THE
SPECIFIC
SOLUTIONS
(From
the Laboratory
BY
ROTATORY
POWER OF GLUCOSE-INSULIN
IN CONTACT
WITH MUSCLE
TISSUE
IN VITRO.
HOWARD H. BEARD
of Biochemistry,
University,
AND
VERNON JERSEY.
of Medicine,
Western
School
Cleveland.)
Reserve
(Received for publication, July 20, 1926.)
In a recent series of papers Lundsgaard
and Holboll (1) have
reported the effects upon the specific rotatory
power of glucoseinsulin solutions in contact with muscle tissue in vitro. They
advance the theory that under the conditions of their experiments
there is a fall in the specific rotation of the glucose solution from
the usual value, 52.5’, for a, @glucose to a new value varying
from 22 to 40”. This form they call new-glucose.
In later papers
(2, 3, 4, 5) they demonstrate
that this new form of glucose is
present in various biological fluids, but is absent from the blood of
diabetic patients.
The results of these investigations
seem to be of considerable
importance
to our understanding
of carbohydrate
metabolism.
However,
Barbour (6) and Paul (7) were unable to confirm the
results of Lundsgaard
and Holboll in this connection.
Soon after the appearance of the second series of papers by the
latter investigators
we began to make a study of the control
experiments
necessary in an investigation
of this nature before
attempting
to repeat their work
on glucose-insulin-muscle
solutions.
Control Experiments.
C.P. glucose was used in all cases.
Its specific
Pfanstiehl’s
rotatory power varied from 49.7 to 53” at equilibrium.
The insulin was the commercial product of Eli Lilly and Company,
containing 20 units per cc. The collodion sacs were prepared
from u.s.p. collodion by filling large test-tubes,
draining 5
167
This is an Open Access article under the CC BY license.
168
Glucose-Insulin
Solutions
minutes to make sure that all the ether had escaped, then adding
80 per cent alcohol for 10 minutes, and after removing same placing the sacs in distilled water with a few drops of toluene until
ready for use. The bags were then tested for leaks.
Preliminary
experiments showed that they were impermeable to protein but
easily permeable to glucose.
Polariscopic readings were made with a Schmidt and Haensch
instrument reading in angular degrees using 2 dm. tubes.
As the
light source a 75 candle power frosted bulb was used, the rays
being filtered through a saturated solution of potassium dichromate. All readings were made at 2O”C., and an average of six
to sixteen determinations
was taken in computing results.
The
reducing value of the dialysates was determined by the HagedornJensen method (8) in quadruplicate
after diluting the samples
so that they contained about 0.1 per cent glucose.
Method.
200 cc. of a 2 per cent glucose solution, ([01]: 49 to 52.3” at
equilibrium)
in 0.9 per cent NaCl were placed in the warm room
at 37°C. for 2 hours, then 50 units of insulin or 15 gm. of fresh
muscle tissue added and allowed to remain for 2 hours longer at
the same temperature.
25 cc. samples were then removed and
dialyzed through collodion tubes against 75 cc. of 0.9 per cent
NaCl for 13 hours.
After the reducing and rotatory powers were
determined the samples were removed and the dialysates allowed
to stand at room temperature for 24 hours for further study.
In a total of 60 determinations
with the glucose-insulin
solutions we found very low values in 9 cases, whereas in the remaining experiments the variations in the specific rotatory power
were within experimental
error.
No change was observed in
the glucose-muscle solutions.
As these studies were in progress there appeared the papers of
Barbour (6) and Paul (7), both of whom were unable to observe
any discrepancy
in the reducing and rotatory
power of glucose
solutions
under various
conditions.
The former investigator
used muscle tissue from various animals, while the latter studied
both the dialysates and ultrafiltrates
from the glucose solutions.
Our next problem was to study the effects upon the specific
rotatory
power of the sugar solutions when both muscle tissue
H. H. Beard and V. Jersey
and insulin were added.
above.
169
We used the same methods as described
EXPERIMENTAL.
962.5 cc. of 2, 4, or 6 per cent glucose in 0.9 per cent NaCl
were placed in the cold room for 24 hours, then at room temTABLE
Specijic
Rotatory
Muscle
Origina
concen
tration
(1)
Glucose
dialysate.
Reduction.
(2)
(3)
Zero
reading
on Contact
with
at
Observed
COJY
rected.
(5)
(6)
(4)
Solutions
Tissue.
Reading
equilibrium.
in
Rotation.
I.
of Glucose-Insulin
Power
OXrected.
(7)
(8)
51.7"
51.7"
51.7"
51.7"
51.7"
50.2"
50.2"
50.2"
50.2"
52.3"
52.3"
52.3'
52.1'
52.1"
52.1'
0.82"
0.80"
0.81"
0.75"
0.84"
0.82"
0.86"
0.84"
0.77"
1.34"
1.41"
1.43"
1.79"
1.82"
1.82"
--
(9)
(10)
0.46"
0.44"
0.45"
0.39"
0.48"
0.46"
0.50"
0.48"
0.41"
0.99"
1.06"
1.07"
1.44"
1.47"
1.47"
49.0"t
46.0"
47.5"
42.4"
51.0"
48.0"
51.2"
50.3"
42.1"
50.8"
53.6"
54.4"
49.7"
51.4"
51.4"
- -__
per ten
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
4.00
4.00
4.00
6.00
6.00
6.00
0.47
0.47
0.47
0.46
0.47
0.48
0.49
0.48
0.47
0.97
0.98
0.98
1.45
1.43
1.40
0.44'
0.43
0.44
0.38
0.46
0.46
0.50
0.48
0.40
0.94
1.02
1.02
1.38
1.41
1.41
0.36" 2.43"
0.36" 2.43"
0.36" 2.43"
0.36" 2.43"
0.36" 2.43"
0.36" 2.37"
0.36" 2.37"
0.36" 2.37"
0.36" 2.37"
0.35" 4.53"
0.35" 4.53"
0.35" 4.53"
0.35" 6.59"
0.35" 6.59"
0.35" 6.59"
2.07"
2.07"
2.07"
2.07"
2.07"
2.01"
2.01"
2.01"
2.01"
4.18"
4.18"
4.18"
6.24"
6.24"
6.24"
100 0.46
X
= 0.444.
