STANDARDIZED METHODS FOR THE DETERMINATION

OF URIC ACID IN UNLAKED BLOOD AND IN URINE

BY OTTO FOLIN
WITH TEE ASSISTANCE OF MARGARET CUSHMAN
(From the Biochemical Laboratory of Harvard Medical School, Boston)

(Received for publication, April 20, 1933)

INTRODUCTION

This paper is the outcome of a comprehensive critical review of

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the calorimetric method for the determination of uric acid both in
blood and in urine. The work began with the observation that we
were no longer able to obtain such complete recoveries of uric acid
added to unlaked blood as had been previously reported (1). The
cause of our failure was soon found. The Folin-Marenzi method
for the preparation of the uric acid reagent free from phenol re-
agent proved inadequate when applied to the Merck’s “reagent
sodium tungstate” now in the market. The reagents invariably
gave some color with tyrosine and some color with the urea-cy-
anide and yielded therefore necessarily only a very narrow range
of true proportionality between different amounts of uric acid.
Apparent lossesof as much as 15 per cent could thus be encoun-
tered, all due to the fact that the range of true proportionality fell
short of what it should have been. It therefore became necessary
first of all to improve the method for the preparation of a depend-
able uric acid reagent. The subsequent check work gradually
developed into revisions of the several uric acid methods sponsored
by this department.

RevisedProcessfor Preparation of Uric Acid Reagent Free from

Phenol Reagent
In the Folin-Marenzi (2) process for the preparation of this
reagent the larger part of the molybdenum sulfides is precipitated
and removed by filtration and the soluble sulfides are removed
from the filtrate by extraction with alcohol. In the process de-
111
112 Uric Acid Determination

scribed below only a minute fraction of the sulfides is rendered

insoluble.
The first step in the preparation should be to dissolve a few
crystals of the tungstate and test the reaction with phenolphthalein
solution to make sure that it is permanent.ly alkaline to phenol-
phthalein. If it is not alkaline, it is apt to be of poor quality in
other ways, especially with respect to its molybdenum content,,
and at all events, it should first be rendered alkaline by boiling
with a slight excess of sodium hydroxide.

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Transfer 100 gm. of sodium tungstate (of the requisite alkaline
reaction) and 150 cc. of water to a 500 cc. Florence flask. Dilute
20 cc. of phosphoric acid with 50 cc. of water and pour this gradu-
ally and with shaking into the tungstate-water mixture. Shake
until the tungstate has dissolved and cool under running water.
Pass H2S into the solution for 10 minutes.
Transfer the solution to a 500 cc. separatory funnel and add
(gradually at first) with gentle shaking a total of 150 cc. of alcohol.
Shake vigorously for 7 to 8 minutes. Let the mixture settle and
then withdraw the more or less yellow bottom layer, the weight
of which should be 160 to 170 gm.
Discard the highly colored upper layer and rinse the separatory
funnel. Return the phosphotungstate solution, together with
100 cc. of rinsing water, to the separatory funnel. Add 75 cc. of
alcohol and shake thoroughly as before. Withdraw the bottom
layer, which should now be substantially colorless, into a weighed
500 cc. Florence flask and dilute the contents to a weight of about
250 gm. Boil vigorously for 5 minutes to remove the H&. Dilute
again to a weight of 250 gm. and add 15 cc. of phosphoric acid (85
per cent). Boil under a reflux condenser for 1 hour. Remove the
condenser, add a little liquid bromine or strong bromine water,
and boil another 5 minutes to remove the surplus bromine. Cool
and dilute to a volume of 500 cc.
The directions given above for the preparation of the uric acid
reagent are the outcome of a prolonged study and a few explana-
tory remarks may prove helpful.
1. The total volume of 85 per cent phosphoric acid used with
100 gm. of sodium tungstate is only 35 cc., instead of 50 cc. The
reasons for this change are as follows: No initial mixture of sodium
tungstate and phosphoric acid could be found in which treatment
 0. Folin 113

with HzS converts all of the molybdenum present into sulfides

which are either insoluble or completely extractable with alcohol.
There always remain some molybdenum compounds which may
be converted into the phenol reagent during the final boiling
period. The greater the acidity or excess of phosphoric acid in
that boiling mixture the more extensively are the molybdenum
compounds present converted into phenol reagent. The uric acid
reagent, however, begins to form in the presence of a ,sufficient
excess of phosphoric acid to give a positive reaction with Congo

