This investigation was undertaken with the purpose of determining whether insulin, alone or in the presence of certain animal fluids, has any influence upon glucose in vitro. The establishment of such an influence might have much significance in relation both to the study of carbohydrate metabolism and to the development of methods of assaying insulin

Alles, Gordon A.

Winegarden, Howard M.

English

Caltech Authors - Main

OXIDATION OF GLUCOSE BY IODINE IN THE PRESENCE 
OF INSULIN. 
BY GORDON A. ALLES AND HOWARD M. WINEGARDEN. 
(From the Gates Cltemical Laboratory of the California Institute of 
Technology, Pasadena.) 
(Received for publication, September 13, 1923.) 
1. Purpose of this Investigation. 
This investigation was undertaken with the purpose of deter- 
mining whether insulin, alone or in the presence of certain animal 
fluids, has any influence upon glucose in vitro. The establishment 
of such an influence might have much significance in relation both 
to the study of carbohydrate metabolism and to the development 
of methods of assaying insulin. 
For this purpose it seemed desirable to use a property for 
determining the glucose content which is highly specific for this 
substance and would not be likely to be shown in at all the same 
degree by products that might result from a change occurring 
under the influence of the insulin. The rate of oxidation of glucose 
(CJ31.20e) to gluconic acid (CaH1207) by iodine in nearly neutral 
solution seemed to be an especially suitable property; preliminary 
experiments on the oxidation of glucose, mannose, fructose, and 
sucrose by iodine having oonlirmed the results of previous inves- 
tigators that the rates are widely different for these different 
compounds. A series of experiments was therefore made on the 
comparative rates of this oxidation, using in one case a pure 
glucose solution, and in other cases glucose solutions which had 
been previously treated with aqueous insulin extract, alone or 
mixed with liver extract, blood serum, or oxalated blood. 
This investigation forms part of a series of researches on the 
chemical nature and behavior of insulin undertaken in this labora- 
tory under the general direction of Prof. A. A. Noyes, to whom 
we desire to express our thanks for advice as to the work and for his 
aid in the preparation of it for publication. This investigation was 
225 
Oxidation of Glucose by Iodine 
assisted on the financial side from the flmds which have been made 
available by Dr. Bertnard Smith for the general prosecution of 
insulin researches in this laboratory. We are also indebted to Dr. 
Smith, and to his associate Dr. Howard West, for cordial coopera- 
tion and assistance on the biological sides of this investigation; 
also to Mr. Albert L. Raymond of this laboratory for many 
valuable suggestions. 
2. Previous Researches. 
Various investigators have already studied the reaction between 
sugars and iodine, with reference to the development of methods 
of analysis. Thus, Romijnl early showed that glucose is quanti- 
tatively oxidized by iodine in alkaline solution to gluconic acid. 
He found that aldoses, in general, ,are oxidized under these con- 
ditions, while ketoses remain unchanged; and he devised a method 
of sugar analysis based on this principle. As the results obtained 
with potassium or sodium hydroxide were rather irregular, hc 
substituted sodium borate for the alkali. The reaction, although 
slow, proved to be quantitative in this weakly alkaline solution. 
Bougault2 substituted sodium carbonate for the borate; and this 
gave a much more rapid oxidation and led to a satisfactory method 
of analysis. Willstatter and Schude13 found that the reaction 
proceeded smoothly to completion when 0.1 N solutions of iodine 
and sodium hydroxide in the proportion of 2:3 were used. Colin 
and Li6vin4 modified Bougauh’s procedure by using disodium 
hydrogen phosphate in the place of the carbonate. Cajori6 has 
recently published an article in which he gives a method of sepa- 
