COLORIMETRIC DETERMINATION OF URIC ACID.

COLORIMETRIC DETERMINATION OF URIC ACID. ESTIMATION OF 0.03 TO 0.5 MG. QUANTITIES BY A NEW METHOD. BY J. LUCIEN MORRIS AND A. GARRARD MACLEOD. (From the Biochemistry Laboratory of the School of Medicine, Western Reserve University, Cleveland.) (Received for publication, October 27, 1921.) Determinations of uric acid, which depend upon the direct weighing of the substance as such or upon an estimation of the nitrogen content of an insoluble salt, have been largely replaced by methods which quantitatively measure its oxidation. Any of these procedures, whether volumetric or calorimetric, are useful in proportion to their success in (a) separating the uric acid from other substances which might give a value in the oxidation reaction and in (b) making the actual conditions of the reaction as specific as possible for uric acid. Volumetric methods, in most cases, have not been sufficiently sensitive for satisfactory application to less uric acid than is found in 100 cc. of urine. Also the means for preliminary precipitation of uric acid made use of in these methods were crude. One of us1 determined conditions for a complete precipitation of uric acid in any amount with zinc salts, and found conditions under which permanganate oxidation could be used successfully on such quantities of uric acid as are found in 5 cc. of urine.* The much smaller amount of uric acid found in blood is below the limit of accuracy which it is possible to attain with the zinc precipitation-permanganate oxidation procedure. Folin and Macallum3 recognized the need of separation of uric acid from interfering substances and attempted the removal of polyphenols from urine residues before oxidizing uric acid with 1 Morris, J. L., J. Biol. Chem., 1916, xxv, 205. ZMorris, J. L., J. Biol. Chem., 1919, xxxvii, 231. 3 Folin, O., and Macallum, A. B., Jr., J. Biol. Chem., 1912-13, xiii, 363 55

Calorimetric Estimation of Uric Acid alkali phosphotungstate. Folin and Denis improved this separation by their adaptation of the SalkowskP precipitation of uric acid to precede the phosphotungstate oxidation. The method described in this paper makes use of the zinc salt separation of uric acid which has proved very satisfactory in our hands and determines the uric acid so separated by a new calorimetric procedure which possesses the double advant,age of greater specificity and greater color obtainable for unit weight of uric acid. With slight variations it is equally applicable to urine and blood. In the latter case we have removed the proteins by the tungstic acid precipitation of Folin and Wu6 and found the procedure satisfactory for our purpose. The results which we have obtained with the new method are, we believe, more dependable and uniform than the usual ones obtained with any of the procedures based upon silver precipitation. One cause for irregularities in t,he results obtained by means of the silver methods is inherent in the metal used. Silver solutions, even when protected from light, soon become clouded with, and eventually precipitate, a form of reduced silver. The acid silver lactate solution used by Folin and Wu,6 is noticeably cloudy soon after preparation and develop s a heavy precipitate on standing. The ammoniacal silver magnesium solution of Benedict and Hitchcock also forms a scum around the neck of bottles, spouts of dropping bottles, etc., which occasionally contaminates the reagent added and thus gives erroneous results. This reduced silver, when taken up in sodium cyanide and the mixture made alkaline with sodium carbonate and phosphotungstic acid reagent added, gives a deep blue color even when taken in small amounts. (The color can also be developed by reversing the order of these reagents.) This possibility of the presence of such a substance is sufficient to cast doubt upon any determination which makes use of a silver precipitation. It is evident that the urine procedure of Folin and Wus is particularly 4 Folin, O., and Denis, W., J. Biol. Chem., 1912-13, xiii, 469. 6 Salkowski, E., Vi~chows Arch. path. Anat., 1870, lii, 58. Ludwig, E., Wien. med. Jahrb., 1884. 6 Folin, O., and Wu, H., J. Biol. Chem., 1919, xxxviii, 81. Benedict, S. R., and Hitchcock, E. H., J. Biol. Chem., 1915, xx, 019. * Folin, O., and Wu, H., J. Biol. Chem., 1919, xxxviii, 459.

