University of Southern California Dissertations and Theses

Adrenalectomy and fat absorption

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Adrenalectomy and fat absorption

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Content ADREN&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
This dissertation, written by
.........LUC1 M . . M 5M I . . M V E T T A ........
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
on Graduate Study and Research, in partial fu l
fillment of requirements fo r the degree of
DOCTOR OF P H IL O S O P H Y
D ea n
S ecretary
D a te ... J. UUE. , . . . 19.42..
1
C o m m ittee on S tu dies
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.
7
At that time the free and bound flavin were found to be approximately equal
in concentration. He consequently postulated the theory that the animal was
unable to phosphorylate riboflavin in the absence of cortical activity.
However, Bruce and Wein (1940) found that the administration of riboflavin
phosphate to adrenalectomized rats had no more beneficial influence upon their
survival period than the use of riboflavin alone. '
In another series of investigations Verzar and Sullman (1937) found that
the intestinal mucosa of rats contains more acid soluble organic phosphorus
when animals absorb glucose, fructose, galactose, mannose and glycerol than
when animals receive HaCl. However, the interesting thing about these experi
ments was the finding that the increased formation of organic phosphorus in
mucosa also occurred after removal of adrenals. The authors made no attempt
to explain this apparent discrepancy in their theory. They also found that
monoiodoaeetic acid prevented the increase in organic phosphate in the mucosa.
However, outside of Verz&r's laboratory the theory that the adrenal
cortical hormones are specifically concerned with phosphorylation in the
intestinal absorption of sugar and fats seem to have made little progress.
The view that adrenalectomized rats can absorb glucose at a normal rate, if
their general condition is maintained by allowing them to drink a saline
solution has been demonstrated by Deuel et al (1937), and further confirmed
by Althausen (1939). Marazzi (1940) believes that the decreased absorption
of carbohydrates by adrenalectomized animals is to be attributed to anorexia.
She found that if she limited the food intake of normal rats to that of the
adrenalectomized ones the decrease in absorption was approximately the same
in both groups of animals. Houssay et al (1941) found that in the toad
adrenalectomy had no influence on the absorption of either glucose, fructose
8
or galactose.
Furthermore, adrenal control of fat absorption has also been disputed and
some evidence has been presented which is opposed to the view of Yerzar.
Barnes, MacKay, Wick and Carne (1939) found no effect on the rate of absorption
of methyl esters of fatty acids or of corn oil itself in adrenalectomized rats.
In another series of experiments Barnes, Miller and Burr (1939) obtained
similar results .by the use of spectroscopically active fatty acids. The rate
of entry of fatty acids was tagged by the formation of conjugated double bonds
which renders them spectroscopically recognizable owing to their high light
absorption coefficient. It was found that in adrenalectomized rats four days
after operation, that there was no decrease in the rate of absorption from the
intestinal tract. There was no significant change from normal controls in the
amount of tagged fat present in the phospholipids of the intestinal mucosa.
Recently, however, Bavetta et al (1941) have reported that there is a
decrease of approximately 24 per cent in the rate of absorption of hydrogenated
cottonseed oil by salt-treated adrenalectomized animals as compared with
normals, also a decrease of 38 per cent in the rate of fat absorption in the
untreated adrenalectomized animals as compared with normals. After adrenalectomy
large amounts of fatty acids accumulated in the intestine than occurred
normally. This greater accumulation of free fatty acids in the gut contents
of adrenalectomized animals probably accounts for the decreased absorption of
fat. Both the decreased absorption of fats and the accumulation of fatty acids
could be corrected by the administration of cortin.
During the course of the experiments described herein, Barnes, Rusoff and
Burr (1942) have reported additional experiments where it was found that emul
sified hydrogenated cottonseed oil was the only fat the absorption of which
9
was depressed following adrenalectomy. These investigators noted no signi
ficant decrease in the absorption of corn, olive or hydrogenated cottonseed
oil or of mutton tallow resulting from ablation of the adrenals. It appears
unreasonable that this inhibition in fat absorption should be limited only to
a special type of fat. These studies were therefore extended to am investigation
in the absorption of corn oil by a technic similar to that of the above inves
tigators. Our results are not in agreement with them.
EXPERIMENTAL PART
General Procedures
The experimental animals were female albino rats from the stock colony
of the Department of Biochemistry, weighing 100 to 160 grams* They were
maintained on the regular stock diet*
The hydrogenated cottonseed oil used was the fat separated from a
commercial margarine (Nucoa) which is composed of partially hydrogenated
cottonseed (about 86 per cent) and peanut (15 per cent) oils. The saponification
number used in the calculation was 194.7, a value repeatedly found for this fat
in the Biochemistry laboratory. The corn oil fed was also a commercial brand
of vegetable oil (Mazola). The saponification value found and used in the
calculations for corn oil was 191.0. Tributyrin was purchased from Eastman
Kodak Company and was further purified and redistilled^. The sodium butyrate
was a 22.45 per cent solution of butyric acid (Eastman Kodak Company) neutral
ized to a pH of 7.4 using the Beckmann pH meter.
Adrenalectomy. The ablation of the adrenals was performed under ether
anesthesia using the lumbar approach. Complete adrenalectomy is much more
easily performed in rats weighing between 100 and 120 grams and whenever
possible such rats were used. The rat is not prone to pyogenic infections
and hence an aseptic technic is unnecessary. The animal is at first placed in
a bell jar containing cotton „saturated with ether and his respiration is care
fully watched. Absolute alertness in regard to ether anesthesia of respiratory
rate is indispensable. There appears to exist only a very small margin between
^The author wishes to thank Dr. Margaret Morehouse for carrying out the
vacuum distillation of the commercial product.
11
the amount needed for adequate anesthesia and that necessary for the death of
the animal. After the stage of adequate anesthesia is reached, the animal is
placed on his stomach on an operating board and the operation performed.
During the surgery the animal is maintained under proper anesthesia by the
use of an ether saturated cone. The back of the animal is then lathered with
green soap and the area over the lumbar region is shaved. The skin over the
spine is now made taut and a single longitudinal incision is made using a
sharp scalpel over the midline. The incision is made at the level of the costal
margin and extending about 2 cm. towards the tail. The looseness of the sub
cutaneous tissue permits the operator to shift this opening over to either the
left or right side. In order to locate the adrenals the operator can utilize
certain landmarks. The spleen is a good landmark in locating the left adrenals.
In young animals the spleen appears as a black shadow through the thin lumbar
muscles. A very small incision is then made in these muscles at the caudal
border of the splenic shadow. The left adrenal of the rat lies superficially
just anterior to the kidney. It comes readily into view when the incision in
the lumbar muscles is held open by spreading the forceps. The gland is grasped
along with the mesentery with a pair of curved forceps. The removal of the
mesentery with the gland makes possible the removal of any accessory cortical
tissue. The gland is now brought forward and the pedicle is cut with scissors
leaving the gland and its attached connective tissue in the grasp of the forceps.
The right adrenal is situated somewhat more anteriorly than the left and
its removal is somewhat more difficult. The incision is made just below the
costal margin. The adrenal may be seen medially anteriorly to the kidney and
covered by a lobe of the liver. In the extirpation of this gland it is imper
ative to grasp it by its pedicle which lies anteriorly to it. This pedicle -is
12
much stronger than the one on the left and unless it is grasped firmly and
securely failure results*
The incisions in the lumbar muscles, which are about 6 mm. long, are
