A MODIFIED FOLIN AND WU BLOOD SUGAR METHOD.

BY VERA E. ROTHBERG AND FRANK A. EVANS.

(From the William H. Singer Memorial Research Laboratory, Pittsburgh.)
(Received for publication, August 7, 1923.)

In the preceding paper data have been presented to emphasize

anew the statement of Folin and Wu that in their method for
quantitative blood sugar estimation* the standard solution must
contain very nearly the same amount of dextrose as the blood
filtrate being tested. Examination of these data shows that if

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approximately correct results on all blood fltrates are to be ob-
tained with this method it would be necessary to have more
standard solutions than can be used conveniently. This is neces-
sary, probably in large part although perhaps not entirely, because
the curve of color dilution of the color developed in this procedure
is irregular and, therefore, if the standard and unknown are not
almost the same color at the time the calorimetric readings are
made, errors are introduced. An error in a method used for
quantitative work is unfortunate under any circumstances, and
particularly so in a blood sugar method of widespread popularity
in these days of insulin therapy of diabetes when correct values
for the blood sugar, especially when low, are so important In
this report a minor modification of the Folin and Wu method
for blood sugar determination is presented whereby the color
developed by the unknown blood filtrate is always the same as
that of the standard dextrose solution at the time the calorimeter
readings are made; and one standard alone may be used for com-
parison with many blood filtrates and two, or at most three
standards, will make possible correct determinations of the amount
of dextrose in any blood filtrate ever encountered.
The modification of the Folin and Wu method for quantitative
blood sugar determination used in this laboratory consists merely
in: (1) Diluting the unknown after the addition of the phos-
1 Folin, O., and Wu, H., J. Biol. Chem., 1920, xli, 367.
443
444 Folin-Wu Blood Sugar Method

phomolybdate-phosphotungstate sugar reagent and development

of color until it is approximately the same shade as the standard,
instead of to a constant amount, before calorimetric comparison
is made; and (2) introducing this variable factor, the amount of
dilution, in the final formula for calculating the amount of dex-
trose in the unknown. The determination is carried out in tubes

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FIG. 1. a, Tube used for the Folin and Wu blood sugar determination.
b, Tube used for the modified Folin and Wu blood sugar determination.

differing from those suggested by Folin and Wu’ only in that they
are longer and graduated in steps of 2.5 cc. from 12.5 to 50.0 cc.,
Fig. L2 In every other particular the Folin and Wu technique
is followed.
* Satisfactory tubes of this character may be purchased fromTheEmi1
Greiner Company, New York.
 V. E. Rothberg and F. A. Evans

This modification will be practical only if the error in the Folin

and Wu method for estimating blood sugar such as was demon-
strated in Tables I and II of the preceding paper was due ent,irely,
or almost entirely, to irregularity in the curve of color dilution
of the color developed in the determination. Experiments pre-
sented in the preceding paper demonstrate that this is so, provided
the instructions of Folin and Wu are carefully followed. Their
statement that the phosphomolybdate-phosphotungstate sugar
reagent must be water-clear is especially important, because if
it has a yellowish tinge an additional source of error may be intro-
duced. Other errors may.also be present; for example, the amount

