.3 16.8 27.3 18.6 10.3 14.3 15.9
Mitochondria 1.5 4.0 0.2 0.4 0.8 1.6 8.8 11A 0.0 8.8 0.0 6.8
Nuclei 5.4 6.9 0.6 0.5 0.9 3.4 8.8 6.9 0.0 2.9 0.0 4.6
J,/ Vitamin A eater:vitamln A alcohol ratio.
2f Expressed as total recovery • 100$ •
-4
vO
80
Beef Liver Fractionation
Fractionation of beef liver homogenates, by means
\
of the modified procedure outlined in Figure i f . , was
attempted, in order to compare these results with those
of rat liver, for beef liver contains, in addition to
vitamin A, ^ -carotene, which is absent from rat liver*
Attempts were also made to gain further insight into the
mechanism of association between the pigments and the
liver tissue by means of various extraction and solubiliza
tion procedures.
Distribution* The first series of experiments were
carried out on beef liver which had been purchased at
various local markets, with an unknown period of time
elapsing between the time of slaughter and fractionation.
The results are presented in Table XIIII They Indicated
that a very similar relationship exists between the
distribution of vitamin A ester and alcohol in both beef
and rat livers. The p-carotene was found widely distribu
ted In the particulate fractions*
TABES XIII
INTRACELLULAR DISTRIBUTION OF p-CAROTENE, VITAMIN A ESTER AND VITAMIN A ALCOHOL
IN BEEF LIVER HOMOGENATES FRCM STORED LIVERS
Fraction
0-Carotene Vitamin A ester Vitamin A alcohol
Experiment number
50 51 52 63 60 51 52 63 60 61 52 53
Amount present, y/gn.
Total homog. 10,2 32.5 4.8 16.1 117 188 42 * 2 138 0,8 1.2 2.0 2.7
Cream 3.1 5.0 0.9 4.1 80.5 143 38.9 125 0.5 0.5 0.5 0.3
Supernatant 1.2 1.5 0.1 0.2 4.0 2.4 0.0 1.0 0.4 0.2 0.3 0.2
Mloro8ome8 2 J L 11.6 1.3 2.4 3.4 15 .9 1.6 3.1 0.0 0.1 0.3 0.5
Mitochondria 1.5 5.6 1.4 5.0 0.9 5.4 0.6 3.8 0.0 0.2 0,0 0.6
Nuclei 0.4 3.5 0.4 3.8 1.0 4.8 0.8 3.8 0.0 0.3 0.0 0.4
Total reoov. 8.4 27.2 4.1 15.6 89.8 171.5 41.9 136.7 0.9 1.3 1.1 2.0
% recovered 82.4 83.6 85.4 96.4 77.0 91.3 99.4 99.0 112 . 5 - 108.1 55.1 74.1
Percentage distribution l/ ‘
Cream 36 .9 18.4 22.0 26.4 89.6 83.2 92.7 91.5 65.6 38.5 45 A 16.0
Supernatant 14.3 5.5 2 . 4 1.3 4.5 1.4 0.0 0.7 44.0 15 A 27.3 10.0
Miorosomss 26.1 42.6 31.8 15.5 3.8 9.3 3.8 2.3 0.0 7.7 27.3 25.0
Mitochondria 17.8 20.6 34.2' 32.3 1.0 3.1 1.4 2.8 0.0 15.4 0.0 30.0
Nuolei 4.8 12 .9 9.8 24.5 1.1 2.8 1.9 2.8 0,0 23.1 0.0 20.0
l/ Expressed on the basis of total reoovery ■ 10C$. CO
H4
82
When fresh livers obtained from an abattoir were
fractionated, the results (Table XIV) were very similar to
those obtained from the fractionation of stored liver* As
will be shown later, however, a difference does exist
between fresh and stored liver, not In the distribution,
but in the relative extractability of the p-carotene and
vitamin A*
TABLE XIV
INTRACELLULAR DISTRIBUTION OP p-CAROTENE, VITAMIN A ESTER AND VITAMIN A ALCOHOL
IN REEP LIVER HOMOGENATES FROM FRESH LIVERS
Fraction
p-Carotene Vitamin A ester Vitamin A aloohol
Experiment number
54 55 56 57 54 65 56 57 54 65 56 57
Amount present, y/gn.
Total homog. 4.8 8.7 8.4 14.0 144 24.5 128 218 0.6 1.4 18.8 8.2
Cream 1.0 0.5 1.6 6.3 120 18.4 115 216 0.2 0.5 11.4 3.6
Supernatant 0.1 0.0 0.0 0.1 1.0 0.0 0.1 1.0 0.1 0.1 0.1 0.1
Mlorosomee 1.1 1.0 1.6 2.9 4.6 0.5 1.4 2.3 0.2 0.1 0.3 0.6
Mltoohondrla 1.0 4.9 2.5 2.0 2.0 0.3 0.5 0.8 0.0 0,2 0.3 0.5
Nuolel O.S 1.2 0.9 1.3 7.3 0.8 1.4 0.9 • 0.1 0,1 0.3 0,2
Total reeov. 5.7 7.6 6.5 12.6 154 ,9 20.0 118.4 221.0 0.6 1.0 12.4 4.9
% reoovered 77 .1 87 .3 77.4 90.0 93.6 81.6 92 .5 101.5 100.0 71,4 66.0 59.8
Percentage distribution l /
Cream 27.0 6.6 24.6 50.0 89.0 92 .0 97.0 97 .7 33.3 50.0 92.0 71.4
Supernatant 2.7 0.0 0.0 0.8 0.7 0.0 0.1 0.4 16.7 10.0 0.8 2.0
Mlorosomee
29.7 13.2 23.1 23.0 3.4 2.5 1.2 1.0 33.3 10.0 2.4 12.2
Mitochondria 27.0 64 .5 38.5 15.9 1.5 1.5 0.4 0.4 0.0 20 .0 2.4 10.2
Nuolel
13.5 15.8 13.8 10.3 5.4 4.0 1.2 0.4 16.7 10.0 2.4 4.1
l/ Expressed on the basis of total reoovery - 1009?.