approximated but it is not necessary to suture them. The skin incision is
closed with skin clips. If one is careful not to use an overdose of the
anesthetic, avoids tearing any of the large, blood vessels, kidney or liver,
the mortality from the operation is practically zero. The animals recover
and within a few hours act perfectly normal. In nearly all cases the com
pleteness of the operation was checked by necropsy upon completion of the
experiment. In all cases the animals were maintained in good physical
condition by giving them Rubin-Krick solution (1931) in place of ordinary
water for drinking. In one group of experiments in which the animals served
as negative controls as well as in one series of absorption tests, the
adrenalectomized rats were given only distilled water. The fact that prac
tically all these animals died within a two-week period indicated the com
pleteness of adrenalectomy. In another experimental group the animals were
given cortin2 (l) in their drinking water. Both the salt-treated and cortin-
treated animals were kept from seven to ten days before being used in the
experiments. At the end of that period most of the animals had shown a gain
in weight or at least were able to maintain their preoperative weight. In all
cases the animals were maintained in an air-conditioned room with the tempera
ture kept between 78° and 80° F.
Procedure for fat absorption. The procedure for the determination of the
rate of fat absorption was similar to that employed by Deuel, Hallman and
Leonard (1940). The method used in the tests on sodium butyrate was the same
o
Cortin was kindly furnished for these studies by Dr. E. C. Kendall of
the Mayo Clinic who suggested the dosage and method of administration.
13
as described by Deuel et al (1941). A fasting period of 24-hours preceded
the tests* The fat was administered on the basis of surface area. In
most cases this amounted to 300 mg. per 100 sq. cm. of body surface. The
latter was calculated by the formula of l*ee (1929). During the tests the
animals were kept in separate cages and any experiments showing evidence of
diarrhea were discarded.
14
Calculations. The fatty acids recovered from the gastrointestinal
tract were estimated by titration of the ether extract with 0.1 N NaOH
using phenolphthalein as an indicator after solution of the fats in
petroleum ether and isopropyl alcohol. The milligrams of fatty acids
were calculated.from the titration by use of fatty acid equivalent based
on the saponification number of the fat. The method used in the tests
on the study of sodium butyrate was the same as that described by Deuel
et al (1941). After subtraction of the value of the ether-soluble
material found in the intestine of control rats fasted a similar period
of 24-hours (Tables I, II and III), the weight of the fats removed from
the intestines are corrected for the amount of that particular fat
recovered when it is fed and immediately removed. These values were 93.6
3
per cent for hydrogenated cottonseed oil , 93.6 per cent for corn oil,
90.2 per cent for tributyrin and 92.5 per cent for sodium butyrate. These
values represent the average percentage of each fat recovered in ten
experiments (Tables XV, V and VI). The amount recovered was then sub
tracted from the amount fed and the difference was considered the amount
absorbed. This value was then used to calculate the amount of fat
absorbed per 100 sq. cm. per hour.
The results obtained above were statistically treated by calculating
the standard error of the mean. This was obtained by use of the following
formulas
2
Based on earlier results from this laboratory (Deuel et al (1940)).
15
Standard Error of Mean =
d = deviation from mean
n = number of observations
The Standard Error of Mean Differences were calculated as
follows from the S. E. M. of the two averages which are being
compared:
S. E. M. D. * | (S.E.Mj^2 - (S.E.Mg)2
The mean difference was then divided by the standard error of the mean
difference. When this value exceeds 3.Ojthe results are considered sig
nificant.
In order to ascertain the lipolytio action in the different groups,
the neutral fat hydrolyzed was calculated. The total neutral fat hydrolyzed
was the sum of the amount of fat absorbed plus the neutral fat equivalent
of amount of the hydrolyzed fat left in the gut. The latter was determined
by titration and expressed as neutral fat by multiplication of the titration
value (corrected for fasting controls) by the saponification equivalent.
16
Results
Control experiments. The amount of ether-soluble material in the
gastro-intestinal tract of normal, sham-operated, and adrenalectomized
rats fasted for 24 hours as well as the titration values for the gastro
intestinal contents are reported in Tables I, II, and III which follow.
These averages were employed for the correction values in the recovery
tests as well as in the absorption experiments.
Results
Control experiments. The amount of ether-soluble material in the
gastro-intestinal tract of normal, sham-operated, and adrenalectomized
rats fasted for 24 hours as well as the titration values for the gastro
intestinal contents are reported in Tables I, II, and III which follow.
A21 average of 26, 34, and 28 mgs. of residual ether-soluble material was
found in the gastro-intestinal contents of normal, sham-operated and
adrenalectomized rats respectively while the mean values for titratable
acidity of the gut contents were 0.88, 0.42 and 0.53 cc. These averages
were employed for the correction values in the recovery tests as well as
the absorption experiments.
17
TABLE I
The weight of fat and the titration values obtained
on the ether extract of the gastro-intestinal tracts
of control female rats fasted one day.
Number height Surface
area
Fat in gut Titration
gm. sq. cm. mg* cc. 0.1N NaOH
14001 136 239 24 0.60
02 124 226 31 1.15
04 132 235 26 1.05
05 124 226 33 0.65
11 128 230 38 1.45
12 130 233 35 0.25
14 128 230 12 0.11
15 114 215 14 0.18
Average 127 229 2 6.6 4 2.I1
0488A0.181
■^Including Standard Error of Mean.
18
TABLE II
The weight of fat and the titration values obtained
on the ether extract of the gastro-intestinal tracts
of control sham-operated female rats fasted one day-
Number Weight Surface
area
Fat in gut Titration
gm. sq. cm. mg. cc. 0.1N NaOH
86 110 210 50 0.45
87 94 192 22 0.20
89 100 199 26 0.20
90 102 201 52 0.55
91 100 . 199 43 0.60
92 112 212 46 0.45
93 115 216 60 0.75
94 112 212 20 0.18
96 102 201 30 0.40
96 116 217 17 0.25
97 118 219 34 0.55
98 92 189 13 0.18.
100 118 219 40 0.60
101 95 192 26 0.60
Average 105 205 34*3.51 0.42*0.05:
including Standard Error of Mean.
TABLE III
The weight of fat and the titration values obtained
on the ether extract of the gastro-intestinal tracts
of adrenalectomized female rats kept on Rubin-Krick
solution and fasted for one day.
Number Weight Surface
area
Fat in gut Titration
gm. sq. cm. mg. cc. 0.1N NaOH
12962 153 256 62 1.41
963 160 264 41 0.79
965 132 235 21 0.50
966 160 264 19 0.39
967 133 236 32 0.62
968 152 256 13 0.20
969 154 258 10 0.17
970 138 241 16 0.28
972 154 258 37 0.60
973 134 237 14 0.16
13043 138 241 53 0.81
044 205 306 12 0.24
045 170 273 32 0.59
046 188 290 57 1.05
047 172 275 39 0.76
048 166 269 28 0.50
_049 202 303 13 0.20
~050 196 298 18 0.31
052 150 254 20 0.49
Average 161 264 2843.61 0.5340.07"
Including standard Error of Mean
20
Recovery experiments. Tables IV to VI record the recovery of corn oil,
tributyrin and sodium butyrate from the gastro-intestinal tracts of fasting
female rats killed immediately after the administration of the fats. The
average recovery in 10 experiments on corn oil was 94.4 per cent, for 10
tests on tributyrin, 90.2 per cent while the values for 15 experiments with
sodium butyrate amounted to 92.3 per cent. The recovery value used for
hydrogenated com oil was 93.6 per cent which was an average value obtained
earlier in this laboratory employing a similar technic. These averages were
used in correcting the values for fat removed from the gut for unavoidable
losses.
21
TABLE IV
The recovery of corn oil from the gastro-intestinal
tracts of female rats previously fasted for one day
and killed immediately after the administration of
0.5 cc. (457 mg.) of corn oil by stomach tube.
Humber Weight Corn oil recovered
Uncorrected Corrected
for blank
Per cent
mg. mg.
285 84 441 414.4 90.7
286 88 446 419.4 91.8
287 88 453 426.4 93.3
288 78 463 436.4 95.4
289 102 481 454.4 99.5
290 98 460 433.4 94.9
291 90 457 430.4 94.2
292 120 441 414.4 90.7
293 90 467 440.4 96.5
294 125 458 431.4 94.8
Average 963 457 430.1 94.4
TABLE V
The recovery of tributyrin from the gastro-intestinal
tracts of female rats previously fasted one day and
killed immediately after administration of 1 cc.
(1,040 mg.) of tributyrin by stomach tube.
Number Weight Tributyrin recovered________
Uncorrected Gorrected Per cent
_________________________________ for blank____________
mg. mg.
