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A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

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A STUDY OF THE EFFECT OF GLYCOGEN
ON THE OXIDATION OF BUTYRATE BY RAT LIVER SLICES
A Dissertation
Presented to
the Faculty of the Department of Biochemistry
University of Southern Califomla
School of Medicine
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
by
Blanche Gauthier Bobbitt
May 1941
UMI Number: DP21531
All rights reserved
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UMI DP21531
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
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This dissertation, w ritten by
under the guidance of h.BT. F a c u lty Com m ittee
on Studies, and app ro ved by a l l its members, has
been presented to and accepted by the C o uncil
on G raduate Study and Research, in p a r t ia l f u l fillm e n t of requirements f o r the degree of
D O C T O R -O F F H IL O S O P H Y
Secretary
D a te
.J.uae_..124l.......
Com m ittee on Studies
#j
fV Z ^
)
j
« /
V
TABLE OF CONTENTS
FOREWORD.........................................
1
HISTORICAL DISCUSSION
...........................
4
EXPERIMENTAL P A R T ...............................
15
Experimental animals
. . .................
. .
Procedures...............................
15
..........
Warburg manometrlc method
15
15
Calibration of apparatus
16
S o l u t i o n s ...............
18
Preparation of tissue slices
........
19
Respiration e x p e r i m e nt s ....................
23
Micro-determination of acetone bodies
Solutions
Determination of acetoacetlc acid
. . . . .
26
. . . . . . .
26
. . . . . .
26
Determination of beta-hydroxybutyric acid
• •
Determination of residual butyric acid . . . . .
29
. .. ..
34
Effect of f a s t i n g .............. ... .........
34
Effect of
glycogen
. . . . . . . . . . . . . . .
36
Effect of
butyrate
plus glycogen...........
38
Effect of
butyrate
plus glycogenontotal
Results
. . . . . . . . . .
ketone production
..........
28
..........................
46
Quantitative studies on the changes in butyrate
with and without g l y c o g e n ...............
•
42
ill
DISCUSSION............, .........................
47
C O N C L U S I O N S ...............
55
BIBLIOGBAFHY
56
...................................
APPENDIX.....................
60
FOREWORD
Ketosis, or the accumulation of ketone bodies, has
been observed to occur In cases of faulty fat metabolism
associated with Insufficient carbohydrate of either exogenous
or endogenous, source or with impaired ability to oxidize
glucose.
The ketone bodies, namely acetoacetlc acid,
beta-hydroxybutyric acid, and acetone are incomplete
oxidation products of fatty acids.
Their existence was
established in studies on diabetic urine during the last
century by Peters, Gerhardt, Kulz, and Stadelmann.
Knoop
(1904-05) showed that the even-chained fatly acids were partly
responsible for the formation of ketone bodies and later
determined that certain amino acids are also ketogenic.
The close correlation between fat and carbohydrate metabolism
is further evident upon the administration of insulin to
diabetics, which results in both improved carbohydrate
utilization and cessation of ketosds.
Since the oxidation of carbohydrate prevents ketosis,
the mechanism of this reaction has been widely investigated.
Two schools of thought have developed! the one proposing
antlketogenesls, the other, ketolysis.
The proponents of
antiketogenesis believe that ketosis is prevented when
carbohydrate Is metabolized because carbohydrate exerts a
fat-sparing action, hence la metabolized in preference to fat*
Under such conditions, ketogenesis, i*e., the formation of
ketone bodies, is suppressed*
Adherents to the theory of
ketolysis affirm that carbohydrate prevents ketosis by combining
with the ketone bodies to form compounds which are then easily
oxidized*
‘
J?he experimental work of Voit (1891), Geelmuyden (1904),
and Magnus-Levy (1908) have been of great value in the develop­
ment of this controversy*
Researches of Zeller (1914), Woodyatt
(1921) and Shaffer (1922) on quantitative aspects of ketogenicketolytic ratios have been of great practical importance*
Evidence accumulated by Embden and Oppenhelmer (1912, 1913)
and by Mir sky and Broh-Kahn (1937) has been interpreted as
upholding the antlketogenesis theory*
On the other hand, the
results of Shaffer (1921), Beuel, Gulick, and Butts (1932),
Deuel, Hallman, and Murray (1938), Shapiro (1935), Butts, Dunn,
and Hallman (1935-36), and Butts, Blunden, and Dunn (1937) (1,2)
have contributed to the evidence in favor of ketolysis.
Since the even-chained fatty acids may be ketogenic in
the absence of carbohydrate, the four carbon acid, butyric, is
convenient to use in ketosis studies*
Heretofore, no quantita­
tive study has been reported on ketone body formation from
butyric acid with a simultaneous determination of residual
butyric acid in the presence and absence of carbohydrate*
3
Since the liver is the chief site of formation of ketone
bodies (Himwich 1931, Chaikoff 1928-29, Mirsky 1936,
Greenberg 1936), liver tissue is very satisfactory to use
in studying ketosis produced by the addition of butyrate
in the absence and in the presence of added glycogen
(Quastel and Wheatley, 1933, 1934).
It is the purpose of this dissertation to study such
a problem employing the Warburg (1926) technique with liver
slices.
Not only have some of the experiments of Quastel
and Wheatley (1933, 1934, 1935) been repeated using liver
slices with butyrate in the presence and absence of
glycogen, but also the ketone bodies formed have been
separated according to the micro-acetone determination of
Edson (1935) employing Rupp*s titration (1906, 1907), and
then the residual butyric acid has been finally determined,
thereby giving information as to whether more butyric acid
is oxidized when carbohydrate is present.
HISTORICAL DISCUSSION
If fats are oxidized when carbohydrate metabolism is
normal, the end products are carbon dioxide and water.
But
if the carbohydrate metabolism is disturbed, as in diabetes
or even in fasting, the fats are oxidized partially to the
acetone bodies.
The presence of the ketone bodies in diabetic
urine was observed in the 19th century,
Peters reported the
presence of acetone in 1857; Gerhardt later discovered acetoacetic acid (1865); and finally Stadelmann (1883) and Kulz
(1884) identified beta-hydroxybutyric acid,
Opie (1900-1901)
confirmed the fact that acetone bodies are present in diabetic
urine.
Hirschfield (1895) noted that ketosis occurs in carbo­
hydrate starvation and disappears upon the administration of
this foodstuff.
The fundamental work of Magnus-Levy (1908)
demonstrated that when beta-oxybutyric acid was fed to
phlorhizinlzed dogs, it was excreted in the urine; Neubauer
(1910) also found when the ketone bodies were administered to
diabetics, that an excretion of these substances occurred in
the urine.
Knoop (1904-05) proved that the precursors of the
acetone bodies are the even-chained fatty acids according to
experiments in which he fed the phenyl derivatives of the lower
fatty acids.
From these observations he advanced his famous
theory of beta oxidation.
work published in 1909.
Dakin supported Knoop*s theory in
Liver perfusion experiments of Embden
5
and Marx (1908) were also in accord with the theory of beta
oxidation.
That beta oxidation Is not the only mechanism of fatty
acid breakdown, however, became evident from various studies
made by Olutterbuck, Verkade, Deuel, Quastel, and others*
Clutterbuck and Raper (1925) reported gamma and delta oxidation
occurred on oxidizing the longer chain fatty acids by hydrogen
peroxide.
In 1935 Butts, Cutler, Hallman, and Deuel added
evidence in support of .both beta and delta oxidation from
feeding experiments using the sodium salts of the fatty acids
which, when fed to fasting rats, caused the excretion of twice
the amount of acetone bodies when caprylic acid was fed than
when butyric, caproic, or acetoacetic was administered.
Morehouse and Deuel (1940) further substantiated delta oxidation
in results obtained by feeding the alpha-beta and the betagamma dideuterocaproic acids.
Jowett and Quastel (1935), as a result of studies with
liver slices, postulated the theory of multiple alternate
oxidation.
Deuel and coworkers (1936) demonstrated that a
ketonuria of greater magnitude obtained after feeding the
ethyl esters of caprylic or caproic acids than after
isomolecular amounts of caproic or butyric acids.
Also a
greater ketonuria obtained after the administration of
lauric than after caprylic acids.
These data were interpreted
to support the theory of multiple alternate oxidation.
6
MacKay, Barnes, Carne, and Wick (1940) believe that
the increased ketonuria which results after the administration
of the long chain fatty acids as compared with that after
butyric acid may be interpreted solely on the basis of the
theory of beta oxidation*
It is postulated that the acetic
acid which is set free by successive beta oxidation® recombines
to form acetoacetlc and beta-hydroxybutyric acids; this theory
is based on a ketogenic action of acetic acid when fed to
phlorhizinized dogs or to fasted rats*
But such a formation
of acetic acid and its recombination to form ketone bodies
is probably not the case as shown by work of Stadie, Zapp, and
Lukens (1941), Toenniessen and Brinkmann (1938), and Deuel,
Hallman, Butts, and Murray (1936)*
Stadie, Zapp, and Lukens
used liver slices from depancreatized cats.
After equilibrating
the tissue in vitro, ketone bodies were produced by partial
oxidation of fatty acids but no formation of acetic or other
steam-volatile acids was found*
Toenniessen and Brinkmann
(1938) could not demonstrate acetic acid formation in the
perfused livers of rabbits, although they stated that they
believed large amounts to be formed by the catabolism of fatty
acids*
Finally the work of Deuel, Hallman, Butts, and Murray
(1936) with the ethyl esters of the fatty acids gives no support
to the oxidation of fatty acids to acetic because the same
amount of ketosis resulted from administering caproic as from
butyric; it was not until caprylic acid was given that an
7
increased ketosis occurred*
Verkade and van der Lee (1934, 1935) suggested that
fatty acids may undergo omega oxidation*
This was based on
the recovery of small amounts of dicarboxylic acids from the
urine of human subjects after triglycerides composed of the
fatty acids from eight to twelve carbon atoms were fed*
Although no single oxidation theory has yet been
accepted, it is, however, well agreed that the liver Is the
chief site of breakdown of fat, since liver fat has a higher
iodine value and phospholipid content than fat stored else­
where in the body*
Hence it is believed that the liver
desaturates fat (Leathes and Wedell, 1909, Schoenheimer and
Hlttenberg, 1936) and such desaturated phospholipids are
transported to the tissues for oxidation.
