Studies on the mechanism of fat absorption using isotope labeled compounds.

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Content STUDIES ON THE MECHANISM OF FAT ABSORPTION
USING ISOTOPE LABELED COMPOUNDS
t>y
Wladimar Pavlovich Skipski
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
(Biochemistry)
June 1956
UMI Number: DP21571
All rights reserved
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U N IV E R S IT Y O F S O U T H E R N C A L IF O R N IA
G R A D U A T E S C H O O L
U N IV E R S IT Y P A R K
L O S A N G E L E S 7
•ph. o' fB > o - S l° S74#
This dissertation, written by
. ... Wladimir-P-^-_Skipski........
under the direction ofifLS-Guidance Committee,
and approved by a ll its members, has been pre
sented to and accepted by the Faculty of the
Graduate School, in partial fu lfillm ent of the
requirements fo r the degree of
D O C T O R O F P H I L O S O P H Y
* Dean
a * 5 ....
Guidance Committee
Ch , ‘
f t . flfedytyMt - Go*
Chairman
(rs fm ±
........
This Dissertation
is
Dedicated to the Memory
of
Dr. Harry J. Deuel, Jr.
! ACKNOWLEDGEMENT
I would like to take this opportunity of
i
; expressing my high appreciation to my Faculty Guidance
i
Committee for their advice during the period in which this j
i
j work was done.
1 I am particularly indebted to Professor Harry J.
i !
t j
[Deuel, Jr., for giving me the financial opportunity to
; undertake my graduate studies.
1 am also pleased to acknowledge the financial
; assistance of Swift and Company, and the laboratory space
made available by the Allan Hancock Foundation.
TABLE OF CONTENTS
! PAGE
f
| INTRODUCTION AND LITERATURE REVIEW ....... I
!
I Early hypothesis of fat absorption ......... 1
Verzar's lipolytic hypothesis of fat
; absorption . ...... ................. 4
| Frazer's partition hypothesis of
1 fat absorption............. 10
Present views on fat absorption ....... 24
j Intraluminar phase of fat absorption . . . 27
Digestion of triglyceride ............. 27
The formation of new ester bonds
during the hydrolysis of glycerides
by the pancreatic lipase..... 37
Absorption and transport of the absorbed
material.......................... 46
Absorption of triglycerides or free
fatty acids and the composition of
lymph lipids.................. 46
Absorption of esters of fatty acids
with alcohols other than glycerol . . 61
.... - - - - — i v
PAGE
Absorption of glycerol ............... 64
Cellular phase of fat absorption ........ 67
Composition of the lipids in the
intestinal tissue after administra
tion of triglyceride or free fatty
acid and the role of the phospho
lipid in fat absorption........ 67
The incorporation of labeled glycerol
into the lipids................ 84
The extent of hydrolysis of the fat
preceding absorption ................... 91
Statement of the problem and plan
of attack........................ 99
MATERIALS AND METHODS...................... 105
Synthesis of labeled compounds .................. 105
Synthesis of l,3-dioleyl-2-deuterio-
stearyl-glyceride-C^............ 106
Synthesis of deuteriostearic acid . . . 107
Synthesis of deuteriostearyl chloride . 107
Synthesis of symmetrical diolein .... 108
PAGE
Synthesis of l,3-dioleyl-2-deuterio- .
stearyl-glyceride-C^............ * * 109
Synthesis of mono-l-deuteriostearyl-
I
glyceride-C^ 112 '
i
Techniques of feeding and isolation of lipid j
samples 114 j
Method of feeding 114 ;
Isolation of tissues ..................... 115
i
Isolation of lipids from.the.intestinal . * 1
tissue, liver and carcass ........... 117
The evaluation of methods of,the extraction
of lipids from the intestinal
tissue and liver ....««». . * * * 118 :
Analytical p r o c e d u r e s 120
Separation of mono-, di- and,triglycerides.
and free fatty acids in the neutral
fat fraction.................. . . . . 120
Chemical determination of monoglyceride . . 124
Determination of free glycerol........ .. 125
Determination of radioactivity and deuterium j
atom per cent of enrichment . .... . . . 126.'
v i
PAGE
Measurement of radioactivity ...... 126
Measurement of the deuterium atom per cent.
of enrichment.............. .......... 132
EXPERIMENTAL RESULTS ................. 137
Extent of the hydrolysis of the digested
fat preceding absorption............ .. 137
Investigation of the role .of lower . . < , . (
glycerides in fat absorption. ....... 149
The investigation of the nature of
glycerides absorbed during the.. . - ...
digestion of triglyceride .......... .. . 160
Incorporation of glycerol-C-^ into
rat lipids ..................... 164
DISCUSSION............................. 187
Evaluation of the data concerning the extent
of hydrolysis of fat preceding absorption . 187
The nature of the glyceride penetrating into
the intestinal tissue during fat
absorption . ........................... 193
“........... " " v i i
PAGE
The composition of the neutral fat fraction
isolated from the different tissues .... 195
The incorporation of glycerol-C^ into .the
intestinal tissue lipids , . . . . . . . . . 199
Pathways of the syntheses of the intestinal 1
tissue phospholipid . . . . ....... . . . . . 207'
A new hypothesis of fat absorption........... 210
SUMMARY, AND CONCLUSIONS . ............ 216
BIBLIOGRAPHY : > .............. . 221'
LIST OF FIGURES
FIGURE PAGE
1. The Extent of Incorporation of Glycerol-
into the Intestinal Lipids
(in Per Cent of Labeling) after
the Administration of Free
Glycerol-C^ with or without
Fat .................................. 184
2. The Possible Pathway of the Inter
conversion of Neutral Fat and
Phospholipid in the Intestinal
Tissue ............................... 211
LIST OF TABLES
TABLE
I.
II.
III.
PAGE
rI4
The —--- Ratios of Neutral Fat Isolated
D
from Intestinal Tissue in Different
Time Periods after the Administration
of l,3-Dioleyl-2-Deuteriostearyl-
14
Glyceride-C with or without Non-
Labeled Free Glycerol................. 140
The Distribution of- Glycerol-C^ and
Deuteriostearic Acid in the Lipids of
Rats 3 Hours after the Administra
tion of 0.50 cc. l,3-Dioleyl-2-
Deuteriostearyl-Glyceride-C^........ 141
The Distribution of Glycerol-C^
and Deuteriostearic Acid in the
Lipids of Rats after the Adminis
tration of 0.50 cc. 1,3-Dioleyl-
14
2-Deuteriostearyl-Glyceride-C
and Glycerol......................... 142
TABLE PAGE
IV. The Distribution of Glycerol-C^4 and
Deuteriostearic Acid in the Lipids of
Rats 6 Hours after the Administration
of 1,3-Dioleyl-2-Deuteriostearyl-
Glyceride-C14 . . . . ................. 143
V. The Distribution of Glycerol-C^4 and
Deuteriostearic Acid in the Lipids
of Rats 20 Hours after the Adminis
tration of l,3-Dioleyl-2-Deuterio-
stearyl-Glyceride-C'*-4 ................. 146
VI. The Composition of Neutral Fat Isolated
from the Intestinal Tissue of 48
Hours Fasted Rats.................. . 132
VII. The Distribution of Glycerol-C*'4 and
Deuteriostearic Acid in the Lipids
of Rats 3 Hours after the Adminis
tration of Monostearin (Mono-1-
Deuteriostearyl-Glyceride-C^4) .... 155
TABLE
VIII.
IX.
PAGE
14
The Distribution of Glycerol-C and
Deuteriostearic Acid in the Neutral
Fat Fractions of the Intestinal
Tissue 3 Hours after the Adminis
tration of Doubly-Labeled Mono-?
stearin (Mono-1-Deuteriosteary1-
Glyceride-C14) ....... ........ 159
The Distribution of Glycerol-C"*"4 and
Deuteriostearic Acid in the Neutral
Fat Fractions of the Intestinal .
Tissue 6 Hours after the Adminis
tration of l,3-?Dioleyl-2-Deuterio-
stearyl-Glyceride-C*-4 ................. 162
The Distribution of Glycerol-C^"4 and
Deuteriostearic Acid in the Lipids of
Rats after the Administration of 0.5 cc.
1,3-Dioley1-2-Deuteriosteary1-Glyceride
and 0.25 cc. of Glycerol-C"*"4 ........ 167
TABLE
XI.
XII.
XIII.
XIV.
PAGE
14
The Distribution of Glycerol-C and
Deuteriostearic Acid in the Lipids
of Rats after the Administration of
0.5 cc. l,3-Dioleyl-2-Deuterio-
steary1-Glyceride and 0.5 cc.cf
Glycerol-C^.............. 168
The Incorporation of Glycerol-C^.
Administered in Different Doses with
or without Simultaneous Adminis- .
tration of Triglyceride into Rat's
Lipid............ 172
The Per Cent of the Exogenous Glycerol-
• C ^ Utilized for the Resynthesis. of . .
Triglyceride in.the Intestinal
Tissue During Fat Absorption ..... 176
The Incorporation of Glycerol-rC-1 -^ into
the Neutral Fat Fractions Isolated
from the Intestinal Tissue after the
Administration of Glycerol-C^ with
or without Triglyceride............... 180
TABLE
XV. The Incorporation of Glycerol-C^*- into
the Neutral Fat Fractions Isolated
from the Intestinal Tissue after the
Administration of Glycerol-C^ with
or without Triglyceride ...............
XVI. The Incorporation, of Glycerol-C?-^,
Administered Intraperitoneally into
the Lipid Fractions of Rats ......
xiii,
PAGE
\
j
i
i
\
181;
i
i
i
186!
INTRODUCTION AND LITERATURE REVIEW
j
!
j The mechanism of fat absorption has been the object
!of study for many years, but, as yet, there is no common
|agreement as to the exact processes involved. Several
i
!hypotheses have been proposed to clarify this mechanism,
I
but in no case has such a hypothesis explained a sufficient
i
I number of the experimental observations to be held com-
l
^pletely valid for any extended period of time.
Early Hypotheses of Fat Absorption
Several hypotheses of the mechanism of fat absorp
tion were postulated in the second half of the nineteenth
century. The principal ideas expressed in these hypothe
ses concerned the problem of whether the complete hydrol
ysis of ingested fat to fatty acids and glycerol is,a
prerequisite for absorption, or whether a triglyceride can
penetrate into the intestinal wall as such in the form of
small droplets.
The appearance of the fine emulsion in the intes-
I
'tinal contents as well as in the lymph of the thoracic
- - 2
duct during fat digestion led Munk (1,2) to the conclusion
i
I that fat was absorbed without any chemical alteration, in
1 the form of fine droplets. Schafer (3) believed that the
leucocytes aided in the process of transfer of the fat into
'the epithelial cells by engulfing fat particles. However,
jbefore Munk offered his hypothesis some experimental data
j indicated that products of triglyceride hydrolysis could
also be absorbed. Radziejewski (4,3) demonstrated that
free fatty acid, fed in the form of soap, was absorbed.
i
Perewoznikoff (6) showed that alkali soap and glycerol
t
were absorbed and synthesized into fat; the lacteals had
the same appearance as after a fat meal. This was con
firmed by Munk and Rosenstein (7) and much later by Bang(8).
A number of workers investigated the absorption of
esters of fatty acids other than triglyceride. Munk and
Rosenstein (7) fed amyl and cetyl esters of fatty acids,
while Frank (9) and Argyris and Frank (10) fed the ethyl
esters of fatty acids and also monoglycerides. In all
these cases the material was easily absorbed and it was not
possible to find any traces of unsplit esters in the lymph.
The fatty acid components appeared in the lymph as
;triglyceride, showing that a synthesis with glycerol had
jtaken place. These data were interpreted as evidence of
i
the complete hydrolysis of the tested esters before their
i
{absorption.
(
On the basis of these experimental results,
Pfluger (11) proposed his hypothesis of fat absorption in
I
1900. He considered that the fat, before absorption,
was completely hydrolyzed by pancreatic lipase to free
fatty acids and glycerol. The liberated free fatty acids
{were neutralized by the alkali present in the bile and
pancreatice juice and formed sodium soaps. Sodium soaps
and glycerol were water soluble compounds and therefore
easily transported across the mucosal wall. The resynthe
sis of triglyceride from these absorbed compounds took
• *
place within the cells of the mucosa. Pfluger denied the
possibility of absorption of fat in the unsplit form, but
none of the work quoted by Pfluger gave proof of his state
ment that all triglyceride must be hydrolyzed before
absorption. Actually, at that time, the experimental
evidence was not sufficient to support or disprove Pfluger's
> soap hypothesis.
4
I
i
■ Verzar's Lipolytic Hypothesis of Fat Absorption
! Gradually, experimental observations which were
•not entirely in agreement with Pfluger's hypothesis began
;to accumulate. The main difficulty in Pfluger's hypothesis
i
l
:arose when the actual reaction of the small intestinal
i
' contents was tested. Pfluger always considered it to be
i
| alkaline.. Although the pancreatice juice has a pH of 8 or
i ' '
even higher, the reaction of the small intestinal contents,
i
1 due to the acidic material entering from the stomach, is
;slightly acid, as has been demonstrated by Kostyal (12) in
Verzar's laboratory. Kostyal demonstrated that the pH of
i
.this area in rats, dogs, guinea pigs and pigeons, was in
the acid region and never rises above pH 7. These data
were substantiated by Robinson (13) who found a reaction
in the duodenum equal to pH 6.3, and in the ileum equal
to pH 7.5 - 8 in rats.
Jarisch (14) demonstrated that sodium salts of
most of the common long-chain fatty acids such as oleic,
palmitic and stearic, are stable only in alkaline solution
above pH 9. At a lower pH, which is normal for the intes-
ltinal contents, they do not exist as soaps but as water-
l
insoluble fatty acids.
I These data demonstrated that Pfluger*s hypothesis
'of absorption of water soluble soaps formed during the
jdigestion of triglyceride was not plausible.
; Revision of Pfluger*s hypothesis was undertaken
i
jby Verzar and his collaborators (15,16,17). They postu
lated a new so-called Lipolytic Hypothesis for the absorp-
jtion of fat. It was originally outlined in 1936 by
j
Verzar and McDougall in their monograph "Absorption from
Ithe Intestine" (16). The authors considered that total
i
'.hydrolysis of fat to free fatty acids and glycerol is a
i
Iprerequisite for absorption. But the proposed mechanism
t
Jof absorption and the factors involved in this process
I
differed essentially from Pfluger*s original hypothesis.
Verzar (15) tested the conditions under which fat
could be absorbed from the washed intestine of dogs with
-t
ligatures closing the common duct and the ileocaecal valve.
i
An appreciable absorption of fat occurred only when tri
glyceride was given together with lipase and sodium
i
taurocholate, or when free fatty acid was administered
I
with sodium taurocholate. In the case where experimental
dogs were given only fat and lipase, the hydrolysis of fat
took place, but the author did not observe any absorption
)
'of the soap formed. Verzar concluded from these results
i
that for the absorption of neutral fat there are two pre
requisites: hydrolysis of fat to free fatty acids and
glycerol, and the presence of bile acids.
The favorable action of bile upon the absorption
J of fat had been known for many years. Nencki (18) and
i Bruno (19) demonstrated that bile increased the action of
! lipase on the fat. They attributed this fact mainly to
j the emulsifying action of the bile. However, Furth and
Schtitz (20), Magnus (21) and Terroine (22) concluded that
the increase of the lipase action by bile cannot be
explained by its emulsifying action, but is due to a spe
cific action as a coenzyme. Verzar and McDougall (16)
attributed the main role of bile in fat absorption to the
hydrotropic action of glyco- and tauro-cholic acids
(so-called conjugated bile acids). It was first pointed
! out by Neuberg (23) that certain substances have the power
to make water-insoluble substances water-soluble. He
termed them hydrotropic substances. Verzar and Kuthy (24)
demonstrated experimentally in vitro the hydrotropic actLcn
7
of conjugated bile acids on fatty acids. The addition,of
conjugated bile acids to a turbid emulsion of fatty acids
in water caused clarification of the solution. It was
shown that the fatty acids in such a solution were in a
diffusible form in an acid as well as in an alkaline
medium. The importance of the conjugated bile acids in
the intestinal canal, therefore, is mainly that they form
water-soluble and diffusible complexes with the water-
insoluble and non-diffusible higher fatty acids.
Verzar and Kuthy (24) performed an experiment to
test how much fatty acid can be absorbed in vivo with a
certain quantity of conjugated bile acid. A definite
amount of fatty acid, together with graded amounts of
taurocholic acid, was injected into an isolated loop of
a dog's small intestine, tfhereas in vitro three times as
much bile acid as fatty acid was needed to make all of it
diffusible, in vivo several times, less bile acid was
required in order to secure the absorption of the same
amount of fatty acid. The authors presumed that the
conjugated bile acids became adsorbed at the intestinal
surface, where they dissolved more and more fatty acid
molecules and brought them into the mucosa cells. It was
demonstrated experimentally that around 50 per cent of the
ibile acids in the intestinal lumen were actually strongly
ibound by the mucosa cells.
i
Another distinct feature of Verzar's hypothesis
*
is the remarkable role played by the phosphorylation in
|mucosa during the process of resynthesis of triglyceride
I
from the absorbed fatty acids. This was based on the
I
following experimental observations. Verzar and Laszt (25)
I
!demonstrated the increase of absorption of oleic acid admin-
' #
i
istered with conjugated bile acids into the isolated
intestinal loop of rats when glycerol and phosphoric acid
{were given simultaneously. An even greater increase of
I
absorption of fatty material occurred when the investiga
tors administered glycerophosphoric acid. However, the
administration of glycerol alone or phosphoric acid alone
did not increase the absorption of fatty acid from the
intestinal loop. Sinclair (26) found that after feeding
animals a fat with a high iodine number, the phospholipid
in the mucosa acquired fatty acids with a high iodine
; number, although the absolute amount of phospholipid in
j
the mucosa was not essentially changed. He concluded
9
that the absorbed fatty acids formed phospholipids, fol
lowed by their breakdown and the formation of neutral fat
and a phosphoric acid - base residue. The latter recom
bined with a new portion of fatty acid to form phospho
lipids which thus continue to assist in the resynthesis
of the absorbed fatty acids into fat.
Reicher (27) found an increase of lecithin in the
blood together with neutral fat during fat absorption.
Sullmann and Wilbrandt (28) and Frolicher and Sullmann
(29) observed the increase of phospholipid in the lymph
collected during fat.absorption. The average phospholipid
content.in the enteric lymph of rabbits during fat absorp
tion was 0.140 per cent, whereas fasting animals had only
0.067 per cent. However, the quantity of phospholipid
in lymph in relation to neutral fat in fasting animals or
during fat absorption was always from 7.3 - 13 per cent
of the total fat. No change in the phospholipid content
was observed in. the portal system during fat absorption.
Several types of experiments were performed in
Verzar's laboratory in order to confirm the importance of
phosphorylation for the absorption of fat. Verzar and
_ , _ ro " 'i
I
Laszt (30) demonstrated the inhibitory effect of monoiodo-
acetic acid and phlorhizin upon the absorption of fat, in-
i
!
spite of the fact that the-action of 'lipase was not ;
inhibited and the extent of hydrolysis of the digested fat j
, ‘ 1
was normal. This action was attributed entirely to the j
inhibition of phosphorylation during fat absorption.
Verzar and Laszt (31,32,33) also showed that rats,
I
following a double adrenalectomy, had a capacity to !
t
absorb fat reduced to 15 per cent of normal. The adminis- j
t
tration of adrenalcortical extracts restored the process j
|of fat absorption in such adrenalectomized animals (34). 1
i 1
»
jln 1941 Bavetta, Hallman, Deuel and Greeley (35) confirmed j
l
that the rate of fat absorption is decreased in adrenal
ectomized rats even when they are properly maintained with ■
salt solution.
j
| Verzar et al. (16) considered that all absorbed
!fat, under normal*physiological conditions, leaves the
!
jintestine by the lymphatic system.
| *
i Frazer’s Partition Hypothesis of Fat Absorption
i The idea that complete hydrolysis of neutral fat
is not an obligatory stage for the absorption of
11
I triglyceride has arisen many times in this century.
1 Mellanby (36), Channon and Collinson (37) and Drummond,
i Bell and Palmer (38) considered that finely emulsified
! neutral fat can be absorbed as such, without hydrolysis.
This view has been further developed by Frazer (39,40,41).
j The first time he postulated his Partition Hypothesis was
| in 1939 (39). Since that time he has attempted to coor
dinate his hypothesis with the progress in the experi-
i
l
j mental work in this field and the hypothesis has therefore
jbeen the subject of many alterations (42,43,44).
i
Frazer originally started to investigate the
factors which were capable of emulsifying fat both in vivo
and in vitro at the slightly acid pH prevailing in the
; lumen of the small intestine. The size of the particles
)
of digested fat in the upper part of the small intestine
is usually less than 0.5 jul in diameter. In seeking the
answer as to what compounds naturally occurring in the
intestine might emulsify the fat to such an extent, Frazer,
Schulman and Stewart (45) investigated the action of fatty
acid (soap), cholesterol, bile salts and monoglyceride.
Analysis of the emulsion from the intestinal lumen,
- 12
I conducted by Frazer et al. (46,47) showed that it contained
<
fatty acid, mono-, di-, and triglycerides, but no phospho-
1 i
j lipid could be demonstrated. The flocculation pattern of
i
1 the intestinal emulsion was that of simple, negatively
i
i
charged particles. These results together with model
! experiments (45) permitted Frazer (40,41) to conclude that
i emulsification in the intestinal lumen is determined by
i
; the triple system of the fatty acid/bile salt/monoglyceride.
; Frazer believed that under the normal conditions prevailing
i
| in the intestinal lumen the hydrolysis of the ingested fat
I
: is only partial and the splitting of the fat molecule was
not regarded as an obligatory step in the absorption pro
cess. It was assumed that about 2/3 of the ingested neu
tral fat is absorbed as triglyceride in the form of a
. very fine emulsion and about 1/3 of the fat incompletely
hydrolyzed to di- and monoglycerides and fatty acids. The
process of such hydrolysis and the formation of the lower
glycerides and fatty,acid are essential for the emulsifi-
cation of the neutral fat. Frazer based these conclusions
on the experiments in vitro with hydrolysis of triglyc
eride by the pancreatic lipase (46,48). The pancreatic
13
lipolysis of the long-chain fatty acid triglyceride is
restricted and comes almost to a halt,in vitro after 20-
!
30 per cent of the combined fatty acid has been liber
ated. The addition of more lipase causes no increase in
the hydrolytic rate, but the addition of more substrate
, may do so, although this would not increase the total
I percentage of the hydrolysis (for further discussion see
i
p.27 ).
1
Frazer (40,41) did not think that in the intes-
i
tinal lumen the removal of fatty acid and glycerol by
absorption would allow hydrolysis to proceed to completion.
It is doubtful whether or not one can justify the applica
tion of the Law of Mass Action to such an inhomogeneous
system where many other factors are involved in the deter
mination of the hydrolytic rate. Thus, according to
Frazer, as hydrolysis proceeds the oil/water interface
must become loaded with oil-soluble products, especially
those which contain hydrophilic groups such as free fatty
acid. The accumulation of fatty acid will be found to
displace the enzyme from the oil/water interface with a
; consequent interference with hydrolysis. This effect can
1 4
! be avoided if the cleavage products are removed from the
i
I interfacial film. However, the fatty acids cannot be
I removed into the aqueous phase because they are not water-
!
, soluble and the prevailing pH excludes the formation of an
i • •
■ appreciable amount of water-soluble soap. The fatty acid
j might form water-soluble complexes with the bile salts,
! but it was pointed out even by Verzar et al. (15,16,17)
i
that the amount of bile salts available in the intestinal
‘ lumen is not sufficient to account for the absorption of
j fatty acid by this mechanism, and it was therefore postu-
i lated that bile salts exerted their hydrotropic action at
the. surface of the intestinal cell. Only in the case of
, short-chain fatty acids, which are water-soluble, can the
interface be cleared.
When pancreatic lipolysis was carried out under
the conditions prevailing in the intestinal lumen, Frazer
i
et al. (46) were unable to demonstrate the liberation of
! glycerol. In other experiments, Frazer (49) showed that
I
complete inhibition of the hydrolysis of fat in the intes
tinal lumen of rats by sodium cetyl-sulfate did not pre
vent its absorption. On the other hand, sodium cetyl-
sulfate is an excellent emulsifying agent for the fat.
15
! Therefore these experiments indicated that triglyceride
I
j may be absorbed without previous hydrolysis if it is pro-
I - . . . . .
! perly emulsified. However, Frazer admitted that if the
! finely dispersed, partially hydrolyzed fat is not removed
■ ( _ . . . . ..
: and the reaction of the aqueous phase becomes more alka-
f
i line, or if the fat is a short-chain triglyceride, such as
tributyrin, more extensive hydrolysis may occur.
i . . . . .
In developing his hypothesis for the absorption
I - .................
< of the triglyceride emulsion, Frazer considered the pos
sibility of an anatomical structure suitable for the
accomplishing of this transfer of small droplets. From
; the study of intestinal tissue, mainly of amphibia, J. R.
Baker (50) came to the conclusion that the outer border
of the intestinal cells contains a system of fine canals,
running at right angles to the surface, through which
particles of rather less than 0. 5 j j. in diameter might
pass.■ Wotton and Zwemer (51) have published photographs
. which they claim show fat passing through canals in the
membrane into the intestinal cell.
Frazer et al. (45,52) tested his hypothesis with
ifinely emulsified paraffin particles. If the partition
16
I
hypothesis is correct and finely dispersed fat particles
,can pass through the intestinal membrane, finely dispersed
iunsaponifiable paraffins must also pass by the same route.
I . . . .
jThe authors demonstrated that when a paraffin emulsion,
jhaving an average particle diameter of less than 0.5 ,
was introduced intraduodenally into rats, extensive absorp-
s
jtion took place.
!
Frazer (53) also investigated the route of absorp-
i
ition of ingested triglyceride and free fatty acid. When
i
I
'triglyceride was fed to rats, the intestinal cells were
i
I . . . . .
i ,
filled with large globules, the lacteals had a milky ap-
pearance, and there was a characteristic post-absorptive
systematic lipemia. In the animals which received fatty
i
acid with glycerol the intestinal cells exhibited only a
fine granular deposition, there was no appreciable milki-
ness in the lacteals and there was no post-absorptive
systematic lipemia. The portal blood, however, showed an
i
increase of fatty material. Frazer also used fat stained
with sudan in long term experiments. When sudanized olive
oil was fed, staining of the fat depots was found, while
| sudanized fatty acid fed over a ten day period resulted
in a striking accumulation of stained fatty material in
the liver. According to Frazer, these observations can
only be explained by the partition hypothesis. The split
and unsplit fractions of neutral fat are absorbed by dif
ferent mechanisms into the intestinal walls, where they
give rise to different histological pictures, and from
which they pass by different routes to different destina
tions. Fatty acid passes by the portal vein to the liver,
while neutral fat goes by the lymphatic route to the blood
and then to the fat depots. In other experiments Frazer
(49) observed that the addition of extra lipase to digested
fat shifted the absorption picture to a situation where
rats absorbed free, fatty acid: resulting in appearance of
fine granular deposition in the intestinal cells, portal
rather than systematic.lipaemia, and deposition of fat in
the liver rather than in the fat depots.
Frazer and Stewart (54,55) have standardized the
chylomicron technique proposed earlier by Gage and Fish
(56) and used it for the demonstration of the quantitative
difference in fat transport depending on whether the
animals ingested triglyceride or free fatty acid. The
18
'number of chylomicrons present runs parallel with the
amount of fat present in the blood. The validity of the
t
ichylomicron technique has recently been confirmed by
'several investigators (57,58). Frazer (49) found high
;chylomicron counts in the systematic blood when rats
received triglyceride and low counts when fatty acid was
I '
fed. Just the opposite picture was observed in the portal
jblood. Addition of lipase to ingested neutral fat
i
idecreased the chylomicron counts, whereas inhibition of
I lipase activity by sodium cetylsulfate increased the
j
chylomicron counts to above normal in the systematic blood.