410 108 1,025 997 95.7
411 104 976 948 91.2
412 110 950 932 89.6
413 106 963 935 89.8
414 94 1,021 993 95.5
415 108 883 855 82.2
416 110 886 858 82.5
444 92 997 969 93.1
445 94 952 924 88.8
446 96 983 955 91.8
Average 102 964 936.6 90.2
23
TABLE VI
The recovery of sodium butyrate from the gastro-intestinal tracts
of female rats previously fasted one day and killed immediately
after the administration of 1 cc. of sodium butyrate (containing
an equivalent of 228 mg. as butyric acid) by stomach tube.
Rat
number
Weight 0.1N NaOH>
distillate
0.1N NaOH
corrected
Theoretical
0 .1N
NaOH required
Percentage
recovery of
theoretical
gnu cc. cc. cc.
133 146 23.55 22.52 25.90 86.9
134 142 24.55 23.52 25.90 90.7
135 148 24.10 23.07 25.90 89.0
136 142 25.38 24.35 25.90 93.8
137 152 25.40 24.37 25.90 93.9
138 152 24.82 23.79 25.90 91.7
139 172 26.28 25.25 25.90 97.5
140 170 24.30 23.27 25.90 89.8
141 130 25.88 24.85 25.90 96.0
142 138 29.80 28.77 25.90 110.9
144 138 23.80 22.77 25.90 87.7
145 146 23.50 22.47 25.90 86.8
146 126 24.40 22.37 25.90 90.0
147 130 24.10 23.07 25.90 89.3
148 138 24.50 23.47 25.90 90.7
Average 145 26.96 23.92 25.90 92.3
24
Absorption of Hydrogenated Cottonseed Oil* The rate of absorption of
hydrogenated cottonseed oil by normal one day-fasted female rats, as recorded
in Table VII, averaged 36.8 mg. per 100 sq. cm. per hour in 17 experiments.
Table VIII gives the results on sham-operated animals where a mean of 34.9 mg.
was found in 16 experiments. The results of 31 absorption tests on adrenal
ectomized rats which received Rubin-Krick solution showed an appreciably lower
rate of 27.6 mg. (Table IX). A somewhat lower level of absorption obtains in
adrenalectomized rats receiving water where the average value was 22.7 mg.
(Table X). Due to the large number of experiments lost because of diarrhea
or death of the animal, it was possible to obtain only 6 successful tests.
It is realized that this last average probably represents the maximum rather
than the mean rate of absorption under these conditions. Cortin restored the
ability of the rats to absorb hydrogenated cottonseed oil at a normal rate,
an average value of 39.7 mg. being found in the 20 experiments recorded in
Table XI. The average amount of 0.1 ¥ NaOH required to neutralize the acids
in the gut contents of the normal, sham-operated and adrenalectomized rats
receiving cortin was 3.0, 2.4 and 1.7 cc. respectively while a much higher
result of 7.9 cc. obtains in the adrenalectomized rats receiving Rubin-Krick
solution. In one case a titration value as high as 17.21 cc. was noted in
this last group. No estimations of free fatty acids were made on the gut
contents of the adrenalectomized rats which received water.
TA&E'VII
The absorption of hydrogenated cottonseed oil over a three hour period by
female rats previously fasted for one day and fed 300 mg. per 100 sq. cm.
Fat in mg.
Number^ Weight Surface Fed Recovered Absorbed
Titration g
area
Total Corrected Corrected Total Per 100 gm./hr. Per 100 sq. (-0.88 cc.)
for blank 93.6 cm. per hr.
per cent
gm. sq. om. cc.0.1 H NaOH
47 (+4) 122 234 695 417 391 417 278 74.40 38.70 3.62
48 (+9) 118 219 623 368 342 365 258 69.40 37.40 3.00
49(fl2) 113 214 642 442 416 444 198 54.80 28.90 2.42
50(+25) 134 237 704 384 358 382 322 70.20 39.80 3.27
53(+31) 118 219 623 347 321 343 280 67.50 36.40 1.52
54(+37) 112 213 642 357 331 354 288 71.00 37.30 .97
55(+42) 110 210 623 329 303 324 299 73.50 38.50 3.02
56(+44) 102 201 596 341 315 336 260 68.20 34.60 2.62
57(+46) 122 234 695 352 326 339 356 77.40 40.40 1.52
58(+55) 114 215 642 388 342 366 276 61.70 32.60 . 3.32
59(+59) 106 206 614 249 223 234 380 81.50 45.10 3.62
60(+6l) 124 226 677 380 354 378 299 61.00 32.90 5.02
61(+62) 126 228 676 332 306 327 349 68.60 37.90 4.32
62(+68) 108 208 623 295 269 288 335 74.30 38.90 3.82
64(+70) 118 219 650 389 363 387 263 53.00 28.80 • 4.82
65(+72) 118 219 650 316 290 310 340 68.00 39.90 1.32
66(+74) 122 224 668 367 341 364 304 59.00 31.10 3*32
Average 117 219 649 356 329 350 299 68.20*1.84 36.24*1.O4 3.03*0.3*
The figures in parentheses are the minutes under or over the three hour period.
2Corrected for recovery of 93.6 per cent* .
^Corrected for titration in fasted controls (Table I).
^Including the Standard Error of Mean
TABLE VIII
The absorption of hydrogenated cottonseed oil over a three hour period by female rats which
were previously sham-operated and previously fasted for one day when fed 300 mg* per 100 sq. cm.
Fat : in mg.
Number^- Weight Surface Fed Recovered Absorbed
Titration g
area
Total Corrected Corrected Total Per 100 gm./hr. Per 100 sq. (-0.42 cc.)
for blank 93.6 cm. per hr.
Per cent
gm. sq* cm.
cc.0.1 If NaOH
67 124 226 676 419 393 419 257 69.00 37.90 0.53
68 118 219 658 461 435 464 194 54.80 29.60 1.98
71(-1) 112 213 641 387 361 386 255 74.20 40.00 2.58
72 (-2) 128 230 687 451 425 454 233 61.30 34.20 2.78
73(-3) 116 217 622 416 390 417 205 60.00 32.10 2.05
74(-2) 131 234 704 407 381 407 297 76.40 42.70 2.98
76(-2) 130 233 696 517 491 524 ‘ 172 44.60 24.90 4.78
77(-1) 94 192 578 435 409 437 141 50.40 24.70 3.18
79 (-3) 110 210 631 371 345 368 243 74.90 39.30 4.08
81 120 222 668 434 408 436 232 64.70 35.00 3.18
69(+3) 110 210 632 403 377 402 230 68.60 36.00 4.18
1:202 122 224 668 . 407 381 407 261
70.40
38.30 0.87
03 142 245 , 731 425 399 426 305 71.30 41.00 0.03
04 142 245 731 509 483 516 215 54.70 31.60 2.57
05 154 256 768 471 .455 486 282 60.70 36.50 0.97
06 150 254 768 500 474 506 262 58.00 34.30 1.97
Average 125 227 698 435 413 441 236 63.30*2.34 34.9*1.04 2.41*0.34
^The figures in parentheses are the minutes under or over the three hour period.
^Corrected for recovery of 93.6 per cent
^Corrected for titration in fasted sham-operated controls (Table II).
^Including the Standard'Error of Mean.
TABLE IX
The absorption of hydrogenated cottonseed oil over a three hour period
by adrenalectomized female rats kept on Rubin-Krick solution and pre
viously fasted for one day when fed 300 mg. per 100 sq. cm.
Fat in mg.
Number^- Weight Surface
area
Fed Recovered
Absorbed
Titratic
Total Corrected
for blank
Corrected
93.6
per cent
Total Per 100 gm./hr. Per 100 sq.
cm. per hr.
(—0.53 i
gm*
sq. cm. cc.0.1 N
12924 +22) 137 240 720 512 484 518 202 43.60 25.00 6.37
925 +27) 188 290 866 570 542 580 286 44.20 28.60 5.97
926 +19) 141 244 736 458 430 460 276 59.00 34.10 11.11
928 +17) 207 308 922 665 637 681 241 34.40 23.80 17.21
929 +18) 190 292 872 688 660 706 166 26.40 17.25 13.67
930 +19) 146 249 744 441 413 442 302 62.40 36.60 7.41
932 +22) 161 264 793 562 534 572 221 40.60 24.80 10.14
952 -6) 175 278 834 605 577 617 217 42.70 26.90 11.78
953 +3) 153 256 769 558 530 567 202 40.70 25.90 7.08
955 +3) 156 260 776 601 573 613 163 34.10 20.50 11.53
957 +5) 184 287 866 579 551 589 277 48.80 31.30 9.37
958 ♦6) 148 252 740 555 527 564 176 38.10 22.50 11.72
961 +5) 125 227 680 429 401 429 251 65.00 35.90 6.00
13023
-4)
162 266 802 639 611 654 148 31.20 18.90 4.42
024 -5) 119 221 664 522 494 588 76 21.90 11.80 2.78
025 -3) 170 273 827 598 570 609 218 42.00 27.10 7.16
026 *-3) 150 254 760 514 486 520 240 54.40 32.10 9.37
ro
-a
TABLE IX (continued)
029( -5) 180 283 849 632 604 646 203 38.80 24.70 13.55
030( -2) 190 292 874 450 422 452 422 74.80 48.70 7.93
033( -8) 150 254 760 608 580 620 140 32.60 19.20 9.18
036 ( -2) 140 243 728 492 464 496 232 56.10 32.30 5.28
14187( 48) 142 245 731 ‘ 544 518 553 178 40.00 23.20 9.47
188(+10) 132 235 703 422 396 423 280 67.10 37.60 7.75
189(+13) 126 228 686 464 438 468 218 53.80 29.70 5.72
19l( +2) 132 234 469 343 317 338 131 32.60 18.40 1.27
192(+15) 160 264 794 508 582 514 280 53.80 32.60 5.67
193 124 226 677 522 496 530 147 39.50 21.70 3.77
194(+17) 125 227 677 541 515 550 127 30.80 16.90 9.72
195(+22) 130 232 695 558 532 567 128 29.20 16.40 4.67
196(+18) 120 222 668 280 254 271 397 100.20 54.30 3.67
197(-15) 128 232 687 449 423 451 236 66.80 37.20 4.07
Average * 146 248 735 509 483 516 217 46.6*2.94 27.6*1.64 7.9*0.24
*The figures in parentheses are the minutes under or over the three hour period.
^Corrected for recovery of 93.6 per cent.
Corrected for titration in fasted adrenalectomized controls (Table III).
^ Including Standard Error of the Mean.
TABLE X
The absorption of hydrogenated cottonseed oil over a three
hour period by adrenalectomized rats kept on water only and
previously fasted one day when fed 300 mg. per 100 sq. cm.
Number* Weight Surface
area
Fat in mg.
Fed Recovered Absorbed
Total Corrected
for blank
Corrected
93.6
per cent
Total Per 100
gm./hr.
Per 100 sq.
cm. per hr.
gm. sq. cm.
12908(-4) 118 220 662 600 572 612 50 9.5 7.8
13008 136 239 720 491 463 495 225 55.2 31.4
009 126 228 689 491 463 495 194 51.3 28.4
01l(+3) ' 159 262 785 576 548 586 199 41.0 24.9
012(+2) 149 - 252 761 546 518 553 208 46.0 27.2
0l3(+2) 162 266 801 653 625 668 133 27.0 16.5
Average 141 244 756 560 532 568 168 38.3*6.53 22.7*3.73