Oxidation may also
occur in the liver*
As to the role of carbohydrate in fatty acid oxidation,
the literature is still inconclusive*
If carbohydrate
metabolism can prevent ketosis, does it do so by sparing fat
(antlketogenesls) or by combining in some way with the fat
to make the latter more readily oxidizable (ketolysis)?
Liver perfusion experiments of Embden and Marx (1908)
were interpreted as indicating that carbohydrates remove fat
from the oxidative processes, therefore are antiketogenic*
But Magnus-Levy (1908) showed that when only a fraction of the
oxidized fat is replaced by carbohydrate, the ketonuria
resulting from carbohydrate fasting is overcome,
Geelmuyden’s
idea (1904) was that some oxidized product of glucose, such
as glycuronic acid, methyl glyoxal, lactic or pyruvic acids,
combines with the acetone bodies to cause their oxidation.
Evidence that such condensation products of acetone bodies
with glucose intermediates may occur has been cited by Henze
(1931) and by Stohr and Henze (1932) in feeding experiments
with “ketol," a product of aldol condensation of acetoacetic
acid and methyl glyoxal followed by decarboxylation,
Ketol
was found to increase the blood sugar level and the liver
glycogen, although it did not apparently form muscle glycogen.
Zeller (1914) concluded from results obtained with high fat,
low carbohydrate and low protein diets that one molecule of
neutral fat is oxidized by one triose molecule, or one molecule
of glucose can oxidize two molecules of fatty acid.
Woodyatt
(1921) and Shaffer (1922) in their studies of ketogenicketolytic ratios confirmed Zeller*s conclusion.
Probably the greatest support for the catalytic role
of glucose in the oxidation of ketone bodies is found in the
in vitro results of Shaffer (1921).
When glucose and aceto­
acetic acid were combined in alkaline solutions, the addition
of hydrogen peroxide caused the acetoacetic acid to disappear
at a rapid rate, increasing with the amount of glucose, with
the alkalinity, and with the temperature.
In the absence of
glucose, however, the acetoacetic acid was oxidized only very
9
slowly*
Fructose and glycerol were likewise ketolytic but
lactic acid was not.
Since the rate of the reaction seemed
to be determined by the conversion of glucose by the alkali
into a derivative which was then oxidized, Shaffer concluded
that some intermediate oxidation product of glucose combined
with acetoacetic acid, the new compound then being oxidized*
Deuel, Gullck, and Butts (1932) reported on the varying
ketolytic ability of carbohydrates, and Shapiro (1935) showed
that only glucose formers can lessen ketonuria when ketogenlc
acids are administered*
Butts, Dunn, and Hallman (1935-36)
and Butts, Blunden, and Dunn (1937) (1, 2) published evidence
also in support of ketolysis from results with various anino
acids and their glycogenic action*
But in 1937 Mirsky and
Broh-Kahn announced that glucose is not ketolytic because they
found no difference in the rate of disappearance of betahydroxybutyric acid injected in the blood of nephrectomized
rabbits with ample carbohydrate reserves as compared with
fasted animals*
Results from this laboratory (Blunden,
Hallman, Morehouse, and Deuel, 1940), however, in rats
administered 1-beta-hydroxybutyrate showed that the rate
of disappearance was significantly lower in experiments
carried on for 75 and 150 minute intervals than for animals
receiving glucose.
Thus, Deuel concluded that the "rate
10
of disappearance of beta-hydroxybutyrate from the tissues
is accelerated when glucose is present.”
Further, in a study
of the comparative action of the fat-sparing substances,
glucose and alcohol, on exogenous and endogenous ketonuria,
Deuel, Hallman, and Murray (1938) demonstrated that only
glucose caused a decrease in acetonuria.
Glucose brought
about an almost complete abolition of the endogenous
ketonuria in fasting rats previously fed a high fat, low
protein diet, while ethyl alcohol was entirely ineffective.
Calculations showed that the extent of fat oxidation was
practically identical in the fasting rats and in those
receiving sufficient glucose to overcome the endogenous
ketonuria.
Also, the amount of glucose which was ketolytic
amounted to only a little more than one per cent of the
total fat oxidation of the day.
Hence, Deuel concluded
that "ketolysis rather than antiketogenesis is the primary
mechanism whereby the metabolism of carbohydrate brings
about the abolition of ketonuria.n
Another method of approach to the antiketogenesisketolysis controversy is via the tissue slice method (Warburg,
1926).
The results obtained by this procedure show considerably
higher and more uniform respiration rates than when hashed
tissues are employed or where perfusion of the excised organ
is used.
This would indicate that the respiration of the
11
intact organ is in the main preserved in the sections anl also
that less variable figures obtain with tissue slice studies
than with perfusion experiments*
Warburg (1926) has compared
respiration under these various procedures as follows:
Authors
Barcroft and
Shore
Usui
Experimental Arrangement
Perfusion with blood
from living animal
(cat)
Liver
Carcinoma
%
S
1*5 - 15
Intact liver lobe
in vitro (mouse)
2.8
Russell and
Gye
Film of tissue pulp
(mouse)
5
-
4
Minaml
Sections of tissue
(rat)
9
>13
1 * 2- 6
7
-11
Outstanding work in the application of tissue slice
technique to fat metabolism has been done by Quastel, Wheatley,
and Jowett*
In 1933 Quastel and Wheatley found that fatty
acids are oxidized at considerable rates by liver slices,
giving acetoacetic a d d as one of the products*
In agreement
with results obtained by perfusion experiments, the fatty
acids with an even number of carbon atoms' produced the
acetoacetic acid.
They found that glucose did not remove
acetoacetic acid formed from butyric but glycogen did,
although in the latter case the respiration of the tissues
was also reduced*
The next year (1934), however, these
12
workers reported their Inability to repeat the experiments
showing the inhibitory action of glycogen on acetoacetic acid
production.
These workers noted that the nutritional state
of the animals used was a very Important factor in the yield
of acetoacetic acid.
In a study of the relation of ascorbic
acid to fatty acid oxidation (1934) they found that ascorbic
acid causes a significant Increase In acetoacetic production
of scorbutic guinea pig liver slices in media containing
butyrate or crotonate.
In 1935 Jowett and Quastel reported
further on the rates of oxidation of butyric# crotonic, and
dl-beta-hydroxybutyric acids and showed the importance of
optimum salt concentration# pH# and nutritional state,
Also
in the same year these men published their classical
quantitative studies on nThe Oxidation of Normal Saturated
Patty Acids in the Presence of Liver Slices" in which they
put forward the hypothesis of multiple alternate oxidation.
Still a third paper in the same year gave evidence that the
kidney# spleen# and testis produce small amounts of
acetoacetic acid from fatty a d d s but the brain gives no
measurable quantity.
They also showed that kidney# spleen#
testis and liver destroy acetoacetic acid aerobically# the
kidney removing it also anaerobically,
Purther studies on
acetoacetic acid breakdown appeared in November 1935 and it
was noted that the rate of beta-hydroxybutyric acid
13
production from acetoacetic acid aerobically was Increased
in the presence of cyanide and still further increased If
glucose or glucose and pyruvate were added to the acetoaeetatecyanide medium.
In a study on the decomposition of butyric acid by
liver slices Ciaranfi (1936) showed that both respiration
and ketone body formation are greater in Ringer*a solution
buffered by phosphate than when buffered by bicarbonate.
Also on the basis of calculations of the respiratory quotients,
both the oxygen consumption and to a less degree the carbon
dioxide formation were increased In the presence of butyrate.
The altered metabolism of fatty livers was shown by Callfano
in 1937 when he produced fatty livers by phosphorus poisoning.
That ketone bodies may be Intermediates not only In
fat metabolism but also In carbohydrate metabolism was
suggested by Krebs and Johnson (1937) from experiments on
the anaerobic oxidation of pyruvic acid wherein they found
that in muscle and in other tissues to a lesser extent
beta-hydroxybutyric acid was the chief end product.
It was
not until 1938 that Gohen and Stark proved conclusively that
a low liver glycogen In rats resulted In greater production
of ketone bodies both In the absence and in the presence of
butyrate as substrate.
Leloir and Munoz (1939) obtained
butyric acid oxidation in a cell-free liver brel and also
14
found that the rate of disappearance of butyric acid with
liver slices was higher than that of any of the other
saturated fatty acids from one to eight carbon atoms inclusive.
As a result of the contributions reviewed, it seemed
possible that additional data on the mechanism whereby glucose
decreases ketonuria might be obtained by the employment of
tissue slices.
In the present investigation it was desired to determine
whether the decreased ketone bodies found when liver slices
are present in a butyrate medium containing glycogen as
contrasted with the level when this polysaccharide is absent
is to be ascribed to a suppression in their formation, which
would support the antiketogenesls theory, or to an acceleration
in their oxidation which would support the idea of ketolysis.
By the determination of the disappearance of butyrate as well
as acetone bodies, it is believed that an answer to this
problem has been obtained.
EXPERIMENTAL PART
Experimental animals
Albino rats from the stock colony of the Department
of Biochemistry of the University of Southern California
were employed for the experiments.
Only adult males were
used, four months or more of age, either well<*fed or fasted
for 24, 48, or 72 hours.
