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The metabolism of the hexitols

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The metabolism of the hexitols

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Content THE METABOLISM OF THE HEXITOLS
A Thesis
Presented to
the Faculty of the Department of Biochemistry
University of Southern California
School of Medicine
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
by
Cornelia Hendrick Johnston
May 1941
UMI Number: EP41275
All rights reserved
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UMI
" OSsssrtatton Pjbfeh'ng
UMI EP41275
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
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This thesis, written by
......................... C m N E LIA ..H E IfflS IC K ..JQ H IST.Q N ..............
under the direction of h Faculty Committee,
and approved by a ll its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fu lfill
ment of the requirem ents f o r the degree of
-MASTEK~GE~SC.imC.E~
D ean
Secretary
1241
Faculty Committee
/J Chairman
The writer gratefully'acknowledges her indebtedness
to Dr. H.J. Deuel, Jr., for his guidance, and to him and
Miss-Lois Hallman for their generous assistance in carrying
out the laboratory work on which this thesis is based.
TABLE OP CONTENTS
CHAPTER PAGE
I. INTRODUCTION............ ...................... 1
II. LITERATURE .......... 4
III.- EXPERIMENTAL ............................ 12
Experiments on the ketolytic action of
the hexitols ........................... 12
Experimental results of the ketolytic
action of the hexitols, ............... 15
Statistical treatment of results . 16
Experiments on glycogen formation ........... 24
Experimental results on the glycogen-
forming ability of sorbitol, mannitol,
and dulcitol .................. 27
■ IV. DISCUSSION ............................... . . 42
¥. SUMMARY ................................ . . . 48-
BIBLIOGRAPHY................................... 49-
LIST OF TABLES
TABLE PAG-E
I. Ketone Bodies in Urine of Fasting Female
Rats Receiving Sorbitol . . .......... 18
II. Ketone Bodies in Urine of Fasting Female
Rats Receiving Mannitol.................. 19
III. Ketone Bodies in Urine of Fasting Female
Rats Receiving Dulcitol........... 20
IV. Ketone Bodies in Urine of Fasting Female
Rats Receiving Glucose ........... 21
V. Ketone Bodies in Urine of Fasting Female
Rats Receiving Sodium Chloride ............ 22
VI. Summary of the Effects of the Hexitols on
Ketonuria................................. 23
VII. The Liver Glycogen in Pasting Male Rats
after the Intraperitoneal Injection of
Different Concentrations of Sorbitol at
Different Time Intervals . . . . . . . . . 1 25
VIII. The Liver and Muscle Glycogen in Fasting
Male Rats 6 Hours after, the Intra
peritoneal Injection of a 25 per cent
Solution of Sorbitol, 1 cc. per 100
sq. cm. ................................. 29
TABLE
ix.
x.
XI,
XII.
i v
PAGE
The Liver,, and Muscle Glycogen in Blasting
Male Rats 6 Hours after the Intra-
peri toneal Injection of a 25 per cent
Solution of M'annitol, 1 cc. per 1QQ
sq. cm, ...................... 30
The Liver and Muscle Glycogen in Fasting
Male Rats 6 Hours after the Intra-
peritoneal Injection of a 25 per cent
Solution of Glucose, 1 cc. per 100
sq. cm.......................................... 31
The Liver and Muscle Glycogen in Fasting
Male Rats 6 Hours after the Intra-
peritoneal Injection of Sodium Chloride,
1 cc. per 100 sq. cm. ...................... 32
The Liver and Muscle Glycogen in Fasting
Female Rats 6 Hours after the Intra-
peri toneal injection of a 25 per cent
Solution of. Sorbitol, 0.5 cc. per 100
sq. cm. ..................... 33
TABLE
XIII.
XIV.
XV.
XVI.
The Liver and Muscle Glycogen in Pasting
Female Rats 6 Hours after the Intra-
peri toneal Injection of a 25 per cent
Solution of Mannitol, 0.5 cc. per 100
sq. cm. .... ... ....................
The Liver and Muscle Glycogen in Fasting
Female Rats 6 Hours after the Intra-
peri toneal Injection of a 25 per cent
Solution of Glucose, 0.5 cc. per 100
sq. cm.......................................
The Liver and Muscle Glycogen in Fasting
Female Rats 6 Hours after the Intra-
peritoneal Injection of a Sodium Chloride
Solution, 0.5 cc. per 100 sq. cm. . . . .
The Liver and-Muscle Glycogen in Fasting
Male Rats 6 Hours after the Intra-
peritoneal injection of a 9 per cent
Solution of Dulcitol, 1 cc. per !GG;
■ vi
TABLE PAGE
XVII. The Liver and Muscle Glycogen in Fasting
Male Rats 6 Hours after the Intra-
peri toneal Injection Of a 9 per cent
Solution of Glucose, 1 cc. per -100
- « -
sq. cm. . . . . . . . . . . . . . . . . -38
XVIII. The Liver and Muscle Glycogen in Fasting
Male Rats 6 Hours after the Intra-
peritoneal Injection of a Sodium
Chloride Solution, 1 cc. per 100
sq. cm. .................... 39
XIX. Summary Table Giving a Comparison of
Liver Glycogen of Fasting Rats 6
Hours after the Intraperitoneal
Injections of the Hexitols.................. 40
XX. Summary Table Giving a Comparison of
Muscle Glycogen of Pasting Rats 6
Hours after the Intraperitoneal
Injections of the Hexitols.................. 41
CHAPTER I
INTRODUCTION
The hexitols are hexahydric alcohols which may be
obtained by reduction of the aldehyde group of a hexose
sugar. Thus sorbitol is the hexitol of glucose, mannitol
of mannose and dulcitol of galactose. These polyhydric
alcohols are widely distributed in plants from which they
can be crystallized. They may also be obtained commercially
by reduction of the corresponding hexose by various reducing
agents such as sodium amalgam. The reaction proceeds very
slowly, two hydrogen atoms being added to the hexose. At
the present time the hexitols are' used in the manufacture
of explosives and are obtained by the electrolytic reduction
of hexose sugars. By this latter process it is possible to
obtain them in large quantities and at a greatly reduced
price.
In recent years the hexitols have become increasingly
prominent, claiming the attention of investigators and filling
various needs of the layman. For example, In Europe, many
clinicians have advocated the use of sorbitol as a carbohy
drate substitute in the treatment of diabetes. Others are
of the opinion that It is to be avoided. Within the past
few years the use of sorbitol in food preparations has been
extended as the price has been greatly reduced. It is now
2
used in the manufacture of candies, and as a moistening
agent in the preparation of bakery products.
A study of the metabolism of the hexitols was there
fore thought to be of interest.
There are several methods that can be used in studying
the metabolism of carbohydrates and glucose producing sub
stances. Probably the most accurate method is to feed the
substance under question, stop the metabolism at a certain
point by means of pancreatectomy or phlorhizin, and investi
gate the elimination products. By this means it is possible
to determine what products are changed to glucose. Since the
animal is unable to utilize glucose, any substance that is
changed to glucose will be excreted quantitatively as such
in the urine. Another method used in metabolism studies is
to follow the blood sugar curve. This also indicates what
substances are changed to glucose. A procedure followed by
some workers is to determine the effectiveness of the material
under study in reviving an animal after insulin shock. A
widely used method is'to fast an’animal until,the liver
glycogen is used-up. The substance under question is then
given and the liver glycogen is determined. Thus any com
pound that is converted to glucose will cause an increase
in the liver glycogen. Another manner of investigating the
ability of a substance to be converted to glucose in the
3
animal body is the study of its effect on ketonuria. If
carbohydrate oxidation is cut to a minimum, the breakdown of
fatty acids, in the animal is disturbed. Their complete
combustion to carbon dioxide and oxygen does not take place
with the result that so-called ketone bodies or acetone
bodies are,eliminated in the urine. Any substance capable
of going to glucose will, however, produce a ketolytic effect
causing a decrease in ketonuria. The last two procedures,
namely, the determination of the ability of the hexitols,
sorbitol, mannitol ‘ and dulcitol, to produce glycogen and of
their effect on ketonuria were carried out.