rately determining glucose, fructose, sucrose, and maltose, in 
mixtures; this method being based on the different behaviors of 
these sugars towards iodine and towards cupric hydroxide. 
The effect of insulin on glucose has been studied by Winter 
and Smith.6 These investigators made a series of experiments 
1 Romijn, G., 2. anal. Chem., 1897, xxxvi, 349. 
% Bougault, J., J. pharm. et chim., 1917, xvi, series 7, 97; Compt. rend. 
Acad., 1917, clxiv, 1008. 
* Willstitter, R., and Schudel, G., Ber. them. Ges., 1918, li, 780. 
4 Colin, H., and LiBvin, O., Bull. Sot. chim., 1918, xxiii, series 4, 403. 
6 Cajori, F. A., J. Biol. Chem., 1922, liv, 617. 
6 Winter, L. B., and Smith, W., Brit. Med. J., 1923, i, 12; cf. J. PhysioZ., 
1922-23, lvii, 100. 
G. A. Alles and H. M. Winegarden 227 
upon the change in optical rotation produced in glucose and fructose 
solutions by the addition of small amounts of insulin and liver 
extracts. They thought they detected, in the case of both sugars, 
an appreciable change, which reached a maximum in 2 to 4 days 
and was greatly increased by the addition of phosphate solution. 
They suggested that the function of insulin might be the activation 
of the enzyme (presumably present in the liver) which is respon- 
sible for the transformation of ordinary (I! /3-glucose into the 7 
form. The y sugars are known to be very reactive chemically: 
in summarizing their properties, Hewitt7 notes their great activity 
toward oxidizing agents and their marked instability in the presence 
of acid and alkali. 
3. Experimental Procedure and Materials. 
The oxidations were carried out in a nearly neutral solution 
with an excess of iodine, so as to insure a nearly complete cover- 
sion of glucose into gluconic acid in a convenient period of time. 
To give the proper acidity, solutions 0.3 molal in NaHC03 or 
0.15 molal in Na2HP04 were used. In these solutions the reaction 
is practically complete at room temperature in 23 hours. About 
five times the theoretical amount of iodine was employed. The 
oxidations were carried on simultaneously in 100 cc. rubber 
stoppered conical flasks. Since the runs compared were always 
parallel, it was not thought necessary to maintain a fixed tem- 
perature. The temperature, however, did not vary over !F’C. 
throughout the entire work; and not more than a few tenths of a 
degree during a given set of oxidations. 
The samples of sugars used were obtained as follows: sucrosefrom 
the U. S. Bureau of Standards; fructose, from Merck; and glucose 
and mannose, from Kahlbaum. Fresh solutions of the glucose 
were made up from time to time. 
4. Rates of Oxidation of Glucose, Mannose, Fructose, and 
Sucrose. 
The chemical equation expressing the oxidation of glucose or 
mannose by iodine is as follows: 
CbHlsOs + 1% + He0 = C&e07 + 2HI 
7 Hewitt, J. A., Brit. lkfed. J., 1923, i, 590. 
228 Oxidation of Glucose by Iodme 
The reaction rate at any moment should therefore be propor- 
tional to the prevailing concentrations both of the sugar and of the 
iodine, provided the temperature remains constant, and the 
hydrogen ion concentration, which has a large effect on the rate, 
also remains unchanged. That is, representing by A and B the 
initial concentrations of the sugar and iodine, respectively, by x 
the fraction of the sugar transformed at any time t, by 2 the 
rate at which this fraction increases with the time, and by k the. 
specific reaction rate at the given temperature and hydrogen ion 
concentration, the rate should be expressed by the equation: 
A$=k(A-Az)(B-AAz);or$=k(l-XT) 
This equation yields on integration the following expression for 
the specific reaction rate. 
B 
0.4343 
k = t (B 
0 
- A) loglo B 
Ti -x 
0 
x (1 - 2) 
Reaction mixtures were made up in the case of each of these 
sugars by mixing the following solutions: 10.0 cc. of 0.1 per cent 
sugar solution; 10.0 cc. of 0.025 molal Iz in 0.06 molal KI; 15.0 cc. 
of 1 molal NaHC03 or of 0.5 molal Na2HP04; and 15.0 cc. of 
water. The mixtures were, therefore, initially 0.00111 molal in 