8k
In order to make a more direct comparison between
fresh and stored beef liver, fresh livers were divided
into two portions, one being fractionated immediately,
and the other stored for a given length of time prior to
fractionation* Two such experiments are shown in Table XV,
Again, very little difference was observed in the dis
tribution of p -carotene or vitamin A, regardless of
whether the liver was fresh or had been stored at -25° or
5° c.
TARLE XV
INTRACBLLTTLAR DISTRIBUTION OP 0-CAROTENE, VITAMIN A ESTER AND VITAMIN A ALCOHOL
IN BEEF. LIVER HOMOGENATES BEFORE AND AFTER STORA® PGR 48 HOURS
Fraetlon
p-Carotene
Vitamin A ester Vitamin A aleohol
Experiment number
68 1 ! 69
S/ 58 59 58 69
Freeh Stored Freeh Stored Fresh Stored Fresh Stored Fresh Stored Fresh Stored
Amount present, r/ffft.
■
Total homog. 5.7 5 a 15.0 15.3 36.9 36.4 211 210 2.2 2.1 3.8 4.0
Cream 1.0 1.0 5.7 3.2 21.9 22.7 210 169 1.6 0.5 0.9 1.0
Supernatant 0 J O 0.1 0.1 0.0 0.3 0.1 0.6 0.4 0.0 0 J O o a o a
Mlerosomes 0.7 0.8 2.6 5.2 1.8 1.1 6.2 9.9 O a O a 1 a i a
Mitoohondrla 0.8 0.6 5.4 5.6 0,3 0.1 1.0 2 .0 0.0 0.0 0.4 o a
Nuolel 0.6 0.7 1.8 1.1 2.7 2.7 2a 3.7 Oa 0.6 0.5 o a
Total reeov. 5.1 Sa 11.6 11.0 27.0 26.7 219.9 185.0 2.3 1A s a 5.7
% reoovered 83.8 91 a 89.5 82.6 73.2 73.4 104.1 88.1 104.7 76^ 89.6 92 a
Percentage distribution
Cream 52.3 31.5 31.8 29.1 81.2 86.0 95.6 91.4 69.6 31.2 26 a 27.0
Supernatant 0.0 5.1 0.9 0.0 1.1 Oa 0.3 0 JS 0 JO 0.0 23 a 10 a
Miorosomes 22.6 26.0 22.4 29.1 6.7 4.1 2.8 6.4 21.7 si a 29.4 40 a
Mitoohondrla 25.8 18.7 29.5 51.8 1.1 0.4 0.5 1.1 0.0 0.0 11.7 10.8
Nuolel 19.4 21.9 16.6 10.0 10.0 10.1 1 J) 2.0 8.7 37a 8.8 10.8
l/ Stored at -25° C.j homogenate not filtered throu£i flannelette.
2/ Stored at 5° C.
7> / Expressed on the basis of total reoovery ” 100$.
OD
vn
8 6
The results of the distribution of j 3-carotene,
vitamin A ester and vitamin A alcohol are summarized in
Table XVI*
TABLE XVI
SUMMARY OF THE INTRACELLULAR DISTRIBUTION OF jP-GAROTENB, VITAMIN A ESTER
AND VITAMIN A ALCOHOL IN BEEF LIVER HOMOGENATES
Fraction
p-Carotene
Vitamin A ester Vitamin A alcohol
Fresh Stored Fresh Stored Fresh Stored
Amount present, Y/gpu
Total homogenate 8.8 13.4 127.1 121.9 5 *6 2.1
Cream 2.4 2.9 116.9 96.5 3.0 0.6
Supernatant 0.1 0.5 0.5 > 1.3 0.2 0.2
Miorosomes 1.6 3.6 2*8 5.8 0,4 0.3
Mitoohondrla 2.4 2.9 0.8 2.1 0.2 0.2
Nuolel 1.0 1.6 2.5 2.8 0.2 0.2
Total recovered 715 11.5 123.5 108.5 4.0 1.5
% recovered 85.4 85.8 97.1 89.0 69.0 71.4
Percentage distribution
l/
Cream 52 .0 25.2 94.6 88.9 75.0 40.0
Supernatant 1.3 4.3 0.4 1.2 5.0 13.3
Miorosomes 21.3 31.3 2.3 5.3 10.0 20.0
Mitoohondrla 52.0 25.2 0.6 1.9 5.0 13.3
Nuolel 13.3 15.9 2.0 2.6 5.0 13.3
l/ Expressed oa the basis of total reoovery « 10($.