CHAPTER II
LITERATURE
In 1883 Jaffe found that the urine of dogs contains
mannitol as a normal constituent. After mannitol was fed to
dogs, large quantities were recovered unchanged in the urine.
Embden and Griesbach (1914) demonstrated that
d-sorbitol is converted into d-lactic acid on perfusion
through the liver of a fasting dog. In the phlorhizinized
dog sorbitol is changed Into a mixture of fructose and
glucose. Mannitol, dulcitol and inositol form neither sugar
nor lactic acid under similar conditions.
Upon plotting blood sugar curves after feeding normal
fasting colored men one hundred grams of various carbohy
drates, Field (1919) found an increase of 10 mg. per cent
in blood sugar when mannitol was fed. There was a 40 mg.
per cent increase when the equal amounts of glucose were fed.
Roth gave the same time curve, however.
Many investigators are of the opinion that sorbitol
can safely be given diabetics as a sweetening agent. Kaufmann
(1929) recommended sorbitol as a carbohydrate substitute in
the treatment of this disease. . He claimed that It was sweet,
easily absorbed, spared protein as It could be oxidized, led
to glycogen formation and did not elevate blood sugar.
Reinwein (1929) found no rise in blood sugar and no decreased.
5
carbohydrate tolerance. He claimed that only traces of sorbi
tol were excreted unchanged. A rise in respiratory quotient
and the disappearance of hypoglucemic symptoms indicated that
the hexitol was utilized.
The effect of sorbitol on blood sugar of two juvenile
diabetics was, studied by Payne, Lawrence and McCance (1933).
The rise in blood sugar after sorbitol administration was
found to be slight compared with that of glucose. The
acetone excreted in the urine was determined by Rotheras'
test before and after feeding sorbitol in order to see if it
produced any effect on ketosis. The differences were not
marked but slightly less acetone was found when sorbitol was.
administered. An attempt was made, to determine the effect
of the sorbitol in relieving insulin hypoglycemia but no
change was noted. Sorbitol also failed to increase liver
glycogen in starved rats. For this reason it is thought
that sorbitol has a place in the diabetic diet.
Roche and Rayboud (1933) are of the opinion that;
sorbitol is not metabolized by normal or diabetic individuals.
Their experimental results on fasting rabbits indicate that
sorbitol is not transformed into glycogen, and has no effect
on insulin hypoglucemia in rabbits. It did appear, however,
to be utilized to some extent by phlorhizinized animals.
On the other hand, Donhofer (1930) gave a normal
fasting individual and a fasting diabetic 50 gm. sorbitol,
and determined the blood glucose and blood sorbitol at
varying- intervals.' In the normal individual approximately
equal gains were observed in both glucose and sorbitol. In
the blood •of' the'diabetic, however, the amount of glucose
increased enormously, but the amount of sorbitol was no
greater than in the normal subject. The conclusions drawn
are that sorbitol is easily synthesized into glycogen and the
sorbitol hyperglucemia seems to result from the same causes
as hyperglucemia following the ingestion of glucose or other
carbohydrates. This is in agreement with the work of Labbe"
(1932) who claims there are no clinical data available to
justify its use in the diabetic diet. In a later paper,
Bertrand and Labbe" (1934) report that sorbitol Is poorly
absorbed and is no better tolerated than glucose. It was
noticed that the Ingestion of sorbitol caused gastric
disturbances. Because of its lower dietetic value Raybaud
and Roche (1934) are likewise of the opinion that sorbitol
can not be used as a substitute for glucose.
Lecoq (1934) fed rations rich In lipids and containing
mannitol and sorbitol to the extent of 35 per cent, of the•
total diet to pigeons. Both sorbitol and mannitol were
completely utilized. When the mannitol diet was given, less
7
than the usual amount of vitamin B was required. When
given a ration containing 66 per cent mannitol, the pigeons
died in 8 to 15 days with symptoms of polyneuritis, even
though extra amounts of yeast -were added. A 66 per cent
sorbitol ration produced the same results except that the
survival period was 17 to .50 days.
Lafon (1937) reports that mannitol is utilized by
mice to a very slight extent. When fed in large quantities
the mannitol proved to be toxic. Up to 30 per cent of the
diet, sorbitol, however, was not toxic and seemed to .be
almost completely metabolized.
In this country as in Europe, there has been great
divergence of opinion concerning the fate of the hexitols.
Carr, Musser, Schmidt' and Krantz (1933) found additional
glycogen storage when fasted rats were given a weighed
supply of cacao-butter and mannitol to the extent of 33
per cent of the diet. The animals were maintained on this
diet for 80 hours and then sacrificed. The controls were
given only the cacao-butter and sacrificed after the same
period. It was found, however, that mannitol has no effect
on the respiratory quotient of rats.
Silberman and Lewis (1933-34), on the contrary,
fasted rats for 24 hours and fed two or four cc. of a
15 per cent solution of manni.tol by stomach tube-. The rats
8
were killed and the liver, glycogen was determined after
absorption periods of 2, 3, 4 and 6 hours. No significant
increases in the glycogen content of the liver after oral
administration of mannitol were noted as compared with the
values obtained for the control series.
Following this work of Silberman and Lev/is, Carr and
Krantz (1938) again endeavored to' study the metabolism of
mannitol. The animals were treated as before— being fed-the
mannitol in cacao-butter. Again they were able to demonstrate
the production of liver glycogen by mannitol. The work of
Silberman and Lewis was repeated and confirmed.
Todd, Myers and West (1939) were able to demonstrate
that in dogs intravenous injections of mannitol cause a drop
in the blood sugar curve. It was suggested' that this drop
might be the result of blood dilution. When mannitol was
given either by stomach tube or by intraperitoneal Injection,
no Increase in liver glycogen was noted. The time interval
between the administration of mannitol and the sacrificing
of the animals was 5 hours. They did find, however, that if
mannitol was fed along with cacao-butter- there was a definite
glycogen increase.
In studying the metabolism of dulcitol, Carr and
Krantz found that when white rats were fed mixtures containing
33 per cent dulcitol with a basal diet of cacao-butter there
y
was an increase in liver glycogen over the controls. The
tissue glycogen was reduced, however, and there was no
noticeable effect on the. respiratory quotient. ’Dulcitol
likewise failed to increase significantly the-blood sugar of
rabbits when administered orally. They were able, however,
to demonstrate an increase in liver glycogen following
administration of dulcitol by stomach tube.
Contradictory to the report of Payne, Lawrence and
McCance (1933) that sorbitol fails to increase liver glycogen
in the rat, Waters (1938) reported an increased liver glyco
gen in rats following intraperitoneal injections of sorbitol.
He was unable to obtain any increase in glycogen following'
administration by stomach tube. Contrary to the results of
Roche and Raybaud (1933) considerable glycogen deposition
in the liver was found in guinea pigs after intraperitoneal
injections of sorbitol, Waters found only a mild transitory
hyperglycemia in normal fasting dogs following the’intra
venous administration of sorbitol. He demonstrated that
intravenous injections markedly depress the glucose tolerance
curve of the normal dog and also of the depancreatized dog
receiving a steady supply of insulin. According to Waters,*
only one-other substance, namely, fructose, has been found so
far to have this effect. It is suggested that perhaps sorbitol
is oxidized to fructose in the liver. This, of course, is
10
compatible with the early perfusion v?ork of Ernbden and
G-riesbach where a mixture of glucose and fructose was shown
to exist after perfusion of sorbitol through a phlorhizin-ized
dog liver. Waters 'seems to feel that the effect of sorbitol
on the glucose tolerance curve is caused by the stimulation
of the glycogen synthesis mechanism.