monosaccharide, 0.00500 molal in Iz, and either 0.3 molal in 
NaHC03 or 0.15 molal in Na2HP04. Seven such mixtures of each 
sugar were placed in 100 cc. rubber stoppered conical flasks, and 
kept at the room temperature of 22-23’. From time to time one 
of the mixtures was removed, and 5 cc. of 6 N HzS04 were added, 
whereby the reaction was stopped; the decrease in the free iodine 
content was then determined by titrating it with a standard 
Na.&03 solution, which was 0.0200 molal in the NaHC03 ex- 
periments and 0.0189 molal in the NsJ!IPOa experiments. 
The results with glucose and mannose are given in Table I. 
In the columns headed 100 x are given the percentage of glucose 
oxidized as calculated from the decrease in iodine content; and in 
those headed k, the values of the specific reaction rate calculated 
by the above equation. The values given for the oxidation in the 
G. A. Alles and H. M. Winegarden 229 
NaHCOa solutions are the average of the three similar runs; those 
in the NasHPOd solutions represent one set of determinations. 
It will be seen from Table I that, as would be expected for 
a bimolecular reaction taking place under constant catalytic con- 
ditions and temperature, the values of the specific reaction rate k 
are fairly constant for both glucose and mannose in the presence 
of the hydrocarbonate. In the presence of the hydrophosphate, 
however, the values of k show a progressive decrease. 
Similar runs with fructose and sucrose showed an iodine con- 
sumption at the end of 160 minutes equivalent to only 6 per cent 
oxidation of the sugar. 
TABLE I. 
Rate of Oxidation of Glucose and Mannose by Iodine at %?-.W. 
Time. 
nztn. 
5 
10 
15 
20 
40 
80 
160 
- 
I 
100 z I k 100 z k 
29.5 14.6 
50.0 14.7 
65.0 15.7 
74.2 15.0 
92.1 13.1 
98.7 
100.5 
- 
Glucose with 
NldIPO4. 
- 
I 
44.8 19.2 
56.1 17.7 
63.9 14.5 
70.6 13.7 
84.6 10.7 
93.5 
100.4 
- 
- 
100 z 
12.3 
22.5 
31.5 
38.5 
59.0 
77.8 
92.5 
5.28 
5.24 
5.23 
5.13 
4.85 
4.23 
100 z k 
~- 
19.4 8.8 
27.9 6.7 
31.2 5.1 
38.9 5.1 
52.1 3.9 
68.0 3.1 
81.7 
These results conform with those of earlier investigators in that 
the aldoses are oxidized, while the ketose and biose remain practi- 
cally unchanged. The small effect which is noted in the fructose 
and sucrose oxidations may well be due to experimental error. 
It is evident that even mannose and glucose differ greatly from 
each other in their rates of oxidation in both the hydrocarbonate 
and hydrophosphate solutions. The difference between the rates 
of glucose, mannose, and fructose should therefore be marked 
enough to indicate any transformation of glucose into isomeric 
hexoses which might occur in the presence of insulin. 
6. Oxidation of Glucose in the Presence of Insulin. 
The insulin used throughout the work was prepared by the proc- 
ess described by Collip8 and was further purified by precipitation 
8 Collip, J. B., Tr. Roy. Sot. Canada, 1922, xvi, series 3, section v. 
230 Oxidation of Glucose by Iodine 
from aqueous solution by changing the hydrogen ion concentration. 
The precipitate was washed with ether and dried over calcium 
chloride. A 0.1 per cent solution of the dried substance was made 
up by dissolving it in approximately 0.001 N hydrochloric acid. 
Previous tests showed that the insulin was stable in this solution. 
The activity of this insulin was tested on rabbits several times: 
0.3 cc. per kilo weight of rabbit lowered the blood sugar of a 
normal rabbit from 110 to 45 mg. per 100 cc. in 1 hour, and the 
convulsive dose was between 0.3 and 0.4 cc. The insulin was 
also found to retain most of its activity on standing for 1 hour in 
solutions with an alkalinity similar to that of the oxidation mix- 
tures. In all the calculations, the iodine taken up by the insulin 
TABLE II. 
Oxidation of Glucose by Iodine in Presence of Insulin. 
Time. 
min. 
5 
10 
15 
20 
40 
80 
160 
Solution 0.30 molsl in NaHCOa. I Solution 0.15 molal in N~zHPOI. 
Without With 
insulin. insulin. 
25.3 23.4 
43.7 40.7 
52.6 53.8 
61.5 60.7 
80.6 80.2 