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red paper (30 cc. of 85 per cent phosphoric acid to 100 gm. of so-
dium tungstate) and a very small excess of phosphoric acid above
that amount is sufficient to yield a practically quantitative con-
version into the uric acid reagent, at the dilution indicated in the
directions. With the slight excess of free phosphoric acid present
when 35 cc. are used, no phenol reagent is formed when only traces
of molybdates are present as is the case after the treatment with
HzS. In the presence of larger amounts of molybdates, however,
even this slight excess of acidity will produce disastrous amounts of
phenol reagent. With Merck’s “reagent sodium tungstate” as it
was a few years ago, for example, a nearly perfect uric acid reagent
can be obtained by boiling 100 gm. with 33 to 35 cc. of phosphoric
acid, but the corresponding product now in the market is unusually
rich in molybdate. It is labeled, “according to Dr. Folin.” The
older good brand carries no such description.
2. The highly concentrated solution of the uric acid reagent
remaining in the boiling flask after the surplus bromine has
boiled off has a lemon-yellow color, but as the solution is cooled
most of the color fades away, so that after diluting to 500 cc., the
solution has only a just perceptible yellow tint-if the solution is
free from phenol reagent. The phosphomolybdates on the other
hand are intensely yellow, especially so in the case of the phenol
reagent, the phospho-18-molybdic acid. Any solution of the uric
acid reagent which has a distinctly yellow color is, therefore, surely
contaminated with phenol reagent, and it will invariably be found
to yield a blank (blue color) when 4 cc. are added to a mixture of
5 cc. of water and 10 cc. of urea-cyanide solution.
Concentrated solutions of the uric acid reagent completely free
from phosphomolybdates are probably quite colorless and the
faint yellowish tint left in the uric acid reagents prepared according
114 Uric Acid Determination
to the directions given above are almost certainly due to traces of
phospho-2kmolybdic acid which, under the given conditions, are
prevented from transformation into the phenol reagent.
3. This uric acid reagent contains no added lithium salt to pre-
vent the formation of turbidities.
Urea-Cyanide Solution-The urea-cyanide solution, described
in 1930, can be used without any change in connection with the
revised method for the direct determination of uric acid in unlaked
blood filtrates, but it does not always yield 100 per cent of the

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uric acid precipitated by means of silver lactate. Accordingly, it
has been modified so as to meet these additional requirements.
The urea-cyanide solution which we now use is prepared as
follows:
Transfer 75 gm. of Merck’s Blue Label sodium cyanide to a 2
liter beaker, add 700 cc. of water, and stir until the cyanide is
completely dissolved. Add 300 gm. of urea and stir. Then add
4 to 5 gm. of calcium oxide and stir for about 10 minutes. Filter,
at once if necessary for immediate use, but preferably not until the
next day. Add to the filtrate about 2 gm. of powdered lithium
oxalate, shake occasionally for 10 to 15 minutes, and filter.
Lithium oxalate is better than the disodium phosphate which
we formerly used for the removal of the dissolved calcium hydrox-
ide. Its solubility in the urea-cyanide solution is slight, yet great
enough to transform the dissolved calcium hydroxide into the
insoluble oxalate. Sodium oxalate powder cannot be used, be-
cause it lacks the required solubility, and potassium oxalate is, of
course, unsuitable.
The lithium oxalate is prepared as follows: Transfer 50 gm. of
lithium carbonate and 85 gm. of oxalic acid to a 3 liter beaker.
Pour on the mixture about 1 liter of hot water (70”). Stir cau-
tiously to avoid loss by foaming until the evolution of CO2 ceases.
Add 1 liter of alcohol and filter on a Buchner funnel.