Before the livers were removed
for sectioning, the animals were anesthetized with sodium
amytal and bled from the throat.
Procedures
The experimental work has involved three definite
procedures: (1) the msnometric method of Warburg with liver
tissue slices, (2) the micro-acetone analysis of Edson
employing Rupp's lodometrlc titration, and (3) the determina­
tion of residual butyric acid by a distillation procedure.
(1) The Warburg manometrlc method
The Warburg manometer described in Warburg* s ”Uber
den Stoffwechsel der Tumoren” (Berlin 1926} and also in
"Manometric Methods'* by Dixon (London 1934), a constant
volume type of respirometer, has been used throughout this
study.
A bank of seven manometers was available.
The
water bath in which the reaction flasks, or vessels, were
16
shaken was maintained at 39°C t 0*02°.
Since one end of
such a manometer tube is open to the air, the manometers
are very sensitive to slight barometric pressure changes,
hence one of the seven was always used as a thermobarometer.
The calibration of the apparatus, that is, the
determination of the vessel constants, was done by liberating
a known amount of carbon dioxide gas in the vessel as a
result of the chemical action between sodium bicarbonate
and sulfuric a d d after temper attire equilibrium had been
attained,
head perchlorate prepared according to Krebs
(1930) was used as the manometer fluid.
The theory of the
apparatus is well discussed in Dixon's "Manometric Methods"
(London 1934).
It might be mentioned that the basis
for calculation of the volume of gas absorbed or evolved
by the surviving tissues is the vessel constant.
This
value represents the dimensions of a surface and actually
is a factor expressed in square millimeters by which the
changes in the height of the manometer fluid may be
multiplied to find the volume or cubic millimeters of gas
17
concerned.
It Is calculated as follows:
Let x s
h x
Vgs
Vfr
T s
P z
p r
Pqs
«*. m
the amount of gas In c.m.m. at H.T.P.
the manometer reading
the volume of the gas space In c.m.m.
the volume of fluid added In c.m.m.
the absolute temperature of the water bath
the Initial pressure In the vessel
the vapor pressure of water at temp. T
the pressure of one atmosphere expressed in
mm. of confining fluid
For example, the lead perchlorate
used had a density of 2.079, hence
760 x 13.59
P0 ■
Z7o7§-the Bunsen absorption coefficient of the
gas concerned.
The total amount of gas finally present in the vessel Is
the sum of the amount initially present and the amount, x,
produced:
iva
~
and x s h
h * (va t 2 *
* *
.. 273 m 'xt of
v0
* VF^
0
The expression in brackets Is the vessel constant far a
given gas, that is,
x - h . kCOg
It may be added that x is positive if gas is formed, but is
negative when gas disappears.
18
When a known amount of gas, as carbon dioxide, is evolved in
determining the vessel constant, the amount of carbon dioxide
gas s x, the difference in manometer readings before and
after evolution of the gas s h, hence kCQg • -j£Calculation of the volume Vg may be done as follows :
0
T
r ~ x 275
From the value V
s
X
-
¥F
the constant for oxygen, or any other
may be determined by applying the appropriate absorption
coefficient in the following formula:
x s h
The data for one calibration using 0.01 M sodium
bicarbonate in the main part of the manometer vessel and
2/3 N sulfuric acid in the side-arm are given in Table A
in the appendix.
The thermobarometer is represented by the
manometer number in parentheses*
A summary of a series of such determinations is:
made in Table B.
The solutions used in the various tissue slice
experiments were prepared as follows:
Salt mixture.
The amounts used are all calculated
19
on the basis of 0*16 M for a salt producing
only two Ions; this figure Is Isotonic with
serum.
Stock solution for use In manometer vessels:
360 ml.
90 ml.
72 ml.
Buffer.
0.16 M KC1
0.107 M CaClg(i.e., SgfeS
0.107 M MgClg
0.10 M NaHgPO^ adjustedto pH
NaOH.
)
of 7.3 with
Indicators..
Brom thymol blue (pH 6.0 yellow
7.6 blue)
B.B.H. ”4.5" (pure grey at pH of 4.5)
Substrates.
Sodium salts of the fatty acids 0.16 M
Glycogen: 0.4 or 0.8 g. dissolved In
25 ml. 0.16 M salt.
Others,
jniline: 5 g. aniline hydrochloride dissolved
in 25 ml. of IN NaOH, prepared fresh dally.
Salt: 0.16 M NaCl
The preparation of the tissue slices for the Warburg
experiments requires the ability to estimate the necessary
tissue slice thickness for satisfactory respiration.
following table is of Importance In this connection:
The
20
(Warburg, “The Metabolism of Tumors," 1926)
Tissue thickness
Oxygen quotient
0*21 mm*
0.30
0.31
0.50
0.95
1.24
A
8*8
8.8
9.4
7.8
5.8
5.9
thickness of the tissue slices ranging between 0.2 and
0.4 mm. la generally accepted for best results.
Tissues
of less thickness tear apart during the shaking operation,
and of greater thickness, show lowered respiration, probably
due to asphyxiation of the Interior cells.
Tissue thickness
may be estimated as follows:
Area Is measured by suspending the slices in
physiological saline in a Petri dish which is
superimposed upon metric ruled paper.
The area
of paper covered by the slice is found by
counting the square millimeters covered.
The
tissue Is then dried to constant weight.
Volume Is estimated by multiplying the dry weight by
5, since tissue is approximately one-fifth
solids and four-fifths water.
Thickness, then, -
YQlume in cubic millimeters
area In square millimeters
21
After a sufficient practice period, the thickness may be
readily estimated from the translucency of the slice*
For sectioning the liver tissue, an alternative
procedure has been devised than the use of a straight razor
as suggested by Warburg (192t>), and Dixon (1934) as well as
employed by Quastel (personal communication from
Professor J. S. Butts).
This Involves the use of a handle
(Figure 1) constructed of spring metal which holds the
ordinary double-edged safety razor blade.
i—f
c
c'
Fig. 1
B and
are grooves.
C and C1 are rests or finger grips
23
The blade is inserted in the handle by bending the grooves
B and B-*- toward each other and introducing them into the
slot of the safety razor blade.
The spring of the handle
causes these to snap into place, thus holding the razor blade
firmly,
C and
are rests or finger grips.
The handle
affords good leverage and requires effort of approximately
the same value from either hand.
This enables a more uniform
effort in cutting than with the straight razor where the
center of gravity lies near the handle.
It has the further
advantage that the blades can be changed for new ones when
dull, by a reversal of procedure.
After the vessel constants have been determined and
the tissue sectioning technique acquired, the Warburg apparatus
is ready to be used for respiration experiments.
Tissue
slices not exceeding 20 mg. dry weight were used in this
study.
The slices prepared from rat liver immediately after
excision of the liver were bathed in 0.9 per cent sodium
chloride and then Immersed in the manometer vessels.
Usually
three slices were used in each vessel so that average thickness
in different vessels would be approximately the same.
The
medium in which the slices were immersed consisted of the
followings
Salt mixture (K, Ca, Mg)
Phosphate buffer
Sodium chloride 0.16 M
0.3 ml.
0.6
2.1
24
When additional metabolites were added, as sodium butyrate or
glycogen, they replaced a similar volume or sodium chloride
solution.
Into the inner cup of each manometer vessel was
inserted a small roll of filter paper moistened with 0,2 ml,
of 2 N sodium hydroxide to absorb carbon dioxide evolved
during respiration of the tissues.
The vessels were
attached to the manometers and gassed for 5 minutes with a
mixture of 95 per cent oxygen and 5 per cent carbon dioxide.
The manometers with vessels attached were then transferred
to the water bath, in which the vessels were shaken usually
for 2 hours.
Ten to twenty minutes were allowed for
temperature equilibrium, after which the readings of oxygen
uptake were taken every thirty minutes.
The difference
between the second and third readings was always used for
calculation of the oxygen quotient.
At the end of the two hour period the slices were
removed from the vessels, washed in distilled water, dried
at about 105° 0, and weighed to constant weight.
each vessel was checked.
The pH of
The filter paper absorbers were
removed from the Inner cups of the vessels, 0,1 ml, of 50
per cent sulfuric acid delivered into each cup, and the cups
finally dried out with filter paper.
Acetoacetic acid was
then determined either manome trie ally or by the micro-acetone
method of Edson (1935), described later.