95.7 96.3 
101.9 101.2 
-7 
'I 
- 
Difference. 
. _ 
.- 
+1.9 34.0 
+3.0 49.3 
-1.2 57.9 
-0.8 64.9 
+0.4 79.5 
-0.6 92.2 
+0.7 96.2 
- 
.- 
- 
With 
insulin. 
34.9 
47.8 
57.2 
64.2 
79.7 
91.6 
96.8 
- 
.- 
- 
DiRermoe. 
-- 
-0.9 
+1.5 
+0.7 
+0.7 
-0.2 
+0.6 
-0.6 
introduced into the reaction mixture was subtracted from the 
total iodine consumed. 
The method of procedure was exactly the same as that described 
above. The reaction mixtures were made tip just as in the experi- 
ments on the oxidation of the sugars alone; except that there were 
always run side by side duplicate mixtures differing only in the 
respect that one contained in the 50 cc. volume 1 cc. of 0.1 per 
cent insulin solution, and the other contained no insulin. The 
temperature was 22-23”. 
Table II contains the results of these experiments. The num- 
bers in the first column show the time elapsed after the mixing 
of the solutions; those in the other columns denote the percentage 
of glucose transformed. 
G. A. Alles and H. M. Winegarden 231 
It is evident from these results that insulin alone has no appre- 
ciable effect upon the oxidation of glucose by transforming it 
into either a more or less chemically reactive form. This is true 
even though a considerable excess of insulin was present over that 
required for the oxidation of the 10 mg. of glucose; thus the quan- 
tity was sufficient to lower the blood sugar of a 3 kilo rabbit 50 mg. 
per 100 cc. of blood in 1 hour. 
6. Oxidation of Glucose in the Presence of Liver Extract and Insulin. 
Both alcoholic and aqueous liver extracts were used in these 
experiments. The method of procedure was the same as that 
followed in previous experiments with the exception that the 
reaction mixtures were allowed to stand for 1 hour with theliver 
extract and insulin, before the addition of the iodine. A tem- 
perature of 22-24’ was maintained by immersing the solutions in a 
water bath. As before, one-half of the solutions contained 1 cc. 
of 0.1 per cent insulin solution (in place of 1 cc. of water), inaddi- 
tion to the other ingredients, which were as follows: 10.0 cc. of 0.1 
per cent glucose solution, 15.0 cc. of a solution 1.0 molal in NaHCOs 
or 0.5 molal in NhHP04, 2.0 cc. of alcoholic or aqueous liver 
extract, 12.0 cc. of distilled water, and 10.0 cc. of a solution 0.05 N 
in 1~ and 0.06 N in KI. 
The alcoholic liver extract was prepared in the following manner. 
Fresh beef liver was ground and intimately mixed with an equal 
volume of 95 per cent alcohol. The juice was pressed out of the 
mass, diluted to 4 volumes, and filtered. The filtered solution vas 
used in the oxidations. 
The aqueous liver extract was prepared as follows: Equal 
volumes of fresh beef liver and 0.9 per cent sodium chloride solu- 
tion were ground together thoroughly with sand in a mortar. 
The juice was then pressed out, diluted to 4 volumes, and filtered. 
Table III contains the results of the experiments in which the 
alcoholic extract was used; and Table IV those in which the 
aqueous extract was used. The figures in the first column show 
the length of time of the oxidations; those in the other columns 
show the cubic centimeters of 0.025 molal Iz consumed in the 
various reaction mixtures. The second pair of experiments in 
each table was made with half the quantity of the liver extract. 
These experiments show that glucose is unaffected by insulin 
in the presence of alcoholic or aqueous liver extract. 
232 Oxidation of Glucose by Iodine 
TABLE III. 
Oxidation of Glucose by Iodine in Presence of Alcoholic Liver Extract and 
Time. 
min. 
5 
10 
15 
20 
40 
80 
160 
5 
10 
15 
20 
40 
80 
160 
- 
- 
.- 
- 
Insulin. 
Solution 0.30 mold in NsHCOa. 
With 
insulin. 
1.63 1.69 
2.37 2.38 
2.86 2.83 
3.48 3.44 
4.23 4.14 
5.34 5.29 
6.23 6.17 
1.75 
2.46 
3.01 
3.26 
4.34 
5.21 
5.41 
1.81 
2.45 
2.97 
3.23 
4.33 
5.21 
5.41 
- 
1 
_- 
- 
xffereme. 
-0.06 
-0.01 
+o .03 
+0.04 
+o .09 
+0.05 
+0.06 
-0.06 
+0.01 
+0.04 
+0.03 
+0.01 
0.00 
0.00 
T -- 
-- 
- 
Solution 0.15 mold in NasHPOd. 