Revised Macromethod for Determination of Uric Acid in Blood

Since it had been seen in the early part of this work what serious
errors can creep into calorimetric analyses merely because the
range of true proportionality in the color reaction is too narrow,
the attempt was made once more to secure a wider range of true
proportionality than has yet been obtained in calorimetric deter-
 0. Folin 115

minations of uric acid in blood, and these endeavors have resulted

in a modification of the method which most workers will probably
accept as an improvement.
The obvious way to try to increase the range of true proportion-
ality, if one has a uric acid reagent and cyanide which give no
blanks, was to use larger quantities of the uric acid reagent and
cyanide for the development of the color. There is a limit, how-
ever, to the amount of uric acid reagent to be used, even with t’he
efficient urea-cyanide solution, without getting turbidities when

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the mixtures are heated. But if the heating is omitted, any de-
sired quantity of the uric acid reagent may be used together with
the urea-cyanide solution without getting turbidities. By the
simple expedient of using more of the reagents and omitting the
heating, the range of true proportionality has been greatly in-
creased. Calorimetric readings between 35 and 10 mm., when the
regular standard is set at 20 mm., have now become dependable,
although it is better to repeat the determination with less than 5
cc. of blood filtrate when the first determination yields colori-
metric readings of 10 mm. or less as indicated on p. 119. While
the determination now takes a little more time, because of the
longer waiting period, it really takes less work and less attention.
We now make the regular (macro-) determination of uric acid
in blood in the following manner: transfer 5 cc. of unlaked blood
filtrate to a test-tube graduated at 25 cc. and transfer to another
similar test-tube 5 cc. of the standard uric acid solution contain-
ing 0.02 mg. of uric acid. With a cylinder, add 10 cc. of the urea-
cyanide solution. Mix by whirling the test-tubes at an angle of
about 60”. Add 4 cc. of uric acid reagent of double the regular
concentration. Let stand for about 20 minutes. Dilute to
volume, mix, and make the color comparison.
(20/x) X 4 gives the uric acid content of the blood in mg. per cent
when the standard is set at 20 mm. 2 represents the calorimetric
reading of the blood filtrate.
The uric acid reagent should be added at the same time to the
standard and the unknown and the tubes should be in a vertical
position when the reagent is added so that it does not flow down
one side.
The maximum obtainable color is not quite reached during a
20 minute waiting period, and if it is more convenient, one can just
116 Uric Acid Determination

as well wait for 40 minutes or longer before finishing the deter-

mination, but the calorimetric readings obtained at the end of 20
minutes are reliable.

Revised Micromethod for Determination of Uric Acid in Blood

While checking up the macromethod, parallel determinations
were also made with the micromethod described by Folin and Sved-
berg (3) and, as was to be expected, the variations and errors in
recovery experiments were distinctly larger than with the macro-

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method. In the micromethod one is working with less than one-
half as much uric acid as in the macromethod, and the errors due
to the color obtained from the reagents alone necessarily become
more significant. These difficulties disappeared after we had
succeeded in obtaining uric acid reagents which gave no color with
the urea-cyanide solution and water.
The modified micromethod corresponding to the macromethod
for the determination of uric acid in blood is as follows:
The blood filtrate for the determination of the uric acid by the
micromethod is obtained by adding 0.2 cc. of blood to 4 cc. of
tungstate-sulfate mixture in a centrifuge tube and 15 minutes
later adding 1 cc. of sulfuric acid and centrifuging.
The sulfate-tungstate solution contains 20 gm. of anhydrous
sodium sulfate and 3 gm. of sodium tungstate per liter. The
sulfuric acid solution is obtained by diluting 12 cc. of f N sulfuric
acid to 100 CC.
Transfer 4 cc. of the extract to a test-tube graduated at 25 cc.
To two other similar test-tubes add 4 cc. and 2 cc. (plus 2 cc. of
water) of a standard uric acid solution containing 1 mg. of uric
acid in 500 cc.
To each of the three tubes add 10 CG. of the same urea-cyanide
solution as is used in the macromethod and mix. Then add 4 cc.
of the concentrated uric acid reagent and let stand for 20 to 30
minutes. Dilute to volume, mix, and make the color comparison.
(20/x) X 5.2 (or 2.6) gives the uric acid in mg. per 100 cc. of
blood.
It will be noted that in this calculation the filtrate is regarded as
representing a dilution of 1 in 26 instead of 1 in 25 as given in the
original method.
 0. Folin

