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The incorporation of deuterated fatty acids into the phospholipids of the liver and intestine during fat transport

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The incorporation of deuterated fatty acids into the phospholipids of the liver and intestine during fat transport

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Content THE INCORPORATION OP DEUTERATED PATTY ACIDS INTO THE
PHOSPHOLIPIDS OP THE LIVER AND INTESTINE
DURING PAT TRANSPORT
A Thesis
Presented to
the Faculty of the Department of Biochemistry
The University of Southern California
School of Medicine
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
By
Ingrid Hellen Kling
January 1949
UMI Number: EP41293
All rights reserved
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UMI EP41293
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T h is thesis, w ritte n by
.............
under the guidance of h.BXt.. Faculty Com m ittee,
and approved by a ll its members, has been
presented to and accepted by the Council on
G raduate Study and Research in p a rtia l fu lfill
ment of the requirements fo r the degree of
.Majs.tBr...oX..B.cienc.e..
......... E.s .&,.B.Qgjar&ufl..
Dean
Date j a im a p - y —, ................
Faculty Committee
Chairman
Acknowledgement
The author wishes to express her sincere ap
preciation to Dr. Margaret Morehouse for her
generous and helpful guidance during the experimen
tal work involved in this thesis; to her entire
thesis committee for their kind advice in writing
this paper; and to Swift and Company for making
this work possible.
TABLE OP CONTENTS
Chapter Page
I. Historical Background 1
The Role of Phospholipids in Fat
Metabolism 1
Comparison of Lecithin and Cephalin
Metabolism 8
The Use of Deuterium for Tagging
Pats 9
II* Plan of Experiment 12
III. Methods 14
Preparation of Deuterated Linseed
Oil 14
Feeding of Animals 15
Extraction of Lipids 16
Absorption of Phospholipids on
Magnesium Oxide 17
Elution of Choline-Containing
Phospholipids 18
Analysis of the Lecithin 19
Analysis of the Neutral Pat 21
Isolation of Total Phospholipid
Patty Acids 21
Isolation of Lecithin Patty Acids 23
, Isolation of Neutral Fat Fatty
Acids 23
Method for Deuterium Analysis 23
Combustion of Patty Acids 23
TABLE OP CONTENTS, con.
Chapter * Page
III. Methods, continued
Purification of Combustion Water 24
Density Measurement 25
IV. Results 26
Deuterium Content of Pat Ingested 26
Analysis of Lecithins arid Neutral
Pat for Purity 28
Deuterium Analyses of Intestinal
Lipid Fractions 29
Comparison of Liver and Intestinal
Lecithins 36
V. Discussion 39
VI. Summary and Conclusions 44
Bibliography 46
LIST OP TABLES
Table
I* Atoms Per Cent D in Deuterated
Linseed Oil
II* Choline;Phosphorus Ratios of
Extracted Lecithins
III. Phosphorus Analysis of Neutral Pat
of Intestine
IV. Atoms Per Cent Deuterium in
Intestinal Lipids — 12 Hour Period
V. Atoms Per Cent Deuterium in
Intestinal Lipids — 9 Hour Period
VI. Atoms Per Cent Deuterium in
Intestinal Lipids --6 Hour Period
VII. Incorporation of Deuterated Fatty
Acids into Intestinal Lipids
VIII. Comparison of Calculated and Pound
Atoms Per Cent in Intestinal
Phospholipid
IX. Atoms Per Cent Deuterium in Lecithin
Patty Acids of Liver
X. Comparison of Atoms Per Gent
Deuterium in Lecithins of Liver -
and Intestine
LIST OP FIGURES
Figure I. Following page 25
Calibration Curve for Falling
Drop Method
CHAPTER I
HISTORICAL BACKGROUND
The Role of Phospholipids In Fat Metabolism. The theory
that phospholipids are instrumental In fat metabolism was
first expressed in 1891 by Loewj(l). H© postulated that
since lecithin is mlscible in water and easily hydrolyzable
in weakly acid solution, fat is changed into lecithin in order
to pass into the tissue cells. The miscibility of lecithin
with water also led Bloor (2) to the view that lecithin
facilitates the transport of fat. He found that when olive
oil was administered to a dog, there was an increase in
lecithin content of both the blood plasma and corpuscles, the
increase being higher in the corpuscles. Bloor felt that the
blood erythrocytes take up the fat from the plasma and trans-
form it into lecithin, which is an intermediate step in the
metabolism of fats.
In 1910 Joannovles and Pick (3) found that food fat
markedly influences the composition of phospholipids in
liver# This was later confirmed by Sinclair (4) for other
organs as well as liver by measuring the change in iodine
number of phospholipid fatty acids of cats on a diet of cod
liver oil. During the absorption of fat, Sinclair (5) found
that there was.a pronounced change in composition but no
change in the amount of the phospholipid fatty acids of the
intestinal mucosa and liver of the cat. He claimed (6) that
2
this phenomenon was distinct from the process of utilizing
the food fat for repair of wear and tear, since the rate of
turnover of these fatty acids in the skeletal muscle of cats
was constant irrespective of the intensity of the metabolism.
These experiments led Sinclair to believe that phospholipids
are necessary intermediates in the metabolism of fat.
Artom also felt that phospholipids play an integral part
in fat transport. He found that injection of iodinized fatty
acids resulted in their incorporation into the phospholipid
fractions of liver and red corpuscles (7). In 1935 Artom and
Peretti (8) found incorporation of iodinized fatty acids Into
the phospholipids of the intestinal mucosa of the rat after
large doses of iodinized fat were given, indicating that pos
sibly phospholipid formation was involved in absorption from
the int e s t ine•
Elaidic acid, the trans isomer of oleic acid, has been
used extensively as a tracer by Sinclair. Although elaidic
acid is apparently never present under normal conditions, its
metabolism is believed to follow the usual course of break
down of fatty acids. It can be detected because, unlike
other unsaturated fatty acids, its lead salt is insoluble in
alcohol. This acid can be incorporated into the phospholipids
of the liver, small intestine, muscle, kidney, and blood cells
of rats that have been fed elaidin for long periods (9). It
was found that while the maximum amount incorporated was about
the same, the rate of entrance of the tagged fatty acids into
3
the phospholipids varied considerably. - In the intestine,
maximum incorporation occurs in less than one day and in the
liver in somewhat more than one day. Sinclair suggested (10)
that there are two classes of phospholipids, one of which
consists of the less unsaturated phospholipids and is postu
lated to function as in intermediary product in the metabolism
of fat. The other class is comprised of the more highly unsat
urated phospholipids and functions in the essential makeup of
the cell.
Barrett and his associates (11) used deuterium for
labeling their fat. They kept rats on a diet containing 20%
deuterated linseed oil for three days and found that the
liver phospholipid fatty acids contained almost 50% > more deu
terium than the neutral fat fatty acids of the liver. Liver
phosphatides contained nearly three times as much deuterium
as body phosphatides.
Work done in 1939 by Cavanaugh and Raper (12) showed
the presence of deuterium-containing phosphatides in liver,
kidney, brain, and plasma a few hours after feeding
deuterated linseed oil. At all intervals (6, 10, and 24 hrs.),
the liver phosphatides contained more deuterium than the
phosphatides of the other organs studied. At the 6 hr. period,
about 14% of the liver phosphatide fatty acids and 18% of the
liver glyceride fatty acids had come from the deutero-fat in
the food. Deuterated linseed oil was also used by Sperry and
coworkers (13). After intervals of 8 to 48 hours, labeled
4
fatty acids were present in liver, intestine, and carcass,
fatty acids in considerable amounts, but only traces were
found in the brain.