The partition hypothesis denied the importance of
phosphorylation in the absorption of fat. The alteration
:of fat absorption caused by adrenalectomy was explained by
! Frazer on the basis of a disturbance of the normal electro**
;lyte balance. Since fat particles are negatively charged,
their absorption should be affected by the disturbance of
the electrolyte balance. Frazer concluded that this
hypothesis was supported by the experiments of Bavetta,
Hallman, Deuel and Greeley (35) and Barnes with collabora
tors (59,60), who demonstrated that adequate salt therapy
j improved the fat absorption. However, Bavetta, Hallman,
JDeuel and Greeley (35) observed that even salt-treated
iadrenalectomized rats still showed some depression of the
! I
I fat absorption. This depression could be completely
j
j restored only by the administration of the adrenal-
|cortical hormone.
In another paper, Bavetta and Deuel (61) demonstra-
1
'ted that the absorption of tributyrin and other butyric
i
esters were not affected by adrenalectomy. Frazer (62)
j explained these last observations as showing that adrenal
ectomy influenced only the absorption of negatively charged
unhydrolyzed particles of the long-chain fatty acid glycer-
!ides. Tributyrin and other short-chain fatty acid glycer-
i
!ides are completely hydrolyzed during digestion to free
j fatty acids and glycerol because these free fatty acids are
«
'water-soluble and easily removed from the water/oil inter
face. Frazer believed that all free fatty acids were ab
sorbed in the soluble form through the portal system.
*
1 In spite of the differences between the lipolytic
and partition hypotheses in explaining the complete mecha-
jnism of fat absorption, there is some general agreement on
20
the many changes which fat may undergo during digestion
and absorption. The occurrence of emulsification, hydrol
ysis of ingested fat, formation of phospholipid in the
intestinal tissue together with the importance of bile
acids and the adrenal cortex in the process of normal
fat absorption are recognized by both hypotheses. The
difference lies in the emphasis placed upon their rela
tionship to the absorption mechanism.
Both hypotheses have undergone, extensive alterations
since they were proposed. At present, Verzar (17) accepts
the importance of the fatty acid/bile salt/monoglyceride
complex as an essential emulsifying agent, but as yet
does.not concede the passage of unhydrolyzed glyceride
through the intestinal mucosa under normal physiological
conditions.
It should be mentioned that according to the
Schmidt-Nielsen (63) electrbmetric titration curve of oleic
acid, about 14 per cent of the acid was present as a soap
at pH 7 and 5 per cent at pH 6. In the presence of dodeca-
nesulfonic acid, which solubilized the fatty acid, the
corresponding fractions were calculated to be about 25
21
and 10 per cent respectively. The author proposed that
bile acids should act in the same manner. This means that
a considerable proportion of the long-chain fatty acid may
t
be present as' the soap even in the upper part of the small
<
i
intestinal lumen. This soap may contribute considerably
to the emulsification of the ingested fat as well as to
the absorption of fat.
Verzar (17) also admitted recently that in experi
ments where animals were fed with pure free fatty acids
the absorption did not necessarily proceed by the physio
logical route since there is an enormous overcrowding of
the product in the intestine, which, under physiological
conditions, appears only very slowly. Under such condi
tions some free fatty acid might escape the normal route
of absorption and appear in the portal system. Verzar
also agrees that occasionally an emulsion of fat may be
i
J
absorbed as such, but, in any case, this is not a normal
process of fat absorption.
On the other hand Frazer et al. (42,43,44) have
recognized the existence of an alternative pathway of fat
absorption. In circumstances where particulate absorption
2 2
!is hindered, further hydrolysis is probable in the ileum,
j and as a result of such a process a higher proportion of
jthe fat may be absorbed in the form of fatty acid and
I glycerol. Recently, Frazer, Pover and Sammons (44)
} . .
'recognized the importance of bile salts as a solubilizing
X
|agent for the otherwise insoluble, long-chain, free fatty
t
|acid.
1
i Frazer et al. (44) have abandoned the idea that
. long-chain fatty acids appearing in the intestinal lumen
i are normally absorbed through the portal system., They
'recognized that some process of resynthesis of triglyceride
from the lower glycerides and the free fatty acids may
occur in the intestinal tissue. These authors even pro
posed that the synthesis of new glycerides from the glycescbL
and free fatty acid in the intestinal tissue might proceed
through the formation of phospholipid. Frazer, French
and Sagrott (64) admitted that the phospholipids synthe-
i
' sized in the intestinal cells are important for stabiliza
tion of the fat particles for their further transport into
the lymph.
Verzar (17) violently criticized Frazer*s
23
staining technique. He insisted that lipid material and
dye-stuff (sudan IV) are absorbed independently. Since
j
Frazer et al. (43,44) do not at the present time emphasize
I this type of experiment and actually abandoned the con-
!
' elusion based on this technique, there is no reason to
I
discuss this question further.
i Nevertheless, the principal difference between the
:two proposed hypotheses remains the same: whether or not
j complete hydrolysis precedes the absorption of ingested
! fat under normal physiological conditions.
One of the basic concepts of the partition hypothe
sis is the presence of the small canals which perforate
the outer border of intestinal cells, as has been stated
iby J. R. Baker (50). Recent investigators have been unable
to confirm the existence of such a structure. Thus, Grapga:
and R. F. Baker (65) studied the structure of the intes
tinal epithelial cells and found no canal structure in
the outer border. Sjostrand and Zetterquist (66) found
that the intestinal surface is thickly covered with finger
like excresences— i.e., the inner border of the cells is
; a brush border. The outer surface is continuously coated
24
by a well-defined cell membrane, which has no pores,
canals, or holes.
At present none of the above discussed hypotheses
can explain all the available experimental material. In
the following chapter will be given the recent developments
of our knowledge concerning the mechanism of fat absorp
tion.
Present Views on Fat Absorption
Approximately six or seven years ago there began
/
a new period in the development of our knowledge of the
• ' i
processes involved in the absorption of fat. This period
is characterized by the introduction of several new
technical approaches to this particular field of physi
ology and biochemistry.
The extensive use of isotope-labeled triglycerides,
free fatty acids, glycerol and phosphoric acid has per
mitted the researchers to design different types of experi
ments which yielded information concerning the extent of
hydrolysis of fat preceding absorption,, the route of
resynthesis of triglyceride and phospholipid in the intes
tinal tissue and their transport to other parts of the
25
body.
Relatively simple methods were worked out in the
various laboratories for analytical estimation of the
. composition of the glyceride mixtures formed during
i
, hydrolysis as well as for the separation of such mixtures
into the free fatty acid, monoglyceride, diglyceride and
i triglyceride (67-74). These new methods permitted the
| study of the mechanism of the lipase action upon the
^ glyceride and determination of the actual composition
; the ingested fat in the intestinal lumen and even in the
intestinal tissue.
The collection of lymph from the intestine is
of fundamental importance for determination of the extent
of hydrolysis preceding absorption and for the determina
tion of the route of transport of the absorbed fat. In
earlier work the investigators were generally limited to
obtaining lymph from anaesthetized animals. As normal
fat absorption is dependent on a normal intestinal mobil
ity, such experiments have given, very often, incomplete
and erroneous results (75,76,77) because the intestinal
mobility is profoundly influenced by anaesthesia and
26
' surgical shock. Therefore a new technique of Bollman,
I
I
; Cain and Grindley (78) for the collection of lymph from
I
l
j the intestinal or thoracic duct of unanaesthetized rats
i
| with a "permanent" cannula is of great importance for the
I '
; investigation of fat absorption. Experimental rats with
!
| such a cannula can remain alive for a week and more. A
i
similar technique was also developed for dogs (79,80,81)
and cats (82) and modified for rats (83). Under this type
; • /
’ of cannulation it is possible to recover up to 90 per cent
I
; of absorbed fat.
i
i The application of these methods together with the
older methods has resulted in the present status of our
knowledge in the field of fat absorption. In presenting
! the more recent experiments the absorption of fat may be
■ considered in four aspects:
1. An intraluminar phase, during which the
glycerides are prepared for absorption.
2. The absorption and the transport of the ab
sorbed material.
3. The cellular phase of fat absorption, which
i
involves changes in composition of lipids in
the intestinal mucosa during the absorption
of triglycerides or free fatty acids, the
role of the phospholipids in fat absorption
.and the incorporation of exogenous glycerol
into the intestinal lipids.
4. The extent of hydrolysis of the fat preceding
absorption.
Intraluminar phase of fat absorption.
A. Digestion of triglyceride.
The most important of the gastro-intestinal
lipases is undoubtedly that supplied by the
pancreas. Some hydrolysis of ingested fat may
occur in the stomach due to gastric lipase (84)
or to pancreatic juice passed into the stomach
from the intestine as a result of antiperistal-
tic movements. The extent of hydrolysis of tri
glyceride in the stomach in any case is negli
gible, due to the strong acid reaction in this
organ (85,86). Recently Herting and Ames (87)
claimed that there is an appreciable hydrolysis
of the completely saturated triglyceride, but not
of the unsaturated triglycerides. However, until
it can be established unequivocally that the
apparent digestion in the stomach is not due to
regurgitation from the intestine into the
stomach, this exceptional observation should be
taken with reservations.
The action of pancreatic lipase was recently
investigated by Borgstrdfrn (88). He obtained
lipase by cannulation of the lower end of the bile
duct of rats. Since the pancreatic ducts in the
rat open into the lower half of the bile duct,
Borgstrom actually had a mixture of bile and
pancreatic juice. In this mixture, adjusted to
pH 6.3 and at 40° C, hydrolysis of corn oil pro
ceeded to nearly 100 per cent in 12 hours. The
rate of liberation of fatty acid as well as the
extent of lipolysis was not affected by the
presence of 21.8 per cent of oleic acid in the
corn oil. The difference between the results
obtained by Borgstrom and those of Frazer et al.
(46,48) must be sought in the activity of the
lipase (89). The addition of trypsin to the active
pancreatic juice preparation obtained by Borgstrom
changed the course of the hydrolysis to that
generally observed when extracts of pancreatic
powder were used. This indicates that the pres
ence of the proteolytic enzymes in active form in
the extracts or suspensions from the pancreas
inactivates the lipase and causes limitation of
the hydrolysis. Lipase, uncontaminated with
active trypsin, can completely hydrolyze neutral
fat in vitro.
Several authors have investigated the course
of hydrolysis of.triglyceride by pancreatic lipase.
Artom and Reale (90) demonstrated in vitro the
formation of lower glycerides during the digestion
of olive oil with extracts from dog pancreas.
Several years later Frazer and Sammons (46)
confirmed the formation of lower glycerides
during the pancreatic lipolysis of olive oil in
vitro and in vivo.
The picture of the formation of lower glycer
ides was further clarified by Desnuelle et al.
in several publications (91-94). These authors
demonstrated that the hydrolysis of coconut,
peanut and sunflower oils, when catalyzed in
vitro by pancreatic lipase, gave rise to only a
minimum quantity of free fatty acid (91). The
action of pancreatic lipase on fat was shown to
be a three-stage reaction (92,93). There first
occurred a rapid formation of diglyceride with
the liberation of one molecule of free fatty
acid. The second and third stages, which
involved the.removal of the second and third
fatty acid molecules from the glycerol, pro
ceeded very slowly. The authors suggested that
the affinity of lipase for the glycerides
decreases with the appearance of the free hydroxyl
groups. The course of the in vitro hydrolysis of
the triolein by a glycerol extract of pig pan
creas was strongly influenced by the composition
of the medium in which the reaction occurred.
In,the presence of bile acids and the absence of
calcium ions a 4 hour hydrolysis of triglyceride
31
resulted in the formation of mainly diglyceride,
a small amount of monoglyceride and only traces
of free glycerol (94). The addition of calcium
ions led to the accumulation of mostly monoglyc
eride. Tri- and diglycerides were practically
degraded within 11/2 hours (95). The amount
of free glycerol liberated was still insignificant.
In the presence of both calcium ions and a large
amount of bile acids approximately 1/3 of the
glycerides were completely hydrolyzed after 11/2
hours of lipolysis, while the remaining glycerides
consisted of a mixture of mono- and diglycerides
(96). Desnuelle and Constantin (97) also demon
strated that the intestinal contents of rats and
dogs contained some lower glycerides as well as
free fatty acid during ingestion of fat. The
presence of calcium ions in the digestive tract
augmented the proportion of monoglyceride and
diminished that of glyceride.
Desnuelle, Naudet and Constantin (96) con
sidered that the hydrolysis of triglyceride is
32
more or less random, resulting in the formation
of two molecules of 1-monoglyceride per molecule
of 2-monoglyceride. However, Desnuelle and Con
stantin (98) recently showed that in in vitro
experiments, for one mole of 1-monoglyceride,
2 to 4 moles of 2-monoglyceride were formed. The
predominance of 2-monoglyceride indicated, accord
ing to the authors,, that lipolysis proceeds
through a 1, 2-diglyceride stage.
Mattson, Benedict, Martin and Beck (99) fed
i .....
rats with 2-oleyldipalmitin. After an absorption
period of three hours, the composition of the
lipids of the intestinal lumen was as follows:
monoglycerides 13.3 per cent, about equally dis
tributed between 1-monoglyceride and 2-monoglyc-
eride isomers; free fatty acid 19.7 per cent;
diglyceride 22 per cent; and triglyceride 45
per cent. Ninety per cent of the monoglyceride
was monoolein. This indicated that; during the
lipolysis 2-monoglyceride was originally formed,
part of which later isomerized to 1-monoglyceride.
3 3
The course of the hydrolysis of triglyceride
in vivo, according to Mattson et al* is a series
of directed stepwise reactions from triglyceride
to 1, 2-diglyceride to 2-monoglyceride. A part
of the 2-monoglyceride isomerized to 1-monoglyc-
eride. This scheme of hydrolysis of long-chain
fatty acid triglyceride was confirmed by Mattson
and Beck (100) in vitro for an aqueous suspension
of steapsin and winterized cottonseed oil. Mat
tson, Benedict and Beck (101) also investigated
in vivo the course of the hydrolysis of tri-
caprin. The results obtained were similar to
those when rats were fed cottonseed oil.
Schonheyder and Volgvartz (102,103) studied
the action of pancreatic lipase on trilaurin and
tripropionin in vitro. They concluded that the
hydrolysis of trilaurin proceeded stepwise from
trilaurin to 1, 2-dilaurin to 2-monolaurin
whereas tripropionin was degraded only to 1, 2-
dipropionin. Then the reaction practically stop
ped. Apparently the pancreatic lipase is not
3 4
able to split off more than one acyl group from
the dipronpionin, as neither 1, 2-dipropionin nor
monopropionin are affected by this enzyme. It
. seems to be a condition for the cleavage of a
triglyceride that the chain length of the fatty
acid is not too short. This assumption was sub
stantiated in experiments where Schonheyder and
Volgvartz investigated the relative rates of
hydrolysis of the different monoglycerides with
pig pancreatic lipase. If one considers the rate
of hydrolysis for 1-monolaurin as 1.00, the rates
for 1-monocaprylin, 1-monobutyrin and 1-mono-
propionin are 1.8, 0.08 and 0.02 respectively.
However,.the triglycerides of short-chain fatty
acid are easily hydrolyzed in the intestinal
lumen.
Borgstrom (104) investigated the mechanism of
the action of lipase of rat bile— pancreatic juice
on olive oil in vitro. The degradation of the
olive oil, mainly to 2-monoglyceride, occurred
via 1, 2-diglyceride. However, the monoglyceride
fraction contained about 20 per cent of 1-mono-
glyceride after 1 hour of incubation. The
author did not determine whether this monoglyc
eride was formed directly from 1, 2-diglyceride
or by isomerization of 2-monoglyceride. Thus,
the majority of the investigators consider that
the hydrolysis of triglycerides of the long-
chain fatty acids is not random, as was originally
postulated by Desnuelle, Naudet and Constantin
(96), but proceeds through the 1, 2-diglyceride
to the 2-monoglyceride. Mattson et al. (99,100)
observed a rapid isomerization of 2-monoglyceride
to 1-monoglyceride whereas Borgstrom (104) and
Schonheyder and Volgvartz (102) did not demon
strate such a phenomenon. The cause of this
discrepancy is not apparent at the present time.
The sequences of the reactions involved in
the lipolysis of fat is very important for the
correct designation and evaluation of the results
where animals have been fed labeled triglyceride
or lower glycerides in order to determine what
products of digestion actually penetrate into the
intestinal mucosa.
Mattson et al. (99) and Borgstrom (88) investi
gated the composition of the intestinal content
of rats at different time intervals after the
administration of vegetable oil. A tendency was
observed for the relative amounts of free fatty
acid and monoglyceride to increase with the time
of digestion. Mattson et al. (101) tested the
effect of the addition of different amounts of
monoglyceride or free fatty acid to the cotton
seed oil fed to rats upon the content of the
intestinal lumen after a digestion period of
3 hours. The results indicated that the presence
of as much as 20 per cent of 1- or 2-monoglyceride
in the dietary fat did not influence the compo
sition of lipids found in the intestinal lumen.
When the amount of free fatty acid added was 27
per cent or more, the content of free fatty acid
in the unabsorbed lipids was higher than normal.
This indicated that at those.higher levels the
rate of entrance into the intestinal lumen of free
fatty acid was greater than the rate of its '
absorption.
t
Qualitatively similar results were obtained by
Borgstrom (88). However, the percentages of mono
glyceride and free fatty acid recovered from the
intestinal lumen after the administration of tri
glyceride or triglyceride together with added
free fatty acid are higher than those of Mattson's
group. This discrepancy is probably due to the
different amounts of fat originally administered
to the rats. Kuhrt, Welch, Blum, Perry, Weber and
Nasset (105), Harris, Chamberlain and Benedict
(106) and Blankenhorn and Ahrens (107) reported
the presence of free fatty acid, monoglyceride
and diglyceride in the intestinal content during
digestion of a fat-rich diet in human beings.
B. The formation of new ester bonds during the
hydrolysis of glycerides by the pancreatic lipase.
The ability of the pancreatic lipase to catalyse
synthetic as well as hydrolytic processes has
3g
been very well known for many years (108). Most
data concerning the synthetic action of the
lipase were obtained under artificial conditions
and therefore had no great physiological signif
icance.
In 1933, for the first time, Fabisch (109)
demonstrated the synthetic effect of pancreatic
lipase under conditions similar to those pre
vailing in the small intestine during the diges
tion of fat. Palmitic, stearic, or oleic acid,
emulsified by sodium oleate or desoxycholate,
was incubated with a large excess of glycerol
and pancreatic powder. The author observed a
decrease of free fatty acid amounting to a maximum
of 18.5 per cent after a 5 hour incubation period.
However, no attempt was made to isolate the
glycerides which were presumed to have been
formed.
Artom and Reale (110) allowed lipase from dog
pancreas to act upon a mixture of oleic acid and
an excess of glycerol in the presence of a small
39
amount of water. After standing 3-10 days at 38°
C, the esterification proceeded to 50-55 per cent
calculated on the basis of the amount of free
fatty acid remaining. The product synthesized
was largely diolein with a small amount of mono
glyceride. When the reaction period was prolonged,
55-65 per cent of the fatty acid became esterified.
The product formed consisted of 40 per cent of
triolein and the remainder was diolein. Thus,
even in the presence of a large amount of the
glycerol, the reaction did not stop at the mono
glyceride stage but proceeded to the formation of
the di- and triglycerides.
Borgstrom (88,111) observed that during the
incubation of corn oil and labeled palmitic
acid with a mixture of bile and pancreatic juice
in vitro the radioactivity appeared in the glyc
eride fraction of the digested mixture. This was
interpreted to mean that simultaneously with the
lipolysis, a resynthesis of the glyceride bonds
occurred with the free fatty acids. The exchange
of the glyceride fatty acid with free fatty acid
was only partial, as the relative specific activ
ity of the glyceride fatty acid at most reached
about 50 per cent of that which should have been
found if a total randomization had taken place.
The system reached equilibrium in approximately
3 hours. There must be a limiting factor for
the exchange which would mean that part of the
glyceride-bound fatty acids are not exchangeable.
A similar phenomenon of the incorporation of
14
C - labeled fatty acid into glycerides was
observed by Borgstrom in the intestinal lumen
when rats were fed a mixture of corn oil with
palmitic acid-C^. Among the glycerides recovered
from the intestinal lumen the highest activity
was in the diglyceride fraction, the lowest in
the monoglyceride fraction. In other experiments
Borgstroxn (111) fed rats with a mixture of 1-mono-
olein, oleic acid and palmitic acid-C^. The
intestinal lumen content was subjected to separa
tion in fractions. Significant amounts of di-
and triglycerices were found. The specific
41 1
activity of these two fractions was much higher
than that of the monoglyceride. These experi
ments, according to Borgstrom, indicated that
the incorporation of free fatty acid into glyc
eride during lipolysis in the lumen of the small
intestine is due to a real synthesis of new glyc
eride ester bonds and not only to a transesteri-
fication process. Some of the radioactivity
found in the fatty acids of the monoglyceride
recovered from the lumen of the small intestine
in these experiments must be due to a continuous
resynthesis of di- and triglycerides with the
I
labeled free acid, followed by a hydrolysis to a
monoglyceride again. Resynthesis of the glyceride
ester bonds during pancreatic lipolysis is limited
to an incorporation of free fatty acid into mono-
and diglycerides and, as soon as the fatty acid
has been split off from a monoglyceride, no
further resynthesis can occur.
In in vitro studies in the presence of large
amount of water, the failure of synthesis of
monoglyceride or other glycerides from glycerol
and free fatty acid was confirmed (89,112). In
the presence of an dxcess of water the reaction
glycerol + free fatty acid monoglyceride +
water must be so far to the left that this reac
tion for practical purposes can be considered as
irreversible. Probably the lipase acts at the
interface of the lipid/water phase and has a
very low affinity for the water soluble free
glycerol. According to Borgstrom's data, the
small amount of the 1-monoglyceride found during
the hydrolysis of triglyceride did not originate
from the direct esterification of glycerol mole
cule, nor from the isomerization of 2-monoglycer
ide, as was considered to be the case by
Mattson, et al. (99,100) but, partly at least,
was formed directly from the hydrolysis of 1,
2-diglyceride by the splitting off of the ester
bond in position 2.
Borgstrom (112) also demonstrated in vitro
that the incorporation of labeled fatty acid into
43
glycerides is not only due to a transacylation
process but also to the formation of new ester
bonds. During the lipolysis of 1, 2-diolein by
bile and pancreatic juice in the presence of
labeled oleic acid, triglyceride has been synthe
sized from the diglyceride and free fatty acid.
About 20 per cent of the original diglyceride
molecules have been converted into triglyceride
by the synthesis of the new glyceride ester
bonds. These findings proved that incorporation
of free fatty acid into glyceride during the
course of hydrolysis by the pancreatic lipase is
due, partly at least, to a synthesis of new
glyceride ester bonds. The high rate of the
incorporation of the labeled free acids into the
glycerides, however, indicated that an exchange
of the esterified fatty acids and free fatty acids
also took place.
Short chain fatty acid such as butyric acid
did not incorporate into glycerides either in
vitro or in vivo.
The in vitro experiments of Borgstrom (111) I
i
I
demonstrated that during the incubation of the j
i
triolein with labeled palmitic acid, bile and ;
i
pancreatic juice and an excess of water, only j
the fatty acids of glycerides in 1- and 3-
position rapidly came into equilibrium with the
palmitic acid-C-^, while the fatty acid in the j
2-position was not exchanged at all during the
course of hydrolysis.
The recent advances in investigation of the
intraluminar phase of fat absorption give the
following picture. The lipolysis of triglyceride
may proceed up to complete hydrolysis to free
fatty acid and glycerol. Lipolysis occurs throtgjb
a stepwise degradation of the ingested material
from triglyceride to diglyceride, to monoglyc
eride and to glycerol. In the intestinal lumen
during fat digestion, triglyceride as well as all
intermediate products of hydrolysis are usually
present. Only the presence of free glycerol has
as yet not been proved experimentally.
.. 45
Simultaneously with lipolysis there is both a
formation of new ester bonds, causing the synthe
sis of some higher glycerides from the lower
glycerides and free fatty acid, and an exchange
reaction between free fatty acid and glyceride
fatty acids, especially in positions 1 and 3.
The rate of hydrolysis is apparently more rapid
in the beginning of digestion because an equi
librium is soon reached between synthesis and
hydrolysis of tri- and diglycerides. The overall
hydrolysis, however, slowly proceeds to comple
tion since monoglycerides are hydrolyzed but
not resynthesized from the glycerol and free
fatty acid in the lumen.
The synthesis of new ester bonds, the
exchange reaction with free fatty acid and pos
sibly the isomerization of formed 2-monoglyceride
may cause the formation of some glycerides,
especially monoglyceride, which one would not
expect to obtain from the fat which was origi
nally fed if hydrolysis alone would occur.
46
This brings up the necessity of great caution
in the interpretation of the results obtained
from feeding experiments using labeled tri
glyceride of a definite structure and subse
quent isolation of the labeled products from
the intestinal lumen, intestinal tissue or
lymph. Actually during the digestion some
randomization of the fed triglyceride (113)
takes place.
Absorption and transport of the absorbed material.
A. Absorption of triglycerides or free fatty acids
and the composition of lymph lipids.
The oldest procedure for the elucidation of
the mechanism of fat absorption was the attempt
to recover the absorbed lipid material from the
lymphatic or blood system following the admin
istration of fat.
Munk (1,2) fed oleic acid to dogs with a
cannulated thoracic duct and found that there
was a considerable increase of the neutral fat
in the lymph. Later Munk and Rosenstein (7)
47
fed olive oil, oleic acid or erucic.acid to a
patient suffering from a lymphatic fistula of
the left leg and recovered up to 60 per cent of
the absorbed material from the lymph in the form
r
of triglyceride. After the administration of the
amyl or cetyl esters of fatty acid the authors
were still able to recover only triglyceride from
the lymph. Frank (9) and Bloor (114) fed mono
glyceride or fatty acid esters other than glyc
erides to animals and also isolated only tri
glyceride from the lymph.
Brockett, Spiers and Himwich (115), Sullman
"i"
et al. (28,29), Eckstein (116) and Little and
Robinson (77) observed the increase of the
neutral fat content of lymph in dogs and rabbits
during fat absorption. However, they were able
to recover only.a small part of the fat admin
istered. The assumption was generally made (114)
that the lymqph lipid represented,that fraction of
the ingested fat which was absorbed into the
circulatory, system via the lymph, whereas the
— - - 48
remainder of the ingested fat was absorbed
directly into the blood. This assumption per
sisted for many years in spite of the evidence
against it. Thus, Zucker (117) reported that
at no time during the course of fat absorption
did the total long-chain fatty acid concentration
in the serum of the portal vein differ to any
extent from the concentration of the same fatty
acid in the serum of the femoral artery. Winter
and Crandall (118) applied the technique of
angiostomy to otherwise normal, unanaesthetized
dogs. No significant difference in the lipid
content of the femoral artery and the portal
vein blood was observed during the ingestion of
olive oil. Winter and Crandall calculated that
if 10 per cent of the fat fed had been absorbed
by way of the portal vein the arterio-portal
difference should have been detected.
At the present time it is obvious that the
early attempts to recover the ingested fat
quantitatively from the thoracic duct lymph was
49
unsuccessful due to the impaired absorption
caused by anaesthesia and surgical shock. Most
of the administered fat remained in the intes
tines .
In 1948 Bollman, Cain and Grindley (78)
proposed a new procedure for the collection of
lymph from unanaesthetized animals. Application
of this new technique of lymph collection
together with the administration to animals of
labeled lipid material secured a reliable
recovery of the absorbed fat from the lymph.