Determination of Uric Acid in Blood by Indirect Method

When uric acid is determined calorimetrically directly on blood
filtrates the process used is called the direct method. The process
involving a preliminary precipitation of the uric acid has therefore
become the “indirect method.” The direct method was first in-
troduced by S. R. Benedict, in 1922, and was promptly adopted by
Folin, but with the reservation that the indirect method should
be retained at least for check purposes. The indirect method is
still described in Folin’s “Laboratory manual,” but as a matter of

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history it fell at once into complete disuse and all subsequent efforts
have aimed at improving the direct method-and this notwith-
standing the fact that uric acid added to blood has never been
quantitatively recovered by the direct method when applied to
the filtrates from laked blood. By the application of a uric acid
reagent free from phenol reagent to the filtrates from unlaked
blood the errors and uncertainties of the direct method were finally
completely removed (if the recovery of added uric acid can be
accepted as an adequate criterion of trustworthiness). But this
time it was decided, nevertheless, to go back once more to the
preliminary precipitation of the uric acid with acid silver solution.
The completeness of the precipitation of uric acid by silver lac-
tate in the presence of a little chloride even from the most dilute
blood filtrates has never been questioned. It is probably the only
strictly quantitative known precipitant for uric acid. One excep-
tion could be taken to these statements. In 1922, it was admitted
that only 90 to 95 per cent of the dissolved urates could be recov-
ered from very dilute solutions in water (4). At that time it was
not so clearly understood as now that such losses as did occur
might be due to some factor interfering with the color reaction
rather than to incomplete precipitation. We have now reexam-
ined that problem and have found that the apparent losses are
due to the depressing effects of the dissolved silver on the color
reaction. By cutting down the chloride or by using a more
effective cyanide solution or by extracting the silver precipitate
with acid chloride the losses vanished.
In applying the indirect method to unlaked blood filtrates we
have used partly the whole silver precipitate dissolved in the urea-
cyanide solution and partly only the acid chloride extract from
the precipitate.
118 Uric Acid Determination

5 cc. of the blood filtrate in a 15 cc. centrifuge tube were pre-

cipitated with 2 cc. of the acid silver solution described below (p.
121) and centrifuged at once. The precipitates were then either
extracted with 1 cc. of a 10 per cent solution of sodium chloride in
0.1 N hydrochloric acid and washed with 4 cc. of water or were
dissolved in 10 cc. of urea-cyanide solution and the tubes rinsed
with 5 cc. of water. In other respects the determinations were
made as described above for the direct macromethod. The ex-

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TABLE I

Showing Recovery of Added Uric Acid and Also Agreement between Results
Obtained by Direct Method, Indirect Method, and Micromethod on
Filtrates from Unlaked Human Blood
The figures ind,icate the uric acid found before and after addin ‘!z 5 mg.
-
A+5 B B+5 C c+5 D D+5
_-
1 3.5 8.3 3.44 8.2 3.5 8.2 3.49 8.44
2 3.2 8.15 3.1 7.85 3.2 8.1 3.1 8.0
3 3.7 8.4 3.55 8.15 3.6 8.3 3.6 8.4
4 2.05 6.95 2.0 7.0 2.1 6.8 2.1 7.0
5* 3.2 8.2 3.2 7.65 3.2 7.65 3.3 8.0
6 3.5 8.36 3.5 8.2 3.5 8.1 3.5 8.4
7* 2.9 7.65 2.9 7.44 2.9 7.65 3.0 7.85
8 2.9 7.8 2.9 7.75 2.9 7.75 3.1 8.05
9 2.05 7.0 2.1 6.9 2.0 7.04 2.05 7.0
10 2.3 7.15 2.3 7.0 213 7.15 2.35 7.2
-
A =_ direct macromethod; B = determination on the silver precipitate;
C = determination on the extract from the silver precipitate; D = micro-
method.
* Not repeated with less than 5 cc. of filtrate.