03
-0
Considerable amounts of £ -carotene, in both the
fresh and stored livers, were present in the particulate
fractions. Although the possibility cannot be excluded
that these fractions were not pure and may have been
mutually contaminated, the gross appearance of these
particulate residues, after centrifugal fractionation,
indicated a fairly sharp separation of the fractions.
Vitamin A ester, as previously described for rat liver,
was found particularly concentrated in the cream fraction,
in both the fresh and stored liver samples, when the
results were expressed on the basis of total recovery. In
those experiments where the per cent recovered was low,
as great a percentage of ester was found In the cream
fraction as was found in the experiments in which satisfac
tory recovery was obtained. The data for vitamin A alcohol
were difficult to evaluate, as only small quantities were
present in these livers, and in most cases the vitamin
present was only detectable at a range where the analytical
reliability Is quite low.
Ether extraction. Although the actual structural
and surface properties of the various particulate com
ponents of liver cells are still unknown, a series of
investigations were conducted in the hope of learning more
about these properties through the relationship between
p -carotene and these particulate fractions* It seemed
difficult to ascribe the high concentrations of ys-carotene
in these fractions t© contamination, for then the question
arose as to the source of the contamination* If the cream
fraction was the contaminant, one would expeet to have
found levels of vitamin A ester associated with the
particulate fractions higher than the traces that were
found. If this association betweenp -carotene and the.
tissue was merely due to adsorption of the carotene on the
surface of the partieulates, then extraction of the
isolated fractions with a non-polar solvent might rupture
this association* For this purpose, the plasma extraction
method of Lever and MacLean (108), using a cold diethyl
ether extraction, was adapted to the extraction of liver
tissue* Using this procedure, the vitamin A ester appears
to have been readily extracted with diethyl ether, for In
the cream fraction, where the vitamin A ester is concentra
ted, at least two-thirds of the vitamin was extracted with
this solvent. The ye-carotene in this fraction was also
easily removed with diethyl ether, although not to the same
extent as the vitamin A ester. However, in the particulate
fractions, much lessyS-carotene could be extracted by this
method. Especially noticeable was the Inability of diethyl
ether to extract p > -carotene from the microsomal fraction
until or unless it had been stored for some time. This
behavior was not always evident in the mitochondrial or
nuclear fractions, although storage did seem to increase
the amount extracted. These results appear in Table XVII
TABLE XVII
ETHER EXTRACT I OH OP fWCAROTENE AND VITAMIN A ESTER FROM BEEF LIVER HOMOCENATBS OF FRESH (F) AND STORED (s) LIVER
Fraction
analysed
Expt. 6S-S Expt. 65-F Expt. 67-F Expt. 58-F Expt, 58-S Expt.59-F Expt. 59-S % Recovery B vs.A
A B A B A B A B A B A B A B Expt.
5S-S
Expt.
65-F
Expt.
S7-F
Expt,
58-F
, Expt
68-S
. Expt.
69-F
Expt.
59-S
p-Carotene in Y/gn,
Whole homogenate 16.1
-
8.7 0.6 14.0 6.9 3.7 0.4 3.5 0.7 13.0 1.0 13.3 1.7 6.9 49.4 10.8 20.0 7.7 12.8
Craa® 4.1 2.9 0.5 0.3 6.3 2.7 1.0 0.3 1.0 0,3 3.7 1.4 3.2 1.9 70.8 60.0 42.9 30.0 30.0 37 .8 69.5
Mierosomea 2.4 2.3 1.0 0.1 2.9 0.3 0.7' 0.0 0.8 0.2 2.6 0.2 3.2 2.5 96.7 10.0 10.3 0.0 25.0 7.7 78.1
Mitoohondri* 5.0 3.2 4.9 0.4 2.0 0.3 0.8 0.3 0.6 0.4 3.4 2.3 3.6 3.2 64.0 8 . 2 15.0 37.6 60.7 67.7 91A
Nuolei 3.8 1.7 1.2 0.1 1.3 0.9 0.6 0.2 0.7 0.2 1.8 0.3 1.1 O.E 44.8 8.3 69.4 33.3 28.6 16.7 45.5
Vitamin A ester in r/gn.
Whole homogenate 136 24.5 4.5 218 137 36.9 11.4 36.4 12.5 211 45.6 210 69.6 18.4 62.8 30.9 34.4 21.6 35.2
Cream 125 102 18.4 11.8 216 143 21.9 15 .3 22 . 7 18.2 210 . 136 169 143 81.6 64.1 66.3 69.9 80.4 64.7 84.6
Microsomes 3.1 3.5 0.5 0.1 2.3 2.1 1.8 0.3 1.1 0.1 6.2 0.8 9.9 7.9 113.0 20.0 91.4 16.7 9.1 12 .9 79.9
Mitoehondria 3.8 2.8 0.3 0.1 0.8 0.2 0.3 0.2 0.1 0.1 1.0 0.7 2.0 1.8 73.7 33.3 26.0 66.7 100.0 70.0 90.0
Nuclei 3.8 3.4 0.8 0.3 0.9 0.6 2.7 2.1 2.7 1.5 2.1 0.9 3.7 1.9 89.5 37.5 66.7 77 .8 56 .6 42 .9 61.4
A » Waring blendor extract,
B * Diethyl ether extract.
vO
H
92
Effect of salts on ether extraction. In several
experiments, the isolated fractions, suspended in distilled
water, were mixed with equal volumes of M potassium chloride
and then extracted with diethyl ether. Although this
procedure was originally used to solubilize proteins from
these fractions, the presence of the salt appeared to
have a definite effect in increasing the amount of pigments
extracted with diethyl ether. In Table XVIII are listed
the results of these experiments.