2xc
100 0.46
X
= 49".
i- l&o =
2 x 0.47
* 51.7 =
perature for 5 hours.
At equilibrium the specific rotatory power
was determined.
In some experiments
25 cc. of phosphate
buffer mixtures of pH 7.38 were added, while no buffers were
used in other cases. The solutions were placed in the warm
room at 37°C. for 1 hour; then 250 units of insulin and 75 gm. of
fresh muscle tissue added. The mixtures were allowed to remain
at 37°C. for 2 hours with continual shaking.
170
Glucose-Insulin
Solutions
The animals, rats or rabbits, were killed by a blow on the back
of the head, and the muscle tissue removed and placed in the
solution.
This operation usually required about 10 minutes.
After 2 hours incubation, 25 cc. samples were dialyzed through
the specially prepared collodion tubes into 75 cc. of 0.9 per cent
NaCl.
The height of the liquids on both sides of the membrane
was the same. The specific rotatory power of the solutions was
determined
(a) after equilibrium was reached, (6) after warming at 37°C. for 2 hours, and (c) after dialysis at room temperature for 13 hours.
The reducing power was determined by the
Hagedorn-Jensen
method (8) in quadruplicate.
A total of 15 experiments was performed.
We did not vary
the conditions, except in one instance, as we were primarily
interested in proving the presence or absence of new-glucose in
the dialysates by means of its low specific rotatory
power.
A
protocol of one typical experiment is given in Table I.
DISCUSSION.
An examination of the table shows that the same small errors
occur here as in the case of the glucose-insulin
solutions.
It
seems to us that the chief source of error lies in the small
dijference between the zero and observed readings.
Using the
dialysates from 2 per cent glucose solutions, we obtained readings
varying from 0.75 to 0.84”; then, after deducting the zero reading,
0.35”, we obtained as our final value 0.40 to 0.49”. Again, we
found that a difference of 0.01” in the observed reading would
correspond to a variation of 0.7” in the specific rotatory
power
and this is the cause of the lowered values in Column 10.
There are also very small differences between the concentration of glucose calculated from reduction and rotation.
Lundsgaard and Holboll (9) in their calculations of the latter value
assume that their glucose had a specific rotation of 52.5’.
This
is true only if they used the purest sugar obtainable.
We determined this value for our solutions before each experiment and
found that it varied from 49 to 53”; hence we used these actually
determined values (Column 7) in calculating the concentration
by rotation
(Column 3). The small differences between the
reducing and rotating figures, therefore, are due to experimental
error and not to the combined action of insulin and a substance
from muscle tissue upon the glucose molecule.
H. H. Beard and V. Jersey
171
In order to eliminate these variations
we also used 4 and 6
per cent glucose solutions, and here the discrepancies between the
reducing and rotatory
values, and also between the specific
rotatory powers, are small and are well within experimental error.
The chief point of interest in these investigations
is that we
did not observe a specific rotatory
power of the glucose-insulinmuscle solutions below 42”. In no case was a value of 22 to 40”,
corresponding
to new-glucose,
obtained.
The dialysates were allowed to stand at room temperature
for
a period of 24 hours, and in every case without exception they
were so cloudy that it was impossible to get a reading on the
polariscope.
CONCLUSIONS.
1. The specific rotatory
power of glucose-insulin
solutions in
contact with fresh muscle tissue is only slightly lower than the
usual value, +52.5”.
These variations are due to experimental
error.
With the use of larger concentrations
of glucose, the
reducing and rotatory
values and also the specific rotatory
powers agree closely.
2. We have been unable to confirm the results of Lundsgaard
and Holboll as to the production of new-glucose in vitro from the
glucose-insulin-muscle solutions.
3. The results obtained are in close agreement with those of
Barbour and Paul.
We wish to thank Eli Lilly and Company for the supply of
insulin used in these investigations.
BIBLIOGRAPHY.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Lundsgaard,
C., and Holb@ll,
S. A., J. Biol.
Chem.,
1924-25,
lxii, 453.
Lundsgaard,
C., and Holb@ll,
S. A., J. Biol. Chem.,
1925, lxv, 305.
Lundsgaard,
C., and Holbell,
S. A., J. Biol.
Chem.,
1925, lxv, 323.
Chem., 1925, lxv, 343.
Lundsgaard,
C., and Holb@ll,
S. A., J. Biol.
Chem., 1925, lxv, 363.
Lundsgaard,
C., and Holb@ll,
S. A., 1. Biol.
53.
Barbour,
A. D., J. BioZ. Chem., 1926, lxvii,
425.
Paul, J. R., J. BioZ. Chem., 1926, Ixviii,
1920, xiii, 347.
H&t,
H. F., and Hatlehol,
R., J. Biol. Chem.,
457.
Lundsgaard,
C., and Holbplll,
S. A., J. Biol. Chem., 1926, lxviii,