THE DETERMINATION OF ASCORBIC ACID IN URINE

WITH THE PHOTOELECTRIC COLORIMETER*

BY KENNETH A. EVELYN, HELGA TAIT MALLOY, AND
CHARLES ROSEN

(Prom the Department of Medicine, McGill University Clinic, Royal Vicloria

Hospital, Montreal, Canada)

(Received for publication, September 8, 1938)

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The main sources of error in the determination of ascorbic acid
in biological fluids by titration with 2,6-dichlorophenol indophenol
are due to difficulties in detecting the somewhat vague end-point,
and to the presence of reducing substances other than ascorbic
acid. The first of these errors may be eliminated by replacing
visual titration by an objective photoelectric measurement of the
amount of dye decolorized when a measured quantity of the solu-
tion reacts with an excess of dye (Rosen and Evelyn (l), Mindlin
and Butler (2), Pijoan, Alexander, and Wilson (3)). In addition
the use of the photoelectric method allows the measurement to be
completed within 5 seconds, thus minimizing errors due to de-
colorization of the dye by interfering substances which react more
slowly than ascorbic acid itself. In fluids such as plasma (2) and
cerebrospinal fluid (3) the amount of interfering material is usually
small, therefore accurate results can be obtained by making a
single measurement at the end of 5 seconds. This is, however,
far from being the case in urine, because non-ascorbic acid reduc-
ing substances often account for more than 90 per cent of the total
indophenol-reducing capacity of normal urine. Attempts have
been made (4-6) to remove the interfering substances by pre-
cipitation with mercuric acetate or barium acetate, but by these
methods only a portion of such substances is removed.
It is the purpose of this paper to describe an alternative method
of differentiating between ascorbic acid and other reducing sub-
* Aided by grants from The Banting Research Foundation, Toronto,
Canada.
645
646 Determination of Ascorbic Acid
stances, which is based on differences in the rate at which the dye
is decolorized. It has been shown that the reaction of ascorbic
acid is almost instantaneous (7), while that of all other known
interfering substances proceeds at a slower rate. This is further
illustrated by the curves of Fig. 1 which show that the ascorbic

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:;Lc; ,5 IO 15 20 25 30 35 40

FIG. 1. Extrapolation of curves obtained from mixtures of reducing

substances in order to obtain the true ascorbic acid content. Abscissa,
time in seconds; ordinate, amount of dye decolorized in arbitrary units.
Curve 1, ascorbic acid 0.0013 mg. per cc., thiosulfate 0.0056 mg. per cc.;
Curve 2, ascorbic acid 0.0013 mg. per cc.; Curve 3, ascorbic acid 0.00143
mg. per cc., cysteine 0.050 mg. per cc .; Curve 4, ascorbic acid 0.00143 mg.
per cc.; Curve 5, ascorbic acid 0.00116 mg. per cc., thiosulfate 0.0040 mg. per
cc., and cysteine 0.0143 mg. per cc.; Curve 6, ascorbic acid 0.00116 mg. per
cc.; Curves 7 to 11, representative samples of urine (Curve 10 was made
with 0.1 cc. of urine of a patient who was undergoing an ascorbic acid
saturation test).

acid reaction is complete within 5 seconds, while the presence of

other reducing substances causes the reaction to continue beyond
this time, In our method an excess of dye is mixed with a meas-
ured aliquot of urine, and the amount of dye decolorized is meas-
ured in the photoelectric calorimeter (8) at 5 second intervals until
 Evelyn, Malloy, and Rosen 647

the reaction is complete. The results are used to plot a reaction

velocity curve from which the amount of decolorization due to
ascorbic acid alone may be determined by extrapolation to zero
time. This method is admittedly empirical and cannot therefore
be expected to give more than approximate results, but the errors
are far less serious than those of the ordinary visual titration
which may often amount to several hundred per cent. Moreover,
by combining the extrapolation procedure with a preliminary
partial chemical purification we believe that measurements on
urine can be made almost as accurately as on plasma.