Todd, Myers and West (1939) demonstrated that follow
ing the. administration of sorbitol to fasted rats by stomach
tube or by intraperitoneal injection, a deposition-of liver
glycogen occurs within 8 hours. When sorbitol was fed with
cacao-butter there was, likewise, an increase In glycogen,
though not so great as deposited after intraperitoneal
injection. An increase in blood sugar was also shown follow
ing the Intravenous injection of sorbitol. Comparing their
studies on sorbitol with those on mannitol, Todd and his
coworkers conclude that sorbitol is much more readily converted
into glucose and glycogen in the animal body than Is mannitol.
Carr and Forman (1939) found that fasting rats fed
one third sorbitol mixed with two thirds cacao-butter have
higher liver glycogen than do the controls' fed on the
cacao-butter alone.
Blatherwick, Bradshaw, Ewing, Larson and Sawyer (1940)
fasted rats for different periods. Various amounts of
sorbitol were given by stomach tube and the effects of
11
3 and. 6 hour absorption periods studied. When the animals
were fasted 48 hours and were then given large doses of sor
bitol male rats appeared to store liver glycogen but the
females did not. According to the.above workers, the' inabil
ity to show glycogen formation after oral administration of
sorbitol is probably due to its low absorption coefficient.
Cacao-butter lessens peristalsis•and prevents the diarrhea
which almost always accompanies the ingestion of large
quantities of aqueous solutions of sorbitol. When fed with
cacao-butter the sorbitol remains1 in the gut for longer
periods and probably for this reason more is absorbed.
CHAPTER III
EXPERIMENTAL
Albino rats from the stock colony weighing between
100 and 215 grams were- used throughout. The animals had
previously been on the stock diet snd were all in good
nutritional condition at the start of the fast. Wherever
possible litter mates were used for comparative tests.
EXPERIMENTS ON KETOLYTIC ACTION OF THE HEXITOLS
The rat differs from man-in that it does not normally
develop ketosis during fasting. An exogenous ketonuria can
be produced, however, by the method of Deuel, Hallman, and
Murray (193b) by feeding the sodium salt of butyric acid.
Deuel, Gulick and Butts (1932) and Butts and Deuel
(1933) demonstrated a marked sex difference in the degree of
ketosis. Because female rats develop ketonuria much quicker
and to a greater degree than males, they were used through
out’ the experiments.
.The metabolism cages.used consisted of round wire
mesh containers which fitted over large 10-inch funnels. A
wire cone fitted into the funnel prevented any fecal or
extraneous material from passing through. A small container,
provided with a stop-cock was attached to the stem of the
funnel. This served to collect and deliver the urine.
13
Approximately one cc. of mineral oil was placed In the
bottom of the container to prevent the loss of acetone bodies
by evaporation.
The animals were fasted for 48 hours, weighed, and
placed in.individual metabolism cages. They were allowed
free access to water throughout the experimental period.
An exogenous ketonuria was produced by the method of Deuel,
Hallman and Murray (1938). The rats were given the sodium
salt of butyric acid in a total amount equivalent to 150 mg.
as acetone per 100 sq. cm. of body surface per day, or
461 mg. of butyric acid per 100 gm. of rat per day. The
desired concentration of the salt was made by neutralizing
S2.5 gm. butyric acid with NaOH and making up to a volume
of 100 cc. with water.
Since general metabolism is thought to be proportional
to the superficial area of an animal, it would seem likely
that the production of ketone bodies is proportional to the
surface area. Butts and Deuel (1933) obtained more uniform
action in animals of different weight where this method was
employed. For this reason, the doses administered were
calculated on the basis of surface area in mg. per square
centimeter. The surface area was determined by the formula-
of Lee, S _ k¥/°*^, where S — surface area in square meters,
W — weight of animal in kilograms, and k = constant which
14
varies In the different .species. For the rat k = 0.125.
Solutions of sorbitol, mannitol, and dulcitol were
made up so as to be the equivalent of 25. mg, of glucose per
0.5 cc. Thus, 5.056 grams of each hexitol was made up to a "
volume of 100 cc. separately.
The rats were divided into five groups corresponding
to the materials■being administered. All received by stomach
tube 0.5 cc. per 100 sq. cm. of the sodium butyrate twice
daily. In addition and Immediately following the butyrate,
part of the rats were given 0.5 cc. of the different hexitol
solutions per 100 sq. cm. Two sets of controls were run: To
one group, 25 mg. of glucose per 100 sq. cm. (0.5 cc.) were
given in addition to the butyrate, and to the other.group
only the butyrate was administered.
The urine was collected the following morning In a
100 cc. volumetric flask and made up to volume by repeated
washings of the funnels. The determination for acetone
bodies was made by the usual Van Slyke technique on an aliquot
of the urine.
The remainders of the urine samples were saved each
day and stored in the ice box. On the last day of the experi
mental period, urinary nitrogens were determined by the
Kjeldahl procedure. In case the nitrogen was abnormally
high, the result on ketone bodies for that particular animal
15
for that day was discarded. (If the urinary nitrogen is
greatly increased the decrease in ketone bodies can not be
attributed solely to the ketolytic effect of the materials
being studied, for in such cases allowance must be made for
the ketolytic effect of the carbohydrate produced by the
breakdown of tissue protein.) If one day's urine were
discarded, Kjeldahls were run on the previous day's urine
sample for that particular animal to determine whether or
not that result■should be discarded as well.
EXPERIMENTAL RESULTS OF THE KETOLYTIC ACTION OF THE HEXITOLS
Tables: I, II, and III give the results of feeding
sorbitol, mannitol, and dulcitol in the doses mentioned above.
Table IV gives the values obtained when an equivalent amount
of glucose was administered, and Table V the degree of
ketosis developed by animals receiving only sodium butyrate.
A summary consisting of a comparison of the average acetone
body excretion In the urine during the administration of the
hexitols with that for.the glucose and fasting controls is
given in Table VI.
16
STATISTICAL TREATMENT OF RESULTS
The reliability of - the results/obtained for the
comparative ketolytic ability of the hexitols studied was
determined by statistical treatment. The mean difference .
of any two groups is significant if the observed difference
is a real one. When the ratio of the difference In the means
obtained In two comparative groups to the standard error of
the mean difference Is 3 or over, the results are considered
significant. Absolute reliability Is obtained if the ratio
is 4 or above.
The method of calculating the ratio is as follows.;
After the mean is calculated, the deviation (d) from the
2
mean is determined for each experiment. Next, d Is
p O
obtained and the sum of the d [Z.& ) is found. The standard
error of the mean (S.E.M.) is calculated by the formula;
S.E.M. =
V
a2:
n in which n is the number of experiments.
\ I n
The standard error of the mean difference (S.E.M.D.) is
equal to the square root of the sum of the squares of the
standard errors of the means: |!( S.E.M. )2_ 2 + (S.E.M.)g2' .
. . M,D.(Mean difference) . .
If the ratio ---- g- ..------- is greater than 3
the results are significant.
17
Another statistical treatment that may be used is the
Fisher t.
■ — _ 1(x-| ) _ _ l(xP )
x 'l n-^ x2 “ ng
* a 2 < * 2 ■
s2 = id! *!■■%
(n-^-1) + (ng-l)
t = *3 I P_i__*_n .£
S V n! + n2
x = mean
x^ and xg = results of individual experiments on the
comparative group.
n = number of experiments.