The figures in parentheses are the minutes under or over the three hour period.
Corrected for recovery of 93.6 per cent.
Including Standard Error of Mean.
TABLE XI
The absorption of hydrogenated cottonseed oil over a three
hour period by adrenalectomized female rats receiving 1 cc*
of cortin daily in drinking water and 0.5 cc. 30 minutes
before fat feeding when fed 300 mg. per 100 sq. cm.
1 •
Fat in mg•
1
Number^- Weight Surface Fed Recovered Absorbed
Titration
area
Total Corrected Corrected Total Per 100 Per 100 sq. (-0.53 cc.)3
for blank 93.6 - gm./hr. cm.» per hr.
oer cent
sq. cm. cc.0.1 N NaOH
102 (- 18) 140 243 731 330 296 316 415 94.50 54.50 0.42
103(*7) 142 245 731 360 326 348 383 86.60 50.20 2.22
104(+7) 134 237 713 389 355 379 334 79.90 45.20 1.47
105(+6) 140 243 730 532 498 532 198 45.60 26.20 3.47
106(*6) 150 253 758 451 417 445 313 67.40 40.00 1.67
107(*7) 132 235 705 344 310 331 374 90.80 51.00 0.97
108(^3) 116 217 648 331 297 317 331 96.60 51.60 0.22
109(42) 132 235 705 488 454 485 220 54.80 30.80 4.37
110(41) 120 222 768 419 385 412 256 70.80 38.30 1.57
lll(-3) 112 213 641 447 413 441 200 60.50 31.80 0.82
112(t3) 130 233 695 321 287 307 388 101.10 56.40 0.97
113(-4) 128 230 685 370 336 359 326 86.70 48.30 2.42
114(tl) 118 219 658 390 356 381 277 77.70 41.80 0.77
116 126 228 677 502 468 500 177 46.80 25.80 0.42
117(45) 120 222 668 457 423 452 216 58.40 31.60 2.02
118(-7) 160 263 785 482 448 478 307 66.60 40.50 • • 2.67
119(-6) 108 208 624 477 443 473 151 47.60 24.80 2.17
120(49) 82 176 524 350 316 338 186 72.10 33.60 0.42
121(49) 112 213 641 422 388 414 227 64.30 33.80 0.17
122(410) 134 237 704 436 402 429 275 64.70 37.60 4.75
Average 127 229 690 415 381 407 278 71.67*3.74 39.69*2.14 1.70±0.34
ljhe figures in parentheses are the minutes under or over the three hour period*
§ Corrected for recovery of 93.6 per cent*
4Corrected for titration in fasted adrenalectomized controls (Table III).
J4 mm ^ Mao r» *
31
Eight hour tests on absorption of hydrogenated cottonseed and corn oils*
In view of the fact that during the course of the present experiments Barnes,
Busoff, and Burr (1942) published a report stating that of the several fats
investigated only hydrogenated cottonseed oil was absorbed at a slower rate
by adrenalectomized rats, it was decided to extend our observation on other
fats composed of long chain fatty acids before proceeding with the neutral
fats such as tributyrin where the short chain fatty acids are water soluble.
Tests were next made on corn oil (in which Barnes et al had found absorbed
at the same rate in normal and adrenalectomized rats) and on hydrogenated
cottonseed oil over an 8 hour period rather than the 3 hour period which had
previously been employed. A similar depression in absorption after adrenal
ectomy was found in the 8 hour tests with hydrogenated cottonseed oil as had
been noted earlier for the three hour e xperiments. The average "value for the
12 tests on normal rats was 42.8 mg. per 100 sq. cm. per hour (Table XIl)r
while the results of 10 experiments on operated animals was 28.7 mg. as
recorded in Table XIII. Similar variations were noted after the administration
of corn oil, the values being 44.9 and 29.3 mg. respectively for averages on
9 and 11 animals (Tables XIV and XV). Probably due to the longer absorption
period, the variations in titration values although significant were not so
marked as in the earlier tests. Thus, the figures for the normal and adrenal
ectomized rats fed hydrogenated cottonseed oil were 2.6 and 5.7 cc. while the
values for the corresponding groups fed corn oil were 2.1 and 5.2 cc. respectively.
I
TABLE XII
The absorption of hydrogenated cottonseed oil over an
8 hour period by normal female rats previously fasted
for one day when fed 451 mg* (0.5 cc.) per 100 sq. cm.
Number Weight Surface
area
Fat in mg•
■
Fed
Recovered Absorbed Titration „
Total Corrected
for blank
Corrected
90.2
per eenti
Total Per 100
gm./hr.
Per 100
cm. per
sq. (-0*88 ce.)“
hr.
££•
sq. cm. cc.0.1 N NaOH
322 88 184 830 336 309 331 499 70.8 33.8 3.90
324 92 189 848 156 129 138 710 96.5 ' 46.9 2.10
325 84 179 812 190 163 175 637 94.5 44.3 2.45
326 84 179 812 190 163 175 637 92.2 43.2 1.55
327 88 184 830 117 90 96 734 103.8 49.7 1.20 .
328 88 184 830 200 173 181 649 91.8 43.9 2.10
329 76 168 758 144 117 125 633 103.8 46.8 2.15
330 75 167 758 124 97 104 654 108.3' 48.8 2.05
331 98 196 884 307 280 ' 303 581
74.2 37.2 2.90
332 86 181 812 234 207 222 590 85.8 40.8 3.50
333 90 186 838 311 284 304 534 74.2 35.9 4.30
334 88 184 830 226 199 213 617 87.8 41.9 2.90
Average 86 182 821 211 185 197 623 90.4-3.4? 42.8*1.4® 2.6*0.8®
Corrected for recovery of 93.6 per cent.
Corrected for titration in fasted adrenalectomized controls (Table III),
including Standard Error of Mean
03
PO
TABLE XIII
The absorption of hydrogenated cottonseed oil over an 8 hour
period by adrenalectomized female rats previously fasted for
one day when fed 451 mg. (0.5 cc.) per 100 sq. cm.
Humber Weight Surface
area
Fat in mg•
Fed Recovered Absorbed Titration „
Total Corrected
for blank
Corrected
93.6
per cent"*-
Total Per 100 Per 100 sq*
gm./4ir. cm./hr.
‘ (-0.53 cc.)
gnu sq. cm. cc.0.1 N NaOH
352 112 213 1010 671 643 686 324 36.2 19.1 12.70
351 96 194 875 578 550 588 287 37.3 18.4 10.50
356 94 191 858 777 749 800 58 7.7 3.8 1.50
364 102 201 912 417 389 415 497 62.1 31.6 3.60
365 100 199 893 212 184 197 696 87.0 43.7 2.20
373 132 230 1056 389 361 386 670 63.9 36.7 4.80
379 112 212 957 454 426 455 502 50.8 29.5 7.15
380 120 222 1002 399 371 397 605 64.2 36.3 6.50
418 110 210 949 417 389 416 533 60.5 31.6 3.50
436 128 230 1056 380 352 376 680 66.4 36.8 4.50
Average 110 210 851 469 441 471 485 53.6*7.1® 28.7*3.6® 5.70*1.7®
Corrected for recovery of 93.6 per cent.
Corrected for titration in fasted adrenalectomized controls (Tables III).
3Including Standard Error of Mean
w
03
TABLE XIV
The absorption of corn oil over an 8 hour period by normal female rats
previously fasted for one day when fed 458 mg. (0.5 cc.) per 100 sq. cm.
Number Weight Surface
area
Fat in mg*
Fed Recovered Absorbed Titration 2
Total Corrected
for blank
Corrected
94.4
per cent
Total Per 100 Per 100 sq.
gm./hr. cm./hr.
(-.88 cc.)
gm. sq. cm. cc.0.1 U NaOH
312 88 184 842 159 132 140 702 100.0 47.6 1.00
313 100 199 915 176 149 159 756 94.5 47.5 3.20
314 96 194- 886 132 105 112 774 101.0 49.9 1.50
315 82 176 805 110 83 89 716 109.0 50.8 0.80
316 82 176 805 144 117 126 680 103.6 48.3 1.80
317 96 194 886 210 183 195 691 90.0 44.5 1.20
318 118 220 1010 389 362 385 625 66.3 35.5 5.90
319 90 186 850 258 321 245 605 84.0 40.6 2.45
320 88 184 840 264 237 252 588 83.6 39.8 1.25
Average 93 168 871 205 188 189 682
92*4±4.1® 44.9*1.63 2.10*0.6®
Corrected for recovery (Table IV).
2corrected for titration in fasted normal female controls (Table I).
^Including Standard Error of Mean.
03
TABLE XV
The absorption of corn oil over an1 8 hour period by adrenalectomized female
rats previously fasted for one day -when fed 458 mg. (0.5 cc.) per 100 sq. cm.
Fat in mg•
Number Weight Surface Fed Recovered Absorbed• Titration „ '
area
Total Corrected Corrected Total Per 100 Per 100 sq.
(-.88 cc.r
for blank 94.4 l gm./hr. cm./hr.
per cent
gm. sq. cm. cc.0.1 N NaOH
336 90 186 841 630 602 638 213 29.8 14.4 9.20
339 92 189 869 279 251 266 603 82.6 40.2 5.90
343 82 176 805 247 219 232 573 87.4 40.7 5.00
346 114 215 1051 788 760 806 245 26.8 14.2 6.50
371 142 245 1116 386 358 380 736 64.8 37.5 1.60
382 120 222 1015 205 177 188 827 86.0 46.4 3.70
388 134 237 1088 682 654 693 395 36.8 20.8 3.88
390 122 224 1025 503 475 503 522 53.4 28.0 3.05
392 132 234 1070 593 565 598 472 44.7 25.2 12.60
393 116 217 988 391 363 385 603 64.9 34.7 2.80
394 100 199 915 502 564 598 317 39.6 19.9 3.20
Average 113 213 981 473 453 481 500 56.1*6.4® 29.3*3.7® 5.22*9.3®
^Corrected for recovery (Table IV).
2corrected for titration in fasted normal female controls (Table I).
^Including Standard Error of Mean.
36
Absorption of tributyrin. Since similar results were obtained in both
the three and eight hour absorption periods, the experiments with tributyrin
were carried out only over a three-hour period. No decrease in absorption
was found after removal of the adrenals. The mean level of 11 tests with
normal rats amounted to 69.1 mg. per 100 sq. cm. per hour while a value of
65.8 mg. was obtained from 16 tests on operated animals. No differences in
titration value of the gut contents were found between the two series. These
values were 0.6 and 0.5 cc. respectively. Because of the difficulty in a
quantitative removal of butyric acid with ether in the presence of water, too
great reliance can not be placed on the titration values. In order to ascertain
whether any retardation resulted in the absorption of the water-soluble butyric
acid, experiments were carried out with sodium butyrate where the material was