The manometrle estimation of acetoacetic acid as
25
developed by Quastel and Wheatley (1935) was accomplished as
follows*
The residual solution (3.0 ml*) after removal of
tissue slices was acidified by adding 0*3 ml* of 1*0 N
acetic acid, which brought the pH approximately to 4*5,
as checked by an indicator*
Into tbs vessel side arm was
run 0*2 ml* of a solution of aniline hydrochloride, prepared
fresh each day*
The vessels, with carbon dioxide as gas
phase, were shaken 10 to 15 minutes in the water bath before
the manometer readings were taken*
As soon as temperature
equilibrium was apparent, the aniline solution was tipped
into the vessel mixture to decompose acetoacetlc acid*
This
reaction was first found by Wohl (1901, 1907), used by
Ostern (1933) and by Krebs (1933) in manometrlc determination
of oxalacetlc acid, and applied by Quastel and Wheatley
(1933) to acetoacetlc acid determinations in tissue slice
studies*
According to Wohl:
COOH.CO.CHg.COOH ♦ NHg.CgHg
oxalacetlc
CgHg.NH.OO.C.O.CHg ♦ HgO ♦ COg
aniline
pyruvanillde
The above reaction is probably analogous with acetoacetlc
acid, that is, 1 mol carbon dioxide is evolved for each mol
acetoacetlc add, the evolution being considered complete
in 45 to 55 minutes*
A typical experiment is given in
detail in the appendix, Table C*
26
The various quotients used to express the results of
the respiration experiments are the following:
Qq o = cubic millimeters of oxygen consumed per
milligram of dry tissue per hour
^Ac - cul3ic millimeters of carbon dioxide equivalent
to acetoacetlc acid formed per milligram of
dry tissue per hour
qq
s
cubic millimeters of carbon dioxide equivalent
to beta-hydroxybutyric acid farmed per milligram
of dry tissue per hour
«Ket * oum of «Ac “ ld ®B-OH
(2) Mloro-determinatlon of acetone bodies*
Edson (1935) has combined the Van Slyke (1917) method
of determining acetone bodies with Rupp’s lodometrlc titration
(1906, 1907) for the mercury*
Solutions:
20$ copper sulfate
10$ calcium hydroxide suspension
Denlges reagent: 10$ mercuric sulfate in
50 vol. % sulfuric acid made as follows: 75
gm. red mercuric oxide dissolved In 1 liter
4 N sulfuric; 500 ml. sulfuric sp. g. 1*835
diluted to 1 liter* Combine 1 liter 50 $
sulfuric with 3*5 liters mercuric sulfate
and 10 liters water*
1 N hydrochloric a d d
35$ formaldehyde
3$ potassium iodide
7*5 N sodium hydroxide
glacial acetic acid
0*01 H iodine
0.01 H thiosulfate
1$ starch solution
5$ potassium dlchromate
Determination of acetoacetlc acid:
After removal of the tissue slices and excess sodium
27
hydroxide from the Warburg vessels, there is run Into each
vessel 0.5 ml, each of 20 per cent copper sulfate and
10 per cent calcium hydroxide suspension.
The solution is
transferred to a 25 ml, Erlenmeyer flask, after which the
Warburg vessel is rinsed twice with 5,0 ml, of distilled
water.
The contents of each Brlenmeyer flask are made up
to 15 ml. volume and allowed to stand 20 minutes before
filtering,
12,5 ml, of the filtrate is pipetted into a
flat bottomed Pyrex glass flask provided with ground glass
connection for a condenser,
4,5 ml, of Deniges combined
reagent is added to each flask and the mixture refluxed
for 30 minutes.
2 ml, distilled water is washed down each
condenser, then the acetone-mercury precipitate Is filtered
with suction on a sintered glass funnel (#4 porosity).
The
flask Is rinsed with two 5 ml. portions- of water which are
also us;ed to wash tbe precipitate.
The filtrate is saved
for the determination of beta-hydroxybutyric acid.
The precipitate is dissolved on the funnel in 10 ml,
warm N hydrochloric acid.
The mercuric chloride solution Is
run into a filter flask by suction.
well with water.
The filter is washed
To the mercuric chloride solution are
added in the following order: 0,5 ml, 35 per cent formaldehyde,
5,0 ml, 3 per cent potassium iodide, and 2,0 ml. 7,5 N
sodium hydroxide.
The solution is allowed to stand for at
28
least 2 minutes, then is shaken.
Finally are added 1.0 ml.
glacial acetic acid and 10.0 ml. 0.01 N iodine solution.
The excess iodine is titrated with 0.01 N thlosulfate.
According to Edson (1935), taking the 12.5 ml. aliquot into
account, 1.0 ml. 0.01 N iodine equals 0.138 mg. acetoacetlc
acid or 30.2 micro liters of carbon dioxide.
The principle of this iodine titration (Rupp 1906,
1907) is as follows.
Metallic mercury will precipitate
from alkaline solution by means of formaldehyde, then may
be changed to mercuric iodide by means of excess n/10 iodine,
the unchanged iodine being titrated with thiosulfate.
To
change any combined mercury in alkaline solution, potassium
iodide, then alkali are used.
The resulting mercury iodide-
potassium iodide is reduced immediately by formaldehyde at
ordinary temperature.
Formaldehyde does not interfere with
the iodine reaction if the solution has previously been made
acid, hence the use of the glacial acetic add.
The
calculation according to Rupp is:
1 Hg ♦ 2 I * 2 K I —
*
K2HgI4
200.4 g. Hg s 2 I; 0.01002 g. Hg a 1 ml. n/10 I.
Determination of beta-hydroxybutyric acid:
The filtrate from the acetoacetlc acid precipitation
is returned to the flat-bottomed flask and brought to
boiling.
1.0 ml. of 5 per cent potassium dicbromate is
29
added through the top of the condenser and boiling is
continued for 90 minutes, after which time 5*0 ml* of water
.t
is run down the condenser, then the acetone-mercury precipitate
is filtered by gravity on a sintered glass funnel (#3 porosity).
The flask is thoroughly rinsed and the washings run through
the funnel until the filtrate appears to be free of dlchromate
as it drops into the filter flask.
The filtrate is saved
for determination of residual butyric acid.
The precipitate
is dissolved in 10.0 nil. of warm N hydrochloric acid and
treated as described above for the acetone-mercury precipitate
due to acetoacetlc acid.
Considering the original 12.5 ml.
aliquot, 1.0 ml. of 0.01 N iodine is equivalent to 0.185 mg.
of beta-hydroxybutyric acid or 39.9 micro liters of carbon
dioxide.
(3) Determination of residual butyric acid
The filtrate obtained after separating the acetonemercury precipitate due to beta-hydroxybutyric acid is
made up to approximately 250 ml. volume and distilled in an
all-glass distillation apparatus consisting of a Kjeldahl
flask, connecting tube with Hopkins* trap, and a condenser
fitted into a filter flask.
The last nemed la always filled
to the same level with distillate, which represents a volume
of 185 ml.
The distillate is immediately titrated with
0.01 N sodium hydroxide using phenolphthalein as indicator.
30
A similar procedure has been used by Deuel, Hallman,
Greeley, Butts, and Halliday (1940) in distilling acetoacetate
and acetone from hashed tissues of the rat*
The apparatus
used was the same except in the determination of butyric acid,
a Hopkins* trap was found necessary to hold back the sulfuric
acid vapors*
The method for butyric acid distillation was
developed on the basis that butyric acid is readily distillable
with steam*
The validity of the method has been tested with
known amounts of butyric acid, recoveries of which are reported
below*
The tltratable acid obtained in the distillate is
concluded to be butyric acid because it may be qualitatively
recognized by odor and was never quantitatively recovered
unless butyrate had been used as substrate in the tissue
slice media*
Five titrations using 0*01 N sodium hydroxide and
phenolphthaleln were made on distillates from water (3) and
from water plus; 1*0 ml. of 60 per cent sulfuric acid (2)*
The readings weres
1.
2.
3.
4.
5.
Average
0*54 ml
0.48
0.58
0.50
0.57
0.53
Five recoveries of butyric acid were made by distilling
31
the following mixture si
1* Water, 1.0 ml* sulfuric,
0.2 ml. butyrate (0.16 M)
3.64 ml.
2. Same as No. 1 with
tissue medium
3.59
3. Water, 4.5 ml. Benig^s,
0.2 ml. butyrate, tissue medium
3.67
4. Same as No. 3 with glycogen
3.69
5. Same as No. 3 with 1.0 ml.
dichr ornate
3.65
Average
3.65
Corrected for water blank
3.12
The average recovery after correction for water blank Is
approximately 97.5 per cent.
Finally, five recoveries of butyric acid were made on
the filtrates obtained after precipitation of the acetone
bodies.
Typical tissue media were made up, the first three
with glycogen, the last two without glycogen.
After copper-
lime precipitation, a 12.5 ml. aliquot was used for the
micro-acetone procedure, followed exactly as previously
described.
1.
2.
3.
4.
5.
Average
3.07 ml.
2.98
3.00
3.04
2.97
3.01
32
Calculation, of the recovery factor
3.01 - 0.53 « 2.48
2.48 x
o
Recovery factor =
= 2.98
s 93.1 per cent
Validity of Edson*s micro-acetone determination of
acetone bodies was checked by usdng a solution of betahydroxybutyric acid (0.55 mg. in 5.0 ml.), the source of
which
was the calcium-zinc salt of 1-beta-hydroxybutyric
acid prepared according to the ^procedure described by
v
Blunden (1938). The recoveries were as follows:
1.
2.
3.
4.
0.56 mg.
0.53
0.55
0.56
These results compare favorably with Edson's recoveries
which ranged from 95 per cent to 110 per cent in 6 trials.
The recovery values tend to be high because Edson used Van
Slyke's factor of 8.45 g. of mercury precipitate equals 1 g.
of beta-hydroxybutyrate.
An experimental study made in
Deuel's laboratory with both the 1- and the dl-beta-hydroxybutyrate shows that this value is too low and that the
factor 9.51 is more nearly accurate (Blunden, Hallman,
33
Morehouse, and Beuel, 1940),
Jowett and Quastel (1935)
also reported a higher value of 9.85 following experiments
using presumably the dl-beta-hydroxybutyrate.
EXPERIMENTAL RESULTS
Effect of fasting
A study on the effect of fasting on acetoacetlc
acid content with and without butyrate substrate was made
with rat liver slices from 10 animals that had been fasted
24, 48, or 72 hours.
A tabulation of these results Is made
In Table I with a comparison of values obtained with unfasted
rats.
Because consistently higher values of acetoacetlc acid
quotients maintained with the 48 hour fasted animals, such
nutritional states were employed in the remaining experiments.
Since the spontaneous acetoacetlc acid content of unfasted
rat liver is generally quite high, fasting brought about much
higher quantities, an advantage when changes in amounts of
ketone bodies are to be studied.
Further, the 48 hour fasting
period was chosen because not such wide-spread changes in the
general condition throughout the animal body might be expected
to occur as after longer fasts.
