A RAPID

MICROMETHOD
FOR THE QUANTITATIVE
ESTIMATION
OF SUGAR ALCOHOLS

BY W. R. TODD,
(From

the Department

JOHANNA
VREELAND,
EDWARD
S. WEST

of Biochemistry,

University

JANE
of Oregon

MYERS,
Medical

AND

School,

Portland)
October 17, 1938)

In studying the metabolism of sorbitol and mannitol in experimental animals (1) it is desirable to have at hand a rapid method
for the estimation of these compounds. Of the various methods
found in the literature none was deemed satisfactory for rapid
routine analysis. A method was therefore developed which lends
itself to rapid and fairly accurate estimations of sugar alcohols.
The method has been applied to the determination of sorbitol,
mannitol, dulcitol, erythritol, pentaerythritol,
and inositol, and
should be applicable to the other sugar alcohols. The reactions
involved are not specific for these compounds and consequently the
method, though satisfactory for the work to which we have
adapted it, has distinct limitations.
The principles of the Hagedorn-Jensen sugar method have been
used with modifications in technique and reagents to meet special
requirements. The method described below represents the best
procedure so far developed.
Method
Reagents1. 1.08 per cent potassium ferricyanide in water.
2. 5 per cent sodium sulfate in 3.33 N sodium hydroxide.
3. 15 per cent zinc acetate and 12 per cent potassium iodide in
combined solution.
4. Glacial acetic acid.
5. 0.005 N sodium thiosulfate.
269

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(Received for publication,

Microestimation

of Sugar Alcohols
Procedure

Titration

TABLE
I
Digerence for Sorbitol and Mannitol
Titration

Concentration

with Varying

difference,

Concentrations

0.005 N thiosulfate

me* 5 ml.
Sorbitol
ml.

0.1
0.3
0.5
0.7

0.92
2.63
4.36
5.83

Mannitol
nil.

0.93
2.73
4.17
4.96

cooled, 5 ml. of this mixed reagent are blown into each tube from a
fast flowing 5 ml. pipette.
The liberated iodine is titrated with
0.005 N thiosulfate, starch indicator being used near the end-point.
Heating Time and Factor Equivalents--In a method to be used
for routine work it is expedient that the time required to complete
an analysis be as short as possible. The length of time allowed
for the reduction is of prime importance in this connection.
Recoveries of known amounts of sugar alcohols in pure solution have
been tried with various heating periods from 10 minutes to 1 hour.
It was found that the shortest time of heating commensurate with
the accuracy desired is 30 minutes.
After this interval the reaction is fairly well completed and little error results from slight discrepancies in timing.

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Pyrex tubes (25 X 200 mm.) are charged with 5 ml. of a solution containing 0.1 to 0.7 mg. of sugar alcohol. Water blanks are
run concurrently.
3 ml. of Reagent 1 are added to each tube. 3
ml. of Reagent 2 are rapidly added to duplicate or triplicate samples as the case may be. The tubes are immediately covered with
glass bulbs and, after the contents are mixed, are placed simultaneously in a boiling water bath. The time required to add Reagent 2 to three tubes and place them in the water bath need not
exceed 1 minute.
After heating for 30 minutes the tubes are removed to a cold water bath in sets and in the same order in which
they were placed in the boiling bath, so that each set of tubes is
subjected to exactly 30 minutes heating.
Reagents 3 and 4 are
mixed in approximately equal portions and, when the tubes have

