Table Of ContentADREN&LECTOMY AND FAT ABSORPTION
A
Dissertation
Presented to
the Department of Biochemistry
School of Medicine
University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
by
Lucien Andrew Bavetta
May 1942
UMI Number: DP21533
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This dissertation, written by
.........LUC1 M . . M 5MI..MVETTA........
under the guidance of hAL.8 Faculty Committee
on Studies, and approved by all its members, has
been presented to and accepted by the Council
1
on Graduate Study and Research, in partial ful
fillment of requirements for the degree of
DOCTOR OF PHILOSOPHY
Dean
Secretary
Date... J.UUE.,... 19.42..
Committee on Studies
i
TABLE OF CONTENTS
INTRODUCTION • • • 1
HISTORICAL DISCUSSION................... 4
EXPERIMENTAL P A R T ......................... 10
General Procedures 10
Adrenalectomy • .................... . . . . . . . . . . 10
Procedure for fat absorption . . • • • . • • • • • • • • 12
Calculations 14
Results 16
Control experiments........................... 16
Recovery experiments 20
Absorption of hydrogenated cottonseed oil . . . . . . . 24
Eight hour tests on absorption of hydrogenated
cottonseed and corn oils 31
Absorption of tributyrin . . . . . . . . . . . . . . . . 36
Absorption of sodium butyrate . . . . . . . . . . . . . 39
Summary table 43
DISCUSSION.................................... 45
SUMMARY............................................ 50
BIBLIOGRAPHY ..................................................... 51
INTRODUCTION
In the absorption of the three fundamental types of food stuffs
consumed by man, fat possesses the potentiality of producing more distur
bance in the orderly function of the gastrointestinal tract than either
protein or carbohydrate. The intestinal absorption of fat has always
presented one of the most interesting problems in physiology. It is a
study in which a great many factors are involved, for not only are the
pancreas, the liver and the intestinal mucosa concerned but there is also
considerable evidence that the adrenal cortex may play a role. In essence,
it involves a study of the circumstances under which a water-insoluble
substance passes in and out of the cell.
Two disorders of fat absorption, celiac disease in infants and non-
tropical sprue in adults, formerly regarded as separate clinical entities,
now appear as one. The portion of the absorptive apparatus to which the
defect lies has not been determined. The symptoms in both children and
adults are large, fatty, foul-smelling stools, anemia and emaciation.
Furthermore, failure to absorb fats results also in a failure to absorb
fat-soluble vitamins, notably A, D and E. This obviously results in the
appearance of deficiency symptoms attributable to lack of these vitamins.
liilhen fats are ingested, they are dealt with, not by the liver, but in
the epithelial cells of the small intestine. In order to understand the
process of fat absorption better it is necessary first to get a clear
picture of the present conception regarding its absorption.
The fats pass through the mouth without any change. In the stomach
there is very little fat hydrolysis as conditions for it are usually
2
extremely adverse. The fat-splitting enzyme, lipase, present in the
pancreatic secretion is essential to the digestion of fat. This enzyme
hydrolyzes the fat present in the small intestine to fatty acid and
glycerol. Some of the acid portion combines with alkali of the intestinal
juices to form soaps. At one time it was generally believed that absorption
of fat took place by means of the soluble soaps formed after its hydrolysis.
This theory was strengthened by the belief that the reaction of the intestine
was highly alkaline. More definite data were only possible with modern
electrometric methods. Kostyal (1926) in Verzar*s laboratory studied the
pH of the intestine of rats, dogs, guinea pigs and pigeons and found it
practically never over a pH of 7.0 and generally under this value. Mehl
and Deuel (unpublished) found the pH of the intestine of rats to be usually
under 7.0 and as low as 4.0 when tributyrin was being absorbed. This is of
extreme importance in formulating any conception of fat absorption as it
appears to exclude the possibility that fats are absorbed in the form of
alkali soaps. It is thought that bile salts form a complex with the fatty
acids and that this complex, being water soluble, is diffused into the cells
of the villi, and there is broken down into its components. Neutral fat is
then again formed and is carried by the lymph to the thoracic duct where it
enters the circulation, beooming available for utilization by the tissues.
The possibility that the adrenals might play a vital role in fat
absorption was first promulgated by Verzar and Laszt (1935). In an
extensive series of investigations these workers found that fat absorption
was inhibited by adrenalectomy in rats and that the normal function could
be restored by the administration of cortical extract. However, since the
publication of these results many other investigators have studied the
3
relationship of the adrenals with conflicting results. The problem was
essentially to find a rationale for these differences. It was also
thought desirable to ascertain whether the water soluble fatty acids are
also similarly influenced by adrenalectomy* In this study a comparison
was made of the rate of absorption of hydrogenated cottonseed oil, corn
oil, tributyrin and sodium butyrate.