Barnes, Miller, and Burr (14) do not accept the view that
formation of phospholipid is an integral step in fat trans
port. Using the methyl esters of conjugated fatty acids which
can be detected spectroscopically, they found incorporation
into both phospholipid and neutral fat fractions of the intes
tinal mucosa. However, maximum incorporation (50$) into
neutral fat occurred at one hour, whereas only 6$ was incor
porated into the phospholipids of the intestine. .At 8 hours,
maximum incorporation of only 15$ into the phospholipids was
found. These workers (15) also observed that in essential
fatty acid deficiency there is a decrease in incorporation of
labeled fatty acids into the phospholipids of the intestinal
mucosa.
Evidence regarding phosphorylation of fat-during absorp
tion has been obtained by observing increases in content or
turnover of phospholipid in intestinal lymph and thoracic
duct lymph. Whereas Eckstein (16) had found no change in
phospholipid content of thoracic duct lymph during absorption
of fat, later Brockett and coworkers (17) found a moderate
rise. Using radioactive phosphorus, Flock, jet al. (18) in
1947 showed that a fat meal produced a much greater increase
in turnover of lymph phospholipid from the intestine than
from the liver.
5
The relationship between phosphorylation and fat absorp
tion has been studied by using inhibitors and accelerators,
Verzar (19) supported the conception of phosphorylation as a
necessary part of fat absorption by showing that the absorption
of fat was inhibited by mono-iodoacetic acid and phloridzin.
However, the doses used were very large and have been shown
by Klinghoffer.(20) and Wood (21) to cause extensive destruc
tion of the Intestinal mucosa. It seems, therefore, that
faulty absorption in this case cannot be attributed with
certainty to faulty phosphorylation. No relation between
fat absorption and iodoacetic acid poisoning was found by
Sinclair (22). When iodoacetic acid was fed, no significant
difference was found In the elaidic acid content of the ti s-
sue lipids of the control and iodoacetic acid poisoned
animals.
It has been shown that fat absorption can be accelerated
by the addition of glycerol and phosphate (25) or glycerophos
phate (24). This has been interpreted by the Verzar school
to mean that during absorption, fat Is transformed in the
intestinal mucosa to phospholipid prior to resynthesis.
The proposal of Verzar (25) that adrenalectomy impedes
fat absorption by interfering with phosphorylation has been
refuted both by the use of labeled fatty acids and radio
active phosphorus. Stillman (26) showed that phosphorylation
of fat in the small intestine, as measured by incorporation
of administered radio phosphorus into the phospholipid
6
molecule, is not Interfered with in the small intestine of
the adrenalectomized rat. Furthermore, according to Barnes
et al. (27), adrenalectomy does not interfere with incorpora
tion of conjugated fatty acids into the intestinal phospho
lipid. These workers also state that adrenalectomy has no
effect on fat absorption (28). This has been questioned by
Bavetta and Deuel (29), who found impaired absorption in
young animals in which a more severe adrenal insufficiency
can be demonstrated. Their work showing inhibition of ab
sorption of long chain fatty acids supports the conclusions
of Verzar.
Work with radioactive phosphorus shows that the intes
tine is one of-the most active tissues concerned-in fat
metabolism (30), not only during fat absorption (31), but
during fasting. In 1938, Fries and his associates (32)
demonstrated phosphatide activity In the small intestine
both in the presence and absence of fat in the diet. Even
in the absence of ingested fat, the small intestine is the
most active part of the gastrointestinal tract, the amount
of phospholipid formed being approximately 30-40$ of that
- formed when fat is fed. Perlman and coworkers (33) found
that maximum formation of radioactive phospholipid occurred
at an earlier time in the liver than in the gastrointestinal
tract when cod liver oil was fed, an observation which cast
some doubt on whether phospholipid is actually synthesized
in the small intestine or transported there from the liver.
7
That the intestine can synthesize phospholipid by itself
was shown in 1938 by Robinson and associates (34). They
found that when slices of intestinal mucosa were incubated
in a Warburg apparatus with radioactive phosphorus, there was
an incorporation of radio P in the mucosal phospholipids.
The following year, Barnes jet al. (35) observed jin vitro
incorporation of conjugated fatty acids of corn oil into
phospholipids of intestinal mucosa. Intestinal phospholipid
synthesis was further substantiated by Fishier and co
workers (36) who demonstrated that hepatectomy did not
reduce the recovery of P3^ kidney and small intestine of
dogs injected with P5^.
Recently, Frazer's theory (37) that fat is absorbed as
unhydrolyzed particles directly into the lacteals, again
seriously questioned the importance of phosphorylation as a
necessary step in fat resynthesis. He believes that phospho
lipids are utilized only for dispersion of the negatively
charged, emulsified fat particles in the intestine.
In the 3qme year, Schmidt-NIelson (38) showed that one
hour after P3^ was administered by intramuscular injection to
a rat, the specific activity of phosphatide P of the intestine
was four times larger after feeding 2.4 grams of peanut oil
by stomach tube than the specific activity of phosphatide P
of the resting intestine. He points out, however, that the
increase is not sufficient to account for the conversion of
absorbed fat to phospholipid unless the phosphorylation pro
cess;, is limited to the epithelial cells*
Similar experiments were reported in 1948 by Zilversmit
et al« (39), who derive conclusions opposite to those of
Schmidt-Nielson. They found that neither the amount or the
turnover of the phospholipid of the mucosa or the villi of
the small intestine is affected measurably by the absorption
of fat. These workers conclude that fat can pass through the
intestinal wall without involving phospholipid as an inter
mediate •
Comparison of Lecithin and Cephalin Metabolism* Very
little is known with regard to the particular phospholipid,
if any, involved in fat transport. This is partially due to
the difficulty of obtaining pure lecithin and cephalin frac
tions. Cephalins have usually been separated from lecithins
by taking advantage of their relative insolubility in alcohol.
Sinclair (40) studied the relative turnover of cephalins
and lecithins in rat liver by the use of elaidin. He found
that the maximum incorporation is somewhat higher in rat
liver lecithins than in the cephalins, but that the rate of
uptake was practically the same. He therefore concluded
then that cephalin as well as lecithin is an intermediate in
the metabolism of fat.
Relative activities of cephalins and lecithins have been
studied by several workers using radioactive phosphorus. In
1940 Hevesy and Hahn (41) found a more rapid renewal of
mucosal cephalins than lecithins at early intervals (four
hours) after P32 administration, but at twelve hours both
fractions had the same activity. After longer intervals,
Chargaff (42) found that incorporation of P32 into intestinal
and liver phospholipid is apparently greater in lecithins
than in cephalins. Hahn and Tyren (43) in 1945 found the
same to be true of cephalins and lecithins extracted from rat
and rabbit liver.
When the lecithin and cephalin turnover of the entire
rat were determined by means of radioactive phosphorus (42),
cephalins appear to be somewhat more active.
The Use Of Deuterium for Tagging Fats. Schoenheimer and
Rittenberg (44) pioneered in the use of deuterium for tagging
organic compounds for use in metabolism studies. It was
found that saturation of unsaturated compounds with deuterium
resulted in a stable product whose metabolism could be
studied provided that the carbon to which the deuterium was
attached does not enter into the reaction.
Work with deuterated fatty acids has been extensive. In
studying incorporation of fatty acids into phospholipids,
however, deuterium has been used only by Cavanaugh and Raper (12),
and Barrett and associates (11).
Several other methods have been used for labeling fats:
(1) iodized fatty acids, (2) elaidic acid and (3) conjugated
fatty acids. All of these have certain very definite
10
shortcomings which mast he noted in evaluating the results
obtained through their use*
The use of iodized fats is unsatisfactory and serves
only to throw light on the metabolism of iodo-fat, but not of
fat in general* Their use has been discontinued entirely in
recent years, since better tools have become available.
Several objections can be raised against the use of
elaidin. First, since elaidin is not a natural fat, the ques
tion of cell permeability must be considered. Secondly, it
is not known whether elaidic acid is transformed in the body
by saturation or desaturation, thus becoming undistinguishable.
Sinclair (45) found that even after a rat had been fed a high
elaidin diet for many months, phospholipid fatty acids of
active tissues like liver and intestine contained not more
than 30$ of elaidic acid. Furthermore, it is not known whether
one or two elaidic acid molecules enter each phospholipid
molecule.