traction method calls for two extra centrifugings and requires neat
work, but if carefully done gives all the uric acid.
All through the rather long period covered by this research, Dr.
C. L. Derick, of the Peter Bent Brigham Hospital, has supplied us
with all the hospital bloods we could use. In view of the very
great demand for space in this Journal the original plan of giving a
comprehensive series of analyses has been abandoned. In Table
I we give therefore in highly condensed form, only a few analyses
of bloods in which the uric acid was determined before and after
the addition of uric acid, by each of the four methods described
 0. Folin 119
above. The significance of these figures is so clear that comments
would seem to be superfluous. It need be stated, however, that
the first macrodeterminations made on the filtrates containing
added uric acid would sometimes be too low by 0.2 to 0.3 mg. per
cent, but these deficits disappeared when the determinations were
repeated with less than 5 cc. of the blood filtrates. In other
words, when 8 mg. or more of uric acid are found by the regular
method involving the use of 5 cc. of blood filtrate and the 0.02 mg.
standard, the determination should be repeated with less than 5

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cc. of blood filtrate.
For the sake of convenience and greater accuracy the added uric
acid was added to the tungstate-sulfate solution used for the pre-
cipitation of the protein and not directly to the blood, but previous
experience has shown that this makes no difference as to the
recovery.
Determination of Uric Acid in Urine
For clinical purposes the determination of uric acid in urine has
been almost entirely replaced by determinations of the uric acid
in blood and for this reason there has been very little recent in-
vestigation or revision of the current methods as applied to urine.
What little check work has been done has consisted of parallel
determinations on urine by the three currently accepted methods,
and since all of these have yielded substantially identical values,
the choice of working method adopted and recommended in differ-
ent laboratories has been based on minor considerations of
convenience or other supposed advantages. The fundamental
question whether the indirect methods involving a preliminary
precipitation do quantitatively recover uric acid from known solu-
tions has seemingly received very little attention. For the quan-
titative (100 per cent) recovery of minute amounts of uric acid
(0.02 mg.) from 5 to 7 cc. of solution the slightly acid silver reagents
are probably the only ones which are strictly dependable. No
figures seem to be available showing the minimum amount of uric
acid which can be quantitatively recovered by other precipitants.
The acid silver precipitation has been criticized from time to
time. Because the uric acid precipitation is always accompanied
by the precipitation of silver chloride it has been thought that the
uric acid is merely carried down by some adsorption process and
120 Uric Acid Determination

does not depend on the insolubility of silver mate in slightly acid

solutions. Silver urate is, however, less soluble in slightly acid
than in strongly ammoniacal solutions, though this advantage
may be lost if too strongly acid silver solutions are used. With
acid silver solutions and 0.5 mg. of uric acid one obtains the same
sort of gelatinous silver urate in the absence of a chloride as is
obtained with ammoniacal silver solutions. The silver chloride
serves two useful purposes. It protects the silver urate from the
oxidizing effects of the surplus silver salt and it makes it possible