TABU m u
ETHER EXTRACTION OP p-CAROTENE AND VITAMIN A ESTER FROM BEEF LIVER HOMO GEN ATES OF FRESH (F) AND STORED (8)
LIVER BEFORE AND AFTER TREATMENT WITH II POTASSIUM CHLORIDE
Fraotion Expt* 56-F Expt. 58-F Expt. 68-S Expt • 59-F Expt. 59-S % Recovery, B . A
analysed
A B A B A B A B A B
Expt. Expt. Expt. Expt e fixpi ,
56-F 68-F 68-S 69-F 68-S
p-Carotene in r/gn.
Whole homogenate 8.7 1.4 5.7 0.6 5.5 0.7 15.0 1.5 15.5 2.3 16.1 16.2 20.0 10.0 17.5
Cream 0.5 0.5 1.0 0.5 1.0 0.3 5.7 1.0 3.2 2.0 100.0 50.0 30.0 27.0 62.6
Microsomea 1.0 - 0.7 0.2 0.8 0.4 2.6 0.5 5.2 2.0 - 28.6 50.0 11.5 62 .6
Mitochondria 4.9 5,7 0.8 0,5 0.6 0.5 3.4 1.5 5.6 2.8 75.4 37.5 50.0 44.2 80.0
Nuclei 1.2 0.6 0.6 0.2 0.7 0.5 1.8 0.7 1.1 2.7 50.0 35.3 42.8 58 .9 63.7
Vitamin A aster In v/gn.
Whole homogenate 24.5 10.8 56.9 13.4 56.4 8,6 211 62.7 210 77.8 44.0 56.4 23.4 29.7 57.1
Cream 18.4 15.5 21.9 17.0 22.7 17.7 210 160 169 161 83.0 77.6 77 .9 71.5 89.3
Microsomes 0.5 - 1.8 0.9 1.1 0.9 6.2 1.0 9.9 7 £ - 50.0 61.9 16.1 72.7
Mitoohondria 0.5 0.2 0.3 0.2 0.1 0.1 1.0 0.6 2.0 1.5 66.7 66.7 ioo jo 60.0 65.0
Nuolei 0.8 0.5 2.7 2.0 2.7 1.8 2.1 0.9 5.7 2.1 62.5 74.0 66.7 42.9 56.8
A • Earing blendor extract.
B • Diethyl ether extract after treatment with M KC1,
vO
*
Butanol extraction. Using the butanol extraction
procedure described by Harris et al. (122) on the mito
chondrial and microsomal fractions of beef liver homogenates,
it was found that essentially all the p-carotene and
vitamin A ester was present in the butanol phase, indi
cating that little or none of these components were
sufficiently tightly bound to resist the lipoprotein
disrupting effect of butanol in the procedure. The results
are presented in Table XIX*
TABLE XIX
BUTANOL EXTRACTION OP ^-CAROTENE AND VITAMIN A ESTER
FROM BEEF LIVER HOMOGENATES
Fraction
Experiment number
analyzed
56 ]/ 57 2/
Control j/ Experimental
y/m» Y/gn. %
Control
r/gm*
Experimental
r/a». %
p-Carotene
Mitochondria 2*5 0 *4 16.0 2.0 2.1 105.0
Microsomes
mm mm
2*9 2.0 69.0
Vitamin A ester
Mitochondria 0*5 0.3 60*0 0.6 0.6 100.0
Microsomes
-
2*0 1.7 85.0
3/ Extracted once with butanol •
2/ Extracted twice with butanol*
3/ Waring blend or extract*
Alkaline digestion* After digesting a microsomal
fraction in strong alkali, the material was acidified,
centrifuged, and the resultant supernatant and residue
were analyzed individually forp-carotene and vitamin A.
The results of this experiment are shown in Table XX*
Neither the p-carotene nor vitamin A ester could be brought
into solution by this procedure*
s TABLE XX
EFFECT OF ALKALINE DIGESTION ON THE DISTRIBUTION OF p-CAROTENE
AND VITAMIN A ESTER IN ISOLATED BEEF LIVER MICROSOMES
Expt* _________ p-Carotene Vitamin A ester
no* Control Residue Supernatant Control Residue Supernatant
Y/gpu y/gau ~ y/ga, j o r/ggu" Y/fflu j * v/gfl* $
52 1.3 0.8 61*5 0.2 15 «4 1.6 1*8 112.5 0.0 0*0
vO
98
Waring blendor homogenization. Recently, parti
culate fractions have been disrupted by homogenization in
a Waring blendor or similar high speed homogenizers. As
seen in Table XXI, this method did not effect a solu
bilization of either ^-carotene or vitamin A*
TABLE XXI
DEGREE OP SOLUBILIZATION OP {3-CAROTENE AND VITAMIN A ESTER FROM ISOLATED BEEP LIVER MITOCHONDRIA
i
..................