Some of the same objections also hold true for the use
of conjugated fatty acids for tagging as done by Barnes and
coworkers (14). These acids, which are detected by their
high ultraviolet absorption, do not occur naturally. There
is again the possibility that they may change in the animal
body into substances of low ultraviolet absorption, undetect
able spectroscopically. Moreover, these fatty acids were fed
as their methyl esters, making the picture even more unnat
ural.
11
On the other hand, deutero-fat is neither chemically nor
physiologically distinguishable from natural fat. In satu
rating linseed oil, for instance, one obtains a deutero-
triglyceride, which the animal body cannot distinguish from a
natural fat. Since the deuterium atoms are attached at
positions where no exchange reactions with hydrogen occur,
these labeled fats are stable. For these reasons, deuterium
is at present the most satisfactory tool for labeling fatty
acids in metabolism studies.
CHAPTER II
PLAN OF EXPERIMENT
The object of these experiments was to study phospho
lipid turnover as related to fat transport, and in this
connection, to compare the turnover of lecithins and cephalins
in the small intestine.
The most useful and convenient tool to employ for a
study of this type is a fat labeled with deuterium. In this
case, deuterated linseed oil was fed to rats in a single dose.
The animals were killed after intervals of 6, 9, or 12 hours,
and the lipids of the Intestine and liver were extracted by
the method of Taurog and associates (46), after the Intestine
had been freed-from extraneous fat.
One of the most important considerations to be determined
in the present study was the proportion of absorbed, fatty
acids which pass through the stage of lecithins and cephalins
respectively. Since the classical method of Bloor (47)
results in rather impure fractions, it was decided to use the
procedure of Taurog et al., which yields a very pure lecithin
and neutral fat fraction. The latter procedure involves
absorption of phospholipids from petroleum ether on light
magnesium oxide and elution of the lecithins with methanol.
The triglyceride fat remains in the petroleum ether. No satis
factory method has been found for eluting the cephalinsor
cephalin fatty acids from the magnesium oxide without losing
13
a considerable amount during the process.. Consequently, it
was decided to make a comparison between the lecithin and
total phospholipid turnover, as indicated by deuterium con
tent. Incorporation of deuterated fatty acids into the
triglyceride fat of the small intestine was also.determined
to make the picture more complete.
Originally, the plan included similar determinations in
the lipid fractions of the liver, but this thesis includes
results only for liver lecithins. To obtain greater accuracy
than that afforded by the falling drop method for deuterium
analysis, the samples of total phospholipid fatty acids are
being saved for analysis with the mass spectrometer.
CHAPTER III
METHODS
Preparation of Deuterated Linseed Oil. Deuterated lin
seed oil was prepared as described by Schoenheimer and
Ritteriberg (48), using deuterium oxide of 99.8$ purity as the
source of heavy hydrogen. Twenty milliliters of boiled
linseed oil (saponification number = 190) were dissolved in
150 ml. of pure, dry, cyclohexane in a 250 ml. round bottom
flask, and 100 mg. of Adams’ platinum oxide catalyst was
added. The flask was attached to the deuteration apparatus
and was shaken in the atmosphere of deuterium until the oil
became solidified and absorbed no more of the gas.
An attempt was made to filter off the platinum catalyst
through various types of filter paper and through sintered
glass crucibles. However, the catalyst had formed such a fine,
stable, colloidal suspension that it could not be filtered
nor coagulated by heat or cold. Therefore, it was necessary
to use the lithium salt method for breaking colloids. A satu
rated solution of lithium nitrate in 95$ ethyl alcohol was
prepared. The fat containing the catalyst, obtained by dis
tilling off the cyclohexane, was dissolved in a minimum amount
of the lithium nitrate-alcohol solution. After agitating
slightly, the platinum coagulated and was filtered off. The
alcohol was removed by distillation under reduced pressure,
leaving the deuterated fat mixed with lithium nitrate. The fat
15
was separated from the salt by extraction with ether. When
the ether was evaporated off, a light yellow fat remained,
which was solid at room temperature.
To this fat, an equal amount of ordinary linseed oil was
added, in order.to obtain a mixture of such consistency that
- * >
it could be drawn up into a syringe at a temperature suitable
for feeding.
The fat was analyzed by methods to be described later
and was found to contain 4,01 atomsper cent deuterium. The
fatty acids of the linseed oil were calculated to contain
4,46 atomsper cent deuterium.
Feeding of Animals. From preliminary experiments, it
was found that from the pooled livers or intestines of three
160-gram rats, enough lipid was obtained to determine the
deuterium content of the fatty acids of the total phospho
lipids, lecithin and neutral fat. Therefore, in order to
insure two or three samples of each lipid fraction, each group
contained six to eight female albino rats, weighing 160-180
grams. The animals, which had been on the stock diet, were
fasted for 26 hours before feeding. To secure accurate
dosage, they were fed 1.5 ml. of the deutero fat by stomach
tube attached to a 2 ml. tuberculin syringe into which the
fat was carefully measured. The table below shows briefly the
plan of experiment.
16
Expt. No. of Absorption
No. Hats Period
I
II
III
7
8
6
12 hrs.
9 hrs.
6 hrs.
The animals were killed at the end of the set time inter
val with an intraperitoneal injection of nembutal. The liver
and small intestine were removed and cleaned of any adherent
fat. Undigested fat in the intestine was flushed out with
water under pressure from a hypodermic syringe. To determine
the amount of unabsorbed fat, the washings were extracted
four times with 40-50 ml. portions of diethyl ether, which
was then dried.for several hours over anhydrous sodium sul
fate. After filtering off the drying agent, the ether extract
was transferred to weighed Ehrlenmeyer flasks and the ether
was evaporated. The residue was dried to constant weight
under vacuum at 60° and weighed. Absorption studies were not
comprehensive, but were done merely to determine that absorp
tion was normal. At the 6-hour period, 10-15$ of the fat
still remained in the small intestine, and at 9 hours the
y
small intestine contained only 3$ of• the fat fed. In all
cases absorption was apparently normal.
Extraction of^Lipids. The cleaned intestines and livers
were divided into two groups of three or four, in order to
obtain two separate samples from each group for each organ.
In extracting the lipids, the method of Taurog ejb al. (46)
was followed. The tissues were ground with sand in a mortar,
17
transferred to a 250 ml* Ehrlenmeyer flask, and extracted
with approximately 150 ml. of alcohol at 55-60° C. for two
hours. The alcohol extract was filtered and the residue was
extracted for a second time with alcohol for one hour, and
after filtering, the extracts were combined. The tissue resi
due was then extracted with anhydrous ether for 6 hours in a
Goldfisch apparatus. This extract was combined with the alco
hol extracts and concentrated to a volume of about 3 or 4 ml.
by evaporation on a hot water bath under reduced pressure and
in an atmosphere of carbon dioxide. The concentrate was
extracted with four 40-50 ml. portions of petroleum ether
(boiling point 50-60°), made up to a volume such that one
milliliter contained 0.06 mg. of phospholipid phosphorus
(usually 200-230 ml.) and transferred to 250 ml. centrifuge
bottles.
Absorption of Phospholipids on Magnesium Oxide. Separa
tion of the phospholipids was accomplished by the procedure
of Taurog ejb al. (46). To absorb the phospholipids in a
petroleum ether extract-from 15-20 grams of liver.or intes
tine, 20 grams of Merck’s light magnesium oxide were added.
The exact amount of MgO was not considered to be of great
importance, since Taurog and his coworkers found that ab
sorption was complete with 0.5 to 1.75 grams of magnesium
oxide per 0.85 mg. of phospholipid P. An attempt was made,
however, to approximate the latter value in order to obtain a
purer choline-containing fraction. The mixture of magnesium
18
oxide and petroleum ether extract was stirred frequently for
15-20 minutes and then centrifuged for 20 minutes at 1500 r.p.m.