Using unanaesthetized rats prepared in this way,
Bloom, Chaikoff, Reinhardt, Entenman and Dauben
(119) fed C-^-labeled palmitic acid either in
the form of tripalmitin or as free fatty acid
dissolved in corn oil. From 70 to 92 per cent
of the absorbed tagged material was recovered
from the thoracic duct lymph and from 69 to 85
per cent from the intestinal lymph. The amount
of the absorbed labeled palmitic acid recovered
from the lymph was apparently not influenced by
50
the form in which the fatty acid was adminis
tered. The smaller recovery of the radioactive
material from the lymph of the main intestinal
duct as compared with that from the thoracic
duct lymph was observed regularly, since some
intestinal lymph also passed via the accessory
intestinal lymphatics to the thoracic duct and
therefore escaped the collection when only the
main intestinal duct was cannulated. However,
even the thoracic duct lymph did not give a
100 per cent recovery of the absorbed palmitic
acid. According to the authors' opinion, there
were two possible explanations for the incomplete
recovery. Either some small amount of the
labeled material was absorbed directly into the
portal vein, or all absorbed fat originally
entered the lymph stream, and that not recovered
in the collected lymph reached the systematic
circulation by way of lymphatic-venous anasto
moses. Several investigators have pointed out
the existence of such anastomotic channels (120,
— 51
121,122). However, the recent paper of Carlsten
and Olin (123) did not confirm these earlier
observations. These authors concluded that the
thoracic duct was the only route available for
the intestinal lymph to reach the blood stream.
Nevertheless some radioactivity was recorded in
the liver of the rats in which thoracic duct
lymph had been diverted by cannulation. Beside
the ways above mentioned by which a part of the
labeled material might escape recovery in the
lymph, it is also possible that some palmitic
acid was metabolically degraded in the intes
tinal tissue and reached the liver as water
soluble compounds through the portal vein.
In a later paper Bloom, Chaikoff, Reinhardt
and Dauben (124) reported that they were able
to recover up to 97 per cent of the absorbed
amount of palmitic acid-C^ from the lymph. The
authors also emphasized that no more than 4
14
per cent of the palmitic acid-C recovered
from the lymph was found incorporated into
~ 52
phospholipid. The rest of the labeled palmitic
acid was recovered in the form of triglyceride.
Cholesterol esters recovered from the lymph did
not contain a detectible quantity of radioactiv
ity. It is interesting to note that from time
to time, the cholesterol esters have been
assigned an essential role in fat absorption.
Schramm and Wolf (125) suggested that fatty acids
set free in the intestinal lumen were esterified
with cholesterol in the intestinal lumen on the
surface of the intestinal mucosa and that these
esters after absorption were split off in the
intestinal cells. The investigation of Bloom
et al. gave no information as to the role played
by the cholesterol esters as an intermediate
stage in fat absorption but they definitely
pointed out that the absorbed fatty acids, in
any case, are not transported as cholesterol
esters. This was confirmed also by Bergstrom
et al. (76) and Borgstrom (126), who found that
a maximum of 2 per cent of the radioactive fatty
53
acid recovered in the lymph was esterified with
cholesterol.
The absorption of palmitic acid-C^- was also
studied by Borgstrom (127,128) and Blomstrand
(129). They fed cannulated rats with palmitic
acid dissolved in corn oil or esterified with
corn oil. The recovery of the radioactivity
from the lymph was, on an average, 60-70 per
cent, no matter in what form the palmitic
acid-C^ was fed. In some experiments the
recovery reached 87.5 per cent. According to
Borgstrom, the phospholipid constituted around
#
11 per cent of the lipids recovered from the
lymph. The specific activity of the recovered
triglyceride was more than 80 per cent of the
activity of the mixtures fed without,any essen
tial differences in all types of experiments.
The specific activity of the phospholipid
was lower than that of triglyceride. It is
apparent from these data that the main route
of the absorption of palmitic acid, whether it
54
is administered as the £ree fatty acid or as
triglyceride, is the lymphatic system. Reiser
(130) and Reiser and Bryson (131) also came to
the conclusion that the route of absorption of
conjugated linoleic acid did not depend on
whether it was administered in the form of tri
glyceride or as free fatty acid to the rats.
Bloom, Chaikoff and Reinhardt (132) undertook
a systematic investigation of the extent to
which the even carbon homologues from the
14
decanoic to stearic acids labeled with C can
be recovered from the thoracic lymph of rats.
These acids were administered as free fatty
acids dissolved in corn oil. From 84 to 95 per
cent of the absorbed stearic acid was recovered
from the lymph. Thus, the extent of the recovery
is practically identical with the recovery of
14
the palmitic acid-C as has been previously
reported (119,124).
From 59 to 82 per cent of the absorbed
myristic acid-C^ was recovered from the lymph.
In the case of fatty acids with a shorter chain,
such as lauric and decanoic, the authors recov
ered from 15 to 55 per cent and from 7 to 19
per cent respectively. The findings were taken
to indicate that the major portion of the absorbed
shorter chain fatty acid is transported from
the intestine via the portal pathway. In order
to substantiate this concept Kiyasu, Bloom and
Chaikoff (133) measured the concentration of
labeled fatty acids in the plasma of the portal
vein and in that of the inferior vena cava in
rats after the administration of palmitic acid-
or decanoic acid-C^^. When palmitic acid
was fed the ratio of the radioactivity in the
plasma of the portal vein to that in the inferior
vena cava was around 1.1, whereas when decanoic
acid was fed the ratio ranged from 1.3 to 9.7 and
in most cases exceeded 2.2.
Borgstrom (134) has extended these studies.
He measured the amount of the labeled decanoic
acid in portal and inferior vena cava blood
during absorption and found that most of the
S 1
decanoic acid was absorbed through the portal
1
system. About two-thirds of the labeled decanoic !
acid was present as free acid and the remainder
was incorporated into the neutral fat and phos
pholipid. The concentration of free fatty acid
in the portal blood reached about 5 mg. per cent, i
Other short chain fatty acids, such as acetic,
propionic and butyric acids, as has been shown by
Hughes and Wimmer (135), are also transported via
the portal system. Fernandes et al. (136,137)
have studied the fate of short chain fatty acids
in human beings. These acids were fed as mixed
triglycerides to a child with the chylothorax.
When fats containing both short and long chain
acids were fed, the former appeared in the lipid
of the chyle in progressively lesser amount the
shorter the chain. When a synthetic triglyceride
was fed, which contained 38 per cent caprylic acid
and the rest long chain fatty acids, the lymph
lipid contained only about 4 per cent caprylic
I
i
acid.
57
Chaikoff, Bloom, Stevens, Reinhardt and Dauben
(138) investigated the route of the absorption of
long chain saturated fatty acid with an odd number
of carbons, a fatty acid which is foreign for the
animal body. The authors administered penta-
decanoic acid-5-C^ dissolved in corn oil to rats
with a cannulated thoracic duct. From 78 to 90
per cent of the administered acid was absorbed.
From 89 to 93 per cent of the absorbed radioactiv
ity was recovered in the lymph as labeled fatty
acids identified as mostly acid with a 15 carbon
chain length.
Bergstrom, Blomstrand and Borgstrom (130) and
Blomstrand (129) investigated the route of the
absorption of the long chain unsaturated fatty
acids in rats. It was found that labeled oleic
and linoleic acids were transported via the thoracic
duct lymph like the saturated long chain fatty
I
acids and appeared in the lymph in the form of
triglyceride and phospholipid. The authors did
not observe any essential difference in the extent
58
of recovery whether these fatty acids were admin
istered as free fatty acids, or in the form of
triglycerides, or as methyl esters. The extent
14
of recovery of oleic acid-C was up to 96 per
cent and linoleic acid-C^ up to 88 per cent.
In summary it can be said that all evidence
indicates that fatty acids with a 14 carbon chain
t
or higher, whether saturated or unsaturated, with
even or odd carbon chain, and whether fed in form
of triglycerides or as free fatty acids, appear
mainly in the lymphatics, largely as triglycerid.e
and in much smaller degree as phospholipid.
The acids with less than 14 carbon atoms are
absorbed via the lymphatic system as well as the
portal system. The distribution of such fatty
acids between.the two routes of transport depends
upon the length of the chain: the shorter the
chain, the more would be absorbed through the
portal system. Bloom et al. (132) considered
that fatty acids with a shorter chain would pass
4
into the lymph to the extent to which they would
.... 59
be incorporated into triglyceride. The capacity
of the mucosa to synthesize glycerol esters is
greater for the long chain fatty acids than for
short ones.
The above cited literature emphasizes that the
extent of recovery of labeled long chain acids
from the lymph collected 18-24 hours is not
affected by the form in which the fatty acids are
administered,— as free fatty acids or as triglyc
erides. In both cases all investigators were able
to recover from the lymph only labeled triglyc
eride, to a much smaller extent phospholipid and
only traces of labeled acids in the cholesterol
esters. No differences in the distribution of
labeled fatty acids among these fractions or the
amount of recovered labeled fatty acids were
observed whether free fatty acids or their triglyc
erides were administered. These experimental data
do not agree with the partition hypothesis, accord
ing to which the ingested free fatty acid and
triglyceride have to take a different route of
_ _ 60
absorption.
However, it seems that the absorption of
ingested free fatty acid is to some extent differ
ent from the absorption of the corresponding amount
of ingested triglyceride. Deuel, Hallman and
Reifman (140) found that the absorption of free
fatty acids with a chain length of up to 13
carbons was slower than with the corresponding
triglycerides. Borgstrom (128) observed a slower
appearance of labeled fat in the lymph after feed
ing free palmitic acid-C^ dissolved in the free
fatty acids obtained by saponification of corn
14
oil than when the palmitic acid-C was incorpor
ated into corn oil.
Such a slower appearance of the fat in the
lymph when free fatty acid was ingested may have
✓
caused lower chylomicron counts in the systemic
blood, and resulted in the erroneous conclusion
drawn by Frazer (49) as. to the. two different
routes for the absorption of ingested triglyceride
or free fatty acid.
61
The slowing of the process of absorption of free
fatty acid, according to Borgstrom (128) is at
least partly due to a decreased rate of emptying
of the stomach. However, the recoveries of the
absorbed radioactivity and the total amount of fat
in the intestinal lymph in an 18-24 hour time
interval was about the same whether animals in
gested palimitic acid-C^ dissolved in free fatty
acid or the acid was incorporated into corn oil.
When a small amount of labeled free fatty acid was
fed dissolved in corn oil, it appeared in the lymph
as triglyceride more rapidly than the main bulk
of the fed triglyceride.
Recently the earlier observations were confirmed
that in animals fed with fat digestion products,
like monoglyceride and diglyceride, only triglyc
eride appeared in lymph (141,142).
B. Absorption of esters of fatty acids with alcohols
other than glycerol.
The results obtained in experiments when
animals were fed with long chain fatty acid esters
62
of other alcohols than glycerol are important for
the evaluation of the validity of the lipolytic
and partition hypothesis. The earlier experiments
of several investigators (7,9,114,143) demonstrated
that long chain fatty acid esters of monohydroxy
alcohols and polyhydroxy alcohols other than
glycerol were absorbed by animals. The authors
were able to isolate only triglycerides from the
lymph or other tissues only triglycerides, but not
the esters administered. Mead, Bennett, Decker
and Schoenberg (144) reinvestigated the absorption
of the fatty acid esters of a monohydroxy alcohol.
They fed mice with methyl esters of oleic and
conjugated linoleic acid mixture. Such fatty acid
esters could not give partially hydrolyzed glycerol
esters like monoglycerides, which are necessary,
according to Frazer, for the emulsification of
the ingested fat and its normal absorption. Mead
et al. observed that the rate of the absorption
of such fatty acid esters was subnormal in fasting
mice in comparison with triglyceride. The
63
addition of 10 per cent of corn oil to the methyl
esters of fatty acids brought the absorption rate
to normal. Simultaneously Mead et al. investigated
the character of the emulsion in the intestinal
lumen. The administration of methyl esters of
fatty acids alone did not give any emulsion in the
small intestine. Addition of the corn oil or
monopalmitin promoted formation of the emulsion.
There was a parallelism between the rate of absorp
tion and the extent of the emulsification of the
intestinal contents. The formation of an emulsion
was evidently necessary for optimum rate of absorp
tion. It has also been found that the rate of
absorption of methyl esters of fatty acids is
normal in animals on a fat-free diet when no forma
tion of monoglyceride would be expected. This
indicated that other dietary components than
partial glycerides may promote the emulsification
and absorption. The data do not necessarily
support Frazer's hypothesis of particulate absorp
tion, since emulsification would also favor the
- - - - - - - 64
action of lipase.
If the partition hypothesis were correct, then
the esters of monohydroxy alcohol, when fed
together with glyceride, would be emulsified and
absorbed in the particulate form without hydrol
ysis. Borgstrom (145) investigated this possibil
ity by feeding ethyl oleate alone or in combination
with glycerides to unanaesthetized rats with a
cannulated main intestinal duct. The author could
not confirm the presence of ethyl esters in the
intestinal lymph even when ethyl oleate was fed
with corn oil. These results provided evidence
against the absorption of ethyl ester in the
particulate form through the intestinal mucosa
to the lymph. It seems that esters of long chain
fatty acids and alcohols other than glycerol were
completely hydrolyzed before absorption and
resynthesized to triglyceride in the mucosa.
C. Absorption of glycerol.
The absorption of glycerol formed during the
ingestion of triglyceride has attracted the
65
attention of investigators much less than the fate
of the fatty acids. Since glycerol is a water-
soluble compound chemically very close to the
carbohydrates, it was assumed that the mechanism
I
of its absorption is very close to that of mono
saccharides .
Proof that glycerol was absorbed was forth
coming from many different experiments. Hirschfeld
(146), Lang (147) and Thomas (148) demonstrated
that the ingestion of glycerol reduced the keto-
nuria in fasting or diabetic human beings.
Voegtlin, Dann and Thompson (149) showed that
glycerol can conteract hypoglycemia after the
injection of insulin. All these authors showed
that glycerol is absorbed and probably converted
into glucose. Chambers and Deuel (150) demonstra
ted a practically quantitative conversion of orally
administered glycerol to glucose in phlorhizinized
dogs. R. Hoeber and- J. Hoeber (151) were able to
demonstrate by direct method the absorption of
glycerol from the ligated rat intestinal loop.
. . . . 66
Laszt and Sullman (152) showed that the absorp
tion of glycerol from the intestinal lumen pre
sumably proceeded through the phosphorylation
stage in a similar way to the absorption of glucose
and fructose. The authors observed the increase
of the acid soluble organic phosphorous compounds
in the mucosa during the absorption of glucose,
fructose, galactose and glycerol. No such increase
of organic phosphorous compounds occurred when
animals absorbed mannose, xylose or arabinose.
The use of glycerol labeled with deuterium or
with permitted the following of the fate of
glycerol in the organism. Favarger, Collet and
Cherbulicz (153) and Buensod, Favarger and Collet
(154) demonstrated that after the administration
of 120-180 mg. of glycerol labeled with deuterium
80-90 per cent was absorbed during a 3 hour
period. Only a small fraction of this glycerol
was incorporated into the lipids. Bernhard and
Wagner (155) fed 600 mg. of deuterium-labeled
glycerol to starved rats and observed an extensive
!
I
III.
- 67
utilization of the absorbed glycerol for the
synthesis of glycogen in the liver. However,
unstarved animals showed only a slight incorpora
tion of deuterium into the liver glycogen. The
incorporation of the labeled glycerol into the
lipids was small (155,156). Gidez and Karnovsky
(157) showed that around 50 per cent of the admin
istered glycerol, independent of .the dose, was
converted to CO2 in several hours. Seventy to
100 per cent of the newly formed blood glucose
and from 15 to 39 per cent of the newly formed
glycogen in the liver were derived from the
labeled glycerol. In a 6 hour period around 7
per cent of the administered radioactive glycerol
was incorporated into the rat lipids. The highest
incorporation .was observed in the liver lipids,
especially in the liver phospholipid.
Cellular phase of fat absorption.
A. Composition of the lipids in the intestinal tissue
*
after administration of triglyceride or free fatty
acid and the role of the phospholipid in fat
✓
. . . . . . . 68
absorption.
The alteration in the composition of the intes
tinal tissue lipids, especially the lipids of the
intestinal mucosa during fat absorption, served
as a clue for the investigation of the mechanism
of fat absorption.
Jeker (158) reported the appearance of fatty
acids in the mucosa of rats within 20 minutes
after the administration of fat. The maximum
amount of free fatty acid was reached at 30 minutes
and then started to decline. At the 6 hour period
the free fatty acid had disappeared from the mucosa
and was replaced by neutral fat. These interesting
data were obtained by the differential staining
of sections prepared from the intestinal tissue.
Lovern and Morton (159) and Lovern, Mead and Morton
(160) analyzed the lipid composition of ..halibut
intestinal tissue. They found a high content of
free fatty acid. The authors attributed the
appearance of this free fatty acid to the autolysis
of the tissue lipids. However, using all possible
precautions, they could not obtain halibut intes
tine oil free from fatty acids.
Most observations concerning the changes of the
intestinal tissue lipids were obtained by the
application of labeled compounds. Sinclair (26)
demonstrated that during fat absorption the phos
pholipid of the intestinal mucosa of cats changed
its fatty acids to correspond with those being
absorbed, but did not change the total amount of
phospholipid present. Artom and Peretti (161)
demonstrated the incorporation of iodinated fatty
acid into the phospholipid of the intestinal muccsa
of the rat after the administration of a large
dose of iodinated fat. Sinclair and Smith (162)
found that the phospholipid of the intestinal
mucosa contained as much as 35 per cent of elaidic
acid after cats were fed trielaidin.
Miller, Barnes, Kass and Burr (163) and Barnes,
Miller and Burr (164) fed conjugated linoleic acid
to animals as the methyl ester or as trilinolein.
They observed the incorporation of this fatty acid
into both phospholipid and neutral fat in the
■ 70
intestinal mucosa. After a 1 hour absorption
period 30 per cent of the neutral fat of the
mucosa consisted of linoleic acid whereas only
6 per cent of the phospholipid was labeled by
the linoleic acid. The labeling of phospholipid
reached a maximum in about 8 hours, when around
15 per cent of the fatty acid of phospholipid was
labeled. They also observed an increase of the
amount of neutral fat in the mucosa of rats, from
72 mg. before feeding to 161 mg. 1 hour after the
administration of trilinolein. The total amount
of phospholipid did not change appreciably. These
results were confirmed by Schmidt-Nielsen (63) .
Kling (165) fed rats with partly deuterized
linseed oil and studied the incorporation of the
labeled acid into the neutral fat, total phospho
lipid and lecithin of the intestinal tissue. The
highest degree of the incorporation of the deuter
ized fatty acid was in the phospholipid, partic
ularly in the lecithin. The neutral fat contained
only about 54 per cent as much deuterium as did
~ 7 1
the lecithin. The amount of deuterized fatty acid
in the cephalin fraction was negligible. However
when Morehouse and Thompson (166) fed a mixed
dioleylstearin with labeled stearic acid to
rats, they observed a greater incorporation of
labeled stearic acid into the neutral fat of the
intestinal tissue and liver than into the phospho
lipid of the same tissues.
Bergstrom, Borgstrom and Rottenberg (167) and
Borgstrom (128) investigated the absorption of
stearic acid-C^ and palmitic acid-C^ when they
were fed to rats either incorporated into corn oil
by transesterification or dissolved in the free
fatty acid from corn oil. The amount of phospho
lipid in the small intestine showed a constant
level during absorption whereas the neutral fat
content increased rapidly. The maximum content
of neutral fat in the intestinal tissue was reached
after 2 hours of absorption and was several times
greater than in fasting conditions. The amount
of free fatty acid present in the intestinal
72
tissue also increased several times during the
fat absorption. It reached a maximum in the first
hour and later started to decrease while the amount
of newly synthesized triglyceride continued to
increase. The amount of the cholesterol did not
change during the fat absorption. The specific
activity of the neutral fat reached a maximum in
2 hours when radioactive acid was fed incorporated
into corn oil, whereas when a mixture of fatty
acids was fed, the maximum specific activity of
neutral fat was reached in.about 4 hours and was
smaller than in the first case. The specific
activity of the isolated phospholipid depended
upon the nature of the fatty acid administered.
Borgstrom (128,168) investigated the extent of the
incorporation of several labeled saturated
fatty acids, namely stearic, palmitic, penta-
decanoic and myristic acids, into the phospholipid
of the small intestinal tissue and the intestinal
lymph. The extent of the incorporation of these
acids depended on the chain length, so that the
longest one, stearic acid, was incorporated to the
73
largest extent, while the shortest one tested,
myristic acid, was the least built into the phos
pholipid. The specific activity curves for the
intestinal phospholipid fatty acid reached a
maximum value 2-3 hours after the administration
of the fat. In the case when the administered
fat contained labeled stearic acid, the specific
activity of the phospholipid of the small intes
tinal tissue and the liver were usually higher
than the specific activities of the neutral fat
from the corresponding organs. Just the opposite
picture was observed after the administration of
fat with labeled palmitic acid. Morehouse, Skipski,
Searcy and Spolter (169) confirmed Borgstrom's
observation concerning the preferential incorpora
tion of stearic acid in comparison with palmitic
acid into the phospholipids of the intestinal
tissue and liver.
Favarger (170) and Favarger and Gerlach (171)
demonstrated that the extent of the incorporation
of fatty acids into the intestinal phospholipid
74
also depends upon the digestibility of the admin
istered fat into which these acids are incorporated.
Various mixtures of triglycerides and free fatty
acids containing deuteriopalmitic, deuteriostearic,
deuteriooleic or elaidic acids were fed. The
extent of the incorporation of these labeled fatty
acids into the intestinal tissue triglycerides
and phospholipids was determined in three succes
sive portions of the small intestines. The dis
tribution of the labeled fatty acids throughout
the gut in the isolated lipids varied with the
composition of the mixture fed and less with the
nature of the labeled fatty acid in such a mixture.
Fat of low digestibility usually exhibited a
higher incorporation of the labeled fatty acid in
the last segment of the intestine whereas the
opposite picture was observed in the case where a
fat of high digestibility with the same labeled
fatty acid was administered. Thus, the deuterio-
stearie acid fed as pure'tristearin showed the
highest incorporation of this acid in the lipids
of the last segment of the intestine. When tri
stearin was fed in a mixture with lard the highest
incorporation of deuteriostearic acid was observed
in the lipids of the first and second segments of
the intestine. The distribution of the labeled
acids between the phospholipid and triglyceride
fractions of the intestinal tissue lipids also
depended upon the digestibility of the lipid
mixture fed. The lipid mixture of low digestibil
ity exhibited, usually, a relatively higher incor
poration of the labeled deuteriostearic acid into
phospholipid than into triglyceride. The lipid
mixture of high digestibility gave a higher
incorporation of deuteriostearic acid into the
neutral fat fraction than in phospholipids. A
similar behavior was observed in a case where
deuteriopalmitic acid was administered in fat
mixtures of different digestibility. Since the
differences in the extent of the labeling were
much greater for triglyceride than for phospho
lipid, the authors considered this as evidence
that phospholipids are not an obligatory inter
mediate for resynthesis of triglyceride in the 1
intestinal tissue. These investigators emphasized
that there are great variations in the rate of
absorption of the same acid and the extent of its
incorporation into different lipids of the intes
tinal tissue, depending on the presence of other
glycerides or free fatty acids.
Favarger, Collet and Veraguth (172) demonstra
ted with the aid of elaidic acid that phospholipid
of the epithelial layer of a dog's small intestinal
mucosa incorporated much more of the fed labeled
fatty acid than the cells of the deep layers. They
also found that the choline containing phospholipid*
of the different parts of the intestinal wall
incorporated more elaidic acid than did the non
choline containing phospholipid.
Until now we have considered the.incorporation
into phospholipids of long chain fatty acids, since
the analysis of the intestinal phospholipids as
well as phospholipids of other organs showed that
77“ "|
they contained predominantly fatty acids with 18 j
or more carbons. Only a small amount of fatty j
I
{
acid with 16 carbons atoms was present in the
I
phospholipid (173,174). j
The incorporation of shorter chain fatty acids
into phospholipids was studied by Stevens and
Chaikoff (175). These authors tested C^-labeled j
lauric and myristic acids. Both acids were found
in the phospholipid fraction of the intestinal
tissue lipids 8 hours after administration of
these acids mixed with corn oil. However, the
majority of counts observed in the phospholipid
were present in longer chain fatty acids. This
indicated the extensive conversion of the lauric
and myristic acids to fatty acids with longer
chains before their incorporation into phospholipid.
Thus, the extent of the incorporation of fatty
acids into the intestinal phospholipids is deter
mined by the nature of the fatty acids as well as
by the digestibility of the administered fat into
which these fatty acids are incorporated.
7 8
Therefore the labeling of the intestinal phospho
lipid by tagged fatty acids after the administra
tion of labeled glycerides or free fatty acids can
be interpreted neither in favor of or against the
hypothesis that phospholipid is an obligatory
intermediate of the resynthesis of triglyceride.
More reliable data concerning the role of phos
pholipid in fat absorption were obtained by apply
ing radioactive phosphorous, labeled glycerol or
glycerides labeled in the glycerol moiety. Artom,
Sarzana and Segre (176) fed radioactive phosphorous
to rats kept on a carbohydrate or fat diet. The
ingestion of fat increased the extent of incorpora
tion of radioactive phosphorous into the liver and
intestinal phospholipid. Perlman, Ruben and
Chaikoff (177) and Fries, Ruben, Perlman and
Chaikoff (178) also demonstrated the incorporation
of radioactive phosphorous into the phospholipid
of the small intestinal tissue as well as of other
tissues in the presence and in the absence of fat
in the diet. The extent of the incorporation of
79
32
P into the phospholipids of the small intestinal
tissue increased 2.5-3 times when fat was ingested.
Schmidt-Nielsen (64) showed that the extent of
incorporation of P32 into the intestinal phospho
lipid was 3-4 times greater in the animals which
had ingested oleic acid than in fasted animals.
Artom and Cornatzer (179) studied the effect of
choline on phospholipid synthesis in the small
32
intestine with the aid of P . A single large
dose of choline administered together with fat
caused the increase both of the amount of the
intestinal phospholipid and its labeling with
P32. This was interpreted to mean that choline
facilitated phospholipid synthesis in the small
intestine. In choline-deficient rats the absorp
tion of fat did not cause an increase in the
formation of phospholipid in the intestinal tissue.
All these data obtained with P32 showed an
increase in the.turnover of the phospholipid in
the intestinal tissue during fat absorption and
were interpreted in favor of the Verzar hypothesis
that the resynthesis of triglyceride in the intes
tinal tissue proceeds through the stage of phos
pholipid. However, several papers have recently
appeared which questioned the necessity of phos
phorylation for the resynthesis of triglyceride
in the intestinal wall.
Zilversmit, Chaikoff and Entenman (180)
removed the surface layer of the mucosa of the
duodenum in several dogs after the injection of
and the ingestion of fat, and determined the
extent of incorporation of radioactive phosphorus
into the lipids of this layer. The ileal part of
intestine, separated from the duodenum by the
ligature, served as a control. No differences
were observed in the rates of the incorporation
32
of P into phospholipid on either sides of the
ligature. However, the authors observed some
32
increase in the incorporation of P into phos
pholipid of rat intestine during the digestion
of different fats, but this increase was too small
to account for the passage of all absorbed fat
. . . . . . 81
by way of phospholipid intermediates.
Reiser and Dickert (181) and Reiser, Bryson,
Carr and Kuiken (182) compared the extent of the
labeling of glycerol and fatty acid moieties of
the phospholipid with the labeling of triglyceride
isolated from the intestinal mucosa or lymph after
the administration of doubly-labeled fat to rats.
As a doubly-labeled fat, these authors used tri-
14
palmitin labeled with C in both the glycerol and
fatty acid moieties or trilinolein labeled with
conjugated double bonds in the fatty acid part
of the molecule and with in the glycerol part
of the molecule. The results showed that the
phospholipids of the mucosa and lymph were less
labeled in both moieties of molecule--in glycerol
and fatty acid--than were the triglycerides iso
lated from the same sources. Especially important
was the degree of labeling of the glycerol moiety
of these two compounds since the glycerol molecule,
once liberated during the lipolysis in the intes
tinal lumen, was shown not to be reutilized to any
extent for the resynthesis of lipids in the intes
tinal tissue. Therefore the authors concluded
that the resynthesized triglyceride in the intes
tinal mucosa must be the precursor of the phos
pholipids of the mucosa and lymph, rather than the
converse.
Tasker (183) could not find any difference in
the rate of absorption of olive oil after the
administration of choline chloride. If the
synthesis of phospholipid is an intermediate stage
in the resynthesis of triglyceride in the intes
tinal tissue, the administration of choline might
favor fat absorption.