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of reduction by more concentrated solutions of dextrose is possibly
not proportional to that by weaker solutions; the degree of re-
oxidation which perhaps takes place in performing the determina-
tion may not be the same for strong and weak solutions of dextrose;
the amount of water added after development of color may intro-
duce an error; and several other features must be considered.
If these errors are present, they are so small that they are within
the limits of error for ordinary calorimetric determinations, so
that if that due to the use of inferior reagents is eliminated, the
only error of consequence remaining is that due to the irregularity
of the curve of color dilution of the color being dealt with. Before
the modification herein suggested was adopted for routine use,
however, several experiments were carried out to prove that it was
practical.
Experiment I.-A specimen of Difco dextrose shown to be pure by polari-
scopic examination was used to make dextrose solutions of the following
strengths: 0.11,0.12,0.13, etc., up to 0.2 mg. of dextrose per cc. of water, the
equivalent of 110,120,130, etc., up to 200mg. of dextrose per 100 cc. of blood.
The routine Folin and Wu blood sugar determination was carried out on
each of these solutions, using 2 cc., exactly as is done with the protein-free
blood filtrate. At the same time the procedure was carried out with these
solutions in the new tubes described above, but diluted, after the addition
of the phosphomolybdate-phosphotungstate sugar reagent and develop-
ment of color, to the amount at which theoretically they would compare
exactly in color with the standard containing 0.1 mg. of dextrose per CC.
diluted, after the addition of the reagent and development of color, to 25
cc. All these dilutions were made promptly, at as nearly the same time as
possible. Each test was compared in the calorimeter with a standard
dextrose solution containing 0.1 mg. of dextrose per cc., the same amount as
is in the filtrate from a blood containing 100 mg. of dextrose per 100 cc., set
at 20 on the calorimeter scale. The results appear in Table I.
 Folin-Wu Blood Sugar Method
Experiment g.-Dextrose solutions of the following strengths were pre-
pared: 0.22, 0.24, 0.26, etc., up to 0.4 mg. of dextrose per cc. of water, the
equivalent of 220,240,260, etc., up to 400 mg. of dextrose per 100 cc. of blood.
Procedures similar to those outlined for Experiment 1 were carried out on
these dextrose solutions and the comparison in the calorimeter was made
against a standard dextrose solution containing 0.2 mg. of dextrose per cc.,
the same amount as is in the filtrate from a blood containing 200 mg. of
dextrose per 100 cc., set at 10 on the calorimeter scale. The results appear
in Table II.
TABLE I.

Determinations according to Determinations according to the modified

original Folin and Wu method. method. Contents of tubes diluted to
Contenta of tubes diluted to volume at which the color of the unknown

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8 volume of 25 co. shouldmatch, theoretically, that of stsnd-
srd.
Equivalent in rohlme to
dextrose Per Average Dextrose which Average l&xtrose
lOo;m&l;;i of ,f colorim- recovered contents of colorim- recovered
eter Error. of tubes eter ElTor.
solutions tested Per Per
readings. 100 00. were readings. 100 cc.
diluted.

Solutions read against the 100 Solutions read against the 100 mg. standard
mg. standard eet at 20 on set at 20 on the calorimeter scale.
the oolorimetm scela.
-
mg. mg. per cent cc. w. per cent
100 20.0 100.0 0 25.0 20.0 100.0 0
110 17.6 113.6 3.3 27.5 19.9 110.5 0.5
120 15.6 128.2 6.8 30.0 19.9 120.6 0.5
130 14.2 140.8 8.3 32.5 19.9 130.7 0.5
140 12.6 158.7 13.3 35.0 20.0 140.0 0
150 11.7 170.9 13.9 37.5 19.9 150.8 0.5
-
Solutions read against the 200
mg. standard set at 10 on
the calorimeter scale.

160 13.8 144.9 9.4 40.0 19.9 160.8 0.5

170 12.4 161.3 5.1 42.5 19.8 171.7 1.0
180 11.7 171.3 4.8 45.0 19.9 180.9 0.5
190 10.7 186.9 1.6 47.5 19.9 191.0 0.5
200 10.0 200.0 0 50.0 19.9 201 .o 0.5

Because of the results obtained in Experiments 1 and 2 we

expected to find that one standard solution would suffice for
determinations on all blood filtrates; that the color developed by
the test on the blood filtrate from a blood containing 400 mg. of
dextrose per 100 cc. when diluted to 50 cc. would match that
developed by the standard dextrose solution equivalent to the
filtrate from a blood containing 100 mg. of dextrose per 100 cc.,
when diluted to 12.5 cc. In other words, we expected to find
 V. E. Rothberg and F. A. Evans 447

that the color developed by a dextrose solution four times as

strong as the standard solution, when diluted to 50 cc., would
match that of the standard solution diluted to 12.5 cc. Such,
however, was found not to be the case. Under these conditions
a plus error of about 8 per cent is introduced so that the color
developed by the stronger solution must be diluted to about
55 cc. before it matches that of the 100 mg. standard diluted to
12.5 cc. Apparently, therefore, the possible sources of error men-
tioned above, other than the irregularity of the curve of color
TABLE II.
-

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1Determinations according to 3eterminations according to the modified
original Folin and Wu method. method. Contents of tubes diluted to
Contents of tubes diluted to volumeat which the color of the unknown
B volume of 25 cc. &;uld match, theoretically, that of stand-

‘olume to
Average Dextrose which Average Dextrose
0 f c&rim- recovered contents of o&rim- recovered
eter Error. of tubes eter Error.
Per
:readings. 100 DC. were readings. IOKO.
diluted.