The supernatant^liquid was saved for.the extraction of fatty
acids of the neutral fat# The residue was washed twice toy
adding 200 ml. of petroleum ether, stirring for five minutes
and' centrifuging as toefore. The washings were combined with
the first, supernatant solution. The neutral fat was obtained
toy distilling off the petroleum ether under reduced pressure.
One-half of the magnesium oxide-phospholipid residue was
used for separation of lecithin by eluting with methanol and
the other half, for isolating total phospholipid fatty acids
toy saponification.
Elution of.Choline-Containing Phospholipids. The choline-
containing phospholipids were eluted from the-magnesium oxide
with methanol. Two hundred milliliters of methanol were added
and the mixture agitated for about 20 minutes. The mixture
was then centrifuged for 20 minutes at 1500 r.p.m. and the
supernatant decanted. This elution procedure was repeated four
more times, and the methanol extracts were.combined. The
methanol which was used for the last three washings was satu
rated with solid sodium chloride in order to prevent cloudiness
of the washings. It was found also that the sodium chloride
prevented the oxidation of the lecithin later when the
methanol was evaporated.
The flask containing the methanol eluate was placed in
a water bath at 60-70° and the methanol was distilled off
19
under reduced pressure, in an atmosphere of carbon dioxide,
leaving a white, wax-like residue of choline-containing phos
pholipids and salt*
No attempt was made to separate any sphingomyelin which
might be present, since according to Hunter (49) and others (50),
sphingomyelin is not completely extracted from tissues unless
chloroform-methanol is used. Moreover, when the tissue lipids
are later extracted with cold petroleum ether, sphingomyelin
is supposedly not dissolved, according to Hahn and Hevesy (51).
Therefore, it was assumed that the methanol eluate contained
practically no sphingomyelin.
Analysis of the Lecithin. Since the lecithin was con
taminated with sodium chloride, the residue was not analyzed
for phosphorus and choline, but rather for the phosphorus to
choline ratio, which indicates the efficacy of separation from
cephalin. The analyses were done in the following manner:
One-fourth of the total methanol eluate of the magnesium
oxide-phospholipid complex of three livers or three intes
tines was evaporated to about 200 ml., and made up to volume
with methanol in a 250 ml. volumetric flask. Aliquots of
100 ml. were taken for the choline determination and trans
ferred to 125 ml. Ehrlenmeyer flasks. This method was used
in order to obtain accurate sampling and to obviate any error
due to loss in transferring solid samples. After evaporation
of the methanol under reduced pressure, 15 ml. of saturated
barium hydroxide solution were added to the residue and the
20
flask was placed on a steam bath. The heating was continued
for two hours with frequent shaking, after which choline was
determined in the hydrolysate by the reineckate method of
Entenman, Taurog, and Chaikoff (52), with a few modifications.
Instead of using a solution of ammonium reineckate in
1.2 N HC1, a saturated solution of the salt in water was used,
as described by Winzler and Meserve (53). Furthermore, be
cause centrifugation of the choline reineckate was found
unsatisfactory, the precipitate was collected on a small
sintered glass crucible of fine porosity prior to dissolving
in acetone.
The methanol-phospholipid solution made up as described
in the previous paragraph was also used for the phosphorus
analyses. Fifteen milliliter aliquots were taken and trans
ferred to 8-inch test tubes. The methanol was distilled off
under reduced pressure and 2 ml. of 50$ HgSO^ were added to
the residue. After complete digestion of the sample with the
aid of a few drops of 30$ hydrogen peroxide, the solution was
made up to a volume of about 20 ml. and heated for 20 minutes
in a boiling water bath to change the pyro-phosphate to the
ortho-phosphate. After cooling, the solution was made up to
volume in a 25 ml. flask. Five ml. aliquots were used for
the colorimetric determination of phosphorus by the molybdi-
vanadate method (54).
These determinations were made in duplicate on one or
two samples of each group and the choline-phosphorus ratio
calculated.
21
Analysis of the Neutral Fat. The neutral fat of the
intestine was analyzed for phosphorus to detect contamination
with phospholipid. The analysis was done as described above
for lecithin. Samples from each group were analyzed and were
found to contain only 51-73 micrograms of phosphorus per
100 mg. of fat, or calculated as lecithin, 1.3 to 1.8 mg. of
lecithin per 100 mg. of fat.
Isolation of Total Phospholipid Fatty Acids. Total
phospholipid fatty acids were obtained by saponification of
the MgO-phospholipid complex. To one-half of the MgO-phos-
pholipid residue resulting from the absorption procedure was
added 200 ml. of 95$ ethyl alcohol and 0.4 ml. of saturated
K0H. The mixture was refluxed for an hour and filtered while
hot through No. 5 filter paper. The magnesium oxide was
washed four times with 125 ml. portions of hot alcohol. The
original filtrate and washings were combined and evaporated to
about 4 ml. on a steam bath under reduced pressure and in an
atmosphere of carbon dioxide. When the volume was reduced to
about 4 ml., 2 ml. of distilled water were added and the
heating continued for a few minutes to dissolve -the salts.
The solution was acidified by adding 1.2 ml. of 25$ (by volume)
of H2SO4. The liberated fatty acids were extracted by boiling
gently with a mixture of chloroform and petroleum ether and
then three more times with petroleum ether alone. The extracts
were combined and allowed to stand over anhydrous copper sul
fate for several hours, after which the copper sulfate was
22
filtered off on No. 5 filter paper. The filtrate was concen
trated under a stream of carbon dioxide to about 3 ml. in a
125 ml. Ehrlenmeyer flask heated on a water bath at 30-35°.
In order to avoid transferring the small amounts of fatty
acids obtained, the final stage of the evaporation was done
in the tube in which the fatty acid was to be combusted for
the deuterium determination. These tubes consisted of a
2-inch piece of 10 mm. Pyrex tubing sealed at one end. The
ether-chloroform extract was transferred from the Ehrlenmeyer
flask to the tube using a small funnel. The flask was washed
out twice with ether and these washings were also transferred
to the tube. A gentle stream of carbon dioxide was passed
into the tube to accomplish the final evaporation. The end
of the tube was then pulled out into a narrow tube 2 mm. in
diameter and one inch in length, which was sealed at the end
until time of combustion. The purpose of the narrow tube is
discussed in the description of the combustion procedure.
Because this experiment was concerned with deuterium
determinations in pure lipid fractions, the amounts of fatty
acids obtained are not absolutely quantitative, since a choice
had to be made between purity and complete extraction. In the
extraction of the total phospholipid fatty acids, the quanti
tative extraction from the large amount of magnesium oxide
used in absorption was practically impossible, unless tre-
' mendous quantities of ethanol were used in the washing. This
difficulty could have been alleviated somewhat by using only
23
one-half as much magnesium oxide, hut according to Taurog and
his associates (46), purer lecithin fractions are obtained by-
using the larger amount of MgO in absorption. We felt that
for this work purity was more important than quantitative
extraction.
Isolation of Lecithin Fatty Acids. The lecithin-salt
mixture was saponified and extracted in the same manner as the
phospholipid fatty acids, except that no filtration was neces
sary after the saponification. In this case also, the final
stage of-the solvent evaporation was done directly in the
tube in which the fatty acids were to be combusted.
Isolation of Neutral Fat Fatty Acids. The neutral fat
remaining in the petroleum ether after absorption of the phos
pholipids was saponified after evaporating off the ether.
Fatty acids were obtained as described for phospholipid fatty
acids.
Method for Deuterium Analysis. The deuterium content of
the fatty acids was analyzed in terms of the density of the
water of combustion. The falling drop apparatus described by
Keston, Rittenberg, and Schoenheimer (55) was used for the
density measurements. The samples used for combustion usually
*
weighed 30-100 mg., but in the case of neutral fat fatty
acids, in which the deuterium concentration was low, larger
samples were used.