Supporting this changing viewpoint, some of
Verzar's evidence that phospholipid is an inter
mediate in fat absorption has lost its force since
the time when the hypothesis was proposed. Kling-
hoffer (184) showed that iodoacetic acid induced
profound pathological changes in the intestine,
so that it was unnecessary to invoke an inter
ference in the phosphorylation to account for a
depressed fat absorption in the animal treated with
this drug. Schmidt-Nielsen (63) showed that
poisoning of the intestinal tissue by phlorhizin
did not decrease the rate of incorporation of
radioactive phosphorous into the intestinal phos
pholipid. Stillman, Entenman, Anderson and
Chaikoff (185) demonstrated that adrenalectomized
rats retained the ability to phosphorylate fat,
as indicated by the extent of incorporation of
P32. It seems that although phospholipid synthesis
does occur in the intestinal mucosa at a greater
rate during fat absorption, it does not proceed
at a sufficient rate to account for the resynthesis
of all of the fat passing through the intestinal
cells. Therefore the role of the phospholipid of
the intestinal tissue in the process of fat absorp
tion stays unclear at the present time. However,
there is no doubt that phospholipids synthesized
in the intestinal tissue play an important role in
the stabilization of the fat droplets, which leave
the intestine by the lymphatic route. Sullmann and
Wilbrandt (28), Frazer (41), Bergstrom, Borgstrom
84
and Carlsten (185), Artom (187), Reiser and
Dickert (181), Tasker (183) and others demonstrated
that the phospholipid content of the intestinal
or thoracic lymph increased during fat absorption,
although such increase was much smaller than the
increase of triglyceride. Such synthesis of
phospholipid in the intestinal tissue anfr the
liberation of it into the lymph may account, at
least partially, for the increase of the turnover
of phospholipid in the intestinal tissue during
fat absorption.
B. The incorporation of labeled glycerol into the
lipids.
The presence in the intestinal lumen of tri
glyceride, diglyceride, monoglyceride and free
fatty acid per se does not give us any information
as to what compounds actually penetrate into the
intestinal wall. Several investigators made an
attempt to elucidate the extent of the hydrolysis
of triglyceride preceding absorption, by the
simultaneous feeding of fat and labeled glycerol
to animals. They considered that the molecules
of triglyceride which were completely hydrolyzed
in the intestinal lumen to fatty acid and glycerol
before absorption, should after the absorption be
reesterified and converted to triglyceride. If
the amount of the labeled glycerol administered
were present in large excess in the mucosa, the
resynthesis of triglyceride should proceed there
with the predominant utilization of labeled glyc
erol. Therefore the extent of the incorporation
of labeled glycerol into the absorbed fat should
indicate the amount of triglyceride which under
went complete hydrolysis before absorption.
Favarger and Collet (188) and Favarger, Collet
and Cherbuliez (153) fed rats with a mixture of
trielaidin and lean meat followed by deuterized
glycerol. The incorporation of the deuterized
glycerol into the neutral fat isolated from the
intestinal tissue amounted to only 1.6-3.7 per
cent of incorporation of elaidic acid into the
triglyceride. The same extent of incorporation
" 8 6
of deuterioglycerol into the absorbed lipid was
obtained by these authors when they fed rats with
lard and deuterioglycerol. A slightly higher
incorporation of labeled glycerol into the absorbed
fat was observed in monkeys. On the basis of the
obtained results and some additional calculations,
the authors concluded that in rats a maximum of
5 per cent of the ingested fat undergoes complete
hydrolysis, in monkeys, probably slightly more.
But, in any case, at least 90 per cent of the fat
is absorbed in the form of tri-and lower glycerides,
hater Collet and Favarger (189) and Buensod,
Favarger and Collet (154) realized that the extent
of the incorporation of the labeled glycerol into
the absorbed neutral fat cannot be considered as
an indication of the amount of the glyceride com
pletely hydrolyzed before absorption, since the
extent of incorporation of deuterioglycerol was
small even when the rats were fed free fatty acid.
The incorporation of labeled glycerol into phos
pholipid was also small. These investigators also
tested whether glycerophosphate is a precursor of i -
the intestinal phospholipid. They administered
*
to rats a mixture of o t and j5 deuterioglycerophos- ;
I
phate simultaneously with lard. The extent of
the incorporation of the deuterium-labeled glycerol
into the intestinal phospholipid was small. The
*
authors concluded that neither exogenous free ;
i
glycerol nor glycerophosphate are important pre
cursors of the intestinal phospholipid. In all
investigations undertaken by the Eavarger group
relatively small doses of labeled glycerol were
used— sometimes only 13 per cent above the amount
of glycerol preformed in the triglyceride which
was fed.
Bernhard, Wagner and Ritzel (156,190) adminis
tered much greater doses of deuterioglycerol
simultaneously with olive oil to cannulated rat.
Only 5 per cent of the fat recovered from the
thoracic duct lymph was labeled with the deuterio
glycerol.
Three years ago Reiser, Bryson, Carr and Kuiken
(182) and Reiser and Williams (141) suggested that i
i
glycerol per se may not be the precursor of glyc- j
eride glycerol in the intestinal tissue during the |
i
resynthesis of triglyceride, but that intermediates'
i
of carbohydrate metabolism, such as dihydroxyacetone
or gj.yceraldeh.yde are the precursors of the glycerol!
J
moiety of the triglyceride. Reiser and Williams i
I
(141) considered that these substances act as
i
precursors by forming the mono- and diesters with
i
fatty acid, then being reduced to glycerol and
finally forming the triglyceride. The authors
tested this idea by feeding rats with 1-palmitoxy- ,
3-hydroxyacetone labeled with in both moieties
[
of the molecule. Only about 10 per cent of the
ingested palmitoxyhydroxyacetone appeared in the
lymph in the form of triglyceride calculated on
the basis of the radioactivity located in the
palmitic acid. The total amount of glycerol-C^
which appeared in the lymph in the form of triglyc
eride after the ingestion of palmitoxyhydroxy
acetone was only 2.6 per cent calculated on the
89
basis of the radioactivity located in the dihydrox-
yacetone. The reduction of small amounts of
dihydroxyacetone ester to the glycerol ester of
fatty acid does not indicate that this is neces
sarily a normal pathway for the resynthesis of
triglycerides in the intestinal tissue. It is
probable that the low incorporation of labeled
exogenous glycerol into the ingested fat is due
to the fact that such compounds as fatty acid and
glycerol or glycerophosphate are located in the
different phases of the mucosa cells. In addition
exogenous glycerol probably enters the capillary
system very Rapidly and is not retained for an
appreciable time in the intestinal cells.
Karnovsky and Gidez (191, 192) and Gidez and
Karnovsky (157) studied the incorporation of
glycerol-C^ into the lipids of different rat
tissues after its intraperitoneal, intravenous
or intragastric administration. In 1 hour after
the administration of glycerol-C^ the specific
activity of the liver triglyceride and phospholipid
_ . . . . 90
reached a maximum. At that point about 20 per
cent of the triglyceride and 7 per cent of the
phospholipid glycerol were derived from the
administered glycerol. Then a rapid drop in the
radioactivity of the liver triglyceride occurred.
The decline of radioactivity in the phospholipid!
was, however, very slow. The fact that the curve
for the incorporation of the glycerol-C^* into
the liver lipid fractions does not satisfy the
precursor-product criteria of Zilversmit et al.
(193) caused the authors to conclude that phos
pholipid glycerol and neutral fat glycerol are
independently derived from the administered radio
active glycerol. The greatest incorporation of
glycerol-C^, 6 hours after its administration,
was in the liver lipids, whereas only around 1/50
of this radioactivity was observed in the intes
tinal wall lipids. The incorporation of radio
active glycerol was higher in the phospholipid of
the intestinal wall than in the triglyceride.
Doerschuk (194) injected glycerol-C^ into rats
intraperitoneally and determined 24 hours later
the incorporation of this labeled glycerol into
the liver lipids. The extent of labeling of phos
pholipid and triglyceride was practically the
same.
These papers do not concern the mechanism of
fat absorption and the resynthesis of triglyceride
in the intestinal tissue, but they definitely
indicate that exogenous glycerol may serve as the
precursor for lipid glycerol.
IV. The extent of hydrolysis of the fat preceding
absorption.
An interesting attempt to determine the extent of
hydrolysis of fat preceding absorption was undertaken
by Borgstrom (126). The author considered it probable
that the fatty acids in cholesterol esters of the
intestine lymph were derived from the free fatty acid
mixture available in the intestinal tissue during
fat absorption. Consequently, if a non-labeled tri
glyceride and a small amount of highly active free
stearic acid-C^ were fed with cholesterol, the
extent of hydrolysis of triglyceride in the intestinal
lumen would then be reflected in the content of
the cholesterol ester fatty acid of the intestinal
lymph. Borgstrom fed cannulated rats with three dif
ferent mixtures: corn oil with a small amount of free
stearic acid-C^ and cholesterol; corn oil trans-
esterified with stearic acid-C^ and cholesterol; or
corn oil and cholesterol esterified with stearic
acid-C^. The amount of the radioactive stearic
acid in the cholesterol ester recovered from the
lymph was about the same regardless of the form of
the stearic acid-C^ fed. These data may be inter
preted in favor of the hypothesis of the complete
hydrolysis of ingested fat in the lumen of the small
intestine previous to absorption and following random
resynthesis. However, the random distribution of
the labeled fatty acid in the cholesterol esters
recovered from the lymph may be due to a transesteri-
fication process and formation of new ester bonds in
the intestinal lumen as well as in the intestinal
cells. This explanation is partly supported by the
... _93_-
observation that the percentage of active fatty acid
recovered in the lymph phospholipid was higher when
corn oil with free stearic acid-C^ was administered.
This can be taken as an indication of an incomplete
hydrolysis of the ingested triglyceride before
absorption as it is difficult to understand how this
difference could arise if the hydrolysis was
complete.
More fruitful results concerning the extent of
fat hydrolysis preceding absorption were obtained in
experiments when the glycerol moiety of the admin
istered triglyceride was labeled or especially when
both glycerol and fatty acid moieties were labeled.
Karnovsky and Gidez (191,192) used tributyrin and
triolein labeled in the glycerol part of the mole
cules with C^. Both triglycerides were administered
orally to rats. During the 4 hour absorption period
O f the triolein and tributyrin 37-77 per cent and
86-100 per cent respectively of these triglycerides
were completely hydrolyzed before absorption. Liver
and blood lipids had higher specific activities than
other lipids isolated from other organs. There is
evidence which indicates that unhydrolyzed triolein
might have been brought directly to the liver and
thus account for the major portion of the radioactive
neutral fat in this organ. The distribution of the
radioactivity in different organs after the admin
istration of tributyrin was very similar to that
where radioactive free glycerol was fed. The authors
concluded, on the basis of the results obtained
when triolein was fed, that free glycerol is an
obligatory intermediate in the transformation of
triglyceride glycerol to phospholipid glycerol.
Bernhard, Wagner and Ritzel (156) fed triacetin
labeled in the glycerol moiety of the molecule with
deuterium. Essentially the same distribution of
deuterium in lipids was observed as when free labeled
glycerol had been fed, indicating that a rapid and
complete hydrolysis of triacetin took place before
its absorption. The lipids isolated from the lymph
showed only a very slight deuterium concentration,
just as after the administration of labeled glycerol.
95
These authors also fed to rats, in other experi
ments, a triglyceride labeled with deuterium in
both glycerol and fatty acid moieties of the molecule.
The neutral fat recovered from the lymph demonstrated
a higher dilution of the deuterium content in the
glycerol moiety than in the fatty acid part of the
molecule. From these data, the authors calculated
that a minimum of from 25 to 53 per cent of the tri
glyceride had been totally hydrolyzed before absorp
tion. Similar experiments were performed by Reiser,
Bryson, Carr and Kuiken (182). They administered
to rats a doubly-labeled triglyceride in which the
glycerol moiety contained and which was esterified
with conjugated linoleic acid. The determination of
the ratio of radioactive glycerol to linoleic acid
in the neutral fat recovered from the thoracic duct
lymph led the authors to conclude that approximately
25 to 45 per cent of the ingested triglyceride was
completely hydrolyzed before absorption. In order
to verify that the radioactive glycerol retained in
the lymph triglyceride had penetrated into the
intestinal tissue in the form of glyceride and to
determine what kind of glyceride, the authors fed
rats with mixtures of the above mentioned, doubly-
labeled, synthetic triglyceride and different amounts
of unlabeled saturated triglyceride. The triglycer
ides recovered from the thoracic duct lymph were
subjected to fractionation with alcohol-acetone
solution at 5°. Under such conditions triglycerides
with two and three saturated fatty acids are relatively
insoluble. Analysis of the soluble ("unsaturated")
and insoluble ("saturated") triglyceride fractions
of the lymph demonstrated that the insoluble ("satu
rated") fraction contained labeled glycerol to a
lesser extent than the soluble ("saturated"). This
excluded the possibility that all absorbed fat was
completely hydrolyzed and then in the intestinal
cells, resynthesized into triglyceride with the
utilization of labeled glycerol, since the distribu
tion of radioactivity should then be more or less
random in both fractions. The formation of "satu
rated" triglyceride containing labeled glycerol was
interpreted in terms of an obligatory absorption of
!
glycerol-labeled monoglyceride, followed by the i
I
I
resynthesis with free saturated acid in the intestinal-
mucosa. However, Borgstrom (111) demonstrated, that
free fatty acid, liberated during hydrolysis, may
I
exchange with the glyceride fatty acid in the lumen
I
of the small intestine. It is apparent that "satu- !
i 1
rated" glycerol-labeled triglyceride could have been
formed also from absorbed di- and triglycerides which j
i
had already exchanged part of their linoleic fatty
I
acid for the saturated acid formed by the hydrolysis
of lard in the intestinal lumen. Therefore, data
from this type of experiment cannot be used as
unequivocal evidence for the species of glycerides
absorbed. In another experiment Reiser and Williams
(141) fed rats with 1-monopalmitin labeled with C ^
in the glycerol and palmitic acid moieties. The
ingested monoglyceride appeared in the lymph as tri
glyceride and the ratio of the radioactivities in the
isolated triglyceride of both moieties of the molecule
indicated that 73 per cent of the monoglyceride was
hydrolyzed before absorption. Probably 27 per cent
of the monoglyceride was absorbed as a unit without
hydrolysis. However, it is not excluded that mono
glyceride may undergo conversion into higher glycer
ides in the intestinal lumen, as has been demonstrated
by Borgstrom (111) and be absorbed in the form of
di- or triglycerides, therefore only the isolation
of monoglyceride from the intestinal cells with the
same ratio of percentage of the labeled glycerol to
the percentage of the labeled fatty acid as in the
compound fed, can be considered as incontrovertible
evidence that monoglyceride was absorbed as such.
Reiser and Williams explained the high degree of
hydrolysis of monoglyceride in contrast to triglyc
eride on the basis that in the absence of free fatty
acid 3 molecules of monoglyceride were required to
form one molecule of triglyceride.
According to Reiser and Williams (141) and Reiser
t
(195196) hydrolysis of the absorbed glycerides may
even take place after their absorption in the mucosa.
Recently Reiser (195) wrote, "... the regularity
99
of the changes after both monoglyceride and triglyc- ,
eride ingestion and the significant differences
,1
between the two suggest that an intracellular mecha- |
nism regulated the hydrolysis." Reiser further stated^
that lipase attacked monoglyceride with difficulty
and therefore the digestion of triglyceride in the
lumen of the intestine proceeded only to the monoglyc-j
eride. The process of the replacement of labeled
glycerol in the absorbed monoglyceride with unlabeled
glycerol took place during triglyceride resynthesis
in the intestinal mucosa. The reason for such
replacement was explained by the author as competition
between the absorbed monoglyceride and endogenous
glycerol for the absorbed free fatty acid. However,
Reiser did not present any experimental evidence to
substantiate this hypothesis (195,196).
V. Statement of the problem and plan of attack.
From the review of the literature in the previous
chapter it is obvious that ingested fat undergoes a
rather profound hydrolysis in the intestinal lumen.
However, it is not clear to what extent ingested fat
is normally hydrolyzed before absorption, since all
available information is based upon a comparison of
the fat in the lymph with the fat fed. Modifications
of the fat which appears in the lymph not only
represent the changes which occurred during absorp
tion, but also changes within the intestinal tissue
itself and perhaps during the passage from the intes
tinal cells to the lymph. Bernhard et al. (156) and
Reiser et al. (182) demonstrated that some glyceride
is absorbed without complete hydrolysis, but the
nature of the glyceride which penetrates into the
mucosa actually is unknown. There is only some .
indirect evidence, that this glyceride might be
monoglyceride, according to Reiser and Williams'
(141) investigation. The pathway of the synthesis
of the triglyceride in the intestinal mucosa from
the absorbed fatty acid and glyceride or glycerides
is also unclear.
On the basis of the experiments of Favarger et al.
(153) and Bernhard et al. (156) in which a relatively
small incorporation of exogenous glycerol into the
intestinal and lymph lipids during fat absorption ;
I
was observed, it was concluded that exogenous glycerol
cannot be utilized for the .resynthesis of the lipids |
in the intestinal tissue. Reiser and Williams (141) j
proposed that dihydroxy acetone, but not glycerol, is
the precursor of the glycerol in the intestinal lipid <
molecules. However, their experiments, as has been !
[
pointed out, are not conclusive and therefore the i
question of the nature of the glycerol precursor in
i
the triglyceride and phospholipid of the intestinal
tissue stays open.
There are controversies as to whether phospholipid
is the precursor of the triglyceride in the intestinal
tissue during fat absorption, or whether the glycer
ides are the precursors of the phospholipid, of they
are both synthesized independently.
The principal object of our investigations was to
determine the nature of the compounds which penetrated
into the intestinal tissue during fat absorption and
to elucidate the pathways of the synthesis of triglyc
eride and phospholipid in the intestinal tissue from
the absorbed material. Attempts have been made to
study these problems by three different approaches. j
1. The extent of hydrolysis of triglyceride has '
«
been investigated by feeding rats with a
synthetic triglyceride labeled in both
I
moieties of the molecule, the subsequent isola- ,
tion of the lipids directly from the intestinal ;
tissue and determination of the changes of the
ratio of labeling in both parts of the isolated j
material. The changes in these ratios indicate
approximately the fraction of glyceride absorbed
without total hydrolysis.
2. A study was made as to whether monoglyceride--
one of the glycerides formed in the intestinal
lumen during normal lipolysis--can penetrate
into the intestinal mucosa without hydrolysis.
This investigation was accomplished by feeding
the rat with a doubly-labeled monoglyceride
followed by the attempt to isolate monoglycer
ide from the intestinal tissue. If the mono
glyceride were absorbed as a unit into the
- 103
intestinal tissue, the ratio of the labeling
in the two parts of the molecule of monoglycer
ide would be expected to stay the same as in
the fed material. An attempt was also made
to isolate monoglyceride from the intestinal
tissue during the ingestion of a doubly-
labeled triglyceride of definite structure
and to elucidate the origin of such monoglyc
eride from the digested triglyceride.
3. For the determination as to whether exogenous
glycerol may be the precursor of the glycerol
moiety of the intestinal triglyceride and phos
pholipid, different doses of glycerol-C^ were
fed to rats during fat ingestion and the extent
of the incorporation of such labeled glycerol
into the above mentioned lipids was estimated.
The possible pathway of the glycerol incorpora
tion was investigated by a comparison of the
distribution of incorporated glycerolinto
the monoglyceride, higher glycerides and phos
pholipid fractions isolated from the intestinal
tissue. It was expected that such experiments
might elucidate the pathways of the synthesis
of glycerides and phospholipid in the intes
tinal tissue.
1
I
MATERIALS AND METHODS
Synthesis of Labeled Compounds
! It was considered that the doubly-labeled triglyc-
i
eride used in the investigation of the extent of hydrolysis
I
!should meet the following requirements:
i
1. A definite chemical structure.
i .
; 2. A different type of isotope labeling in both
i fatty acid and glycerol moieties of the
l
i
1 molecule.
j
1 3. A content of long chain fatty acids normally
occurring in natural fats.
4. A high digestibility--therefore the melting
point of the synthetic triglyceride should be
I not too high.
5. The presence of at least two different acids
in the triglyceride molecule.
After the evaluation of all these requirements, as
well as the availability of the initial compounds for
synthesis in the laboratory or market, it was decided that
t
I
lone of the most suitable triglycerides would be 1,3-
j dioleyl-2-deuteriostearyl-glyceride-C^4.
( In order to determine whether the intermediate
I
I products of triglyceride hydrolysis like monoglyceride
i
!can penetrate into the intestinal mucosa as a unit without
further hydrolysis, 1-monostearin was used. The use of
2-monostearin was excluded since such a compound is
j
I unstable and would very easily isomerize to 1-monostearin
I ■
■in the acid pH prevailing in the stomach (197). According
to Mattson et a_l. (99,100) the 1-monostearin would be the
.naturally formed intermediate during lipolysis of the
I
jtriglyceride l,3-dioleyl-2-stearyl-glyceride in the intes-
I
I
:tinal lumen. 1-monostearin was labeled with deuterium
|in the fatty acid moiety and with C^4 in the glycerol
imoiety. This doubly-labeled monoglyceride was termed
mono-l-deuteriostearyl-glyceride-C^4.
r
I. Synthesis of 1,3-dioleyl-2-deuteriostearyl-glyceride-
C14.
I
l,3-dioleyl-2-deuteriostearyl-glyceride-C^4 was pre
pared by the coupling of deuteriostearyl chloride
with 1,3-dioleyl-glyceride-C^4. This synthesis pro
ceeded through the steps described below.
107
A. Synthesis of deuteriostearic acid.
The synthesis of deuterium labeled stearic
acid was carried out according to the method of
Rittenberg and Schoenheimer (198,199). The prin
ciple of this method consists of electrolysis of
deuterium oxide to yield deuterium and the sub
sequent deuterization of an ester of an unsaturated
fatty acid in the presence of a catalyst. Methyl
or ethyl linoleate (Hormel Foundation, Austin,
Minnesota) was used as the initial material. The
deuteriostearic acid had a melting point of 69-
70° C. The reported melting point for stearic
acid is 69.6 (200). Upon analysis the mean deute
rium content was shown to be 11.07 atom per cent.
In the case of a fully saturated pure linoleic acid
the content of deuterium should be 11.11 atom
per cent. The yield of deuteriostearic acid was
80-85 per cent of the theoretical in the several
syntheses.
B. Synthesis of deuteriostearyl chloride.
Stearyl chloride was synthesized by the method
of Adams and Ulrich (201). When aliphatic acids,
according to this method, are warmed with oxalyl
chloride, they are converted into the corresponding
acid chloride. The reaction requires an excess
of oxalyl chloride in the ratio 2.5 moles to 1
mole of acid. It was found that even better
results were obtained when the amount of oxalyl
chloride was increased to 3.5 moles. A yield
approximating 70 per cent was obtained in the
several syntheses required for this problem.
. Synthesis of symmetrical diolein.
1,3-dioleyl-glyceride-C^ was synthesized by
direct esterification of glycerol-C^ (Isotope
Specialties Company) with the equivalent amount
of oleic acid (Reagent, Eimer and Amend) according
to a modified method as cited by Kawai, Nobori
and Yamada (202). Fischer (203), Jackson and
King (204) have shown that the acyl group of the
secondary hydroxyl group of glycerol possesses the
tendency at the elevated temperature to be shifted
to a free primary hydroxyl. The secondary hydroxyl
1 0 9
group also tends not to be esterified due to its
chemical sluggishness. Therefore when oleic acid j
l
i
was used in the amount of 2 moles of acid to 1 mole!
of glycerol the majority of the product formed !
i
should be the symmetrical diolein. The final prod-
1
uct obtained by us had an iodine number of 84.0 and-
' i
an acid number of 0.8. According to theoretical j
calculations and literature data the iodine value
for diolein is 80.4-81.7, for triolein 97.3 and ;
1 » *
monoolein 71.2 (205). It was considered that the
product obtained was predominantly diolein and
therefore it was used for the synthesis of 1,3-
dioleyl-2-deuteriostearyl-glyceride-C^.
D. Synthesis of l,3-dioleyl-2-deuteriostearyl-glycer-
ide-C^.
The esterification of the secondary hydroxyl
group in the diolein was accomplished by the
reaction with deuteriostearyl chloride according
to Averill, Roche and King (206). The total yield
of the product was 45 per cent of theoretical and
the physico-chemical constants were as follows:
1 1 0
Melting Iodine Acid Saponifica-
Point Number Number tion Number
l,3-dioleyl-2-
deuteriostearyl- i)
glyceride-C^ 36-44 C 53.8 0.3 192.8
Theoretical
Standard
Values - 57.3 0 193.0
^At 36° C triglyceride started to melt and had
a semi-liquid consistency. At 44° C it was completely
melted and exhibited a good transparency.
The determination of the deuterium content in 1,
14
3-dioleyl-2-deuteriostearyl-glyceride-C was
performed according to the method described
below. Several samples diluted by a non-
deuterized compound of the same structure were
analyzed. The mean deuterium content in 1,
14
3-dioleyl-2-deuteriostearyl-glyceride-C were
found to equal 4.14 atom per cent. The theoretical
content for absolutely pure l,3-dioleyl-2-deute-
14
riostearyl-glyceride-C should be equal to 3.81
atom per cent.
II. Synthesis of 1,3-dioley1-2-deuteriosteary1-glyceride.
The synthesis of l,3-dioleyl-2-deuteriostearyl-
glyceride was accomplished in the same manner as the
synthesis of the doubly-labeled triglyceride 1,3-
14
dioleyl-2-deuteriostearyl-glyceride-C . In this
case ordinary glycerol was used instead of glycerol-
C^. The total yield of the product obtained was
43 per cent of the theoretical. The physico
chemical constants of 1,3-dioley1-2-deuteriostearyl-
glyceride are as follows:
Melting Iodine Acid Saponifica-
Point Number Number tion Number
1,3-dioley1-2-
deuteriostearyl- i)
glyceride 37-46 C 65.0 0 193.5
Theoretical
Standard
Values - 57.3 0 193.0
^At 37° triglyceride started to melt and had a
semi-liquid consistency. At 46° C it was completely
melted and exhibited a good transparency.
The determination of the deuterium content in 1,3- )
dioleyl-2-deuteriostearyl-glyceride was performed
according to the method described below. The mean
deuterium content in the 1,3-dioley1-2-deuterio-
stearyl-glyceride was equal to 3.59 atom per cent.
" " " " ' 112
III. Synthesis of mono-l-deuteriostearyl-glyceride-C^.
The synthesis of the doubly labeled monostearin,
14
which has a C labeled glycerol moiety and stearic
acid labeled with deuterium, was performed by the
coupling of deuteriostearyl chloride with free
glycerol.
The synthesis of the deuteriostearic acid as
well as its chloride was accomplished as before. The
deuteriostearyl chloride, dissolved in chloroform,
14
was slowly added to glycerol-C , dissolved in the
mixture of chloroform and quinoline. The ratio of
reacting compounds was 2.5 moles of glycerol to 1
mole of stearyl chloride. The reacting mixture was
heated to 50° C and dried nitrogen bubbled through
it.during 16 hours. Then the reacting mixture was
washed with ice cold 0.5 N sulfuric acid until the
test for quinoline was negative (207). This was
followed by washing with a 10 per cent solution of
potassium bicarbonate to remove traces of sulfuric
acid. The excess of alkali was then removed by
washing with water. The chloroform solution was
evaporated slowly in vacuum at low temperature and
the residue dried. The product obtained was dis
solved in minimal amounts of warm ether-alcohol
mixture (3:1) and titrated with an alcoholic solution
of sodium hydroxide to a permanent pink withphenol-
phthalein. A large excess of water was then added
and the soaps of the free fatty acids were thus
removed from the synthesized product. The ether
fraction was washed several .times with water, dried
with sodium sulfate, filtered and.the ether evapor
ated. The product obtained had an acid value equal
to zero and was presumably composed of monoglyceride
and some higher glyceride. The monoglyceride was
separated by the. systematic multiple fractional
extraction procedure described below. The monoglyc
eride fraction obtained contained 91 per cent of
1-monostearin as determined by the periodic acid
oxidation method (208). Additional recrystallization
of the monoglyceride was performed by dissolving it
i
in hot ether and allowing it to crystallize in cold
ether. The recrystallization increased the purity
■ ' 114
of the monoglyceride to 99 per cent. The melting
point of final monoglyceride obtained was 73.5° C
whereas, according to Malkin and el Shurbagy (209)
it should be 74.0.