3olutions read against the 200 3olutions read against the 203 mg. standard
mg. standard set at 10 on set at 10 on the calorimeter wale.
the oolorimeter scale.
-
mu. WI. p&r cent cc. m7. per cent
200 10.0 200.0 0 25.0 10.0 200.0 0
220 8.6 232.6 5.7 27.5 10.0 220.0 0
240 7.7 259.7 8.2 30.0 9.9 242.4 1
260 7.0 285.7 9.9 32.5 9.9 262.6 1
280 6.0 333.3 19.0 35.0 10.0 280.0 0
300 5.6 357.1 19.0 37.5 10.0 300.0 0
320 5.7 350.9 9.7 40.0 9.9 323.2 1
340 5.1 392.1 15.3 42.5 9.9 343.4 1
360 4.9 408.1 13.4 45.0 9.9 363.6 1
380 4.3 465.1 22.4 47.5 9.8 387.7 2
400 4.1 487.8 21.9 50.0 10.0 400.0 0
- - -
dilution of the color developed, do come into play. When the
unknown does not differ more than 100 per cent from thestandard,
however, the error introduced by these factors is too small to be
demonstrated by ordinary calorimetric determinations, but when
there is a difference much greater than 100 per cent an appreciable
error creeps in. When the standard dextrose solution equivalent
to the filtrate from a blood containing 100 mg. of dextrose per
100 cc. is used for comparison by this revised method with the
448 FobWu Blood Sugar Method

filtrate from a blood containing 400 mg. of dextrose per 100 cc.
the error is less than when the determination on a similar filtrate
diluted to 25 cc. is read against a 200 mg. standard, diluted to
the same amount as ordinarily done with the method of Folin and
Wu. And, inasmuch as absolute values for the sugar of the blood
when in such great concentration are not necessary in routine
clinical work, one standard may be used with this revised method
for all blood filtrates and fairly satisfactory results be obtained.
If results closely approaching absolute values for all blood filtrates
are desired, however, standard solutions differing from the sugar
concentration in the unknown not more than 100 per cent must

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be used.
To perform the test according to the modification above sug-
gested the Folin and Wu blood sugar determination on the
standard dextrose solution (or solutions) chosen and on the
unknown blood lilt.rates is carried out in tubes described above.
After the addition of the phosphomolybdate-phosphotungstate
sugar reagent and development of color, the test on the st,andard
solution is diluted to 25 cc. and those on the unknowns are diluted
to the cubic centimeter mark on the tube at which the color in-
tcnsity most closely resembles that of the standard. The amount
of dilution of each unknown is recorded and they are then com-
pared in the calorimeter with the standard preparation. If the
dilutions of the unknowns in the tubes have been made care-
fully, they should read on the calorimeter scale very nearly that,
figure at which the standard preparation was set.
When, as here obtains, the dilution of the unknown is not the
same as the standard solution, the formula presented by Folin
and Wu for calculating the final results by their method, namely

rsg X 100 = mg. of dextrose per 100 cc. of blood,

must be multiplied by the fraction

dilution of unknown
dilution of standard (25 CC~’

We have, therefore,
20 dilution of unknown
- x 100 x
reading 25
 V. E. Rothberg and F. A. Evans 449

or simplifying,
80 X dilution of unknown
= mg. of dextrose per 100 cc. of blood.
reading

If the amount of dilution necessary to make the color developed

by t.he unknown blood filtrates match that of the standard is
considered in relation to the strength and dilution of the standard
used, a rough idea of the amount of dextrose in the bloods in
question may be obtained. For instance, if the standard dextrose
solution, equivalent to t,he filtrate from a blood containing 100 mg.
of dextrose per 100 cc., diluted to 25 cc., is used, the values are

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as follows:
Dilution of unknown Dextrose per 100 cc. Dilution of unknown
solution. of blood. II solution.

cc. mg. cc. mg.

12.5 50 32.5 130

15.0 60 35.0 140
17.5 70 37.5 150
20.0 80 40.0 160
22.5 90 42.5 170
25.0 100 45.0 180
27.5 110 47.5 190
30.0 120 50.0 200

If the standard dextrose solution, equivalent to the filtrate from

a blood containing 200 mg. of dextrose per 100 cc., diluted to
25 cc., is used, the values are as follows:

D&c;,,,,100 cc. Dilution of unknown Dext;;; o;dlOO cc.

solution. P .
-
CC. mg. cc. mg.

