A RAPID MICROMETHOD FOR THE QUANTITATIVE

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Jul 27, 2018 - 5 per cent sodium sulfate in 3.33 N sodium hydroxide. 3. 15 per ... Titration difference,. 0.005 N thiosulfate. Concentration me* 5 ml. 0.1. 0.3. 0.5.
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) (Received for publication,

October 17, 1938)

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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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.

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

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). Downloaded from http://www.jbc.org/ by guest on July 27, 2018

A RAPID MICROMETHOD FOR THE QUANTITATIVE ESTIMATION OF SUGAR ALCOHOLS W. R. Todd, Johanna Vreeland, Jane Myers and Edward S. West J. Biol. Chem. 1939, 127:269-273.

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