removed with water rather than ether.
TABLE XVI
the absorption of tributyrin over a 3 hour period by normal female rats
previously fasted for one day when fed 300 mg. of tributyrin per 100 sq. cm.
Fat in mg.
Number^ Weight Surface Fed Recovered Absorbed Titration
area
Total Corrected Corrected Total Per 100 Per 100 sq.
(-0.88 cc.)3
for blank 90.2 gm./hr. cm. /hr.
per cent2
gta.
sq. cm. cc.0.1 N NaOH
421 132 235 706 272 244 270 436 110.1 61.8 1.20
422 118 219 655 350 322 357 298 84.2 45.3 0.85
423(-5) 118 219 655 256 228 253 402 116.9 63.0 0.50
424(-4) 120 222 666 244 216 240 426 121.1 65.4 0.35
425 114 215 645 156 128 142 503 146.9 77.8 0.85
426(-2) 118 219 655 146 118 131 524 149.9 80.8 0.45
427 112 217 645 226 198 219 426 127.1 65.6 1.20
428 82 176 531 48 20 22 509 207.0 96.4 0.30
429(-1) 104 203 614 176 148 164 450 143.1 73.4 0.15
430 82 176 531 193 165 183 348 141.5 65.8 0.50
431 90 186 562 209 181 200 362 134.1 64.8 0.65
Average 108 208 688 207 179 198 426 143.8*9.34 69.1*3.7* 0.64*»34
■^The figures in parentheses are the minutes under or over the three hour period.
^Corrected for recovery (Table V).
^Corrected for titration in fasted normal female controls (Table I).
^Including the Standard Error of Mean.
w
->3
TABLE XVII
The absorption of tributyrin over a 3 hour period by adrenalectomized female
rats fasted for one day when fed 300 mg. of tributyrin per 100 sq. cm.
Fat in mg•
Humber Weight Surface Fed Recovered Absorbed Titration
1 1 O
area
Total Corrected
for blank
Corrected
90.2
per cent
Total Per 100
gm./hr.
Per 100 sq.
cm./hr.
(-.53 cc.)
397
J5E*
130
sq. cm.
233 697 265 237 262 435 111.8 62.4
cc.0.1 H HaOH
0.30
398 118 219 655 197 169 187 468 133.0 71.4 0.65
402 122 224 676 248 220 244 432 117.9 64.4 0.50
406 136 239 718 251 223 247 471 115.5 65.8 0.65
408 116 217 644 196 168 186 458 131.8 70.4 0.75
432 116 217 644 202 174 193 451 129.8 69.4 0.70
433 96 194 583 248 220 244 339 117.8 58.2 0.50
434 112 213 635 264 236 262 373 111.1 58.4 0.40
435 130 233 697 277 249 276 421 108.1 60.4 0.70
438 100 199 593 199 171 189 404 134.9 67.7 0.50
439 124 226 676 291 263 291 385 103.2 56.8 0.25
440 128 230 686 319 291 323 363 94.4 52.5 0.60
437 124 226 676 241 213 236 440 118.5 65.0 0.40
443 104 203 614 144 116 129 485 155.5 79.7 0.60
448 118 219 655 158 130 144 511 144.4 77.8 0.35
449 106 206 614 178 150 166 448 141.0 72.6 0.50
Average 117 218 654 229 202 225 440 124.0*3.83 65.8*1.83 0.52*0.63
^■Corrected for recovery (Table V)
^Corrected for titration in fasted adrenalectomized female controls (Table III).
3Including the Standard Error of Mean.
39
Absorption of sodium butyrate. Because of the rapidity of its absorption,
the experiments with sodium butyrate were conducted for only 1.5 hours. As in
the tests with tributyrin, no decrease in rate of absorption of sodium butyrate
followed adrenalectomy when the animals were kept in good nutritional condition
with Rubin-Krick solution. The average rate of absorption of sodium butyrate
in 16 normal rats was 45.0 mg. (as butyric acid) per 100 sq. cm. per hour
(Table XXVIII) while the mean of 14 tests on adrenalectomized rats was 42.8 mg.
as recorded in Table XIX. However, as in the case of glucose, an appreciable
decline in absorption was noted in operated animals in poor nutritional condi
tion because they received only water instead of salt solution. Under such
circumstances an average level of 33.4 mg. was obtained for 11 experiments
(Table XX).
TABLE XVIII
The absorption of butyric acid over a 90 minute period by normal
female rats and previously fasted for one day when fed 114 mg.
(0.5 cc. of sodium butyrate solution) per 100 sq. cm.
Number Weight Surface
area
Fed
Corrected Fatty acid in mg.
titration of
Recovered Absorbed
distillate
(-1.10 cc.)
Total Corrected
92.5
per cent1
Total Per 100
gm./hr.
Per 100 sq.
cm./hr.
gm. sq. cm. mg. cc. 0.1 N NaOH
164 134 237 292 14.57 128.0 138.4 153.6
76.4 43.2 .
165 144 247 292 10.47 91.9 39.4 192.6 89.2 51.9
166 128 230 252 4.57 40.3 43.5 218.5 114.2 63.4
167 147 250 284 13.07 115.0 124.2 159.8 72.4 42.6
168 138 241 273 8.17 71.9 77.7 195.3 94.4 52.4
169 129 232 264 11.47 100.8 108.9 155.1 80.1 43.2
170 136 239 272 8.72 76.6 82.9 189.1 93.0 50.5
171 130 232 264 17.07 150.0 162.1 101.9 52.2 27.9
183 114 215 244 8.67 76.4 82.6 161.4 94.6 47.3
184 116 217 246 5.57 49.0 53.0 193.0 111.8 58.6
185 114 215 244 12.12 106.8 115.2 ■ 128.8 75.1 39.9
186 102 201 228 12.17 107.0 115.8 112.2 73.3 37.2
187 110 210 240 17.57 156.0 168.5 71.5 43.3 22.7
188 112 213 242 7.19 63.2 68.3 173.7 103.2 54.3
189 108 208 237 9.47 83.1 89.9 147.1 90.3 47.2
190 90 186 214 11.32 98.2 106.1 107.9 . 77.6 38.6
Average 122 223 255 10.76 94.6 102.3 153.9 84.0*4.62 45.0*2.62
^Corrected for recovery (Table VI).
2Including Standard Error of Mean
TABLE XIX
The absorption of butyric acid over a 90 minute period by adrenalectomized
female rats kept on Rubin-Krick solution and previously fasted one day vdien
fed 114 mg. (0.5 cc. of sodium butyrate solution) per 100 sq. cm.
Number Weight Surface
area
Fed
Corrected Fatty acid in mg.
titration of
Recovered Absorbed
distillate
(-1.10 cc.)
Total Corrected
92.5
per cent^
Total Per 100
gm./hr.
Per 100 sq.
cm./hr.
gm. sq. cm. mg. cc. Oa N NaOH
172 120 222 253 7.85 69.0 74.6 178.4 99.2 53.6
175 124 226 258 11.00 96.8 104.6 153.4 82.5 45.3
176 114 215 244 18.50 158.6 171.5 72.5 42.3 22.4
177 122 224 256 10.40 91.6 98.9 157.1 85.9 46.8
178 106 206 235 11.65 102.8 110.9 124.1 77.7 40.1
179 104 203 230 9.15 80.6 87.3 142.7 91.3 46.8
180 128 230 262 7.30 64.4 69.6 192.4 100.2 55.8
181 122 222 253 7.75 68.3 73.8 179.2 98.2 53.8
182 124 226 258 10.95 96.5 104.2 153.8 82.6 45.4
191 126 228 260 11.85 104.2 112.9 147.1 77.8 42.9
192 135 238 272 12.90 113.3 122.2 149.8 73.8 41.9
193 122 224 254 16.10 141.9 153.1 100.9 54.8 30.0
194 130 233 264 12.85 113.0 122.0 142.0 72.6 40.6
196 100 199 226 12.50 110.0 119.0 107.0 71.2 55.6
Average 119 221 252 11.48 100.8 108.9 142.8 79.3*4.22 42.8*2.32
^Corrected for recovery (Table VI).
^Including Standard Error of Mean.
TABLE XX
The absorption of butyric acid over a 90 minute period by adrenalectomized
female rats kept on water and previously fasted one day when fed 114 mg.
(0.5 cc. of sodium butyrate solution) per 100 sq. cm.
Corrected ________________ Fatty acid in mg.
lumber Weight Surface Fed titration of Recovered Absorbed
area distillate
(-1.10 cc.)
Total Corrected Total
92.5
per cent
Per 100
gm./hr.
Per 100 sq.
cm./hr.
m-* ia* «£•
mg. cc. 0.1 N NaOH
197 104 203 320 13.37 115.9 125.2 104.8 67.1 34.4
198 118 219 246 19.27 169.4 184.2 61.8 39.0 18.8
199 106 206 234 10.17 89.4 96.7 137.7 86.6 ;44.6
200 112 213 242 18.17 160.0 173.1 68.9 40.8 21.9
202 110 210 240 12.97 114.0 123.2 116.8 70.7 37.1
203 88 184 209 10.05 88.5 95.7 113.3 78.3 41.2
204 101 200 228 13.67 120.1 129.8 98.2 64.8 32.7
208 92 189 214 14.67 129.0 139.4 74.6 54.2 26.4
210 98 196 224 11.89 104.2 113.0 111.0 75.6 37.7
212 .102 201 228 12.39 108.9 117.8 110.2 72.1 36.8
213 101 200 228 9.32 82.0 88.6 139.4 92.1 46.4
Average 103 202 229 13.27 116.5 126.1 103.3 67.4*4.72 33.4*2.52
■^Corrected for recovery (Table VI).
2Including Standard Error of Mean.
43
Summary Table. In Table XXI "the averages in the various tests are
summarized while in Table XXII a statistical evaluation is made in such a way
that the means of the various series can be compared with each other to ascer
tain whether significant differences obtain between them. Thus it is shown
that the average rate of absorption.both of hydrogenated cottonseed and corn
oils is significantly depressed by adrenalectomy. Moreover, in the adrenalec
tomized animals receiving hydrogenated cottonseed oil, the results are signifi
cantly lower than the animals which underwent a mock operation. Furthermore,
the administration of cortin resulted in a rise in absorption of fat which was
significantly higher than found in the adrenalectomized rats. In all cases
where tests were made, the increase in fatty acid content was shown to be
significant after the ablation of the adrenals.
Although this thesis reports experiments carried out on 278 rats, many
additional tests which failed are unrecorded. Although obviously there were no
experiments discarded in the 41 control tests or the 35 recovery tests due to
diarrhea, a number of absorption experiments were not considered because
diarrhea had obtained during the absorption period. Out of a total of 93
experiments on normal rats, only 12 (12.8 per cent) had to be discarded because
of diarrhea while out of a total of 298 animals adrenalectomized, successful
tests resulted in only 119 cases. In the operated animals receiving Rubin-Krick
solution, only 37 per cent were successful, 42.4 per cent being lost because of
diarrhea and 20.4 per cent on account of death during the absorption tests.
When water instead of salt solution was employed with the adrenalectomized
