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A RAPID MICROMETHOD FOR THE QUANTITATIVE

ESTIMATION OF SUGAR ALCOHOLS

BY W. R. TODD, JOHANNA VREELAND, JANE MYERS, AND

EDWARD S. WEST

From the Department

of

Biochemistry, University of Oregon Medical Scho ol,

Portland)

Received for publication, October 17, 1938)

In studying the metabolism of sorbitol and mannitol in experi-

mental 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

Reagents-

1. 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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Microestimation of Sugar Alcohols

Procedure

Pyrex tubes 25 X 200 mm.) are charged with 5 ml. of a solu-

tion 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 sam-

ples as the case may be.

The tubes are immediately covered with

glass bulbs and, after the contents are mixed, are placed simul-

taneously in a boiling water bath. The time required to add Re-

agent 2 to three tubes and place them in the water bath need not

exceed 1 minute. After heating for 30 minutes the tubes are re-

moved 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

TABLE

I

Titration Digerence for Sorbitol and Mannitol with Varying Concentrations

Titration difference, 0.005 N thiosulfate

Concentration me* 5 ml .

0.1

0.3

0.5

0.7

Sorbitol

Mannitol

ml. nil.

0.92 0.93

2.63 2.73

4.36

4.17

5.83 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. Re-

coveries 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 reac-

tion is fairly well completed and little error results from slight dis-

crepancies in timing.

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Todd, Vreeland, Myers, and West 271

The relation between sorbitol concentration and titration differ-

ence is not linear and a curve must be prepared for the calcula-

tion of results. This is true for all of the alcohols investigated.

Table I gives our values for sorbitol and mannitol in varying con-

centrations 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. Treat-

ment 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-Scharles-

Peterson 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 equiv-

alent in blood filtrates free of sugar alcohol and then determining

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272 Microestimation of Sugar Alcohols

the reduction in another filtrate of the same blood after the addi-

tion of known amounts of a sugar alcohol. By simple calculation

a factor for the compound in question is obtained. Large varia-

tions in the glucose content of blood samples under experimental

conditions necessitate the determination of such factors con-

currently with sugar alcohol estimations.

DISCUSSION

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 solu-

tion 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 potas-

sium 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 pur-

pose. Because the sugar alcohols do not reduce the ordinary cop-

per reagents, it is possible to determine blood sugar in the presence

of these compounds.

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Todd, Vreeland, Myers, and West 273

SUMMARY

A rapid method for the estimation of sugar alcohols is pre-

sented.

0.1 to 0.7 mg. in 5 ml. of solution may be determined in

properly clarified filtrates of blood, urine, etc.

BIBLIOGRAPHY

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).

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