J. L. Morris and A. G. Macleod 57 open to criticism on this point for the reason that the entire precipitate is dissolved in sodium cyanide previous to the development of color. The extraction step of their blood procedure to a large extent eliminates this error. Myers9 suggestion in regard to this method that cyanide be added before the second centrifuging can hardly be accepted as an improvement when considered in the light of these facts. We have found that the reliability of these methods is greatly increased when care is taken t,o use only perfectly clear silver reagents. Another possible source of error in the Folin and Wu procedure is a common impurity found in many of the best grades of sodium sulfite we could obtain. Three out of four lots of this salt, from as many sources, gave a react ion with the uric acid reagents. The color which developed in each instance was sufficient to introduce a considerable error if the su1fit.e had been used in the amount prescribed for the determination. Neither silver nor sulfite is used in the new method described in this paper. The new method requires the use of no metal which may exist in a reduced form or reagent which is likely to contain any interfering impurity. Fortunately, in addition to their adaptability to the purpose, there is the additional advantage that all the reagents used are relatively inexpensive. The use of potassium cyanide suggested by Benedict and Hitchcock for tying up the silver greatly improved the earlier Folin-Denis method. We successfully applied cyanide for the purpose of forming a double radical with zinc and found it useful as well in two other particulars. Used in larger amounts its alkalinity is sufficient for the complete and rapid development of the color when the actual molecular concentration of alkali is still less than one-third that used in the Benedict-Hitchcock procedure. The second advantage in the use of cyanide is the very marked increase of color which is obtained from a given amount of uric acid. That this increase is due to the uric acid reduction of phosphotungstate, while the cyanide only accelerates the reaction, is shown by results of the kind typified in Table I. The negative result of Flask 10 shows that the cyanide will not act directly on the phosphotungstate. The 18 per cent increase of color in 9 Myers, V. C., J. Lab. and Clt n. Med., 1919-20, v, 499.

58 Calorimetric Estimation of Uric Acid TABLE I. Effect of Sodium Cyanide upon Phosphotungstate Oxidation of Uric Acid. Flask No. Uric acid. 5 per cent sodium cyanide. Value found. 1 2 3 4 5 6 7 8 9 10 ma. cc. mm. ml 0.25 None. 30.0 1.00 X 0.25 0.25 0.05 25.5 1.18 X 0.25 0.25 0.25 21.1 1.42 X 0.25 0.25 0.50 20.4 1.47 X 0.25 0.25 1.00 19.2 1.56 X 0.25 0.25 2.00 16.1 1.86 X 0.25 0.25 4.00 14.1 2.13 X 0.25 0.25 8.00 12.5 2.40 X 0.25 0.25 12.00 12.2 2.46 X 0.25 None. 10.00 No color. None. Color developed in each case by addition of 1 cc. of phosphotungstic acid reagent and 10 cc. of 20 per cent sodium carbonate. All made up to 50 cc. volume after 10 minutes and compared with No. 1. TABLE Effect of Sodium Cyanide upon Arsenotun@ate Oxidation of Uric Acid. Flask No. Uric acid. 10 per cent Calorimeter iodium cyanide. reading. Value found. m0. cc. mm. ml?. 1 0.4 1.0 148.0 0 054 2 0.4 1.5 52.0 0 156 3 0.4 2.0 34.4 0.236 4 0.4 2.5 30.0 0.266 5 0.4 3.0 27.1 0.287 6 0.4 3.5 25.9 0.309 7 0.4 4.0 24.7 0.324 8 0.4 4.5 23.5 0.340 9 0.4 5.0 21.3 0 376 10 0.4 7.5 20.2 0 396 11* 0.4 10.0 20.0 0 400 12 0.4 15.0 20.0 0 460 13 None. 10.0 I No color. None. II. - Color developed by addition of 2 cc. of arsenotungstic uric acid reagent and the amount of cyanide indicated for each flask. * No. 11 was used as a standard and the others compared with it.

J. L. Morris and A. G. Macleod 59 Flask 2, due to 1 drop of cyanide, recalls an increase of the same amount which Benedict and Hitchcock observed under similar conditions (2 drops of cyanide and 15 cc. of carbonate in a 50 cc. flask). Flasks 8 and 9, with 50 per cent more cyanide in the latter, showed nearly a constant value with two and a half times as much color as Flask 1. Obviously the cyanide causes displacement of the equilibrium so as to approach the maximum amount of the blue compound for these conditions. Attempts to determine the full extent of this effect met with little success at first for the decrease of carbonate and increase of cyanide was required to accomplish further deepening of the color, and this change in the alkalies caused precipitation before the time for development of color had passed. Also we found that a certain amount of carbonate was necessary to prevent the blue color which otherwise develops when phosphotungstic acid reagent is made alkaline with sodium cyanide. We had previously made many conjugated tungstic acids, substituting analogous acids for phosphoric, concerning which we expect to make a more extended report soon. Upon trial we found that one of these, arseno-18-tungstic acid, in addition to other desirable properties, gave absolutely no color with sodium cyanide even when no other alkali than the cyanide was present, and did not precipitate in the presence of large amounts of cyanide. By its use we made further observations of the effect of cyanide upon the oxidation of uric acid by tungsten compounds and secured the results recorded in Table II. It will be noted that the amount of uric acid used in each flask was 0.4 mg., for our experience had shown that the color obtained from that quantity was of a desirable depth for calorimetric comparisons. It is also apparent that the color in Flasks 1 to 10 increased with additional quantities of cyanide, the increments being progressively smaller. In Flasks 11 and 12 there was practically no increment, though the quantities of cyanide were, respectively, 133 and 200 per cent of that in Flask 10. Evidently the conditions had been reached by which the oxidation of uric acid by a tungsten compound was complete. The determination of relations between the amount of color obtainable by the new arsenotungstate-cyanide method and the former phosphotungstate-carbonate procedures could not be abso-