1.50 
2.09 
2.36 
2.75 
3.36 
4.14 
4.93 
1.46 
1.91 
2.23 
2.43 
2.97 
3.71 
4.46 
- 
_- 
- 
With 
insulin. 
1.50 
2.08 
2.38 
2.71 
3.37 
4.12 
4.84 
1.45 
1.90 
2.20 
2.42 
2.94 
3.69 
4.39 
- 
-- 
- 
Difference. 
0.00 
+0.01 
-0.02 
+0.04 
-0.01 
+0.02 
+o .09 
+0.01 
+0.01 
+0.03 
+o .Ol 
+0.03 
+0.02 
+0.07 
TABLE IV. 
Oxidation of Glucose by Iodine in Presence of Aqueous Liver Extract and 
Insulin. 
Time. - 
- 
With 
insulin. 
_- 
min. 
5 
10 
15 
20 
40 
80 
160 
3.96 
4.98 
5.35 
5.67 
6.44 
7.07 
7.39 
3.93 
4.98 
5.66 
6.43 
7.09 
7.45 
5 2.26 2.25 
10 2.71 2.72 
15 3.10 3.10 
20 3.47 3.48 
40 4.00 4.02 
80 4:61 4.64 
160 4.88 4.92 
T Solution 0.30 mold in NaHCO:. 
Ditkrence. 
- 
- 
- 
$0.03 
0.00 
+o .Ol 
$0.01 
-0.02 
-0.06 
+0.01 1.52 
-0.01 1.93 
0.00 2.26 
-0.01 2.55 
-0.02 2.89 
-0.03 3.24 
-0.04 3.59 
T 
- 
Solution 0.15 molsl in NarHPOa. 
3.51 
4.10 
4.40 
4.74 
5.46 
6.25 
7.02 
- 
_- 
- 
With 
insulin. Difference. 
3.49 
4.09 
4.38 
4.72 
5.27 
6.24 
7.01 
1.49 
1.90 
2.23 
2.53 
2.88 
3.24 
3.60 
- 
-- 
- 
+0.02 
+0.01 
+0.02 
.+0.02 
+0.19 
+o .Ol 
+o .Ol 
+0.03 
$0.03 
+0.03 
+0.02 
+0.01 
0.00 
-0.01 
G. A. Alles and H. M. Winegarden 233 
7. Oxidation of Glucose in the Presence of Insulin and Blood. 
In order to make the work more complete, the effect of insulin 
on the oxidation of glucose in the presence of the blood serum 
and oxalated blood was studied. As in the other experiments, the 
results consistently indicated no change in the glucose on the 
addition of insulin. 
The method of procedure was exactly the same as that followed 
in the work with liver extract. The reaction mixtures were 
changed only by substituting blood or blood serum for the liver 
extract. 
Table V contains the results of the set of experiments in which 
0.2 cc. of oxalated blood was added to each of the reaction mix- 
tures. The temperature was 23.2-23.4” 
TABLE V. 
Oxidation of Glucose in Presence of Oxalated Blood and Insulin. 
Time. 
min. 
5 
10 
15 
20 
40 
80 
160 
Solution 0.30 molal in NaHCO8. I 
Without 
insulin. 
With 
insulin. 
2.92 2.91 +0.01 
3.48 3.48 0.00 
3.83 3.89 -0.06 
4.07 4.12 -0.05 
4.75 4.77 -0.02 
5.20 5.23 -0.03 
5.64 5.64 0.00 
- 
- 
Difference. 
-I- 
_ _ 
- 
Solution 0.15 molel in NazHPOc. 
Without With 
insulin. insulin. Difference. 
2.51 
2 .,J-kl 
3.24 
3.44 
4.01 
4.60 
5.03 
2.48 +0.03 
2.92 +0.02 
3.22 +0.02 
3.42 +0.02 
4.00 +o .Ol 
4.57 +o .03 
5.05 -0.02 
SUMMARY. 
It was first shown by this investigation that, in confirmation 
of the results of others, the rate of oxidation of various sugars by 
iodine in solutions of NaHC03 or N&HP04 varies greatly with the 
nature of the sugar; thus mannose was oxidized only about one- 
third as fast as glucose, and fructose and sucrose were scarcely 
oxidized at all, under the conditions of the experiments. This 
indicated that a study of the relative rates of oxidation of glucose 
before and after treatment of it with insulin would furnish a 
sensitive means of determining whether any of the glucose had 
been transformed by it into any other substance, even into a stereo- 
merit hexose. 
234 Oxidation of Glucose by Iodine 
Strictly comparable experiments were therefore made with glu- 
cose alone, with mixtures of it with insulin, with insulin and liver 
extract, or with insulin and blood serum or oxalated blood. In no 
case was any difference detected in the rate at which the iodine 
is consumed. This shows that no appreciable reaction takes place 
between glucose and insulin even in the presence of the animal 
fluids mentioned. It indicates, therefore, that the metabolic 
process must be more complicated in chamcter; also that there is 
little promise of developing a method of assay for insulin on the 
basis of its action on glucose in glass. 


Oxidation of glucose by iodine in the presence of insulin

https://authors.library.caltech.edu/4246/1/ALLjbc23.pdf

Oxidation of glucose by iodine in the presence of insulin

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main

Caltech Authors - Main