Method Expt.
^-Carotene
Vitamin A ester
no*
Control Residue Supernatant Control Residue Supernatant i
r/gm. r/gm. % 't/m* %
r/gm. r/gm. % r/gm. %
Equilibrate against M
KC1. “
Cent • at 5,000 x
55 4*9 4*6 93*9 0.3 6*1 0.3 0.2 66.7 0.1 35.3
Equilibrate against
dist • H2O.
Cent. at 5,000 x _g.
56
2.5 1.5 60.0 0.0 0.0 0.7 0.4 57.1 0.1 14.3
Homogenise for 2 min*
in Waring blendor •
Cent • at 500 x j>.
56 2.5 1.5 60.0 O.0 0.0 0.7 0.5 71.4 0.2 28.6
i
!
•
s O
v O
100
Hypertonic and hypotonic extractions. Another
procedure which has found some success involves disrupting
the particulate fractions by placing them in either
hypertonic or hypotonic solutions*. This procedure, using
isolated mitochondria, proved unsuccessful, as is seen in
Table XXI.
Nitrogen distribution. Table XXII lists the
distribution of total nitrogen in the various fractions
obtained from beef liver homogenates. Although the cream
fraction invariably contained some nitrogen, it cannot be
concluded that this fraction actually contains protein, as
contamination from the supernatant fraction, which contains
the majority of the tissue nitrogen, was almost unavoidable.
101
TABLE XXII
DISTRIBUTION OP NITROGEN IN BEEP LIVER HOMO SENATES
Fraction
Experiment number
analyzed
55 55 56 57 58 ±/
•
59
Presh Stored Presh Stored
-
Per cent of total recovered
Cream 9*9 1.8 6 .8
1
7.1 6.6 8.3 7.0
Supernatant 30*9 44*6 43 *2i
> 50.4
42.7 36.1 42 .5 45.3
Mieroeomes 16.4 23.0 25*4 27.9 23 .4 26 A 23.8 26.3
Mitochondria 20*2 17 *5 13*9 8.0 8.9 5.9 11.6 8.2
Nuolei 22*7 13*2 10*6 13 .6 17.9 25.0 13.8 13.2
Total .
recovery 2/ 90*8 88*4 88 *3 92.6 93 .9 94.7 97.0 100.0
1/ Witfe the exception of experiment 58, all homogenates were
filtered throng flannelette*
2/ Per cent of total homogenate*
University of Southern Coli&SCfa
DISCUSSION
The results of the fractionation of plasma by
different procedures not only indicated that the carote
ne id pigments and vitamin A were associated with the plasma
proteins, but that they were associated with different pro
tein fractions. Whether the association occurs by means
of adsorption or by chemical binding cannot be deduced at
this time, although Bull (129) states that:
"It is now believed that there is no funda
mental difference between adsorption and
chemical binding; identical types of forces
are operating."
On the basis of the results presented here, it is
proposed that these pigments are associated in plasma
with rather specific proteins. In studying the distribu
tion of the carotenolds and vitamin A in the various plasma
protein fractions obtained through salt fractionation or
dialysis, it is apparent that the vitamin A ester was in
variably associated with a.less soluble or more readily
precipitable protein fraction than either the.free vitamin
or any of the carotenoids. As the vitamin A ester was
usually found only in those animals that had been supple
mented with vitamin A and were still in the process of
! 103 |
jactive absorption, it is possible that this material was j
i j
istill present in the plasma in the ehylomieron-like parti- i
i 1
cles that enter the blood stream through the thoracic duct# j
This is very probable in chickens, for Kon and Thompson <
(130) have mentioned the much slower absorption of vitamin
A in chickens than in such animals as the rat# This would
I
then serve as an explanation for the solubility behavior
jof the ester, for the chylomicra have been shown to be
readily salted out of plasma, and in general have pro
perties similar to the plasma globulin fraction (131)«
1
I
This raises a very interesting point# Lutein
appears to be associated with the most soluble protein
i
fraction. If its method of absorption is similar to that j
I
of vitamin A, one might expect to find it in the same
jfraction that contains vitamin A ester# Instead its dis
tribution is the same regardless of whether the lutein had
been administered six to eight hours before analysis, or
had accumulated in the plasma through a normal diet. Thus,
it appears that lutein is absorbed directly in association ]
with the protein with which it is normally found in the ;
jcirculation, whereas vitamin A is absorbed by a different j
F ’ [
Imethod, and presumably must first be metabolized before it ;
I I
lean be found as part of the normal plasma vitamin A. The
[difference between the lutein and vitamin A may even ex- I
lo if
tend to the path of absorption, for if lutein is absorbed
in direct combination with its plasma protein acceptor it
would be more likely to occur by means of the portal route,
as has been postulated earlier (13, l i j . , 15# 1&), than by
the lymphatics, through which vitamin A is absorbed (3©.» 31
32).