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Method
Freshly voided urine acidified with 5 per cent of glacial acetic
acid by volume is used for all the determinations.
Reagents and Apparatus-
1. Aqueous solution of 2,6-dichlorophenol indophenol. No
standardizat,ion of this solution is required other than to adjust
its concentration so that 9 cc. of dye plus 1 cc. of 5 per cent acetic
acid give a reading of about 30 on the photoelectric calorimeter
with Filter 52O.l The solution should be filtered, brought to
approximately pH 7 by addition of a few drops of ~/15 phosphate
buffer, and stored in a brown bottle with a glass stopper.
2. A rapid delivery 9 cc. pipette is made by cutting off a suitable
length from the lower end of an ordinary 10 cc. pipette, and re-
calibrating it to deliver 9 cc. The bore of the tip should be about
3 mm., so that the emptying time is about 1 second. A No. 6
rubber stopper is attached to the outside of the pipette at such a
height that when it is inserted into a calorimeter tube the tip will
be about 2 inches from the bottom of the tube (Fig. 2). The
pipette must be rinsed after each determination to avoid contam-
ination of the dye solution by traces of acid which may adhere to
the tip.
Procedure
The initial optical density2 of the dye solution is measured by
adding 1 cc. of 5 per cent acetic acid to 9 cc. of dye in a calorimeter
1 The calorimeter which we have used is manufactured by the Rubicon
Company, 29 North Sixth Street, Philadelphia. Filter 520 is supplied with
the instrument as standard equipment.
2 The term optical density is used here to refer to the average optical
648 Determination of Ascorbic Acid
tube and reading immediately in the photoelectric calorimeter with
Filter 520, after the galvanometer is adjusted to 100 with a tube
containing water only. The corresponding optical density is
recorded as LI. The galvanometer is then adjusted to 100 with a
blank tube containing 9 cc. of water and 1 cc. of acidified urine; a
sample tube containing 1 cc. of acidified urine is placed in the
instrument, and 9 cc. of dye are run in from the special pipette
which is held in place as shown in Fig. 2. The galvanometer is
read at 5, 10, 20, and 30 seconds measured from the time at which
addition of the dye is begun, the readings are plotted against time

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on graph paper, and the curve extrapolated by a smooth free-hand

FIG. 2. Rapid delivery 9 cc. pipette shown in place in the calorimeter

tube just before delivery of the dye.

curve to intersect the axis of ordinates. The optical density

corresponding to this extrapolated galvanometer reading is re-
corded as Lz, and the concentration of ascorbic acid in mg. per 100
cc. of acidified urine is calculated from the equation
x = 10.8(h - Lz)
A
0)

density which is measured in the photoelectric calorimeter, as distinct

from the true monochromatic optical density which is measured by the
spectrophotometer. This approximate optical density is represented by
the symbol L, and is similar to true optical density in that it obeys the
relation L = K X C where K is a constant and C is the concentration of the
colored substance in the solution.
 Evelyn, Malloy, and Rosen 649

where A cc. is the volume of acidified urine used in the test, and
10.8 is the numerical value of the calibration constant which has
been determined by measurements on solutions of pure ascorbic
acid.3
In measurements on urine it is very seldom necessary to use
more than 1 cc. of urine, but the urine may have to be diluted
before the test is made in order to limit the excursion of the gal-
vanometer during the first 5 seconds to a maximum of 20 mm.4 If
the ascorbic acid content of the sample solution is below 0.5 mg.
per 100 cc., as for example in plasma filtrates, 1 cc. of a 10 times

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stronger dye solution should be placed in the calorimeter tube,
and 9 cc. of the sample solution run in from the pipette. Equation
1 is of course accurate only as long as the final volume is 10 cc.,
and the 9: 1 ratio is necessary to insure adequate mixing. In
adapting the method to other fluids the solutions should be acidi-
fied or buffered so that the final pH of the dye mixture is about 3.

Results
Recovery of Ascorbic Acid-Table I illustrates the accuracy with
which the true ascorbic acid content of mixtures containing vary-
ing amounts of interfering substances can be determined by the
extrapolation method described above. The close agreement
between the results obtained by independent observers shows that
the somewhat empirical process of extrapolation can be carried
out with a relatively small personal error. The complete failure
of the titration method to differentiate between ascorbic acid and
thiosulfate is well shown by the figures in the last column. It

3 The numerical value of this constant applies only to the particular

type of photoelectric calorimeter used by us (Rubicon Company, 29 North
Sixth Street, Philadelphia), but no difficulty would be experienced in
adapting the method to any other type of instrument, other than the
necessity for redetermining the value of the constant.
4 This precaution is necessary to allow for the lag in the galvanometer
response due to inertia of the moving system. With the instrument used
by us a movement of 20 mm. is the maximum which can be followed faith-
fully in 5 seconds. It is important that each new instrument used for this
technique be checked by making a measurement on a solution of pure
ascorbic acid of such a strength that a movement of 20 mm. is obtained.
There must be no movement of the galvanometer spot or needle after the
first 5 seconds.
650 Determination of Ascorbic Acid
TABLE I
Recovery Acid from Mixtures
of Ascorbic Containing Other
Reducing Substances
The figures in the two columns headed “Photoelectric” are the results
obtained by two independent observers. In order to give the titrimetric
method every advantage all titrations were carried out in the photoelectric
calorimeter to an end-point in exactly 30 seconds.