2 •
d = square of the deviation from the mean.
The significance of the t value found is then
interpreted by reading from the Table' of t of Fisher when
the jo value is 0.01, or when there is one chance in 100.
of the difference being due to experimental error.
Each investigator may choose the value for jo which
he considers significant. In many cases a value of 0.05
(one chance in 20) is accepted as satisfactory. However,
by choosing the value of 0.01, there can be no question
about the significance of the results.
The use of the Fisher t method for statistical
evaluation in cases where the number of observations is
1 7 A
small (under 30) is generally considered to tie more
reliable than the first procedure described (determination
of ratio of mean difference to standard error of mean
difference), while the latter procedure is equally
acceptable if the number of cases exceeds 30.
TABLE I
Ketone Bodies in Urine of Fasting Female Bats Receiving Sorbitol
Expt. Uo. Body Wt. Surface Urinary E f per Acetonuria per 100 s^. cm. as acetone
Area 100 sq. cm. 1st day . 2nd day 3rd day 4th day
gms. sq.. cm. mg. mg. mg. mg.
« m8 S h
1 125 228 94.6 78.7
--- ----
2 105 205 35.0 109.0 79.0
---
9 156 260 35.5 73.8 68.2 60.7
----
10 141 244 37.3 77.0 60.2 74.0
19 144 247 28.3 131.5 88.6 88.3 —— * *
20 154 257 33.8 78.6
----
29 157 261 36.3 94.7 96.4 76.2 59.6
30 143 246 40.2 56.7 58.5 51.2 51.8
38 196 298 30.9 73.8 67.2 75.2
---
39 186 288 26.7 80.4 63.3 83.7
----
Average 151 253 30.4 87.0 73.3 72.8 55.7
±6.4 ±3.8 ±4.0 ±2.8
V ♦ 4
3
11
12
13
21
22
31
40
41
076
077
088
TABLE IX
Ketone Bodies in Urine of Fasting Female Hats Receiving Mannitol
Body, Wt. Surface Urinary, I T per _______Acetonuria per 100 sq. cm. as acetone
_________ Area 100 sq. cm.______1st day,_____2nd day, 3rd day, 4th day.
gms. sq. cm. mg.
”g» ■ M i M i ‘
mg.
135 238 32.0 184.0 131.0
-----
155 259 33.5 111.0 114.0 131.0
138 241 32.0 112.4 102.2 101.0
136 239 29.8 90.5 94.2 108.0
152 256 29.9 105.2 95.7 .95.6
214 314 29.1 79.6 68.4 71.0
-----
116 217 40.3 77.2 82.3 ■ 84.5 73.7
172 275 29.5 95.5 68.4 79.3
184 287 •25.4 83.9 90.5 57.0
-----
154 258 99.0 67.6
'
165 268
----
76.5 93.7
145 348
----
84.6 92.5
157 258 31.2 99.9 91.7 90.9 73.7
-
+8.1 +4.9 +7.7
TABLE III
Ketone Bodies in Urine of Fasting Female Eats Eeceiving Dulcitol
Expt. Ho. Body, Wt. Surface Urinary, N per Acetonuria per 100 sq. cm. as acetone
Area 100 sq. cm. 1st day. 2nd day, 3rd day 4th day,
gms. sq. cm. mg. mg. mg. • mg. mg.
23 174 377 31.0 91.5 73.7 89.0
----
34 165 369 27.6 115.0 144.0 141.5
33 133 335 38.0 97.7 105.2 98.0 ■ ' . 90.7
33 134 336 40.8 75.4 84.2 85.6 74.0
34 118 330 34.9 57.2 64.3
---- ----
43 134 337 36.7 109.8 89.5 104.7
43 116 317 38.7 82,0 84.7 82.3
----
44 136 328 38.9 80.8 69.8 71.4
Average 135 237 35.9 88.7 89.4 96.1 82.4
♦7.4 £8.4 ♦7.9 ♦5.9
TABLE IV
Ketone Bodies in Urine of Easting Female Bats Receiving Glucose
Expt. Ko. Body Wt, Surface Urinary, N per Acetonuria per 100 sq. cm. as acetone
Area 100 sq. cm. 1st day, 2nd day 3rd day, 4th day.
gms. sq. cm. mg.
SSL
mg. mg. mg.
5 108 208 84.8 83.7
---- ----
6 130 233 31.5 80.4 61.7 - — -
14 181 284 29.4 39.5 47.7 50.4
----
15 156 260 27.0 15.0 28.3 12.1
25 166 269 26.6 66.4 47,4 63.0
----
26 168 271 32.4 54.3 46.2 47.2
35 123 225 36.4 45.6 32.5 18.5
----
45 118 220 39.5 60.0 34.9 33.8 — -
Average 148 251 31.8 55.8 47.8 37.5
• • • ■ w e e
±7.5 ±5.9 ±7.3
TABLE V
Ketone Bodies in Urine of Pasting Female Bats Receiving Sodium Chloride
Expt. No. Body Wt. Surface Urinary N per Acetonuria per 100 sq. cm. as acetone
Area 100 sq. cm. . 1st day, 2nd day 3rd day. . 4th day,
gms. sq. cm.
mg.
mg. mg. mg.
7 112 213 117.0 117.8
16 174 277 37.5 108.0 115.5 124.0
17 168 271 35.6 116.2 143.2 139.4
18 134 237 36.7 64.2 78.1 104.3
27 . 171 274 31.6 101.7 105.0 101.7
28 172 275 31.6 135.5 114.0 103.7
36 107 207 37.2 109.8 101.2 112.0 98.2
37 125 227 34.2 80.1 85.3 83.4
46 134 226 40.5 123.5 109.3 113.3
47 ‘ 135 238 42.4 104.2 88.3 98.4
-----
083 130 232 98.8 108.9
Average 141 243 36.4 105.4 106.1 108.9 98.2
*5.7 ±5.4 ±4.9
' TABLE VI
Summary, of the Effects of the Hexitols on Ketonuria
Substance Acetonuria per 100 sq. cm,, as acetone
Average
Acetonuria Differ M.D.
t
Cal
t
Theo
Fed 1st day 2nd day 3rd day 4th day, over 4-day,
period
ence S.Jj.M.D, cu
lated
ret
ical"
Ho.
Expts.
mg.
Ho.
Expts.
mg. Ho.
Expts.
Ho.
Expts.
mg. mg. mg.
Controls 11 105.4
±5.7
11 106.1
±5.4
9 108.9
±4.9
1 98.2 106.5
±2.9
— — .
Sorbitol 10 87.0
*6.4
9 73.3
±3.8
7 72.8
±4.0
2 55.7
±2.8
76.9
±3.3
29.6 6.53 6.53 2.58
Maanitol 12 99.9
±8.1
12 91.7
±4.9
8 90.9
±7.7
1 73.7 93.9
±3.9
12.6 2.57 2.51 2.58
Dulcitol 8 88.7
±7.4
8 89.4
±8.4
7 96.1
±7.9
2 82.4
±5.9
90.5
±4.1
16.0 3.19 3.14 2.58
Glucose 8 55.8
±7.5
8 47.8
±5.9
6 37.5
±7.3
0
---
47.9
± 4.3
58.6 11.32 11.20 2.58
»
♦With £»0.01
24
EXPERIMENTS ON GLYCOGEN FORMATION
Determinations were made of the amount of glycogen
deposited in the liver and the gastrocnemius muscle of rats
fasted 48 hours and-receiving intraperitoneal injections of
the hexitols. Groups of rats used in these experiments were
either all male or all female, since it was thought best
not to use mixed groups, as Deuel, Samuels, and Gulick
(1932-33) reported that there is a sex difference in the
glycogen content of the livers of fasting animals.