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to isolate by means of the centrifuge the silver urate in such min-
ute quantities that they could not possibly be so isolated in the
absence of the silver chloride. The absence of the latter condi-
tion sets a limit to the minimum amount of uric acid which can
be isolated by means of the ammoniacal silver precipitation.
The amount of silver chloride precipitated together with the
uric acid must not be too large. It should not exceed that derived
from 10 mg. of NaCl and, preferably, it should be somewhat less,
because much silver tends to depress the color obtained from the
uric acid.
The zinc hydroxide precipitation resembles the acid silver pre-
cipitation in that it also yields a urate enclosed in another precipi-
tate which can be isolated by help of the centrifuge. But the zinc
urate is apparently somewhat more soluble than the silver urate
and hence does not yield 100 per cent recovery wit.h as little as 0.02
mg. of uric acid (from 5 cc. of the standard uric acid solution).
The fact that the precipitation of silver urate and zinc urate is
due to the insolubility of these salts rather than to adsorption
phenomena does not exclude the possibility that these precipitates
may contain materials, other than uric acid, capable of reacting
with t,he uric acid reagent. In the course of this work we have
in fact satisfied ourselves that the silver precipitates as heretofore
obtained from practically undiluted urine must have contained
some other reacting material besides uric acid (see p. 123). We
have not investigated the zinc precipitate or the ammoniacal silver
precipitate from this point of view.
Another series of criticisms of the acid silver precipitation of uric
acid is based on the darkening of the silver precipitates. It is
certainly true that this darkenin,, u if pronounced, is apt to involve
destruction and loss of uric acid. With a modicum of care this
 0. Folin 121
darkening is not encountered when working with blood filtrates or
diluted urine, but when working with 1 cc. of urine the silver pre-
cipitates are often dark gray in color, even when no undue lapse of
time or exposure to light intervenes between the precipitation and
the solution of the precipitate in the cyanide solution. Some
urines contain reducing materials which have a very rapid reduc-
ing effect on the surplus silver lactate and if delay or more than a
minimum exposure to light occurs, the precipitates will be black;
in such cases loss of uric acid is an inevitable result, as has been

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pointed out recently by Christman and his coworkers (5). If the
centrifuging is made immediately (as it always should be since the
uric acid precipitation is instantaneous) some black sediment may
form during the centrifuging process and it will be deposited partly
on top of the nearly white silver precipitate. The black sediment
so formed has little if any effect on the uric acid.
These facts simply constitute an unavoidable limitation on the
acid silver precipitation of uric acid in urine. They mean that
the centrifuging must be begun immediately after the addition
of the silver and that no undue delay may intervene from the time
the silver has been added until the precipitate has been completely
dissolved in the cyanide solution. Directions of this sort are just
as legitimate as are the directions to wait a certain length of time
for the development of the color reaction.
The degree of acidity at which the silver precipitate is produced
affects to a considerable extent the speed with which the surplus
silver is reduced, but there is a limit to the permissible acidity if
one does not w,ant to encroach in the slightest degree on the in-
solubility of silver urate.
Our uric acid precipitations on blood filtrates were made mostly
with a reagent, 1 liter of which contained 25 gm. of silver nitrate
and 5 cc. of lactic acid which had been partly neutralized by boil-
ing with 5 gm. of Na2C03. After a few days exposure to sunlight
and filtering, this reagent keeps fairly well. When used only for
occasional determinatiqns, it would not be safe to use this reagent
without filtering, and many efforts were made to find a suitable
silver solution of unlimited keeping quality, but no solution could
be found which was entirely satisfactory. The conditions were
altered, therefore, so as to permit the use of silver nitrate without
the addition of any organic acid,
122 Uric Acid Determination
One objective in this study was to adapt the method to the
needs of hospital laboratories where they now make at least
twenty-five uric acid determinations on blood for every such
determination made on urine. To this end we have sought to
make the method as nearly identical as possible with the method
used for blood, including the use of the same utensils and reagents,
except for one separate step, namely the preliminary precipitation
of the uric acid.
Of course, we have not been unmindful of the possibility that the