animals, 28.6 per cent were successful while 42.6 and 28.6 per cent were lost
because of diarrhea or death. After cortin 74 per cent of the tests were
satisfactory; only 25.9 per cent had diarrhea while none died. No diarrhea
whatsoever obtained when sodium butyrate was fed in any case while some diarrhea
43 A
occurred in both groups receiving tributyrin.
From these data it is evident that diarrhea occurs much more frequently
in the absence of the adrenal cortex. The average of successful tests on
operated rats in all probability represents the maximum value. If all the
tests where diarrhea obtained were also included in these averages, it is quite
probable that they would be markedly lowered. In most cases practically no
absorption obtains where diarrhea supervenes. The rather high incidence of
death occurring in adrenalectomized rats after fat feeding is further evidence
of the fact that these animals can not handle the fatty acids formed as a result
of lipolytic activity in the gut.
TABLE XXX
Summary table showing absorption rates and acid in gut contents of normal (N)
and adrenalectomized (a ) female rats receiving various fats and sodium buty
rate. Adrenalectomized rats received Rubin-Krick solution except where noted.
Fat fed Absorp
tion
period
Number of tests Absorbed in mg. per
100 sq. cm. per hr.2
Titration in cc.
0.1 N NaOH2
Natural fat hydro
lyzed per hr. in Remarks
mg. per 100 sq. cm.^
N1 Al N A N A N A
hrs.
Hydro 5 17(1)
31(11) 36.3*1.0 27.6*1.6 3.0*1.3 7.9*0.2 46.2*1.5 54.1*2.4
genated
cotton 3 16(111) 34.9*1.0 2.4*0.3 46.0*1.7 Sham-operated
seed oil
3 6(IV) 22.7*3.7 45.7*4.5 Received water
3 20(V) 39.7*2.1 1.7*0.3 47.7*2.7 Received water
plus cortin
8 12(VI) 10(VII) 42.8*1.4 28.7*3.6 2.6*0.1 5.7*1.7 52.5*0.8 50.0*4.7
Corn oil 8 9(VIII) 11(IX) 44.9*1.6 29.3*3.7 2.1*0.8 5.2*0.9 53.0*1.5 48.8*3.7
Tri 3 11(X) 16(XI) 69.1*3.7 65.8*1.8 0.6*0.3 0.5*0.1
-
butyrin
Sodium
1.5 16(XII) 14(XIII) 45;0*2.6 42.8*2.3
butyrate
1.5 11(XIV) 33.4*2.5 Received water
^The values in parentheses are used to identify the groups for statistical analysis in Table XXII*
^Including Standard Error of Mean.
43 B
44
TABLE XXII
The statistical evaluation of the absorption rate and fatty acids
of gut contents of averages of various groups given in Table XXI.
Groups
compared
Absorption rates Titration of gut contents
Statistical evaluation
M.D.sS.E.M.D.1 Method II2 M.D.zS.E »M «D Method II2
per cent per cent
I
«
• II 4.53 90 13.62 100
I
•
• IV 3.55 100
II
•
• IV 1.17 67
II
•
« V 4.58 94 17.24 100
III
•
• II 3.84 81 15.32 100
III
•
• IV 3.21 100
IV
•
• V 3.89 100
VI : VII 3.65 90 1.84 100
VIII : IX 3.87 91 2.88 89
XII i XIV 3.21 91
XIII • XIV 2.75 82
*Mean Difference: Standard Error of Mean Difference* TOien this
exceeds 3.00, the differences are significant.
2
feThe percentage of tests of series having lower average which do
not overlap group with higher average. A value of 90 per cent
is considered significant.
45
DISCUSSION
These experiments further support the earlier work of Verzar and LAszt
(1935) in demonstrating that the adrenal glands are involved in the normal
absorption of fat. They do not, however, offer any proof as to the mechanism
by which the adrenals exert their influence. Verzar's theory of the control
of phosphorylation by the adrenals is still a debatable question, with much
of the more recent evidence apparently against it.
However, there seems little doubt from the data presented here that the
adrenal glands.play an important role in fat absorption. The average rate
of absorption of hydrogenated cottonseed oil was 36.3 mg. per 100 sq. cm.
per hour in the 3 hour testsand 42.8 mg. for the 8 hour tests which compare
well with the values obtained earlier by Deuel, Hallman and Leonard (1940)
of 39.7 mg. This was lowered to 27.6 mg. for the 3 hour tests and 28.7 mg.
for the 8 hour tests on adrenalectomized rats kept in good condition by the
administration of Rubin-Krick solution. The value of 22.7 mg. per 100 sq. cm.
per hour was found with adrenalectomized rats which had only water to drink.
That the lowering in rate of absorption after adrenalectomy is not an
effect specific for hydrogenated cottonseed oil, as suggested by Barnes
et al (l942), is indicated by the parallel results on the absorption of corn
oil. The rate of absorption of this vegetable oil in normal and adrenalec
tomized female rats in 8 hour tests was found to be 44.9 and 29.3 mg. per
100 sq. cm. per hour respectively. Adrenalectomy caused an average decrease
of 30 per cent in the speed of absorption of cottonseed oil and a decline of
35 per cent in the rate at which corn oil was absorbed.
46
The lowered effect in adrenalectomy is also not to be ascribed to
the trauma attendant on the operation. In the first place, the experi
ments were not conducted until at least a week had elapsed following the
operation. A similar length of post-operative period was sufficient so
that normal rates of absorption had been shown to obtain in-adrenalectomized
animals administered glucose (Deuel et al (l937)). However, the most cogent
evidence that trauma did not cause the lowering in the adrenalectomized
animals is that there was no appreciable decline in the fat absorption of
rats which had undergone a sham operation. Both groups of adrenalectomized
rats (II and IV) absorbed fat at a rate which was significantly slower from
a statistical standpoint than these sham-operated animals.
The lowered absorption rate is also not to be ascribed to a lowered
speed of lipolysis. In Table XXI a calculation indicates that the neutral
fat hydrolyzed (that absorbed plus the quantity of neutral fat equivalent to
the fatty acid in gut contents) is practically identical after, hydrogenated
cottonseed or com oil in normal and adrenalectomized rats.
The differences after ablation of the adrenals is to be traced to the
failure in removing at a normal rate the fatty acids formed and an accumula
tion of fatty acid occurs. In practically all experiments both with corn
and hydrogenated cottonseed oils the level of free fatty acids in the intes
tinal contents (as determined by titration ) were two .to three times as high
in the adrenalectomized animals as in the normal ones. The differences also
are shown to be statistically significant (Table XXII).
That the adrenal cortical hormone is responsible for the alterations
would seem to be proved by the fact that not only was the level of absorption
of hydrogenated cottonseed oil returned to normal after administration of the
47
cortical hormone (39.7 mg. compared with a normal of 36.3 and a value of
27.6 on adrenalectomized rats), but also the free fatty acid content of the
gut contents drops to the normal value (1.7 cc. compared with 3.0 cc. in
normal rats and 7.9 cc. in adrenalectomized^animals).
The most probable explanation for the discrepancies between our tests
and those of Barnes et al (1942) appears to lie in the fact that they used
much larger and older rats. Their animals weighed somewhat over 300 grams
while the average weight of the rats used in the present tests approximate
110 grams. It is well known that cortical deficiency is much more critical
in young animals. Also it is known that accessory tissue may also occur in
older animals. Moreover, the complete removal of the adrenal gland is much
more difficult in older rats principally because of the large amount of fat
in which the suprarenals become imbedded.
The differences in procedure between the two laboratories are probably
not the reason for the variable results. Thus while in the earlier experi
ments, the length of the absorption period was shorter and the dose of fat
fed smaller than employed by Barnes et al (l942), later tests were conducted
in which these procedures were identical. At first it was thought that the
difference between this work and that of Barnes et al might be due, to the
slightly different technic employed by the latter. However, .this cannot
explain why the results with normal animals are in agreement, while only the
values on the adrenalectomized animals are not in harmony. The rate of
absorption per 100 sq. cm. per hour by Barnes et al (l942) and that obtained
in the present investigations was 39.8 mg. and 42.8 ing. respectively for
hydrogenated cottonseed oil (8 hour tests) while those for corn oil were
42.4 mg. and 44.9 mg. respectively. This is a rather remarkable agreement.
48
Thus after a careful study of the many possible reasons for the different
experimental results obtained here with those of Barnes et al, one is forced
back again to the only logical explanation, namely, the differences in the
sizes of the animals. It should also be noted that actually it appears from
Barnes' data that a lower absorption obtains in at least three of the five
fats to be studied. The differences probably would be significant if there
were enough rats to make a statistical evaluation instead of 5 or 6 in a group.