Material in sample Ascorbic acid determined

-
Sodium Ascc$ic
Urine Photoelectric Titration
thiosulfate

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microgTaml cc. micrograms micrograms micrograms micrograms
23 0 0.4 0 13.9
25 0 0.6 1.2 16.0
26 0 0.3 0.7 16.0
37 0 1.2 1.2 18.0
41 0 0.8 0.4 24.2
41 0 2.0 2.5 25.2
25 15.8 13.1 13.1 31.6
40 9.1 7.2 9.5 26.4
40 19.6 23.2 20.2 47.0
143 0 0 0 2.4
153 0 0 0 1.6
153 0 0 0 2.0
286 0 0.2 0 3.9
143 10.8 10.7 9.3 16.8
286 15.8 15.9 15.7 39.0
306 14.5 13.7 13.1 14.5
40 143 10.8 7.2 11.3 36.5
24.8 153 12.5 14.3 13.7 31.0
24.8 153 11.0 7.2 9.5 28.6
24.8 153 8.5 11.9 10.5 27.2
24.8 153 14.5 16.7 14.6 21.4
1.0 19.0 16.7 18.0
1.0 19.0 15.9 16.7
0.5 10.4 9.2 9.2
1.0 10.4 10.8 10.5
1.0 10.4 13.2 13.3
0.5 10.4 9.8 9.8
-

should be mentioned at this point that, although we have used

cysteine and thiosulfate as examples of typical interfering sub-
stances, we do not believe that they are necessarily the most im-
portant factors in human urine, because even when these two
 Evelyn, Malloy, and Rosen 651
substances are removed there still remains a variable amount of
non-ascorbic acid reducing material.
Comparison of Titration and Photoelectric Methods in Urine-
Table II shows the results obtained by measurements on fresh
samples of urine from forty-five normal individuals whose diets
were apparently adequate in ascorbic acid content. The figures

TABLE II
Comparison of Photoelectric and Titrimetric Methods of Determining Ascorbic
Acid Concentration in Urine

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The conditions of the experiments were the same as in the experiments
reported in Table I. The results are expressed in arbitrary units.
Photoelectric Titmtion Photoelectric Titration
-
85 85 114 4 8 104
40 40 106 22 25 100
2 3 30 35 33 94
19 19 51 38 35 120
30 29 85 32 38 114
28 28 61 10 9 85
170 170 202 9 9 64
65 65 109 31 40 97
38 38 73 98 97 178
50 50 74 76 74 157
100 100 141 125 135 271
13 13 33 43 40 89
140 140 172 63 62 120
50 50 113 90 89 211
23 23 75 90 111 229
130 130 160 75 75 170
100 100 167 20 16 58
32 32 79 0 10 148
23 23 60 0 40 315
2 0 25 85 100 297
170 170 179 60 50 179
25 28 111

show that the ratio of titrimetric to photoelectric values varies

from almost 1:l to 10: 1 and more. In view of the fact that a
considerable percentage of normal individuals seems to excrete
only traces of ascorbic acid in the urine, in spite of the apparent
presence of relatively large amounts as determined by titration,
it seems necessary to reexamine the significance of the urinary
652 Determination of Ascorbic Acid

output of ascorbic acid as an index of “vitamin C subnutrition.”

Our findings in this connection will be reported elsewhere.
Removal of Interfering Substances-In view of the magnitude of
the errors caused by the presence of interfering substances in
urine, and realizing that the extrapolation correction can only
partially eliminate this type of error, we tried to combine the
extrapolation procedure with the mercuric acetate and barium

TABLE III
E$ect of Removal of Interfering Substances by Precipitation by Mercuric

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Acetate and by Solid Barium Acetate
All measurements were made by the photoelectric method. The figures
in the columns headed “Ascorbic acid” were obtained by the extrapolation’
procedure described in this paper. The figures under the heading “Inter-
fering substances” represent the ascorbic acid equivalent of the amount of
dye decolorized during the first 30 seconds after the end of the true ascorbic
acid reaction. The values are given in mg. per 100 cc.
Before precipitation Solid barium acetate
I Mercuric acetate