The concentration of the materials administered was
determined by preliminary experiments. Different concentra
tions were injected and different time intervals allowed to
elapse before determination of the liver glycogen. The
results of these experiments are listed in Table VII. From
these results, the optimum dose was found to be a 25 per cent
solution, administered in two doses three hours apart with a
6 hour time interval between the first dose and the removal
of the liver. Dosages were based on surface area here as
in the ketosis work for the reasons previously given.
TABLE VII
The Liver Glycogen in Pasting Male Rats
after the Tntraperitoneal Injection of
Different Concentrations of Sorbitol
at Different Time Intervals
Expt.
No.
Rat
No.
Body
Wt.
Surface
Area
Cone, of
Solution
Adm.
Doses
Given
Time
interval
after
1st dose
Liver
Wt. Glyco
gen
gms. sq.cm. per cent No. hours gms. per cent
3 600. 130 233 25 1 3 5.21 1.80
4 648 190 292 25 1 3' 5.88 1.34
5 . 610 165 268 50 1 3 6.58 2.12
6 601 165 268 50 1 3 4.91 1.88
7 669 175 278 25 1 4 5.79 2.21
8 620 180 283 25 2 6 5.31 3.70
9 695 170 273 50 1 ■ 6 5.80 2.72
26
After the preliminary experiments, one cc. per 100
sq. cm. of rat of a 25 per cent solution of sorbitol and
mannitol was injected intraperitoneally into two different
groups of animals. As a comparison with' the effect produced
by glucose, a similar concentration of this material was
injected into another group of fas'.ting rats. The control
animals received one cc. of physiological saline per 100
sq. cm.
The injections were started in the morning and
spaced 10 minutes apart for the different animals. Six
hours later, one cc. of a one per cent arnytal solution per
100, grams rat was1 injected into the peritoneal cavity.
After several minutes, the animals were completely anesthe
tized. The muscle was exposed without stimulation, frozen
in situ in solid carbon dioxide-ether mixture; the liver
was then removed followed by the frozen muscle. The
removed parts were quickly placed in the freezing mixture,
to prevent glycogenesis. After quickly weighing the dried
frozen tissue, potassium hydroxide was added to it and the
mixture digested in a water bath. After digestion, the
glycogen was precipitated by alcohol, centrifuged, reprecipi
tated and recentrifuged. For the determination of glycogen
the method of Good, Kramer and Somogyi (1933) was followed.
After the glycogen was hydrolyzed with 0.6 N.HC1 for 3 hours,
the glucose was determined by the Shaffer-Hartman method.
27
EXPERIMENTAL RESULTS ON THE GLYCOGEN-FORMING
' ABILITY OP SORBITOL, MANNITOL, AND DULOITOL
Tables VIII and IX give the results of the intra-
peri tone al injections of one cc. per 10Q sq. cm. of surface
area of a 25 per cent solution of sorbitol and mannitol on
the liver and muscle glycogen of male rats after a fasting
period of 48 hours. Table X gives the values obtained when
the same amount of glucose is administered by the same route
while Table XI shows the amount of glycogen that has been
retained by the fasting, control animals.
Another set of experiments was carried out on female
rats receiving 0.5 cc. per 100 sq. cm. surface area instead
of one cc. as previously given. The concentrations of the
solutions were the same, however. The results of this work
are given in Tables XII through XV.
As it was impossible to obtain more than a 9 per cent
aqueous solution of dulcitol because of its relative insolu
bility, two groups of animals were given one cc. per 100
■sq. cm. surface area of 9 per cent solutions of dulcitol
and glucose, respectively. A control group was also ruin
which received NaCl. The results are found in Tables XVI
through XVIII.
Summaries of the comparison of liver and muscle
28
glycogen of rats receiving intraperltoneal Injections of the
above mentioned dosages and'concentrations of the different
hexitols with those receiving like amounts of glucose are
given in Tables XIX and XX,
The only statistical treatment used was the Fisher jb
as the number of experiments was too small to apply the
ratio of the difference in the means to the standard error
of the mean difference.
TABLE VIII
The Liver and Muscle Glycogen in Pasting Male Rats
6 Hours after the Intrap'eritoneal Injection of
25 per cent Solution of Sorbitol,
1 cc. per 100 sq. cm.
Expt. Rat Body ‘ Surface Liver Muscle
No. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
gms. sq. cm. gms. per cent gms. per cent
8 1120 180 283 5-. 31 3.70
---- ----
15 1051 225 322 6.50 - 4.37
---- ----------------
16 1046 120 221 4.27 3.51
---- ----
17 1067 110 210 4.56 5.47
---- ----
18 1057 110 210 4.57 3.86
---- ----------------
19 1157 160 264 4.96 3.38
--------------- _ _ _ _
14170 8678 140 243 • 6.13 3.14
--------------- ----------------
171 8687 141 244 5.51 3.86 0.92 0.37
172 8696 165 268 7.21 1.87 1.04 0.29
173 8700 134 237 5.28 3.40
--------------- — - —
174 8709 185 288 6.43 3.79
---------------- ----------------
175 8718 137 240 5.69 4.07
---------------- ----------------
176 8727 153 256 6.47 4.21 0.89 0.39
177 8740 137 ' 240 5.78 4.51
--------------- ----------------
178 8736 141 244 4.89 3.05 0.69 0.32
Average 149 250 5.57 3.75 0.88 0.34
±0.20 ±0.02
TABLE IX
The Liver and Muscle Glycogen in Pasting Male Rats
6 Hours after the Intraperitoneal Injection of
25 per cent Solution of Mannitol,
1 cc. per 100 sq. cm.
Bxpt. Rat Body Surface Liver Muscle
. Ho. ' • No... Wt. Area Wt. Glyco
gen
. Wt. Glyco
gen
gms. sq. cm. gms. per cent gms. per cent
20 1050 235 332 5.30 1.39
---- ---- _
21 897 210 310 4.82 0.42
---- ----
22 769 115 216 3.98 0.31
---- ----
23 1060 113 214 4.66 0.67
----
24 1148 120 220 4.12 0.40
---- ----
25 1061 170 273 3.99 0.08
---- ----
14179 8679 140 243 4.56 0.44
---- ----
180 8688 137 240 4.69 0.23 0.82 0.20
181 8697 160 264 6.82 0.71 0.78 0.26
182 8706 128 230. 4.33 0.33 0.76 0.30
183 8710 133 236 5.08 0.38 1.03 0.26
184 8719 168 271 5.51 0.15 1.01 0.18
185 8728 142 245 5.14 0.26 0.82 0.28
186 8737 141 244 5.44 0.85
Average 151 252 4.88 ,0.47 0.87 0.25
tO.09 lO.02
TABLE X
The Liver and Muscle Glycogen in Fasting Male Rats
6 Hours after the Intraperitoneal Injection of
a 25 per cent Solution of Glucose,
,1 cc. per 100. sq. cm.
Expt. Rat Body Surface Liver Muscle
No. 'No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
gms. sq. cm. . gms. per cent gms. per cent
30 1151 225 323 6.45 1.64
---- ----
31 1176 100 199 3.58 3.07
---- ----
32 1149 235 332 6.52 0.98
---- ----
33 749 120 220 4.41 3.45
---- ----
.34 768 120 220 4.80 3.69
---- ----
35 1164 190 292 6.07 3.32
---- ----
L4161 8677 157 260 6.51 2.83 0.69 0.63
162 8686 150 254 6.17 4.03
---- ----
163 8690 145 248 6.88 3.67
---- ----
164 8699 130 233 5 . 60 3.06 0.68 0.68
165 8708 153 256 5.72 3.09 0.94 0.59
166 8717 137 240 6.20^ 4. 37 . 1.00 0.44
167 8726 138 241 5.57 3.87
---- ----
168 8730 187 289 7.85 3. 50 1.50 0.60
169 8739 160 264 5.83 3.11
---- ----
Average 154 255 5.90 3.18
±0.20
0.96 0.59
+ 0 * 04
TABLE XI
The Liver and Muscle Glycogen In Fasting Male Rats
6 Hours after the Intraperitoneal Injection of
a Sodium Chloride Solution,
1 cc. per 100 sq. cm.
