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A METHOD FOR THE DETERMINATION OF MANNITOL

IN PLASMA AND URINE*

BY

A. C. CORCORAN

AND

IRVINE H. PAGE

(From the Research Division of the Cleveland Clinic Foundation, Cleveland)

(Received for publication, June 20, 1947)

Concurrent analyses of mannitol in urine solutions and protein-free

plasma filtrate are used in the measurement of renal function.

Two types of procedures have been used in determining mannitol con-

centration. One depends on the reduction of ferricyanide by mannitol in

strongly alkaline solution and requires a correction for the glucose content

of the sample 2). The other 3) disposes of glucose by treatment with

yeast. In it the total periodate consumed after oxidation in hot acid solu-

tion is determined by titration. This method is more specific, as periodic

acid oxidizes only compounds with 2 adjacent partially oxidized carbon

atoms.

It has the further advantage that it does not require a separate

determination of glucose. Correction for non-mannitol reducing substance

is made from a mannitol-free blank sample. However, the high blank

obtained from materials treated with yeast and the fact that the oxidation

consumes only 1 of the 4 titratable oxygen atoms of periodate impair its

sensitivity.

There are many substances oxidizable by periodate 4).

Among them,

those with 2 adjacent partially oxidized carbon atoms at the end of the

chain yield formaldehyde.

Thus, oxidation of 1 mole of mannitol with

periodic acid yields 2 moles of formaldehyde and 4 of formic acid. Mac-

Fadyen 5) has recently developed a sensitive procedure for determination

of formaldehyde. It therefore seemed easible to develop a more sensitive

and specific method for the determination of mannitol in biological media,

in which the determination is based on measurement of formaldehyde

produced during periodic acid oxidation.

In attempting to apply this principle, there are serious drawbacks to

treating the plasma filtrates and urine dilutions with yeast.

Wijth plasma

this treatment increased the blank rather than decreased it, and the

blank of glucose solutions so treated is also materially increased 6).

Moreover, in our hands, mannitol was sometimes lost, apparently by

absorption on the yeast. We therefore altered the conditions of the

oxidat,ion in order to make it more specific and thus to diminish the plasma

and glucose blanks.

Mannitol is oxidized by periodic acid at room temperature in neutral or

* A preliminary report has been published (1).

165

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166

DETERMlNATION OF MANNITOL

acid solution, while the oxidation of many other substances, including

glucose, proceeds rapidly only under other conditions. We therefore

selected a concentration of periodic acid sufficient to oxidize mannitol in the

concentrations best suited for color measurement, allowing for the presence

of oxidizable, non-chromogenic material, chiefly glucose.

Method

Principle-Mannitol is oxidized by periodic acid to formic acid and

formaldehyde. The conditions of the oxidation are such that glucose,

although attacked, produces little formaldehyde. The periodic acid is

reduced to iodide by stannous chloride and the formaldehyde produced is

determined by the method of MacFadyen.

Reagents-

1. Periodic acid reagent.

Potassium periodate, 0.03 M in 0.25 M sulfuric

acid.

2. Stannous chloride. Prepared freshly every day as approximately

0.125 M in 0.3 N HCl. This solution should be titrated by the periodate

reagent immediately before use and so adjusted that 10 cc. of SnC& reagent

titrate 10.2 cc. of HI04 reagent.

For the titration, 5 cc. of concentrated

HCl are added to the SnClz and starch is used as the indicator.

3. Chromotropic acid reagent. 0.2 gm. of chromotropic acid (l, di-

hydroxynaphthalene-3,6-disulfonic acid) is dissolved in 4 cc. of water in a

100 cc. volumetric flask and made up to volume with 15 M sulfuric acid.

Procedure-Pipette into a tall test-tube graduated at 25 cc., 2 cc. of

plasma filtrate or urine dilution. Plasma filtrat,e is prepared as a 1:20

dilution of plasma by the method of Somogyi (7).

Filtrates made with

zinc sulfate and sodium hydroxide or cadmium sulfate and sodium hydrox-

ide are equally satisfactory. Urine dilutions are made with water. In

both cases the 2 cc. should contain 0.5 to 3.0 mg. of mannitol per 100 cc.

and not more than 7 mg. of glucose per 100 cc.

A reagent blank is made

containing 2 cc. of distilled water. From a fiitrate of mannitol-free

plasma two tubes are prepared. One (oxidized blank) 4s carried through

the procedure as described below. The other (unoxidized blank) is treated

with stannous chloride prior to addition of periodic acid.

0.5 cc. of periodic acid reagent is added to the oxidized blank and sample

tubes and the contents mixed and allowed to stand at room temperature

for 8 to 10 minutes.

Add 0.5 cc. of stannous chloride and shake well. The stannous chloride

is oxidized by the periodate to stannic acid, which appears as a milky

precipitate in the tube. This precipitate is soluble in the strong acid to be

added in the next step and does not interfere with the determination.

There may also be a momentary appearance of elementary iodine in the

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A. C. CORCORAN AND I. H. PAGE 167

tube as stannous chloride is being added. The iodine color should not

persist if the proper amount of stannous chloride has been added.

Add 5 cc. of chromotropic acid reagent from an automatic pipette with

free delivery. The reagent should be added rapidly with vigorous shaking

to get complete mixing of the contents of the tube. The tube is then

placed in a boiling water bath for 30 minutes. Remove the tube, cool,

make up to 25 cc. with distilled water, and allow the temperature to

stabilize at 25 in a water bath. At this temperature the color is stable

for several ho&s.