Before continuing, it might be well to point out
the following facts. Vitamin A is normally found in plasma
in the free form, with little or no ester present. In
addition, plasma appears to maintain a rather steady level
of vitamin A alcohol (70* 7 i j - ) which might be due to the
saturation of a specific protein carrier. The ester form,
on the other hand, can reach exceedingly high concentra
tions during absorption, but soon returns to its normally
low level. In those conditions where an increase in plasma
vitamin A has been observed, such as nephrosis (132),'
steroid hormone treatment (133# 13^1 -» 135) and following
the administration of ethyl alcohol (136), the Increase
was due to the presence of additional vitamin A ester in
the plasma, and not to increased vitamin A alcohol. This
same behavior is not seen with the carotenoids. Even if
they are associated with specific protein acceptors in
plasma, their concentrations can be markedly increased by
dietary means, as opposed to the difficulty of increasing
1 0 5
the plasma vitamin A alcohol level (70)*
The results obtained by fractionating hog and beef
plasma indicate that vitamin A alcohol is associated in
both these species with protein fractions having similar
solubility characteristics. In the case of beef plasma*
the f-carotene appeared in a more soluble fraction than the
one containing vitamin A. Lutein, which is present in
beef plasma at a concentration of only 2% of that of
P-earotene, was apparently present due to its fortuitous
absorption, and not associated with a specific protein.
i
If it is associated with a specific protein, one might
expect to find higher concentrations In the plasma, due
to its preponderance over ^ -carotene in the diet, and to
the fact that these carotenoid specific protein acceptors
do not appear to limit to any great extent the concentration
of these pigments in plasma. Another possibility, however,
is that a specific acceptor protein does exist, but the
lutein Is rejected before It comes in contact with this
protein, possibly within the Intestinal well.
Due to the large losses, the results of the dialysis
of rat plasma are difficult to interpret. However, vitamin
A alcohol could be found In the precipitated protein frac
tion only during the period of active absorption of the
vitamin. The dialysis was also carried out during this
! 106
!period, at which time the alcohol concentration rises above j
1 its usual constant level (7i * - ) and may be an indication that
I
this added alcohol is not associated with the same protein
fraction in which the alcohol is normally carried.
That the large losses encountered during the di
alysis experiments may be due to an actual loss into the
dialysate and not merely to an oxidation or to a destruc-
; tion is suggested by the observations of Ray ^t al. (137)
j who reported that dialysis of lipoprotein preparations
resulted in the release of lipid material from the lipo
protein, This could be prevented by the addition of a
Mstabilizing factor” obtained from bovine or rabbit serum.
In addition, Roberts and Szego (138) claimed that other j
i
lipoidal substances In addition to estrogens dialyzed out
1 of ^-lipoprotein fractions, for the concentrated ether
I
extracts of the dialysate were of a distinct yellow hue.
With the finding that a difference exists in the
distribution of the carotenoids and vitamin A between the
plasma proteins, it became of Interest to investigate the
.possibility that this difference might also be found in
i j
!the storage of these components. Although there have been
J reports of the localization of vitamin A in the mitochondria
jof liver cells (96, 97, 98, 100, 101, 102, 103) none of
j
3 • ^
j these investigators made any attempt at separating the free i
107
and esterified forms of the vitamin.
The method chosen for fractionation of liver cells
was that of differential centrifugation, first introduced
by Bensley and Hoerr (139) and subsequently extended by
Claude (119, 120) and Schneider and Hogeboom (128). However,
before it could be applied to the investigation of the
distribution of vitamin A, the modification described in
the experimental section had to be introduced. During the
course of these studies a very similar procedure was
described by Chauveau and Clement (ll|.0) •
Very little is known about the composition of the
eentripetally-migrating or cream fraction which contains
almost all of the vitamin A ester found in the liver cell.
Hogeboom et al. (109) have observed that the number and
amount of lipid droplets that rose to the top of their
tubes during centrifugation of rat liver homogenates in
creased if the animals were fasted more than 18 hr., an
observation which correlates with the gross increase of
lipids in the livers of starved animals, Palade and
Claude (lij.1, llt2-) have attempted to correlate this centri-
petally-migrating material with the myelin body associated
with the G-olgi apparatus. They differentiated histologi
cally the small peripheral fat globules in rat liver epi
thelial cells of the order of magnitude of 0.5 to 1.0 jx in
diameter, which stain with neutral red, indicating phospho
lipids, from the larger centrally-located droplets, 1 to
in diameter which gave the characteristic staining re
actions of neutral fat. The droplets in the Kupffer cells
also appeared to he composed of phospholipid complexes.
When viewed in a microscope, and under certain conditions
these droplets and the Golgi apparatus appeared quite
similar. When the tissue was homogenized in a hypertonic
sucrose solution, these droplets would migrate eentri-
petally in a centrifugal field. No analysis of these
components was attempted.
Prom the results reported by Popper (82), it can be
concluded that the majority of the vitamin A fluorescence
observed in normal rat liver was associated with the
smaller peripheral fat droplets, these being apparently
phospholipid complexes; In addition, some fluorescence
was associated with the larger neutral fat droplets and
in the cytoplasm. ,
Chauveau and his associates (143) were unable to
find any protein nitrogen in their eentripetally migrating
fractions from rat liver homogenates. As reported here,
a small but nevertheless significant amount of nitrogen -
was always found in the cream fraction of beef liver
homogenates. As the possibility of contamination from the
109
supernatant fraction cannot be neglected, the question of
whether protein is actually present In the cream fractions
must await further investigations.