Expt:. Rat Body Surface Liver Muscle
NO. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
gms. sq. cm. gms. per cent gms. per cent
1 1087 168 271 4.50 0.14
----
2 1091 150 254 4.52 0.05
---- ----
11 1096 190 292 4.34. 0.00
----
12 1170 100 199 3.74 0.53
---- ----
13 1158 110 210 '4.03 0.12
---- ----
14 1150 120 222 3.90 0.39
----
L4152 8676 135 238 5.10 0.26 0.50 0.25
153 8680 167 270 7.25 0.23
--- ----
154 8689 150 254 6.01 0.12
---- .----
155 8698 151 254 5.48 0.12 1.26 0.13
156 8707 162 266 4.83 0.17 ----- — —
157 8716 193 295 7.30 0.27 0.61 0.36
158 8720 135 238 5.15 0.16
---- ----
159 8729 129 232 5.27 0.20 0.81 0.24
160 8738 135 238 5.35 0.36 0.92 0.34
Average 146 -249 5.12 0.21
±0.03
0.82 0.26
±0.04
TABLE XII
The Liver and Muscle Glycogen in Pasting Female Rats
6 Hours after the Intraperitoneal Injection of
a 25 per cent Solution of Sorbitol
0.5 cc. per 100 sq.' cm.
Expt. Rat Body Surface Liver Muscle
No. No. Wt. Area. Wt. Glyco
gen
Wt. Glyco
gen
gms. sq. cm. gms. per cent gms. per cent:
.4122 10365 84 179 3.60 2.56
---- ----
123 10264 113 214 4.76 2.55 0.80 0.24
124 10272 118 220 5.07 2.53 0.91 0.36
125 . ■10311 132 235 5.70 2.27 0.80 0.28
126 " 10315 126 .228 4.40 2.19 0.59 0.44
127 10323 97 195 4.80 2.07 0.58 0.32
128 10333 114 215 4.58 1.61 0.72 0.27
129 10342 107 207 4.56 2.96 0.60 0.32
130 10351 111 .212 5.02 3.02 0.78 0.25
131 10355 117 218 4.33 1.78 • 0.80 0.26
Average 112 212 4.68 2.35
±0.14
0.76 0.30
±0.01
TABLE XIII
The Liver and Muscle Glycogen in Pasting Female Rats
6 Hours after the Intraperitoneal Injection of
a 25 per cent Solution of Mannitol
9
0.5 cc. per 100 sq. cmi .
Expt. Rat Body Surf ace Liver Muscle
No. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
. Sms*.
sq. cm. gms. p■er cent gms. per cent
L4132 10364 93 . 190 3.74 0.00 0.51 0.26
133 10263 102 201 4.11 0.46 0.78 0.54
134 10271 135 238 4.67 0.09 0.77 0. 10
135 10275 132 235 ■ 4.82 . 0.11
-- ----
136 10314 •90.. 186 4.12 0.17 0.60 0.20
137 10321 103 202 3.93 0.23
---- ----
138 10332 94 192 3.55 0.00 0.54 0.29
139 10341 105 205 3.88 0.00
-- --
140 10345 101 200 3.70 0.09
-- --
141 10354 100 199 4.06 0.16
---- ----
Average 106 205 4.06 0.13
±0.05
0.64 0.28
±0.07
TABLE XIV
The Liver and Muscle Glycogen in Fasting Female Rats
6 Hours after .the Intraperitoneal Injection of
a 25 per cent Solution of Glucose,
0.5 cc. per 100 sq. cm.
Expt. - Rat Body Surface Liver Muscle
No. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen.
gms. sq. cm. gms. per cent gms. per cent
.4112 10261 109 209 4. 65 2.53 0.87 0.28
113 10265 119 221 4.78
----
0.93 0.33
114 10273 136 239 5.48 2.25
---- ----
115 10312 118 • 220 5.16 2.03 0.72 0.36
116 10321 107 207 •4.90 1.36 0.62 0.40
117 10325 96 194 4.00 1.22 ' 0.42 0.46
118 10334 107 207 4.21 1.47 0.70 0.40
119 10343 116 217 4.41 2.39 0.66 0.28
120 10352 107 207 4.30 2.24 0.66 0.34
121 10361 97 195 3.77 2.23 0.58 0.47
Average 111 212 4.57 1.97
±0.15
0.71 0.57
±0.02
TABLE XV
The Liver and Muscle Glycogen in Fasting Female Rats
6 Hours after the Intraperitoneal Injection of
a Sodium Chloride Solution,
0.5 cc. per 100 sq. cm.
Expt. Rat Body Surface Liver Mus cle
Ho. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
gms. sq. cm. gms. per cent gms. per cent
.4142. 10563 ■ 89 185 3.47 0.53
---- ----
145 10262 107 207 4.67 0.00 0.65 0.21
144 10274 130 233 4.90 0.00 0.73 0.24
145 10313 130 233 4.77 0.05 0.83 0.20
146 ' 10325 108 208
■---- ---- _ _ _ _ ----
147 10332 96 194 3.53 ' 0.00 0.53 0.22
148 10335 111 212 4.50 0.00
---- ----
149 10544 108 208 4.38 0.12 0.76 0.09
150 10353 114 215 4.31 .0.00 0.76 0.21
151 10363 89 185 3.20, 0.00 0.70 0.33
Average 108 208 4.19' ' ' 0.08• ■ ■
'±0.06
0.71 0.21
±0.03
TABLE XVI
The Liver and Muscle Glycogen in Pasting Male Rats.-.
6 Hours after the Intraperitoneal Injection of
a 9 per cent Solution of Dulcitol,
1 cc. per 100 sq. cm.
Expt. Rat Body Surface. Liver Muscle
No. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
m s-
sq. cm. gms. per cent gms. p er c ent
L4217 8680 223 322 7.68 0.43
----- ----
218 8337 281 370 • 8.67 0.68 . 1.65 0.26
219 8349 223 322 7.27 0.26
---- ----
220 1084 243 339 6.38 0.09 1.26 0.23
221 1119 144 247 5.00 0.39 0.90 0.29,
222 8356 291 377 7.84 0.46
---- ----
223 8340 254 348 7.26 0.30 . 1.50 0.28
' 224 1093 . 210 310 5.75 0.10 1.22 0.22
225 1138 150 254 4.88 0.30 1.01 0.14
226 8360 216 316 6.25 0.00 1.35 0.16
Average 224 321 6.70 0.30
±0.06
1.27 0.23
±0.02
TABLE XVII
The Liver and Muscle Glycogen in Pasting Male Rats
6 Hours after the intraperitoneal Injection of
a 9 per cent Solution of Glucose
• - 1 cc. per.100 sq. cm. .
Exp t. Rat Body Surface Liver Muscle
No. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
gms. sq.'cm. gms. per cent gms. per cent
14207 8333 244 339 7.46 1.66 1.55 0.43
208 8336 273 263 9.00 1.29 1.66 0.33
209 8346 207 308 6.57 1.73 1.00 0.33
210 8047 237 334 7.21 1.74 1.37 0.38
211 1115 148 251 5.82 2.41
---- ----
212 8356 233 330 6.60 1.28 1.31 0.36
213 8338 225 313 6.32 1.28 1.35 0.31 -
214' 8348 231 329 7.65 .1.59 1.53 0.26
215 1168 202 303 7.64 0.94 . 1.33 0.35;.