12.5 loo 32.5 260

15 .o 120 35.0 280
17.5 140 37.5 300
20.0 160 40.0 320
22.5 180 42.5 340
25.0 200 45.0 360
27.5 220 47.5 380
30.0 240 50.0 400

Folin and Wu point out clearly that in their method for blood
sugar determinations it is necessary, after the addition of the
 Folin-Wu Blood Sugar Method

phosphomolybdate-phosphotungstate sugar reagent and develop-

ment of the color, to carry out the subsequent dilution on the
standard solution and on the unknown blood filtrate at the same
time. Experiments in this laboratory demonstrated the impor-
tance of this instruction, for if the dilutions of the standard a,nd
unknown are made 5 minutes apart an appreciable error is intro-
duced. This observation is of importance in relation to the
modification of the Folin and Wu blood sugar method here pre-
sented, because the dilution of the test on the unknown blood
filtrate is not made all at once, but in two, or more, steps. If one
keeps in mind the necessity for dilution of the standard and

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unknown at approximately the same time, and works rapidly,
it is possible to dilute at least six unknowns to match the standard
promptly enough to eliminate this source of error. The method
of procedure which has been devised with this point in mind is
as follows: After the addition of the phosphomolybdate-phospho-
tungstate sugar reagent and development of color-but before
dilution-the tests on unknown blood filtrates are compared with
that on the standard dextrose solution. One can tell whether the
color developed in the unknowns is less than, about the same as,
or more than that of the standard dextrose solution; and depending
on this observat,ion the unknowns are immediately diluted to a
mark of less than 25 cc. to the 25 cc. mark, or to a mark of more
than 25 cc. The standard is then diluted to 25 cc., the contents
of each tube are thoroughly mixed, and they are compared again.
Any further diMon of the tests on the unknown blood filtrates
which are necessary to make them about the same color as the
standard is carried out promptly. If it is expected that any of
the protein-free filtrates being tested are from bloods containing
more than 200 mg. of dextrose per 100 cc., it is wise to carry out
at the same time a preparation not only on a standard dextrose
solution containing 0.1 mg. per cc. of solution, but also on one
containing 0.2 mg. per cc. of solution; for, as stated above, accurate
results cannot be obtained by comparing the test on such a blood
filtrate with the weaker standard diluted to 12.5 cc. With a, little
practise on the procedure outlined, it is possible to match the
color of the test on unknown blood filtrates with that of the
standard dextrose solution diluted to 25 cc. very rapidly, and in
most instances in no more than two steps.
 V. E. Rothberg and F. A. Evans 451

Since, in the procedure suggested, the total dilution of the test

on unknown blood filtrates is not carried out all at once, but by
the addition of smaller amounts of water several times, it is
necessary in mixing the contents to invert the tube more often
than in the original method. It seemed possible that, under these
circumstances, enough fluid would be lost on the stopper and
around the mouth of the tube to introduce an appreciable error.
This, however, was not found to be so. A tube filled with water
inverted fifteen times, the stopper being removed after each third
inversion, showed no diminution in the volume of its contents
that could be made out on gross inspection. This number of

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inversions is sufficient to mix thoroughly with its contents water
added to a tube five different times, more than is ever necessary
in carrying out the procedure presented above.

SUMMARY.

If correct values are to be obtained with the Folin and Wu blood

sugar determination it is essential, for the oolorimetric comparison,
to have the color finally developed by the blood filtrate being
tested very nearly the same as that developed by the standard
dextrose solution with which it is to be compared. This neces-
sitates for accurate results the use of more standard solutions than
is convenient. In this paper the details of a technique are pre-
sented in which the color developed by the unknown blood
filtrate with the Folin and Wu method is the same as that of the
standard dextrose solution when the calorimetric readings are
made; and one standard may be used for many bloods, and two
or at most three make possible accurate determinations of the
dextrose in any blood every encountered. The modification here
presented consists simply in diluting the unknown in the test-
tube after the addition of the phosphomolybdate-phosphotung-
state sugar reagent and development of color until it is approxi-
mately the same color as the standard, and including this variable
factor in the formula for calculating the final results.
A MODIFIED FOLIN AND WU BLOOD
SUGAR METHOD
Vera E. Rothberg and Frank A. Evans
J. Biol. Chem. 1923, 58:443-451.

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