TABLE I
THE EFFECT OF FASTING ON THE Q0g AND Q ^ OF THE LIVER SLICES OF MALE RATS
Unfasted
Fasted 24 hours
0.01M
0
0.01H
$02 §AC Q°2 Qac
Q02 $AC
Fasted 48 hours
Fasted 72 hours
0
0.011
0
0.01M
$02 ^AC Q02 $AC QG2 Qac
Qp2 Qac
9.1 1.14 10.0 4.34 8.0 1.99 10.0 5.58
8.9 1.13 9.9 4.21 8.5 1.96 9.9 5.34
8.2 2.64 12.4 5.16 7.1 2.19 10.6 5.59
0
Butyric-ConC•
Quotient
^02 Qac
5.3 1.19
5.6 1.08
7.5 3.33 8.5
8.8 4.47
7.6 1.08
8.0 1.12
9.3 2.96
7.8 2.77
0,
8.5 2.32
11.7 1.46 10.2 4.48 8.0 1.77
7.1 1.79
7.9 1.55
7.9 1.28
7.3
8.6
8.2
7.0
3.37 10.9 7.37
4.04 11.0 6.72
3.48
2.14
8.8 2.14 13.7 5.32
8.2 2.14 13.9 6.04
9.2 2.04 13.9 6.15
7.4 1.12
8.9 3.68 8.3 1.32 10.0 5.46
Grouped experiments are on the liver slioes of the same rat.
7.0 1.95 12.6 6.25
7.4 2.15 11.9 6.12
8.4 2.51 12.3 5.89
9.7 3.38
9.5 2.66
7.8 4.38
36
Effect of glycogen.
Using the livers from unfasted and from 48 hour fasted
animals, the effect of the addition of gLycogen to the
tissue medium was studied.
In Table II.
The quotients found are listed
Because greater lowering of the acetoacetlc
acid quotients occurred when the glycogen concentration
was one per cent of the medium, this amount was used In
later experiments.
Seventeen rats were used.
TABLE II
THE EFFECT OF GLYCOGEN ON THE
fionc. in per oent
0
Ao
Quotient
Oj>
% z AND
QAC OF THE LITER SLICES OF MALE RATS
Unfasted
0*5
O2
Ac
°2
Ac
7.6
7.9
7.4
9.0
0
0
7.9
7.4
2.79
8.2
8.3
8.1
0.77
2.82
0.80
1.49
1.82
1.0
Fasted 48 hours
0
1.0
02
Ao
Ac
02
6.6
8.3
1.99
6.9
6.3
1.77
1.25
8.6
7.4
1.64
1.54
7.0
3.05
6.0
7.6
2.41
2.78
10.2
10.4
1.31
1.09
10.6
12.8
9.9
10.3
1.30
0.52
0.30
1.23
7.2
7.1
1.49
1.09
8.3
9.3
7.9
10.3
1.40
0
0.15
0.55
9.3
8.5
1.30
1.14
9.0
9.6
8.5
8.5
0.21
0.23
0.56
1.69
7.92 3.05
7.76 2.65
8.21
10.75
8.37
8.42
0.19
0.95
1.50
0
13.68 1.80
12.12 1.82
10.0 1.79
7.73 1.47
8.52 1.42
8.68 1.41
10.22
9.46
8.84
0.29
0.38
1.12
8.88 2.14
9.34
0.49
8.2
8.3
8.1
0.77
2.82
0.80
8.0
7.6
2.41
2.78
7.59 2.07
7.48
1.26
9.03 2.20
8.02
1.72
6.91 2.72
7.56
2.04
7.9? 1.86
10.39 1.85
10.63 2.02
8.6
7.4
1.64
1.54
38
Effect of butyrate plus glycogen
Since glycogen was found usually to lower the
endogenous acetoacetlc acid content, it was decided to study
the effect on exogenous ketosis, employing butyrate as
substrate*
The values from these experiments are reported
in Table III.
TABLE III
THE EFFECT OF BUTYRATE WITH AND WITHOUT GLYCOGEN ON THE Q0j>
AND Qag OF THE LIVER SLICES OF MALE RATS FASTED 48 HOURS
Substrate
Quotient
0
Og
Ao
Glycogen 1%
0<>
Ao
Butyrate O.OIM
Ao
___ Sa.....
Glycogen
O2
1$
+ Butyrate O.OIM
Ac
7.9
7.4
2.79
8.2
8.3
8.1
0.77
2.82
0.80
11.8
5.87
7.0
3.05
8.0
7.6
2.41
2.78
13.5
10.9
, 5.06
4.61
7.59
2.07
7.48
1.26
13.21
13.76
5.86
5.36
10.79
12.26
3.88
4.37
9.02
2.20
8.02
1.72
13.22
15.57
6.57
6.68
13.64
12.48
4.98
5.77
6.91
2.72
7.56
2.04
13.85
13.32
7.17
6.38
12.31
12.52
4.88
5.44
40
Effect of butyrate plus glycogen on total ketone production
The presence of glycogen having been found so often
to lower the acetoacetlc acid content, it seemed necessary
to determine whether such an effect were at the expense of
the beta-hydroxybutyric acid.
The micro-acetone determina­
tion of Edson was used in order to recover the ketone bodies
separately, the sum of such recoveries being total ketones
found.
The quotients obtained in these experiments are
shown in Table IV,
TABLET 17
EFFECTS OF BUTYRATE AND GLYCOGEN ON TOTAL KETONE PRODUCTION
OF LITER SLICES OF MALE RATS FASTED 48 HOURS
Substrate_________ 0
B-OH
Ao
itient °2
i* No.
1
7.02 2.65
2
3
4
5
6
7
8
9
10
11
12
13
Ket
Glycogen 1% ____
Ac B-OH Ket
°2
Butyrate O.OIM
Glycogen
B-OH Ket
Ao
°2
°s
1.64 4.29 6.20 3.18 2.65 5.83 10.28 8.54
11.89 9.08
7.64 5.07 2.20 7.27 7.17 4.28 1.89 6.17 12.02 12.54
13.70 9.01
6.19 3.07 0.90 3.97 5.22 3.18 0.75 3.93 9.64 6.13
13.94 8.63
6.76 0.68 1.39 2.07 8.33 1.13 1.18 2.31 10.33 9.66
15.12 9.66
7.57 0.98 0.99 1.87
10.95 5.21?
6.83 1.23?
14.33 7.13
3.66
12.12 6.23
6.58 1.16 , 2.50
8.88 0.96 2.25 3.21
13.48 6.81
7.04 1.88 1.50 3.38
12.61 6.09
7.13 0.77 2.86 3.63
11.85 6.15
2.99
7.57 0.78 2.21
10.73 5.85
7.49 0.81 2.07 2.88
11.37 5.64
7.73 0.94 1.51 2.45
10.34 5.21
8.51 0.95 1.48 2.43
11.46 5.26
2.01
2.97
9.07
0.96
10.01 5.01
8.44 2.34 1.15 3.49
11.42 5.31
7.33 3.98
7.70 0.87 0.74 1.61
8.26 0.86 0.64 1.50
10.22 5.73
10.49 3.21
6.46 0.75 2.85 3.60
13.22 6.08
7.18 1.15 1.87 3.02
11.51 4.76
9.29 1.14 2.18 3.32
3.53
14.27
3.16
0.91
2.25
8.96
4.54
2.16
3.45
4.13
3.38
2.92
3.37
1.77
3.02
3.20
3.75
2.85
2.66
2.29
3.94
2.64
2.90
2.>91
2.94
1.05
1.26
1.43
3.17
1.39
2.78
Butyrate Q«0121
Ao B-OH Ket
lf> f
13.08 10.54 0.34
11.24 9.84 0.14
15.99 14.22 11.05
13.14 13.09 12.08
9.51 12.18 10.01
11.55 9.66 18.75
13.03 12.38 6.70
11.43 11.13 6.11
12.42 5.59
10.15 11.35 6.05
9.43 12.68 5.59
10.56 12.86 6.17
8.94 12.82 4.91
8.81 13.25
8.14 11.13 7.37
9.58 13.29 5.36
7.85 10.75 5.06
8.16 12.93 5.54
7.92 11.26 4.35
8.25 11.52 4.38
5.03 9.73 4.20
6.99 11.30 4.24
4.64 10.94 3.49
9.25 11.48 5.62
6.15 12.29 3.45
6.31 14.10 3.42
0.15
0.29
3.91
3.92
3.07
0.79
1.94
1.57
2.41
2.52
2.76
2.60
2.68
2.29
2.23
5.55
2.14
2.08
1.78
1.71
0.92
0.79
1.43
1.23
1.29
1.66
*?atty liver
**Diarrhoea
0.49
0.43
14.96*
16.00
13.08*
19.54
8.64
7.68
8.00
8.57
8.35
8.77
7.59
9.60**
10.91
7.20
7.62
6.13
6.09
5.12
5.03
4.92
6.85
4.74
5.08
TABLE 17 (cont.)