Techniques of Feeding and Isolation of Lipid Samples
I. Method of feeding.
The procedure of, the feeding of the animals was
a variation of that used by Morehouse and Thompson
(166). Female rats, 180-210 g. weight, were fasted
for 48 hours before the experiment. Each rat was
fed by stomach tube 0.5 cc. of slightly warmed 1,3-
dioleyl-2-deuteriostearyl-glyceride-C^. In the
experiments where the incorporation of glycerol-C^*
was studied, the feeding procedure was somewhat
14
altered. The dose of glycerol-C intended for
feeding was divided into two portions. Each portion
was administered by stomach tube with a 1 hour time
interval. In the case when animals were also fed
14
triglyceride (0.5 cc*) the glycerol-C was adminis
tered 1 and 2 hours after the administration of the
fat.
_115
Isolation of tissues.
The rats were sacrificed under sodium nembutal
anaesthesia at the end of 3 hours after the admin
istration of triglyceride or at the end of 2 hours
after the administration of the first portion of
14
glycerol-C . The abdominal cavity was opened, the
liver removed and frozen in an alcohol-ether (3:1)
and dry ice mixture. In each experiment the livers
of two animals were combined. The small intestine
was removed, washed.out with 60 cc. distilled water
at 50-60° C. All outside fat was removed, the
intestine cut open and washed three times with
ether-alcohol solution (9:1) and then again with
warm water. In the experiments where animals were
fed monoglyceride, or when it was intended to isolate
monoglyceride from the intestinal lumen, warm ether
instead of ether-alcohol mixture was used. The
ether-alcohol mixture was combined with the water
washing. The water layer was extracted three times
with fresh ether and all extracts were combined. In
some experiments, where the quantitative recovery
of all free fatty acid and monoglyceride from the
intestinal lumen was important, the combined water
wash was acidified with HC1, extracted with ether,
the ether washed with water two times and added to
the main ether extract. After drying with sodium
sulphate, the mixture was filtered and the solvent
evaporated off. The residue is referred to as the
"unabsorbed fat."
The intestinal tissue was frozen in the alcohol-
ether (3:1) and dry ice mixture. For each experiment
the tissues of two rats were combined. The carcasses
of two rats were ground up together and two aliquots
of 40-50 g. each were taken for the determination of
the lipid content.
In some experiments, as will be indicated below,
where the fractionation of the intestinal tissue
neutral fat into free fatty acid, mono-, di-, and
triglycerides was intended to be performed, the
washing out of the intestines was done jLn situ. Such
a change of procedure was necessary in order to avoid
some hydrolysis of the intestinal tissue lipids
... 117
during the washing and removing of outside fat on
the excised intestine. In these experiments the
abdominal cavity was opened, the intestine was cut
just below the pyloric opening of the stomach and
above the ileocecal valve and washed out three times
with a 0.9 per cent solution of NaCl or distilled
water warmed to 50-55° C and then three times with
an ether: alcohol (1:1) mixture. After washing,
the intestine was quickly removed from the organism
and immediately frozen in an ether-alcohol mixture
of the same composition. No removing of outside
fat from the intestine was attempted in such experi
ments .
III. Isolation of lipids from the intestinal tissue, liver
and carcass.
The extraction of lipids from the intestine and
liver was performed according to Goldman, Chaikoff,
Reinhardt, Entenman and Dauben (210). The lipid
fraction insoluble in acetone is referred to as
phospholipid, and the acetone soluble fraction as
neutral fat. The amount of lipid fractions isolated
1I8„
was determined by weight. The determination of the
I
phosphorous content in the lipids isolated was :
carried out by the method of Fiske and Subbarow (211).
The content of phosphorous in the liver phospholipid
<
ranged from 3.02 to 3.30 per cent. The neutral fat j
I
fraction was found to have no phosphorous content. |
The extraction of lipid in the carcass was performed i
i
according to Stevens and Chaikoff (175) with some j
alterations. The amount of lipid isolated was
1
determined by weighing. 1
In the experiments, where glycerol-C^ was
administered, the isolated lipids dissolved in the
petroleum ether were extensively washed with water
containing a small amount of non-labeled glycerol. j
In order to facilitate the washing free from non
incorporated glycerol-C^ several drops of non
labeled glycerol also were added to the tissue during
the mincing in the Waring blender.
IV. The evaluation of methods of the extraction of lipids
from the intestinal tissue and liver.
In the experiments where the incorporation of
glycerol-C"^ into the rat lipids was studied, contam
ination of the neutral fat and phospholipids by
radioactive glycerol not incorporated into the mole
cule is a possibility. Therefore control experiments
were performed,in which the efficiency of washing
out of all non-incorporated glycerol was tested.
Several drops of highly active glycerol-C*"^ were
added to the frozen small intestine and liver of
rats. These,organs were then minced in the Waring
blender with the ether-alcohol mixture and further
processed as in the experiments where rats were fed
with glycerol-C*- ^. The samples of neutral fat and
phospholipid isolated were subjected to radioactivity
determination. Since no appreciable activity was
found in these lipid fractions, it was concluded
that the washing procedure used was sufficient to
ensure that thd radioactivity recorded in lipids in
14
the experiments where rats ingested glycerol-C was
due to the incorporation of this component into
neutral fat and phospholipid.
In the experiments where rats were fed the
! doubly-labeled monoglyceride (mono-l-deuteriostearyl-
i 14
j glyceride-C ) and later monoglyceride was isolated
| from the intestinal tissue, there was a danger that
| the unabsorbed monoglyceride might not be washed out
entirely. Therefore the following type of control
i
j experiments were performed. Normal, starved rats
I
j were placed under sodium nembutal anaesthesia, the
I abdominal cavity opened, the small intestine cut
j just below the stomach and a suspension of the
; doubly-labeled monoglyceride in water introduced
i
t
into the intestinal lumen. Then the experiment pro- «
ceeded with the washing out of the intestine as
usual; later the intestine was frozen, minced, and
the lipids extracted from this tissue. No radio
activity was found in the neutral fat and phospholipid
isolated from this intestine. Thus, all monoglycer
ide introduced into the intestinal lumen was removed
by the washing procedure ordinarily used.
Analytical Procedures
*• Separation of mono-, di- and triglycerides and free
i
i fatty acids in the neutral fat fraction.
^ “ 121 1
i
In some experiments the neutral fat isolated from i
the intestinal tissue, as well as ingested fat, was !
i
fractionated into free fatty acid, mono-, di- and j
l
triglycerides. The separation of the free fatty |
i
acid was accomplished with Amberlite IRA-400 accord
ing to the method described by Cason, Sumrell and
Mitchel (70). The Mattson et al. modification (100,
i
212) of the method of "completion of squares" (213), j
I
j
by solvent extraction (multi-stage separatory funnel
separation), was employed for the separation of the
mono-, di- and triglycerides from each other. For
the isolation of monoglyceride, the ether soluble
material from which the free fatty acid had been
I
removed by Amberlite treatment, was passed through I
a 4 x 4 "completion of squares" process using 80
per cent ethanol-petroleum ether (Skelly A) as the
solvent pair. Under these ;conditions the monoglycer
ide was concentrated in the fraction 1 and 2 of the
alcohol, whereas the di- and triglycerides remained
in the petroleum ether of the first funnel. By
this procedure both isomers of monoglyceride,
1 2 2
1-monoglyceride and 2-monoglyceride, were separated
together, since they have essentially the same parti
tion coefficient.
The separation of the diglyceride and triglyceride
mixture was accomplished by similar procedures. The
solvent pair, however, used here was isooctane and
methanol. The ratio of the volumes was 1 to 2.5
respectively. The extraction proceeded through 10
separatory funnels. Under these conditions the
triglyceride was concentrated in the isooctane
fractions from the first to the fifth funnels while
the diglyceride spread from the first through the
sixth fractions of methanol.
The reliability of the separation technique was
checked on artificial mixtures where one component
of the system was a radioactive compound.
Three types of such mixtures were employed:
1. Palmitic acid-C^, triolein and monostearin.
2. Palmitic acid-C^, monopalmitin and triolein.
3. Stearic acid, triolein and monostearin with
the glycerol moiety of the molecule labeled
by C^,
After the separation of these mixtures into
fractions by the procedures described above, 98-99
per cent of the radioactivity was recovered in the
corresponding fractions. Other fractions usually
had an insignificant number of counts. The procedure
of the separation of the neutral fat into fractions
was also checked on the lipids isolated from the
intestinal tissue. Doubly-labeled monoglyceride
(mono-l-deuteriostearyl-glyceride-C^) or a singly
labeled triglyceride (dioleyl-stearyl-C^-glyceride)
was added to the frozen intestines. These organs
were then minced, the lipids extracted and separated
as has been described previously. Essentially all
of the radioactivity was recovered in the monoglycer
ide or triglyceride fraction depending upon which
labeled material had been added. All other fractions
had only insignificant radioactivity. The reliability
of the separation technique was thus proven by these
experiments. The control procedures also demonstra
ted that no appreciable hydrolysis of added material
occurred during the extractions.
124
The neutral fat isolated from the intestinal
tissue contained, besides free fatty acids and
glycerides, cholesterol and cholesterol esters.
During the fractionation of the neutral fat by the
methods described, free cholesterol as well as
cholesterol esters entered into the higher glyceride
fraction. However, the content of free or esterified
cholesterol was not routinely determined since the
relative amounts of these compounds in the relation
to the total amount of higher glyceride were small
and were considered insignificant for the type of
investigation undertaken.
II. Chemical determination of monoglyceride.
The purity of the monoglyceride fraction isolated
from the intestinal neutral fat, as well as from the
synthetic product, was determined by the periodic
acid oxidation method. The Pohle and Mehlenbacker
modification (208) of this method was used for the
determination of the purity of the synthetic 1-
monostearin. The determination of 1-monoglyceride
and the total monoglyceride in the samples isolated
125
from the intestinal tissue or the intestinal lumen
was carried out according to the microprocedure
proposed by Martin (69). The isomerization of the
2-monoglyceride to the 1-monoglyceride necessary for
the determination of total amount of monoglyceride
was performed by^hydrochloric acid (197) or by the
FeCl3 (104) procedure. The actual content of mono
glyceride in the "monogly'ceride fraction” isolated
from the neutral fat of the intestinal tissue was
93-96 per cent. From 20 to 40 per cent of this
monoglyceride was 1-monoglyceride and the rest 2-
monoglyceride.
III. Determination of free glycerol.
The benzoate derivative of the free labeled
glycerol liberated during the lipolysis of doubly-
labeled triglyceride was prepared by a slightly
modified method of Huntrees and Mulliken (214). The
water wash fraction of the intestinal lumen after
the ingestion of l,3-dioleyl-2-deuteriostearyl-glyc-
-« s
eride-C was extracted 4-5 times with a small amount
of petroleum ether. Two to three drops of non-labeled
; glycerol was added to the water fraction as a carrier
i
j and mixed well. Fresh benzoyl chloride in the amount
| of 0.4 cc. together with 5 cc. of 15 per cent of NaOH
i
were added and the mixture left for 2-3 days in
order that crystallization at room temperature might
occur. The melting point of the glyceroltribenzoate
| was 70-71° C whereas according to the literature
I
: data it should be 71-72° C (214).
i
The radioactivity of the glyceroltribenzoate was
j determined by the procedure described below. Simul-
i
I
taneously the radioactivity of glyceroltribenzoate
1 14
prepared from the glycerol-C with known activity
was measured. All countings were performed at
| infinite thickness. On the basis of the comparison
p
of the activities of these glyceroltribenzoates the
14
amount of glycerol-C originating from the 1,3-
14
dioleyl-2-deuteriostearyl-glyceride-C during the
lipolysis in the intestinal lumen was calculated.
Determination of Radioactivity and Deuterium -Atom Per Cent
of Enrichment
! I. Measurement of radioactivity.
127
The radioactivity was measured by direct planchet-
ing of the isolated lipids and counting at infinite
thickness using a windowless counter with a constant
flow of a quenching gas. A Berkeley scaler was used
in these measurements.
Standard curves had been prepared to obtain
infinite thickness for radioactive lipids and radio
active glycerol. A value of 80 mg. per planchet was
obtained for the infinite thickness of the neutral
fat and phospholipid. For glycerol, which also was
counted by direct plancheting, the infinite thick
ness was 100 mg. per planchet. Samples of high
activity were counted at least 10 minutes, samples
with low activity were counted 30 minutes. However,
where the amount of total counts in 30 minutes was
less than 3000, the counting was continued until
3000 counts were obtained. In the case where the
amount of the isolated lipids was less than required
for infinite thickness, samples were diluted by the
corresponding compounds.
The results of radioactivity determination are
- 128
expressed as the relative specific activity or, in
other words, as the per cent of the labeled molecules
in the fraction. The activity of the material fed
was considered as 100 per cent. Radioactivity is
also expressed in mg. of the material labeled.
Thus, the per cent of neutral fat molecules
14
labeled with glycerol-C is calculated as the ratio
of the specific activity of the isolated fat to the
specific activity of the fat fed times 100.
Specific activity of
Per cent of neu- isolated neutral fat
tral fat labeled _ _______________________ X 100
with glycerol- Specific activity of
C . triglyceride fed
It was assumed that the neutral fat fraction con
sisted of triglyceride of the same composition as
the fat fed. The per cent of the labeled neutral
fat, or relative specific activity, indicates the
per cent of the glycerol of the neutral fat fraction
which originated from the material ingested.
For the calculation of the per cent of phospho-
14
lipid labeled with glycerol-C , a theoretical
129
standard specific activity of phospholipid was
estimated. Here it was assumed that the phospholipid
fraction consisted of lecithin. The calculation was
performed in the following way:
Molecule weight
of dioleyl-deuter-
iostearyl-glyc-
Correction eride 891.6
= 1.104
factor “ Molecular weight ~ 809.3
of lecithin with
one deuteriostearic
acid and one oleic
acid
Theoretical standard specific activity of the
phospholipid = specific activity of triglyceride
fed x 1.104.
Specific activity of
Per cent of isolated phospholipid
phospholipid = x 100
labeled wi|£i Theoretical standard
glycerol-C specific activity of
lecithin
The chemical meaning of the per cent of phospho-
14
lipid labeled with glycerol-C is the same as for
the neutral fat and indicates the per cent of labeled
molecules of glycerol in the phospholipid isolated
- - 130
originating from the triglyceride fed.
In the same way the theoretical standard specific
activity and the per cent of labeling with glycerol-
C14 for the monoglyceride originating from the
14
l,3-dioleyl-2-deuteriostearyl-glyceride-C were
calculated. Similar computations were applied to
the diglyceride, triglyceride and phospholipid origi-
14
nating from the mono-l-deuteriostearyl-glyceride-C
fed.
In the series of the experiments where rats were
fed free glycerol-C14 the theoretical standard
specific activity of the triglyceride and the per
cent of labeling of triglyceride were calculated
as it is shown below. The theoretical standard
«
specific activity of the triglyceride is equal to:
Molecular weight of
glycerol Specific activity
5 of glycerol
Molecular weight of
dioleylstearyl
glyceride
The per cent of neutral fat labeled with glycerol-
was calculated as follows:
131
Specific activity of
Per cent of isolated neutral fat
neutral fat ______________________
x 100
labeled with
glycerol-C^
standard
specific activity of
dioleylstearyl^glyceride
In a similar way the standard specific activities
for monoglyceride and phospholipid and the corre-
glyceride and phospholipid isolated from tissues
14
after the feeding animals with glycerol-C were
calculated. Since the incorporation of glycerol into
fat causes a smaller decrease of radioactivity than
can be expected by dilution, a correction factor was
determined by measuring the activity of the free
glycerol and chemically synthesized fat derived from
it. This factor is equal to 1.35. Therefore the
theoretical standard specific activity, in the
experiments where the incorporation of glycerol-C"^
was studied, should be multiplied by this factor.
However, the relative values of the incorporation of
glycerol-C^ into lipids were considered more impor
tant. Since the value of such a factor for
sponding va!lues of per cent of labeling of the mono-
- ------- ------- — 1.32
phospholipid and monoglyceride was not known, such
a correction was not applied.
II. Measurement of the deuterium atom per cent of
enrichment.
The determination of deuterium enrichment (excess)
in the material fed, as well as, in the isolated glyc
erides and phospholipid from rat tissues consisted
of the following steps (198,215):
1. Oxidation of organic substances and conversion
of their hydrogen and deuterium atoms to water
and heavy water molecules respectively. Oxida
tion (combustion) was carried out by the ordi
nary method of elementary organic analysis
used for the determination of carbon and
hydrogen (216). The water formed during the
combustion was collected by freezing in the U-
tube immersed in a mixture of alcohol and dry
ice. About 20-25 mg. of lipid material were
oxidized.
2. Reduction of the water and heavy water mixture
by zinc and the collection of the hydrogen-
deuterium sample.
The U-tube containing the frozen water was
joined to the reduction apparatus and the
reduction was carried out according to the
method of Sprinson and Rittenberg (215) and
Alfin-Slater et al. (217,218). The resulting
deuterium-hydrogen mixture was collected in a
vessel over mercury. When the reduction was
completed the hydrogen-deuterium gas was trans
ferred into a sample container by a modified
Toepler pump.
The measurement of the deuterium/hydrogen ratio
was performed on a Consolidated-Nier Mass Spectro
meter, Model 21-201 (217,219,220,221,222). The ratio
of the mass 3 (HD+) to the mass 2 (HH+) was measured.
Since the mass 2 and mass 3 ion beams are too far
apart to be simultaneously collected, the mass 2 was
first focused and then the mass 3. A calibration
curve was prepared for the conversion of the ratios
obtained on the Mass Spectrometer to the atom per
cent of enrichment of deuterium.
' 134
The results of the deuterium enrichment of the
lipids isolated from the tissues are expressed in mg.
of material labeled with deuteriostearic acid as well
as the per cent of the lipid fraction tagged with
this acid.
For the triglyceride fraction (and also crude
neutral fat) isolated after the administration of 1,
3-dioleyl-2-deuteriostearyl-glyceride-C^ the per
cent of labeling with deuteriostearic acid was cal
culated as follows:
Atom per cent of deuter
ium excess in the iso-
Per cent of neutral lated neutral fat
fat labeled with = -------------------------- x 100
deuteriostearic Atom per cent of deuter-
acid ium excess in the doubly-
labeled triglyceride fed
In all other cases the per cent of any compound
labeled with deuteriostearic acid was calculated as
follows. The theoretical standard atoms per cent
was computed with the assumption that one deuterio
stearic acid molecule was incorporated into the com
pound. Then the ratio of atom per cent of deuterium
actually observed in the isolated compound to the atom
per cent of deuterium in the theoretical standard
compound was then determined and the product multi
plied by 100. It was realized that the calculations
of the per cent of molecules labeled by glycerol-C*4
or deuteriostearic acid given above included many
assumptions. Nevertheless such treatment of the
data is more convenient since it gives a chance to
compare results obtained after the administration
of varied labeled compounds in the different experi
ments. The difference of the incorporation of
14
glycerol-C and deuteriostearic acid into lipids
is given by applying the ratio — which is
equivalent to the following:
C
Per cent of compound
^ l^ijpeled with glycerol-
D Per cent of compound
labeled with deuterio
stearic acid
C14
As evidence that this g— ratio does reflect the
14
real difference of the incorporation of glycerol-C
and deuteriostearic acid into the isolated lipid
fraction, the simple ratio of the specific activity
to atom per cent of deuterium excess was also
~ ----- 136
calculated. This ratio, does not include any assump
tions and is the result of experimental measurements,
The results obtained by both approaches showed the
same trends. Therefore, only the --- ratio is
D
given in the tables.
L. EXPERIMENTAL RESULTS
t f i
)
t
! Extent of the Hydrolysis of the Digested Fat Preceding
i Absorption
i
1 The extent of the hydrolysis of triglyceride pre-
I
I ceding absorption was investigated by feeding 0.5 cc. of
l
! the doubly-labeled triglyceride, l,3-dioleyl-2-deuterio-
i
1 - j j
| stearyl-glyceride-C . If this triglyceride were not
t
| hydrolyzed in the intestinal lumen and were absorbed as
1 14
p
I an emulsion, the ratio _ of the isolated neutral fat
' D
from intestinal tissue should be the same as in the mate-
t
1 rial fed and therefore equal to 1.00. In the event that
J100 per cent of the doubly-labeled fat should undergo
complete hydrolysis in the intestinal lumen and if the
:liberated glycerol-C^ were not used for the resynthesis
r14
of the fat in the intestinal wall, then the ratio ___
D
'would be 0.
! nl4
In our experiments (Tables I, II) the mean __
D
ratio for the neutral fat isolated from the small intes
tinal tissue is equal to 0.60 after a 3 hour absorption
j
I period. If the glycerol liberated during the lipolysis
| of fat is not utilized in the intestinal mucosa for the
i
I
| resynthesis of triglyceride, this ratio indicates that
i :
i around 60 per cent of fat was absorbed in the form of
j
iglycerides without complete hydrolysis and 40 per cent
I
was completely hydrolyzed to glycerol and free fatty acids
: preceding absorption. The range of variation of the
i
I amount of fat absorbed in the form of glycerides in the
individual experiments is from 47 to 75 per cent.
That the glycerol-C14 found combined could not
have first been split from the glyceride and again esteri-
fied with the free fatty acid is shown by the results
obtained in the following experiments. Rats were fed
with the same amount of l,3-dioleyl-2-deuteriostearyl-
glyceride-C14 and 1 and 2 hours later with 0.25 cc.
C14
of unlabeled glycerol each time. The mean —— ratio
obtained under this condition is equal to 0.61 (Tables I,
III). If the retention of radioactive glycerol in the
triglyceride isolated from the intestinal wall was due
to an appreciable reutilization of absorbed glycerol-C14
the minimum 14 fold dilution of this radioactive glycerol
( should essentially decrease the ratio. Actually
,no such decrease in the ratio was observed (Table I).
,Thus, glycerol liberated during hydrolysis in the intes-
!
tinal lumen is not reutilized to an appreciable extent
for the resynthesis of triglyceride in the mucosa. Hence
all glycerol-C^ found in the neutral fat isolated from
f
.the. intestinal tissue penetrates the mucosa in some form
i
»
:of glyceride, with at least one hydroxyl group of glycerol
I
esterified with fatty acid.
I
t
; The amount and the composition of the neutral fat
I -
I
in the intestinal tissue are determined by three processes
proceeding simultaneously: absorption of fat, transport
of fat through the lymphatic system and metabolic pro
cesses in the intestinal tissue. -Therefore the
D
ratio only approximately indicates the extent of hydrolysis
preceding the time of sacrificing the rats.
[ In rats killed after a 6 hour absorption period
the mean ratio is equal to 0.45 (Tables I, IV). It
D
seems that the lipolysis of ingested doubly-labeled fat
in the intestinal lumen proceeded further and relatively
more material was absorbed in the form of free fatty acid.
1 rl4
iThe change of ~--- ratios of unabsorbed lipids isolated
I D
TABLE I
rl4
THE il— RATIOS OF NEUTRAL FAT ISOLATED FROM INTESTINAL TISSUE IN DIFFERENT TIME PERIODS
AFTER THE ADMINISTRATION OF l,3-DI0LEYL-2-DEUTERI0STEARYL-GLYCERIDE-C14 WITH OR WITHOUT
NON-LABELED FREE GLYCEROLU
Amount of 1,3-dioleyl-
2-deuteriostearyl-
glyceride-Cl4 fed
to one rat
mg.
Absorp
tion time
hours
Per cent
of lipid
absorbed
Amount of
non-labeled
glycerol fed
mg.
C ratio
®of neutral
fat from
, intestinal
tissue
Mean
c1 4
D
457 3 40.0 0.56
457 3 41.4
-
0.47
457 3 59.6 -
0.75 0.60
457 3 42.8 — 0.55
457 3 31.4 630 0.64
-
457 3 25.6 630 0.56 0.61
457 6 66.7
_
0.44
457 6 68.8 - 0.46 0.45
457 20 96.5
' _
0.23
457 20 94.2 — 0.30 0.27
J^Each experiment was perforaed on two rats. The values are expressed on a per
rat basis.
^These values were calculated on the basis of the recovery of the total lipids
from the intestinal lumen. ^
v
140
TABLE II
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE LIPIDS OF RATS 3 HOURS
AFTER THE ADMINISTRATION OF 0.50 cc. l,3-DIOLEYL-2-DEUTERIO^TEARYL-GLYCERIDE-C14.1'
Tissue and Material Sample
weight
mg.
Glycerol-
C14 Labeled
Lipid
mg.
Per cent
Glycerol-
C Labeled
Lipid
Deuterio
stearic
Acid
Labeled
Lipid
mg.
Per cent
Deuterio
stearic
Acid
Labeled
Lipid
Cl4
D
Material fed: l,3-dioleyl-2-
deuteriostearyl-glyceride-
ci4
457 457 100.0 457 100.0 1.00
Unabsorbed Fat 236 174.0 73.7 186.0 . 78.8 0.94
Small Intestinal Tissue
Neutral Fat
Phospholipid
239
133
24.9
2.7
10.4
2.0
41.1
4.9
17.2
3.7
0.60
0.54
Liver
Neutral Fat
Phospholipid
164
296
4.0
4.3
2.4
1.4
6.8
18.4j
4.1
6.2
0.58
0.23
Carcass
Total Lipid 11250 18.0 0.16 27.0 0.24 0.682)
^The results are tabulated as the average of 4 groups; each group contains two rats. The
values are expressed on a per rat,basis. 51.0 per cent of the material fed was absorbed
during 3 hours. This value is calculated on the basis of the recovery of the total lipids
from the intestinal lumen.
'Average of two groups of animals only.
141
TABLE III
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE LIPIDS OF RATS AFTER THE
ADMINISTRATION OF 0.50 cc. l,3-DIOLEYL-2-DEUTERIOSTEARYL-GLYCERIDE-C AND GLYCEROL '
Tissue and Material
isolated
. . . . . i ,
Sample
weight
mg.
Glycerol-
C14Labeled
Lipid
mg.
Per cent
Glycerol-
Cl4 Labeled
Lipid
Deuterio
stearic
Acid Label
ed Lipid
mg.
Per cent
Deuterio
stearic
Acid Label
ed Lipid
C14
D
Material fed: 1,3-dioleyl-
2-£euteriostearyl-glyceride-
457 457 100.0 457
s
100.0 1.00
Glycerol 630 0 0 0
Unabsorbed Fat 327 264 80.7 270 82.8 0.99
Small Intestinal Tissue
Neutral Fat 329 28.3 8.6 46.4 14.1 0.61
Phospholipid 134 1.7 1.2 3.6 2.6 0.47
Liver
Neutral. Fat 253 1.1 0.4 5.1 2.0 0.21
Phospholipid 257 0.8 0.3 13.4 5.2 0.06
Carcass
'
Total Lipid 14525 11.6 0.08 17.4 0.12 0.63
^The results are tabulated as the average of 2 groups; each group contains two rats. ^
The values are expressed on a per rat basis. 29 per cent of the fat fed was absorbed during
3 hours. This value is calculated on the basis of the recovery of total lipids from the
intestinal lumen.
142
TABLE IV
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE LIPIDS OF RATS 6 HOURS
AFTER THE ADMINISTRATION OF 1,3-DIOLEYL- 2-DEUTERIOSTEARYL-GLYCERIDE-C14
Tissue and Material
isolated
Sample
weight
mg.
Glycerol-
C14 Labeled
Lipid
mg.
Per cent
Glycerol-
C14 Label
ed Lipid '
Deuterio
stearic
Acid Label
ed Lipid
mg.