216 8357 368 434 10.57 1.38
Average 227 320 7.48 1.53
±0.12
1.38 0.34
±0.02
TABLE XVIII
The Liver and Muscle Glycogen in Pasting Male Rats
6 Hours alter the Intraperitoneal Injection of
a Sodium Chloride-Solution,
1 cc. per 100 sq. cm.
Expt. Rat Body Surface Liver Muscle
No. No. Wt. Area Wt. Glyco
gen
Wt. Glyco
gen
gms. sq. cm.
£2S *_
per cent gms. per cent
.4227
----
227 325 6.93 0.49 1.63 0.18
228 8339 263 355 8.04 0.20 1.11 0.29
229 8350 222 321 6. 61 0.00 1.35 0.22
230 1057 264 356 8.56 0.14
---- .
231 1135 134 237 4.85 0.40 ■
Average 222 319 6.99 0.22 1.36 0.23
±0.08 ±0.03
TABLE XIX
Summary, Table Giving a Comparison of Liver Glycogen of Pasting Hats
6 Hours after the Intraperitoneal Injection of the Hexitols
Material
Injected
Concen
tration
Amt. per
100 sq.cm.
Ho.
Expts.
Sex Liver
Glycogen Difference
t
Calculated
t
Theoretical*
per cent cc. per cent per cent
Sodium chloride 0.9 1.0 15 Male 0.21 * 0.03
----
Sorbitol 25 1.0 15 Male 3.75 ± 0.20 3.54
0.57**
16.40
1.96**
2.76
2.76**
Mannitol 25 1.0 14 Male 0.47 ± 0.09 0.26 2.70 2.77
Glucose 25 1.0 15 Male 3.18 ± 0.20 2.97 12.98 2.76
Sodium chloride 0.9 0.5 9 Female 0.08 4 0.06
----
— —
Sorbitol 25 0.5 10 Female 2.35 k 0.01 2.27
0.38**
13.88-
1.77**
2.90
2.90**
Mannitol 25 0.5 10 Female 0.13 k 0.05 0.05 0.61 2.90
Glucose 25 0.5 9 Female 1.97 * 0.15 1.89 11.15 2.92
Sodium chloride 0.9 1.0 5 Male 0.22 ± 0.08
----
Dulcitol 9 1.0 10 Male
0.30 k 0.06 0.08 • 0.73 3.01
Glucose
*9 1.0 10 Male 1.53 * 0.12 1.31 6.75 3.01
♦With p * 0.01
♦♦Compared with glucose. Other comparisons are with sodium chloride controls.
TA3LE XX
Summary Table Giving a Comparison of Muscle Glycogen of Fasting Hats
6 Hours after the Intraperitoneal Injection of the Hexitols
Material
Injected
Concen
tration
Amt. per Ho.
100 sq. cm. Expts. Sex
Muscle
Glycogen Difference
t
Calculated
1
Theoretical*
per cent cc. per cent per cent
Sodium chloride 0.9 1.0 5 Male 0.26 i 0.04
Sorbitol 25 1.0 4 Male 0.34 ± 0.02 0.08 1.54 3.50
Mannitol 25 1.0 6 Male 0.25 i 0.02
_ ---
Glucose 25 1.0 5 Male 0.59 ± 0.04 0.33
0.25***
5.73
5.05***
3.36
3.50***
Sodium chloride 0.9 0.5 7 Female 0.21 * 0.03
--- ---
Sorbitol 25 0.5 9 Female 0.30,i 0.01 0.09 3.53 2.98
Mannitol 25 0.5 5 Female 0.28 i 0.07 0.07 1.02 3.17
Glucose 25 .0.5 9 Female 0.37 i 0.02 0.16
0.07***
6.12
2.90***
2.80
2.92***
Sodium chloride 0.9 1.0 3 Male 0.23 £ 0.03 — —
---
Dulcitol 9 1.0 7 Male 0.23 ± 0.02
___ ---
Glucose 9 ' 1.0 8 Male 0.34 ± 0.02 0.11 3.17 3.25
•With £ = 0,01
••Values too low to compare
•••Compared with sorbitol.
with control.
Other comparisons are with sodium chloride controls.
CHAPTER IV
DISCUSSION
If a substance can be demonstrated to relieve ketosis
in an animal and to cause an increase in live? arid muscle
glycogen, then the particular substance must have been
absorbed from the intestine, gone into the blood stream, and
entered into the metabolism of the animal. Since it has
been demonstrated by many workers that only carbohydrates
or carbohydrate-producing substances are capable of cutting
down the ketone-body excretion of an animal, It seems fairly
certain that any substance which acts in a ketolytic capacity
is convertible to glucose in the animal organism. If the
material under study Is shown to cause an increase in liver
and muscle glycogen in a fasting animal, It is a confirmation
that the substance in question has been converted to glucose,
which in turn has been changed to glycogen.
In the experiments, that were carried out on fasting
female rats receiving sodium butyrate alone, a high degree
of ketosis was obtained. - This was maintained throughout
the experimental period with' an average daily excretion of
106.5 + 2.9 mg. total acetone bodies per 100 sq. cm.
Acetonuria of the animals receiving the butyrate and glucose
was greatly reduced as was expected owing to the ketolytic
43
effect of the glucose. The average dally excretion was
47.9 ± 4.3 mg. per 100 sq. cm.
While the Retone-body output of the animals receiving
sorbitol was greater than that of those receiving equimolec-
ular amounts of glucose, it was, none the less, much lower
than that of the controls, viz., 76.9 * 3.29 mg. per 100
sq. cm. Mannitol, on the contrary, was not found to produce
the ketolytic effect that sorbitol did, as the average daily
excretion of ketone bodies was 93.9 i 3.94 v/hen mannitol
was fed. While this value is lower than that of the controls
by 12.6 mg. per 100 sq. cm., it is not statistically
'significant, as the ratio of the difference in the means
of the controls and the animals receiving mannitol, to the
standard error of the mean difference is 8.57. Neither
is it significant if the Fisher _t is applied when the jo
value is 0.01 (one chance in 100 of the difference being
due to experimental error), as the t value found is 2.51
while the theoretical t value is 2.58. If, however, a j> ■
value of 0.02 is used (one chance in 50 of the difference
being due to experimental error), mannitol is significantly
ketolytic as the t - value Is 2.35. While dulcitol was like
wise found to be inferior to sorbitol in relieving acetonuria,
it appears to be a slightly better ketolytic substance than
mannitol. The average acetonuria over the 4-day period is
44
90.5 £ 4.1 mg. per 100 sq. cm. which is statistically
significant. The ratio of the mean difference to the
standard error of the mean difference is 3.19 and the _t
value calculated is 3.14 while the theoretical t value of
Fisher for a jc value of 0.01 is 2.58. ■
The experimental results of the effect of the
hexitols on the formation of liver and muscle glycogen
after intraperitoneal administration are much more striking
with respect to sorbitol. The liver glycogen in the fasting,
control male animals was found to be 0.21 ± 0.03 per cent.
The value obtained for glucose is 3.18 t 0.20 per cent 6
hours after the injection of one cc. per 100 sq. cm. of a
25 per cent solution. Glucose, of course, is highly
significant in causing glycogenesis. The calculated t
value is 12.98, while the theoretical _t value for a p
value of 0.01 is 2.76. After the administration of a like
amount of sorbitol, the liver glycogen values obtained are
even higher than those for glucose, being 3.75 t 0.20. In
this case the calculated t value is 16.40 while the p value
is the same as for that of glucose. When 0.5 cc. per 1QQ
sq. cm.’ of a 25 per cent solution of glucose Is given, the
liver glycogen Is 1.97 + ■ 0.15 per cent with a calculated
t value of 11.15 while the theoretical t_ value is 2.92.