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preliminary precipitation of the uric acid might be omitted, thus
making its determination in urine completely similar to the deter-
mination in blood filtrates. Past efforts in this direction, made in
our laboratory, have always yielded unacceptable results, but it
seemed possible that by working with no more than 0.015 to 0.04
mg. of uric acid, the various disturbing factors might be reduced
to the vanishing point. These most favorable conditions have
not been used in any previously published direct method for the
determination of uric acid in urine. From theoretical considera-
tions both the positive and the negative sources of error, recently
elucidated by Christman and his coworkers (5), might become
negligible under the new conditions for the determination of minute
amounts of uric acid.
The indirect method, described below, should yield results, the
accuracy of which should be beyond question, and we hoped to
use it as a standard by which to judge the validity of the direct
method. Parallel determinations were made also by a suitable
modification of Folin’s method based on the use of 1 (or 2) cc. of
undiluted urine.
Conflicting results were obtained. The direct method and
Folin’s macromethod usually gave identical values with normal
urines, but these values were nearly always from 5 to over 10 per
cent higher than those obtained by the indirect method applied
to the diluted urines.
In order to settle the difficulty represented by these discrepan-
cies, we have adopted a procedure by which the validity of any
method for the determination of uric acid in human urine may be
controlled, without having recourse to the unconvincing process of
merely checking one method by another which may or may not be
more dependable.
 0. Folin 123
Transfer 0.3 gm. of oxalic acid and 3 gm. of Lloyd’s reagent
t,o a small flask, add 25 to 30 cc. of urine, shake immediately
for about 2 minutes, and filter. All of the uric acid (up to 25
mg.), nearly all of the creatinine, and very little else, are removed
by this treatment.
(a) If 2 cc. of this uric acid-free urine be diluted to 100 cc. with
water, 5 cc. will yield considerable color with 10 cc. of the cyanide
solution and 4 cc. of the uric acid reagent, whereas the silver pre-
cipitate from 5 cc. (plus 0.5 cc. of acid chloride solution) will yield

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no color.
(b) If 2 cc. of the undiluted uric acid-free urine be added to 5
cc. of water in a centrifuge tube and precipitated with acid silver
solution or silver nitrate, the precipitate will yield a color with
cyanide-carbonate-urea solution and uric acid reagent.
(c) If suitable amounts of standard uric acid solution are added
together with the water in these tests with uric acid-free urine, a
correct and dependable method must yield 100 per cent recovery
(and no more), just as it would, or would be expected to do, if no
uric acid-free urine were present. The validity of these tests is
increased, if one works, as we have done, with twice as much uric
acid-free urine as the volume of untreated urine actually used in a
determination.
By the application of these tests to the three methods included
in this investigation, it was found that the indirect method de-
scribed below, and that method only, yields 100 per cent recovery
(and no more) of uric acid added to uric acid-free urine. We are
therefore inclined to consider this as a standard method, the first
really standardized method for the determination of uric acid in
urine. Other methods in current use could of course be subjected
to the same test, but it has seemed better to leave that work to
those who are more interested in the other methods. In such
tests, the amount of uric acid to be added and recovered is of
course different for different methods.
In these experiments with uric acid-free urines, as in actual uric
acid determinations, the direct method, described below, has
yielded recoveries and values of about the same order of accuracy
as the corresponding figures by Folin’s older method based on un-
diluted urine. But, as already indicated, these figures are apt to
be too high by from 5 to 10 per cent and occasionally even more.
 Uric Acid Determination

And these errors are likely to be larger still, if the uric acid reagent
is not completely free from phenol reagent.
Indirect Method-Two solutions, in addition to those used in
blood analysis, are required; both keep indefinitely.
1. A 5 per cent solution of silver nitrate. This solution, even
if perfectly clear when first prepared, may develop a slight color on
standing. This color is most quickly produced by heating to 100”
for 2 hours in a flask covered with a beaker. After cooling, add a
few cc. of a solution containing 50 mg. of sodium chloride, shake