HISTORICAL DISCUSSION
The problem of fat digestion is a very old one. The chief difficulty
has been in explaining how a water-insoluble substance could pass into the
mucosa of the intestinal epithelium. The results of investigations by many
workers, especially those of Munk (1880-1890), have shown that ingested fat
is not directly absorbed into the lymphatic system but is first hydrolyzed
and then resynthesized into glycerides. This was further proven by experi
ments of Frank (1898) who showed that glycerides could be recovered in the
chyle after the administration of waxes such as cetyl palmitate or of the
ethyl esters of fatty acids. Recent work by Frazer (1938) suggests the
possibility of an alternate procedure. Frazer devised a method of tracing
the glycerides present in the blood by microscopic observation, employing
dark ground illumination which permitted the particles of unhydrolyzed
fat to be detected and counted. In this way the sequence of events con
sequent to the ingestion of fats was followed. By the use of this qualitative
method this investigator believes that hydrolysis takes place but only
partially. It is his belief that the unhydrolyzed fat is absorbed by way
of the lacteal-lymphatic system while the fatty acids pass by the capillaries
and the portal vein to the liver. However, evidence along these lines is not
yet completely clear.
The influence of the adrenals on fat absorption has been supported by
the experiments of Verzar and Laszt (1935) who also postulated a somewhat
similar mechanism for carbohydrate absorption (judovits & Verzar, 1937).
These investigators believed that there is a special selective absorption
of both carbohydrates and fats. This specificity was attributed to a
5
process which they termed phosphorylation, a mechanism which can be
inhibited by monoiodoaeetic acid. This acid prevented phosphorylation and
consequently inhibited fat absorption. The rats were studied six hours
after feeding 3.5 gr. of olive oil. After this interval of time the
intestine of the normal rat was quite white from the enormous number of
lymphatics which were filled with fat. The large lymphatic vessels in
the mesentery were also strikingly white. When the animals were poisoned
with monoiodoaeetic acid, the intestines had the color in the fasting rat,
and no white lymphatics showed in the mesentery. Recovery experiments
also showed that practically no fat was absorbed. Similar results were
obtained by the use of phlorhizin. Verzar and Laszt (1935) then tested
the absorption of olive oil by adrenalectomized rats three to six days
after surgery. After twenty-four hours fasting they were given 3 cc. of
olive oil by a stomach tube and killed six to eight hours later. Similar
experiments with normal rats showed that after this time about 1.2 gr. of
fat was absorbed. In the adrenalectomized rats amazingly little fat was
absorbed during the first six to eight hours. The intestines of these
animals looked much the same as after poisoning with monoiodoaeetic acid,
or as in normal animals not fed with fat. When the adrenalectomized rats
were fed great quantities (3.4 gr.) of olive oil, all the animals died within
twelve to twenty-four hours. The authors attributed this to the toxic action
of the fatty acids. Normal control animals fed the same way survived. They
also showed that if sua adrenalectomized rat received 1 cc. of cortin per
100 gr. body weight, twelve hours and one hour before and once during the
absorption test, the absorption of fat could be restored to normal. The
activity of the adrenals was attributed to its control of the phosphorylating
6
process# This phenomenon was postulated as a prerequisite for the absorption
of the fatty acid portion of the fat molecule. The results of Artom and
Peretti (1935) using iodized fats, and of Sinclair (1936) employing elaidic
acid, indicate that the ingested fatty acids do become incorporated into the
phospholipids of the intestinal mucosa# It is also interesting to note that
according to the work of Clark (1939) and of Stein and Wertheimer (1941) it
would seem to indicate that this gland also controls the absorption of
sodium chloride. These latter investigators found that the absorption of
NaCl in adrenalectomized rats was twenty per cent to eighty per cent less than
in normal animals. The administration of desoxycorticosterone reestablished
normal absorption of chlorides. The use of monoiodoaeetic acid also resulted
in inhibition of salt absorption.
The inability to produce fatty livers after adrenalectomy was used as
further proof by Verzar (1936) for his theory of adrenal control. He found
the adrenalectomy abolished fatty livers produced by phosphorus poisoning
or fasting in the rat# Thus, the livers of adrenalectomized rats five days
after injections of 0.5 mg. phosphorus had a fat content of 4 per cent. This
was taken to be roughly parallel to the inhibition of fat absorption after
adrenalectomy. However, when cortin was injected, fat again accumulated very
intensively in the livers and fatty livers with 8.2 per cent fat were seen.
This effect on fatty livers has been further confirmed by MacKay and Barnes
(1937). As still further evidence of his theory of phosphorylation Verzar
(1937) compared the flavin content of livers of adrenalectomized and normal
rats. He found the liver of normal rats to contain about 1000 gamma of
flavin, 95 per cent of which was found to the yellow enzyme. After adrenal
ectomy the total liver flavin decreased by about 50 per cent in four days.