Todd, Vreeland,

Myers, and West

271

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The relation between sorbitol concentration and titration difference is not linear and a curve must be prepared for the calculation of results. This is true for all of the alcohols investigated.
Table I gives our values for sorbitol and mannitol in varying concentrations of pure solutions.
It is a relatively simple matter for
anyone wishing to make use of the method to develop curves for
his own use.
Recovery of Sorbitol and Mannitol--Recovery
of known amounts
of sorbitol and mannitol ranged from 95 to 110 per cent. Treatment with HgS04 and BaC03 (vide infra) does not interfere with
recoveries. From blood and urine 85 to 105 per cent of added
sorbitol or mannitol has been recovered regularly.
Determination in Urine and Blood-The
procedure given below
has been used in following the excretion of sorbitol in urine and
its concentration in blood after intravenous injection (1). Since
glucose and other substances reduce the ferricyanide reagent, it
is necessary to determine the sorbitol-reducing
equivalent of the
fluids before giving the sorbitol and to subtract these values from
those found following its administration.
The West-ScharlesPeterson (2) method of clarification has been used for blood and
urine.
Urine-10 ml. of urine are added to 75 ml. of water in a 250 ml.
Erlenmeyer Aask and 15 ml. of HgSOd reagent (28 per cent HgSOc
in 2 N HzS04) are added; the mixture is neutralized with BaC03
(about 28 gm.) and filtered.
1 gm. of zinc dust per 15 ml. of
filtrate is added to remove traces of mercury.
After filtering
again through a fine paper such as Whatman No. 42, the filtrate
is diluted according to the concentration of sorbitol present and
the reduction estimated as outlined.
Blood-5 ml. of blood are added to a flask containing 90 ml. of
water. After laking is complete 5 ml. of HgSOh reagent are added
and the mixture neutralized with about 9 gm. of BaC03. Zinc
dust is employed as in the case of urine filtrates.
The final filtrate
is diluted as necessary before analysis. The reducing equivalent
of sorbitol and the other alcohols is changed by the presence of
glucose in the blood. Consequently corrections must be made
for this contingency.
We have accomplished this by determining the glucose equivalent in blood filtrates free of sugar alcohol and then determining

272

Microestimation

of Sugar Alcohols

the reduction in another filtrate of the same blood after the addition of known amounts of a sugar alcohol. By simple calculation
a factor for the compound in question is obtained.
Large variations in the glucose content of blood samples under experimental
conditions necessitate the determination
of such factors concurrently with sugar alcohol estimations.
DISCUSSION

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Potassium ferricyanide is not stable in strong alkali; a slow

decomposition
occurs with the production of ferric hydroxide.
The ferricyanide reagent is thus prepared in aqueous solution.
Sugar alcohols react in the cold with ferricyanide in alkaline solution and for this reason samples for analysis are placed in the water
bath immediately
following the addition of the alkali.
Another
point that must be considered is the reaction between the potassium iodide-zinc acetate mixture and glacial acetic acid. When
these are mixed, iodine is slowly liberated and it is essential that
this solution be prepared immediately
before use. The end-point
in the titration is well defined and little difficulty is encountered
if good lighting is provided.
The method outlined has proved very satisfactory for biological
work even in view of the limitations
resulting from the fact that
many substances in physiological solutions besides sugar alcohols
react with the ferricyanide solution in the strongly alkaline
medium employed.
In blood filtrates, for instance, recoveries of
added sorbitol were apparently very poor before it was ascertained
that sorbitol exhibits less reduction in the presence than in the
absence of glucose. The reason for this we believe is that glucose
is attacked far more readily at the high pH by the ferricyanide
reagent than is sorbitol.
The result is that less ferricyanide is
present after a short initial period of reduction to oxidize the
sorbitol.
It is, therefore, necessary in blood work to determine a
factor for sorbitol in the presence of glucose. After this rather
simple expedient was worked out, it was found that recoveries of
added sorbitol from blood were sufficiently accurate for our purpose. Because the sugar alcohols do not reduce the ordinary copper reagents, it is possible to determine blood sugar in the presence
of these compounds.

Todd, Vreeland,

Myers, and West

273

SUMMARY

A rapid method for the estimation of sugar alcohols is pre0.1 to 0.7 mg. in 5 ml. of solution may be determined in
sented.
properly clarified filtrates of blood, urine, etc.
BIBLIOGRAPHY

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1. Todd, W. R., Myers, J., and West, E. S., J. Biol. Chem., 137,275 (1939).
2. West, E. S., Scharles, I?. H., and Peterson, V. L., J. Biol. Chem., 82,
137 (1929).