Per cent
Deuterio
stearic
Acid Label
ed Lipid
• C14
D
Material Fed: 1,3-dioleyl-
2-deuteriostearyl-
glycerol-Cw 457 457 100.0 457 100.0 1.00
Unabsorbed Fat 148 64.0 43.3 76.0 51.5 0.84
Small Intestinal Tissue
Neutral Fat 267 8.9 3.3 19.9 7.4 0.45
Phospholipid 130 2.0 1.5 5.2 4.0 0.37
Liver
Neutral Fat 134 3.4 2.5 7.5 5.6 0.45
Phospholipid 265 3.8 1.4 29.4 11.1 0.13
!)The results are tabulated as the average of 2 groups; each group contains two rats.
The values are expressed on a per rat basis. 67.7 per cent of material fed was absorbed
during 6 hours. This value is calculated on the basis of the recovery of total lipids
from thfe intestinal lumen.
143
144
from the intestinal lumen pointed in the same direction.
14
The ^__ _ ratio of the total unabsorbed lipid material
D
from the intestinal lumen after 3 hours ingestion period
was 0.94 (Table II) and after 6 hours ingestion period
r14
was 0.84 (Table IV). Such a change in the ____ ratio
D
demonstrated the more extensive hydrolysis of ingested
triglyceride with subsequent loss of radioactive glycerol.
0
In rats killed 20 hours after the administration
of fat, when the process of absorption is completed, the
mean ratio of neutral fat from small intestinal
D
tissue is equal to 0.27 (Tables I, V).
All these data indicate that in the first hours
more fat is absorbed in the glyceride form, whereas at
the end of absorption it penetrates into the intestinal
mucosa predominantly as free fatty acid. When the main
bulk of the ingested fat has been absorbed, the extent of
complete hydrolysis is in the range from 25 to 56 per
cent. The mean value of the extent of complete hydrolysis
would be slightly less than 50 per cent. In other words
around half the amount of ingested fat is absorbed in the
form of glyceride and the other half, after complete
- 145
hydrolysis, as glycerol and fatty acid.
)
I
In the experiments where rats were killed after
j
3 hours absorption period, the unabsorbed fat from the
I
intestinal lumen was recovered and subjected to fractiona
tion. The composition was as follows:
i
Free fatty acid 16.0 per cent
i
i
| Monoglyceride 7.3 per cent
Di- and Triglycerides 76.7 per cent
At the same time free glycerol, recovered as the
!tribenzoate from the intestinal lumen water washing,had
i .
radioactivity in an amount equal to 0.2 per cent of the
i . 1 A
glycerol-C administered in the doubly-labeled fat. This
is the first experimental demonstration of the formation of
free glycerol during the lipolysis of triglyceride in vivo.
Phospholipid isolated from the intestinal tissue
was labeled with in the glycerol moiety and contained
deuteriostearic acid. The amount of labeled phospholipid
3 and 6 hours after the ingestion of doubly-labeled tri
glyceride was much smaller than the amount of neutral fat
C14
isolated from the same tissue. The ---- ratios of
lphospholipid were also slightly less than those for neutral
TABLE V
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE LIPIDS OF RATS 20 HOURS
AFTER THE ADMINISTRATION OF 1,3-DIOLEYL-2-DEUTERIOSTEARYL-GLYCERIDE-C14.
Tissue and Material
isolated
Sample
' weight
Glycerol-
C14 Label-
. ed Lipid
mg.
Per cent
Glycerol-
C14 Labeled
Lipid
Deuterio
stearic
Acid Label
ed Lipid
mg.
Per cent
Deuterio
stearic
Acid Label
ed Lipid
Cl4
Material Fed: 1,3-dioleyl-
2-deuteriostearyl-
glyceride-C14 457 •457 100.o'
(
457 100.0 ; 1.00
Small Intestinal Tissue
Neutral Fat 156 0.78 0.50 2.94 1.89 0.27
Phospholipid 114 1.27 1.11 7.60 6.67 0.17
Liver
Neutral Fat 170 1.18 0.6S 5.87 3.45 ‘ 0.20
Phospholipid 227 ‘ 2.45 1.08 22.90 • 10.12 ' 0.11
Carcass
Total Lipid 8180 14.72 ’ 0.18 42.45 . 0.52 0.35
^The results are tabulated as the average of 2 groups} each group contains twb rats.
The values are expressed on a per rat basis. 95 per cent of material fed was absorbed
during 20 hours. This value is calculated on the basis of the recovery of total lipids
from the intestinal lumen.
“ 147
fat (Tables II, IV). This gives rise to the question
whether the labeling of phospholipid is due to utilization
of free glycerol-C^ and free deuteriostearic acid or to
the formation of phospholipid from some absorbed glycer
ides. The simultaneous administration of doubly-labeled
triglyceride and non-labeled glycerol and the resulting
high dilution of glycerol-C^ liberated during lipolysis
rl4
did not decrease the ~__ ratio of phospholipid to a
D
great extent, as it should be if the incorporation of
glycerol-C^ into phospholipid proceeded through the
stage of free glycerol (Tables II, III). Thus, phospho
lipid is formed directly from the absorbed glycerides.
However, what form of glyceride (monoglyceride, diglyceride
or triglyceride) is actually the precursor of phospholipid,
is impossible to conclude from our experiments. Some
c14
smaller value for the z . ratios in phospholipid than
D
that for neutral fat, observed in our experiments, probably
may be explained on the basis of the selective incorpora
tion of labeled stearic acid into phospholipid, as has
been demonstrated by Borgstrom (128).
The simultaneous administration of doubly-labeled
....................... " " 1 4 8
triglyceride with non-labeled glyeerold decreased the 1
rl4 rl4 !
^ ratios of liver lipids. The z ratio of neutral i
D D
fat dropped from 0.55, when only doubly-labeled triglycer- I
ide was fed, to 0.21 when non-labeled glycerol was simul- !
j
taneously administered (Tables II, III). In phospholipid
r14 !
the ---- ratio decreased from 0.23 to 0.06. This indicates
that the liver extensively utilized exogenous glycerol for
pl4
the formation of phospholipid and neutral fat. The ---
ratio of liver phospholipid was usually much smaller than i
for liver neutral fat (Tables II, III, IV). Such a
r14 .
smaller —--- ratio of liver phospholipid is probably
D
due to a selective incorporation of stearic acid into
phospholipid, since the per cent of labeling of liver
phospholipid by deuteriostearic acid was always higher
than labeling of the neutral fat. However, a much more
interesting observation is the ability of the liver to
utilize free glycerol extensively for the synthesis of
both neutral fat and phospholipid. The presented data
do not permit us to conclude whether the synthesis of both
of these compounds occurs independently or whether the
high turnover of one of these compounds affects the
• " " ' ' “ 149
i
t
! con-position of another, since they both are probably in
j a condition of: f lexible equilibrium.
! The comparison of the ratios, as well as the ■
D i
extent of labeling of liver lipids isolated from the rats
20 hours after the administration of doubly-labeled tri-
■ glyceride with those at 3 and 6 hours demonstrated a more
; rapid loss of the labeled glycerol moiety of lipid 1
; molecules than of deuteriostearic acid. It is apparent
j that the rate of the turnover of these two moieties of
i
! lipids is different and it is much higher for the glycerol
i part of the molecules (Tables II, IV, V).
i
i
! Investigation of the Role of Lower Glycerides in Fat
' Absorption
1 In the previous chapter it was shown that from
75 to 46 per cent of the ingested fat was absorbed in the
form of glycerides. However, the method applied did not
permit any conclusions to be drawn concerning the nature
♦
of these glycerides. In the intestinal lumen during the
jdigestion of triglyceride, as has been demonstrated, there
are present all forms of glycerides: monoglyceride, diglyo-
| eride and triglyceride. Theoretically any form of glycecide
150
can penetrate into the intestinal mucosa. It was consid
ered that the most reliable data concerning the nature
of the glyceride absorbed would be obtained by feeding
experiments with doubly-labeled monoglyceride, namely
with mono-l-deuteriostearyl-glyceride-C^. According
to Mattson et al. (99) this monostearin would be the
normal intermediate product of lipolysis in the intestinal
lumen of the doubly-labeled triglyceride, l,3-dioleyl-2-
deuteriostearyl-glyceride-C^.
Jeker (158) and BorgstrSm (128) demonstrated the
presence of free fatty acid in the intestinal tissue
during fat absorption. If monoglyceride were also
absorbed as a unit, one would expect to find this compound
in the intestinal tissue lipids. The monoglyceride which
penetrates into the intestinal tissue as such should have
the same ratio as the monoglyceride fed.
D
As a preliminary step to the feeding of rats
with doubly-labeled monostearin and the attempt to isolate
this compound from the intestinal tissue, it was decided
to fractionate neutral fat from normal, fasted rats. The
fractionation was performed by the techniques of Cason
_ ... 151
j et al. (70) and Mattson (212). In order to eliminate the
J possibility of hydrolysis of the intestinal lipids in the
• i
tissue, the intestines were washed out in situ, removed
Jfrom the animals and immediately frozen. Hence in these
/
! experiments the intestinal tissue neutral fat was diluted
j to some extent by the mesenterial fat, since the latter
i
was removed together with the intestines. The fractiona-
j tion of the neutral fat isolated in such a way from the
I
I intestinal tissue showed that this fat was actually a
i
mixture of triglyceride and its derivatives. (Table VI)
From one rat was isolated, on an average, 286 mg. of
1 neutral fat, which contained 9.8 mg. of free fatty acid
(3.42 per cent), 4.2 mg. of monoglyceride (1.47 per cent)
i
. The remainder consisted of di- and triglycerides,
’ cholesterol and cholesterol esters.
i
The doubly-labeled monostearin was fed to rats in
I the form of a water suspension in the amount of 175 mg.
j per rat. This 175 mg. of monostearin is approximately
I
: equivalent to the amount of monostearin which may have
formed during the hydrolysis of doubly-labeled triglycer-
{ ide fed in the previous experiments.
4
TABLE VI
THE COMPOSITION OF NEUTRAL FAT ISOLATED FROM THE INTESTINAL
TISSUE OF 48 HOUR FASTED RATS
Number
Rats in
. Total
Amount
Free Fatty Acids Monoglyceride Diglyceride Triglyceride
Experi
ment
of Neu
tral Fat
mg. mg. Per cent mg. Per cent mg. Per cent mg. Per cent
3
957
34; 6 3.62 13.1^ 1.37 74.4 7.78 835.0 87.23
5 1295 38.0 2.90 20.3 1.60 285.0 22.00 952.0 * 73.50
3 898 35.2 3.92 12.5 1.40 76.0 8.41 773.0 86.27
Average
per one
rat 286 9.8 3.42 4.2 1.47 39.6 13.85 232.4 81.26
152
I The administered monostearin was diluted in the
i
j intestinal lumen by metabolic fat, probably excreted by
the intestinal tissue. On an average, 67.5 mg. of lipid
: material per rat was identified as the endogenous fat
l
j present in the small intestinal lumen. A slightly smaller
'amount, 59.3 mg., of metabolic fat was estimated during
the ingestion of doubly-labeled triglyceride dioleylstearyl4
i glyceride. An average 28.7 mg. per rat of metabolic fat
was recovered from the small intestinal lumen of rats
j fasted for 48 hours. It seems that the ingestion of
I
| monostearin and dioleostearin increased the amount of
metabolic fat present in the small intestinal lumen.
Around 50 per cent of the doubly-labeled mono-
. glyceride was absorbed during a 3hhour period, as was
calculated from the amount of the deuteriostearic acid
t
, (free and esterified) recovered from the small and large
. intestinal lumen and feces. From 20 to 62 per cent
j
| (a mean 33 per cent) of unabsorbed, labeled material was
J
; recovered in the form of free fatty acids. Four and four
; tentts per cent of the administered radioactivity was
recovered in the water wash of the small intestine. No
154
further identification of this fraction was attempted, but
it may, at least partially, be attributed to free glycerol
liberated during hydrolysis of the monoglyceride.
The distribution of the glycerol-C^ and deuterio
stearic acid in the lipids isolated 3 hours after the
administration of monoglyceride is given in Table VII.
r14
The — ratios for neutral fat and phospholipid isolated
from the intestinal tissue were 0.46 and 0.37 respectively.
Thus, an average of 54 per cent of monoglyceride was
14
absorbed after complete hydrolysis to glycerol-C and
deuteriostearic acid, and 46 per cent penetrated into the
intestinal tissue in the form of monoglyceride.
^ 14
The higher value of the ____ ratio for liver
D
neutral fat (0.57) than for the same lipid fraction from
the intestinal tissue indirectly showed that at the
beginning of the ingestion a relatively more unhydrolyzed
form of monoglyceride had been absorbed. However, it is
not excluded, but is hardly probable, that the absorbed
free glycerol-C^ was utilized, in a greater extent, for
the synthesis of liver lipids than in the case of ingestion
of triglyceride and therefore increased the value of the
TABLE VII
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE LIPIDS OF RATS 3 HOURS
AFTER THE ADMINISTRATION OF MONOSTEARIN (M0N0-1-DEUTERI0STEARYL-GLYCERIDE-C14) '
Tissue and Material
isolated
Sample
weight
mg.
Glycerol-
C1? Label
ed Lipid
mg.
Per cent
Glycerol^
C14 Label
ed Lipid
Deuterio
stearic
Acid Label
ed Lipid
mg.
Per cent
Deuterio
stearic
Acid Label
ed Lipid
Cl4
D
Material Fed: Mono-1-
deuteriostearyl-
glyceride-C14 175 175 100.0 175 100.0 1.00
Small Intestinal Tissue
Neutral Fat 197 4.10 2.08 9.00 4.60 0.46
Phospholipid 143 0.9 0.63 2.48 . 1.73 0.37
Liver
Neutral Fat 104 1.6 1.55 2.8 2.70
V
0.57
Phospholipid 267 1.6 0;58 6.5 2.44 0.24
^The results are tabulated as the average of 4 groups of rats; each group contains two animals.
The values are expressed on a per rat basis.' 50 per cent of the fed material was absorbed during
a 3. hour period. This value is calculated on the basis of the recdvery of total deuteriostearic
acid from the intestinal lumen and feces.
; ratio. In any case, the ratios of neutral
D D
t fat and phospholipid isolated from liver were identical
i
iwhether rats were fed monoglyceride or triglyceride
| r14
|(Tables II, VII). The ____ ratios of neutral fat and
j D
!phospholipid isolated from the intestinal tissue were
t
I
I slightly higher where rats ingested triglyceride than
f 14
where they ingested monoglyceride. The z . ratio of
! 5
|neutral fat from the intestinal tissue after a 3 hour
absorption period with doubly-labeled triglyceride was
;0.60 whereas doubly-labeled monoglyceride under the same
i
.conditions gave a ratio of 0.46. If during the ingestion
'of triglyceride a large amount of di- and triglyceride
penetrated into mucosa, as was originally postulated by
rl4
Frazer (40-42), the difference in the :r— — ratios
u
obtained for the intestinal tissue neutral fat should be
greater. This suggests the conclusion that during the
absorption of triglyceride the main bulk of glycerides
.which penetrate into mucosa as a unit are monoglyceride.
,If the higher glycerides also penetrate into the intes
tinal mucosa, their relative amount is small. It is
I realized that these conclusions are of a preliminary
i
:nature and require further experimental support.
157
i The fractionation of neutral fat isolated from the
intestinal tissue 3 hours after the administration of
i
I doubly-labeled monostearin demonstrated that this fat was
'composed of free fatty acid and monoglyceride as well as
jdi- and triglycerides. The amount of free fatty acid and
monoglyceride was higher than in starved animals. The
jC^ ratio for the isolated monoglyceride was equal to
!B
,1.08 (Table VIII). Such a ratio indicates that monoglycer-
I
iide penetrated into the intestinal tissue as a unit without
pl4
jhydrolysis. In addition to the ^___ ratio, the extent
of tagging of monoglyceride and other fractions pointed
in the same direction. The monoglyceride fraction was
the fraction most highly labeled by and deuterium and
therefore it could not have originated either by direct
esterification of glycerol-C^ and deuteriostearic acid
or by hydrolysis of the di- or triglycerides. The slightly
II * i c •
higher ___— ratio of the isolated monoglyceride than the
theoretically expected 1.00, may be explained not only by
an error of measurement, but also may be due to the process
of partial transesterification of monoglyceride in the
I intestinal lumen, as has been observed by Borgstrom (111).
r 14
The g— ratio for the diglyceride and triglyceride
|fraction is equal to 0.47. The further separation of the
di- and triglycerides mixture into its components and the
: p l ^ j .
jdetermination of ^--- ratios for the separated compounds
‘shows the following changes of such ratio:
! Monoglyceride 1.08
| Diglyceride 0.60
[
; Triglyceride 0.48
The relative amount of deuteriostearic acid in
these esters increases from monoglyceride to triglyceride
due to the incorporation of absorbed free deuteriostearic
acid formed during the lipolysis of monoglyceride in the
i
lumen. Thus, di- and triglycerides appear to be derived
from the absorbed monoglyceride.
; r14
The g ratio for phospholipid isolated from
the intestinal tissue after the ingestion of doubly-labeled
i
j monoglyceride is equal to 0.37 (Table VII) . Here, again,
j as in the case when rats were fed doubly-labeled triglyc-
c14
ieride, the ratio is slightly lower for the phospho-
D
' lipid than for the neutral fat isolated from the same
i
tissue. Nevertheless both moieties of phospholipid mole
cules are labeled and it seems that the intestinal wall
TABLE VIII
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE NEUTRAL FAT FRACTIONS OF
THE INTESTINAL TISSUE 3 HOURS AFTER THE ADMINISTRATION OF DOUBLY-LABELED MONOSTEARIN
(MONO-1-DEUTERIOSTEARYL-GLYCERIDE-C14)11
Sample Glvcerol-
Cw Label
Per cent Deuterio Per cent
Material isolated Weight Per cent Glycerol-
C14 Label
stearic Labeled
C14
from total ed Lipid Acid Label with Deuter
Neutral ed Lipid ed Lipid iostearic
D
Fat Acid
mg. ing. mg.
-
Neutral Fat
Free Fatty Acid
197.0
19.4
100.0
9.9
4.10. 2.08 9.00
0.45
4.60
2.27
0.46
(0.402-0.526)
Monoglyceride 11.2 5.7 0.50 4.48 0.46 4.13 1.08
(1.080-1.085)
Di- and Tri
glycerides^' 165.4 84.4 2.00 1.21 4.27 2.58 0.47
(0.432-0.555)
^The results are tabulated as the average of 2 groups of rats; each group contains 4 animals.
The values are expressed on a per rat basis.
^Calculated as triglyceride.
phospholipid originated from glycerides. However, on the
basis of the available data it is impossible to conclude
whether monoglyceride, diglyceride or triglyceride is the
direct precursor of the intestinal phospholipid. In any
case, it is obvious that the monoglyceride which pene
trated into the intestinal mucosa as a unit plays the role
i
|of a frame for the formation of triglyceride as well as
i
,phospholipid.
i
i
The Investigation of the Nature of Glyceride Absorbed
\
'During the Digestion of Triglyceride
Borgstrom (104); Mattson, Benedict, Martin and
Beck (99); Mattson and Beck (100) considered that the
hydrolysis of triglyceride in the intestinal lumen was
not random, but followed a definite pattern. First there
is formed predominantly 1,2-diglyceride which is
further hydrolyzed to 2-monoglyceride. According to
Mattson and his collaborators (99,100) this 2-monoglycer-
ide is isomerized to 1-monoglyceride. If such a scheme
of intestinal hydrolysis were to be applied to the
14
l,3-dioleyl-2-deuteriostearyl-glyceride-C , one would
161
expect to obtain predominantly mono-2-deuteriostearyl-
glyceride-C*-4 Qr mono-l-deuteriostearyl-glyceride-Cl4.
Since the monoglyceride can be partly absorbed as such
without hydrolysis, as has been demonstrated in the
earlier experiments, it was expected that during the
ingestion of l,3-dioley-2-deuteriostearyl-glyceride-C^
cl4
a monoglyceride with a __ ratio equal to 1.00 or
D
slightly higher would be isolated from the intestinal
tissue. Four rats were fed with l,3-dioleyl-2-deuterie-
stearyl-glyceride-C^*Y and 6 hours later the animals
were sacrificed. The lipids from the intestinal tissue
were extracted as usual and separated into neutral fat and
phospholipid fractions. The neutral fat, isolated in
such way, was subjected to further fractionation to free
fatty acid, monoglyceride and higher glycerides, and
the labeling of each fraction was determined. The results
of this experiment are given in Table IX. Whereas the
ratio of the total neutral fat was equal to 0.45,
D
the same ratio for isolated monoglyceride was 1.0. Thus,
TABLE IX
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE NEUTRAL FAT FRACTIONS OF
THE INTESTINAL TISSUE 6 HOURS AFTER THE ADMINISTRATION OF 1,3-DIOLEYL-
2-DEUTERIOSTEARYL-GLYCERIDE-C14.
Sample
Glycerol-
C14 Label
ed Lipid
mg.
Per cent Deuterio- Per cent
Material Isolated
Weight
mg.
Per cent
from total
Neutral Fat
Glycerol-
C14 Label
ed Lipid
stearic
Acid Label
ed Lipid
mg.
Deuterio-
iostearic v
Acid Label
ed Lipid
C*4
D
Material Fed: 1,3-
dioleyl-2-deuterio-
8|j?aryl-glyceride-
457.0 457 100 457 100 1.00
Neutral Fat
Free Fatty Acid
Monoglyceride
Di- and Tri
glycerides '
267.0
37.7
14.3
215.0
100.0
14.1
5.4
80.5
8.9
. 0.22
9.40
3.3
1.51
4.37
19.9
0.82
0.22
18.5
7.4
2.17
1.50
8.59
0.45
1.01
0.51
^The results are tabulated for a group of 4 rats. The values are expressed on a per rat basis.
67.7 per cent of the material fed was absorbed. This value is calculated on the basis of the
recovery of total lipids from the intestinal lumen.
163
the monoglyceride isolated from the intestinal tissue
originated from the ingested doubly-labeled triglyceride
and is a doubly-labeled monostearin.
This experiment supports the scheme of intestinal
lipolysis of triglyceride proposed by Borgstrom (104) and
Mattson et al. (99,100) and is evidence that monoglyceride
formed during this lipolysis actually penetrates as a unit
into the intestinal tissue.
It is realized that the isolation of a monoglycer
ide with ratio equal to 1.01 does not necessarily
D
indicate that only doubly-labeled monostearin has pene
trated into the intestinal tissue during the digestion of
l,3-dioleyl-2-deuteriostearyl-glyceride-C^. An extensive
transesterification of the digested glycerides or their
random lipolysis in the intestinal lumen may result in
f 9
the formation of a monoglyceride with a ~___ ratio much
D
greater than 1.01, and which subsequently will penetrate
into the intestinal tissue. In addition, simultaneous
synthesis of a monoglyceride in the intestinal tissue
from endogenous glycerol and from absorbed deuteriostearic
acid may form a mixture of monoglycerides with the
I pl4
I resulting ____ ratio equal to 1.01. However, the chances
I D C1^
i are very small of obtaining a value of _____ ratio equal
I D
'to 1.01 under these circumstances.
In this experiment no precautions were taken to
eliminate the hydrolysis of fat during the isolation of
I
|
! neutral fat, therefore the absolute amount of each fraction
I isolated, such as free fatty acid and monoglyceride, cannot
! rl4
; be considered as correct. Probably the ____ ratio of
! D
| monoglyceride is decreased to some extent by the hydrolysis
| of higher glycerides and phospholipid with a smaller
i ^
I ratio than in the monoglyceride fraction. However, this
!
I
experiment did prove that some monoglyceride formed during
, the hydrolysis of triglyceride were absorbed as a unit
I
without hydrolysis.
Incorporation of Glycerol-C^ into Rat Lipids
t
i
It was reasoned originally, that the mechanism of
, fat absorption, especially the extent of hydrolysis of
|
i triglyceride preceding absorption, could, possibly, be
i
. clarified by the simultaneous feeding of triglyceride
1 and labeled glycerol to the animals. The molecules of
i
i triglyceride which were completely hydrolyzed in the
... " ■ ' • 165
jintestinal lumen to fatty acid and glycerol, should after
jabsorption be re-esterified. If an amount of glycerol-C^
I i
jwere .present in the intestinal tissue in great excess
I !
in comparison with the glycerol formed from hydrolysis !
of fat and the endogenous glycerol which might be formed
i
:in the intestinal mucosa, the resynthesis of triglyceride
j in the intestinal wall should proceed with predominant
i ;
!utilization of the labeled glycerol. Thus, the extent of
i i ;
I tagged glycerol.into the absorbed fat should indicate the
amount of triglyceride which underwent complete hydrolysis..
f
I At the time when this work was started only the paper of
Favarger, Collet and Cherbuliez (153) was available. The
.failure to observe any appreciable incorporation of deu-
terioglycerol into the absorbed fat was attributed to the
; small dosage of tagged glycerol used by these authors.
In order to aid in the evaluation of the quantita-
I
■tive relationship between the total amount of absorbed fat
j present in the intestinal wall and the amount which had
been formed through complete hydrolysis and the incorpora-
; i
1 tion of glycerol-G*-^, a triglyceride labeled with deterio- ,
stearic acid (l,3-dileyl-2-deuteriostearyl-glyceride) was
•' 1 6 6
administrated simultaneously with glycerol-C^. The proce-
I
;dure of feeding the experimental rats was a variation of
ithat used for doubly-labeled triglyceride. Each rat was
I ‘ i
i ;
'fed by stomach tube with 0.5 cc. of 1,3-dioleyl-2-deuterio-
steary1-glyceride and 1 and 2 hours later with an equal
amount of glycerol-C^. The animals were sacrificed at the I
I
iend of 3 hours following their feeding with triglyceride. 1
i t
In Tables X and XI are presented the results when !
457 mg. of l,3-dioleyl-2-deuteriostearyl-glyceride and 315
jor 630 mg. glycerol-C^ were administered respectively. The
Iradioactive glycerol was incorporated in both the neutral
fat and phospholipid fractions isolated from the intestinal
tissue and liver, as well as in the lipids isolated from
the carcass. If all of the absorbed fat molecules were
to undergo complete hydrolysis, and if the administered
iglycerol-C^ were used preferentially for the resynthesis,
t^ie ratio of the neutral fat from the intestinal
D
tissue should approach a value of 1.00. A smaller ratio
would be expected if only a portion of the ingested
fat was completely hydrolyzed. In the experiments
where rats were fed doubly-labeled triglyceride, it
TABLE X
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE LIPIDS OF RATS AFTER THE ADMIN
ISTRATION OF 0.5 cc. I,3-DIOLEYL-2-DEUTERIOSTEARYL-GLYCERIDE AND 0.25 cc. OF GLYCEROL-C14.
Tissue and Material
isolated
Sample
Weight
mg.
Glycerol-
C Label
ed Lipid
mg.
Per cent
Glycerol-
C14 Label
ed Lipid
Deuterio-
stearic
Acid Label
ed Lipid
mg.
Per cent
Deuterio-
stearic Acid
Labeled
Lipid
olo
1
Material Fed: 1,3-
dioleyl-2-deuterio-
stearyl-glyceride
Glycerol-C
457
315
0 0 457 ‘ 100.00 0
Small Intestinal
Tissue
Neutral Fat 284 7.1 2.48 39.8 10.31 0.24
Phospholipid 112 9.7 8.6 4.80 3.93 2.19
Liver
•
Neutral Fat 198 18.2 9.20 5.5 2.78 3.29
Phospholipid 240 22.8 9.49 12.7 5.28 1.80
Carcass
x
Total Lipid 15813 135.6 0.86 33.4 0.211 4.07
The results are tabulated as the average of 2 groups; each group contains two rats. The
values are expressed on a per rat basis. 58 per cent of the fat fed and 74 per cent of the
glycerol-C14 fed were absorbed. The value of the per cent of the fat absorbed is calculated
on the basis of the recovery of total lipids, from the intestinal lumen.
167
TABLE XI
THE DISTRIBUTION OF GLYCEROL-C14 AND DEUTERIOSTEARIC ACID IN THE LIPIDS OF RATS AFTER THE ADMIN- .
ISTRATION OF 0.5 cc. 1,3-DIOLEYL-2-DEUTERIOSTEARYL-GLYCERIDE AND 0.5 cc. OF GLYCEROL-C .