Calorimetric Estimation of Uric Acid lute, owing to the great difference in the concentrations of the reaction liquids. Approximate cpmparisons show that the new uric acid method gives 3.3 times the color of the Folin-Macallum- Denis method, 2.8 times that of the Benedict-Hitchcock procedure, and 2.5 times that of the Folin-Wu method. There is, of course, a great mechanical advantage in being able to get three times as much color from the very limited amount of uric acid in blood and other body fluids. In addition, there is greater satisfaction in the realization that the deeper color made possible by the new method represents a truer value of the uric acid present in that it is the result of a complete chemical action and not subject to the disturbing variations which may occur in procedures which depend upon artificially maintained equilibria. Finally, the use of sodium cyanide in the manner described in this paper possesses another advantage in the selective application of its driving power. How exclusive this selective action of cyanide, whether there are other substances present in body fluids upon which it will act, and what the nature of the driving power may be are questions now being investigated further in this laboratory. Method. Reagents. Preparation of Arseno-lbtungstic Acid SoZ ution.-boil a mixture of 100 gm. of hydrated sodium tungstate (Naz W04.2H~ 0), 125 gm. of arsenic acid anhydride (As2 Q6), and 650 cc. of water for 2 to 4 hours in a flask. If the reagent so formed has a blue or green color after it has boiled the required time, it should be decolorized by boiling with sufficient bromine water to make the color a clear yellow or yellowish brown.10 After boiling off any excess bromine add distilled water to make the volume 1 liter. The arsenotungstic acid reagent so prepared is a somewhat lighter color than the phosphotungstic acid reagent. Other Reagents Required.-2.5 per cent zinc chloride solution; 10 per cent sodium carbonate solution (if monohydrated sodium carbonate is used, allowance must be made for the water of crystallization); 10 per cent hydrochloric acid solution; 10 per cent sodium cyanide solution; standard uric acid solution (phosphate solution of Benedict-Hitchcock). For Removal of Proteins.6-10 per cent sodium tungstate; +$ N sulfuric acid, within 5 per cent by titration; solid potassium oxalate. lo Decolorization in this way is desirable for any conjugated tungstic acid which is to be used for calorimetric work. A dark blue or green reagent (either phospho- or arsenotungstic acid) introduces a very noticeable error when used where the color to be read is light.

J. L. Morris and A. G. Macleod 61 Determination. The method is essentially the same when used in uric acid solutions of such different concentration as urine and blood. Convenient quantities of reagents and choice of volumetric flasks which facilitate calorimetric comparison are the principal point)s of difference in the procedures described. Procedure as Used in Urine.-Pipette 1 cc. of urine into a 50 cc. centrifuge tube and dilute with distilled water to about 40 cc. Add 1 cc. of 2.5 per cent zinc chloride and mix with a stirring rod. Add 1.0 cc. of 10 per cent sodium carbonate which should make the solution alkaline to litmus and stir thoroughly. Centrifuge for about 2 minutes, drain off, and discard the supernatant liquid. Dissolve the residue, with stirring, in 3 or 4 drops of 10 per cent hydrochloric acid, dilute with 5 cc. of water, add 10 cc. of 10 per cent sodium cyanide, and transfer quantitatively to a 100 cc. volumetric flask, and dilute to about 60 cc. If 1 cc. of urine contains more than 0.5 mg. of uric acid the amount of cyanide should be doubled (20 cc.) and a 200 cc. flask used. In this case dilute to about 120 cc. To prepare a standard containing 0.2 mg. in 50 cc. pipette 1 cc. of the phosphate standard solution into a 50 cc. volumetric flask and 25 to 30 cc. of distilled water and 5 cc. of 10 per cent sodium cyanide. Develop the color in both by addition of the arseno-1%tungstic acid reagent, 1 cc. to the standard (50 cc. flask), 2 cc. to the unknown if in 100 cc. flask or 4 cc. if in the 200 cc. flask. Shake, dilute to volume, let stand 2 or 3 minutes, and compare in the calorimeter. The color develops with such rapidity that the time interval indicated is sufficient if the standard and the unknown are made simultaneously. If, for any reason, they are not so prepared, it is best to allow 10 minutes to elapse before making the color comparison. Procedure as Used in Blood.-Collect oxalated blood in the usual manner, drawing the biood from a vein into a weighed flask containing 2 mg. of potassium oxalate for each cubic centimeter of the sample taken. After determining the amount of blood by weight, pour it into seven times its volume of distilled water, add 1 volume of 10 per cent sodium tungstate solution and then, while shaking, run in slowly 1 volume of $ N sulfuric acid. Shake for several minutes and filter (precipitation method of Folin and WU~). Pipette 25 cc. of the clear filtrate (corresponding to 2.5 cc. of blood) into a 50 cc. centrifuge tube and dilute with distilled water to about 40 cc. Add 1 cc. of 2.5 per cent zinc chloride and mix with a stirring rod. Add 1.0 cc. of 10 per cent sodium carbonate to make just alkaline to litmus and stir thoroughly. Centrifuge for about 2 minutes, drain off and discard the supernatant liquid. Dissolve the residue with stirring in 3 or 4 drops of 10 per cent hydrochloric acid, dilute with 5 cc. of water, and add 2.5 cc. of 10 per cent sodium cyanide and transfer quantitatively to a 25 cc. volumetric flask. Prepare two standards containing 0.1 and 0.2 mg. in 50 cc., by pipetting 0.5 and 1 cc. of the phosphate standard solution into two 50 cc. volumetric flasks. Add about 30 cc. of distilled