Combustion of Fatty Acids. The fatty acid samples were
contained in a small pyrex tube, 10 mm. In diameter and
24
about 3*5 cm. in length, one end of which was pulled out into
a narrow tube of 2 ram. in diameter and 2 cm. in length. This
narrow constriction was found to be absolutely necessary if
excessively rapid burning and flashing of the fat was to be
avoided. The seal at the narrow end of the tube was broken
off just before inserting into the combustion tube of an ordi
nary combustion apparatus. The temperature of the furnace
was set at 500°. A U-tube immersed in a dry ice-alcohol bath
was connected to the end of the combustion tube to collect
the water. The combustion tube was heated gently with a
microburner just below the tube containing the sample in such
a way that the fatty acid was forced out of the narrow tube
in very small droplets and burned. Heating with the micro
burner was continued until only a carbonized residue remained;
the complete combustion was accomplished by sliding the sample
into the furnace, where the residue burned slowly, since the
opening at the end of the tube was very small. The time for
complete combustion was usually 2 hours for a 100 mg. sample
of fatty acid. The weight of the sample was determined by
weighing the tube containing the fatty acids before and after
combustion.
Purification of Combustion Water. The combustion water
was purified by an alkaline permanganate oxidation, a chromic
acid oxidation, and a re-distillation performed in a train of
TJ-shaped tubes connected by ground glass joints. A dry ice-
alcohol bath was used for condensing the water vapor
25
during the distillations. The weight of the water of combus
tion was found by weighing the collecting tube before and
after distillation of the water. A known amount of pure water,
freshly distilled from alkaline permanganate, was added to
the purified sample to give a final heavy water concentration
of about 0.1-0.2$. It was necessary to obtain a total amount
of at least 0.3 ml. of water for use in the falling drop
apparatus.
Density Measurement♦ The heavy water content of the
/
sample was. determined by the falling drop method using the
micropipetfce described by Keston and associates (52). The
time necessary for a drop of the water to fall between two
lines in a tube containing m-fluorotoluene in a water bath at
19.600 ± 0.002°C. was determined. Then the water was repurified
and the drop time checked.
A calibration curve for the falling drop apparatus was
constructed, using standard solutions of heavy water freshly
prepared from 99.87$ DgO. A graph relating atomsper cent
heavy water to the reciprocal of drop time 13 shown in Pig. I.
Standards were run periodically in order to check the con
stancy of the calibration curve.
12
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No 6203, U niversity Bookstore, Los AngeJes
CHAPTER IV
RESULTS
Deuterium Content of Fat Ingested. Analysis of the
deuterated linseed oil which was fed is shown in Table I.
The calculation of samples 1 and 2 are based on calibration
curve A, whereas sample 3 was calculated from curve B.
The deuterium analyses of the triplicate samples check
very well, being If.02, If.03, and 3.98, giving an average of
if.01 atoms per cent. Since the subsequent analyses on the
intestinal and liver lipids were done on the fatty acids
themselves, the per cent D was recalculated in terms of the
fatty acids instead of the triglycerides. Since we know .that
all of the deuterium was contained in the fatty acid portion
of the molecule, we can use the equation:
Atoms % D in fatty acids % D in !inseedJIMol.Wt*of linseed oil
oil Mol.Wt.of fatty acids
in oil
Atoms % D in fatty acids if.01 x 882
of linseed oil 793
Atoms % D in fatty acids lf.lf6
of linseed oil
The molecular weight of linseed oil was calculated from
the saponification number of 190 which had been determined.
TABLE I
Atoms Per Cent D in Deuterated Linseed Oil
Sample
Number
Weight of
Sample
(mg.)
Weight of
Combustion
Water
(mg.)
Dilution
Factor
Average
Drop Time
(t)
Reciprocal
of Drop
Time (3/t)
x 10-3
$ D20 in
Dilution
I 95.7 103.2 9.56 117.6 8,50 0.420
II 140.5 146.1 6.89 96.8 10.32
0.585
III 90.5 93.7 11.90 128.4 7.78 0.334
4.02
4. OS
3.98
w
28
Analysis of Lecithins and Neutral Fat for Purity. The
purity of the lecithins extracted, expressed as the choline:
phosphorus ratio is shown in Table II. The ratios range from
0.90,to 1.03 and agree with those obtained by Taurog (46).
Actually, the lowest ratio of 0.90 was obtained in the pre
liminary experiments; higher purity was obtained later.
TABLE II
Choline:Phosphorus Ratios of Extracted Lecithins
Source of Moles Choi. Moles P Moles Choline
Lecithins in Aliquot in Aliquot Moles P
Control Group
Liver 2.23 2.47 0.90
12 hr. Group
Intestine 3.88 4.08 0.95
6 hr. Group
Liver and
Intestine 4.28 4.16 1.03
Extent of contamination of neutral fat with phospholipid
was determined by analyzing for phosphorus. As shown in
Table III, the amount of phospholipid remaining in the
neutral fat is very small; expressed as milligrams of lecithin
per 100 milligrams of fat, contamination amounts to less
than 2% in every case.
29
TABLE III
Phosphorus Analysis of Neutral Fat of Intestine
Sample
; ^ P per 1%, of Lecithin
100 Mg. of Fat per 100 Mg. of Fat
12 hr, - Group I
12 hr. - Group II
9 hr. - Group II
6 hr. - Group I 72.8
72.0
62.2
50. o
1.81
1.80
1.55
1.26
Deuterium Analyses of Intestinal Lipid Fractions.-
Tables IV, V, AND VI give the deuterium analyses of the vari
ous lipid fractions in detail. Usually, the entire amount of
choline and phosphorus analyses were made, and in a few cases
where a part of the sample is being saved for analysis on the
mass spectrometer.
It can be observed that in every instance the atoms per
cent D is the highest in the lecithin fatty acids, the lowest
in the neutral fat fatty acids, with the total phospholipid
fatty acids in an intermediate position. This difference is
significant and consistent in all' groups. The average per
centage of deuterium at 12, 9, and 6 hours respectively in
lecithin fatty acids is 1.80, 1.48, and 1.83, for phospholipid
fatty acids, 1.34, 1.04, 1.16, and for triglyceride acids,
0.97, 0.69, and 1.00. The amount of deuterium in the phospho
lipid fraction is about 74$ of that in the lecithin fraction,
fatty acid obtained was used for the combustion except where
TABLE IY
Atoms Per Cent Deuterium in Intestinal Lipids
Experiment No. 1
12 Hour Period
Average
Fatty Acid Combustion Dilu- Drop
in Sample Water tion Time $ DgO in Atoms $ D
Group I (4 rats); (mg.) (mg.) Factor (t) 1/t x 10“^ Dilution in Sample
Leoithin fatty
Acids 118.9 127.0 8.93 156.8 6.33 0.184 1.64
Total Phospho
lipid Fatty
Acids 66.2 71.1 13.5 191.7 5.22 0.085 1.15
Neutral Fat
Fatty Acids 154.8 175.8 2.91 132.8 7.53 0.31 0.90
Group II (3 rats);
Lecithin Fatty
Acids 56.4 55.6 15.1 174.6 5.73 0.130 1.96
Total Phospho
lipid Fatty 59.1 59.4 8.21 158.6 6.30 0.185 1.52
Acids - 17.6* 14.0 14.2 187.8 5.32 0.090 1.28*
Neutral Fat
Fatty Acids 99.5 114.8 4.29 146.1 6.84 0.24 1.03
* Composite sample of phospholipid fatty acids of both Group I and Group II
ca
o
TABLE V
Atoms Per Cent Deuterium in Intestinal Lipids
Experiment No. 2
9 Hour Period
Average
Patty Acid Combustion Dilu- Drop
in Sample Water tion Time „ % D2O in
Group I (4 rats); (mg.) (mg.) factor (t) l/t x 10 Dilution
Lecithin Patty 95.1 100.3 6.05 141.7 7.05 0.262
Acids 38.9 40.6 7.07 151.3 6.60 0.215
1
Total Phospho
lipid Patty 63.8 67.7 6.83 157.0 6.37 0.194
Acids 27.6 25.2 10.55 187.4 5.33 0.091
Neutral Pat
Patty Acids 89.5 98.8 4.00 159.0 6.28 0.185
Group II (3 rats);
Lecithin Patty
Acids 66.8 72.6 6.55 152.1 6.57 0.214
Total Phospho
lipid Patty 54.0 61.9 6.54 175.4 5.70 0.127
Acids 25.2 26.7 7.70 173.0 5.77 0.134
Neutral Pat
Patty Acids 175.0 204.2 2.67 146.0 6.84 0.24
Atoms $ D
in Sample
1.59
1.52
1.33
0.96
0.74
1.40
0.83
1.03
0.64
03
H
TABLE 71
Atoms Per Cent Deuterium in Intestinal Lipids
Experiment No. 3
6 Hour Period
Average
Patty Acid Combustion Dilu- Drop
in Sample Water tion Time $ D£0 in Atoms % D
Group I (3 rats): (mg.) (mi.) Pactor (t) 1/t x 10“^ Dilution in Sample
Lecithin Patty
Acids 63.9 61.1 10.98 166.3 6.01 0.158 1.73
Total Phospho
lipid Patty *
Acids 26.7 26.3 16.54 195.0 5.13 0.72 1.20
Neutral Pat
Patty Acids 137.3 142.8 2.13 105.7 9.45 0.50 1.05
Group IX {3 rats):
Lecithin Patty
Acids 56.1 61.3 9.59 166.0 6.02 0.160 1.53
Total Phospho
lipid Patty
Acids 68.7 56.1 9.92 180.9 5.53 0.112 1.12
Neutral Pat *
Patty Acids " 36.3 32.8 6.03 163.4 6.11 0.157 0.95
* Only about one-half the sample was used. The remainder is being' saved
for analysis with the mass spectrometer.