From the data of Collins (105)* one can conclude
that he also has observed the majority of vitamin A ester
in a cream fraction, even though he did not separate his
supernatant fraction into fractions comparable to the
supernatant and cream fractions reported here.
Combining the observations of these previous in
vestigators (82, 105, lip-* llj. 2, 1^3) with the results re
ported here, they would seem to indicate that vitamin A
ester is primarily associated in the liver cell with a
phospholipid complex. This possibility is based on the
assumption that the Isolated fractions exist preformed
within the liver cell, for otherwise, the distribution
observed could be due to artifacts developed In the
fractionation procedure. Evidence that these fractions may
not exist preformed has been presented by Porter (lijlp who
has concluded that the fraction referred to here as the
microsomal fraction exists within the liver cell as a
fine reticulum of strands, which vesiculates into parti
cles corresponding to microsomes only during the actual
isolation procedure. Whether this association between
vitamin A ester and the cream fraction involves any of the
110
liver proteins remains to be determined*
The presence of vitamin A in the isolated mito
chondria of rat livers as suggested by previous investi
gators (101, 102, 103> 10£) could not be confirmed (1 1|5).
Their findings appear to be due to contamination from the
cream fraction which, as reported here, was observed in
those fractionations carried out before the introduction
of the modified procedure. Unless precautions are taken
during the initial steps of the fractionation, the parti
culate material, especially the nuclear and mitochondrial
fractions, can be readily contaminated*
That a difference in distribution of the alcohol and
ester form of vitamin A exists in rat liver homogenates can
be seen from both the percentage distribution and the
ratio of these two components in the isolated cell frac
tions, shown in Table XII. Whereas the ester is almost
quantitatively located in the cream fraction, the alcohol
in addition to being present in this fraction is also
present in appreciable amounts in the supernatant and
microsomal fractions*
The presence of vitamin A alcohol in the same
fraction as that in which the ester is concentrated is
similar to the finding of vitamin A alcohol in the preci
pitate obtained by dialyzing rat plasma during the period
Ill
«
of active absorption following vitamin A supplementation.
This cream fraction may not be the normal storage depot
for the free vitamin. Another possibility is that its
presence is due only to a transitory association caused
by the action of vitamin A esterase on the vitamin A ester
in the cream fraction, the free alcohol formed awaiting
transfer to another fraction for eventual entrance into the
circulation and utilization. It is possible that the same
protein is associated with vitamin A alcohol in plasma and
liver.
The eoncept of protein and vitamin A complexes in
liver was first suggested by Abels jet .al. (9^) and by
Baumann and his associates (95)* More recently Glover*
Goodwin and Morton (71) suggested that vitamin A alcohol
was dispersed iri an aqueous medium as a protein complex,
while the ester was in the fat droplets. These results
reported here agree well with their suggestions. Further
more, it has been observed (T^i-) that following the oral
administration of large quantities ©f vitamin A, the rat
liver tends to resist an increase in vitamin A alcohol
even while the vitamin A ester continues to be deposited.
It seems possible that this resistance Is due to the
limited ability of the liver to form a protein complex
with further vitamin A alcohol.
112
Somewhat similar results were obtained from the
fraetionation of beef liver. The ester appeared to be
located exclusively in the cream fraction in both fresh and
stored liver samples. In the case of the free vitamin, a
difference was observed between its distribution in the
fresh and stored beef liver. In the fresh liver, the
alcohol was found concentrated in the cream fraction,
whereas, in the stored samples, it appeared to be much
more widely distributed. The concentrations found of the
free vitamin however, were very low and therefore were less
reliable than the ester values.
The p-carotene was found distributed almost Uni
formly throughout the particulate fractions and cream
fraction of beef liver homogenates, in both fresh and
stored livers. At first glance, it might appear that this
distribution is due merely to a rather non-specific ad
sorption or absorption phenomenon, such as the one postu
lated by Weiss (II4 . 6) involving the ionization of those
hydrocarbons with conjugated double bond systems and
subsequent formation of salts with tissue proteins. This
view of a non-specific phenomenon must be modified however,
for it appears that the pigment will only associate with
cell fractions that eontain some particulate component, as
little p -carotene was found in the supernatant fraction,
113
which contains the majority of the cellular protein, but no
known structural components. This is based on the as
sumption that the lipid particles which give rise to at
least a part of the cream fraction do not coalesce and lose
their structural integrity in the.isolated fraction, as it
is already known that the particulate fractions, may be
somewhat altered but still maintain some degree of structur
al Integrity when Isolated. Another objection to viewing
the distribution as being due to a non-specific type phe
nomenon is that it would indicate that the fractions con
taining the pigment have a common characteristic to form
this function. This however, cannot be reconciled with
the biochemical and structural heterogeneity which these
fractions display between themselves (llj.7, II4 . 6) •
The ether extraction procedure employed with the
beef liver fractions has been shown to extract the added
lipids from plasma in certain hyperlipemic conditions,
without affecting the normal lipid content (108). In the
present report, the vitamin A ester was readily extracted
from the eream fraction, where it is concentrated. The
^-carotene in this fraction was also readily extracted
although not to the extent that the vitamin A ester was.