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thoroughly, and filter through a double layer of quantitative filter
paper until crystal-clear. Thereafter the solution will remain
perfectly colorless and need not be kept in brown bottles.
2. A solution containing 1 per cent of sodium chloride, 2 per cent
of crystallized sodium acetate, and 1 volume per cent of concen-
trated acetic acid (99 per cent).
Half fill a 100 cc. volumetric flask with water. With a Folin-
Ostwald pipette introduce 1 cc. of the urine. Add 10 cc. of the
chloride acetate solution and then, without shaking, so as to avoid
foaming, dilute to the mark with water and mix.
From this diluted urine transfer 5 cc. and 3 cc., plus 2 cc. of
water, to 15 cc. centrifuge tubes. Add to each 3 cc. of the silver
nitrate solution and centrifuge at once fairly rapidly, for 4 to 5
minutes, so as to get perfectly clear supernatant solutions. A few
tiny flakes may float on the surface, but these contain no uric acid.
Decant and drain over a sink. It is permissible to let cold tap
water rinse the mouth of the tube during the draining. Add to
each tube 10 cc. of the urea-cyanide solution described on p. 114.
Stir immediately (and simultaneously) with glass rods until the
two sediments have completely dissolved. Transfer the silver
cyanide solutions to test-tubes graduated at 25 cc., and rinse with
exactly 5 cc. of water. Mix by whirling at an angle of about 60”
until the solutions are visibly uniform. In another graduated
test-tube place 5 cc. of the standard uric acid solution containing
0.02 mg. of uric acid together with 10 cc. of the urea-cyanide solu-
tions and mix.
Add to each of the three tubes 4 cc. of the uric acid reagent de-
scribed on p. 111 and let stand for 15 to 25 minutes. Dilute to
volume, mix, and make the color comparison between the standard
and the unknown which is nearest to it in depth of color. When
 0. Folin 125

the standard is set at 20 mm., calorimetric readings between 35

and 10 mm. are acceptable.
Direct Method-Half fill a 100 cc. volumetric flask with water.
With a Folin-Ostwald pipette introduce 1 cc. of urine, dilute to
volume, and mix. Introduce into test-tubes graduated at 25 cc.
5 cc. of the diluted urine and 3 cc. of diluted urine plus 2 cc. of
water. To another graduated test-tube add 5 cc. of the standard
uric acid solution. Add 10 cc. of the urea-cyanide solution to
each, mix, and add 4 cc. of the uric acid reagent. Then finish the

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determination as in the indirect method.
The merely diluted urine used in the direct method can be used
also for the indirect method, by adding 0.5 cc. of the chloride-ace-
tate solution, before precipitating with the silver nitrate.
In conclusion, it must be stated that dependable uric acid values
cannot be obtained from urines which contain much bile. Neither
the indirect method described in this paper nor any other known
method will yield exactly 100 per cent recovery of uric acid added
to such urines after the preliminary removal of the preformed uric
acid. After a number of unsuccessful attempts were made to
produce a special method for use with such urines, the project was
abandoned, at least for the present. Taken by itself, the problem
represented by icteric urines is probably of slight importance, but
it makes one wonder whether there may be other urines, the uric
acid content of which cannot be determined with precision. Urines
of some herbiverous animals may well be a case in point. The
identification of such urines can probably always be made by
means of recovery experiments such as have been outlined in this
paper.
BIBLIOGRAPHY

1. Folin, O., J. Biol. Chem., 86, 186 (1930).

2. Folk, O., and Marenzi, A. D., J. Biol. Chem., 83, 109 (1929).
3. Folin, O., and Svedberg, A., J. Biol. Chem., 88,239 (1930).
4. Folin, O., J. Biol. Chem., 64, 169 (1922).
5. Christman, A. A., and Ravwitch, S., J. BioZ. Chem., 96, 115 (1932).
Christman, A. A., and Mosier, E. C., J. BioZ. Chem., 83, 11 (1929).
STANDARDIZED METHODS FOR THE
DETERMINATION OF URIC ACID IN
UNLAKED BLOOD AND IN URINE
Otto Folin and With the assistance of Margaret
Cushman
J. Biol. Chem. 1933, 101:111-125.

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