33
whereas the amount in the triglyceride fat is only about 54$
of the deuterium content of the lecithins.
These differences in deuterium content apparently have
no definite relationship with time in the intervals used
..here, since the atoms per cent deuterium of the various lipid
fractions seems to be almost the same in the 12 hour period
as in the 6 hour period. Group II in the 12 hour interval
seems to have incorporated more deuterium into the Intestinal
lipids than any other group. But since only 3 rats were used,
and there seems to be considerable variation between animals,
this result is probably not significant.
Table VII gives the average values for deuterium content
in atoms per cent and also a calculation of the per cent
incorporation of ingested fatty acids, based on the fact that
the fat fed contained 4.46 atoms per cent deuterium in its
fatty acids. An average of 37$ is baleen up by the lecithins,
26$ by the total phospholipids, and 20$ by the neutral fat.
The extent of this incorporation into the lecithin fraction
would indicate that lecithin formation is involved in fat
absorption from the intestine.
Comparison of the deuterium content of the lecithins and
total phospholipids (lecithins and cephalins, in this case)
shows that the lecithins must have carried the larger portion
of the ingested acids. Actual comparison of lecithins with
cephalins is difficult since the relative amounts of these
phosphatides in the intestine are uncertain. The literature
TABLE VII
Incorporation of Deuterated fatty Acids into Intestinal Lipids
Time After feeding
Group Number
Atoms f o D in Leci
thin fatty Acids
Atoms % D in Total
Phospholipid fatty
Acids
Atoms % D in Tri
glyceride fatty
Acids
* f > Incorporation
into Lecithin
fatty Acids
** % Incorporation
into Phospholipid
fatty Acids
*** % Incorporation
into Triglyceride
fatty Acids
12 Hours
I II Average
1.64 1.96 1.80
1.15 1.52 1.34
0.90 1.03 0.97
36.8 43.9 40.3
25.8 34.0 29.9
20.2 23.1 21.7
_____ 9 Hours_____
I II Average
1.56 1.40 1.48
1.15 0.93 1.04
0.74 0.64 0.69
35.0 31.4 33.2
25.8 20.8 23.3
16.6 14.3 15.5
6 Hours
I II Average
1.73 1.53 1.63
1.20 1.12 1.16
1.05 0.95 1.00
38.8 34.3 36.6
26.9 25.1 26.0
23.5 21.3 22.4
* f o D in lecithin fatty acids ** f o D in total phospholipid fatty acids
f o D in fatty acids fed f > D in fatty acids fed
*** ^ D in triglyceride fatty acids
f > D in fatty acids fed
35
contains practically no quantitative data on lipids of the
small intestine. Chargaff and associates (56) report ex
traction of 26.0 milligrams of lecithin compared to 9.2 milli
grams of cephalin from the intestine of a male rat weighing
approximately 300 grams. Using this value, the ratio of
lecithin to cephalin was calculated to be 2.82. On this
basis the ratio of lecithin s total phospholipid would be
2.82 : 2.82 +1, or 2.82 : 3.82. From this we can find the
amount of deuterium in the total phospholipid fraction in the
event that all of the deuterium is contained in the lecithin
fraction by making the following calculation (calculation A):
$ D in total phospholipid = $ D in lecithins x 2.82.
3.82
In an earlier paper, Chargaff (42) gave a lecithin :
cephalin ratio of 172 : 95, or 1.81. Using this ratio, cal
culations (calculation B) similar to the one above were made.
Both calculations and a comparison to values actually found
are shown In Table VIII.
There are two possible deductions from Table VIII;
1) Cephalins take no part in the reaction of fat resynthesis
in the intestine, at least In the case of saturated fats, or
2) according to Calculation B, based on a lower lecithin :
cephalin ratio, only 15$ of the deuterated fatty acids of
the total phospholipid could be in the cephalin, since 85$
of them are accounted for by the deuterium content of lecithin.
This shows that cephalins may play a relatively minor part
56
TABLE VIII
Comparison of Calculated and Found
Atoms Per Cent D in Intestinal Phospholipid
Time After Feeding
12 Hours
(Average)
9 Hours
(Average)
6 Hours
(Average)
Atoms % D in Phospholipid
(Calculation A) 1.33 1.09 1.20
Atoms % D in Phospholipid
(Found) 1.33 i.09
, 9
1.16
Atoms % D Unaccounted for
by Lecithins 0.00 0.00 0.00
Atoms % D in Phospholipid
(Calculation B) 1.16 0.95 1.05
Atoms % > D Unaccounted for
by Lecithins 0.17 0.14 0.11
in absorption compared to the le c ithins. However, a single
analysis of cephalin fatty acids from the intestines of
rats in the 6 hour group showed the atoms per cent D to be
about one-half that in lecithin, or about 33% > of the total
phospholipid. It must be noted that this analysis was done
on only a single sample, which was so small that only an ap
proximate analysis could be made.
Comparison of Liver and Intestinal Lecithins. Most of
the samples extracted from the liver are being saved for
analysis on the mass spectrometer, because determinations
with our falling drop apparatus were found to be rather inac
curate in the range lower than 0, 08%. Although the results on
liver lecithins shown in Table IX are not complete, It is
37
interesting to note (Table X) tbat at the 6 and 12 hour
intervals, the incorporation (9-17$) of ingested acids is
much lower in liver than in intestine. This would render
unlikely any possibility that the labeled lecithins which
were found in the intestine were transported there from the
liver.
