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Mannitol Determination in Plasma and Urine
Transcript of Mannitol Determination in Plasma and Urine
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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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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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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
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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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