14
15
16
17
18
19
20
21
22
23
8.38
7.73
7.10
7.50
6.96
7.13
7.43
8.32
7.39
8.47
7.14
7.43
8.37
9.86
7.98
7.33
7.01
7.85
6.51
7.98
0.75
0.79
1.53
1.52
1.27
1.43
1.27
1.32
0.83
0.79
0,88
0.89
3.29
3.31
0.83
0.96
0.84
0.82
0.82
1.12
2.15
1.89
1.47
1.44
1.17
1.04
1.56
1.57
1.26
1.19
1.24
1.44
2.08
1.72
1.71
2.45
1.17
1.52
0.95
1.45
2.90
2.68
3.00
2.96
2.44
2.47
2.83
2.89
2.09
1.98
2.12
2.33
5.37
5.03
2.54
3.41
2.01
2.34
1.77
2.57
7*73
7.47
7.54
8.39
7.33
8.27
8.32
9.87
7.33
8.58
7.19
10.53
8.75
9.45
9.67
9.63
7.68
8.48
7.72
8.15
1.15
1.28
0.65
1.06
0.74
0.85
1.22
1.34
1.33
1.50
1.24
1.50
1.92
1.27
1.01
1.49
1.00
1.31
0.77
0.97
0.61
0.38
0.68
0.78
0.58
0.68
1.08
0.81
1.06
1.01
0.93
1.09
1.88
1.48
1.45
0.97
0.83
0.96
1.06
1.00
1.76
1.66
1.33
1.84
1.32
1.53
2.30
2.15
2.39
2.51
2.17
2.59
3.80
2.75
2.46
2.46
1.83
2.27
1.83
1.97
11.37
5.37
2.57
7.94
9,77
4.30 2.08
6.38
11.17
4.02
1.76
5.78 11.82
3.14 1.43
4,57
12.43
3.44
1.84.
5.28 11.42
2.92 1.07
3.99
11.37
4.69
2.01
6.70 11.90
4.26 1.64
5.90
12.86
4,77
2.18
6.95 11.43
4.34 1.77
6.11
9.78
2.38
1.36
3.74
9.09
2.04 1.11
3.15
12.40
6.67
1.83
8.50 11.13
3.24 1.52
4.76
13.18
5.40- 3.41
8.81 13.16
4.05 2.84
6.89
12.32
5,91
1.97
7.88 11.53
5.27 2.78
8.05
13.12
6.33
2.99
9.32 11.09
4.62 2.43
7.05
42
Quantitative studies on the changes in butyrate with and
without glycogen
Although in the majority of experiments a marked
decrease in the quantity of ketone bodies was noted in the
presence of glycogen, it may be argued that this results
from a suppression in the transformation of butyric acid
to these compounds because of the preferential oxidation
of the glycogen.
Under such, conditions, therefore, the
butyrate remaining in the medium should be greater when
i
glycogen
Isb
in the substrate than when it is absent.
In order to determine this fact, the butyrate
remaining in the tissue medium at the end of the respiration
experiments was determined after acetoacetate and betahydroxybutyrate had been quantitatively removed and
determined.
The speed of disappearance of butyrate is
calculated from the difference between the quantity of the
original butyrate added and the sum of the butyrate
recovered at the end plus the amount of acetoacetate and
beta-hydroxybutyrate (calculated as butyrate) also present.
Because the latter compounds are formed when butyrate is
absent from the medium, correction la made for the amounts
of these which were found in the control tests.
results are summarised in Table V,
These
43
It is also recognized that the decreased ketone body
recovery in the glycogen-butyrate medium may partly result
from the decrease in the endogenous content.
In Table V
the corrections are based on control experiments where
the tissues were suspended in the basal salt solution
without either butyrate or glycogen.
TABLE 7
THE COMPARATIVE ACETOACETATE AND HYDROXYBUTYRATE EEC0VERY ABB BUTYRATE
OXIDATION OP LIVER SLICES IK SUBSTRATES OP BUTYRATE ALONE OR BUTYRATE ANB GLYCOGEN
Expt.
•oUH
JB
£
NO.
Butyrate and & tones reooyerec
Butyrate added
Butyrio as such
ml N/1001 p gras Aeetoaeetic as butyric B*!QH butyric as
NaOH
«1. corrected 1^grams /ul. corrected 1 ^ grams ml N/100 1 f^gma
NaOH
Total
//gms
Butyric oxidized
beyond aoetone
bodies
Total
Total per
mg tissue
5 B
BG
BG
2*20
3,20
3.20
2818
2818
2818
126.6
99.7
143.6
497.6
391.7
564.2
42.6
31.5
44.4
167.5
123.9
174.4
1.99(2.14)
1.90(2.04)
1.61(1.73)
1883.2.
1795.2
1522.4
2548.3
2310.8
2261.0
269.7
507.2
557.0
25.7
45.7
38.2
6 B
B
BG
BG
3.20
3.20
3.20
3.20
2818
2818
2818
2818
101.3
103.5
87.9
97.1
398.2
406.8
345.4
381.6
16.0
24.7
7.4
4.2
63.2
96.9
29.0
16.4
1.96(2.11)
2.01(2.16)
1.98(2.13)
1.94(2.08)
1856.8
1900.8
1874.4
1830.4
2318.2
2404.5
2248.8
2228.4
509.8
413.5
569.2
589.6
52.0
45.9
58.6
62.1
7 B
B
BG
3.20
3.20
3.20
2818
2818
2818
75.4
75.4
54.6
296.2
296.1
214.5
10.6
7.5
7.6
41.6
29.4
29.9
2.02(2.17)
2.04(2.19)
1.88(2.02)
1909.6
1927.2
1777.6
2247.4
2252.7
2022.0
570.6
565.3
796.0
72.2
72.3
104.7
*8 B
B
BG
BG
3.20
3.20
3.20
3.20
2818
2818
2818
2818
87.9
83.2
131.4
93.0
345.3
327.2
516.4
365.6
2.6
31.0
1.8
69.6
10.3
121.7
7.1
273.4
1.98(2.13)
2.02(2.17)
1.75(1.88)
1.68(1.81)
1874.4
1909.6
1654.4
1592.8
2230.0
2358.5
2177.9
2231.8
588.0
459.5
640.1
586.2
67.6
53.4
64.0
57.5
9 B
B
BG
BG
3.20
3.20
3.20
3,20
2818
2818
2818
2818
105.9
107.1
105.5
108.0
416.2
421.1
414.5
404.9
28.3
34.7
16.4
13.0
111.1
136.4
64.4
51.0
2.22(2.39)
2.11(2.27)
1.97(2.12)
1.94(2.09)
2103.2
1997.6
1865.6
1839.2
2630.5
2555.1
2344.5
2295.1
187.5
262.9
473.5
522.9
15.1
21.2
36.9
46.7
10 B
B
BG
BG
3.20
3.20
3.20
3.20
2818
2818
2818
2818
87.7
88.9
61.6
62.9
344.8
349.3
242.2
247.3
57.4
53.4
20.4
18.6
225.4
210.8
80.4
73.1
1.96(2.11)
1.98(2.13)
1.80(1.94)
1.78(1.92)
1856.8
1874.4
1707.2
1689.6
2427.0
2434.5
2029.8
2010.0
391.0
383.5
788.2
808.0
25.2
27.0
55.5
56.5
TABLE 7 (eont.)
11 B
B
BG
BG
3.20
3.20
3.20
3.20
2818
2818
2818
2818
112.3
155.8
108.2
111.5
441.4
612.4
425.3
438.4
13.0
18.2
7.4
3.3
50.9
71.7
29.3
13.0
2.00(2.15)
1.84(1.98)
1.62(1.74}
1.72(1.85)
1892.0
1742.4
1531.2
1628.0
2384.3
2426.5
1985.8
2079.4
433.7
391.5
832.2
738.6
24.1
24,5
51.4
44.8
12 B
B
BG
BG
3.20
3.20
3.20
3.20
2818
2818
2818
2818
56.0
160.1
68.6
129.8
220.0
629.2
269.6
510.1
-23.1
25.3
-25.1
-31.4
-90.8
99.4
-98.6
-123.4
1.86(2.00)
1.78(1.92)
1.71(1.84)
1.67(1.80)
1760.0
1689.6
1619.2
1584.0
1889.2
2418.2
1790.2
1970.7
928.2
399.8
1027.8
847.3
74.9
25.6
76.1
61.0
13 B
B
BG
BG
3.20
3.20
3.20
3.20
2818
2818
2818
2818
111.4
67.8
70.0
65.8
437.8
266.4
275.1
258.6
-24.7
15.1
-26.8
-15.3
-97.1
59.3
-105.3
-60.1
1.73(1.86)
1.64(1.76)
1.60(1.72)
1.60(1.72)
1636.8
1548.8
1513.6
1513.6
1977.5
1874.5
1683.4
1712.1
840.5
943.5
1134.6
1105.9
56.4
69.9
78.8
80.7
*Diarrhoea
Calculations made as follows:
Acetoacetic
^AG o ^ ain©d“SV»Q^Q of tissue alone x mgs. tissue x 2 hrs. = ^lCOg
Hydrosybutyrate
Qb-oh ottained-av.Qb_gh of tissue alone x mgs. tissue x 2 hrs. = /*lC0g
Micrograms
/<ie02 % 22 400 000 " r e 108110110 bod7 as ^utyrate
Butyric as such
ml. 0.01N KaOH cor. for 93$ recovery
1 ml. 0.01N Ha0H^:880 /*g Butyric
B = butyrate in substrate (0.2 ml. of 0.16 1}
BG = butyrate and glyoogen in substrate
In the following series of tests, recorded In Table VI,
the corrections on the butyrate experiments are based on
values obtained as above, while the corrections in the
butyrate-glycogen tests were obtained from values found
for liver slices in the salt solution containing glycogen.
TABLE 71
THE COMPARATIVE ACETOACETATE AND HYDROXYBUTYRATE RECOVERY AND BUTYRATE OXIDATION OF
LIVER SLICES IN SUBSTRATE OF BUTYRATE ALONE, OF GLYCOGEN ALONE, OR OF BUTYRATE AND GLYCOGEN
Expt.
No.