TABLE IX
Atoms Per Cent Deuterium in Lecithin Fatty Acids of Liver
Time Weight Combustion Average
After Group Sample Water Dilution Drop $ DgO in Atoms f > D
Feeding Humber (mg.) (mg.) Factor Time (t) l/t x 10“5 Dilution in Sample
I
12 (4 rats) 132.4 133.4 6.84 190.7 5.24 0.082 0.56
Hours II
(3 rats) 60.2 62.5 16.67 214.9 4.65 0.024 0.38
I
6 (3 rats) 75.2 78.7 8.72 187.9 5.32 0.090 0.77
Hours II
(3 rats) 56.1 59.5 8.19 212.4 4.70 0.028 0.23*
TABLE X
Comparison of Atoms Per Cent Deuterium in Lecithins of Liver and Intestine
Time After Feeding 12 Hours 9 Hours 6 Hours
Group Number I II I II I II
Atoms % D in Intestinal Lecithin Fatty Acids 1.64 1.96 1.56 1.40 1.73 1.53
Per Cent Incorporation 36.8 45.8 35.0 31.4 38.8 34.5
Atoms f > D in Liver Lecithin Fatty Acids 0.56 0.38 * * 0.77 *0.23
Per Cent Incorporation 12 9 — - — 17.2 *5
* Samples are being saved for analysis on the mass spectrometer
CHAPTER V
DISCUSSION
The large amount (37$, average) of the fat ingested
which was Incorporated into the intestinal lecithins throws
light on the question of phospholipid formation as a neces
sary step in fat absorption, especially when compared to the
average incorporation of only about 20$ into the neutral fat.
This finding is not in agreement with the work of Barnes,
et al, (15), who found the maximum incorporation of conjugated
fatty acids into intestinal phospholipids of the rat to be
only 18$ compared to 50$ maximum incorporation into the neu
tral fat. Our work therefore refutes their contention that
phospholipid is not involved as an intermediate in fat ab
sorption because of the small amount of labeled acids which
they found in the phospholipid fraction. It must be noted
that work done with the methyl esters of conjugated fatty
acids as carried out by Barnes and coworkers (15) is open to
many criticisms, particularly since these acids are not present
in natural fats. It might be that these acids are not found
in the phospholipids in larger amounts because they have
undergone some change to become spectroscopically undetectable*
Zilversmit and coworkers (39) argue in a recent paper that
some incorporation of ingested fatty acids into the intestinal
phospholipids is to be expected since their presence in the
small intestine makes them available as building blocks for
40
the molecules of structural phospholipids. However, it is
hardly probable that the rate of wear and tear on structural
phospholipids is such that 6 hours after feeding labeled fat,
about 37# of it is taken up by the intestinal lecithins. We
believe that the extent of incorporation of deuterated fatty
acids into the lecithins of the intestine indicates that leci
thins are involved in the mechanism of fat absorption. This
is further borne out by the fact that the amount of deuterated
acids in the neutral fat is much smaller than in the lecithins
of the intestine. That the deuterated lecithins * are hot trans
ported to the small intestine from the liver is indicated by
the fact that the liver lecithins contain only a fraction as
much deuterium as the intestinal lecithins during the same
time intervals. Cavanaugh and Raper (12) found incorporation
of deuterated acids into liver phospholipids of the rat to be
approximately 14# at 6 hours, which also is much smaller than
the amount we find in the intestinal lecithins. In view of
these considerations, we feel that lecithins function as inter
mediates in fat resynthesis in the intestinal wall.
Whether there is an increase in amount as well as turn
over of intestinal phospholipid during fat absorption was not
investigated since many workers, including Schmidt-Nielson (38),
have found no increase in quantity of phosphatides during the
absorption period. Presumably, then, the newly formed phos
pholipid molecule which we feel is involved in fat transport
is split up again near the place of formation and the residual
41
part of the molecule is re-used for transport of additional
fatty acid* If this is so, it would be of great interest to
know whether the phospholipid molecule is broken up entirely
into its fundamental components, and what the relative rates
of formation are of the phosphate-glycerol bond, the fatty
acid-glyeerol bond, and the nitrogen base-phosphate bond.
The data from these experiments show no definite rela
tionship between incorporation of deuterated fatty acids and
the time interval after feeding; to clarify the process of
phospholipid formation, further experiments should be conduc
ted during the earlier stages of absorption. The higher amount
of deuterium in the intestinal lecithins from rats killed
after the 12 hour interval is probably not significant. How
ever, it corresponds to the work of Perlman et; al. (33), who
found maximum incorporation of radio phosphorus at the 12
hour period in fasting rats which had been fed and cod
liver oil. Sperry, Waelsch, and Stoyanoff (13) determined
deuterated fatty acid content only in the total lipid fraction
of the rat intestine. They used 8, 16, 24, and 48 hour time
intervals and found the highest amount of deuterium after
8 hours. Barnes and coworkers (15) found maximum incorpor
ation of conjugated fatty acids into intestinal phospholipids
at the 8 hour period.
A comparison of the deuterium content of lecithins and
total phospholipid fractions indicates that the lecithins con
tain;. most, if not all, of the labeled fatty acids. Of the
42
fatty acids fed, an average of 37$ is found in the lecithins,
whereas only 27$ is found in the total phospholipid fraction,
which, in this case, includes only lecithins and cephalins.
Calculations based on the lecithin:cephalin ratios reported
in Chargaff's publications (42) (56) show that only a small
fraction of the ingested deuterated fatty acids could be
present in the cephalins, since the deuterium content of the
lecithin portion accounts for all, or nearly all, of the deu
terium found in the total phospholipid. This would indicate that
lecithins play the major part in fat transport, whereas the
role of cephalins is minor, at least in the absorption of
saturated fats. More conclusive evidence regarding the role
of cephalins in fat metabolism will be obtained from experiments
now in progress which will compare the turnover of these
phospholipids in liver. Further experiments should be per
formed during the earlier stages of absorption.
No other work has been done in comparing the rate of
turnover of lecithin and cephalin fatty acids of the intestine.
Sinclair (40) compared the rate of turnover of lecithins and
cephalins in the liver of the rat, using elaidic acid as an
indicator. Separation was accomplished by Bloor's method (47).
Incorporation of elaidic acid into the lecithins was only
slightly higher than in cephalins and the rate of uptake was
about the same. Sinclair feels then that cephalins as well
as lecithins are intermediates in fat metabolism. His work
does not necessarily mean, however, that the picture is the
43
same in the small intestine. Experiments on intestinal cepha-
lin and lecithin turnover have heen done with radioactive
phosphorus (41) (42), but the data are contradictory and the
determinations were not done during fat absorption.
While we cannot entirely rule out the role of cephalins
in fat absorption from the intestine, cephalin turnover in
the intestine as indicated by the deuterated fatty acid con
tent, appears to be only a very small fraction of the leci
thin turnover at the time intervals used in these experiments.
The question arises whether this apparent difference in incor
poration of labeled fatty acids may be due to the fact that
the deuterated fatty acids of linseed oil are not suitable
indicators for this purpose. In this fat, only the saturated
acid (stearic acid) is labeled, so we have the usual disad
vantage that not all of the fatty acids incorporated into the
phospholipids are tagged. This difficulty can be overcome
only by feeding a fat in which both the saturated and unsatu
rated acids are labeled. On the other hand, stearic acid is
a component of natural fat and has long been known as a com
ponent of both lecithins (57) and cephalins (58); it should,
therefore, be as satisfactory a label for the cephalin mole
cule as it is for lecithin. That the deuBero-stearic acid is
incorporated into the intestinal cephalins to a lesser degree
than into the lecithins indicates that the cephalins probably
serve mainly as structural phospholipids, rather than metabolic
intermediate s•
CHAPTER VI
SUMMARY- AND CONCLUSIONS
!• Incorporation of ingested deuterated fatty acids
into the intestinal lipids 6, 9, .and 12, hours after feeding
was found to be of the following order of magnitude:
lecithin > total phospholipid > neutral fat,
2, The high percentage (approximately 37$) of incorpo
ration of labeled acid into the intestinal lecithins as early
as 6 hours after feeding would indicate that this phospholipid
is involved as an intermediate in fat resynthesis in the small
intestine. This is further borne out by the fact that the
neutral fat contains only about 54$ as much deuterium as do
the lecithins,
3, That the lecithins found in the small intestine are
not transported there after synthesis in the liver is shown
by the comparatively small amount of deuterated acids in the
liver lecithins after 6 and 12 hours,
4, Lecithins, rather than cephalins, seem to be the
phospholipid fraction primarily involved in fat transport.