62 Calorimetric Estimation of Uric Acid water and 5 cc. of 10 per cent sodium cyanide to each. Develop the color by the addition of the arseno-18-tungstic acid reagent, 0.5 and I cc. respectively into the unknown and standards. If the color has been developed simutaneously, shake, dilute to volume, let stand a minute or two, and compare in the calorimeter; if not, the same lapse of time should be allowed as indicated in the case of urine. Both procedures are adapted to the quantities of uric acid found in the largest number of urine and blood samples analyzed by us. In a very few cases we found it advantageous to choose volumetric flasks of a larger or smaller size to contain the unknown. This may be done with good results if the concentrations of arseno-l8-t,ungstic acid and sodium cyanide are kept comparable. For this purpose the following simple rule must be observed : 100 cc. flask contains 10 cc. of 10 per cent sodium cyanide and 2 cc. of arseno-1%tungstic acid reagent; 50 cc. flask contains 5 cc. of 10 per cent sodium cyanide and 1 cc. of arseno-1%tungstic acid reagent; 25 cc. flask contains 2.5 cc. of 10 per cent sodium cyanide and 0.5 cc. of arseno-18- tungstic acid reagent. TABLE III. Urine Comparative Estimations of Uric Acid in Urine. m0. 390 752 514 547 Folin-Wu method. New method. m0. mo. 375 388 625 702 492 533 498 508 Table III presents comparative uric acid results for several urine specimens. In their analyses we used the method described here, the Folin-Wu method, and the Benedict-Hitchcock procedure. Precaution to use only the clearest possible, silver reagents had the effect of practically eliminating irregularities due to reduced silver. In spite of similar precautions in work with blood specimens there were marked irregularities. Upon undertaking the work of their explanation we were led into various problems connected with the chemistry of the methods and the chemical nature of the uric acid present in blood. Some of the results obtained appear in the following paper.l 11 Morris, J. L., and Mncleod, A. G., J. Biol. Chcm., 19 22, 1, 65.

J. L. Morris and A. G. Macleod 63 SUMMARY. Combination of zinc precipitation with a new calorimetric method has made possible the estimation of very small quantities of uric acid. Arseno-1%tungstate proves a great improvement over phospho-1%tungstate of earlier methods. Sodium cyanide is used as the oniy alkali for development of coior and serves to bring about the complete oxidation of uric acid. In comparison with the amount of color obtained in the methods which depend upon oxidation to a point of equilibrium, the new conditions of complete oxidation permit the development of three times as much color per unit weight of uric acid. The same conditions that bring about completion of the reaction are also responsible for greater speed in reaching the maximum color and in a very marked permanency of the color. The use of cyanide as alkali practically eliminates the precipitation of various compounds in the colored liquid which, next to fading, was the most serious difficulty accompanying the use of carbonate. Precipitation of uric acid with zinc salts lends itself just as well to the subsequent formation of a double radical with cyanide as in the case with silver methods. In addition there is excluded all possibility of a reduced metal giving erroneous results in the later oxidation reaction. Finally, the number of reagents required is small, they are easily prepared and the use of inexpensive zinc chloride instead of expensive silver salts makes the determination much more desirable, especially where many analyses are run, as in medical classes and extended research work on purines.

COLORIMETRIC DETERMINATION OF URIC ACID: ESTIMATION OF 0.03 TO 0.5 MG. QUANTITIES BY A NEW METHOD J. Lucien Morris and A. Garrard Macleod J. Biol. Chem. 1922, 50:55-63. Access the most updated version of this article at http://www.jbc.org/content/50/1/55.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/50/1/55.citation.full.html #ref-list-1