standardized methods for the determination of uric … · 2003-03-12 · this uric acid reagent...

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 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 reagent 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-cyanide and yielded therefore necessarily only a very narrow range of true proportionality between different amounts of uric acid. Apparent losses of as much as 15 per cent could thus be encountered, 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 dependable uric acid reagent. The subsequent check work gradually developed into revisions of the several uric acid methods sponsored by this department.

Revised Process for 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-

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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 phenolphthalein. 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.

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 gradually 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


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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 red paper (30 cc. of 85 per cent phosphoric acid to 100 gm. of sodium 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


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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 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 hydroxide. 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, because 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-


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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 proportionality, 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, however, to the amount of uric acid reagent to be used, even with t’he efficient urea-cyanide solution, without getting turbidities when 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 increased. 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 containing 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


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as well wait for 40 minutes or longer before finishing the determination, 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 macromethod. 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.


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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 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 lactate 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 recovered 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.


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

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.

1 3.5 8.3 3.44 8.2 3.5 8.2 2 3.2 8.15 3.1 7.85 3.2 8.1 3 3.7 8.4 3.55 8.15 3.6 8.3 4 2.05 6.95 2.0 7.0 2.1 6.8 5* 3.2 8.2 3.2 7.65 3.2 7.65 6 3.5 8.36 3.5 8.2 3.5 8.1 7* 2.9 7.65 2.9 7.44 2.9 7.65 8 2.9 7.8 2.9 7.75 2.9 7.75 9 2.05 7.0 2.1 6.9 2.0 7.04

10 2.3 7.15 2.3 7.0 213 7.15

A+5 B B+5 C c+5 D D+5 _ -

3.49 3.1 3.6 2.1 3.3 3.5 3.0 3.1 2.05 2.35

-

8.44 8.0 8.4 7.0 8.0 8.4 7.85 8.05 7.0 7.2

-

A =_ direct macromethod; B = determination on the silver precipitate; C = determination on the extract from the silver precipitate; D = micromethod.

* 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


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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 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 precipitation 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 investigation 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 different 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 solutions has seemingly received very little attention. For the quantitative (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


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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 to isolate by means of the centrifuge the silver urate in such minute 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 precipitation in that it also yields a urate enclosed in another precipitate 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


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darkening is not encountered when working with blood filtrates or diluted urine, but when working with 1 cc. of urine the silver precipitates 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 reducing 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 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 insolubility 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 boiling 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,


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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 preliminary precipitation of the uric acid might be omitted, thus making its determination in urine completely similar to the determination 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 considerations 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.


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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 precipitate from 5 cc. (plus 0.5 cc. of acid chloride solution) will yield 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 described 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 undiluted urine. But, as already indicated, these figures are apt to be too high by from 5 to 10 per cent and occasionally even more.


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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 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 concentrated 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 solutions and mix.

Add to each of the three tubes 4 cc. of the uric acid reagent described 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


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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 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-acetate 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).


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CushmanOtto Folin and With the assistance of Margaret

UNLAKED BLOOD AND IN URINEDETERMINATION OF URIC ACID IN

STANDARDIZED METHODS FOR THE

1933, 101:111-125.J. Biol. Chem.

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