On the other hand a most striking fact was noted in the absorption of
tributyrin and of sodium butyrate. The rate of absorption of tributyrin per
100 sq. cm. per hour for normal and adrenalectomized rats was 69.1 mg. and
65.8 mg. respectively. These are the same as reported earlier by Deuel
and Hallman (1940) where a value of 65.0 mg. per 100 sq. cm. per hour were
found. This appears to indicate that the soluble butyric acid formed as a
result of the hydrolysis of this simple triglyceride is not influenced by
absence of the adrenals. Further proof of this is shown by the experiments
in which sodium butyrate was fed. The rate of absorption per 100 sq. cm. per
hour for sodium butyrate is normal and adrenalectomized animals was 45.0 mg.
and 42.8 mg. respectively. This is slightly higher than the value of 39.7
reported by Deuel, Hallman, and Reiftaan (1941). One must conclude from such
results that the adrenals apparently do not influence the absorption of these
water-soluble fatty acids.
As in the absorption of glucose, the rate may also be adversely affected
in the case of sodium butyrate by secondary effects following adrenalectomy
rather than as a result of a primary action. In any instance where the salt
balance is not maintained by keeping adrenalectomized animals on a salt solution
which insures an adequate salt intake, hemoconcentration with resulting poor
49
circulation occurs. In the groups where water alone was fed, the absorption
of sodium butyrate was lowered just as glucose is lowered. Also the absorp
tion of the hydrogenated cottonseed oil declines to a greater degree than occurs
when salt solution is given to the adrenalectomized rats. However, this is not
a specific result of absence of the cortical hormone but only an indirect one.
A possible explanation for the effect of adrenalectomy on the absorption
of corn and hydrogenated cottonseed oils could be the effect of the adrenals
on the secretion of bile and bile acids rather than because of any phosphory-
lating action as postulated by Verzar (1935). It is generally agreed that
the bile acids form water-soluble complexes with the higher fatly acids and
thus make possible their absorption. Thus the absorption both of hydrogenated
cottonseed oil and corn oil was inhibited because of the high content of water
insoluble fatty acids, such as palmitic, in both of these oils. This was not
the case with tributyrin and sodium butyrate where the butyric acid formed
was already water-soluble and consequently easily absorbed by both normal and
adrenalectomized animals.
50
SUMMARY
After adrenalectomy an inhibition in the rate of absorption of corn oil
and of hydrogenated cottonseed fat from the gastro-intestinal tract occurred
in female rats. This resulted even when the animals were kept in good
physical condition by the administration of Rubin-Krick solution in place of
drinking water. Neutral fats therefore differ from glucose in requiring the
cortical hormones for absorption.
After fat feeding much larger quantities of fatty acids were recovered
from the gastro-intestinal contents of the adrenalectomized rats than from
that of normal rats. Since the rate of lipolysis is similar in the two
groups, it is concluded that the lowering in absorption rate is to be
ascribed to a retardation in the rate of absorption of the fatty acids.
Whether this is related to a failure in phosphorylation as suggested by
Verzar or to an effect of the adrenal on bile secretion is not known.
On the other hand, tributyrin and sodium butyrate are absorbed as
rapidly after adrenalectomy as in the normal. It is believed that the cortical
hormones are not directly related to the absorption of such fatty acids as
are water-soluble. However, in adrenalectomized rats given water instead of
salt solution, a slower rate of absorption of sodium butyrate obtains; this
is related to secondary effects as circulation rather than to a primary action.
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T H E E F F E C T O F A D R E N A L E C T O M Y O N F A T A B S O R P T IO N
BY
L U C I E N B A V E T T A , L O IS H A L L M A N , H A R R Y J . D E U E L , J R .
a n d P A U L O. G R E E L E Y
F ro m the D e p a rtm e n ts o f B io c h e m is tr y a n d P h y s io lo g y , U n iv e r s ity o f S o u th e rn
C a lifo r n ia M e d ic a l S ch o o l, L o s A n g e le s
. R e p r i n t e d f r o m T h e A m e r i c a n J o u r n a l o f P h y s i o l o g y
V o l. 134, N o . 3 , O c to b e r , 1941
M a d e i n the U n ite d S ta te s o f A m e r ic a
Reprinted from The American Journal op Physiology
Vol. 134, No. 3, October, 1941
T H E E F F E C T O F A D R E N A L E C T O M Y O N F A T A B S O R P T IO N 1
L U C I E N B A V E T T A , L O IS H A L L M A N , H A R R Y J . D E U E L , J R .
a n d P A U L O . G R E E L E Y
F r o m the D e p a r tm e n ts o f B io c h e m is tr y a n d P h y s io lo g y, U n iv e r s ity o f S o u th e r n
C a lifo r n ia M e d ic a l S ch o o l, L o s A n g e le s
A c c e p te d fo r p u b lic a tio n J u ly 26, 1941 . .
T h e stu d y of in testin al absorption has alw ays presented one of th e m ost
interesting problem s in p h ysiology. N o t on ly are th e pancreas^ th e liver
and th e in testin al m u cosa in volved b u t also there is considerable eviden ce
th a t th e adrenal cortex m a y p lay a role. . ■ .
T h e influence pf th e adrenals on fa t absorption has b een supported by
th e experim ents of Verzar and L aszt (1935), w ho also p ostu lated a som e
w h at sim ilar m echanism for carbohydrate absorption (Ju d ovits .'and Ver
zar, 1937). In an exten sive series of in vestigation s, th ese workers found
th a t fa t absorption w as inh ibited b y adrenalectom y in rats and th a t th e
norm al fu nction could be restored by th e adm inistration of cortical extract.
In th e absence of th e adrenal cortex th is effect w as attrib u ted to th e
failure of phosphorylation, w hich phenom enon is p ostu lated as a prerequi
site for th e absorption of th e fa tty acid portion of th e fa t m olecule. T h e
results of A rtom and P eretti (1935) using iodized fa ts and of Sinclair (1936)
w ho em ployed elaidic acid in d icate th a t th e ingested fa tty acids do becom e
incorporated in to th e phospholipids of th e in testin al m ucosa. N o t only
w ould it appear th a t th e adrenal glands regu late fa t and carbohydrate ab
sorption b u t th e w ork of Clark (1939) and of Stein and W ertheim er (1941)
w ould seem to ind icate th a t th is gland also controls th e absorption of
sodium chloride.
On th e other hand, evidence from th is laboratory (D euel, H allm an,
M urray and Sam uels, 1937) indicates th a t th e role of th e adrenal cortex in
carbohydrate absorption is a secondary one. T he low ered absorption of
glucose did. n o t occur if dehyd ration and th e consequent circulatory dis
turbances w ere avoided b y adm inistration of R ubin-K riek or sodium chlo
ride solution follow ing adrenalectom y. T h a t a sim ilar exp lan ation for the
low ering of fa t absorption in adrenalectom ized anim als m a y obtain, is indi
cated b y th e report of B arnes, M acK ay, W ick and C arne (1939) w ho found
1 T h e s e d a ta are fro m a th e s is to b e p r e s e n te d b y L u c ie n B a v e t t a to th e G r a d u a te
S c h o o l o f th e U n iv e r s ity o f S o u th e r n C a lifo r n ia in p a r tia l fu lfilm e n t fo r th e d e g r e e o f
D o c to r of P h ilo s o p h y .
619
620 BAVETTA, HALLMAN, DEUEL AND GREELEY
no effect on th e rate of absorption of m eth yl esters of fa tty acids or of corn
oil itself in adrenalectom ized rats and also b y th a t of B arnes, M iller and
Burr (1939) w ho used spectroscopically a ctiv e fa tty acids.
B ecau se of th ese divergent results it seem ed desirable to rein vestigate
th e effects of adrenalectom y on th e absorption of fats. In th is stu d y a com
parison has been m ade of th e rate of absorption of h yd rogenated cotton seed
oil in norm al, sham -operated, and adrenalectom ized rats w hich w ere given
either R ubin-K rick solution alone, w ater, or w ater and cortin.
M e th o ds. T h e experim ental anim als w ere fem ale albino rats from our
sto ck colony w eighing 120 to 160 gram s k ep t on our regular sto ck diet.
T h ey w ere adrenalectom ized under ether anesth esia using th e lum bar ap
proach. E xperim ents on fa t absorption w ere m ade 7 to 10 d ays after th e
operation. In m ost cases th e com pleteness of operation w as checked post
m ortem .
T h e procedure for th e determ ination of th e rate of fa t absorption w as
sim ilar to th a t em ployed earlier (D euel, H allm an and Leonard, 1940) and
th e p h ysical and chem ical con stan ts of th e fa t w ere iden tical w ith th ose of
th e hydrogenated cotton seed oil reported in our earlier work. A fastin g
period of 24 hours preceded th e tests. T h e fa t w as adm inistered a t a level