Butyrate added
Butyrate and ketones recovered
ml N/lOOl ^ugma|Acetoacetic as butyric(B-OH butyric as butyrio
Butyric as such
NaOH
jii.% corrected Jy«graras| y<l. corrected I^tgrams
y^gms
ml N/lOO
NaOH
Total
p gms
Butyric oxidized
beyond acetone
bodies
Total Total per
mg tissue
14 B
BG
3.20
3.20
2818
2818
130.6
78.8
513.2
309.7
15.6
40.4
61.3
158.7
1.93(2.08)
1.85(1.99)
1830.4
1751.2
2404.9
2219.6
413.1
598.4
29.1
46.8
15 B
BG
3.20
3.20
2818
2818
68.0
53.4
267.2
209.9
8.2
8.2
32.2
32.2
1.89(2.03)
1.89(2.03)
1786.4
1786.4
2085.8
2028.5
732.2
789.5
53.8
67.5
16 B
BG
3.20
3.20
2818
2818
61.0
63.6
239.7
249.9
21.6
13.2
84.9
51.9
1.90(2.04)
1.73(1.86)
1795.2
1636.8
2119.8
1938.6
698.2
879.4
47.8
58.6
17 B
BG
3.20
3.20
2818
2818
109.2
88.2
429.2
346.6
14.5
20.7
57.0
81.4
1.76(1.89)
1.71(1.84)
1663.2
1619.2
2149.4
2047.2
668.6
770.8
41.5
52.1
18 B
BG
3.20
3.20
2818
2818
118.0
36.2
463.7
142.3
28.6
19.0
112.4
74.7
1.92(2.06)
1.86(2.00)
1812.8
1760.0
2388.9
1977.0
429.1
841.0
28.8
64.7
19 B
BG
3.20
3.20
2818
2818
40.5
15.1
159.2
59.3
0.5
2.3
2.0
9.0
2.31(2.48)
2.27(2.44)
2182.4
2147.2
2343.6
2215.5
474.4
602.5
35.1
53.3
20 B
BG
3.20
3.20
2818
2818
64.7
-1.5
252.3
-5.9
-1.3
-4.6
-5.1
-18.1
2.08(2.24)
1.93(2.08)
1971.2
1830.4
2218.4
1806.4
599.6
1011.6
62.5
82.9
fABLET VI (cont.)
21
B
BG
3.20
3.20
2818
2818
73.8
48.7
290.0
191.4
21.8
28,4
85.7
111.6
1.98(2.13}
1.82(1.96}
1874.4
1724.8
2250.1
2027.8
567.9
790.2
69.2
90.8
22
B
BG
3.20
3.20
2818
2818
79.2
79.7
311.2
313.2
9.8
36.5
1.98(2.13)
38.5
143.4 . 1.97(2.12)
1874.4
1865.6
2224.1
2322.2
593.9
495.8
76.1
51.1
23
B
BG
3.20
3.20
2818
2818
90.0
79.5
353.7
312.4
30.1
29.7
118.3
116.7
1909.6
1812.8
2381.6
2241.9
436.4
576.1
52.0
54.4
2.02(2.17)
1.92(2.06)
Calculations:
Same as for Table V, except butyrate quotients corrected
by quotients from tissues alone; quotients for butyrate
with glycogen corrected by quotients from tissue plus
glycogen.
TABLE VII
SUMMARY TABLE OF COMPARATIVE ACETOACETATE AND HYLROXYBUTYRATE RECOVERY
AND OF BUTYRATE OXIDATION OF LIVER SLICES IN BUTYRATE ALONE OR WITH GLYCOGEN
Exp. 1
Acetone bodies recovered per 10 mg. dry tissue per Hour
Acetoacetate
B
BG
14
15
16
17
18
19
20
21
22
23
361.3
196.4
164.2
266.5
311.1
118.0
262.9
353.8
399.0
420.9
242.2
179.5
166.7
234.3
109.4
52.5
-4.8
220.1
322.9
294.6
Hydroxybutyrate
B
BG
43.2
23.7
58.2
35.4
75.4
. 1.5
-5.3
104.6
49.4
140.6
124.1
27.5
34.6
55.0
57.4
8.0
-14.8
128.3
147.8
110.0
Butyrate oxidized per 10 mg.
dry tissue per hour
Total
B
BG
B
BG
404.5
220.1
222.4
301.9
386.5
119.5
257.6
458.4
448.4
561.7
366.3’
207.0.
201.3
289.3
166.8
60.5
-19.6
348.4
470,7
404.6
290.8
538.2
478.4
415.2
287.9
351.5
624.6
692.5
761.4
519.5
467.9
675.0
586.6
521.1
646.7
533.2
829.1
908.3
511.1
543.5
338.1
251.5
.496.0
622.2
DISCUSSION
The present experiments offer cogent proof that the
presence of carbohydrate accelerates the rate at which butyric
acid disappears from a medium In which liver slices are
suspended.
Thus It was shown In every case that the total
butyrate (as acetoacetate, hydroxybutyrate, and unchanged
butyrate) remaining in the medium at the end of the tests
was invariably less, if glycogen had been added to the
butyrate substrate.
In a series of ten tests in which slices from the
same liver were placed In respirometers containing respectively
phosphate buffer solution alone, plus glycogen, plus butyrate,
and plus butyrate and glycogen, it was found that the average
butyrate which disappears beyond the ketone body stage after
the addition of this component alone was 496.0 micrograms
per 10 milligrams dry liver per hour, while the level found
when glycogen as well as butyrate was added amounted to
approximately 622.2 micrograms per 10 milligrams per hour,
which corresponds with an increased rate of disappearance
of butyric acid of 25.4 per cent over the basal level.
Although a considerable variability obtains in the
basal rate of disappearance of butyrate in different livers,
the comparative results with and without glycogen were all
48
carried out simultaneously on slices from the same livers.
Despite the considerable differences obtained, the results
are significant.
This is shown by the fact that in 9 out of
10 cases the basal rate of butyrate disappearance does not
equal the level found when glycogen and butyrate were both
present in the medium.
In addition to the greater rate of butyrate disappear­
ance exhibited by the sections treated with glycogen, there
is also a marked decrease in the level of total ketone
bodies which remain at the end of the test.
Although the
decrease is not always noted in both of the separate fractions
(acetoacetate and hydroxybutyrate), it was found that the
total ketones in all cases except experiment No. 22, Table VII,
were lower than those found in the vessel with butyrate but
without glycogen.
The average total ketone bodies in the
liver slices immersed in butyrate were 338.1 micrograms per
10 milligrams of dry liver tissue, while the mean for those
tests with slices of corresponding livers to which glycogen
had been added had been decreased to 251.5 micrograms.
The decreased amount of the ketone bodies with the
addition of glycogen must be due to a more rapid disappear­
ance of these constituents.
If It were due to a suppression
in the formation from exogenous butyric acid, then the
butyric acid left at the end of the tests In those cases
49
should be Increased.
Actually, the total butyrate which
still remained unchanged at the conclusion of the experiments
was usually slightly less In the glycogen-butyrate medium
than In the tests vihere butyrate alone was used.
Another possible reason which may be advanced to
explain the lowered amount of ketone bodies in the carbohydrate-supplemented media is that a decrease in the endogenous
ketosia takes place.
Since less ketone bodies would then
result spontaneously, a lowered content of the acetone bodies
might be found even though the rate of production from butyric
acid were unaltered.
However, in both groups of experiments
a correction has been made for the rate of the endogenous
content by subtracting from the butyrate and butyrate-glycogen
tests, the values of ketone bodies found respectively with
liver slices in the basal medium alone and with glycogen.
The validity of the experiments reported here is also
indicated by the fact that the results with butyrate closely
approximate those of Quastel both in regards to oxygen and
acetoacetlc acid quotients.
in Table VIII which follows.
The close comparison is evident
TABLE VIII
THE VALUES FOE Q02 ^
$AG OBTAINED WITH LIVER SLICES OF RATS
IMMERSED IN VARIOUS CONCENTRATIONS OF BUTTRATE COMPARED WITH
RESULTS REPORTED BY JOWETT AND QUASTEL (1935).
Biochem. J. Z& 2159 (1935)
Glycerophosphate Buffer
Jowett and Quastel
Rat liver. Oxygen
Sodixun phosphate Buffer
Fatty Aoid
Butyrie
0
Cone. H
QOj
‘AC
9.1,
5.3,
7.6,
. *
8.9
5.6
8.0
1.14, 1.13
1.19, 1.08
1.08, 1.12
0.01
10.0,
7.5,
9.3,
9.9
8.8
7.8
4.34, 4.21
3.33, 4.47
2.96, 2.77
0.01
0.02.
12.0,
7.8,
10.7,
9.9
8.1
9.6
4.39, 3.99
4.38, 4.37
4.02, 3.34
9.77(P.2165)
9.43(P.2166)
0.96(P.2165)
1.00(P.2166)
12.74(P.2165)
12.34(P.2166)
4.55(P.2165)
4.13(P.2166)
3.42-5.68(9- exp.)
(P.2165)
51
The results also are supported by the experiments of Quastel
(1935), where it was demonstrated that the addition of glyco­
gen decreases the aeetoacetic acid quotient although he also
found decreased oxygen quotients (from 9*9 to 7*0)•
However,
an attempt to repeat this work (1934) was unsuccessful.
Several reasons may be suggested for this difficulty.
The
animals used were presumably unfasted, hence the liver
glycogen was probably normal.
more glycogen.
There was then no need of
In fact, excessive glycogen might have
interferred with the oxidative process, a suggestion which
might also explain the decrease in oxygen quotients of
approximately 30 per cent.
Differences in oxygen quotients,
of course, occur with different tissue slices, but 30 per
cent is a very significant figure.
Further, no mention is
made as to the method of preparing the glycogen solution.
In order to be certain that changes in osmotic pressure
would be minimized, in this study the glycogen was prepared
in 0.16 M sodium chloride.
Also, glycogen equivalent to
one per cent of the tissue medium was used instead of 0.5
per cent (Quastel, 1933), and was prepared 48 hours before
use and refrigerated.