However, in the particulate fractions (especially in the
microsomal fraction), theyS-carotene could not be readily
l l l j .
extracted, unless the tissue had been modified by storing
it or adding a potassium chloride solution just before
extracting with diethyl ether. These two procedures would
usually render the £ -carotene more extraetable In all the
fractions in which it was found.
This apparent laek of specificity in the distribution
of ^-carotene in beef liver homogenates may be correlated
i
in some manner with the fact that the liver apparently does
* ,
not serve as a storage depot for the various carotenoids
even in those species capable of absorbing them. When a
high degree of specificity is observed, as in the case of
the vitamin A ester concentrated in the cream fraction,
then the liver or tissue involved apparently does serve as
a storage depot. In beef, the corpus luteum and plasma
can be considered the main storage depots for carotenoids
rather' than the liver.
This lack of specificity Is In sharp contrast with
the recent reports of Hubbard and Wald (llj-9* l£0) who
observed that a stereochemical specificity existed for
the synthesis of rhodopsin from the protein, opsin and the
carotenoid, retinene. This specificity so limited the
reaction that only a single geometrical isomer of retinene
could combine with the protein acceptor*
SUMMARY AMD CONCLUSIONS
Bj means of both salt fractionation and dialysis,
I
the distribution of vitamin A ester, vitamin A alcohol and
lutein was studied in fractions of chicken plasma proteins
6 to 8 hours after the oral administration of vitamin A and
lutein in an oily medium. Despite losses in these com
ponents during the fractionation, the following conclusions
seem possible. Vitamin A ester is associated with the
least soluble fraction, which may be composed of ehylomlera,
whereas vitamin A alcohol and lutein are found with a more
soluble fraction. The presence of lutein In the soluble
fraction even during active absorption suggests that
vitamin A and lutein have different mechanisms of absorption
and possibly even different pathways of transport to the
liver; the vitamin A ester enters the circulation through
the lymphatics, while lutein Is believed to be carried
through the portal blood.
In beef plasma, the p-carotene appeared to be In a
slightly more soluble fraction than the vitamin A alcohol.
The small amount of lutein present was also found in the
same fraction that contained fl-earotene. The vitamin A
r i
alcohol in hog plasma appeared to be in an almost identical
fraction as the alcohol in beef plasma.
116
In rat plasma, vitamin A alcohol was found in th©
precipitate brought down by dialysis only during the period
of active absorption of vitamin A* It is proposed that the
rise In the alcohol form of the vitamin during this period
occurs in a fraction other than the on© which normally
transports the free vitamin*
It is' suggested that the carotenoid pigments and
vitamin A are associated with specific protein carriers
in plasma, which is the case of vitamin A alcohol also
seems to limit the concentration in plasma* It Is further
suggested that protein acceptors are responsible for the
specificity of absorption of the carotenoids and vitamin A
in different species*
These observations were extended to Include an
investigation of the storage mechanism of these components.
The procedure of differential centrifugation of liver
homogenates was modified to permit the quantitative iso
lation of the centripetally-migrating or cream fraction*
In rat liver homogenates and homogenates prepared from
both fresh and stored beef liver, the vitamin A ester was
found concentrated almost exclusively In this cream
fraction* It is proposed that the esterified vitamin is
stored in the liver in association with the small phospho
lipid-containing droplets in the cell*
117
The vitamin A alcohol in rat liver homogenates was
found concentrated in the cream, supernatant and microsomal
fractions. It is suggested that specific protein acceptors
are involved in this association possibly the same protein
being associated with vitamin A alcohol both in plasma and
in the supernatant fraction of rat"liver homogenates..
The presence of vitamin A in the mitochondrial
fraction, as reported previously, could not be confirmed.
These earlier findings were probably due to the contami
nation of this fraction by the cream fraction.
The vitamin A alcohol appeared to be concentrated
in the cream fraction of fresh beef liver homogenates but
was more widely distributed in the stored samples. In
addition to vitamin A, beef liver also contains £ -carotene.
This pigment was found distributed almost equally between
the nuclear, mitochondrial, microsomal and cream fractions.
That this wide distribution is not due to a non-specifie
association with either proteins or lipids is seen by the
absence of ^-carotene in the supernatant fraction which
contains the majority of the cellular proteins, and its
lack of concentration in the cream fraction which contains
the majority of the neutral fat. In addition, diethyl
ether extraction of the isolated fractions from fresh beef
liver homogenates indicated that there was a difference
118
In affinity between these fractions and p-carotene. This
was particularly notieable in the microsomal fraction,
where p-carotene could not be extracted by diethyl ether
unless the fraction had been altered by storage or the
addition of potassium-chloride. The vitamin A ester was
readily extracted by diethyl ether from the cream fraction
where it was concentrated*
BIBLIOGRAPHY
>A- ___________________________________________________________________________________________________________________ — -- 1
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Asset Metadata

Creator Krinsky, Norman Irving (author)

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

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Nutrition

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-601785

Unique identifier UC11354698

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Legacy Identifier DP21553.pdf

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Document Type Dissertation

Rights Krinsky, Norman Irving

Type texts

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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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