Interfering Interfering Interfering

substances substances substsnoes
.-
0.3 1.5 0.2 0.7 0.5 1.4
0.3 1.3 0.2 0.7 0.4 0.8
0.4 0.8 0.3 0.5 0.3 0.4
0.5 0.3 0.4 0.2 0.5 0.1
0.8 0.7 0.7 0.2 0.6 0.4
1.0 0.8 0.9 0.4 1.1 0.3
1.1 1.6 1.0 0.3 0.9 0.3
1.2 2.8 1.4 1.2 1.2 1.7
2.2 0.7 1.9 0.5 2.3 0.6
2.9 2.2 3.2 1.7 2.8 0.9
4.2 0.5 3.7 0.3 4.4 0.4
7.0 0.3 6.6 0.3 7.0 0.3

acetate purifications of van Eekelen and his coworkers (4-6).

The shape of the reaction velocity curves of the filtrates obtained
by these procedures showed that they still contained a variable
(though usually much smaller) amount of non-ascorbic acid re-
ducing substances. Moreover, the mercuric acetate filtrates
which had been reduced with hydrogen sulfide occasionally con-
tained more of these interfering substances than the original
 Evelyn, Malloy, and Rosen 653
urine, no doubt due to the reduction by the hydrogen sulfide of
substances present in the original urine (other than dehydroascor-
bit acid) in a harmless oxidized form. From Table III it may be
concluded that the barium acetate precipitation is of definite
value, since it always removes a portion of the interfering sub-
stances without causing any loss of ascorbic acid. There seems
to be no advantage in using the mercuric acetate-hydrogen sulfide
procedure, since in addition to the possibility of errors arising in
the manner described above, it is tedious and may easily cause
loss of ascorbic acid unless it is carried out very rapidly. Reduc-

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tion with hydrogen sulfide must, however, be carried out on any
solution which is known to contain appreciable amounts of dehy-
droascorbic acid.
In conclusion it may be mentioned that one of the most im-
portant applications of the method described in this paper is
qualitative rather than quantitative. A study of the shape of
the reaction velocity curve obtained from any solution affords a
sensitive test of the ratio of interfering substances to true ascorbic
acid, and may therefore be employed as a test of the efficiency of
any method suggested for the removal of interfering substances.
We recommend that this test be applied to all solutions, even those
such as plasma filtrates in which, as a rule, the interfering sub-
stances are relatively unimportant.

SUMMARY

1. A method is described for the determination of ascorbic acid

in urine and other biological fluids, in which titration has been
replaced by an objective photoelectric measurement of the amount
of dye decolorized when a measured quantity of urine reacts with
an excess of dye.
2. This method does not require standardization of the dye
solution, eliminates errors due to interfering colored substances,
and allows the measurement to be completed within 5 seconds
after addition of the dye. This greatly reduces errors due to
non-ascorbic acid reducing substances, and a simple extrapolation
procedure makes it possible to reduce this error still further.
3. As compared with the values obtained by the new method
the Ctration values for normal urine are apparently often from 2
654 Determination of Ascorbic Acid
to more than 10 times too high, although the titrimetric method is
quite accurate when the ascorbic acid content of the urine is very
high.
4. The mercuric acetate purification of Emmerie and van
Eekelen has not been found of value in connection with the
photoelectric method of measurement, but preliminary precipita-
tion with solid barium acetate is apparently both safe and desir-
able although it only removes a fraction of the interfering mate-
rial.
5. With the new method 24 hour ascorbic acid excretions of

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less than 5 mg. are often found in normal healthy individuals on
adequate well balanced diets.

BIBLIOGRAPHY

1. Rosen, C., and Evelyn, K. A., Proc. Ann. Meeting Roy. Sot. Canada, sect.
5, 61 (1937).
2. Mindlin, R. L., and Butler, A. M., J. Biol. Gem., 122, 673 (1938).
3. Pijoan, M., Alexander, L., and Wilson, A., J. Clin. Inv., 17, 169 (1938).
4. Emmerie, A., and van Eekelen, M., Biochem. J., 28,1153 (1934).
5. van Eekelen, M., and Emmerie, A., Biochem. J., 30,25 (1936).
6. van Eekelen, M., and Heinemann, M., J. CZin. Inv., 17,293 (1938).
7. Millikan, G. A., Biochem. .I., 29,2817 (1935).
8. Evelyn, K. A., J. Biol. Chem., 116, 63 (1936).
THE DETERMINATION OF ASCORBIC
ACID IN URINE WITH THE
PHOTOELECTRIC COLORIMETER
Kenneth A. Evelyn, Helga Tait Malloy and
Charles Rosen
J. Biol. Chem. 1938, 126:645-654.

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