Tissue and Material
isolated
Sample
Weight
mg.
Glycerol-
C14 Label
ed Lipid
mg.
Per cent
Glycerol-
C14 Label
ed Lipid
Deuterio-
stearic
Acid Label
ed Lipid
mg.
Per cent
Deuterio-
stearic Acid
Labeled
Lipid
c14
P
Material Fed: 1,3-
dioley1-2-deuterio-
stearyl-glyceride
Glycerol-C14
457
630
0 0 457 100.0 0
Small Intestinal
Tissue
Neutral Fat 222 19.1 8.61 63 28.39 0.30
Phospholipid 124 8.9 7.18 6.4 5.16 1.39
Liver
Neutral Fat 98 28.0 28.59 6.1 6.22 4.60
Phospholipid 226 32.1 14.23 9.5 4.21 3.38
Carcass
Total Lipid 13958 169.7 1.217 59.1 0.423 2.88
The results are tabulated as the average of 2 groups; each group contains two rats. The
values are expressed on a per rat basis. 58.2 per cent of the administered triglyceride and
68.9 per cent of the administered glycerol-C14 were absorbed. The value of the per cent of
the fat absorbed is calculated on the basis of the recovery of total lipids from the
intestinal lumen.
*
168
| was observed that in a 3 hour absorption period around
40 per cent of the ingested fat was completely hydrolyzed
I
i
. before absorption. Under such conditions, had the rats
! 14
. been fed glycerol-C and triglyceride labeled with
! o 14
deuteriostearic acid, the ---- ratio of the neutral fat
D
| isolated from the intestinal tissue should be slightly
| less than 0.40 if the labeled glycerol in the intestinal
| tissue were in large excess over the non-labeled endo-
I
i
j genous glycerol and glycerol formed during the ingestion
r14
: of the fat fed. Actually the _--- ratios obtained for
! D
1 the intestinal neutral fat were 0.24 and 0.30 where 315
mg. and 63Q;mg. of glycerol-C^ were administered respect-
: ively. However, these ratios as well as the per cent of
*
^ glycerol-C-^ labeling of the isolated neutral fat from
i
: the intestinal wall cannot be interpreted as an indication
i
of the extent of hydrolysis of the triglyceride preceding
absorption. This conclusion was reached following the
i
observation of the incorporation of radioactive glycerol
: into the other lipid fractions: intestinal tissue phos
pholipid, liver neutral fat and phospholipid and total
r 14
| carcass lipid. The ---- ratios for all these fractions
! D
were much higher than it is possible to explain on the
i '
basis of incorporation of glycerol-C^ during the resynthe
sis of absorbed fat in the intestinal tissue (Tables X,X1).
iThe observed ratios for the intestinal wall phospholipid,
liver neutral fat, liver phospholipid and carcass total
:lipid were 2.19, 3.29, 1.80, and 4.07 respectively where
i
,315 mg. of gf.ycerol-C^ was administered. In the case when
i -l t , r «14
630 mg. of glycerol-Cwas administered the ^— ratios for
r ■
!the same fractions mentioned in the same order were 1.39,
|4.60, 3.38 and 2.28. Saponification of the neutral fat and
j • -
phospholipid and the subsequent separation of free fatty
acid demonstrated that practically no incorporation of C ^
occurred into the fatty acid moieties of these isolated
t
lipids. All the radioactivity was located in the glycerol
moieties of these lipids. It is important to note that the
14
incorporation of glycerol-C into the intestinal tissue
phospholipid occurred much more readily than into neutral
fat from the same tissue in comparison with the deuterio-
stearic acid.
The high j j —--- ratios observed in all fractions
except the intestinal tissue neutral fat indicated an
incorporation of glycerol-C3-^ into these preformed lipids
171
|which might be due to the turnover of the tissue lipids.
I In order to clarify whether the incorporation of glycerol-
jc^ into lipids, is only connected with the fat metabolism
|
in the tissue designated as turnover or whether it is also
related to the resynthesis of triglyceride in the intes-
|tinal tissue during fat absorption, a series of experiments
|was performed.
Three different levels of glycerol-C*-^--80 mg.,
'315 mg. and 630 mg. per rat— were fed with or without
i
jsimultaneous administration of fat. Glycerol-C*-^ was
divided into two equal portions and was given with a 1
hour interval. One hour after the last feeding the
i
animals were sacrificed. In the experiments where tri
glyceride was also administered, rats were fed with 0.5 cc.
of partly hydrogenated triolein 1 hour before the admin
istration. of the first portion of glycerol-C**^. In
Table XII is given a summary of the results of this
series of experiments.
The amount of the intestinal tissue neutral fat
labeled with glycerol-C**^ was increased with increasing
dose of glycerol-C**^. Where equal doses of glycerol-C^
1 <
i
TABLE XII
THE INCORPORATION OF GLYCEROL-C14 ADMINISTERED IN DIFFERENT DOSES WITH OR WITHOUT SIMULTANEOUS
ADMINISTRATION OF TRIGLYCERIDE INTO RAT'S LIPID.1'
(Absorption Period for Triglyceride is 3 Hours.)
No. Material Fed Small Intestinal Tissue Liver
of _____________________ ' __________________ ’ ________________________
Rats Glyc- Tri- ' ” ! ' '
erol- glyc- Neutral Fat Phospholipid Neutral Fat < Phospholipid
• C14 ' eride ___ • ■ _________________
Total Glycerol-C14 Total Glycerol-C Total Glycerol-C1^ Total Glycdrol-C14
Amt. Labeled Amt. Labeled Amt. Labeled Amt. Labeled
mg. mg. mg. mg. per cent mg. mg. per cent mg. mg. per cent mg. mg. per cent
4 80 - 239 1.2 0.50 109
I-4
1.24 179 17.8 9.95 260 13.9 5.39
4 80 472 274 3.4 1.25 129 0.9 0.69 132 12.5 9.45 236 7.5 3.24
6 315
- 177 3.7 2.11 110 6.9 6.28 180 34.2 18.95 217 35.6 16.53
2 315 457 284 7.1 2.48 112 9.7 8.62 . 198 18.2 9.20 240 22.8 9.49
8 630 - 231 4.2 1.82 104 5.8 5.57 208
$7.5
27.60 230 58.2 25.30
12
630 464 271 13.2 4.88 130 10.0 7.69 182 56.2 30.90 255 48.1 18.86
I)Results are tabulated as the average for a single rat. The administered triglyceride
absorbed averages 52 per cent. 50 to 95 per cent of the administered glycerol was absorbed.
172
• were administered, the amount of labeled neutral fat
isolated from the intestinal tissue was higher if the rats
<
l 14
j ingested fat. For 80 mg. glycerol-C administered, 1.2
! mg. of the neutral fat from the intestinal tissue was
1 labeled with glycerol-C^. However, if 1 hour before the
! 14
j administration of the same amount of glycerol-C , 0.5 cc.
i
j of fat was administered, 3.4 mg. of neutral fat was
J labeled with radioactive glycerol. With a higher dose of
! 14
; the administered glycerol-C , e._g., 630 mg. per rat,
j 4.2 mg. and 13.2 mg. of the intestinal tissue neutral fat
I
; were labeled where rats obtained only glycerol or glycerol
; and triglyceride respectively. From these data it is
obvious that the organism is able to utilize exogenous
glycerol to some extent for the resyhthesis of triglyceride
in the intestinal tissue. When a relatively small dose
of the labeled glycerol was administered, it probably very
quickly proceeded to the portal blood system, was not
present in appreciable amounts in the mucosa cells and
therefore escaped utilization in the intestinal tissue.
Where a greater amount of glycerol-C^ was administered
;and absorbed, a higher concentration might occur in the
. . 174 ' ;
i
intestinal wall cells and therefore the chances for its
I
j partial utilization were increased.
I ;
| In our experiments, where rats were fed triolein,
! I
; around 52 per cent of the ingested triglyceride was absorbed
t . •
i •
! during 3 hours. According to the previous experiment
iwith doubly-labeled triglyceride approximately 40 per
} |
J j
i cent of this amount was completely hydrolyzed before I
; !
i
absorption or 89 mg. In order to esterify this amount of [
fat there was required only 9.3 mg. of glycerol. A dose
! 1
i
68 times this administered orally (630 mg. per rat) seems
I not to secure the re-esterification of triglyceride with
i
jonly labeled glycerol in the intestinal tissue. Under
I
, normal conditions of lipolysis of the ingested fat only
j a small amount of glycerol is liberated. Therefore the
chances for its reutilization in the intestinal tissue
! are very small. From the quantitative point of view it
t
: is correct to consider that the glycerol molecule split
i
I off from the fat during digestion is not utilized for
the resynthesis of triglyceride in the intestinal tissue.
However, when there is a great excess of available glycerol
I in the intestinal lumen, the organism is able to utilize
175
absorbed exogenous glycerol for the esterification pro
cesses in the intestinal wall. The quantitative evaluation!
of the extent of the utilization of exogenous glycerol-C^ ;
I
for the resynthesis of triglyceride is given in Table X1I1.J
From the calculations presented in this Table it follows
that a minimum of 40 per cent of the glycerol used for
the resynthesis of absorbed fat is of exogenous nature,
i
where 630 mg. of glycerol was administered. However, it ;
is obvious that the extent of incorporation of labeled
glycerol cannot be considered as a tool for the investiga
tion of the extent of hydrolysis of ingested fat. The
amount of glycerol-C^ incorporated into the intestinal
phospholipid also has a tendency to increase where greater
doses of labeled glycerol were administered. At higher
doses of glycerol-C^ administered the amount of such
glycerol incorporated into phospholipid is greater where
the rats ingested fat.
The extent of labeling with glycerol-C^ of the
intestinal tissue neutral fat is smaller than the phospho
lipid isolated from the same tissue. It seems that under
conditions where the synthesis of neutral fat and
176
TABLE XIII
APPROXIMATE PER CENT OF THE EXOGENOUS GLYCEROL-C14 UTILIZED
FOR THE RESYNTHESIS OF TRIGLYCERIDE IN THE INTESTINAL
TISSUE DURING FAT ABSORPTION
j (3 Hours Absorption Period) ,
Material
Glycerol-C14
Fed
Non-Labeled
Triglyceride
Amount of neutral fat isolated
from the intestinal tissue
labeled with glycerol-C14
ms. ms. ms.
630 464 13.21)
630 0 4.2
Amount of neutral fat labeled with
glycerol-C14 due to utilization of the
radioactive exogenous glycerol for the
resynthesis of the completely
! hydrolyzed part of absorbed f a t ................ 9 mg.
Amount of this neutral fat corrected by
the factor eliminating the relative
increase of the specific activity of
glycerol-C14 after its incorporation
into triglyceride^) ..... .................. 6.7 mg.
Total amount of absorbed; neutral fat
present in the intestinal tissue3) ............. 41.1 rag.
Amount of absorbed neutral fat present
in the intestinal tissue, which proceeded
through the stage of complete hydrolysis
(40 per cent of total amount ') ....... 16.6 mg.
Per cent of resynthesized absorbed
neutral fat with utilization of
exogenous glycerol-C1 4 .......................... 40 per cent
1^From Table XII.
2)
See page 131.
3)From Table II.
; phospholipid occur from free glycerol and fatty acid, the
: neutral fat is not the precursor of phospholipid, as has
t
j been observed where both compounds were synthesized from
I
I
|absorbed glycerides. It is more probable that both
1
:compounds— neutral fat and phospholipid--independently
i
incorporating exogenous glycerol-C-^ or that labeled
phospholipids are the precursors of the labeled neutral
jfats. The total amounts of neutral fat and phospholipid
isolated from the intestinal tissue were higher in rats
which absorbed fat and glycerol than in the animals which
i
'were fed only glycerol.
The extent of the incorporation of glycerol-C^
into liver lipids was much higher than into intestinal
i
tissue lipids (Table XII). This agrees well with the
• i
I results obtained in experiments where rats were fed
!doubly-labeled triglyceride. In those experiments the
high ability of the liver lipids to incorporate absorbed
I
small doses of free glycerol-C-^ formed during hydrolysis
of the doubly-labeled triglyceride in the intestinal lumen
was demonstrated. From the experiments where animals were
j fed different amounts of free glycerol-C^ with or w&fchhut
178
|non-labeled triglyceride it follows that the incorporation
of labeled glycerol into liver lipids had a tendency to be
jhigher when only glycerol-C^* was administered (Table XII).
jProbably under such conditions more liver fat is involved
i
i
in the metabolic transformation and therefore easily incor
porated the glycerol-C^ due to mobilization of the endo-
jgenous lipid for metabolic purposes. High incorporation
'of glycerol-C^ into liver lipids indicated their tre-
imendously high rate of turnover. Thus, according to
t
iZilversmit et ai. (193), at one turnover of material 63.2
,per cent of material becomes labeled if there is an
!
adequate supply of the labeled direct precursor. In the
I
icase of liver lipids, in a 2 hour period after the admin-
■istration of glycerol-C^ up to 31 per cent of material
i
l
becomes labeled. Such high incorporation of radioactive
f
: carbon into the liver lipids is actually due to the
I
1 incorporation of labeled glycerol as a unit, since only
!2-4 per cent of the observed radioactivity in the liver
lipids was located in the fatty acid and 98-96 per cent
was located in the glycerol moieties of the lipids.
I
j In order to investigate the mechanism of the
" " “ •' "179
incorporation of glycerol-C^ into the neutral fat of the
intestinal tissue, the fat isolated from this tissue was
subjected to fractionation in six experiments. In three
experiments animals were fed only glycerol-C^, in the
other three experiments, 1 hour before the administration
of glycerol-C^ partly hydrogenated triolein was also
given. In Tables XIV and XV the results of these experi
ments are given.
In the first series of the experiments (Table
XIV) an attempt was made to remove all mesenterial fat
from the outside of the small intestine. Apparently
during such handling of the organs some hydrolysis of
lipids in the intestinal tissue occurred, since the
isolated fractions of free fatty acid and monoglyceride
were higher than in the experiments where all precautions
were taken.
In another series of experiments (Table XV) some
mesenterial fat located outside of the intestines was not
removed. The intestines were washed out in situ by the
method described above, rapidly removed and immediately
frozen. It is expected that under such conditions no
TABLE XIV
THE INCORPORATION OF GLYCEROL-C14 INTO THE NEUTRAL FAT FRACTIONS ISOLATED FROM THE INTESTINAL
TISSUE AFTER THE ADMINISTRATION OF GLYCEROL-C14WITH OR WITHOUT TRIGLYCERIDE. '
Material Total Neutral Free Fatty Monoglyceride Di-and Triglycerides
Fed Fat Acid
Glyc-
3 * 1-
Tri- Amoun t
glyc
eride
Label- Amount
ed with
Glyc-
'IS1-
Per Cent Amount
from
total
neutral
fat
Per cent
from
total
neutral
fat
Label
ed with
Glyc-
'IS1'
Per cent
labeled
with
Glyc-
'IS1-
Amount Per cent Label-
from to- ed
tal Neu- with
tral fat Glyc-
'is1-
Per cent
labeled
with
Glyc-
erol-
c1 4
mg. mg. mg. mg. mg. mg. mg. mg.
mg.
80 - 239 1.2 17.8 7.45 16.8 7.04 0.13 0.80 204.4 85.51 0.86 0.42
80 472 274 3.4 36.9 13.35 22.8 8.34 0.15 0.64 214.3 78.31 3.03 1.40
630 - 198 3.8 18.3 9.21 10.6 5.33 0.26 2.45 169.2 85.46 2.82 1.67
630 472 243 7.0 26.5 10.90 20.8 8.55 0.47 2.26 195.7 80.55 5.74 2.93
1^Four rats were used in each experiment. The values are expressed on a per rat basis.
TABLE XV
THE INCORPORATION OF GLYCEROL-C14 INTO THE NEUTRAL FAT FRACTIONS ISOLATED FROM THE INTESTINAL
TISSUE AFTER THE ADMINISTRATION OF GLYCEROL-C14 WITH OR WITHOUT TRIGLYCERIDE.
Material Total Neutral Free Fatty Acid Monoglyceride Di- and Triglycerides
Fed
Glyc-
erol-
C14
Tri- Amount
glyc
er
ide
Label
ed
with
Glyc-
erol-
Cl4
Amount Per cent
from
total
neutral
fat
Amount Per cent
from
total
neutral
fat
Label
ed
with
Glyc-
erol-
C14
Per cent Amount
labeled
with
Glyc-
3 * 1'
Per cent Label-
from ed
total with
neutral Glyc-
fat er£1-
Per cent
labeled
with
Glyc-
erol-
C»
mg. mg. mg. mg. mg. mg. mg. mg. mg.
630 289 3.9 9.6 3.31 6.6 2.28 0.12 1.96 272.8 94.41 3.38 1.21
630 472 348 13.7 12.4 3.55 8.8 2.51 0.13 1.53 326.8 93.94 13.60 4.21
l)Four rats were used in each experiment. The values are expressed on a per rat basis.
| appreciable hydrolysis of the intestinal tissue lipids
i *
i
can take place. ■
i
Although there were some differences in the amounts'
i
j of the fractions isolated in these two series of experi- |
^ ments, the general picture was the same in both cases.
I
j The amount of free fatty acid as well as monoglyceride
I
| isolated from the intestinal tissue had a tendency to
! increase during fat absorption. Thus, in the group of
I :
! animals where all precautions were taken to avoid hydrol-
i
! ysis of the intestinal lipids, the following data were
' 14
obtained. In the rats which ingested glycerol-C and
i
i triglyceride the amounts of isolated free fatty acid and
i
' monoglyceride were 12.4 mg. and 8.8 mg. respectively. In
!
1 the rats which ingested only glycerol-C^, the amounts
i
' of free fatty acid and monoglyceride were 9.6 mg. and
j ■
6.6 mg. (Table XV). The extent of labeling of the mono
glyceride with glycerol-C^ was only slightly less where
: the animals absorbed fat. However, the extent of labeling
of di- and triglycerides fraction was much higher when the
organism absorbed fat. This fraction of higher glycerides
■ was also more labeled than the monoglyceride fraction
isolated from the same tissue. Thus, the di- and triglyc
erides fractions contained 4.21 per cent of lipid labeled
with glycerol-C^ whereas the monoglyceride fraction con
tained 1.53 per cent of material! labeled with glycerol-
c!4. seems that the data obtained do not permit us to
. explain the incorporation of glycerol-C^ into glyceride
i
|as the result of direct esterification of glycerol with
jfree fatty acid and the formation of monoglyceride which
Jare then further esterified to di- and triglycerides. It
i
is more likely that the incorporation of glycerol-C^ into
! higher glycerides occurred at least in part by another
I
I pathway than through the monoglyceride stage. The phos-
t
j pholipids isolated from the intestinal tissue were usually
; more highly labeled with glycerol-C^ than were the mono-
I
glyceride and higher glycerides fractions (Figure 1).
t
; The incorporation of glycerol-C^ into rat lipids
■ after an intraperitoneal injection was also studied. Three
i
I hundred and fifteen mg. of glycerol-C^ was injected per
i rat. Two hours after the injection the animals were
; sacrificed and the separation of lipids proceeded according
to the usual method. The results of this experiment are
P E R C E N T O F MOLECULES LABELED W ITH GLYCEROL
184
MONOGLYCERIDE
^ D I- S TRIGLYCERIDE
PHOSPHOLIPID
o J
2 °
cc
o U
< 0 o
o -J
20
ou
now
(O >- r-
FIGURE I
THE E X TE N T OF INCORPORATION OF GLYCEROL-C 1 4
INTO TH E IN TESTIN A L LIPIDS (iN PER CENT OF
LABELING) A FTER THE ADM INISTRATIO N OF FREE
G LYC ER O L-C 14, WITH OR W ITHOUT FAT.
given in the Table XVI. The general picture of the dis
tribution of the incorporation of the glycerol-C^ is
similar to that observed after the oral administration of
glycerol-C^. However, the incorporation of labeled
glycerol into the intestinal tissue lipids was smaller
than in the experiments where the same amount of glycerol-
was fed. This is probably due to the lower concentra
tion of glycerol-C^ in the intestinal tissue after such
a route of administration of exogenous glycerol. The
ex&ent of the incorporation of glycerol-G^ into the
liver lipids was high, as is usually so after the admin
istration of a large dose of labeled glycerol.
TABLE XVI
THE INCORPORATION OF GLYCEROL-C14 ADMINISTERED INTRAPERITONEALLY INTO THE LIPID FRACTIONS OF RATS1)
Tissue and Material Sample ' Glycerdl-C Per cent
isolated Weight Labeled Lipid Glycerol-C
Labeled Lipid
mg. mg.
Small Intestinal Tissue
Neutral Fat 401 2.2 0.54
Phospholipid
126
5.9 4.67
Liver
Neutral Fat 213 19.3 9.06
Phospholipid
267
61.0 22.85
^315 mg. of Glycerol-C*4 was injected. Results were obtained from two rats. The values
are expressed on a per rat basis.
DISCUSSION
i ;
1 Evaluation of the Data Concerning the Extent of Hydrolysis ;
i i
! ]
J of Fat Preceding Absorption j
i
So far the extent of hydrolysis of ingested triglyc-
i eride preceding absorption has been studied successfully by
* /
, two different procedures: Bernhard et al. (156) and i
: Reiser et al. (182) fed cannulated animals with doubly- i
: i
j labeled triglyceride and recovered fat from the lymph.
! The change of the ratio of the extent of labeling of the
1 glycerol moiety to the fatty acid moiety in the recovered
lymph triglyceride in comparison with the material fed
indicated the extent of hydrolysis of the fat before ab
sorption. The other procedure was that employed in the
pr&sent study, in which animals were fed with doubly-
: labeled triglyceride and 3 or 6 hours later neutral fat
was isolated from the intestinal tissue. The change of
, ratio of the extent of labeling of the glycerol moiety to
that of the fatty acid moiety of the isolated intestinal
tissue neutral fat served for the estimation of the extent
i of hydrolysis of triglyceride preceding absorption. Both
l ‘
these approaches have their advantages and disadvantages.
It is considered that the ratio of labeled glycerol to ]
labeled fatty acid of isolated neutral fat from lymph or j
j intestinal tissue is roughly affected by the extent of
i '
J |
j hydrolysis of fat in the intestinal lumen before absorption.j
However, Frank (9,223), Bloor (224,22$ and Artom and j
Peretti (226,227) observed an essential difference in the [
i j
!melting point and iodine number between the fat ingested by I
I !
|animals and the fat recovered from the lymph. Recently !
<22,, .Oil-C"
j to the animals in the form of stearyl diacetin was partly j
I converted into palmitic acid and an unsaturated fatty acid j
by the time it reached the lymph. All these experiments
indicated that some of the absorbed material underwent
! metabolic changes in the intestinal tissue. There may
i
;also occur some selection in the nature of the fat composi-:
I
I
;tion even among the triglycerides of long chain fatty
i
acids which preferentially enter the lymphatic system and
ileave the intestinal tissue. Both these processes might
!affect the ratio of labeled glycerol to labeled fatty acid
of the absorbed fat recovered either from the lymph or from
|the intestinal tissue. In the case of lymph fat, even more
i_p_rofound changes in the ratio may_ occur, since intestinal 1
189
tissue fat, in order to enter the lymphatic system, has to
pass through an additional membrane. Actually the nature
of the process involved in passing lipids from the intes
tinal tissue to lymph is not known. Therefore the ratio
of neutral fat recovered from the lymph is the resulting
ratio of the extent of hydrolysis of the ingested fat
before absorption, some metabolic changes in the intes
tinal tissue and the processes of passing lipid from the
intestinal cells to the lymph. The ratio of lipid recov
ered from the intestinal tissue is the result of the
first two processes only.
The application of linoleic acid with conjugated
double bonds as a tracer for the tagging of lipid per
formed by Reiser et^ al. (182) requires some comment.
Linoleic acid with conjugated double bonds does not occur
in the organism and a considerable portion of such absorbed
acid probably undergoes metabolic changes in the intes
tinal tissue before entering the lymph. Miller, Barnes,
Kass and Burr (163) pointedubut fifteen years ago that
linoleic acid with conjugated double bonds can be used as
a tracer only in very short experiments, for a maximum of
_____ _____----------- — _— ; ------ 190
\
several hours, whereas Reiser et: al- (182) used this acid
for experiments lasting up to 28 hours. It is much better
to use deuterium or labeled fatty acids as a tracer.
However, intestinal tissue has the ability to dehydro-
genate fatty acids, as has been shown by Mattson (228) and
Artom and Peretti (226,227). Bernhard et al. (229)
observed the process of dehydrogenation of the deuterium
labeled hydrocarbons in the intestinal tissue. Carboxyl-
labeled fatty acids are also not ideal as a tracer
because, according to Mattson (228) part of the stearic
acid fed lost a two carbon fragment in the intestinal
tissue. It seems that the best type of tagging fatty
acid used as a tracer for the investigation of fat absorp-
i
tion and transport would be labeled fatty acid when
j the radioactive carbon occupied one of the central posi-
i * ■
tions in the carbon chain. However such a type of
labeling has as yet been used very seldom, due to the
difficulties involved in synthesis.
Bernhard ej: al. (156) and Reiser et al. (182)
t
recovered a maximum of 60 per cent of absorbed fatty acid
in the lymph lipids after the administration of
! doubly-labeled fat. In some experiments, however, only a
I
j few per cents of absorbed material were recovered. It is
i
difficult to judge how representative such fat samples
obtained from the lymph are, and how the calculated
' ratios of the extent of labeling of two moieties of neutral
|
! fat reflect the extent of hydrolysis preceding absorption.
In any case all data as yet obtained by a single method
I « ■
I concerning the extent of hydrolysis of fat before absorp
tion should be accepted with some reservation. Bernhard
J jet al. (156) calculated that a minimum of from 25 to 53
| per cent of the triglyceride had been totally hydrolyzed
i
jbefore absorption. Reiser, et al. (182) considered that
1 from approximately 25 to 45 per cent of the ingested tri-
I glyceride was completely hydrolyzed preceding absorption.
! rl4
' From our determinations of the ~___ ratio of the neutral
| . D
i fat present in the intestinal tissue it was concluded
t « .
1
that from 25 to 56 per cent of the ingested fat was hydro-
i
jlyzed before absorption. On the average, slightly less-
;than 50 per cent of the fat was completely hydrolyzed in
;the period of time during which the main bulk of the
i
'ingested fat was absorbed. The agreement between the
• - 192
j
|results obtained by the two different approaches and with
I
|the different labeled compounds is very good. Such agree-
j
I ment between the results also indicates that the relative
» i
I ' ;
1 labeling of the glycerol and fatty acid moieties is not
t
; substantially affected by the process of passing triglycer
ide from the intestinal tissue to the lymph and therefore
, i
|this process does not presumably involve hydrolysis to an i
i
appreciable extent. It seems also that no selection takes
iplace in the nature of the fat which leaves the intestinal
jtissue in all the above described experiments. However,
the good agreement of the results obtained by those two
different approaches does not exclude the possibility
' that the absorbed fat in the intestinal tissue was affected
to some extent by metabolic changes. Nevertheless the
fact of the mutual conformation of the results obtained
by these two different approaches excluded many possible
errors and makes the data quite reliable.