Finally, to show real differences In
ketone body content, the 48 hour fasted livers which exhibit
greater spontaneous ketosia would be preferable.
Therefore,
in the remainder of the experiments reported here the livers
52
from 48 hour fasted animals were used.
Consideration of
Table IV shows that on the whole the oxygen quotients were
comparable In the presence and absence of glycogen.
The
respiration, therefore, was not appreciably altered, but the
acetoacetic acid quotient was.
Further support of the work is to be found in the
fact that the results on the ketolytic effect of carbohy­
drate also agree with those reported by Cohen and Stark
(1938).
However, these investigators failed to report
their oxygen quotients which are necessary to evaluate the
validity of the experiments.
Lowered ketone body content
may originate as readily with a poorly metabolizing or
oxygen-starved tissue as by a ketolytic effect.
Moreover,
for a complete solution of the problem, a simultaneous
determination of butyrate is also necessary, a procedure
not carried out by any of the other investigators.
The variability in the amount of butyric acid presum­
ably completely oxidized by the tissue from one liver as
compared with that of other livers indicates that the metabolic
processes Involved are specific for the individual liver.
other words, each liver Is a law unto itself.
In
Hence, this
type of data is not subject to statistical treatment.
Calcula­
tion by tha Fisher "t" (1934) method gives a value of 1.35,
which is not significant.
Nutritional condition is probably
53
involved, and the storage of fat and other substances as well
as glycogen are important factors.
Some evidence for altered
metabolism may be seen from experiments Nos. 2 and 3, in
Table IV, in which very fatty livers were employed.
The
increase in ketosis when glycogen was added to butyrate is
apparent from the extremely high total ketone quotients found,
16.00 and 19.54.
Respiration studies with tissue from fatty
livers seem certainly worthy of some future investigation.
As to the method of analyzing for residual butyric
acid, further comments should be made.
The acid obtained
by distilling the filtrate remaining after precipitation of
the ketone substances beyond any question was butyric.
It
is true that such acids as lactic might be thought to interfere.
If this were the case, then the distillates obtained from
tissue media to which no substrates were added should show a
titration value much higher than that for distilled water.
This, however, was not the case.
No tltratable acid was
found to be present in the tissue medium which lacked butyrate.
In fact, the titration readings from the media in which tissue
had metabolized without added substrate or with glycogen were
always comparable with those from an equal volume of distilled
water.
Also, when the ketone quotients from added butyrate
were low, more residual butyric acid was found.
However, to
make corrections that would be beyond any criticism, the
54
titration values of the tissue media with added glycogen
were always subtracted from the corresponding values of media
with added butyrate and glycogen, while those of media without
any substrate were subtracted from the corresponding values
for media with added butyrate.
Prom such calculations! the
disappearance of more butyric acid occurred when glycogen had
been added to the medium.
The fact that no volatile acid is found in the liver
tissue when butyrate is absent indicates the improbability
that butyric acid is an Intermediate in the normal oxidation
of
tax.
If the ketone bodies originate by multiple alternate
oxidation, there would be no necessity for butyrate to be
postulated as an intermediate.
The explanation of the increased disappearance of
butyric acid in the presence of glycogen can only be made
on the basis of ketolysis.
Glycogen usually decreased the
endogenous ketone body content, true, but it also decreased
the exogenous.
This must have occurred by oxidizing butyric
acid beyond the ketone body stage.
Hence evidence is
hereby added to that already accumulated which supports the
theory that “ketolysis; rather than antiketogenesis is the
primary mechanism” whereby carbohydrates effect a decrease
in the ketone body content of liver tissue.
CONCLUSIONS
1*
The effects of fasting on the acetoacetate content
of liver tissue from male rats have been investigated by use
of the Warburg technique.
The highest and least variable
values were reached after a fasting period of 48 hours.
2.
For sectioning liver tissue an alternative procedure
has been devised which involves the use of a handle constructed
of spring metal which holds the ordinary double-edged safety
razor blade.
3.
& quantitative study has been made of the effect
of glycogen on the oxidation of butyrate by liver slices.
Butyrate has been recovered as acetoacetlc.jrcid, betahydroxybutyric, acid (both according to the method of Edson) ,
and as the residual, unchanged acid.
4. ^A^method for the determination of residual butyric
acid in tissue media has been developed, a recovery value of
better than 93 per cent having bean shown.
5.
Experiments have been reported which show that
glycogen added to tissue media lowers both the endogenous
and the exogenous content of rat liver sections.
6.
Evidence has been presented which supports the
theory that the effect of carbohydrate on fat oxidation is
ketolytic.
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TABLE A.
DATA FOR ONE D E TE R M IN A TIO N OF VE SS EL CONSTANTS
Manometer
. #1
:- #2 ...
#3_
#4
#5
_
#6__
(#7)
2.0
1.1
0.1
3.2
2.0
1.1
0.1
3.2
2.0
1.1
0.1
3.2
2.0
1.1
0.1
3.2
2.0
1.1
0.1
3.2
2.0
.1
0.1
3.2
3.1
0.1
3.2
42.8
36.0
34*8
34-9
35.6
61.8
58.9
58.2
58.7
59.7
76.0
76.3
76.1
76.8
77.9
65.5
65.4
65.5
65.9
67.2
69.2
69.9
69.4
69.8
70.7
62.2
64.O
63.9
64.2
65.1
92.0
94*3
94.9
95.4
96.7
252.0
249.0
248.9
248.3
246.9
246.8
280.0
279.6
279.0
266.3
264.8
264.3
250.8
249.8
249.3
248.2
247.2
246.9
111.5
113.2
113.8
Difference
213.3
187.1
201.1
197.1
178.6
181.8
17.1
Diff. cor.
196.2
170.0
I84.O
180.0
161.5
164.7
0•OlMNaHCO^-ml.
h 2o
2/3NH2SO^-ml.
Readings
Acid tipped in
Readings
TABLE B
SUMMARY OF A S E R IE S OF D E TER M IN A TIO N S OF VESSEL CONSTANTS
Manometer
Differences, cor.
#1
#2
195.0
194*1
195*3
195*0
170.6
170.1
171.0
170.6
#3
•
182.9
184.4
I84.O
182.9
198.6
197.2
198.8
196.9
198.1
Average
#4
196.2
172.5
172.6
169.6
161.8
168.9
168.6
170.0
180.8
181.2
184.0
196.5 .
169.7
182.9
183.8
185.4
183.6
181.7
#5
#6
#7
160.9
I64.4
158.3
176.1
175.7
175.1
176.3
163.4
163.3
163.1
162.9
163.5
159.1
180.0
161.1
159.1 •
161.5.
158.8
158.4
160.8
160.0
164.7
182.9
161.8
160.8
176.2
172.0
174-6
172,3
174*8
3200
2.269
2.627
2.438
2.438
2.756
2.773
2.551
kQ2 VF = 3200
1.955
2.292
2.102
2.102
2.420
2.437
2.215
kC02 VF = 3500
2.268 •
2.606
2.416
2.416
2.733
2.750
2.529
kC02 ^F
~
TABLE C
TYPICAL RESPIRATION EXPERIMENT
Rat #7909, 300 gms., fasted 48 hours.
Manometer
Flask:
Salt Mixture
Buffer
Glycogen
Butyrate, 0.16M
NaCI, 0.16M
Inner oup:
NaOH, 2N
#1
#2
#3
0.3
0.6
0.3
0.6
1.9
0.3
0.6
2.1
0.2
0.2
0.2
+3
Liver Slices
39°C * o.02°
+3
(#4)
#5
#6
#7
0.3
0.6
0.3
0.6
0.2
1.9
0.3
0.6
1.9
0.2
2.1
0.2
1.9
0.3
0.6
1.9
0.2
0.2
0.2
0.2
0.2
0.2
+2
*3
+3
43
In bath
Readings
5:35
5:50
6:20
6:50
7:20
Tissue out
7:35
Flasks returned
Readings
8:10
8:25
8:28
8:31
8:34
8:37
70.0
65.0
62.8
62.2
63.8
58.3
58.6
59.8
61.0
62% 6
63.9
62.8
62.9
63.7
64.8
77.8
76.7
76.8
77.8
79.2
65.2
65.9
66.8
67.1
67.7
43.6
41.8
41.0
41.0
41.0
69.9
70.9
72.0
73.6
74.8
Aniline tipped in
Readings
8:40
9:20
9:25
9:30
89.8
91.2
92.9
83.8
83.8
84.7
118.5
118.9
119.8
91.9
92.2
92.9
114.8
114.7
114.7
82.1
81.9
82.1
115.5
115.8
116.9
285.9
259.6
238.9
218.9
278.0
256.0
238.0
219.8
224.6
188.0
157.5
128.4
232.7
224.8
221.0
217.2
221.8
185.9
157.9
132.1
224.6
193.4
168.1
141.9
264.3
232.1
205.6
180.3
pH checked. Alkali washed out. 0.3ml. of l.GN acetic acid added to flask.
Change in pH checked. 0.2ml aniline run into side arm. V™ = 3500.
'TABLE G (oont.)
Tissue wt. mg*
A \
A hA -oor.
8.7
8.7
8.5
20.7
18.0
30.5
16.9
14.2
26.7
3.8
8.5
9.7
8.2
28.0
25.3
26.5
24.2
21.5
22.7
°2
ko -Vp = 3200
1.955
2.292
Q°2
7.59
7.48
A
hG02
^
A ha02-oor.
w
qac
»
2.102
2.42
13.21
29.1
22.1
55.0
15.4
8.4
41.3
13.7
2.437
2.215
13.76
10.79
12.26
47.0
41.1
42.1
33.3
27.4
28.4
2.268
2.606
2.416
2.733
2.75
• 2.529
2.07
1.26
5.86
5.36
3.88
4.37
= 3500
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