of 300 m gm . per 100 sq. cm . of b od y surface. T h e la tter w as calcu lated by
th e form ula of L ee (1929). D uring th e tests th e anim als w ere k ep t in
separate cages and a n y experim ents show ing evidence of diarrhea w ere dis
carded. T h e fa tty acids recovered from th e gastro-in testin al con ten ts were
estim ated b y titration of th e ether extract w ith 0.1 N N aO H using phenol-
ph thalein after solu tion in petroleum ether and isopropyl alcohol. T h e
m illigram s of fa tty acids w ere calcu lated from th e titration b y u se of a fa tty
acid eq u ivalen t based on th e saponification num ber of th e fat.
C ortin2 w as adm inistered in th e drinking w ater for 4 d ays prior to th e
absorption tests in am ou n ts of 1 cc. daily. T h is am ou n t of horm one w as
added each 'd ay to approxim ately th e volum e of w ater ta k en b y th e rat th e
previous d ay. One hour before th e fa t feeding, each anim al w as given an
ad d ition al 0.5 cc. of cortin b y stom ach tube.
R e su l t s. A sum m ary tab le show ing th e rate of absorption of norm al
and adrenalectom ized rats is recorded in tab le 1 w hile th e control tests on
th e fa sted anim als are g iv en in tab le 2.
T h e fa t absorbed is calculated from th e difference b etw een th e am ou nt
fed and th e corrected am ou nt recovered from th e gu t. T h e q u a n tity actu
a lly recovered from th e g u t is first corrected b y su b traction of th e am ou nt
of ether-soluble m aterial w hich w as rem ovable from th e gastro-in testin al
tracts of rats fa sted for a sim ilar period b u t fed n o fa t (table 2 ). A further
correction is applied for th e ex ten t of recovery based on th e valu es obtained
2 C o r tin w a s k in d ly fu r n ish e d fo r th e s e s tu d ie s b y D r . E . C. K e n d a ll o f t h e M a y o
C lin ic w h o s u g g e s te d th e d o sa g e a n d m e th o d of a d m in is tr a tio n .
ADRENALECTOMY AND EAT ABSORPTION 621,
w hen know n am ou nts of fa t are g iv en and th e gastro-in testin al tract re
m oved im m ed iately. T h e la tter v a lu e em ployed w as for a 93.6 per c en t
recovery.
T A B L E 1
S u m m a r y ta b le sh o w in g th e f a t a b so rb ed i n th ree hou r p e r io d 'b y f e m a le 'r a ts f a s te d
one d a y a n d f e d 300 m g m . o f h y d ro g e n a te d co tto n seed o i l p e r 100 sq.' cm . o f
su rfa c e a re a ; , .
. NUM
BER OE
EX
AVER
AGE
SUR
FAT ABSORBED IN MGM.
PER HOUR* .
TITRA
TION OF
CATTY'ACII>
IN-ETHER
EXTRACT
EXPERIMENTAL
AVER
AGE
Per 100 sq. cm.
Per.
cent
iof:
total
lip id ’
PERI
MENTS
WEIGHT FACE
AREA
Per 100
g m .
- Total
Q
..s
EX
TRACT
Total
c '
gram s s'q. cm .
C C . 0.1 N
N a O H
m g m .
Normal (a) 17 117 219 68.2±1.8 36.3±1.0 3.0=1=0.3 83.3 24.5
Normal—Sham-
operated (b)
16 125 ,227 63.3±2.3 34.9±1.0 2.4±0.3 66.5 15.6:
Adrenalectomized-—
Rubin-Krick C c)
31 ' 146 248 46.6±2.9 27.6=1=1.6 4.53(a)
3.88(b)
7.9=1=0.2 217.0 39.9'
Adrenalectomized—
' Water only (d)
6 141 244 38.3±6.5 22.7=1=3.7 3.57(a)
3.19(b)
6.3±0.6 170.0 30.3
Adrenalectomized—
Cortin (e)
20 127 229 71.7±3.7 39.7±2.1 4.25(c)
4.00(d)
1.7±0.3 47.1 12.9
NEUTRAI*
P A T
H T D R O -
L T Z E D
. PER
H O X JR *
m g m . p e r
100 sq. cm .
46.2±1,5
46.0=fcl.7
54.1=fc2,4
45.7=1=4.5
•47.7db2.7
* Including standard error of mean
d = deviation from mean
n — number of observations
t Mean difference: standard error of mean difference. When this value exceeds 3.0 the results are considered
significant. The letter in parentheses indicates groups with which comparisons are made.
T A B L E 2
S u m m a r y ta b le sh o w in g e th e r-so lu b le m a te r ia l in fe m a le r a ts fa s te d one d a y w ith o u t
f a t fe e d in g
EXPERIMENTAL CONDITION
NUMBER
OF EXPERI
MENTS
AVERAGE
WEIGHT
TOTAL FAT
IN GUT*
TITRATION
OF ETBER
EXTRACT
ETHER SOLU
BLE EXTRACT'
AS FATTY ACID*
\
N o r m a l..............................................
S h a m -o p e r a te d .............................
A d r e n a le c to m iz e d — R u b in -
K r ic k .............................................
8
14
19
gram s
127
105
161
m g m .
26. 6=1=2.1
34.0=1=3.5
28.0=1=3.6
cc. 0.1 N
N a O H
0 .8 8
0 .4 2
0 .5 3
m g m .
1 8 .5 ± 4 .6 ,
11.7=1=1.3
1 4 .6 = fcl.8
* I n c lu d in g s ta n d a r d erro r o f m e a n c a lc u la te d as in ta b le 1.
T h e neu tral fa t h yd rolyzed w as calcu lated to determ ine th e com parative
lip olytic action in th e different groups. T h e to ta l n eu tral fa t hyd rolyzed
w as th e sum of th e am ou n t of fa t absorbed plus th e am ou n t of th e hyd ro-
622 BAVETTA, HALLMAN, DEUEL AND GREELEY
lyzed fa t left in th e gut_. T he latter w as determ ined b y titration and ex
pressed as neutral fa t b y m u ltiplication of th e titration valu e (corrected for
fastin g controls) b y th e saponification equivalen t.
D i s c u s s i o n . T here is a decrease of app roxim ately 38 per cen t in th e
rate of fa t absorption in th e untreated adrenalectom ized rats as com pared
w ith norm als. W hen salt solution is adm inistered to operated anim als,
th e depression is som ew h at less (24 per cen t) b u t th e difference is still high ly
sign ifican t from a sta tistica l stand point. A lth ou gh th e rate of absorption
of norm al rats w as sligh tly lower on an average: after undergoing a sham
operation, it w as significan tly higher th a n th a t of th e adrenalectom ized
anim als. T h a t th e depression is to be ascribed to th e absence of th e adrenal
cortex is ev id en t from th e fa ct th a t absorption could be com p letely restored
b y th e adm inistration of cortin. T h is fa ct is corroborated b y th e observa
tion th a t th e in testin al lym p hatics appeared practically w h ite in th e norm al
an d cortin-treated rats killed a t th e height of fa t absorption w hile th is w as
n o t ev id en t in th e adrenalectom ized anim als to w hich cortin w as n o t given.
T h e decreased absorption apparently results from a failure to rem ove
fa tty acid a t a norm al rate as evidenced b y greater accum ulation of free
fa tty acids in th e g u t conten ts of th e adrenalectom ized anim als n o t receiv
ing cortin. On th e other hand, there is no evidence of an y decrease in
lip olytic a c tiv ity associated w ith th e rem oval of th e adrenal gland. T hese
results w ould seem to support th e th eory of Verzar and L aszt (1935), al
th ou gh th ey do n o t give a n y evidence as to w hether phosphorylation is
in volved .
SUM M ARY
A definite inh ibition in fa t absorption w as n o ted in adrenalectom ized
rats. I t w as on ly slig h tly im proved b y th e adm inistration of R ubin-K rick
solu tion . A fter adrenalectom y larger am ou n ts of fa tty acids accu m ulated
in th e in testin e th a n occurred norm ally. B o th of th ese ph en om ena w ere
restored to norm al b y th e adm inistration of cortin.
T here is no evidence th a t adrenalectom y alters lip olytic a ctiv ity .
R E F E R E N C E S
A r t o m , C . a n d G . P e r e t t i . A rch . In te r n . P h y s io l. 42: 61, 1935.
B a r n e s , R . H ., A . N . W ic k , E . S. M i l l e r a n d E . M . M a c K a y . P ro c. S o c. E x p er.
B io l, a n d M ed . 40: 651, 1939.
B a r n e s , R. H., E. S . M i l l e r a n d G . O. B u r r . T h is J o u r n a l 126: P 427, 1939.
C l a r k , W . G . P ro c. S o c . E x p e r . B io l, a n d M e d . 40: 468, 1939.
D e u e l , H . J ., J r ., L . H a l l m a n a n d A . L e o n a r d . J . N u t r it io n 20 : 215, 1940.
D e u e l , H . J ., J r ., L . H a l l m a n , S. M u r r a y a n d L . T . S a m u e l s . J. B io l. C h em .
119: 607, 1937.
J u d o v i t s , N . a n d F . V e r z a r . B io c h e m . Z tsch r. 292: 182, 1937.
L e e , M . O. T h is J o u r n a l 89: 24, 1929.
S i n c l a i r , R . G . J. B io l. C h em . 115: 211, 1936.
S t e i n , L . a n d E . W e r t h e i m e r . P r o c . S o c. E x p e r . B io l, a n d M ed . 46: 172, 1941.
V e r z a r , F . a n d L . L a s z t . B io c h e m . Z tsc h r . 276: 11, 1935; 278: 396, 1935.
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Asset Metadata

Creator Bavetta, Lucien Andrew (author)

Core Title Adrenalectomy and fat absorption

School School of Medicine

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1942-05

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Deuel, H.J. (committee chair), [illegible] (committee member), Copeland, C.S. (committee member), Harrison, Bruce M. (committee member), Kessel, John F. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-605474

Unique identifier UC11355044

Identifier DP21533.pdf (filename),usctheses-c17-605474 (legacy record id)

Legacy Identifier DP21533.pdf

Dmrecord 605474

Document Type Dissertation

Rights Bavetta, L. A.

Type texts

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

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

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