Probably the slightly less extensive hydrolysis
of ingested fat preceding absorption observed by Reiser.
et al. (182) was due to some decrease of the denominator
iof the ratio since a part of the linoleic acid with
!conjugated double bonds was metabolized.
i
i
r
! The Nature of the Glyceride Penetrating into the Intestinal
I -
I Tissue During Fat Absorption
It was considered that the labeled glycerol, which
vas found in the neutral fat isolated from the intestinal
tissue during ingestion of doubly-labeled triglyceride,
i
! penetrated into the intestinal mucosa in the form of
|glycerides. Analyses of the ingested triglyceride per-
jformed by Artom and Reale (90), Frazer and Sammons (46),
iDeanuelle et al. (91,92,95,96,97), Mattson et al. (99,100,
|101), Borgstrom (88,104) and the author demonstrated that
!during lipolysis in vivo as well as in vitro diglyceride
;and monoglyceride are formed. Hence in the intestinal
; lumen there are available, during absorption, all three
types of glycerides. However, this does not indicate
, what glyceride actually penetrates into the intestinal
I
i mucosa. In order to elucidate this problem Reiser and
;Williams (141) fed rats with 1-monopalmitin labeled in
I
both moieties of the molecule with C^. The ingested
monoglyceride appeared in the lymph as triglyceride and
j
the ratio of the extent of labeling of the glycerol moiety
- - _ _ 194
|to the fatty acid moiety of the triglyceride recovered
j 1
jfrom the lymph indicated that 73 per cent of the monoglyc-
eride was hydrolyzed before absorption. However, the '
,authors had no direct evidence that there was actually
j
any penetration of monoglyceride into the intestinal
tissue, since some of the ingested monoglyceride may
.undergo conversion to higher glycerides in the intestinal
lumen, as has been demonstrated by Borgstrom (111), and be
1 absorbed in the form of higher glycerides. Therefore
the actual isolation of monoglyceride from the intestinal
tissue with the same ratio of extent of labeling of the
glycerol moiety to the extent of labeling in the fatty
acid moiety of molecule as in the ingested monoglyceride,
is the first convincing evidence that monoglyceride does
penetrate as a unit into the intestinal mucosa. There
v C1^
was also demonstrated a gradual decrease of the g---
ratio starting with monoglyceride to the triglyceride
isolated from the intestinal tissue during ingestion of
monoglyceride. This indicates that the absorbed monoglyc
eride plays the role of acceptor for the absorbed free
( fatty acid. Apparently similar processes occurred during
-------------- ' - .- - 195
j the ingestion of doubly-labeled triglyceride. Lipolysis
i
jof triglyceride in the intestinal lumen caused the f orma-
|tion of monoglyceride, part of which penetrated into the
!intestinal tissue as a unit and there was further esteri-
I
1fied by the available free fatty acid and converted to
jhigher glycerides. All these experiments proved that
i
\
monoglyceride can and does penetrate into the intestinal
;tissue and plays a key role in the processes of conversion
of absorbed lipid material to triglyceride. However these
experiments did not answer the question whether di- and
triglycerides can also be absorbed as a unit.
The Composition of the Neutral Fat Fraction Isolated from
the Different Tissues
It was shown that neutral fat isolated from the
; intestinal tissue in a fasting condition is actually not
1 a triglyceride, but a mixture of free fatty acid, mono
glyceride, diglyceride and triglyceride. During the absocp
tion of fat the amounts of free fatty acid and monoglycer
ide have a tendency to increase. The presence of free
long chain fatty acid in animal tissue has been reported
| in the literature. Bloor and Snider (230) found free
196
fatty acid in the liver and blood. Hilditch and Shorland
(231) confirmed these results concerning liver and Kelsey
and Longenecker (232) concerning blood. Jeker (158) demon
strated the presence of free fatty acid in the intestinal
mucosa. Borgstrom (128) confirmed the presence of free
fatty acid in the intestinal tissue. The amount of free
fatty acid in the intestinal tissue increased several
times, according to Borgstrom, during the first hour of
ingestion of triglyceride or free fatty acid by rats.
Fairbairn (233) investigated the,origin of free
fatty acid in mouse and cat livers. The author considered
that a rapid autolysis of lipid material occurred immedi
ately upon removal of the liver. This autolysis mostly
affected the tissue phospholipid. Within a few minutes
in the isolated, intact liver around 8 per cent of phos
pholipid hydrolyzed with the formation of free fatty acid.
If the liver was ground, hydrolysis increase to about 15
per cent. The author concluded that only tissue immedi?-
ately frozen in an alcohol-dry ice mixture and ground in
such a condition would yield correct values for the free
fatty acid content. Fairbairn gave as the normal value
of free fatty acid per gram of fat free dry weight of j
[mouse tissue 2.2 mg., or 2.3 per cent of the acetone- !
soluble lipid fraction in cat liver. Our figures obtained
with intestinal tissue which had been immediately frozen |
i
(
are slightly higher than the corresponding figures obtained,
by Eairbairn for cat liver, but in any case are of the
I
same order.
!
I
Much less is known concerning the presence of mono-;
I
glyceride in animal tissues. Reichstein (234) and Winter- !
f
steiner and Pfiffher (235) identified 1-monopalmitin as i
i
a minor constituent of the adrenal lipids. Jones, Koch, I
| '
iHeath and Munson isolated 1-monopalmitin from hog pancreas
i
j
(236). The 1.0 to 1.2 per cent of the fresh weight of
pancreas or around 10 per cent of the alcohol-soluble !
j extractives consisted of monopalmitin. The authors were
!
: unable to isolate monoglyceride from brain, liver and
j adrenal tissue of hog. However, their analyses indicated
i
| the existence of monoglyceride in the above mentioned
i
1 tissues in amount not more than 0.11 per cent of the fresh
! tissue weight.
Apparently in several of our experiments performed
without adequate precautions to prevent hydrolysis of the |
; intestinal lipids, the isolated fractions of monoglyceride ;
i
and free fatty acid were higher (Tables IX,.XIV) than in j
the experiments where tissues were frozen immediately 1
after their removal from the organism by a dry ice-
i !
I ether-alcohol mixture (Tables VI, XV). The distribution
' ■ I
I 1 / . !
I of the deuteriostearic acid and glycerol-C ^ in the
| different fractions--free fatty acid, monoglyceride,
f
i higher glyceride and phospholipid--was not uniform. Thus,
|
in cases when animals were fed with doubly-labeled mono-
!
i
' glyceride (Table VIII), the monoglyceride, fraction isolated
; from the intestinal tissue contained at least 4 times
i
j more of incorporated glycerol-C^ than did the di- and
i
triglyceride fraction and approximately 8 times more than
i
| the phospholipid. Hence the monoglyceride could not be
; likely to originate by hydrolysis of higher glycerides or
of phospholipid. In those cases where the incorporation
j of free glycerol-C^ simultaneously fed with the non
labeled triglyceride was studied,, the extent .of labeling
of higher glycerides and phospholipid isolated from
j intestinal tissue was much greater than the extent of
199
' labeling of the monoglyceride fraction.
* r
I 1 A
Such distribution of the incorporated glycerol-C
i
i
I among the lipid fractions indicated that, monoglyceride
i
• was preformed originally in the living tissue and it
; could not originate from other lipid material by its
|
, post mortem hydrolysis. Hence, the isolated fractions
!
i of monoglyceride and free fatty acid are present in the
!
intact animal tissue. However, in some experiments these
; fractions are quantitatively increased to some extent by
! the hydrolysis of other lipids.
I
; The Incorporation of Glycerol-C^ into the Intestinal
: Tissue Lipids
The origin of the glycerol moiety of the triglyc-
t
eride resynthesized in the intestinal tissue during fat
absorption was studied by several investigators. Favarger
et al. (154,189) concluded that the organism does not
utilize exogenous glycerol formed during the lipolysis of
fat for the resynthesis of triglyceride in the intestinal
tissue, since the incorporation of the labeled glycerol
fed to animals into the intestinal lipid was small,
jBernhard et all (156,190) came to a similar conclusion.
! Reiser with his collaborators (182,141) suggested that
I
i glycerol may not be the precursor of glyceride glycerol,
!
I
| but that some intermediates of carbohydrate metabolism
j
j like dihydroxyacetone or glyceraldehyde are the precursors
I of the glycerol moiety of the triglyceride formed in the
1
intestinal tissue. The experimental testing of this idea
: by feeding rats with doubly-labeled l-palmitoxy-3-hydroxy-
i
; acetone showed that only about 10 per cent of the labeled
i
! palmitic acid appeared in the lymph in the form of tri-
t
I
j glyceride and 2.6 per cent of the administered dihydroxy-
i
| acetone was recovered in the form of the triglyceride
glycerol. Such results cannot be considered as conclusive
! evidence in favor of Reiser’s et al. suggestion. However
I Doerschuk (237) demonstrated that a relatively large
! quantity of was found in the secondary alcohol carbon
l
of the carcass triglyceride glycerol 24 hours after the
i
administration of glycerol labeled only in the primary
: alcohol carbon. Such partial randomization of labeling
suggests that one of the possible routes for glycerol
incorporation to triglyceride is by the reduction to
I glycerol or to a substituted glycerol of an intermediate
_ 201
in a higher state of oxidation, since all the randomizing
processes accepted at the present time involve intermedi
ates in a higher state of oxidation than glycerol. But
it is not excluded that the administered glycerol gave
glucose which served as the precursor of glycerol. Our
results obtained by two new different.approaches also
indicated that the glycerol liberated during hydrolysis
in the intestinal lumen is practically not reutilized
for the synthesis of triglyceride in the intestinal mucosa
because the concentration of such glycerol in the intes
tinal tissue is apparently very low. However, it was
demonstrated that if glycerol is administered in high
dosage (80-630 per rat) appreciable utilization of exo
genous glycerol occurs for the resynthesis of triglyceride
in the intestinal tissue (Table XIII). The extent of
the incorporation of labeled glycerol into triglyceride
increases with the increase of the dose of glycerol admin
istered. Thus, exogenous glycerol can serve as the
precursor of triglyceride glycerol. However, the fact
that it has relatively low incorporation--40 per cent of
the maximum theoretically possible--demands explanation.
Three different explanations may be proposed for
this phenomenon. The possibility is not excluded that
14
the concentration of glycerol-C in the locus of resyn
thesis of triglyceride from the glycerol and free fatty
14
acid, even when a high dose of glycerol-C is adminis
tered, is not sufficient to dilute the endogenous
glycerol to such an extent that the majority of the mole
cules of triglyceride would be synthesized from a labeled
exogenous compound. The second explanation, proposed
by Favarger ejt al. (154,189) is that endogenous glycerol
formed from carbohydrate is transformed directly from the
enzymatic system which synthesized it to the enzymatic
system which esterifies the fatty acid. Apparently some
exogenous glycerol in some way may also form such an
enzyme-glycerol complex and participate in the synthesis
of triglyceride. But the amount of labeled exogenous
glycerol combined with the esterifing enzyme is relatively
low. The third possibility is the previously discussed
hypothesis of Reiser et al. (141) and of Doerschuk (237)
that the precursor of triglyceride glycerol is not glycerol
itself, but its derivative. Therefore exogenous glycerol,
in order to be utilized for the resynthesis of triglyceride,
tshould be converted to such a derivative.
i
I
From our experimental data it seems that the
1 incorporation of the exogenous free glycerol-C^ into the
i neutral fat of the intestinal tissue proceeded through
1 the stage of phospholipid. It has been pointed out pre-
I
! viously that the extent of labeling of monoglyceride
j
, isolated from the intestinal tissue of rats during the
j ingestion of glycerol-G^ and non-labeled triglyceride is
I lower than the higher glycerides isolated in the same
1 conditions (Tables XIV, XV). Such a higher concentration
1 of the incorporated glycerol-C-^ in di- and triglycerides
than in monoglyceride indicates that monoglyceride is not
, the precursor of the higher glycerides, when the formation
of glycerides occurred from free glycerol and fatty acid.
Figure 1 is a summary of data from Tables XIII, XIV and
XV, concerning the extent of the labeling of phospholipid,
T /
monoglyceride and di- and triglycerides with glycerol-C
after the administration of different doses of radioactive
glycerol with or without fat. The extent of labeling of
, phospholipid is much higher, except in one experiment,
than the extent of labeling of glycerides. Since the
i monoglyceride cannot be the precursor of higher glycerides
|it is logical to conclude that di- and triglycerides are
;formed from the phospholipid by splitting off of the
i
phosphoric acid-base group. The result of such a reaction
I
would be the formation of diglyceride. This diglyceride
may be esterified and converted to triglyceride or two
j molecules of diglyceride may undergo transesterification
jand would give one molecule of triglyceride and one mole
cule of monoglyceride. This monoglyceride labeled with
glycerol-C^ enters the pool of monoglyceride in the
intestinal tissue. Therefore the original radioactivity
of this monoglyceride becomes diluted. ,
There are two sources for the replenishment of
the monoglyceride pool, namely the absorption of the
exogenous monoglyceride from the intestinal lumen and
the synthesis of monoglyceride through phospholipids. The
monoglyceride of the pool is further esterified by the
free fatty acid and converted to triglyceride.
In the case when no absorption of fat takes place
and only glycerol-C^ enters the intestinal mucosa the
| formation of labeled phospholipid still occurs with endo-
j genous free fatty acid. Such phospholipid may be partly
t
| decomposed to diglyceride, which is further converted to
(
I
i mono- and triglycerides. Since practically no exogenous
I
i
monoglyceride has entered into the intestinal tissue, the
f
i labeled monoglyceride would be less diluted. Monoglycer-
I
; ide is further esterified and converted to triglyceride.
!
: Under conditions where no absorption to free fatty acid
i
■ takes place the process of decomposition of phospholipid
i
. to monoglyceride and the conversion of monoglyceride to
: higher glyceride is limited since no free fatty acid enters,
the intestinal tissue. In such circumstances, the mono
glyceride fraction should be more highly labeled than the
i
di- and triglyceride fraction. This was actually observed
when only glycerol-C^ was administered (Figure 1) .
In one experiment where a1small dose of glycerol-
C1^ (80 mg.) was administered together with triglyceride,
the extent of the intestinal phospholipid labeling by
glycerol-C1^ was smaller than the neutral fat fraction
isolated from the same tissue. This can be explained on
I ;
; the basis of the assumption of Artom et al. (176,187) that
two kinds of phospholipid exist: lipometabolic phospho
lipid, especially concerned with fat metabolism, and
icytoplasmic phospholipid, concerned primarily with the
structure of protoplasm and cell membranes. The total
Jfraction of phospholipid, under such conditions, should
' 1 / .
jnot necessarily be more highly labeled with glycerol-C
I
(than the neutral fat fraction. The principal, role of
f
!lipometabolic phospholipid, according to the postulated
i
.hypothesis, is the synthesis of monoglyceride necessary
I
I
jfor the esterification of free fatty acid absorbed, since
i
ithe amount of monoglyceride absorbed is not sufficient
i
to have esterified all the free fatty acid which formed
i
|during lipolysis in the intestinal lumen and entered the
i
|intestinal mucosa. It is obvious that under normal condi-
I
tions such synthesis of phospholipid proceeds by the
^utilization predominantly of endogenous glycerol.
In connection with this suggested role of phos-
i
ipholipid in fat absorption it is important to mention
the recently published work of Dickie, Robinson and Tuba
(238). These authors studied the effect of fatty acids
jon the intestinal alkaline phosphatase. Ingestion of
207 ;
■ fatty acid by rats caused the elevation of intestinal
I
I
alkaline phosphatase. Since alkaline phosphatase is ;
known to function in a number of tissues as a dephos-
phorylating enzyme, and because it has been shown to
l
participate in the absorption of certain monoaccharides
: from the intestine, it seems probable, according to Tuba
j and Dickie (239) that at least some fat is absorbed by
i
I
the mechanism which involves phosphorylation-dephosphoryla-
i
i tion. However, choline fed simultaneously completely
■ obliterated the effect of fatty acid on the intestinal
i
, phosphatase.
Pathways of the Syntheses of the Intestinal Tissue
Phospholipid
In the review of the literature the concepts
concerning the role of phospholipid in the process of
fat absorption were discussed. Upon examination of our
i
, data, especially the extent of labeling of phospholipid
and neutral fat isolated from the intestinal tissue as
r14
well as the ____ ratios of these fractions when animals
D
were fed with doubly-labeled triglyceride or monoglyceride,
I
j it was concluded that phospholipid originated from some
! form of glyceride. In favor of such a conclusion was the
1 observation that both moieties of phospholipid were
i
' labeled, but the extent of the labeling of phospholipid
»
! during fat absorption was usually much less than of the
!
^14
1 corresponding neutral fat. The ^___ ratio of the
D
j phospholipid was relatively close to the ratio for
j neutral fat, but slightly smaller. The administration of
t
i
; non-labeled glycerol together with the doubly-labeled
triglyceride did not essentially alter the observed rela
tionship. Thus, absorbed glycerides can serve as the
precursor of phospholipid.
14
The examination of the data where glycerol-C
’ was administered simultaneously with labeled triglyceride
(Tables X, XI), or non-labeled triglyceride (Table XII),
or without any triglyceride (Table XII) showed that the
extent of incorporation of the labeled glycerol into the
phospholipid of the intestinal tissue has a tendency to
be higher than that into neutral fat. However, there
were two exceptions i.e., when 80 mg. of glycerol-C^
was administered together with partly hydrogenated triolein
! (Table XII) and where l,3-dioleyl-2-deuteriostearyl-
' glyceride was administered together with 630 mg. of
' glycerol-C^ (Table XI). In both these cases the extent
I of the labeling of neutral fat was slightly higher than
!
Jof the phospholipid. Nevertheless the incorporation of
| free glycerol-C^ into neutral fat and phospholipid as a
I
iwhole indicates that glycerol labeled glycerides are not
the precursors of the glycerol labeled phospholipid. There
i
I are alternative explanations. Phospholipid and neutral
j
fat may be synthesized independently from glycerol-C^
I
1 and fatty acid, as has been postulated by Gidez and
1 Karnovsky (157); or phospholipid labeled with glycerol-C-^
; or its derivatives, are the precursors of_the glycerol
i
labeled neutral fat. In order to accept the last possi
bility it is also necessary to accept Artom's concept of
' the existence of two types of phospholipids: metabolic
(lipometabolic) and structural (cytoplasmic) (176,187).
, Then the total labeling of phospholipid might be less than
of neutral fat, but metabolic phospholipid would have a
higher labeling and would be the source of the precursor
1 of neutral fat. The distribution of the glycerol-C-^
incorporated into different fractions of glycerides is
j an indication in favor of the last explanation, that the
i formation of glycerides from free glycerol and fatty acid
i
I proceeds through the stage of phospholipid. Thus, the
I synthesis of phospholipid in the intestinal tissue may
| occur by two pathways. It can be synthesized directly
i
j from the absorbed glycerides or from the free glycerol
\
j (normally endogenous) and absorbed free fatty acid. Hence,
I
I
Jthe processes of interconversion of neutral fat into
i
I
J phospholipid and vice versa may occur in the intestinal
; tissue. The scheme of such interconversion is given in
!Figure 2.
1
! The formation of new ester bonds in the intestinal
i
. tissue during the formation of glycerides as well as phos
pholipid probably occurs through the formation of fatty
i ■
acid-CoA intermediate, as has been demonstrated for liver
phospholipid by Kornberg and Pricer (240,241).
A New Hypothesis of Fat Absorption
Summarizing all the processes which have been
discussed (Figure 2), it xis possible to picture the absorp
tion and resynthesis of lipids in the intestinal tissue
in the following way. Approximately 50 per cent of the
ingested neutral fat is completely hydrolyzed to free
211
LUMEN IN TES TINA L TISSUE
TRIGLYCERIDE
GLYCEROL ♦ PH.A ♦ CHOLINE
GLYCEROL - PH.A - CHOLINE
DIGLYCERIDE
PHOSPHOLIPID
+ PH.A. ♦ CHOLINE
- PH.A. - CHOLINE
MONOGLYCERIDE + F. A.
MONO- ♦ F A.
GLYC- 1 — -
ERIDE - F.A.
DIGLYCERIDE
GLYCEROL * F A.
TRIGLYCERIDE
PREDOMINATLY TO
PORTAL VEIN
ABBREVIATIONS:
F.A .-FA TTY ACID
RH. A .— PHOSPHORIC ACID
FIGURE 2
THE POSSIBLE PATHWAY OF THE INTERCONVER
SION OF NEUTRAL FAT & PHOSPHOLIPID IN
THE INTESTINAL TISSUE
i
|glycerol and free fatty acid. Another 50 per cent of
!ingested triglyceride is hydrolyzed to monoglyceride and
|
:free fatty acid. Monoglyceride, free fatty acid and
j
jglycerol (probably as glycerol phosphate) enter the intes-
j
‘tinal mucosa and mix with the corresponding compounds of
;the intestinal tissue pools. Monoglyceride provides the
acceptor for the esterification of free fatty acid, which
probably must be converted into a CoA-acyl compound before
esterification. The main pathway of the esterification
of monoglyceride follows through diglyceride to triglycer
ide. Since the organism requires phospholipid for the
stabilization of the triglyceride emulsion during transport
!
|through the lymphatic system, part of the absorbed mono-
»
!glyceride or higher glyceride is converted to phospholipid.
The exogenous glycerol, formed during fat digestion
in the lumeh and having entered into the intestinal tissue,
<
.is practically not utilized for the resynthesis of tri-
i
'glyceride or phospholipid and passes through the portal
vein into the liver. However, when the amount of the
i
absorbed glycerol is high and therefore its concentration
in the intestinal mucosa is also high, exogenous glycerol
213
is utilized to some extent for the synthesis of intestinal
phospholipid. Thus, an alternative p'athway of the syn
thesis of phospholipid proceeds through the esterification
of endogenous (and in special cases also exogenous)
glycerol with phosphoric acid and the corresponding organic
base (choline) causing the formation of glyceryl phospho-
rylcholine. Schmidt, Hecht, Fallot, Breebaum and Thann-
hauser (242) demonstrated the presence of such a compound
in lamb liver. The glyceryl phosphorylcholine further
esterifies with fatty acid-CoA and forms phospholipid.
Part of this phospholipid may be used for the stabiliza
tion of the triglyceride emulsion transported through
the lymphatic system. When the amount of free fatty acid
absorbed is in excess of the amount of monoglyceride
available for- the synthesis of triglyceride, some amount
of phospholipid splits off phosphorylcholine and a diglyc
eride is formed. Two molecules of diglyceride undergo
transesterification with the formation of one molecule of
triglyceride and one molecule of monoglyceride, which is
further used as an acceptor for free fatty acid and is
also converted to a molecule of triglyceride.
' 2 1 4
l
j It is realized that this scheme is to some extent
i
speculative, but it explains the pattern of distribution
!
lof the tracers observed in our experiments.
| Exogenous labeled glycerol is much more easily
|incorporated into the liver lipids than into the intestinal
i
lipids. Probably even some of the glycerol liberated
during the digestion of triglyceride in the intestinal
i
I
tissue is incorporated into the liver lipids, since the
t
administration of non-labeled glycerol together with
doubly-labeled triglyceride caused a decrease in the
■____ ratio in the liver lipids in comparison with the
D
same ratio obtained after the administration of doubly-
labeled triglyceride only. The extensive labeling of
!liver lipids— neutral fat and phospholipid--after the
i
f
administration of glyeerol-C-^ indicates the very high
rate of the turnover of these lipids. Our results con
cerning the large incorporation of glycerol-C^ into liver
lipids are in good agreement with the recently published
data obtained by Gidez and Karnovsky (157). The authors
observed a higher incorporation of glycerol-C^ into
; liver lipids than in lipids isolated from other organs,
including the intestine, after the injection of labeled
glycerol into rats.
SUMMARY AND CONCLUSIONS
The processes involved in fat absorption have
been investigated using isotope-labeled compounds. Rats
were fed the doubly-labeled triglyceride l,3-dioleyl-2-
14
deuteriostearyl-glyceride-C , the monoglyceride mono-1-
deuteriostearyl-glyceride-C^, and glycerol-C^ together
with labeled triglyceride, or non-labeled triglyceride
or without triglyceride. Lipid material was isolated
from the intestinal tissue, liver and carcass after the
administration of the above-mentioned labeled compounds
and fractionated into its components. The distribution
of glycerol-C^ and deuteriostearic acid as well as their
ratio in all isolated lipid fractions was determined. The
experiments where doubly-labeled triglyceride was admin
istered showed that free fatty acid, monoglyceride,
diglyceride and also some amount of free glycerol were
formed during the digestion of this fat in the intestinal
lumen. From 25 to 56 per cent of the ingested fat was
completely hydrolyzed before absorption. From 44 to 75
per cent fat was absorbed in the form of glyceride. The
relative amount of fat absorbed in the form of glyceride
' was larger in the first hours of absorption. Later, in
i
i a 6 hour absorption period, relatively more fat was
»
! absorbed after complete hydrolysis. The nature of this
i
; glyceride which penetrated into the intestinal tissue
| was elucidated by the fractionation of the neutral fat
isolated from the intestinal tissue after a 6 hour
; absorption period. It was apparent that monoglyceride
! formed during the digestion of triglyceride was partly
: absorbed as a unit without hydrolysis. There was no
direct evidence whether higher glycerides were also
absorbed without hydrolysis.
The neutral fat isolated from the intestinal
, tissue during the ingestion of fat or in fasting animals
\
contained small amounts of free fatty acid, monoglyceride
and diglyceride. The amount of free fatty acid and mono
glyceride had a tendency to increase during fat absorption.
The key role of monoglyceride in fat absorption
‘ was substantiated by the experiments in which rats were
fed with doubly-labeled monoglyceride mono-l-deuterio-
j stearyl-glyceride-C^. About half of the absorbed
1 monoglyceride penetrated into the intestinal tissue as a
i
junit without hydrolysis. This was proven by the isolation
|
|of doubly-labeled monoglyceride from the intestinal tissue
; with the same ■ —■ A-.-— ratio as in the
I deuteriostearic acid
compound fed. The absorbed monoglyceride served in the
{ intestinal tissue as an acceptor for the absorbed free
i
i fatty acid, which stepwise converted it to diglyceride
!
i
' and then to triglyceride, as demonstrated by the change
' * * | ^
i of £--- ratios in the three fractions of isolated
D
j glycerides.
/
Glycerol liberated during the lipolysis of glyc
erides was practically not reutilized in the intestinal
tissue for the synthesis of lipids. However, when the
i
glycerol-C^ was administered in much larger doses than
the amount of glycerol liberated during the hydrolysis
of fat, an appreciable amount was used to resynthesize
triglyceride. The extent of the utilization of the
exogenous glycerol for the resynthesis of triglyceride
depended upon the amount of the ingested glycerol. Where
the amount was small, practically no incorporation of
I exogenous glycerol into triglyceride was observed, but
where the dose was 630 mg. per rat a minimum of 40 per
cent of the resynthesized triglyceride isolated from the
t
i intestinal tissue contained exogenous radioactive glycerol.
i
f
j The fractionation of neutral fat isolated from
14
| the intestinal tissue of rats which ingested glycerol-C
i
! simultaneously with non-labeled triglyceride showed that
i
i the monoglyceride was relatively less labeled than the
t
higher glycerides. Therefore, it seems that the incor-
; poration of glycerol-C^* into triglyceride does not
: proceed through the direct esterification of glycerol
i
with one fatty acid and the formation of monoglyceride
which is further esterified to triglyceride, but occurs
by another pathway.
Experimental evidence was presented for the fact
I
' that the synthesis of monoglyceride from free glycerol
: (endogenous and exogenous) and absorbed fatty acid in the
intestinal tissue proceeded through the stage of phospho
lipid.
Phospholipid in the intestinal tissue can be
synthesized directly from the absorbed glycerides or from
the free glycerol and absorbed fatty acid. There was a
|
tendency to incorporate exogenous free glycerol into
220
! phospholipid to a greater extent than into triglyceride.
Exogenous labeled glycerol was also incorporated
i
into the lipids of the intestinal tissue and liver when
I the organism did not absorb fat. Such incorporation of
I
j glycerol is attributed to the natural turnover of lipids.
; The turnover of the liver lipids is very high. Two hours
| after the administration of 630 mg. of glycerol-C^ to
; rats up to 30.9 per cent of neutral fat and up to 25.3
; per cent of phospholipid became labeled by isotopic
' < glycerol in the glycerol moieties of the molecules.
i
1 In the light of the distribution of labeled
glycerol and stearic acid after five different types of
. feeding of experimental rats the relationship between
phospholipid and glycerides and their interconversion
in the intestinal tissue is discussed.
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University of S o u t h e r n California

Asset Metadata

Creator Skipski, Wladimar Pavlovich (author)

Core Title Studies on the mechanism of fat absorption using isotope labeled compounds.

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

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-c17-603723

Unique identifier UC11354382

Identifier DP21571.pdf (filename),usctheses-c17-603723 (legacy record id)

Legacy Identifier DP21571.pdf

Dmrecord 603723

Document Type Dissertation

Rights Skipski, Wladimar Pavlovich

Type texts

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