This was demonstrated by showing that all, or practically all,
of the deuterated fatty acid content of the intestinal phos
pholipid fraction could be accounted for by the lecithin. .
The amount of labeled acids calculated to be in the cephalin
45
portion is negligible or very small, indicating that this
phospholipid plays only a minor role, if any, in fat absorp
tion from the intestine.
5. More extensive and conclusive evidence to support
these contentions should be obtained by experiments during
the earlier stages of absorption.

BIBLIOGRAPHY
1. Loew, Biol. Zentr. 11:269. 1891, cited by R. G . Sinclair,
J. Biol. Chem. 95:393, 1932
2. Bloor, W. R., J. Biol. Chem., 2k:hll7, 19l6
x 3« Joannovics and Pick, Wien. Klin. 7/och 23:573, 1910, cited
by R. G. Sinclair, J. Biol. Chem.; 82:131, 1929
ip. Sinclair, R..G., J. Biol. Chem., 82:117, 1929; Ibid.,
86:579, 1930
5. Sinclair, R. G., Ibid., 82:117, 1929
6. Sinclair, R. G., Ibid., 95:393, 1932
7. Artom, C., Arch, intern, physiol., 36:101, 1933; Chem.
Abstr. . 27T5821, 1933
8. Artom, C. and G. Peretti, Arch, intern, physiol. . l}.2:6l,
1935; Chem. Abstr. . 30:Ip20lj;
9. Sinclair, R. G., Biol. Symposia. 5:82, 19^4-1
10. Sinclair, R. G., J. Biol. Chem.. 111:515, 1935
11. Barrett, H. H., C. N. Best, and J. Ridout, J.. Physiol.
93:367, 1936
12. Cavanaugh, B. and H. S. Raper, Biochem. J., 33:17, 1939
13. Sperry, W. M., H. vYaelsch, and V. A. Stoyanoff, J. Biol.
Chem. . 135:281, 19if0
lip. Barnes, R. H., E. S. Miller, and G. 0. Burr, Ibid.,
1^0:233, 19lpl
15. Barnes, R. H., E. S. Miller, and G . 0. Burr, Ibid.,
UpO:773, 191}-!
16. Eckstein, H. C., Ibid., 62:7lp3, 192lp-25
17* Brockett, S. R., M. A. Spiers, and H. E. Himmwich, Am. J.
Physiol. , 110:31+2, 1934
h-7
18. Flock, E. V., J. C. Cain, J. H. Grindlay, and J.,L. Bo Hr
man, Federation Proceedings. 6:252, 19^4-7
19. Verzar, F., Biochem. Ztschr. . 270:35, 193^-
20. Klinghoffer, K. A., J. Biol. Chem. . 126:201, 1 9 3 8
21. Wood, H.O., Proc. Soc. Exper. Biol., April, I9I 4I 4, cited
by A. C. Frazer, Physiol. Rev. . 2o:llij., 19I 40
22. Sinclair, R. G., J. Biol. Chem. . ll|.0:cxvi, I9I 4I
2 3. Verzar, E. and L. Laszt, Bio chem. 2., 270:2l[., 193k-
2I 4. . Cera, B. and L. Bellini, Pathologica, 32:375? 19^ 4 - 0; Chem.
Abstr. , 35:l ^ h 19*4-1
25. Verzar, F. and L. Laszt, Bjochem. _2., 276:11, 1935
26. Stillman, N., C. Sntenman, E. Anderson, and I. L. Chaikoff,
Endocrinology, 31: *4-81, 19*1-2
27. Barnes, R. H., I. I. Rusoff, and G. 0. Burr, Proc. Soc.
Exptl. Biol, and Med. , Ii9: 8I 4, 19* 4-2
28. Barnes, R. H . , E. S. Miller, and G. 0. Burr, J. Biol. Chem, .
1I 4. 0: 2I 4. 1, I9I 4I
2 9. Bavetta, L. A. and H. J. Deuel, Jr., Am. J. Physiol. .
136:712, 1914-2
30. Chaikoff, I. L., Physiol. Rev^ 22:291, I9I 42
31. Artom, G., G. Sarzana, and E. Segre, Arch, intern, physiol*,
1 4. 7:2145, 1938,* Chem. Abstr* 32:9202, 193B
32. Fries, B. A., S. Ruben, I. Perlman, and I. L. Chaikoff,
J. Biol. Chem. , 123:587, 1938
33. Perlman, J., S, Ruben, and I. L. Chaikoff, Ibid., 122:169,
1937-38
3I 4. Robinson, A., I. Perlman, S. Ruben, I. L. Chaikoff,
nature', 1* 41: 1 1 9, 1938
1 ^ 8
35. Barnes, R. H., E. S, Miller, and G. 0. Barr, Proc. Soc.
Exper. Biol, and Med. , 1939
3 6. Fishier, M. C., C. Entenman, M. L. Montgomery, and I. L.
Chaikoff, J. Biol. Chem. , l50:l{.7, 19M-3
37* Frazer, A. C., Physiol. Rev. , 26:103, 19[f-6
3 8. Schmidt-Nielson, K., Acta. Physiol. 8cand. , Vol. 12,
Suppl. 37, 19^6
39* Zilversmit, D. B., I. L. Chaikoff, and C. Entenman, J.
Biol. Chem. , 172:637, 19^8
I 4O. Sinclair, R. G., Ibid., 13^ 4- 583» 19^1-0
1 4.1. Hevesy, G. and L. Hahn, Kgl. Danske Videnskab. Selskab.
Biol. Medd„ 15,. No. "WT 19li0; Chem. Abstr. , 3 6:1 3 8, 1^2
1 4. 2. Chargaff, E., J. Biol. Chem. . 128:587, 1939
1 4 - 3 • Hahn, L. and H. Typgn, Arkiv. Kemi. Mineral Geol. 21A,
No. 11:1, 1 9 l . | . 5 ; chem. Abstr „ L>-0:6599, I9EF”
i p i - l . Schoenheimer, R. and D. Rittenberg, Physiol. Rev^ 20:218,
I9I 4- O
45. Sinclair, R. G., J. Biol. Chem. , 111:515, 1935
l j - 6 . Taurog, A., C. Entenman, B. A. Fries, and I. L. Chaikoff,
Ibid., 155:19, 1944
I 4. 7. Bloor, W. R., Ibid., 2l+:i^7, 1916
l j . 8. Schoenheimer, R. and D. Rittenberg, Ibid., 111:169, 1935
1l9. Hunter, F. E., Ibid., l i | J 4:k3 9, I9I 4.2
50. Haven, F. L. and S. R. Levy, Ibid., llpl:lj-17, 19^1
51. Hevesy, G. andHahn, L. K., Danske Vidensk Selsk. Biol.
Medd. 15:5, 19^-0, cited by A. C Taurog, C. Entenman,
B. A. Fries, and I. L. Chaikoff, J. Biol. Chem.,
155:19, 19Mi
>9
52. Entenman, C., A. Taurog, and I. L. Chaikoff, J. Biol.
Chem. . 155:13,
53. Winzler, R. and E. Meserve, Ibid., 159:395, 19^5
5k • Simonson, D. G-., M. Wertman, L. M. Westover, and J. W.
Mehl, Ibid., 166:7^7, 19¥>
55. Keston, A. S., D. Rittenberg, and R. Schoenheimer, Ibid.,
122:227, 1937
56. Chargaff, E., K. B. Olson, and P. P. Partington, Ibid.,
13^:505, 19i +0
57* Levene, P. A. and H. S. Simms, Ibid., 51:285, 1922
58. Parnas, J., Biochem. Z., 56:17, 1913
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Creator Kling, Ingrid Hellen (author)

Core Title The incorporation of deuterated fatty acids into the phospholipids of the liver and intestine during fat transport

Degree Master of Science

Degree Program Biochemistry

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-776174

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