When the same amount of sorbitol is.given, the liver
45
glycogen Is again greater than that of glucose, viz.,
2.35 ± 0.01. Here the calculated _t value is 13.88 while
the theoretical t value is 2.90. Although the values found
following sorhitol are higher than those obtained when
glucose was given the difference is not quite significant
if the Fisher _t is applied and a jo value of 0.G1 used. But
the slight superiority of sorbitol over glucose as a glyco
genic substance does not hold true for the percentage of
muscle glycogen found after administration of the above
amounts of the two substances. In this case it was found
that the muscle glycogen formation in the fasting animal
receiving sorbitol Is not statistically different from that
of the controls, while glucose is highly glycogenic In
the muscle when one cc. per 100 sq. cm. of a 25 per cent
solution is administered. If, however, doses of 0.5 cc.
per 100 sq. cm. are given fasting females, the muscle glyco
gen following sorbitol is statistically significant when
compared with the controls. In this case, the calculated
j; value Is 3.53 with a theoretical _t value of 2.98. For
the same amounts of' glucose, the calculated t . value is
6.12 with a theoretical t value of 2.80. The muscle glycogen
for glucose administered animals Is statistically higher than
for those receiving sorbitol when the t formula of Fisher is
applied.
46
Thus it would appear that in the doses given follow
ing a 6-hour time interval between the first dose and the
glycogen determinations, more liver glycogen is present
when sorbitol is given than when glucose is given. There
is, however, more muscle.glycogen following glucose than
following sorbitol. Prom this it would appear that the liver
glycogen is more rapidly formed in the case of glucose with
the result that after a 6-hour period the liver glycogen
has undergone a certain amount of glycogenolysis resulting
in an increased amount in the muscle.
Although there was some increase in the liver glycogen
following mannitol and dulcitol, the increases were not
significant statistically. An increase in muscle glycogen
is found in fasting female rats given 0.5 cc. per 100 sq. cm.
of a 25 per cent solution of mannitol.
It is difficult to compare the results on glycogen
formation of dulcitol with those of the other two hexitols,
as the concentration used was so much less, owing to its
insolubility. It did appear to have a positive effect on
relieving ketonuria, however. As dulcitol is the hexitol
that is converted to galactose on oxidation, it is interesting
to compare the metabolism of these two, substances. In
contrast to the limited ability of dulcitol to relieve
ketosis, Deuel, Gulick, and Butts (1932) found that on
human subjects galactose exhibited a superior ketolytic
'action.to that of glucose. Clark and Mur1in (1936) demon
strated that galactose has a greater ketolytic ability than
glucose on ketosis produced by prolonged high-fat .diet and
injection of anterior pituitary extract. Butts (1934)
found that galactose exhibits a superiority to glucose in
ketolytic action, in rats exhibiting ketonuria produced by
the administration'of sodium acetoacetate. The glycogenic
superiority of galactose over dulcitol is likewise an
argument against the ready convertibility of dulcitol to
galactose. Deuel, MacKay, Jewel, Gulick, and Grunewald'(1933)
showed that the glycogen retention in the livers of rats
fasted for intervals up, to 72 hours was actually greater if
the previous diet had been high in galactose than if glucose
had been the predominating dietary sugar. Higher values in
liver glycogen also were found in dogs when galactose wa.s
given than when glucose was fed. Thus the metabolism of
these two chemically similar compounds does not appear to be
related in the animal body.
The relative inability of mannitol to relieve ketosis
would not seem caused by its failure to be absorbed from the
intestine alone, as it was likewise unable to increase
48
glycogenesis to any appreciable extent following intra-
■ peritoneal.injection. In some respects mannitol appears to
behave much like mannose. In studies on this monosaccharide,
Deuel, Hallman, Murray, and Hilliard found that mannose was
absorbed at a rate of'12.3 compared with glucose as 100, 'It
was also able to form small amounts of glycogen. Even when
glucose and mannose were absorbed at the same rate, glucose
■was definitely a better glycogen former. When mannose was
fed to fasting rats, having an endogenous ketonuria produced
by a previous high-fat diet, the ketone body excretion was
only-slightly lowered, while glucose depressed it to about
one-third its original level. From this it would appear
that mannitol and,mannose behave similarly when taken into
the animal body.
In general a parallelism between the ability to reduce,
ketonuria and the ability to be deposited as liver glycogen
has been observed in earlier experiments from this laboratory
when various metabolites were fed. In the present tests,
greater amounts of glycogen in the liver follow the administra
tion of sorbitol while the ketolytic effect is decidedly less
than after glucose. There are two possible explanations for
this discrepancy. In the first place the rise in muscle
glycogen is much slower with sorbitol than with glucose. If
the disappearance in butyric acid is primarily related to the
49
level of glucose precursors In the muscle, then this would
answer the discrepancy. One must then conclude that a
ketolytic effect is related to the glycogenic action of an
intermediate in the muscle rather than in .the liver. In
general the increases in muscle and liver glycogen are parallel
but this apparently does not always occur,- as in. the case of
sorbitol.
A second possible explanation is that the rate of-
absorption of glucose Is much greater than that of sorbitol.
In the exogenous type of ketonuria, the butyric acid may be
completely metabolized before sorbitol is absorbed; on the
other hand glucose will already have been completely utilized.
The best method to obviate the difficulties in the discrepancies
in absorption rate is to compare the ketolytic activity of these
substances in an endogenous, ketonuria when a constant production
of ketone bodies -Is taking place. Such experiments are now in
progress.
Prom all of these results, it might be concluded that
of the three hexltols studied, sorbitol is more completely
metabolized than are mannitol and dulcitol. If sorbitol is
so readily convertible to glycogen following an intraperitoneal
injection, but is not as ketolytic as glucose following oral
administration, It.would seem that'sorbitol Is not as readily
absorbed from the intestine as is glucose. These results may
50
be explained by postulating an enzymatic change ’ taking place
in the liver. The. liver may contain'an oxidase which is
capable of oxidizing’sorbitol to glucose. It is also
possible that sorbitol is oxidized in the liver to fructose.
The work of Waters (1938) demonstrating the similar effects
of sorbitol and fructose on the glucose tolerance curve, .
and the early perfusion work of Embden and Greisbach (1913)
where a mixture of glucose and fructose is shown to exist
after perfusion of sorbitol through a phlorhizinized dog
liver would make It seem highly probable 'that sorbitol Is
converted Into fructose in the liver.
CHAPTER V
SUMMARY
Exogenous ketonuria In fasting male rats Is appreciably
decreased following 'the oral administration of sorbitol.
Dulcitol was found to relieve ketosis but to a lesser degree
•than sorbitol. Mannitol, while causing a somewhat lower
level of ketosis, was not statistically significant as a
ketolytic agent.
Six hours following the intraperitoneal injection of
a 25 per cent sorbitol solution, the values for liver
glycogen were greater following the administration of
sorbitol than for like amounts of glucose in both fasting
male and female rats. The muscle glycogen, on the contrary,
was higher following glucose. Although-mannitol caused a
slight increase in liver glycogen, and, in some cases, of
muscle glycogen, the difference was not great enough to be
significant. In the concentrations administered, dulcitol
failed to show glycogenic activity.
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Asset Metadata

Creator Johnston, Cornelia Hendrick (author)

Core Title The metabolism of the hexitols

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Degree Program Biochemistry

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-775222

Unique identifier UC11347776

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Legacy Identifier EP41275.pdf

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Rights Johnston, C. H.

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...

Repository Name University of Southern California Digital Library

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