When the temperature of the tube is stabilized at 25, read the optical

density (D) of each tube at 570 rnp in a No. 6-300 cuvett,e of a Coleman

model GA clinical spectrophotometer, setting the galvanometer at zero with

air in the cuvette holder. The reagent blank is measured at the same time;

this consists of a tube containing 2 cc. of distilled water instead of the

sample, and treated throughout in the same manner as the sample tubes.

It is also advisable to run standard tubes containing from 0.5 to 3.0 mg.

per cent of mannit. until t.he observer is thoroughly familiar with the

conditions of the procedure.

Calculation-AD is found for each urine or mater sample tube by sub-

tracting the D of the reagent blank from the D found for the sample.

For plasma samples AD is found as the difference in D between the unoxidized

blank and tinknowns, including the oxidized blank.

The apparent man-

nitol concentration is then found by reference to a calibration curve of AD

against mannitol concentration. Levels of plasma mannitol are computed

by subtracting the apparent mannitol content of the oxidized plasma blank

from the apparent mannitol content of the sample tubes and mtiltiplying

the mannitol content by the dilution factor.

Apparent urinary mannitol

is calculated as mg. per minute.

The value found before mannitol is given

is subtracted from that observed during the clearance determination.

An alternative is to increase plasma and urinary mannitol levels to con-

centrations in which the expected urinary blank (averaging 0.5 mg. per

100 cc.) is less than 1 per cent of the apparent urinary mannitol.

Results

Recovery of mannitol added to water or urine dilutions is regularly

obtained, with an error not exceeding 2 per cent when al l precautions are

carefully observed.

Incomplete mixing of reagents, improper timing of the

development of color, failure to adjust the temperature at the time of

reading, and failure to get uniform drainage of the automatic pipette at the

time of addition of the chromotropic acid reagent all contribute to errors.

Recovery from plasma filtrates is satisfactory (98 to 100 per cent) when the

glucose content is within the specified limits (Table I).

A comparison of

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168

Mannitol added

m g. per 100 cc.

mg. WlOOcc.

0 3.8

20 23.6

40 43.6

60 63.8

0 4.0

20 23.6

60

63.4

0 2.4

20

22.0

40 42.4

60 62.4

0 3.8

20 23.6

40 43.6

60 63.2

DETERMINATION OF MANNITOL

TABLE I

Recovery of

Mannitol

Added to Plasma

Apparent mannitol Mannitol recovered

mg.gcr 100cc.

19.8 99

39.8 99.5

60.0 100

19.6 98

59.4 99

19.6 98

40.0 100

60.0

100

19.8 99

39.8

99.5

59.4 99

Recovery

per cent

TABLE

II

Ratios of Simultane ous

Plasma

Clearances of Mannitol, Inulin, Creatinine, and

Thiosulfate in Dogs and

Human

Beings

The simultaneous plasma clearances of substances believed to be excreted by

glomerular filtration are compared in observations made in dogs and human beings,

during intravenous infusion of the substances studied. Inulin was determined by

the method of Corcoran and Page (8), creatinine by the method of Folin as adapted

to the Coleman clinical spectrophotometer, and thiosulfate by the method of Gilman,

Philips, and Koelle (9). The data indicate that, unlike inulin, creatinine, and

thiosulfate, the clearance ratios of which approximate unity, mannitol is reabsorbed

or destroyed in the kidney to the extent of about 10 per cent of that which is filtered.

The data therefore confirm unpublished observations of Earle (personal commun-

ication) comparing mannitol-inulin clearance ratios and Hoobler (personal com-

munication) as concerns mannitol-thiosulfate clearance ratio in human beings.

Clearances Mean ratio

Mannitol-inulin, dogs and hu- 0.902

man beings

Mannitol-creatinine, dogs 0.872

Mannitol-thiosulfate, dogs 0.89

Inulin-creatinine, dogs 0.98

Inulin-thiosulfate 1.04

Creatinine-thiosulfate 1.03

No. of Standard Standard de-

observations deviation viation of mean

42

35

12

12

7

12

fO.113

10.079

10.017

fO.013

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DETERMINATION OF MANNITOL

nit01 is complete in .3 to 5 minutes.

While the reaction could be checked

at this point, 10 minutes are chosen as a more convenient interval (Table

III). If oxidation is prolonged beyond 10 minutes, the shifting of the

TABLE

III .

Relationship between Time of Oxidation, Temperature, and Optical

Density in a 6-309 Cuvette

Tiie

Temperature

Mamitol added

Optical density D)

min. C. m g. per 100 CC.

10 9 1

20 9 1

30 9 1

40 9 1

60 9 1

10 9 4

20 9 4

30 9 4

40 9 4

60 9 4

4 26 1

6 26 1

8 26 1

10 26 1

4 26 4

6 26 4

8 26 4

10 26 4

0.191

0.191

0.191

0.191

0.193

0.707

0.702

0.702

0.704

0.707

0.192

0.196

0.192

0.193

0.701

0.701

0.701

0.696

Mean....................................................... rto.1

per cent

0

0

0

0

+0.6

0

-0.7

-0.7

-0.4

0

-0.5

+2.1

-0.5

0

+0.7

+0.7

+0.7

0

TABLE

IV

Effect of Temperature on Optical Density in a No. 6-301 Cuvette

Mannitol concentration

-

mg. ger 100 cc.

0.5

2.0

4.0

-

AD at 20

0.100 0.108 1.08

0.360

0.398

1.10

0.672 0.732 1.09

AD at 0

AD 30

AD 20

equilibrium mixture of glucose isomers results in higher yields of formal-

dehyde from glucose.

Determination of Formaldehyde-In applying MacFaclyens procedure

under these conditions, we reinvestigated the effect of the time of heating

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